# Patent

## Title
Severe acute respiratory syndrome DNA compositions and methods of use

## Abstract

The present invention is directed to raising a detectable immune response in a vertebrate by administering in vivo, into a tissue of the vertebrate, at least one polynucleotide comprising one or more regions of nucleic acid encoding a SARS-CoV protein or a fragment, a variant, or a derivative thereof. The present invention is further directed to raising a detectable immune response in a vertebrate by administering, in vivo, into a tissue of the vertebrate, at least one SARS-CoV protein or a fragment, a variant, or derivative thereof. The SARS-CoV protein can be, for example, in purified form. The polynucleotide is incorporated into the cells of the vertebrate in vivo, and an immunologically effective amount of an immunogenic epitope of a SARS-CoV polypeptide, fragment, variant, or derivative thereof is produced in vivo. The SARS-CoV protein is also administered in an immunologically effective amount.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 60/470,820, filed May 16, 2003, and U.S. Provisional Application Ser. No. 60/482,505, filed Jun. 26, 2003, which are both incorporated herein by reference in their entirety.


BACKGROUND OF THE INVENTION
The present invention relates to a novel coronavirus (referred to herein as SARS-CoV) and to SARS-CoV vaccine compositions and methods of treating or preventing SARS-CoV infection and disease in mammals. SARS-CoV was discovered in March of 2003, in association with Severe Acute Respiratory Syndrome (SARS), a newly emerging infectious disease of global importance.
The recognition of SARS has led to activation of a global response network, with resultant travel restrictions, major quarantine, and closure of health care facilities. As of May 14, 2003, 7628 cases and 587 deaths from SARS have been reported from 29 countries. Initial reports of an atypical pneumonia began to surface in November of 2002 from the Guangdong province of China. This early outbreak reportedly involved 305 people, many of whom were healthcare workers. On Feb. 21, 2003, a healthcare worker from Guangdong traveled to Hong Kong, where his pre-existing cold symptoms escalated and he was hospitalized for acute respiratory distress. From Hong Kong, the illness spread rapidly throughout Southeast Asia and to Canada from this one index case. Seven individuals can be linked to the index case through a stay on the ninth floor of the hotel he occupied during his first night in Hong Kong. Infected persons from three hospitals in the Hong Kong metropolitan area are traceable to this index case as well. The primary mode of transmission has been either person-to-person contact or droplet transmission. Two notable exceptions to this are the hotel in Hong Kong, where direct human contact cannot be established for all those infected, and the Amoy Garden apartment buildings where more than 221 residents have been infected. In the outbreak at the Amoy Garden apartments, an unknown environmental factor is suspected of playing a role in transmission.
The incubation period ranges on average between two and seven days. Onset of symptoms begins with a high fever associated with chills and rigors. Additional symptoms at onset may include headache, malaise, myalgia, mild respiratory symptoms and more rarely common cold symptoms such as sore throat and runny nose. After this initial three to seven day period, additional lower respiratory symptoms appear including dry, non-productive cough and dyspnea. Initial chest x-rays reveal small, unilateral, patchy shadowings that progress quickly to bilateral, diffuse infiltrates. Preliminary. Outbreak news: severe acute respiratory syndrome (SARS). Wkly. Epidemiol. Rec., 2003: 81-88 (2003). The median duration of symptoms in a small epidemiologic study was 25.5 days. Tsang, K.W., et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong, N. Engl. J. Med. (2003). The severity of illness can range widely from a mild illness to acute respiratory failure resulting in death. Patients with a significant co-morbidity, such as diabetes, or who are older, are more likely to suffer from a severe form of the disease. Questions remain as to why some patients become infected, while others who have intimate contact with infected individuals are spared. It does appear that patients are very contagious at the onset of symptoms. Studies from hospitals in Hong Kong and Hanoi have shown attack rates >56% among healthcare workers caring for SARS patients. It is unclear at this time whether individuals are contagious during the incubation phase.
Important Features of Coronaviruses
Coronaviruses are large, enveloped, positive-stranded RNA viruses, and they are known to elicit coincident diseases in animals and humans. Mature human coronavirus (HCoV) virions are approximately 100 nm-diameter enveloped particles exposing prominent spike (S), hemagglutinin-esterase (HE) (in some types of coronaviruses), envelope (E) and membrane (M) glycoproteins. Each particle contains an approximately 30 kilobase (kB) RNA genome complexed with an approximately 60 kilodalton (kD) nucleoprotein (N). Fields, B.N. VIROLOGY New York: Lippincott, Williams & Wilkins, (Fields, B.N., ed. 2001). All of the above references are herein incorporated by reference in their entireties.
The S proteins of HCoV's have two large domains, the variable S1 domain responsible for host cell binding, Breslin, J.J. et al. J Virol. 77: 4435-8 (2003), and the S2 domain containing a heptad coiled-coiled structure reminiscent of those involved in fusion in HIV and influenza. Yoo, D.W. et al. Virology 183: 91-8 (1991). The HCoV-229E, group I S protein appears to bind to the human aminopeptidase N glycoprotein, Yeager, C.L., et al. Nature 357: 420-2 (1992); Bonavia, A. et al. J. Virol. 77: 2530-8 (2003), whereas the HCoV-OC43 strain (HCoV-OC43, group II) may bind via sialic acid moieties. Vlasak, R. et al. Proc. Natl. Acad. Sci. USA 85:4526-9 (1988). The genetic variability between strains of coronavirus has not been thoroughly evaluated, although only minor variability has been observed in the S protein in the small number of strains sequenced. Hays, J.P. and Myint, S.H. J. Virol. Methods 75: 179-93 (1998); Kunkel, F. and Herrler, G. Arch. Virol. 141: 1123-31 (1996). Most coronaviruses are not only species specific, but also somewhat tissue tropic. This tropism is mostly related to changes in the S protein. Sanchez, C.M. et al. J Virol. 73: 7607-18 (1999). Examples of such coronavirus tropism changes are the in vitro demonstration that tropism can be experimentally manipulated by genetically replacing a feline S protein with a mouse S protein, and the natural emergence of the porcine respiratory coronavirus (PRCoV) from the transmissible gastroenteritis virus of swine (TGEV) strain merely through a deletion of a region in the S protein. Haijema, B.J. et al. J. Virol. 77:4528-38 (2003); Page, K.W. et al. J. Gen. Virol. 72:579-87 (1991); Britton, P. et al. Virus Res. 21:181-98 (1991). All of the above references are herein incorporated by reference in their entireties.
The recently discovered novel coronavirus, SARS-CoV, appears to be a new member of the order Nidovirales. Concerted efforts by many laboratories worldwide has led to the rapid sequencing of various strains of SARS-CoV, including CUKH-Su10 (GenBank Accession No. AY282752), TOR2 (GenBank Accession No. AY274119 and NC.sub.-004781), BJ01 (GenBank Accession No. AY278488), CUHK-W1 (GenBank Accession No. AY278554), Urbani (GenBank Accession No. AY278741) and HKU-39849 (GenBank Accession No. AY278491). The Urbani strain of SARS-CoV, sequenced by the Centers for Disease Control in Atlanta, Ga., is a 29,727-nucleotide, polyadenylated RNA with a genomic organization that is typical of coronaviruses: 5′-replicase, spike (S), envelope (E), membrane (M)-3′. Rota et al., Science 300:1394-1399 (2003), (hereinafter “Rota et al.”). In addition, there are short untranslated regions at both termini, and open reading frames (ORFs) encoding non-structural proteins located between S and E, between M and N, or downstream of N. Rota et al. The hemagglutinin-esterase (HE) gene found in group 2 and some group 3 coronaviruses was not found in SARS-CoV. Rota et al. Sequencing of the Tor2 SARS-CoV strain by a collaboration of researchers in British Columbia, Canada, yielded a genomic sequence that differed from the Urbani SARS-CoV strain by eight nucleotide bases. Marra et al., Science 300:1399-1404 (2003), (hereinafter “Marra et al.”). A comparison of the HKU-39849 and CUHK-W1 SARS-CoV strains also differed from the Urbani sequence by 10 or fewer nucleotide bases. Rota et al. All of the above references are herein incorporated by reference in their entireties.
Phylogenetic analyses indicate that, based on the genetic distance between SARS-CoV and other known coronaviruses in all of their genetic regions, no large region of the SARS-CoV genome was derived from other known viruses, and that SARS forms a distinct group within the genus Cornavirus. Rota et al.; Marra et al. The analyses also showed greater sequence conservation among enzymatic proteins of SARS-CoV than among the S, N, M, and E structural proteins; and, while there were regions of amino acid conservation within each protein as between SARS-CoV and other coronaviruses, the overall similarity was low. Rota et al. All of the above references are herein incorporated by reference in their entireties.
A virus, almost identical to the human SARS-CoV virus, has been isolated from rare Chinese masked palm civet cats. This virus is believed to be identical to human SARS-CoV except for a 29 nucleotide deletion in the region encoding the N protein of the virus. Walgate, R. “Human SARS virus not identical to civet virus” The Scientist, May 27, 2003, incorporated herein by reference in its entirety.
Coronavirus Vaccine Candidates
Because SARS-CoV was so recently discovered, there are no vaccines against the virus. The approach to vaccine development can, however, be partially guided by the results of past studies in animals, of which three diseases have received the greatest attention. These are transmissible gastroenteritis virus (TGEV) in swine, feline infectious peritonitis virus (FIPV), and avian infectious bronchitis virus (IBV). Of note, none of the vaccines, most of which have been attenuated vaccines, have proven to be highly efficacious except for inactivated IBV. Enjuanes, L. et al., Adv. Exp. Med. Biol. 380: 197-211 (1995). The FIPV vaccine is a live attenuated virus that has provided minimal efficacy in field trials, and the TGEV vaccine has also been problematic. Scott, F. W., Adv. Vet. Med. 41:347-58 (1999); Sestak, K. et al., Vet. Immunol. Immunopathol. 70:203-21 (1999). All of the above references are herein incorporated by reference in their entireties.
In the TGEV model, the major focus has been on neutralizing antibody directed at the S glycoprotein. Sestak, K. et al., Vet. Immunol. Immunopathol. 70: 203-21 (1999); Tuboly, T. et al. Vaccine 18: 2023-8 (2000); Shoup, D.I. et al. Am. J. Vet. Res. 58: 242-50 (1997). Protection has also been associated with antibodies in IBV and bovine coronavirus. Mondal, S. P. et al. Avian. Dis. 45:1054-9 (2001); Yoo, D.W. et al. Virology 180: 395-9 (1991). In fact, in most of the animal models, control of coronavirus infection can be due to antibodies reactive to the N-terminal region of the S protein. Gallagher, T.M. and Buchmeier, M.J. Virology 279: 371-4 (2001); Tuboly, T. et al. Arch. Virol. 137: 55-67 (1994). In one study of respiratory bovine coronavirus, antibody appearance to the S and N proteins was correlated with recovery. Lin, X.Q. et al. Arch. Virol. 145: 2335-49 (2000); Passive transfer studies have also been successful and demonstrated the value of humoral immune responses. Enjuanes, L. et al., Adv. Exp. Med. Biol. 380: 197-211 (1995); Spaan, W.J. Adv. Exp. Med. Biol. 276: 201-3 (1990). All of the above references are herein incorporated by reference in their entireties.
Cell-mediated immune responses have been most clearly detected in coronaviruses against the S, M and N proteins. Spencer, J.S. et al. Adv. Exp. Med. Biol. 380: 121-9 (1995); Collisson, E.W. et al. Dev. Comp. Immunol. 24: 187-200 (2000); Stohlman, S.A. et al. Virology 189: 217-24 (1992). In one study, the use of a DNA vaccine encoding the carboxyl terminus of the N gene of IBV, which induced cytotoxic T cell (CTL) activity, was able to decrease virus titers by 7 logs in target organs. Seo, S.H. et al. J. Virol. 71: 7889-94 (1997). Some protection was also noted in a DNA vaccine encoding the N protein in the Mouse Hepatitis Virus (MHV) model. Hayashi, M. et al. Adv. Exp. Med. Biol. 440:693-9 (1998). There is also some evidence that CTL may be involved in the control of MHV, and prevent the development of persistent infection and neuropathology. Pewe, L. and Perlman, S. Virology 255: 106-16 (1999); Pewe, L. et al. J. Virol. 71: 7640-7 (1997). All of the above references are herein incorporated by reference in their entireties.
A large number of coronavirus challenge studies have been conducted in humans by Tyrrell and colleagues, in which the subjects were inoculated intranasally and followed. Callow, K.A. et al. Epidemiol. Infect. 105: 435-46 (1990); Bende, M. et al. Acta Otolaryngol. 107: 262-9 (1989). Such challenge studies will clearly be impossible for the much more serious SARS-CoV virus. The presence of antibodies to the challenge strain did not prevent infection or disease, even in the face of rising neutralizing antibody titers. However, a second infection with similar strains led to decreased symptoms, revealing persistence of immunity against homologous challenge. Reed, S.E. J. Med. Virol. 13: 179-92 (1984). Also, the 2-4 year cyclical nature of the disease points to some persistence of immune response over time. Reed, S.E. J. Med. Virol. 13: 179-92 (1984); Hendley, J.O. et al. Am. Rev. Respir. Dis. 105: 805-11 (1972); Evans, A.S. and Kaslow, R.A. VIRAL INFECTIONS OF HUMANS. 4th ed. New York and London: Plenum Medical Book Company, (Evans, A.S. and Kaslow, R.A., eds., 1997). All of the above references are herein incorporated by reference in their entireties.
Heterologous “prime boost” strategies have been effective for enhancing immune responses and protection against numerous pathogens. Schneider et al., Immunol. Rev. 170:29-38 (1999); Robinson, H.L., Nat. Rev. Immunol. 2:239-50 (2002); Gonzalo, R.M. et al., Vaccine 20:1226-31 (2002); Tanghe, A., Infect. Immun. 69: 3041-7 (2001). Providing antigen in different forms in the prime and the boost injections appears to maximize the immune response to the antigen. DNA vaccine priming followed by boosting with protein in adjuvant or by viral vector delivery of DNA encoding antigen appears to be the most effective way of improving antigen specific antibody and CD4+ T-cell responses or CD8+ T-cell responses respectively. Shiver J.W. et al., Nature 415: 331-5 (2002); Gilbert, S.C. et al., Vaccine 20:1039-45 (2002); Billaut-Mulot, O. et al., Vaccine 19:95-102 (2000); Sin, J.I. et al., DNA Cell Biol. 18:771-9 (1999). Recent data from monkey vaccination studies suggests that adding CRL1005 poloxamer to DNA encoding the HIV gag antigen enhances T-cell responses when monkeys are vaccinated with an HIV gag DNA prime followed by a boost with an adenoviral vector expressing HIV gag (Ad5-gag). The cellular immune responses for a DNA/poloxamer prime followed by an Ad5-gag boost were greater than the responses induced with a DNA (without poloxamer) prime followed by Ad5-gag boost or for Ad5-gag only. Shiver, J.W. et al. Nature 415:331-5 (2002). U.S. Patent Appl. Publication No. US 2002/0165172 A1describes simultaneous administration of a vector construct encoding an immunogenic portion of an antigen and a protein comprising the said immunogenic portion of an antigen such that an immune response is generated. The document is limited to hepatitis B antigens and HIV antigens. Moreover, U.S. Pat. No. 6,500,432 is directed to methods of enhancing an immune response of nucleic acid vaccination by simultaneous administration of a polynucleotide and polypeptide of interest. According to the patent, simultaneous administration means administration of the polynucleotide and the polypeptide during the same immune response, preferably within 0-10 or 3-7 days of each other. The antigens contemplated by the patent include, among others, those of Hepatitis (all forms), HSV, HIV, CMV, EBV, RSV, VZV, HPV, polio, influenza, parasites (e.g., from the genus Plasmodium), pathogenic bacteria (including but not limited to M tuberculosis, M leprae, Chlamydia, Shigella, B. burgdorferi, enterotoxigenic E. coli, S. typhosa, H. pylori, V. cholerae, B. pertussis, etc.). All of the above references are herein incorporated by reference in their entireties.
SUMMARY OF THE INVENTION
The present invention is directed to compositions and methods for raising a detectable immune response in a vertebrate against the infectious agent transmitting Severe Acute Respiratory Syndrome (SARS), by administering in vivo, into a tissue of a vertebrate, at least one polynucleotide comprising one or more nucleic acid fragments, wherein each nucleic acid fragment is a fragment of a coding region operably encoding a polypeptide, or a fragment, variant, or derivative thereof, or a fragment of a codon-optimized coding region operably encoding a polypeptide, or a fragment, variant, or derivative thereof, from a coronavirus which causes SARS (SARS-CoV). The present invention is also directed to administering in vivo, into a tissue of the vertebrate the above-described polynucleotide and at least one isolated SARS-CoV polypeptide, or a fragment, variant, or derivative thereof. The isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof can be, for example, a recombinant protein, a purified subunit protein, a protein expressed and carried by a heterologous live or inactivated or attenuated viral vector expressing the protein. According to either method, the polynucleotide is incorporated into the cells of the vertebrate in vivo, and an amount of the SARS-CoV protein, or fragment or variant encoded by the polynucleotide sufficient to raise a detectable immune response is produced in vivo. The isolated protein or fragment, variant, or derivative thereof is also administered in an amount sufficient to raise a detectable immune response. The polynucleotide may be administered to the vertebrate either prior to, at the same time (simultaneously), or subsequent to the administration of the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof.
Also within the scope of the present invention are combinations of SARS-CoV polypeptides and polynucleotides that encode SARS-CoV polypeptides that assemble into virus-like particles (VLP). One such combination is, but is not limited to a combination of SARS-CoV S, M, and E polypeptides or fragments, variants, or derivatives thereof, and polynucleotides encoding SARS-CoV S, M, and E polypeptides or fragments, variants, or derivatives thereof.
In a specific embodiment, the invention provides polynucleotide (e.g., DNA) vaccines in which the single formulation comprises a SARS-CoV polypeptide-encoding polynucleotide vaccine as described herein. An alternative embodiment of the invention provides for a multivalent formulation comprising several (e.g., two, three, four, or more) SARS-CoV polypeptide-encoding polynucleotides, as described herein, within a single vaccine composition. The SARS-CoV polypeptide-encoding polynucleotides, fragments or variants thereof may be contained within a single expression vector (e.g., plasmid or viral vector) or may be contained within multiple expression vectors.
In a specific embodiment, the invention provides combinatorial polynucleotide (e.g., DNA) vaccines which combine both a polynucleotide vaccine and polypeptide (e.g., either a recombinant protein, a purified subunit protein, a viral vector expressing an isolated SARS-CoV polypeptide) vaccine in a single formulation. , The single formulation comprises a SARS-CoV polypeptide-encoding polynucleotide vaccine as described herein, and optionally, an effective amount of a desired isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof. The polypeptide may exist in any form, for example, a recombinant protein, a purified subunit protein, or a viral vector expressing an isolated SARS-CoV polypeptide. The SARS-CoV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide vaccine may be identical to the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof. Alternatively, the SARS-CoV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide may be different from the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof.
The present invention further provides a method for generating, enhancing, or modulating a protective and/or therapeutic immune response to SARS-CoV in a vertebrate, comprising administering to a vertebrate in need of therapeutic and/or preventative immunity one or more of the compositions described herein.
The invention also provides for antibodies specifically reactive with SARS Co-V polypeptides which have been produced from an immune response elicited by the administration, to a vertebrate, of polynucleotide and polypeptides of the present invention.
In one embodiment, purified monoclonal antibodies or polyclonal antibodies containing the variable heavy and light sequences are used as therapeutic and prophylactic agents to treat or prevent SARS-CoV infection by passive antibody therapy. In general, this will comprise administering a therapeutically or prophylactically effective amount of the monoclonal antibodies to a susceptible vertebrate or one exhibiting SARS Co-V infection.



BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
FIG. 1 shows the protocol for the preparation of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a final volume of 3.6 ml, through the use of thermal cycling.
FIG. 2 shows the protocol for the preparation of a formulation comprising 0.3 mM BAK, 34 mg/ml or 50 mg/ml CRL 1005, and 2.5 mg/ml DNA in a final volume of 4.0 ml, through the use of thermal cycling.
FIG. 3 shows the protocol for the simplified preparation (without thermal cycling) of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml DNA.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to compositions and methods for raising a detectable immune response in a vertebrate against the infectious agent transmitting Severe Acute Respiratory Syndrome (SARS), by administering in vivo, into a tissue of a vertebrate, at least one polynucleotide comprising one or more nucleic acid fragments, wherein each nucleic acid fragment is a fragment of a coding region operably encoding a polypeptide, or a fragment, variant, or derivative thereof, or a fragment of a codon-optimized coding region operably encoding a polypeptide, or a fragment, variant, or derivative thereof, from a coronavirus which causes SARS (SARS-CoV). The present invention is also directed to administering in vivo, into a tissue of the vertebrate the above-described polynucleotide and at least one isolated SARS-CoV polypeptide, or a fragment, variant, or derivative thereof. The isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof can be, for example, a recombinant protein, a purified subunit protein, a protein expressed and carried by a heterologous live or inactivated or attenuated viral vector expressing the protein. According to either method, the polynucleotide is incorporated into the cells of the vertebrate in vivo, and an amount of the SARS-CoV protein, or fragment or variant encoded by the polynucleotide sufficient to raise a detectable immune response is produced in vivo. The isolated protein or fragment, variant, or derivative thereof is also administered in an amount sufficient to raise a detectable immune response. The polynucleotide may be administered to the vertebrate either prior to, at the same time (simultaneously), or subsequent to the administration of the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof.
In certain embodiments, the present invention provides for methods for raising a detectable immune response to polypeptides from a SARS-CoV virus, comprising administering to a vertebrate a polynucleotide which operably encodes a SARS-CoV polypeptide, wherein said polynucleotide is administered in an amount sufficient to elicit a detectable immune response to the encoded polypeptide.
The nucleotide and amino acid sequences of several SARS-CoV polypeptides have recently been determined. Several strains of human SARS-CoV (hSARS-CoV) have been sequenced. Sequences available on GenBank include the complete genomic sequences for SARS coronavirus strains CUKH-Su1, TOR2, BJ01, CUHK-W1, Urbani, and HKU-39849. SARS-CoV polypeptides from any of these strains are within the scope of the invention. Non-limiting examples of SARS-CoV polypeptides within the scope of the invention include the Spike (S), Nucleocapsid (N), Envelope (E), and Membrane glycoprotein (M) polypeptides, fragments, derivatives, (e.g., a TPA-S fusion), and variants thereof. As shown in Table 1 below, adapted from Rota et al., the various SARS-CoV strains that have been sequenced differ in various nucleotide base positions, some of which, as shown in Table 2 below, adapted from Marra et al., may result in a different amino acid residue. Thus, also within the scope of the invention are polypeptides that have different amino acids at those positions. The SARS-CoV polypeptide examples described below are from the Urbani strain of SARS-CoV, and are not meant to be limiting in terms of the scope of the invention.







TABLE 1







Comparison of Genomic Sequences of SARS-CoV Strains












Nucleotide







Positiond
Consensus
HKU-39849
CUHK-W1
Urbani
TOR2















2,601
T
C
*
*
*


7,746
G
*
T
*
*


7,919
C
*
*
T
*


7,930
G
A
*
*
*


8,387
G
C
*
*
*


8,417
G
C
*
*
*


9,404
T
*
C
*
*


9,479
T
*
C
*
*


13,494
G
A
*
*
*


13,495
T
G
*
*
*


16,622
C
*
*
T
*


17,564
T
*
G
*
*


17,846
C
*
T
*
*


18,065
G
A
*
*
*


19,064
R
A
G
G
A


21,721
G
*
A
*
*


22,222
T
*
C
*
*


23,220
T
*
*
*
G


24,872
T
*
*
C
*


25,298
G
*
*
*
A


25,569
T
A
*
*
*


26,600
C
T
*
*
*


26,857
T
*
*
C
*


27,827
T
*
C
*
*
















TABLE 2







Comparison of Tor2 and Urbani Strains of SARS-CoV


and Corresponding Amino Acid Substitutions












Nucleotide
Tor2
Corresponding
Urbani
Corresponding



Position
Base
Amino Acid
Base
Amino Acid
Protein















7,919
C
A
T
V
Rep1A


16,622
C
A
T
A
Rep1B


19,064
A
E
G
E
Rep1B


19,183
T
V
C
A
Rep1B


23,220
G
A
T
 S*
Spike (S)


24,872
T
L
C
L
Spike (S)


25,298
A
R
G
 G*
ORF 3


26,857
T
S
C
 P*
M





*Non-conservative Amino Acid Substitution






From about nucleotide 21492 to about 25259 of the Urbani strain of the SARS-CoV genome encode the Spike (S) protein. (Bellini et al. SARS Coronavirus Urbani, complete genome. GenBank Accession No. AY278741.) The complete S protein is about 1255 amino acids in length (139.12 kDa) and is predicted, by analogy to other coronaviruses, to be a surface projection glycoprotein precursor. The S protein has several important biologic functions. Monoclonal antibodies against S can neutralize virus infectivity, consistent with the observation that S protein binds to cellular receptors. The S glycoprotein has several important biologic functions. Monoclonal antibodies against S can neutralize virus infectivity, consistent with the observation that S protein binds to cellular receptors. The S protein is encoded by the following polynucleotide sequence in the Urbani strain and is referred to herein as SEQ ID NO:22.







ATGTTTATTTTCTTATTATTTCTTACTCTCACTAGTGGTAGTGACCTTGA


 


CCGGTGCACCACTTTTGATGATGTTCAAGCTCCTAATTACACTCAACATA


 


CTTCATCTATGAGGGCGGTTTACTATCCTGATGAAATTTTTAGATCAGAC


 


ACTCTTTATTTAACTCAGGATTTATTTCTTCCATTTTATTCTAATGTTAC


 


AGGGTTTCATACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTA


 


AGGATGGTATTTATTTTGCTGCCACAGAGAAATCAAATGTTGTCCGTGGT


 


TGGGTTTTTGGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTAT


 


TAACAATTCTACTAATGTTGTTATACGAGCATGTAACTTTGAATTGTGTG


 


ACAACCCTTTCTTTGCTGTTTCTAAACCCATGGGTACACAGACACATACT


 


ATGATATTCGATAATGCATTTAATTGCACTTTCGAGTACATATCTGATGC


 


CTTTTCGCTTGATGTTTCAGAAAAGTCAGGTAATTTTAAACACTTACGAG


 


AGTTTGTGTTTAAAAATAAAGATGGGTTTCTCTATGTTTATAAGGGCTAT


 


CAACCTATAGATGTAGTTCGTGATCTACCTTCTGGTTTTAACACTTTGAA


 


ACCTATTTTTAAGTTGCCTCTTGGTATTAACATTACAAATTTTAGAGCCA


 


TTCTTACAGCCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCA


 


GCCTATTTTGTTGGCTATTTAAAGCCAACTACATTTATGCTCAAGTATGA


 


TGAAAATGGTACAATCACAGATGCTGTTGATTGTTCTCAAAATCCACTTG


 


CTGAACTCAAATGCTCTGTTAAGAGCTTTGAGATTGACAAAGGAATTTAC


 


CAGACCTCTAATTTCAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCC


 


TAATATTACAAACTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAAT


 


TCCCTTCTGTCTATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCT


 


GATTACTCTGTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTA


 


TGGCGTTTCTGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATG


 


CAGATTCTTTTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGA


 


CAAACTGGTGTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCAT


 


GGGTTGTGTCCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAACTG


 


GTAATTATAATTATAAATATAGGTATCTTAGACATGGCAAGCTTAGGCCC


 


TTTGAGAGAGACATATCTAATGTGCCTTTCTCCCCTGATGGCAAACCTTG


 


CACCCCACCTGCTCTTAATTGTTATTGGCCATTAAATGATTATGGTTTTT


 


ACACCACTACTGGCATTGGCTACCAACCTTACAGAGTTGTAGTACTTTCT


 


TTTGAACTTTTAAATGCACCGGCCACGGTTTGTGGACCAAAATTATCCAC


 


TGACCTTATTAGAACCAGTGTGTCAATTTTAATTTTAATGGACTCACTGG


 


TACTGGTGTGTTAACTCCTTCTTCAAAGAGATTTCAACCATTTCAACAAT


 


TTGGCCGTGATGTTTCTGATTTCACTGATTCCGTTCGAGATCCTAAAACA


 


TCTGAAATATTAGACATTTCACCTTGCTCTTTTGGGGGTGTAAGTGTAAT


 


TACACCTGGAACAAATGCTTCATCTGAAGTTGCTGTTCTATATCAAGATG


 


TTAACTGCACTGATGTTTCTACAGCAATTCATGCAGATCAACTCACACCA


 


GCTTGGCGCATATATTCTACTGGAAACAATGTATTCCAGACTCAAGCAGG


 


CTGTCTTATAGGAGCTGAGCATGTCGACACTTCTTATGAGTGCGACATTC


 


CTATTGGAGCTGGCATTTGTGCTAGTTACCATACAGTTTCTTTATTACGT


 


AGTACTAGCCAAAAATCTATTGTGGCTTATACTATGTCTTTAGGTGCTGA


 


TAGTTCAATTGCTTACTCTAATAACACCATTGCTATACCTACTAACTTTT


 


CAATTAGCATTACTACAGAAGTAATGCCTGTTTCTATGGCTAAAACCTCC


 


GTAGATTGTAATATGTACATCTGCGGAGATTCTACTGAATGTGCTAATTT


 


GCTTCTCCAATATGGTAGCTTTTGCACACAACTAAATCGTGCACTCTCAG


 


GTATTGCTGCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTCAAGTC


 


AAACAAATGTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTTAATTT


 


TTCACAAATATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTTTATTG


 


AGGACTTGCTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCATGAAG


 


CAATATGGCGAATGCCTAGGTGATATTAATGCTAGAGATCTCATTTGTGC


 


GCAGAAGTTCAATGGACTTACAGTGTTGCCACCTCTGCTCACTGATGATA


 


TGATTGCTGCCTACACTGCTGCTCTAGTTAGTGGTACTGCCACTGCTGGA


 


TGGACATTTGGTGCTGGCGCTGCTCTTCAAATACCTTTTGCTATGCAAAT


 


GGCATATAGGTTCAATGGCATTGGAGTTACCCAAAATGTTCTCTATGAGA


 


ACCAAAAACAAATCGCCAACCAATTTAACAAGGCGATTAGTCAAATTCAA


 


GAATCACTTACAACAACATCAACTGCATTGGGCAAGCTGCAAGACGTTGT


 


TAACCAGAATGCTCAAGCATTAAACACACTTGTTAAACAACTTAGCTCTA


 


ATTTTGGTGCAATTTCAAGTGTGCTAAATGATATCCTTTCGCGACTTGAT


 


AAAGTCGAGGCGGAGGTACAAATTGACAGGTTAATTACAGGCAGACTTCA


 


AAGCCTTCAAACCTATGTAACACAACAACTAATCAGGGCTGCTGAAATCA


 


GGGCTTCTGCTAATCTTGCTGCTACTAAAATGTCTGAGTGTGTTCTTGGA


 


CAATCAAAAAGAGTTGACTTTTGTGGAAAGGGCTACCACCTTATGTCCTT


 


CCCACAAGCAGCCCCGCATGGTGTTGTCTTCCTACATGTCACGTATGTGC


 


CATCCCAGGAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATGAAGGC


 


AAAGCATACTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCTTG


 


GTTTATTACACAGAGGAACTTCTTTTCTCCACAAATAATTACTACAGACA


 


ATACATTTGTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTAACAAC


 


ACAGTTTATGATCCTCTGCAACCTGAGCTCGACTCATTCAAAGAAGAGCT


 


GGACAAGTACTTCAAAAATCATACATCACCAGATGTTGATCTTGGCGACA


 


TTTCAGGCATTAACGCTTCTGTCGTCAACATTCAAAAAGAAATTGACCGC


 


CTCAATGAGGTCGCTAAAAATTTAAATGAATCACTCATTGACCTTCAAGA


 


ATTGGGAAAATATGAGCAATATATTAAATGGCCTTGGTATGTTTGGCTCG


 


GCTTCATTGCTGGACTAATTGCCATCGTCATGGTTACAATCTTGCTTTGT


 


TGCATGACTAGTTGTTGCAGTTGCCTCAAGGGTGCATGCTCTTGTGGTTC


 


TTGCTGCAAGTTTGATGAGGATGACTCTGAGCCAGTTCTCAAGGGTGTCAA


 


ATTACATTACACATAA






The S protein has the following amino acid sequence and is referred to herein as SEQ ID NO:23.







MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD


 


TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG


 


WVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHT


 


MIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGY


 


QPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAA


 


AYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIY


 


QTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVA


 


DYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPG


 


QTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRP


 


FERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLS


 


FELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQ


 


FGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQD


 


VNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDI


 


PIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNF


 


SISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALS


 


GIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFI


 


EDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDD


 


MIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE


 


NQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSS


 


NFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEI


 


RASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYV


 


PSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTD


 


NTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGD


 


ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWL


 


GFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGV


 


KLHYT






The S protein can be divided into three structural domains: a large external domain at the N-terminus, a transmembrane domain and a short carboxyterminal cytoplasmic domain. These domains within the S protein of SARS-CoV Urbani strain have been identified using the program TMHMM2.0. (Sonnhammer et al. Proc. Of 6th Int. Conf On Intelligent Systems for Molecular Biology. AAAI Press:175-182 (1998). Based on this algorithm, amino acids about 1 to about 1195 comprise an extracellular domain; amino acids about 1196 to about 1218 are part of a transmembrane domain; and amino acids about 1219 to about 1240 comprise the cytoplasmic domain. Removal of residues comprising the transmembrane domain and optionally, the cytoplasmic domain, results in a soluble protein that can be used in the compositions of the invention.
The large external domain of the S protein is further divided into two sub-domains, S1 and S2. The S1 sub-domain (amino acids about 1 to about 683) includes the N-terminal half of the molecule and forms the globular portion of the spikes. This region contains sequences that are responsible for binding to specific receptors on the membranes of susceptible cells. S1 sequences are variable, containing various degrees of deletion and substitutions in different coronavirus strains or isolates. Mutations in S1 sequences have been associated with altered antigenicity and pathogenicity of the virus. The receptor-binding domain of the S protein of murine hepatitis virus (MHV) is localized within the N-terminal 330 amino acids of the S1 domain. Consequently, the amino acid sequences of the S1 domain may determine the target cell specificity of coronaviruses in animals.
The S2 sub-domain comprises amino acids about 684 to about 1210 of the S protein. In coronaviruses, the S2 sub-domain of the S protein is usually acylated and contains two heptad repeat motifs. The motifs suggest that this portion of the S protein may assume a coiled-coil structure. The mature S protein forms an oligomer, which is most likely a trimer based on the spike proteins of other coronaviruses. Thus, the S2 subdomain probably constitutes the stalk of the viral spike.
Non limiting examples of nucleotide sequences encoding the S protein are as follows. It should be noted that S sequences vary between SARS-CoV strains. Virtually any nucleotide sequence encoding a SARS-CoV S protein is suitable for the present invention. In fact, S polynucleotide sequences included in vaccines and therapeutic formulations of the current invention may change from year to year, depending on the prevalent strain or strains of SARS-CoV.
From about nucleotide 21492 to about 25080 of the Urbani strain of the SARS-CoV genome encode a soluble extracellular portion of the S protein (Bellini et al. SARS Coronavirus Urbani, compete genome, Genbank accession number AY278741) and has the following sequence, referred to herein as SEQ ID NO: 1:







ATGTTTATTTTCTTATTATTTCTTACTCTCACTAGTGGTAGTGACCTTGA


 


CCGGTGCACCACTTTTGATGATGTTCAAGCTCCTAATTACACTCAACATA


 


CTTCATCTATGAGGGGGGTTTACTATCCTGATGAAATTTTTAGATCAGAC


 


ACTCTTTATTTAACTCAGGATTTATTTCTTCCATTTTATTCTAATGTTAC


 


AGGGTTTCATACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTA


 


AGGATGGTATTTATTTTGCTGCCACAGAGAAATCAAATGTTGTCCGTGGT


 


TGGGTTTTTGGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTAT


 


TAACAATTCTACTAATGTTGTTATACGAGCATGTAACTTTGAATTGTGTG


 


ACAACCCTTTCTTTGCTGTTTCTAAACCCATGGGTACACAGACACATACT


 


ATGATATTCGATAATGCATTTAATTGCACTTTCGAGTACATATCTGATGC


 


CTTTTCGCTTGATGTTTCAGAAAAGTCAGGTAATTTTAAACACTTACGAG


 


AGTTTGTGTTTAAAAATAAAGATGGGTTTCTCTATGTTTATAAGGGCTAT


 


CAACCTATAGATGTAGTTCGTGATCTACCTTCTGGTTTTAACACTTTGAA


 


ACCTATTTTTAAGTTGCCTCTTGGTATTAACATTACAAATTTTAGAGCCA


 


TTCTTACAGCCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCA


 


GCCTATTTTGTTGGCTATTTAAAGCCAACTACATTTATGCTCAAGTATGA


 


TGAAAATGGTACAATCACAGATGCTGTTGATTGTTCTCAAAATCCACTTG


 


CTGAACTCAAATGCTCTGTTAAGAGCTTTGAGATTGACAAAGGAATTTAC


 


CAGACCTCTAATTTCAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCC


 


TAATATTACAAACTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAAT


 


TCCCTTCTGTCTATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCT


 


GATTACTCTGTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTA


 


TGGCGTTTCTGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATG


 


CAGATTCTTTTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGA


 


CAAACTGGTGTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCAT


 


GGGTTGTGTCCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAACTG


 


GTAATTATAATTATAAATATAGGTATCTTAGACATGGCAAGCTTAGGCCC


 


TTTGAGAGAGACATATCTAATGTGCCTTTCTCCCCTGATGGCAAACCTTG


 


CACCCCACCTGCTCTTAATTGTTATTGGCCATTAAATGATTATGGTTTTT


 


ACACCACTACTGGCATTGGCTACCAACCTTACAGAGTTGTAGTACTTTCT


 


TTTGAACTTTTAAATGCACCGGCCACGGTTTGTGGACCAAAATTATCCAC


 


TGACCTTATTAAGAACCAGTGTGTCAATTTTAATTTTAATGGACTCACTG


 


GTACTGGTGTGTTAACTCCTTCTTCAAAGAGATTTCAACCATTTCAACAA


 


TTTGGCCGTGATGTTTCTGATTTCACTGATTCCGTTCGAGATCCTAAAAC


 


ATCTGAAATATTAGACATTTCACCTTGCTCTTTTGGGGGTGTAAGTGTAA


 


TTACACCTGGAACAAATGCTTCATCTGAAGTTGCTGTTCTATATCAAGAT


 


GTTAACTGCACTGATGTTTCTACAGCAATTCATGCAGATCAACTCACACC


 


AGCTTGGCGCATATATTCTACTGGAAACAATGTATTCCAGACTCAAGCAG


 


GCTGTCTTATAGGAGCTGAGCATGTCGACACTTCTTATGAGTGCGACATT


 


CCTATTGGAGCTGGCATTTGTGCTAGTTACCATACAGTTTCTTTATTACG


 


TAGTACTAGCCAAAAATCTATTGTGGCTTATACTATGTCTTTAGGTGCTG


 


ATAGTTCAATTGCTTACTCTAATAACACCATTGCTATACCTACTAACTTT


 


TCAATTAGCATTACTACAGAAGTAATGCCTGTTTCTATGGCTAAAACCTC


 


CGTAGATTGTAATATGTACATCTGCGGAGATTCTACTGAATGTGCTAATT


 


TGCTTCTCCAATATGGTAGCTTTTGCACACAACTAAATCGTGCACTCTCA


 


GGTATTGCTGCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTCAAGT


 


CAAACAAATGTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTTAATT


 


TTTCACAAATATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTTTATT


 


GAGGACTTGCTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCATGAA


 


GCAATATGGCGAATGCCTAGGTGATATTAATGCTAGAGATCTCATTTGTG


 


CGCAGAAGTTCAATGGACTTACAGTGTTGCCACCTCTGCTCACTGATGAT


 


ATGATTGCTGCCTACACTGCTGCTCTAGTTAGTGGTACTGCCACTGCTGG


 


ATGGACATTTGGTGCTGGCGCTGCTCTTCAAATACCTTTTGCTATGCAAA


 


TGGCATATAGGTTCAATGGCATTGGAGTTACCCAAAATGTTCTCTATGAG


 


AACCAAAAACAAATCGCCAACCAATTTAACAAGGCGATTAGTCAAATTCA


 


AGAATCACTTACAACAACATCAACTGCATTGGGCAAGCTGCAAGACGTTG


 


TTAACCAGAATGCTCAAGCATTAAACACACTTGTTAAACAACTTAGCTCT


 


AATTTTGGTGCAATTTCAAGTGTGCTAAATGATATCCTTTCGCGACTTGA


 


TAAAGTCGAGGCGGAGGTACAAATTGACAGGTTAATTACAGGCAGACTTC


 


AAAGCCTTCAAACCTATGTAACACAACAACTAATCAGGGCTGCTGAAATC


 


AGGGCTTCTGCTAATCTTGCTGCTACTAAAATGTCTGAGTGTGTTCTTGG


 


ACAATCAAAAAGAGTTGACTTTTGTGGAAAGGGCTACCACCTTATGTCCT


 


TCCCACAAGCAGCCCCGCATGGTGTTGTCTTCCTACATGTCACGTATGTG


 


CCATCCCAGGAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATGAAGG


 


CAAAGCATACTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCTT


 


GGTTTATTACACAGAGGAACTTCTTTTCTCCACAAATAATTACTACAGAC


 


AATACATTTGTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTAACAA


 


CACAGTTTATGATCCTCTGCAACCTGAGCTCGACTCATTCAAAGAAGAGC


 


TGGACAAGTACTTCAAAAATCATACATCACCAGATGTTGATCTTGGCGAC


 


ATTTCAGGCATTAACGCTTCTGTCGTCAACATTCAAAAAGAAATTGACCG


 


CCTCAATGAGGTCGCTAAAAATTTAAATGAATCACTCATTGACCTTCAAG


 


AATTGGGAAAATATGAGCAATATATTAAATGGCCTTGG






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response. The present invention is also directed to raising a detectable immune response with or without a wildtype or other secretory leader sequence as described below.
The amino acid sequence of the soluble S protein encoded by SEQ ID NO:1 has the following sequence shown below and is referred to herein as SEQ ID NO:2:







MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD


 


TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG


 


WVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHT


 


MIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGY


 


QPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAA


 


AYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIY


 


QTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVA


 


DYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPG


 


QTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRP


 


FERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLS


 


FELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQ


 


FGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQD


 


VNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDI


 


PIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNF


 


SISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALS


 


GIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFI


 


EDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDD


 


MIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE


 


NQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSS


 


NFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEI


 


RASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYV


 


PSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTD


 


NTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGD


 


ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2, wherein said polypeptide raises a detectable immune response. The present invention is also directed to raising a detectable immune response with or without a wildtype or other secretory leader sequence as described below.
A conserved protein domain program on the National Center for Biotechnology Information's web site (www.ncbi.nlm.nih.gov) was used to predict domains within the SARS-CoV S protein. Two domains, S1 and S2, were predicted within the soluble portion of the S protein. The S1 domain spans from amino acids about 1 to about 683 of the S protein. The nucleotide sequence encoding the soluble S1 domain from SARS-CoV Urbani strain has the following sequence and is referred to herein as SEQ ID NO:3: TABLE-US-00007







ATGTTTATTTTCTTATTATTTCTTACTCTCACTAGTGGTAGTGACCTTGA


 


CCGGTGCACCACTTTTGATGATGTTCAAGCTCCTAATTACACTCAACATA


 


CTTCATCTATGAGGGGGGTTTACTATCCTGATGAAATTTTTAGATCAGAC


 


ACTCTTTATTTAACTCAGGATTTATTTCTTCCATTTTATTCTAATGTTAC


 


AGGGTTTCATACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTA


 


AGGATGGTATTTATTTTGCTGCCACAGAGAAATCAAATGTTGTCCGTGGT


 


TGGGTTTTTGGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTAT


 


TAACAATTCTACTAATGTTGTTATACGAGCATGTAACTTTGAATTGTGTG


 


ACAACCCTTTCTTTGCTGTTTCTAAACCCATGGGTACACAGACACATACT


 


ATGATATTCGATAATGCATTTAATTGCACTTTCGAGTACATATCTGATGC


 


CTTTTCGCTTGATGTTTCAGAAAAGTCAGGTAATTTTAAACACTTACGAG


 


AGTTTGTGTTTAAAAATAAAGATGGGTTTCTCTATGTTTATAAGGGCTAT


 


CAACCTATAGATGTAGTTCGTGATCTACCTTCTGGTTTTAACACTTTGAA


 


ACCTATTTTTAAGTTGCCTCTTGGTATTAACATTACAAATTTTAGAGCCA


 


TTCTTACAGCCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCA


 


GCCTATTTTGTTGGCTATTTAAAGCCAACTACATTTATGCTCAAGTATGA


 


TGAAAATGGTACAATCACAGATGCTGTTGATTGTTCTCAAAATCCACTTG


 


CTGAACTCAAATGCTCTGTTAAGAGCTTTGAGATTGACAAAGGAATTTAC


 


CAGACCTCTAATTTCAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCC


 


TAATATTACAAACTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAAT


 


TCCCTTCTGTCTATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCT


 


GATTACTCTGTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTA


 


TGGCGTTTCTGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATG


 


CAGATTCTTTTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGA


 


CAAACTGGTGTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCAT


 


GGGTTGTGTCCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAACTG


 


GTAATTATAATTATAAATATAGGTATCTTAGACATGGCAAGCTTAGGCCC


 


TTTGAGAGAGACATATCTAATGTGCCTTTCTCCCCTGATGGCAAACCTTG


 


CACCCCACCTGCTCTTAATTGTTATTGGCCATTAAATGATTATGGTTTTT


 


ACACCACTACTGGCATTGGCTACCAACCTTACAGAGTTGTAGTACTTTCT


 


TTTGAACTTTTAAATGCACCGGCCACGGTTTGTGGACCAAAATTATCCAC


 


TGACCTTATTAAGAACCAGTGTGTCAATTTTAATTTTAATGGACTCACTG


 


GTACTGGTGTGTTAACTCCTTCTTCAAAGAGATTTCAACCATTTCAACAA


 


TTTGGCCGTGATGTTTCTGATTTCACTGATTCCGTTCGAGATCCTAAAAC


 


ATCTGAAATATTAGACATTTCACCTTGCTCTTTTGGGGGTGTAAGTGTAA


 


TTACACCTGGAACAAATGCTTCATCTGAAGTTGCTGTTCTATATCAAGAT


 


GTTAACTGCACTGATGTTTCTACAGCAATTCATGCAGATCAACTCACACC


 


AGCTTGGCGCATATATTCTACTGGAAACAATGTATTCCAGACTCAAGCAG


 


GCTGTCTTATAGGAGCTGAGCATGTCGACACTTCTTATGAGTGCGACATT


 


CCTATTGGAGCTGGCATTTGTGCTAGTTACCATACAGTTTCTTTATTACG


 


TAGTACTAGCCAAAAATCTATTGTGGCTTATACTATGTCTTTAGGTGCT






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S1 polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:3, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response. The present invention is also directed to raising a detectable immune response with or without a wildtype or other secretory leader sequence as described below.
The amino acid sequence of the soluble S1 protein encoded by SEQ ID NO:3 has the following sequence shown below and is referred to herein as SEQ ID NO:4:







MFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD


 


TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG


 


WVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHT


 


MIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGY


 


QPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAA


 


AYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIY


 


QTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVA


 


DYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPG


 


QTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRP


 


FERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLS


 


FELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQ


 


FGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQD


 


VNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDI


 


PIGAGICASYHTVSLLRSTSQKSIVAYTMSLGA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S1 polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4, wherein said polypeptide raises a detectable immune response. The present invention is also directed to raising a detectable immune response with or without a wildtype or other secretory leader sequence as described below.
The S2 domain spans from amino acids about 684 to about 1210 of the S protein. The nucleotide sequence encoding the soluble S2 domain from SARS-CoV Urbani strain has the following sequence and is referred to herein as SEQ ID NO:5:







GATAGTTCAATTGCTTACTCTAATAACACCATTGCTATACCTACTAACTT


 


TTCAATTAGCATTACTACAGAAGTAATGCCTGTTTCTATGGCTAAAACCT


 


CCGTAGATTGTAATATGTACATCTGCGGAGATTCTACTGAATGTGCTAAT


 


TTGCTTCTCCAATATGGTAGCTTTTGCACACAACTAAATCGTGCACTCTC


 


AGGTATTGCTGCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTCAAG


 


TCAAACAAATGTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTTAAT


 


TTTTCACAAATATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTTTAT


 


TGAGGACTTGCTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCATGA


 


AGCAATATGGCGAATGCCTAGGTGATATTAATGCTAGAGATCTCATTTGT


 


GCGCAGAAGTTCAATGGACTTACAGTGTTGCCACCTCTGCTCACTGATGA


 


TATGATTGCTGCCTACACTGCTGCTCTAGTTAGTGGTACTGCCACTGCTG


 


GATGGACATTTGGTGCTGGCGCTGCTCTTCAAATACCTTTTGCTATGCAA


 


ATGGCATATAGGTTCAATGGCATTGGAGTTACCCAAAATGTTCTCTATGA


 


GAACCAAAAACAAATCGCCAACCAATTTAACAAGGCGATTAGTCAAATTC


 


AAGAATCACTTACAACAACATCAACTGCATTGGGCAAGCTGCAAGACGTT


 


GTTAACCAGAATGCTCAAGCATTAAACACACTTGTTAAACAACTTAGCTC


 


TAATTTTGGTGCAATTTCAAGTGTGCTAAATGATATCCTTTCGCGACTTG


 


ATAAAGTCGAGGCGGAGGTACAAATTGACAGGTTAATTACAGGCAGACTT


 


CAAAGCCTTCAAACCTATGTAACACAACAACTAATCAGGGCTGCTGAAAT


 


CAGGGCTTCTGCTAATCTTGCTGCTACTAAAATGTCTGAGTGTGTTCTTG


 


GACAATCAAAAAGAGTTGACTTTTGTGGAAAGGGCTACCACCTTATGTCC


 


TTCCCACAAGCAGCCCCGCATGGTGTTGTCTTCCTACATGTCACGTATGT


 


GCCATCCCAGGAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATGAAG


 


GCAAAGCATACTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCT


 


TGGTTTATTACACAGAGGAACTTCTTTTCTCCACAAATAATTACTACAGA


 


CAATACATTTGTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTAACA


 


ACACAGTTTATGATCCTCTGCAACCTGAGCTCGACTCATTCAAAGAAGAG


 


CTGGACAAGTACTTCAAAAATCATACATCACCAGATGTTGATCTTGGCGA


 


CATTTCAGGCATTAACGCTTCTGTCGTCAACATTCAAAAAGAAATTGACC


 


GCCTCAATGAGGTCGCTAAAAATTTAAATGAATCACTCATTGACCTTCAA


 


GAATTGGGAAAATATGAGCAATATATTAAATGGCCTTGG






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S2 polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response. It should be noted that in order to achieve a polynucleotide “operably encoding” a SARS-CoV S2 polypeptide, at least a methionine codon (ATG) would need to be included, in frame, upstream of the polynucleotide presented herein as SEQ ID NO:5. An example of such a polynucleotide includes, but is not limited to the following, presented herein as SEQ ID NO:54.







ATGGATAGTTCAATTGCTTACTCTAATAACACCATTGCTATACCTACTAA


 


CTTTTCAATTAGCATTACTACAGAAGTAATGCCTGTTTCTATGGCTAAAA


 


CCTCCGTAGATTGTAATATGTACATCTGCGGAGATTCTACTGAATGTGCT


 


AATTTGCTTCTCCAATATGGTAGCTTTTGCACACAACTAAATCGTGCACT


 


CTCAGGTATTGCTGCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTC


 


AAGTCAAACAAATGTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTT


 


AATTTTTCACAAATATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTT


 


TATTGAGGACTTGCTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCA


 


TGAAGCAATATGGCGAATGCCTAGGTGATATTAATGCTAGAGATCTCATT


 


TGTGCGCAGAAGTTCAATGGACTTACAGTGTTGCCACCTCTGCTCACTGA


 


TGATATGATTGCTGCCTACACTGCTGCTCTAGTTAGTGGTACTGCCACTG


 


CTGGATGGACATTTGGTGCTGGCGCTGCTCTTCAAATACCTTTTGCTATG


 


CAAATGGCATATAGGTTCAATGGCATTGGAGTTACCCAAAATGTTCTCTA


 


TGAGAACCAAAAACAAATCGCCAACCAATTTAACAAGGCGATTAGTCAAA


 


TTCAAGAATCACTTACAACAACATCAACTGCATTGGGCAAGCTGCAAGAC


 


GTTGTTAACCAGAATGCTCAAGCATTAAACACACTTGTTAAACAACTTAG


 


CTCTAATTTTGGTGCAATTTCAAGTGTGCTAAATGATATCCTTTCGCGAC


 


TTGATAAAGTCGAGGCGGAGGTACAAATTGACAGGTTAATTACAGGCAGA


 


CTTCAAAGCCTTCAAACCTATGTAACACAACAACTAATCAGGGCTGCTGA


 


AATCAGGGCTTCTGCTAATCTTGCTGCTACTAAAATGTCTGAGTGTGTTC


 


TTGGACAATCAAAAAGAGTTGACTTTTGTGGAAAGGGCTACCACCTTATG


 


TCCTTCCCACAAGCAGCCCCGCATGGTGTTGTCTTCCTACATGTCACGTA


 


TGTGCCATCCCAGGAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATG


 


AAGGCAAAGCATACTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACT


 


TCTTGGTTTATTACACAGAGGAACTTCTTTTCTCCACAAATAATTACTAC


 


AGACAATACATTTGTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTA


 


ACAACACAGTTTATGATCCTCTGCAACCTGAGCTCGACTCATTCAAAGAA


 


GAGCTGGACAAGTACTTCAAAAATCATACATCACCAGATGTTGATCTTGG


 


CGACATTTCAGGCATTAACGCTTCTGTCGTCAACATTCAAAAAGAAATTG


 


ACCGCCTCAATGAGGTCGCTAAAAATTTAAATGAATCACTCATTGACCTT


 


CAAGAATTGGGAAAATATGAGCAATATATTAAATGGCCTTGG






The present invention is also directed to raising a detectable immune response with or without a wildtype or other secretory leader sequence as described below.
The amino acid sequence of the soluble S2 protein encoded by SEQ ID NO:5 has the following sequence shown below and is referred to herein as SEQ ID NO:6







DSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECAN


 


LLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFN


 


FSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLIC


 


AQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQ


 


MAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDV


 


VNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRL


 


QSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS


 


FPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTS


 


WFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEE


 


LDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQ


 


ELGKYEQYIKWPW






The amino acid sequence of the soluble S2 protein encoded by SEQ ID NO:54 has the following sequence shown below and is referred to herein as SEQ ID NO:56







MDSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECA


 


NLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGF


 


NFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLI


 


CAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAM


 


QMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQD


 


VVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGR


 


LQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGOSKRVDFCGKGYHLM


 


SFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGT


 


SWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKE


 


ELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL


 


QELGKYEQYIKWPW






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S2 polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6, wherein said polypeptide raises a detectable immune response. The present invention is also directed to raising a detectable immune response with or without a wildtype or other secretory leader sequence as described below.
In one embodiment, soluble S, soluble S1 and soluble S2, described herein, are encoded by a polynucleotide which contains the wild-type S secretory leader peptide sequence. The secretory leader peptide of the S protein in SARS-CoV Urbani strain comprises about the first 13 residues of the protein. Marra et al. The present invention is also directed to raising a detectable immune response with or without amino acids about 1 to about 10, about 1 to about 11, about 1 to about 12, about 1 to about 13, about 1 to about 14, about 1 to about 15, about 1 to about 16, about 1 to about 17, about 1 to about 18, about 1 to about 19, about 1 to about 20, about 1 to about 21, about 1 to about 22, about 1 to about 23, about 1 to about 24, and about 1 to about 25 of the secretory leader peptide sequence.
In an alternative embodiment, the secretory leader peptide of soluble S, soluble S1 and soluble S2 can be replaced by the secretory leader peptide of human Tissue Plasminogen Activator (TPA). The polynucleotide sequences encoding the various S polypeptides with the TPA secretory leader peptide are shown below.







Soluble TPA-S


(SEQ ID NO: 7)


ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGC


 


AGTCTTCGTTTCGCCCAGCGCTAGAGGATCGGGAAGTGACCTTGACCGGT


 


GCACCACTTTTGATGATGTTCAAGCTCCTAATTACACTCAACATACTTCA


 


TCTATGAGGGGGGTTTACTATCCTGATGAAATTTTTAGATCAGACACTCT


 


TTATTTAACTCAGGATTTATTTCTTCCATTTTATTCTAATGTTACAGGGT


 


TTCATACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTAAGGAT


 


GGTATTTATTTTGCTGCCACAGAGAAATCAAATGTTGTCCGTGGTTGGGT


 


TTTTGGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTATTAACA


 


ATTCTACTAATGTTGTTATACGAGCATGTAACTTTGAATTGTGTGACAAC


 


CCTTTCTTTGCTGTTTCTAAACCCATGGGTACACAGACACATACTATGAT


 


ATTCGATAATGCATTTAATTGCACTTTCGAGTACATATCTGATGCCTTTT


 


CGCTTGATGTTTCAGAAAAGTCAGGTAATTTTAAACACTTACGAGAGTTT


 


GTGTTTAAAAATAAAGATGGGTTTCTCTATGTTTATAAGGGCTATCAACC


 


TATAGATGTAGTTCGTGATCTACCTTCTGGTTTTAACACTTTGAAACCTA


 


TTTTTAAGTTGCCTCTTGGTATTAACATTACAAATTTTAGAGCCATTCTT


 


ACAGCCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCAGCCTA


 


TTTTGTTGGCTATTTAAAGCCAACTACATTTATGCTCAAGTATGATGAAA


 


ATGGTACAATCACAGATGCTGTTGATTGTTCTCAAAATCCACTTGCTGAA


 


CTCAAATGCTCTGTTAAGAGCTTTGAGATTGACAAAGGAATTTACCAGAC


 


CTCTAATTTCAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCCTAATA


 


TTACAAACTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAATTCCCT


 


TCTGTCTATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCTGATTA


 


CTCTGTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTATGGCG


 


TTTCTGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATGCAGAT


 


TCTTTTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGACAAAC


 


TGGTGTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCATGGGTT


 


GTGTCCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAACTGGTAAT


 


TATAATTATAAATATAGGTATCTTAGACATGGCAAGCTTAGGCCCTTTGA


 


GAGAGACATATCTAATGTGCCTTTCTCCCCTGATGGCAAACCTTGCACCC


 


CACCTGCTCTTAATTGTTATTGGCCATTAAATGATTATGGTTTTTACACC


 


ACTACTGGCATTGGCTACCAACCTTACAGAGTTGTAGTACTTTCTTTTGA


 


ACTTTTAAATGCACCGGCCACGGTTTGTGGACCAAAATTATCCACTGACC


 


TTATTAAGAACCAGTGTGTCAATTTTAATTTTAATGGACTCACTGGTACT


 


GGTGTGTTAACTCCTTCTTCAAAGAGATTTCAACCATTTCAACAATTTGG


 


CCGTGATGTTTCTGATTTCACTGATTCCGTTCGAGATCCTAAAACATCTG


 


AAATATTAGACATTTCACCTTGCTCTTTTGGGGGTGTAAGTGTAATTACA


 


CCTGGAACAAATGCTTCATCTGAAGTTGCTGTTCTATATCAAGATGTTAA


 


CTGCACTGATGTTTCTACAGCAATTCATGCAGATCAACTCACACCAGCTT


 


GGCGCATATATTCTACTGGAAACAATGTATTCCAGACTCAAGCAGGCTGT


 


CTTATAGGAGCTGAGCATGTCGACACTTCTTATGAGTGCGACATTCCTAT


 


TGGAGCTGGCATTTGTGCTAGTTACCATACAGTTTCTTTATTACGTAGTA


 


CTAGCCAAAAATCTATTGTGGCTTATACTATGTCTTTAGGTGCTGATAGT


 


TCAATTGCTTACTCTAATAACACCATTGCTATACCTACTAACTTTTCAAT


 


TAGCATTACTACAGAAGTAATGCCTGTTTCTATGGCTAAAACCTCCGTAG


 


ATTGTAATATGTACATCTGCGGAGATTCTACTGAATGTGCTAATTTGCTT


 


CTCCAATATGGTAGCTTTTGCACACAACTAAATCGTGCACTCTCAGGTAT


 


TGCTGCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTCAAGTCAAAC


 


AAATGTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTTAATTTTTCA


 


CAAATATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTTTATTGAGGA


 


CTTGCTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCATGAAGCAAT


 


ATGGCGAATGCCTAGGTGATATTAATGCTAGAGATCTCATTTGTGCGCAG


 


AAGTTCAATGGACTTACAGTGTTGCCACCTCTGCTCACTGATGATATGAT


 


TGCTGCCTACACTGCTGCTCTAGTTAGTGGTACTGCCACTGCTGGATGGA


 


CATTTGGTGCTGGCGCTGCTCTTCAAATACCTTTTGCTATGCAAATGGCA


 


TATAGGTTCAATGGCATTGGAGTTACCCAAAATGTTCTCTATGAGAACCA


 


AAAACAAATCGCCAACCAATTTAACAAGGCGATTAGTCAAATTCAAGAAT


 


CACTTACAACAACATCAACTGCATTGGGCAAGCTGCAAGACGTTGTTAAC


 


CAGAATGCTCAAGCATTAAACACACTTGTTAAACAACTTAGCTCTAATTT


 


TGGTGCAATTTCAAGTGTGCTAAATGATATCCTTTCGCGACTTGATAAAG


 


TCGAGGCGGAGGTACAAATTGACAGGTTAATTACAGGCAGACTTCAAAGC


 


CTTCAAACCTATGTAACACAACAACTAATCAGGGCTGCTGAAATCAGGGC


 


TTCTGCTAATCTTGCTGCTACTAAAATGTCTGAGTGTGTTCTTGGACAAT


 


CAAAAAGAGTTGACTTTTGTGGAAAGGGCTACCACCTTATGTCCTTCCCA


 


CAAGCAGCCCCGCATGGTGTTGTCTTCCTACATGTCACGTATGTGCCATC


 


CCAGGAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATGAAGGCAAAG


 


CATACTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCTTGGTTT


 


ATTACACAGAGGAACTTCTTTTCTCCACAAATAATTACTACAGACAATAC


 


ATTTGTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTAACAACACAG


 


TTTATGATCCTCTGCAACCTGAGCTCGACTCATTCAAAGAAGAGCTGGAC


 


AAGTACTTCAAAAATCATACATCACCAGATGTTGATCTTGGCGACATTTC


 


AGGCATTAACGCTTCTGTCGTCAACATTCAAAAAGAAATTGACCGCCTCA


 


ATGAGGTCGCTAAAAATTTAAATGAATCACTCATTGACCTTCAAGAATTG


 


GGAAAATATGAGCAATATATTAAATGGCCTTGG


 


Soluble TPA-S1


(SEQ ID NO: 9)


ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGC


 


AGTCTTCGTTTCGCCCAGCGCTAGAGGATCGGGAAGTGACCTTGACCGGT


 


GCACCACTTTTGATGATGTTCAAGCTCCTAATTACACTCAACATACTTCA


 


TCTATGAGGGGGGTTTACTATCCTGATGAAATTTTTAGATCAGACACTCT


 


TTATTTAACTCAGGATTTATTTCTTCCATTTTATTCTAATGTTACAGGGT


 


TTCATACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTAAGGAT


 


GGTATTTATTTTGCTGCCACAGAGAAATCAAATGTTGTCCGTGGTTGGGT


 


TTTTGGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTATTAACA


 


ATTCTACTAATGTTGTTATACGAGCATGTAACTTTGAATTGTGTGACAAC


 


CCTTTCTTTGCTGTTTCTAAACCCATGGGTACACAGACACATACTATGAT


 


ATTCGATAATGCATTTAATTGCACTTTCGAGTACATATCTGATGCCTTTT


 


CGCTTGATGTTTCAGAAAAGTCAGGTAATTTTAAACACTTACGAGAGTTT


 


GTGTTTAAAAATAAAGATGGGTTTCTCTATGTTTATAAGGGCTATCAACC


 


TATAGATGTAGTTCGTGATCTACCTTCTGGTTTTAACACTTTGAAACCTA


 


TTTTTAAGTTGCCTCTTGGTATTAACATTACAAATTTTAGAGCCATTCTT


 


ACAGCCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCAGCCTA


 


TTTTGTTGGCTATTTAAAGCCAACTACATTTATGCTCAAGTATGATGAAA


 


ATGGTACAATCACAGATGCTGTTGATTGTTCTCAAAATCCACTTGCTGAA


 


CTCAAATGCTCTGTTAAGAGCTTTGAGATTGACAAAGGAATTTACCAGAC


 


CTCTAATTTCAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCCTAATA


 


TTACAAACTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAATTCCCT


 


TCTGTCTATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCTGATTA


 


CTCTGTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTATGGCG


 


TTTCTGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATGCAGAT


 


TCTTTTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGACAAAC


 


TGGTGTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCATGGGTT


 


GTGTCCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAACTGGTAAT


 


TATAATTATAAATATAGGTATCTTAGACATGGCAAGCTTAGGCCCTTTGA


 


GAGAGACATATCTAATGTGCCTTTCTCCCCTGATGGCAAACCTTGCACCC


 


CACCTGCTCTTAATTGTTATTGGCCATTAAATGATTATGGTTTTTACACC


 


ACTACTGGCATTGGCTACCAACCTTACAGAGTTGTAGTACTTTCTTTTGA


 


ACTTTTAAATGCACCGGCCACGGTTTGTGGACCAAAATTATCCACTGACC


 


TTATTAAGAACCAGTGTGTCAATTTTAATTTTAATGGACTCACTGGTACT


 


GGTGTGTTAACTCCTTCTTCAAAGAGATTTCAACCATTTCAACAATTTGG


 


CCGTGATGTTTCTGATTTCACTGATTCCGTTCGAGATCCTAAAACATCTG


 


AAATATTAGACATTTCACCTTGCTCTTTTGGGGGTGTAAGTGTAATTACA


 


CCTGGAACAAATGCTTCATCTGAAGTTGCTGTTCTATATCAAGATGTTAA


 


CTGCACTGATGTTTCTACAGCAATTCATGCAGATCAACTCACACCAGCTT


 


GGCGCATATATTCTACTGGAAACAATGTATTCCAGACTCAAGCAGGCTGT


 


CTTATAGGAGCTGAGCATGTCGACACTTCTTATGAGTGCGACATTCCTAT


 


TGGAGCTGGCATTTGTGCTAGTTACCATACAGTTTCTTTATTACGTAGTA


 


CTAGCCAAAAATCTATTGTGGCTTATACTATGTCTTTAGGTGC


 


Soluble TPA-S2


(SEQ ID NO: 11)


ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGC


 


AGTCTTCGTTTCGCCCAGCGCTAGAGGATCGGGAGATAGTTCAATTGCTT


 


ACTCTAATAACACCATTGCTATACCTACTAACTTTTCAATTAGCATTACT


 


ACAGAAGTAATGCCTGTTTCTATGGCTAAAACCTCCGTAGATTGTAATAT


 


GTACATCTGCGGAGATTCTACTGAATGTGCTAATTTGCTTCTCCAATATG


 


GTAGCTTTTGCACACAACTAAATCGTGCACTCTCAGGTATTGCTGCTGAA


 


CAGGATCGCAACACACGTGAAGTGTTCGCTCAAGTCAAACAAATGTACAA


 


AACCCCAACTTTGAAATATTTTGGTGGTTTTAATTTTTCACAAATATTAC


 


CTGACCCTCTAAAGCCAACTAAGAGGTCTTTTATTGAGGACTTGCTCTTT


 


AATAAGGTGACACTCGCTGATGCTGGCTTCATGAAGCAATATGGCGAATG


 


CCTAGGTGATATTAATGCTAGAGATCTCATTTGTGCGCAGAAGTTCAATG


 


GACTTACAGTGTTGCCACCTCTGCTCACTGATGATATGATTGCTGCCTAC


 


ACTGCTGCTCTAGTTAGTGGTACTGCCACTGCTGGATGGACATTTGGTGC


 


TGGCGCTGCTCTTCAAATACCTTTTGCTATGCAAATGGCATATAGGTTCA


 


ATGGCATTGGAGTTACCCAAAATGTTCTCTATGAGAACCAAAAACAAATC


 


GCCAACCAATTTAACAAGGCGATTAGTCAAATTCAAGAATCACTTACAAC


 


AACATCAACTGCATTGGGCAAGCTGCAAGACGTTGTTAACCAGAATGCTC


 


AAGCATTAAACACACTTGTTAAACAACTTAGCTCTAATTTTGGTGCAATT


 


TCAAGTGTGCTAAATGATATCCTTTCGCGACTTGATAAAGTCGAGGCGGA


 


GGTACAAATTGACAGGTTAATTACAGGCAGACTTCAAAGCCTTCAAACCT


 


ATGTAACACAACAACTAATCAGGGCTGCTGAAATCAGGGCTTCTGCTAAT


 


CTTGCTGCTACTAAAATGTCTGAGTGTGTTCTTGGACAATCAAAAAGAGT


 


TGACTTTTGTGGAAAGGGCTACCACCTTATGTCCTTCCCACAAGCAGCCC


 


CGCATGGTGTTGTCTTCCTACATGTCACGTATGTGCCATCCCAGGAGAGG


 


AACTTCACCACAGCGCCAGCAATTTGTCATGAAGGCAAAGCATACTTCCC


 


TCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCTTGGTTTATTACACAGA


 


GGAACTTCTTTTCTCCACAAATAATTACTACAGACAATACATTTGTCTCA


 


GGAAATTGTGATGTCGTTATTGGCATCATTAACAACACAGTTTATGATCC


 


TCTGCAACCTGAGCTCGACTCATTCAAAGAAGAGCTGGACAAGTACTTCA


 


AAAATCATACATCACCAGATGTTGATCTTGGCGACATTTCAGGCATTAAC


 


GCTTCTGTCGTCAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTCGC


 


TAAAAATTTAAATGAATCACTCATTGACCTTCAAGAATTGGGAAAATATG


 


AGCAATATATTAAATGGCCTTGG






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S, S1, or S2 polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs:7, 9, or 11, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response.
The amino acid sequences of the soluble S protein, S1 and S2 proteins with the TPA secretory leader peptide are shown below.







Soluble TPA-S


(SEQ ID NO: 8)


MDAMKRGLCCVLLLCGAVFVSPSARGSGSDLDRCTTFDDVQAPNYTQHTS


 


SMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKD


 


GIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDN


 


PFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREF


 


VFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAIL


 


TAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAE


 


LKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFP


 


SVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYAD


 


SFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGN


 


YNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYT


 


TTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKMQCVNFNFNGLTGT


 


GVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVIT


 


PGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGC


 


LIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADS


 


SIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLL


 


LQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFS


 


QILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQ


 


KFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMA


 


YRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVN


 


QNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQS


 


LQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFP


 


QAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWF


 


ITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELD


 


KYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL


 


GKYEQYIKWPW


 


Soluble TPA-S1 protein


(SEQ ID NO: 10)


MDAMKRGLCCVLLLCGAVFVSPSARGSGSDLDRCTTFDDVQAPNYTQHTS


 


SMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKD


 


GIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDN


 


PFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKMLREF


 


VFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAIL


 


TAFSPAQDIWGTSAAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAE


 


LKCSVKSFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFP


 


SVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYAD


 


SFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGN


 


YNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYT


 


TTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGT


 


GVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVIT


 


PGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGC


 


LIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGA


 


Soluble TPA-S2 protein


(SEQ ID NO: 12)


MDAMKRGLCCVLLLCGAVFVSPSARGSGDSSIAYSNNTIAIPTNFSISIT


 


TEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAE


 


QDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLF


 


NKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAY


 


TAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQI


 


ANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAI


 


SSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN


 


LAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQER


 


NFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVS


 


GNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGIN


 


ASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV S, S1, or S2 polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs:8, 10, or 12, wherein said polypeptide raises a detectable immune response.
In a further embodiment, the present invention provides for methods for raising a a detectable immune response to the SARS-CoV polypeptides, comprising administering to a vertebrate a polynucleotide which operably encodes polypeptides, fragments, variants, or derivatives thereof as described above.
The S protein of some coronaviruses contain an Fcγ-like domain that binds immunoglobulin. Data from the FIPV immunization suggests that high levels of potentially neutralizing antibody may be bound by the Fc-mimicking region of the S protein. Scott, F. W. Adv. Vet. Med. 41: 347-58 (1999). Thus, modification or deletion of an Fcγ region of the SARS-CoV S protein may be useful in the compositions of the present invention.
The nucleocapsid protein (N) is encoded by about nucleotides 28120 through about 29388 of the Urbani strain of SARS-CoV. (Bellini et al. SARS Coronavirus Urbani, complete genome. GenBank Accession No. AY278741).
The protein is a phosphoprotein of 50 to 60 kd that interacts with viral genomic RNA to form the viral nucleocapsid. N has three relatively conserved structural domains, including an RNA-binding domain in the middle that binds to the leader sequence of viral RNA. N protein in the viral nucleocapsid further interacts with the membrane protein (M), leading to the formation of virus particles. N is also suggested to play a role in viral RNA synthesis, by a study in which an antibody directed against N inhibited an in vitro coronavirus RNA polymerase reaction. Marra et al. N protein also binds to cellular membranes and phospholipids, a property that may help to facilitate both virus assembly and formation of RNA replication complexes.
From about nucelotides 28120 to about 29388 of the Urbani strain of the SARS-CoV genome encode the N protein. (Bellini et al. SARS Coronavirus Urbani, complete genome. GenBank Accession No. AY278741) and has the following sequence, referred to herein as SEQ ID NO:13:







ATGTCTGATAATGGACCCCAATCAAACCAACGTAGTGCCCCCCGCATTAC


 


ATTTGGTGGACCCACAGATTCAACTGACAATAACCAGAATGGAGGACGCA


 


ATGGGGCAAGGCCAAAACAGCGCCGACCCCAAGGTTTACCCAATAATACT


 


GCGTCTTGGTTCACAGCTCTCACTCAGCATGGCAAGGAGGAACTTAGATT


 


CCCTCGAGGCCAGGGCGTTCCAATCAACACCAATAGTGGTCCAGATGACC


 


AAATTGGCTACTACCGAAGAGCTACCCGACGAGTTCGTGGTGGTGACGGC


 


AAAATGAAAGAGCTCAGCCCCAGATGGTACTTCTATTACCTAGGAACTGG


 


CCCAGAAGCTTCACTTCCCTACGGCGCTAACAAAGAAGGCATCGTATGGG


 


TTGCAACTGAGGGAGCCTTGAATACACCCAAAGACCACATTGGCACCCGC


 


AATCCTAATAACAATGCTGCCACCGTGCTACAACTTCCTCAAGGAACAAC


 


ATTGCCAAAAGGCTTCTACGCAGAGGGAAGCAGAGGCGGCAGTCAAGCCT


 


CTTCTCGCTCCTCATCACGTAGTCGCGGTAATTCAAGAAATTCAACTCCT


 


GGCAGCAGTAGGGGAAATTCTCCTGCTCGAATGGCTAGCGGAGGTGGTGA


 


AACTGCCCTCGCGCTATTGCTGCTAGACAGATTGAACCAGCTTGAGAGCA


 


AAGTTTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAA


 


TCTGCTGCTGAGGCATCTAAAAAGCCTCGCCAAAAACGTACTGCCACAAA


 


ACAGTACAACGTCACTCAAGCATTTGGGAGACGTGGTCCAGAACAAACCC


 


AAGGAAATTTCGGGGACCAAGACCTAATCAGACAAGGAACTGATTACAAA


 


CATTGGCCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCTTTGG


 


AATGTCACGCATTGGCATGGAAGTCACACCTTCGGOAACATGGCTGACTT


 


ATCATGGAGCCATTAAATTGGATGACAAAGATCCACAATTCAAAGACAAC


 


GTCATACTGCTGAACAAGCACATTGACGCATACAAAACATTCCCACCAAC


 


AGAGCCTAAAAAGGACAAAAAGAAAAAGACTGATGAAGCTCAGCCTTTGC


 


CGCAGAGACAAAAGAAGCAGCCCACTGTGACTCTTCTTCCTGCGGCTGAC


 


ATGGATGATTTCTCCAGACAACTTCAAAATTCCATGAGTGGAGCTTCTGC


 


TGATTCAACTCAGGCATAA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV N, polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:13, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response.
The amino acid sequence of the N protein encoded by SEQ ID NO:13 has the following sequence shown below and is referred to herein as SEQ ID NO:14







MSDNGPQSNQRSAPRITFGGPTDSTDNNQNGGRNGARPKQRRPQGLPNNT


 


ASWFTALTQHGKEELRFPRGQGVPINTNSGPDDQIGYYRRATRRVRGGDG


 


KMKELSPRWYFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTR


 


NPNNNAATVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRGNSRNSTP


 


GSSRGNSPARMASGGGETALALLLLDRLNQLESKVSGKGQQQQGQTVTKK


 


SAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYK


 


HWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDN


 


VILLNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAAD


 


MDDFSRQLQNSMSGASADSTQA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV N polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:14, wherein said polypeptide raises a detectable immune response.
The N protein contains a nuclear localization sequence (NLS) which directs the protein to the nucleus infected cells or cells in which the protein is expressed. The sequence of the NLS is KTFPPTEPKKDKKKKTDEAQ (underlined above) and is referred to herein as SEQ ID NO:17. For purposes of the invention, the NLS may be deleted from the protein to obtain a non-nuclear localized version of the protein. The nucleotide sequence of an N protein lacking the NLS is referred to herein as SEQ ID NO:15 and is shown below.







ATGTCTGATAATGGACCCCAATCAAACCAACGTAGTGCCCCCCGCATTAC


 


ATTTGGTGGACCCACAGATTCAACTGACAATAACCAGAATGGAGGACGCA


 


ATGGGGCAAGGCCAAAACAGCGCCGACCCCAAGGTTTACCCAATAATACT


 


GCGTCTTGGTTCACAGCTCTCACTCAGCATGGCAAGGAGGAACTTAGATT


 


CCCTCGAGGCCAGGGCGTTCCAATCAACACCAATAGTGGTCCAGATGACC


 


AAATTGGCTACTACCGAAGAGCTACCCGACGAGTTCGTGGTGGTGACGGC


 


AAAATGAAAGAGCTCAGCCCCAGATGGTACTTCTATTACCTAGGAACTGG


 


CCCAGAAGCTTCACTTCCCTACGGCGCTAACAAAGAAGGCATCGTATGGG


 


TTGCAACTGAGGGAGCCTTGAATACACCCAAAGACCACATTGGCACCCGC


 


AATCCTAATAACAATGCTGCCACCGTGCTACAACTTCCTCAAGGAACAAC


 


ATTGCCAAAAGGCTTCTACGCAGAGGGAAGCAGAGGCGGCAGTCAAGCCT


 


CTTCTCGCTCCTCATCACGTAGTCGCGGTAATTCAAGAAATTCAACTCCT


 


GGCAGCAGTAGGGGAAATTCTCCTGCTCGAATGGCTAGCGGAGGTGGTGA


 


AACTGCCCTCGCGCTATTGCTGCTAGACAGATTGAACCAGCTTGAGAGCA


 


AAGTTTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAA


 


TCTGCTGCTGAGGCATCTAAAAAGCCTCGCCAAAAACGTACTGCCACAAA


 


ACAGTACAACGTCACTCAAGCATTTGGGAGACGTGGTCCAGAACAAACCC


 


AAGGAAATTTCGGGGACCAAGACCTAATCAGACAAGGAACTGATTACAAA


 


CATTGGCCGCAAATTGCACAATTTGCTCCAAGTGCCTCTGCATTCTTTGG


 


AATGTCACGCATTGGCATGGAAGTCACACCTTCGGGAACATGGCTGACTT


 


ATCATGGAGCCATTAAATTGGATGACAAAGATCCACAATTCAAAGACAAC


 


GTCATACTGCTGAACAAGCACATTGACGCATACCCTTTGCCGCAGAGACA


 


AAAGAAGCAGCCCACTGTGACTCTTCTTCCTGCGGCTGACATGGATGATT


 


TCTCCAGACAACTTCAAAATTCCATGAGTGGAGCTTCTGCTGATTCAACT


 


CAGGCATAA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV N, polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response.
The amino acid sequence of the N protein without the NLS sequence is encoded by SEQ ID NO:15 has the following sequence shown below and is referred to herein as SEQ ID NO:16:







MSDNGPQSNQRSAPRITFGGPTDSTDNNQNGGRNGARPKQRRPQGLPNNT


 


ASWFTALTQHGKEELRFPRGQGVPINTNSGPDDQIGYYRRATRRVRGGDG


 


KMKELSPRWYFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTR


 


NPNNNAATVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRGNSRNSTP


 


GSSRGNSPARMASGGGETALALLLLDRLNQLESKVSGKGQQQQGQTVTKK


 


SAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYK


 


HWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDN


 


VILLNKHIDAYPLPQRQKKQPTVTLLPAADMDDFSRQLQNSMSGASADST


 


QA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV N polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16, wherein said polypeptide raises a detectable immune response.
The membrane glycoprotein (M) is encoded by about nucleotides 26398 to about 27063 of the Urbani strain of SARS-CoV. (Bellini et al. SARS Coronavirus Urbani, complete genome. GenBank Accession No. AY278741). The M protein differs from other coronavirus glycoproteins in that only a short amino terminal domain of M is exposed on the exterior of the viral envelope. This domain is followed by a triple-membrane-spanning domain, an α-helical domain, and a large carboxylterminal domain inside the viral envelope. In some coronaviruses, such as transmissible gastroenteritis coronavirus (TGEV), the carboxylterminus of the M protein is exposed on the virion surface. Glycosylation of the aminoterminal domain is O-linked for MHV and N-linked for infectious bronchitis virus (IBV) and TGEV. Monoclonal antibodies against the external domain of M neutralize viral infectivity, but only in the presence of complement. M proteins of some coronaviruses can induce interferon-α. The M proteins are targeted to the Golgi apparatus and not transported to the plasma membrane. In TGEV and MHV virions, the M glycoprotein is present not only in the viral envelope but also in the internal core structure. (Field's Virology, B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, eds., 4th Edition. Lippincott-Raven, Philadelphia, Pa.).
From about nucelotides 26398 to about 27063 of the Urbani strain of the SARS-CoV genome encode the M protein, Bellini et al. SARS Coronavirus Urbani, complete genome, GenBank Accession No. AY27874, and has the following sequence, referred to herein as SEQ ID NO:18:







ATGGCAGACAACGGTACTATTACCGTTGAGGAGCTTAAACAACTCCTGGA


 


ACAATGGAACCTAGTAATAGGTTTCCTATTCCTAGCCTGGATTATGTTAC


 


TACAATTTGCCTATTCTAATCGGAACAGGTTTTTGTACATAATAAAGCTT


 


GTTTTCCTCTGGCTCTTGTGGCCAGTAACACTTGCTTGTTTTGTGCTTGC


 


TGCTGTCTACAGAATTAATTGGGTGACTGGCGGGATTGCGATTGCAATGG


 


CTTGTATTGTAGGCTTGATGTGGCTTAGCTACTTCGTTGCTTCCTTCAGG


 


CTGTTTGCTCGTACCCGCTCAATGTGGTCATTCAACCCAGAAACAAACAT


 


TCTTCTCAATGTGCCTCTCCGGGGGACAATTGTGACCAGACCGCTCATGG


 


AAAGTGAACTTGTCATTGGTGCTGTGATCATTCGTGGTCACTTGCGAATG


 


GCCGGACACCCCCTAGGGCGCTGTGACATTAAGGACCTGCCAAAAGAGAT


 


CACTGTGGCTACATCACGAACGCTTTCTTATTACAAATTAGGAGCGTCGC


 


AGCGTGTAGGCACTGATTCAGGTTTTGCTGCATACAACCGCTACCGTATT


 


GGAAACTATAAATTAAATACAGACCACGCCGGTAGCAACGACAATATTGC


 


TTTGCTAGTACAGTAA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV M, polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response.
The amino acid sequence of the M protein encoded by SEQ ID NO:18 has the following sequence shown below and is referred to herein as SEQ ID NO:19:







MADNGTITVEELKQLLEQWNLVIGFLFLAWIMLLQFAYSNRNRFLYIIKL


 


VFLWLLWPVTLACFVLAAVYRINWVTGGIAIAMACIVGLMWLSYFVASFR


 


LFARTRSMWSFNPETNILLNVPLRGTIVTRPLMESELVIGAVIIRGHLRM


 


AGHPLGRCDIKDLPKEITVATSRTLSYYKLGASQRVGTDSGFAAYNRYRI


 


GNYKLNTDHAGSNDNIALLVQ






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV M polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:19 wherein said polypeptide raises a detectable immune response.
The small envelope protein (E) is encoded by about nucleotide 26117 to about 26347 of the Urbani strain of SARS-CoV (Bellini et al. SARS Coronavirus Urbani, complete genome, GenBank Accession No. AY278741), and has the following sequence, referred to herein as SEQ ID NO: 20:







ATGTACTCATTCGTTTCGGAAGAAACAGGTACGTTAATAGTTAATAGCGT


 


ACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTCACACTAGCCATCC


 


TTACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGT


 


TTAGTAAAACCAACGGTTTACGTCTACTCGCGTGTTAAAAATCTGAACTC


 


TTCTGAAGGAGTTCCTGATCTTCTGGTCTAA






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV E, polypeptide, wherein said polynucleotide is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,. 99% or 100% identical to SEQ ID NO:20, or a codon-optimized version as described below, and wherein said polynucleotide encodes a polypeptide that elicits a detectable immune response
Based on protein comparisons with other coronaviruses, the SARS-CoV E protein shares conserved sequences with TGEV and MHV. For some coronaviruses, such as TGEV, the E protein is necessary for replication of the virus, while for others, such as MHV, loss of the E protein merely reduces virus replication without eliminating it completely. Marra et al. The protein sequence is shown below and referred to, herein as SEQ ID NO:21.







MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVS


 


LVKPTVYVYSRVKNLNSSEGVPDLLV






In a further embodiment the methods of the present invention provide for administering a polynucleotide which operably encodes a SARS-CoV E polypeptide comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21 wherein said polypeptide raises a detectable immune response.
It should be noted that nucleotide sequences encoding various SARS-CoV polypeptides may vary between SARS-CoV strains. Virtually any nucleotide sequence encoding a SARS-CoV protein is suitable for the present invention. In fact, polynucleotide sequences included in vaccines and therapeutic formulations of the current invention may change from year to year, depending on the prevalent strain or strains of SARS-CoV.
Further examples of SARS-CoV polypeptides within the scope of the invention are multimerized fragments of SARS-CoV polypeptides and polynucleotides that encode multimerized fragments of SARS-CoV polypeptides. The polypeptide fragments of the invention contain at least one antigenic region. The SARS-CoV polypeptide fragments are fused to small assembly polypeptides. Non-limiting examples within the scope of the invention include coiled-coiled structures such as: an amphipathic helix, the yeast CGN4 leucine zipper, the human p53 tetramerization domain, and synthetic coil polypeptides. The SARS-CoV and assembly peptide fusion proteins self-assemble into stable multimers forming dimers, trimers, tetramers, and higher order multimers depending on the interacting amino acid residues. These multimerized SARS-CoV polypeptide fragments have increased local epitope valency which functions to more efficiently activate B lymphocytes, thereby producing a more robust immune response. Also within the scope of the invention are multimerized SARS-CoV polypeptide fragments that maintain conformational neutralizing epitopes.
Also within the scope of the present invention are combinations of SARS-CoV polypeptides and polynucleotides that encode SARS-CoV polypeptides, where the polypeptides assemble into virus-like particles (VLP). One such combination is, but is not limited to a combination of SARS-CoV S, M, and E polypeptides or fragments, variants, or derivatives thereof, and polynucleotides encoding SARS-CoV S, M, and E polypeptides or fragments, variants, or derivatives thereof. Combinations of SARS-CoV polypeptides that form VLPs may be useful in enhancing immunogenicity of SARS-CoV polypeptides and in eliciting a detectable immune response to the SARS-CoV virus. Also within the scope of the present invention are methods of producing SARS-CoV VLPs in vitro by using protocols that are well known in the art. The production of VLPs may be performed in any tissue culture cell line that can tolerate expression of SARS-CoV polypeptide. Examples of cell lines include, but are not limited to, fungal cells, including yeast cells such as Saccharomyces spp. cells; insect cells such as Drosophila S2, Spodoptera Sf9 or Sf21 cells and Trichoplusa High-Five cells; other animal cells (particularly mammalian cells and human cells) such as Vero, MDCK, CV1, 3T3, CPAE, A10, Sp2/0-Ag14, PC12, CHO, COS, HeLa, Bowes melanoma cells, SW-13, NCI-H295, RT4, HT-1376, UM-UC-3, IM-9, KG-1, R54;11, A-172, U-87MG, BT-20, MCF-7, SK-BR-3, ChaGo K-1, CCD-14Br, CaSki, ME-180, FHC, HT-29, Caco-2, SW480, HuTu80, Tera 1, NTERA-2, AN3 CA, KLE, RL95-2, Caki-1, ACHN, 769 P, CCRF-CEM, Hut 78, MOLT 4, HL-60, Hep-3B, HepG2, SK-HEP1, A-549, NCI-H146, NCI-H82, NCI-H82, SK-LU-1, WI-38, MRC-5, HLF-a, CCD-19Lu, C39, Hs294T, SK-MEL5, COLO 829, U266B1, RPMI 2650, BeWo, JEG-3, JAR, SW 1353, MeKam, and SCC-4; and higher plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art.
De Haan et al., J. Virol. 72: 6838-50 (1998), describe the assembly of coronavirus VLPs from the coexpression of mouse hepatitis virus M and E genes in eukaryotic cells. Bos et al., J. Virol. 71: 9427-33 describe the role of the S protein in infectivity of coronavirus VLPs produced by coexpression of mouse hepatitis virus S, M, and E proteins. These references are hereby incorporated by reference in their entireties.
In another embodiment, the VLP comprising SARS-CoV polypeptides S, M, and E provides a method for mimicking a SARS-CoV infection without the use of the actual infectious agent. In addtion, the VLP provides a method for eliciting a detectable immune response to multiple antigens in a confirmation similar to the actual virus particle thereby enhancing the immunogenicity of the SARS-CoV polypeptides.
The VLP's of the invention can be produced in vivo by delivery of S, M or E polynucleotides or polypeptides, described herein, to a vertebrate wherein assembly of the VLPs occurs with the cells of the vertebrate. In an alternative embodiment, VLPs of the invention can be produced in vitro in cells that have received the S, M, and E polynucleotides described herein and express said proteins. VLPs are then purified from the cells using techniques known in the art for coronavirus particle purification. These purified particles can then be administered to a vertebrate to elicit a detectable immune response or to study the pathogenesis of the SARS-CoV infection without the need of the actual infectious agent.
The combination of S, M and E to create virus like particles in the previous examples is not meant to be limiting. Other SARS-CoV polypeptides, which assemble into, or are engineered to assemble into virus like particles, may be used as well.
The present invention also provides vaccine compositions and methods for delivery of SARS-CoV coding sequences to a vertebrate. In other embodiments, the present invention provides vaccine compositions and methods for delivery of SARS-CoV coding sequences to a vertebrate with optimal expression and safety conferred through codon optimization and/or other manipulations. These vaccine compositions are prepared and administered in such a manner that the encoded gene products are optimally expressed in the vertebrate of interest. As a result, these compositions and methods are useful in stimulating an immune response against SARS-CoV infection. Also included in the invention are expression systems, delivery systems, and codon-optimized SARS-CoV coding regions.
In a specific embodiment, the invention provides polynucleotide (e.g., DNA) vaccines in which the single formulation comprises a SARS-CoV polypeptide-encoding polynucleotide vaccine as described herein. An alternative embodiment of the invention provides for a multivalent formulation comprising several (e.g., two, three, four, or more) SARS-CoV polypeptide-encoding polynucleotides, as described herein, within a single vaccine composition. The SARS-CoV polypeptide-encoding polynucleotides, fragments, or variants thereof may be contained within a single expression vector (e.g., plasmid or viral vector) or may be contained within multiple expression vectors.
In a specific embodiment, the invention provides combinatorial polynucleotide (e.g., DNA) vaccines which combine both a polynucleotide vaccine and polypeptide (e.g., either a recombinant protein, a purified subunit protein, a viral vector expressing an isolated SARS-CoV polypeptide) vaccine in a single formulation. The single formulation comprises a SARS-CoV polypeptide-encoding polynucleotide vaccine as described herein, and optionally, an effective amount of a desired isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof. The polypeptide may exist in any form, for example, a recombinant protein, a purified subunit protein, or a viral vector expressing an isolated SARS-CoV polypeptide. The SARS-CoV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide vaccine may be identical to the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof. Alternatively, the SARS-CoV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide may be different from the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
It is to be noted that the term “about” when referring to a polynucleotide, coding region or any nucleotide sequence, for example, is understood to represent plus or minus 1 to 30 nucleotides on either end of the defined coding region, polynucleotide or nucleotide sequence. It is to be noted that when referring to a polypeptide, or polypeptide sequence, that the term “about” is understood to represent plus or minus 1 to 10 amino acids on either end of the defined polypeptide or polypeptide sequence. It should be further noted that the term “about,” when referring to the quantity of a specific codon in a given codon-optimized coding region has a specific meaning, described in more detail below.
The term “polynucleotide” is intended to encompass a singular nucleic acid or nucleic acid fragment as well as plural nucleic acids or nucleic acid fragments, and refers to an isolated molecule or construct, e.g., a virus genome (e.g., a non-infectious viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles as described in Darquet, A-M et al., Gene Therapy 4:1341-1349 (1997)) comprising a polynucleotide. A nucleic acid or fragment thereof may be provided in linear (e.g., mRNA), circular (e.g., plasmid), or branched form as well as double-stranded or single-stranded forms. A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
The terms “nucleic acid” or “nucleic acid fragment” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide or construct.
As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, and the like, are not part of a coding region. Two or more nucleic acids or nucleic acid fragments of the present invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate polynucleotide constructs, e.g., on separate (different) plasmids. Furthermore, any nucleic acid or nucleic acid fragment may encode a single SARS-CoV polypeptide or fragment, derivative, or variant thereof, e.g., or may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides. In addition, a nucleic acid may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator, or may encode heterologous coding regions fused to the SARS-CoV coding region, e.g., specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
The terms “fragment,” “variant,” “derivative,” and “analog,” when referring to SARS-CoV polypeptides of the present invention, include any polypeptides which retain at least some of the immunogenicity or antigenicity of the corresponding native polypeptide. Fragments of SARS-CoV polypeptides of the present invention include proteolytic fragments, deletion fragments, and in particular, fragments of SARS-CoV polypeptides which exhibit increased secretion from the cell or higher immunogenicity or reduced pathogenicity when delivered to an animal. Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three-dimensional epitopes. Variants of SARS-CoV polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally, such as an allelic variant. By an “allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome or genome of an organism or virus. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985), which is incorporated herein by reference. Naturally or non-naturally occurring variations such as amino acid deletions, insertions or substitutions may occur. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of SARS-CoV polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. An analog is another form of a SARS-CoV polypeptide of the present invention. An example is a proprotein which can be activated by cleavage of the proprotein to produce an active mature polypeptide.
The terms “infectious polynucleotide” or “infectious nucleic acid” are intended to encompass isolated viral polynucleotides and/or nucleic acids which are solely sufficient to mediate the synthesis of complete infectious virus particles upon uptake by permissive cells. Thus, “infectious nucleic acids” do not require pre-synthesized copies of any of the polypeptides it encodes, e.g., viral replicases, in order to initiate its replication cycle in a permissive host cell.
The terms “non-infectious polynucleotide” or “non-infectious nucleic acid” as defined herein are polynucleotides or nucleic acids which cannot, without additional added materials, e.g, polypeptides, mediate the synthesis of complete infectious virus particles upon uptake by permissive cells. An infectious polynucleotide or nucleic acid is not made “non-infectious” simply because it is taken up by a non-permissive cell. For example, an infectious viral polynucleotide from a virus with limited host range is infectious if it is capable of mediating the synthesis of complete infectious virus particles when taken up by cells derived from a permissive host (i.e., a host permissive for the virus itself). The fact that uptake by cells derived from a non-permissive host does not result in the synthesis of complete infectious virus particles does not make the nucleic acid “non-infectious.” In other words, the term is not qualified by the nature of the host cell, the tissue type, or the species taking up the polynucleotide or nucleic acid fragment.
In some cases, an isolated infectious polynucleotide or nucleic acid may produce fully-infectious virus particles in a host cell population which lacks receptors for the virus particles, i.e., is non-permissive for virus entry.
Thus viruses produced will not infect surrounding cells. However, if the supernatant containing the virus particles is transferred to cells which are permissive for the virus, infection will take place.
The terms “replicating polynucleotide” or “replicating nucleic acid” are meant to encompass those polynucleotides and/or nucleic acids which, upon being taken up by a permissive host cell, are capable of producing multiple, e.g., one or more copies of the same polynucleotide or nucleic acid. Infectious polynucleotides and nucleic acids are a subset of replicating polynucleotides and nucleic acids; the terms are not synonymous. For example, a defective virus genome lacking the genes for virus coat proteins may replicate, e.g., produce multiple copies of itself, but is NOT infectious because it is incapable of mediating the synthesis of complete infectious virus particles unless the coat proteins, or another nucleic acid encoding the coat proteins, are exogenously provided.
In certain embodiments, the polynucleotide, nucleic acid, or nucleic acid fragment is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally also comprises a promoter and/or other transcription or translation control elements operably associated with the polypeptide-encoding nucleic acid fragment. An operable association is when a nucleic acid fragment encoding a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide-encoding nucleic acid fragment and a promoter associated with the 5′ end of the nucleic acid fragment) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid fragment encoding a polypeptide if the promoter were capable of effecting transcription of that nucleic acid fragment. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.
A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g. promoters inducible by interferons or interleukins).
Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, elements from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
A DNA polynucleotide of the present invention may be a circular or linearized plasmid, or other linear DNA which may also be non-infectious and nonintegrating (i.e., does not integrate into the genome of vertebrate cells). A linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease. Linear DNA may be advantageous in certain situations as discussed, e.g., in Cherng, J. Y., et al., J. Control. Release 60:343-53 (1999), and Chen, Z. Y., et al. Mol. Ther. 3:403-10 (2001), both of which are incorporated herein by reference.
Alternatively, DNA virus genomes may be used to administer DNA polynucleotides into vertebrate cells. In certain embodiments, a DNA virus genome of the present invention is nonreplicative, noninfectious, and/or nonintegrating. Suitable DNA virus genomes include without limitation, herpesvirus genomes, adenovirus genomes, adeno-associated virus genomes, and poxvirus genomes. References citing methods for the in vivo introduction of non-infectious virus genomes to vertebrate tissues are well known to those of ordinary skill in the art, and are cited supra.
In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). Methods for introducing RNA sequences into vertebrate cells are described in U.S. Pat. No. 5,580,859, the disclosure of which is incorporated herein by reference in its entirety.
Polynucleotides, nucleic acids, and nucleic acid fragments of the present invention may be associated with additional nucleic acids which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a nucleic acid fragment or polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native leader sequence is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian leader sequence, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.
In accordance with one aspect of the present invention, there is provided a polynucleotide construct, for example, a plasmid, comprising a nucleic acid fragment, where the nucleic acid fragment is a fragment of a coding region operably encoding an SARS-CoV-derived polypeptide. In accordance with another aspect of the present invention, there is provided a polynucleotide construct, for example, a plasmid, comprising a nucleic acid fragment, where the nucleic acid fragment is a fragment of a codon-optimized coding region operably encoding an SARS-CoV-derived polypeptide, where the coding region is optimized for expression in vertebrate cells, of a desired vertebrate species, e.g., humans, to be delivered to a vertebrate to be treated or immunized. Suitable SARS-CoV polypeptides, or fragments, variants, or derivatives thereof may be derived from, but are not limited to, the SARS-CoV S, Soluble S1, Soluble S2, N, E or M proteins. Additional SARS-CoV-derived coding sequences, e.g., coding for S, Soluble S1, Soluble S2, N, E or M, may also be included on the plasmid, or on a separate plasmid, and expressed, either using native SARS-CoV codons or one or more codons optimized for expression in the vertebrate to be treated or immunized. When such a plasmid encoding one or more optimized SARS-CoV sequences and/or one or more optimized SARS-CoV sequences is delivered, in vivo to a tissue of the vertebrate to be treated or immunized, one or more of the encoded gene products will be expressed, i.e., transcribed and translated. The level of expression of the gene product(s) will depend to a significant extent on the strength of the associated promoter and the presence and activation of an associated enhancer element, as well as the degree of optimization of the coding region.
As used herein, the term “plasmid” refers to a construct made up of genetic material (i.e., nucleic acids). Typically a plasmid contains an origin of replication which is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells comprising the plasmid. Plasmids of the present invention may include genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in eukaryotic cells. Also, the plasmid may include a sequence from a viral nucleic acid. However, such viral sequences normally are not sufficient to direct or allow the incorporation of the plasmid into a viral particle, and the plasmid is therefore a non-viral vector. In certain embodiments described herein, a plasmid is a closed circular DNA molecule.
The term “expression” refers to the biological production of a product encoded by a coding sequence. In most cases a DNA sequence, including the coding sequence, is transcribed to form a messenger-RNA (mRNA). The messenger-RNA is then translated to form a polypeptide product which has a relevant biological activity. Also, the process of expression may involve further processing steps to the RNA product of transcription, such as splicing to remove introns, and/or post-translational processing of a polypeptide product.
As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and comprises any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term further includes polypeptides which have undergone post-translational modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
Also included as polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. Polypeptides, and fragments, derivatives, analogs, or variants thereof of the present invention can be antigenic and immunogenic polypeptides related to SARS-CoV polypeptides, which are used to prevent or treat, i.e., cure, ameliorate, lessen the severity of, or prevent or reduce contagion of infectious disease caused by the SARS-CoV.
As used herein, an antigenic polypeptide or an immunogenic polypeptide is a polypeptide which, when introduced into a vertebrate, reacts with the vertebrate's immune system molecules, i.e., is antigenic, and/or induces an immune response in the vertebrate, i.e., is immunogenic. It is quite likely that an immunogenic polypeptide will also be antigenic, but an antigenic polypeptide, because of its size or conformation, may not necessarily be immunogenic. Examples of antigenic and immunogenic polypeptides of the present invention include, but are not limited to, e.g., S or fragments, derivatives, or variants thereof; N or fragments, derivatives, or variants thereof; E or fragments, derivatives, or variants thereof; M or fragments, derivatives, or variants thereof; other predicted ORF's within the sequence of the SARS-CoV viruses which may posses antigenic properties, for example, an ORF which may encode for the hemagglutinin-esterase or fragments, derivatives, or variants thereof; or any of the foregoing polypeptides or fragments, derivatives, or variants thereof fused to a heterologous polypeptide, for example, a hepatitis B core antigen. Isolated antigenic and immunogenic polypeptides of the present invention in addition to those encoded by polynucleotides of the invention, may be provided as a recombinant protein, a purified subunit, a viral vector expressing the protein, or may be provided in the form of an inactivated SARS-CoV vaccine, e.g., a live-attenuated virus vaccine, a heat-killed virus vaccine, etc.
By an “isolated” SARS-CoV polypeptide or a fragment, variant, or derivative thereof is intended a SARS-CoV polypeptide or protein that is not in its natural environment. No particular level of purification is required. For example, an isolated SARS-CoV polypeptide can be removed from its native or natural environment. Recombinantly produced SARS-CoV polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant SARS-CoV polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique, including the separation of SARS-CoV virions from tissue samples or culture cells in which they have been propagated. In addition, an isolated. Thus, isolated SARS-CoV polypeptides and proteins can be provided as, for example, recombinant SARS-CoV polypeptides, a purified subunit of SARS-CoV, or a viral vector expressing an isolated SARS-CoV polypeptide.
The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in a vertebrate, for example a human. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an immune response in an animal, as determined by any method known in the art. The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody or T-cell receptor can immunospecifically bind as determined by any method well known in the art. Immunospecific binding excludes non-specific binding but does not exclude cross-reactivity with other antigens. Where all immunogenic epitopes are antigenic, antigenic epitopes need not be immunogenic.
The term “immunogenic carrier” as used herein refers to a first polypeptide or fragment, variant, or derivative thereof which enhances the immunogenicity of a second polypeptide or fragment, variant, or derivative thereof. Typically, an “immunogenic carrier” is fused to or conjugated to the desired polypeptide or fragment thereof. An example of an “immunogenic carrier” is a recombinant hepatitis B core antigen expressing, as a surface epitope, an immunogenic epitope of interest. See, e.g., European Patent No. EP 0385610 B 1, which is incorporated herein by reference in its entirety.
In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 8 to about 30 amino acids contained within the amino acid sequence of a SARS-CoV polypeptide of the invention, e.g., an S polypeptide, an N polypeptide, an E polypeptide or an M polypeptide. Certain polypeptides comprising immunogenic or antigenic epitopes are at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenic as well as immunogenic epitopes may be linear, i.e., be comprised of contiguous amino acids in a polypeptide, or may be three dimensional, i.e., where an epitope is comprised of non-contiguous amino acids which come together due to the secondary or tertiary structure of the polypeptide, thereby forming an epitope.
As to the selection of peptides or polypeptides bearing an antigenic epitope (e.g., that contain a region of a protein molecule to which an antibody or T cell receptor can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, e.g., Sutcliffe, J. G., et al., Science 219:660-666 (1983).
Peptides capable of eliciting an immunogenic response are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20 peptides designed according to these guidelines, containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
Codon Optimization
“Codon optimization” is defined as modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g., human, by replacing at least one, more than one, or a significant number, of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that vertebrate. Various species exhibit particular biases for certain codons of a particular amino acid.
In one aspect, the present invention relates to polynucleotides comprising nucleic acid fragments of codon-optimized coding regions which encode SARS-CoV polypeptides, or fragments, variants, or derivatives thereof, with the codon usage adapted for optimized expression in the cells of a given vertebrate, e.g., humans. These polynucleotides are prepared by incorporating codons preferred for use in the genes of the vertebrate of interest into the DNA sequence. Also provided are polynucleotide expression constructs, vectors, and host cells comprising nucleic acid fragments of codon-optimized coding regions which encode SARS-CoV polypeptides, and fragments, variants, or derivatives thereof, and various methods of using the polynucleotide expression constructs, vectors, and/or host cells to treat or prevent SARS disease in a vertebrate.
As used herein the term “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in the cells of a given vertebrate by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that vertebrate.
Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code,” which shows which codons encode which amino acids, is reproduced herein as Table 3. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six triplets, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.







TABLE 3







The Standard Genetic Code












T
C
A
G
















T
TTT Phe (F)
TCT Ser (S)
TAT Tyr (Y)
TGT Cys (C)




TTC Phe (F)
TCC Ser (S)
TAC Tyr (Y)
TGC



TTA Leu (L)
TCA Ser (S)
TAA Ter
TGA Ter



TTG Leu (L)
TCG Ser (S)
TAG Ter
TGG Trp (W)


 


C
CTT Leu (L)
CCT Pro (P)
CAT His (H)
CGT Arg (R)



CTC Leu (L)
CCC Pro (P)
CAC His (H)
CGC Arg (R)



CTA Leu (L)
CCA Pro (P)
CAA Gln (Q)
CGA Arg (R)



CTG Leu (L)
CCG Pro (P)
CAG Gln (Q)
CGG Arg (R)


 


A
ATT Ile (I)
ACT Thr (T)
AAT Asn (N)
AGT Ser (S)



ATC Ile (I)
ACC Thr (T)
AAC Asn (N)
AGC Ser (S)



ATA Ile (I)
ACA Thr (T)
AAA Lys (K)
AGA Arg (R)



ATG Met (M)
ACG Thr (T)
AAG Lys (K)
AAG Arg (R)


 


G
GTT Val (V)
GCT Ala (A)
GAT Asp (D)
GGT Gly (G)



GTC Val (V)
GCC Ala (A)
GAC Asp (D)
GGC Gly (G)



GTA Val (V)
GCA Ala (A)
GAA Glu (E)
GGA Gly (G)



GTG Val (V)
GCG Ala (A)
GAG Glu (E)
GGG Gly (G)









Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the “Codon Usage Database,” available at www.kazusa.or.jp/codon/ (visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). As examples, the codon usage tables for human, mouse, domestic cat, and cow, calculated from GenBank Release 128.0 (15 Feb. 2002), are reproduced below as Tables 4-7. These tables use mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. The tables have been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.







TABLE 4







Codon Usage Table for Human Genes (Homo sapiens)












Amino Acid
Codon
Number
Frequency















Phe
UUU
326146
0.4525



Phe
UUC
394680
0.5475



Total

720826




Leu
UUA
139249
0.0728



Leu
UUG
242151
0.1266



Leu
CUU
246206
0.1287



Leu
CUC
374262
0.1956



Leu
CUA
133980
0.0700



Leu
CUG
777077
0.4062



Total

1912925




Ile
AUU
303721
0.3554



Ile
AUC
414483
0.4850



Ile
AUA
136399
0.1596



Total

854603




Met
AUG
430946
1.0000



Total

430946




Val
GUU
210423
0.1773



Val
GUC
282445
0.2380



Val
GUA
134991
0.1137



Val
GUG
559044
0.4710



Total

1186903




Ser
UCU
282407
0.1840



Ser
UCC
336349
0.2191



Ser
UCA
225963
0.1472



Ser
UCG
86761
0.0565



Ser
AGU
230047
0.1499



Ser
AGC
373362
0.2433



Total

1534889




Pro
CCU
333705
0.2834



Pro
CCC
386462
0.3281



Pro
CCA
322220
0.2736



Pro
CCG
135317
0.1149



Total

1177704




Thr
ACU
247913
0.2419



Thr
ACC
371420
0.3624



Thr
ACA
285655
0.2787



Thr
ACG
120022
0.1171



Total

1025010




Ala
GCU
360146
0.2637



Ala
GCC
551452
0.4037



Ala
GCA
308034
0.2255



Ala
GCG
146233
0.1071



Total

1365865




Tyr
UAU
232240
0.4347



Tyr
UAC
301978
0.5653



Total

534218




His
CAU
201389
0.4113



His
CAC
288200
0.5887



Total

489589




Gln
CAA
227742
0.2541



Gln
CAG
668391
0.7459



Total

896133




Asn
AAU
322271
0.4614



Asn
AAC
376210
0.5386



Total

698481




Lys
AAA
462660
0.4212



Lys
AAG
635755
0.5788



Total

1098415




Asp
GAU
430744
0.4613



Asp
GAC
502940
0.5387



Total

933684




Glu
GAA
561277
0.4161



Glu
GAG
787712
0.5839



Total

1348989




Cys
UGU
190962
0.4468



Cys
UGC
236400
0.5532



Total

427362




Trp
UGG
248083
1.0000



Total

248083




Arg
CGU
90899
0.0830



Arg
CGC
210931
0.1927



Arg
CGA
122555
0.1120



Arg
CGG
228970
0.2092



Arg
AGA
221221
0.2021



Arg
AGG
220119
0.2011



Total

1094695




Gly
GGU
209450
0.1632



Gly
GGC
441320
0.3438



Gly
GGA
315726
0.2459



Gly
GGG
317263
0.2471



Total

1283759




Stop
UAA
13963




Stop
UAG
10631




Stop
UGA
24607
















TABLE 5







Codon Usage Table for Mouse Genes (Mus musculus)












Amino Acid
Codon
Number
Frequency















Phe
UUU
150467
0.4321



Phe
UUC
197795
0.5679



Total

348262




Leu
UUA
55635
0.0625



Leu
UUG
116210
0.1306



Leu
CUU
114699
0.1289



Leu
CUC
179248
0.2015



Leu
CUA
69237
0.0778



Leu
CUG
354743
0.3987



Total

889772




Ile
AUU
137513
0.3367



Ile
AUC
208533
0.5106



Ile
AUA
62349
0.1527



Total

408395




Met
AUG
204546
1.0000



Total

204546




Val
GUU
93754
0.1673



Val
GUC
140762
0.2513



Val
GUA
64417
0.1150



Val
GUG
261308
0.4664



Total

560241




Ser
UCU
139576
0.1936



Ser
UCC
160313
0.2224



Ser
UCA
100524
0.1394



Ser
UCG
38632
0.0536



Ser
AGU
108413
0.1504



Ser
AGC
173518
0.2407



Total

720976




Pro
CCU
162613
0.3036



Pro
CCC
164796
0.3077



Pro
CCA
151091
0.2821



Pro
CCG
57032
0.1065



Total

535532




Thr
ACU
119832
0.2472



Thr
ACC
172415
0.3556



Thr
ACA
140420
0.2896



Thr
ACG
52142
0.1076



Total

484809




Ala
GCU
178593
0.2905



Ala
GCC
236018
0.3839



Ala
GCA
139697
0.2272



Ala
GCG
60444
0.0983



Total

614752




Tyr
UAU
108556
0.4219



Tyr
UAC
148772
0.5781



Total

257328




His
CAU
88786
0.3973



His
CAC
134705
0.6027



Total

223491




Gln
CAA
101783
0.2520



Gln
CAG
302064
0.7480



Total

403847




Asn
AAU
138868
0.4254



Asn
AAC
187541
0.5746



Total

326409




Lys
AAA
188707
0.3839



Lys
AAG
302799
0.6161



Total

491506




Asp
GAU
189372
0.4414



Asp
GAC
239670
0.5586



Total

429042




Glu
GAA
235842
0.4015



Glu
GAG
351582
0.5985



Total

587424




Cys
UGU
97385
0.4716



Cys
UGC
109130
0.5284



Total

206515




Trp
UGG
112588
1.0000



Total

112588




Arg
CGU
41703
0.0863



Arg
CGC
86351
0.1787



Arg
CGA
58928
0.1220



Arg
CGG
92277
0.1910



Arg
AGA
101029
0.2091



Arg
AGG
102859
0.2129



Total

483147




Gly
GGU
103673
0.1750



Gly
GGC
198604
0.3352



Gly
GGA
151497
0.2557



Gly
GGG
138700
0.2341



Total

592474




Stop
UAA
5499




Stop
UAG
4661




Stop
UGA
10356
















TABLE 6







Codon Usage Table for Domestic Cat Genes (Felis cattus)












Amino Acid
Codon
Number
Frequency of usage















Phe
UUU
1204.00
0.4039



Phe
UUC
1777.00
0.5961



Total

2981




Leu
UUA
404.00
0.0570



Leu
UUG
857.00
0.1209



Leu
CUU
791.00
0.1116



Leu
CUC
1513.00
0.2135



Leu
CUA
488.00
0.0688



Leu
CUG
3035.00
0.4282



Total

7088




Ile
AUU
1018.00
0.2984



Ile
AUC
1835.00
0.5380



Ile
AUA
558.00
0.1636



Total

3411




Met
AUG
1553.00
0.0036



Total

1553




Val
GUU
696.00
0.1512



Val
GUC
1279.00
0.2779



Val
GUA
463.00
0.1006



Val
GUG
2164.00
0.4702



Total

4602




Ser
UCU
940.00
0.1875



Ser
UCC
1260.00
0.2513



Ser
UCA
608.00
0.1213



Ser
UCG
332.00
0.0662



Ser
AGU
672.00
0.1340



Ser
AGC
1202.00
0.2397



Total

5014




Pro
CCU
958.00
0.2626



Pro
CCC
1375.00
0.3769



Pro
CCA
850.00
0.2330



Pro
CCG
465.00
0.1275



Total

3648




Thr
ACU
822.00
0.2127



Thr
ACC
1574.00
0.4072



Thr
ACA
903.00
0.2336



Thr
ACG
566.00
0.1464



Total

3865




Ala
GCU
1129.00
0.2496



Ala
GCC
1951.00
0.4313



Ala
GCA
883.00
0.1952



Ala
GCG
561.00
0.1240



Total

4524




Tyr
UAU
837.00
0.3779



Tyr
UAC
1378.00
0.6221



Total

2215




His
CAU
594.00
0.3738



His
CAC
995.00
0.6262



Total

1589




Gln
CAA
747.00
0.2783



Gln
CAG
1937.00
0.7217



Total

2684




Asn
AAU
1109.00
0.3949



Asn
AAC
1699.00
0.6051



Total

2808




Lys
AAA
1445.00
0.4088



Lys
AAG
2090.00
0.5912



Total

3535




Asp
GAU
1255.00
0.4055



Asp
GAC
1840.00
0.5945



Total

3095




Glu
GAA
1637.00
0.4164



Glu
GAG
2294.00
0.5836



Total

3931




Cys
UGU
719.00
0.4425



Cys
UGC
906.00
0.5575



Total

1625




Trp
UGG
1073.00
1.0000



Total

1073




Arg
CGU
236.00
0.0700



Arg
CGC
629.00
0.1865



Arg
CGA
354.00
0.1050



Arg
CGG
662.00
0.1963



Arg
AGA
712.00
0.2112



Arg
AGG
779.00
0.2310



Total

3372




Gly
GGU
648.00
0.1498



Gly
GGC
1536.00
0.3551



Gly
GGA
1065.00
0.2462



Gly
GGG
1077.00
0.2490



Total

4326




Stop
UAA
55




Stop
UAG
36




Stop
UGA
110
















TABLE 7







Codon Usage Table for Cow Genes (Bos taurus)












Amino Acid
Codon
Number
Frequency of usage















Phe
UUU
13002
0.4112



Phe
UUC
18614
0.5888



Total

31616




Leu
UUA
4467
0.0590



Leu
UUG
9024
0.1192



Leu
CUU
9069
0.1198



Leu
CUC
16003
0.2114



Leu
CUA
4608
0.0609



Leu
CUG
32536
0.4298



Total

75707




Ile
AUU
12474
0.3313



Ile
AUC
19800
0.5258



Ile
AUA
5381
0.1429



Total

37655




Met
AUG
17770
1.0000



Total

17770




Val
GUU
8212
0.1635



Val
GUC
12846
0.2558



Val
GUA
4932
0.0982



Val
GUG
24222
0.4824



Total

50212




Ser
UCU
10287
0.1804



Ser
UCC
13258
0.2325



Ser
UCA
7678
0.1347



Ser
UCG
3470
0.0609



Ser
AGU
8040
0.1410



Ser
AGC
14279
0.2505



Total

57012




Pro
CCU
11695
0.2684



Pro
CCC
15221
0.3493



Pro
CCA
11039
0.2533



Pro
CCG
5621
0.1290



Total

43576




Thr
ACU
9372
0.2203



Thr
ACC
16574
0.3895



Thr
ACA
10892
0.2560



Thr
ACG
5712
0.1342



Total

42550




Ala
GCU
13923
0.2592



Ala
GCC
23073
0.4295



Ala
GCA
10704
0.1992



Ala
GCG
6025
0.1121



Total

53725




Tyr
UAU
9441
0.3882



Tyr
UAC
14882
0.6118



Total

24323




His
CAU
6528
0.3649



His
CAC
11363
0.6351



Total

17891




Gln
CAA
8060
0.2430



Gln
CAG
25108
0.7570



Total

33168




Asn
AAU
12491
0.4088



Asn
AAC
18063
0.5912



Total

30554




Lys
AAA
17244
0.3897



Lys
AAG
27000
0.6103



Total

44244




Asp
GAU
16615
0.4239



Asp
GAC
22580
0.5761



Total

39195




Glu
GAA
21102
0.4007



Glu
GAG
31555
0.5993



Total

52657




Cys
UGU
7556
0.4200



Cys
UGC
10436
0.5800



Total

17992




Trp
UGG
10706
1.0000



Total

10706




Arg
CGU
3391
0.0824



Arg
CGC
7998
0.1943



Arg
CGA
4558
0.1108



Arg
CGG
8300
0.2017



Arg
AGA
8237
0.2001



Arg
AGG
8671
0.2107



Total

41155




Gly
GGU
8508
0.1616



Gly
GGC
18517
0.3518



Gly
GGA
12838
0.2439



Gly
GGG
12772
0.2427



Total

52635




Stop
UAA
555




Stop
UAG
394




Stop
UGA
392









By utilizing these or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons more optimal for a given species. Codon-optimized coding regions can be designed by various different methods.
In one method, termed “uniform optimization,” a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 4 above, the most frequent codon for leucine in humans is CUG, which is used 41% of the time. Thus, all of the leucine residues in a given amino acid sequence would be assigned the codon CUG. A coding region for SARS-CoV soluble S protein (SEQ ID NO:1) optimized by the “uniform optimization” method is presented herein as SEQ ID NO:25.
In another method, termed “full-optimization,” the actual frequencies of the codons are distributed randomly throughout the coding region. Thus, using this method for optimization, if a hypothetical polypeptide sequence had 100 leucine residues, referring to Table 4 for frequency of usage in humans, about 7, or 7% of the leucine codons would be UUA, about 13, or 13% of the leucine codons would be UUG, about 13, or 13% of the leucine codons would be CUU, about 20, or 20% of the leucine codons would be CUC, about 7, or 7% of the leucine codons would be CUA, and about 41, or 41% of the leucine codons would be CUG. These frequencies would be distributed randomly throughout the leucine codons in the coding region encoding the hypothetical polypeptide. As will be understood by those of ordinary skill in the art, the distribution of codons in the sequence can vary significantly using this method, however, the sequence always encodes the same polypeptide.
As an example, a nucleotide sequence for soluble S (SEQ ID NO:1) fully optimized for human codon usage, is shown as SEQ ID NO:24.
In using the “full-optimization” method, an entire polypeptide sequence may be codon-optimized as described above. With respect to various desired fragments, variants, or derivatives of the complete polypeptide, the fragment, variant, or derivative may first be designed, and is then codon-optimized individually. Alternatively, a full-length polypeptide sequence is codon-optimized for a given species, resulting in a codon-optimized coding region encoding the entire polypeptide; then nucleic acid fragments of the codon-optimized coding region, which encode fragments, variants, and derivatives of the polypeptide, are made from the original codon-optimized coding region. As will be well understood by those of ordinary skill in the art, if codons have been randomly assigned to the full-length coding region based on their frequency of use in a given species, nucleic acid fragments encoding fragments, variants, and derivatives would not necessarily be fully codon-optimized for the given species. However, such sequences are still much closer to the codon usage of the desired species than the native codon usage. The advantage of this approach is that synthesizing codon-optimized nucleic acid fragments encoding each fragment, variant, and derivative of a given polypeptide, although routine, would be time consuming and would result in significant expense.
When using the “full-optimization” method, the term “about” is used precisely to account for fractional percentages of codon frequencies for a given amino acid. As used herein, “about” is defined as one amino acid more or one amino acid less than the value given. The whole number value of amino acids is rounded up if the fractional frequency of usage is 0.50 or greater, and is rounded down if the fractional frequency of use is 0.49 or less. Using again the example of the frequency of usage of leucine in human genes, for a hypothetical polypeptide having 62 leucine residues, the fractional frequency of codon usage would be calculated by multiplying 62 by the frequencies for the various codons. Thus, 7.28 percent of 62 equals 4.51 UUA codons, or “about 5,” ie., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e., 7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or “about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13 CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons, and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e., 24, 25, or 26 CUG codons.
In a third method termed “minimal optimization,” coding regions are only partially optimized. For example, the invention includes a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide in which at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codon positions have been codon-optimized for a given species. That is, they contain a codon that is preferentially used in the genes of a desired species, e.g., a vertebrate species, e.g., humans, in place of a codon that is normally used in the native nucleic acid sequence. Codons that are rarely found in the genes of the vertebrate of interest are changed to codons more commonly utilized in the coding regions of the vertebrate of interest.
Thus, those codons which are used more frequently in the SARS-CoV gene of interest than in genes of the vertebrate of interest are substituted with more frequently-used codons. The difference in frequency at which the SARS-CoV codons are substituted may vary based on a number factors as discussed below. For example, codons used at least twice more per thousand in SARS-CoV genes as compared to genes of the vertebrate of interest are substituted with the most frequently used codon for that amino acid in the vertebrate of interest. This ratio may be adjusted higher or lower depending on various factors such as those discussed below. Accordingly, a codon in a SARS-CoV native coding region would be substituted with a codon used more frequently for that amino acid in coding regions of the vertebrate of interest if the codon is used 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3. times, 3.4 times, 3.5 times, 3.6 times. 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times, 13.5 times, 14.0 times, 14.5 times, 15.0 times, 15.5 times, 16.0 times, 16.5 times, 17.0 times, 17.5 times, 18.0 times, 18.5 times, 19.0 times, 19.5 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, or greater more frequently in SARS-CoV coding regions than in coding regions of the vertebrate of interest.
This minimal human codon optimization for highly variant codons has several advantages, which include but are not limited to the following examples. Since fewer changes are made to the nucleotide sequence of the gene of interest, fewer manipulations are required, which leads to reduced risk of introducing unwanted mutations and lower cost, as well as allowing the use of commercially available site-directed mutagenesis kits, and reducing the need for expensive oligonucleotide synthesis. Further, decreasing the number of changes in the nucleotide sequence decreases the potential of altering the secondary structure of the sequence, which can have a significant impact on gene expression in certain host cells. The introduction of undesirable restriction sites is also reduced, facilitating the subcloning of the genes of interest into the plasmid expression vector.
In a fourth method, termed “standardized optimization,” a Codon Usage Table (CUT) for the sequence to be optimized is generated and compared to the CUT for human genomic DNA (see, e.g., Table 8 below). Codons are identified for which there is a difference of at least 10 percentage points in codon usage between human and query DNA. When such a codon is found, all of the wild type codons for that amino acid are modified to conform to predominant human codon.
The codon usage frequencies for all established SARS-CoV open reading frames (ORFs) is compared to the codon usage frequencies for humans in Table 8 below.







TABLE 8







SARS CoV Urbani Codon Frequencies using all established ORFs















Urbani

Human


Amino

Urbani
Frequency
Human
Frequency


Acid
Codon
Number
of usage
Number
of usage















Phe
UUU
272
0.6154
326146
0.4525


Phe
UUC
170
0.3846
394680
0.5475


Total

442

720826


Leu
UUA
150
0.1777
139249
0.0728


Leu
UUG
150
0.1777
242151
0.1266


Leu
CUU
254
0.3009
246206
0.1287


Leu
CUC
119
0.1410
374262
0.1956


Leu
CUA
90
0.1066
133980
0.0700


Leu
CUG
81
0.0960
777077
0.4062


Total

844

1912925


Ile
AUU
262
0.5784
303721
0.3554


Ile
AUC
98
0.2163
414483
0.4850


Ile
AUA
93
0.2053
136399
0.1596


Total

453

854603


Met
AUG
212
0.0005
430946
1.0000


Total

212

430946


Val
GUU
299
0.4194
210423
0.1773


Val
GUC
126
0.1767
282445
0.2380


Val
GUA
152
0.2132
134991
0.1137


Val
GUG
136
0.1907
559044
0.4710


Total

713

1186903


Ser
UCU
202
0.3328
282407
0.1840


Ser
UCC
41
0.0675
336349
0.2191


Ser
UCA
176
0.2900
225963
0.1472


Ser
UCG
20
0.0329
86761
0.0565


Ser
AGU
118
0.1944
230047
0.1499


Ser
AGC
50
0.0824
373362
0.2433


Total

607

1534889


Pro
CCU
163
0.4405
333705
0.2834


Pro
CCC
38
0.1027
386462
0.3281


Pro
CCA
156
0.4216
322220
0.2736


Pro
CCG
13
0.0351
135317
0.1149


Total

370

1177704


Thr
ACU
275
0.4264
247913
0.2419


Thr
ACC
86
0.1333
371420
0.3624


Thr
ACA
257
0.3985
285655
0.2787


Thr
ACG
27
0.0419
120022
0.1171


Total

645

1025010









The present invention provides isolated polynucleotides comprising codon-optimized coding regions of SARS-CoV polypeptides, e.g., S, S1, S2 N, E, or M, or fragments, variants, or derivatives thereof.
Additionally, a minimally codon-optimized nucleotide sequence can be designed by changing only certain codons found more frequently in SARS-CoV genes than in human genes. For example, if it is desired to substitute more frequently used codons in humans for those codons that occur at least 2 times more frequently in SARS-CoV genes.
In another form of minimal optimization, a Codon Usage Table (CUT) for the specific SARS-CoV sequence in question is generated and compared to the CUT for human genomic DNA. Amino acids are identified for which there is a difference of at least 10 percentage points in codon usage between human and SARS-CoV DNA (either more or less). Then, the wild type SARS-CoV codon is modified to conform to the predominant human codon for each such amino acid. Furthermore, the remainder of codons for that amino acid are also modified such that they conform to the predominant human codon for each such amino acid.
In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:2 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:2 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:2, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:2 is shown in Table 9.









TABLE 9






AMINO
Number in



ACID
SEQ ID NO: 2

















A
Ala
81


R
Arg
39


C
Cys
30


G
Gly
74


H
His
14


I
Ile
74


L
Leu
92


K
Lys
56


M
Met
18


F
Phe
81


P
Pro
56


S
Ser
91


T
Thr
96


W
Trp
10


Y
Tyr
52


V
Val
86


N
Asn
81


D
Asp
70


Q
Gln
55


E
Glu
40









Using the amino acid composition shown in Table 9, a human codon-optimized coding region which encodes SEQ ID NO:2 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:2 as follows: the 81 phenylalanine codons are TTC, the 92 leucine codons are CTG, the 74 isoleucine codons are ATC, the 18 methionine codons are ATG, the 86 valine codons are GTG, the 91 serine codons are AGC, the 56 proline codons are CCC, the 96 threonine codons are ACC, the 81 alanine codons are GCC, the 52 tyrosine codons are TAC, the 14 histidine codons are CAC, the 55 glutamine codons are CAG, the 81 asparagine codons are AAC, the 56 lysine codons are AAG, the 70 aspartic acid codons are GAC, the 40 glutamic acid codons are GAG, the 30 cysteine codons are TGC, the 10 tryptophan codon is TGG, the 39 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 74 glycine codons are GGC. The codon-optimized coding region designed by this method is presented herein as SEQ ID NO:25.








ATGTTCATCTTCCTGCTGTTCCTGACCCTGACCAGCGGCAGCGACCTGGA



 


CCGGTGCACCACCTTCGACGACGTGCAGGCCCCCAACTACACCCAGCACA


 


CCAGCAGCATGCGGGGCGTGTACTACCCCGACGAGATCTTCCGGAGCGAC


 


ACCCTGTACCTGACCCAGGACCTGTTCCTGCCCTTCTACAGCAACGTGAC


 


CGGCTTCCACACCATCAACCACACCTTCGGCAACCCCGTGATCCCCTTCA


 


AGGACGGCATCTACTTCGCCGCCACCGAGAAGAGCAACGTGGTGCGGGGC


 


TGGGTGTTCGGCAGCACCATGAACAACAAGAGCCAGAGCGTGATCATCAT


 


CAACAACAGCACCAACGTGGTGATCCGGGCCTGCAACTTCGAGCTGTGCG


 


ACAACCCCTTCTTCGCCGTGAGCAAGCCCATGGGCACCCAGACCCACACC


 


ATGATCTTCGACAACGCCTTCAACTGCACCTTCGAGTACATCAGCGACGC


 


CTTCAGCCTGGACGTGAGCGAGAAGAGCGGCAACTTCAAGCACCTGCGGG


 


AGTTCGTGTTCAAGAACAAGGACGGCTTCCTGTACGTGTACAAGGGCTAC


 


CAGCCCATCGACGTGGTGCGGGACCTGCCCAGCGGCTTCAACACCCTGAA


 


GCCCATCTTCAAGCTGCCCCTGGGCATCAACATCACCAACTTCCGGGCCA


 


TCCTGACCGCCTTCAGCCCCGCCCAGGACATCTGGGGCACCAGCGCCGCC


 


GCCTACTTCGTGGGCTACCTGAAGCCCACCACCTTCATGCTGAAGTACGA


 


CGAGAACGGCACCATCACCGACGCCGTGGACTGCAGCCAGAACCCCCTGG


 


CCGAGCTGAAGTGCAGCGTGAAGAGCTTCGAGATCGACAAGGGCATCTAC


 


CAGACCAGCAACTTCCGGGTGGTGCCCAGCGGCGACGTGGTGCGGTTCCC


 


CAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAAGT


 


TCCCCAGCGTGTACGCCTGGGAGCGGAAGAAGATCAGCAACTGCGTGGCC


 


GACTACAGCGTGCTGTACAACAGCACCTTCTTCAGCACCTTCAAGTGCTA


 


CGGCGTGAGCGCCACCAAGCTGAACGACCTGTGCTTCAGCAACGTGTACG


 


CCGACAGCTTCGTGGTGAAGGGCGACGACGTGCGGCAGATCGCCCCCGGC


 


CAGACCGGCGTGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCAT


 


GGGCTGCGTGCTGGCCTGGAACACCCGGAACATCGACGCCACCAGCACCG


 


GCAACTACAACTACAAGTACCGGTACCTGCGGCACGGCAAGCTGCGGCCC


 


TTCGAGCGGGACATCAGCAACGTGCCCTTCAGCCCCGACGGCAAGCCCTG


 


CACCCCCCCCGCCCTGAACTGCTACTGGCCCCTGAACGACTACGGCTTCT


 


ACACCACCACCGGCATCGGCTACCAGCCCTACCGGGTGGTGGTGCTGAGC


 


TTCGAGCTGCTGAACGCCCCCGCCACCGTGTGCGGCCCCAAGCTGAGCAC


 


CGACCTGATCAAGAACCAGTGCGTGAACTTCAACTTCAACGGCCTGACCG


 


GCACCGGCGTGCTGACCCCCAGCAGCAAGCGGTTCCAGCCCTTCCAGCAG


 


TTCGGCCGGGACGTGAGCGACTTCACCGACAGCGTGCGGGACCCCAAGAC


 


CAGCGAGATCCTGGACATCAGCCCCTGCAGCTTCGGCGGCGTGAGCGTGA


 


TCACCCCCGGCACCAACGCCAGCAGCGAGGTGGCCGTGCTGTACCAGGAC


 


GTGAACTGCACCGACGTGAGCACCGCCATCCACGCCGACCAGCTGACCCC


 


CGCCTGGCGGATCTACAGCACCGGCAACAACGTGTTCCAGACCCAGGCCG


 


GCTGCCTGATCGGCGCCGAGCACGTGGACACCAGCTACGAGTGCGACATC


 


CCCATCGGCGCCGGCATCTGCGCCAGCTACCACACCGTGAGCCTGCTGCG


 


GAGCACCAGCCAGAAGAGCATCGTGGCCTACACCATGAGCCTGGGCGCCG


 


ACAGCAGCATCGCCTACAGCAACAACACCATCGCCATCCCCACCAACTTC


 


AGCATCAGCATCACCACCGAGGTGATGCCCGTGAGCATGGCCAAGACCAG


 


CGTGGACTGCAACATGTACATCTGCGGCGACAGCACCGAGTGCGCCAACC


 


TGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAACCGGGCCCTGAGC


 


GGCATCGCCGCCGAGCAGGACCGGAACACCCGGGAGGTGTTCGCCCAGGT


 


GAAGCAGATGTACAAGACCCCCACCCTGAAGTACTTCGGCGGCTTCAACT


 


TCAGCCAGATCCTGCCCGACCCCCTGAAGCCCACCAAGCGGAGCTTCATC


 


GAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTCATGAA


 


GCAGTACGGCGAGTGCCTGGGCGACATCAACGCCCGGGACCTGATCTGCG


 


CCCAGAAGTTCAACGGCCTGACCGTGCTGCCCCCCCTGCTGACCGACGAC


 


ATGATCGCCGCCTACACCGCCGCCCTGGTGAGCGGCACCGCCACCGCCGG


 


CTGGACCTTCGGCGCCGGCGCCGCCCTGCAGATCCCCTTCGCCATGCAGA


 


TGGCCTACCGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAG


 


AACCAGAAGCAGATCGCCAACCAGTTCAACAAGGCCATCAGCCAGATCCA


 


GGAGAGCCTGACCACCACCAGCACCGCCCTGGGCAAGCTGCAGGACGTGG


 


TGAACCAGAACGCCCAGGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGC


 


AACTTCGGCGCCATCAGCAGCGTGCTGAACGACATCCTGAGCCGGCTGGA


 


CAAGGTGGAGGCCGAGGTGCAGATCGACCGGCTGATCACCGGCCGGCTGC


 


AGAGCCTGCAGACCTACGTGACCCAGCAGCTGATCCGGGCCGCCGAGATC


 


CGGGCCAGCGCCAACCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTGGG


 


CCAGAGCAAGCGGGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCT


 


TCCCCCAGGCCGCCCCCCACGGCGTGGTGTTCCTGCACGTGACCTACGTG


 


CCCAGCCAGGAGCGGAACTTCACCACCGCCCCCGCCATCTGCCACGAGGG


 


CAAGGCCTACTTCCCCCGGGAGGGCGTGTTCGTGTTCAACGGCACCAGCT


 


GGTTCATCACCCAGCGGAACTTCTTCAGCCCCCAGATCATCACCACCGAC


 


AACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCATCAACAA


 


CACCGTGTACGACCCCCTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGC


 


TGGACAAGTACTTCAAGAACCACACCAGCCCCGACGTGGACCTGGGCGAC


 


ATCAGCGGCATCAACGCCAGCGTGGTGAACATCCAGAAGGAGATCGACCG


 


GCTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGCAGG


 


AGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGG






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:2 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:2 as follows: about 37 of the 81 phenylalanine codons are TTT, and about 44 of the phenylalanine codons are TTC; about 7 of the 92 leucine codons are TTA, about 12 of the leucine codons are TTG, about 12 of the leucine codons are CTT, about 18 of the leucine codons are CTC, about 7 of the leucine codons are CTA, and about 36 of the leucine codons are CTG; about 26 of the 74 isoleucine codons are ATT, about 35 of the isoleucine codons are ATC, and about 13 of the isoleucine codons are ATA; the 18 methionine codons are ATG; about 15 of the 86 valine codons are GTT, about 40 of the valine codons are GTG, about 10 of the valine codons are GTA, and about 21 of the valine codons are GTC; about 17 of the 91 serine codons are TCT, about 20 of the serine codons are TCC, about 14 of the serine codons are TCA, about 5 of the serine codons are TCG, about 13 of the serine codons are AGT, and about 22 of the serine codons are AGC; about 16 of the 56 proline codons are CCT, about 18 of the proline codons are CCC, about 16 of the proline codons are CCA, and about 6 of the proline codons are CCG; about 23 of the 96 threonine codons are ACT, about 35 of the threonine codons are ACC, about 27 of the threonine codons are ACA, and about 11 of the threonine codons are ACG; about 21 of the 81 alanine codons are GCT, about 33 of the alanine codons are GCC, about 18 of the alanine codons are GCA, and about 9 of the alanine codons are GCG; about 23 of the 52 tyrosine codons are TAT and about 29 of the tyrosine codons are TAC; about 6 of the 14 histidine codons are CAT and about 8 of the histidine codons are CAC; about 14 of the 55 glutamine codons are CAA and about 41 of the glutamine codons are CAG; about 37 of the 81 asparagine codons are AAT and about 44 of the asparagine codons are AAC; about 24 of the 56 lysine codons are AAA and about 32 of the lysine codons are AAG; about 32 of the 70 aspartic acid codons are GAT and about 38 of the aspartic acid codons are GAC; about 17 of the 40 glutamic acid codons are GAA and about 23 of the glutamic acid codons are GAG; about 14 of the 30 cysteine codons are TGT and about 16 of the cysteine codons are TGC; the 10 tryptophan codons are TGG; about 3 of the 39 arginine codons are CGT, about 7 of the arginine codons are CGC, about 4 of the arginine codons are CGA, about 8 of the arginine codons are CGG, about 9 of the arginine codons are AGA, and about 8 of the arginine codons are AGG; and about 12 of the 74 glycine codons are GGT, about 25 of the glycine codons are GGC, about 19 of the glycine codons are GGA, and about 18 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:2, optimized according to codon usage in humans is presented herein as SEQ ID NO:24.








ATG TTT ATC TTC CTC CTC TTC CTG ACG CTC ACT AGC



 


GGA TCC GAC TTA GAT CGG TGT ACC ACT TTC GAC GAC


 


GTC CAG GCC CCT AAC TAT ACT CAA CAT ACC TCC AGT


 


ATG CGC GGG GTG TAC TAT CCA GAT GAG ATT TTT CGG


 


AGC GAC ACT CTG TAC TTA ACA CAG GAC CTG TTT CTA


 


CCG TTT TAT TCA AAT GTA ACC GGC TTC CAC ACC ATT


 


AAC CAT ACA TTT GGC AAT CCC GTG ATA CCA TTC AAA


 


GAC GGC ATT TAC TTC GCC GCA ACA GAA AAG AGC AAT


 


GTT GTG AGG GGG TGG GTC TTC GGC TCC ACA ATG AAC


 


AAT AAA TCT CAG TCT GTC ATC ATC ATC AAT AAC AGC


 


ACT AAC GTG GTA ATC CGT GCC TGC AAT TTC GAG CTT


 


TGT GAC AAC CCA TTC TTC GCC GTG TCT AAG CCT ATG


 


GGC ACC CAG ACT CAC ACA ATG ATC TTT GAC AAT GCT


 


TTC AAC TGC ACC TTC GAA TAC ATA TCA GAT GCA TTC


 


TCT TTG GAT GTC AGT GAA AAG TCT GGA AAC TTT AAA


 


CAT CTG AGA GAG TTT GTC TTC AAA AAC AAG GAC GGC


 


TTT CTC TAC GTT TAC AAG GGT TAT CAG CCC ATT GAT


 


GTG GTG CGG GAC CTC CCT TCA GGG TTT AAC ACA TTG


 


AAA CCA ATA TTC AAA CTG CCC CTG GGT ATC AAT ATT


 


ACT AAC TTT CGA GCC ATC TTG ACC GCC TTT TCC CCC


 


GCG CAA GAC ATA TGG GGA ACC AGC GCG GCA GCC TAT


 


TTC GTC GGT TAT CTG AAG CCC ACT ACA TTT ATG CTG


 


AAG TAC GAC GAG AAC GGA ACC ATT ACC GAT GCT GTC


 


GAT TGT TCA CAG AAT CCA CTG GCT GAA TTG AAA TGC


 


TCC GTG AAG AGC TTT GAG ATC GAT AAG GGG ATT TAC


 


CAG ACG TCT AAT TTT CGA GTG GTT CCC TCA GGA GAT


 


GTG GTT AGA TTC CCC AAT ATC ACA AAT TTG TGC CCC


 


TTC GGT GAA GTG TTC AAT GCC ACA AAG TTC CCG TCT


 


GTC TAC GCT TGG GAG CGG AAA AAG ATA AGC AAC TGT


 


GTC GCG GAT TAC AGT GTC CTA TAT AAC TCG ACC TTT


 


TTT AGC ACG TTC AAG TGT TAC GGG GTG AGT GCT ACT


 


AAA CTG AAT GAT TTA TGT TTT AGT AAC GTT TAT GCA


 


GAC TCC TTT GTT GTA AAG GGT GAT GAC GTG CGC CAA


 


ATT GCA CCT GGG CAG ACC GGA GTG ATG GCA GAT TAT


 


AAC TAC AAA CTT CCA GAC GAC TTT ATG GGA TGC GTG


 


CTC GCC TGG AAC ACT CGC AAC ATC GAC GCA ACC AGC


 


ACC GGG AAC TAT AAT TAC AAA TAC AGA TAC CTC AGG


 


CAC GGC AAG CTG CGG CCT TTT GAG CGG GAT ATC TCA


 


AAC GTC CCA TTT AGC CCG GAC GGC AAG CCC TGT ACT


 


CCT CCC GCA CTT AAC TGT TAC TGG CCA CTG AAC GAT


 


TAT GGC TTT TAT ACC ACA ACC GGC ATC GGC TAC CAG


 


CCC TAC CGG GTG GTG GTG CTA TCT TTC GAG CTG CTG


 


AAC GCG CCT GCC ACC GTA TGT GGG CCC AAG CTT TCG


 


ACA GAT CTC ATC AAG AAC CAA TGC GTA AAT TTC AAT


 


TTC AAT GGC CTT ACA GGA ACC GGT GTG CTG ACA CCC


 


TCC TCC AAG AGG TTT CAA CCT TTC CAG CAG TTT GGA


 


CGT GAC GTC TCA GAC TTT ACT GAC AGT GTG AGG GAT


 


CCT AAG ACC TCT GAA ATC CTG GAT ATA TCT CCC TGT


 


TCC TTC GGT GGG GTT AGT GTG ATA ACC CCT GGG ACA


 


AAT GCT AGT TCC GAA GTG GCC GTA CTC TAT CAA GAC


 


GTG AAC TGC ACA GAC GTG TCA ACC GCC ATC CAC GCT


 


GAT CAA CTC ACA CCG GCT TGG CGG ATC TAT AGC ACT


 


GGC AAT AAC GTG TTC CAA ACG CAG GCC GGC TGC CTT


 


ATA GGG GCA GAG CAT GTC GAC ACT TCT TAC GAG TGT


 


GAT ATA CCA ATC GGA GCC GGC ATC TGC GCC TCA TAC


 


CAC ACG GTG AGC TTG CTG CGC TCC ACC AGT CAG AAG


 


AGT ATT GTC GCA TAC ACC ATG TCA CTC GGC GCA GAT


 


TCA AGT ATC GCC TAC AGC AAT AAC ACT ATC GCT ATT


 


CCT ACC AAC TTT TCC ATT TCC ATC ACA ACT GAG GTT


 


ATG CCT GTC TCC ATG GCT AAG ACT TCC GTG GAC TGC


 


AAT ATG TAC ATT TGT GGG GAC TCT ACC GAG TGC GCT


 


AAC CTT TTA CTG CAG TAT GGC TCC TTC TGC ACA CAG


 


CTG AAT AGA GCC CTG AGC GGA ATT GCC GCT GAG CAG


 


GAT AGA AAT ACG AGA GAA GTG TTT GCC CAG GTG AAA


 


CAG ATG TAT AAG ACT CCA ACC TTG AAG TAT TTC GGA


 


GGG TTC AAT TTT AGC CAG ATC CTT CCT GAC CCC TTG


 


AAG CCG ACC AAA AGG ACC TTC ATC GAA GAT CTT CTG


 


TTC AAC AAA GTT ACT TTA GCG GAC GCC GGG TTC ATG


 


AAA CAG TAT GGC GAG TGT CTC GGG GAT ATT AAT GCC


 


CGC GAT CTC ATC TGT GCT CAG AAA TTC AAC GGC CTC


 


ACA GTG CTC CCC CCA CTT CTG ACG GAT GAT ATG ATC


 


GCC GCT TAC ACA GCC GCA CTC GTG AGC GGC ACC GCC


 


ACA GCC GGT TGG ACA TTC GGA GCT GGA GCC GCA TTA


 


CAG ATT CCA TTC GCT ATG CAG ATG GCG TAC AGG TTC


 


AAC GGA ATA GGC GTG ACC CAG AAC GTG TTG TAT GAA


 


AAT CAG AAG CAG ATT GCG AAC CAG TTC AAC AAA GCC


 


ATT TCT CAA ATC CAG GAG TCC CTG ACC ACC ACA AGC


 


ACG GCA CTG GGA AAG CTG CAA GAC GTG GTC AAC CAG


 


AAC GCC CAA GCC CTA AAT ACC CTG GTT AAG CAG CTG


 


TCT AGC AAT TTT GGA GCG ATT TCA TCT GTC CTT AAC


 


GAT ATA CTA TCA AGA CTG GAC AAA GTG GAG GCA GAG


 


GTC CAA ATC GAC CGC CTG ATT ACG GGC CGC CTC CAG


 


AGC CTT CAG ACG TAT GTG ACA CAG CAG CTG ATA AGA


 


GCT GCT GAA ATA CGA GCC TCG GCT AAT CTG GCC GCA


 


ACC AAA ATG TCC GAA TGC GTC CTG GGG CAG TCC AAA


 


CGT GTC GAT TTC TGC GGC AAA GGT TAC CAT TTG ATG


 


TCA TTT CCA CAG GCG GCT CCT CAC GGC GTA GTG TTT


 


CTG CAC GTG ACT TAT GTA CCT TCG CAG GAA AGG AAC


 


TTC ACA ACT GCC CCA GCC ATC TGC CAT GAG GGA AAA


 


GCA TAT TTC CCC CGA GAA GGT GTT TTC GTT TTC AAC


 


GGG ACA AGC TGG TTC ATT ACT CAA AGG AAT TTT TTT


 


TCG CCA CAG ATC ATT ACC ACT GAT AAC ACA TTT GTA


 


TCT GGT AAC TGC GAC GTA GTT ATC GGG ATT ATC AAT


 


AAT ACG GTC TAT GAC CCC TTG CAA CCT GAG CTG GAT


 


AGC TTT AAG GAA GAG CTG GAC AAG TAC TTT AAG AAT


 


CAC ACC TCT CCA GAC GTG GAC CTG GGA GAC ATC TCC


 


GGC ATT AAT GCA AGT GTT GTG AAT ATT CAG AAA GAG


 


ATT GAT AGA CTA AAC GAA GTT GCT AAG AAC TTG AAT


 


GAG AGT TTA ATT GAC CTA CAG GAG CTC GGT AAG TAC


 


GAA CAG TAC ATC AAA TGG CCG TGG






Another representative codon-optimized coding region encoding SEQ ID NO:2 is presented herein as SEQ ID NO: 44.








ATG TTT ATC TTC CTG CTG TTT CTG ACA CTG ACA AGC



 


GGC AGT GAC CTG GAT AGA TGC ACA ACG TTT GAC GAC


 


GTG CAG GCC CCC AAC TAC ACC CAG CAT ACA TCC AGC


 


ATG AGG GGC GTT TAC TAC CCC GAT GAG ATC TTT AGA


 


AGT GAT ACT CTG TAT CTG ACT CAG GAC CTG TTT CTG


 


CCC TTC TAT TCT AAC GTT ACT GGC TTC CAT ACA ATC


 


AAC CAC ACC TTC GGC AAC CCC GTA ATA CCC TTT AAG


 


GAT GGC ATC TAC TTT GCC GCC ACC GAG AAG TCT AAC


 


GTA GTG AGA GGC TGG GTG TTC GGC AGT ACT ATG AAC


 


AAC AAG TCT CAG TCT GTG ATA ATA ATC AAC AAC TCC


 


ACT AAC GTC GTC ATC AGA GCC TGT AAC TTC GAG CTG


 


TGC GAT AAC CCC TTC TTC GCC GTT TCG AAG CCC ATG


 


GGC ACT CAG ACC CAT ACA ATG ATC TTT GAT AAC GCC


 


TTC AAC TGC ACC TTT GAG TAT ATC TGC GAT GCC TTC


 


AGT CTG GAT GTG TCC GAG AAG TCA GGC AAC TTC AAG


 


CAT CTG AGA GAG TTT GTG TTC AAG AAC AAG GAT GGC


 


TTT CTG TAC GTC TAC AAG GGC TAC CAG CCC ATA GAT


 


GTG GTA CGT GAC CTG CCC AGC GGC TTC AAC ACT CTG


 


AAG CCC ATA TTC AAG CTG CCC CTG GGC ATA AAC ATT


 


ACC AAC TTT AGA GCC ATT CTG ACG GCC TTC TCC CCC


 


GCC CAG GAT ATC TGG GGC ACA AGT GCC GCC GCC TAC


 


TTC GTG GGC TAC CTG AAG CCC ACA ACT TTT ATG CTG


 


AAG TAC GAC GAG AAC GGC ACC ATA ACA GAT GCC GTG


 


GAC TGT TCT CAG AAC CCC CTG GCC GAG CTG AAG TGC


 


TCA GTT AAG AGT TTT GAG ATA GAT AAG GGC ATC TAT


 


CAG ACA AGC AAC TTC CGC GTG GTC CCC AGC GGC GAT


 


GTG GTG AGG TTT CCC AAC ATT ACC AAC CTG TGC CCC


 


TTC GGC GAG GTA TTC AAC GCC ACA AAG TTC CCC TCC


 


GTT TAC GCC TGG GAG AGG AAG AAG ATT TCA AAC TGC


 


GTG GCC GAC TAC TCG GTG CTG TAT AAC TCT ACT TTC


 


TTC AGT ACC TTT AAG TGC TAC GGC GTG TCT GCC ACA


 


AAG CTG AAC GAT CTG TGC TTT AGC AAC GTG TAT GCC


 


GAT AGC TTC GTC GTC AAG GGC GAC GAC GTC AGA CAG


 


ATC GCC CCC GGC CAG ACA GGC GTC ATC GCC GAC TAC


 


AAC TAC AAG CTG CCC GAC GAT TTC ATG GGC TGC GTG


 


CTG GCC TGG AAC ACG AGG AAC ATA GAT GCC ACC AGC


 


ACT GGC AAC TAC AAC TAC AAG TAC AGA TAT CTG CGG


 


CAC GGC AAG CTG AGG CCC TTC GAG AGA GAC ATC TCT


 


AAC GTT CCC TTT TCC CCC GAT GGC AAG CCC TGC ACT


 


CCC CCC GCC CTG AAC TGC TAC TGG CCC CTG AAC GAC


 


TAT GGC TTC TAC ACC ACA ACT GGC ATC GGC TAT CAG


 


CCC TAC CGC GTA GTC GTG CTG TCG TTC GAG CTG CTG


 


AAC GCC CCC GCC ACA GTC TGC GGC CCC AAG CTG TCC


 


ACT GAC CTG ATT AAG AAC CAG TGT GTG AAC TTC AAC


 


TTT AAC GGC CTG ACT GGC ACC GGC GTG CTG ACA CCC


 


AGC AGC AAG CGG TTC CAG CCC TTC CAG CAG TTT GGC


 


AGA GAC GTG TCT GAT TTC ACA GAT TCC GTG AGA GAT


 


CCC AAG ACT TCC GAG ATA CTG GAT ATC AGT CCC TGC


 


TCC TTC GGC GGC GTG TCA GTT ATT ACA CCC GGC ACT


 


AAC GCC TCG TCC GAG GTA GCC GTT CTG TAT CAG GAC


 


GTG AAC TGC ACT GAT GTG AGT ACA GCC ATC CAC GCC


 


GAC CAG CTG ACC CCC GCC TGG CGG ATT TAT AGT ACG


 


GGC AAC AAC GTC TTT CAG ACT CAG GCC GGC TGC CTG


 


ATC GGC GCC GAG CAT GTA GAT ACG TCT TAT GAG TGC


 


GAC ATC CCC ATC GGC GCC GGC ATC TGC GCC AGC TAT


 


CAC ACC GTT TCT CTG CTG CGA AGT ACT TCT CAG AAG


 


TCT ATA GTG GCC TAC ACC ATG TCT CTG GGC GCC GAT


 


AGC TCT ATC GCC TAT AGC AAC AAC ACT ATA GCC ATC


 


CCC ACA AAC TTC TCT ATT TCT ATC ACT ACA GAG GTG


 


ATG CCC GTC TCC ATG GCC AAG ACC AGC GTT GAT TGC


 


AAC ATG TAC ATC TGC GGC GAT AGT ACA GAG TGC GCC


 


AAC CTG CTG CTG CAG TAT GGC AGC TTC TGC ACC CAG


 


CTG AAC AGA GCC CTG TCT GGC ATC GCC GCC GAG CAG


 


GAT AGG AAC ACA AGA GAG GTT TTC GCC CAG GTT AAG


 


CAG ATG TAC AAG ACT CCC ACT CTG AAG TAC TTT GGC


 


GGC TTT AAC TTT TCT CAG ATT CTG CCC GAT CCC CTG


 


AAG CCC ACT AAG AGG AGT TTC ATA GAG GAC CTG CTG


 


TTC AAC AAG GTG ACT CTG GCC GAC GCC GGC TTT ATG


 


AAG CAG TAC GGC GAG TGC CTG GGC GAT ATC AAC GCC


 


AGA GAC CTG ATC TGT GCC CAG AAG TTT AAC GGC CTG


 


ACA GTA CTG CCC CCC CTG CTG ACT GAT GAC ATG ATT


 


GCC GCC TAT ACG GCC GCC CTG GTG TCT GGC ACT GCC


 


ACC GCC GGC TGG ACC TTT GGC GCC GGC GCC GCC CTG


 


CAG ATA CCC TTT GCC ATG CAG ATG GCC TAC CGA TTC


 


AAC GGC ATA GGC GTA ACC CAG AAC GTT CTG TAT GAG


 


AAC CAG AAG CAG ATA GCC AAC CAG TTC AAC AAG GCC


 


ATC TCT CAG ATT CAG GAG TCT CTG ACC ACT ACA TCT


 


ACT GCC CTG GGC AAG CTG CAG GAC GTA GTG AAC CAG


 


AAC GCC CAG GCC CTG AAC ACC CTG GTT AAG CAG CTG


 


TCA AGT AAC TTC GGC GCC ATC TCT AGC GTT CTG AAC


 


GAT ATA CTG AGT CGG CTG GAT AAG GTG GAG GCC GAG


 


GTG CAG ATT GAC AGA CTG ATC ACA GGC AGA CTG CAG


 


TCT CTG CAG ACA TAT GTT ACT CAG CAG CTG ATA AGG


 


GCC GCC GAG ATT AGA GCC AGT GCC AAC CTG GCC GCC


 


ACT AAG ATG TCC GAG TGC GTC CTG GGC CAG AGT AAG


 


AGG GTA GAC TTT TGT GGC AAG GGC TAT CAC CTG ATG


 


TCC TTC CCC CAG GCC GCC CCC CAC GGC GTC GTG TTT


 


CTG CAT GTC ACT TAT GTT CCC TCA CAG GAG AGG AAC


 


TTC ACG ACC GCC CCC GCC ATC TGC CAC GAG GGC AAG


 


GCC TAT TTC CCC AGG GAG GGC GTC TTC GTA TTC AAC


 


GGC ACG AGT TGG TTC ATC ACC CAG CGA AAC TTC TTT


 


TCG CCC CAG ATA ATT ACA ACG GAC AAC ACT TTT GTA


 


AGT GGC AAC TGC GAT GTC GTC ATC GGC ATA ATC AAC


 


AAC ACG GTT TAC GAC CCC CTG CAG CCC GAG CTG GAT


 


TCA TTC AAG GAG GAG CTG GAC AAG TAC TTC AAG AAC


 


CAT ACT AGC CCC GAC GTT GAT CTG GGC GAC ATA AGC


 


GGC ATC AAC GCC AGT GTA GTC AAC ATA CAG AAG GAG


 


ATC GAT AGA CTG AAC GAG GTG GCC AAG AAC CTG AAC


 


GAG TCT CTG ATA GAC CTG CAG GAG CTG GGC AAG TAC


 


GAG CAG TAC ATC AAG TGG CCC TGG






A representative codon-optimized coding region encoding SEQ ID NO:2 according to the “standardized optimization” method is presented herein as SEQ ID NO: 67.








ATG TTC ATC TTC CTG CTG TTC CTG ACC CTG ACC AGC



 


GGC AGC GAC CTG GAT CGC TGC ACC ACC TTC GAT GAC


 


GTG CAG GCC CCC AAC TAC ACC CAG CAT ACC AGC AGC


 


ATG CGC GGC GTG TAC TAC CCC GAT GAG ATC TTC CGC


 


AGC GAC ACC CTG TAC CTG ACC CAG GAC CTG TTC CTG


 


CCC TTC TAC AGC AAC GTG ACC GGC TTC CAC ACC ATC


 


AAC CAT ACC TTC GGC AAC CCC GTG ATC CCC TTC AAG


 


GAC GGC ATC TAC TTC GCC GCC ACC GAG AAG AGC AAC


 


GTG GTG CGC GGC TGG GTG TTC GGC AGC ACC ATG AAC


 


AAC AAG AGC CAG AGC GTG ATC ATC ATC AAC AAC AGC


 


ACC AAC GTG GTG ATC CGC GCC TGC AAC TTC GAG CTG


 


TGC GAC AAC CCC TTC TTC GCC GTG AGC AAG CCC ATG


 


GGC ACC CAG ACC CAT ACC ATG ATC TTC GAT AAC GCC


 


TTC AAC TGC ACC TTC GAG TAC ATC AGC GAC GCC TTC


 


AGC CTG GAC GTG AGC GAG AAG AGC GGC AAC TTC AAG


 


CAT CTG CGC GAG TTC GTG TTC AAG AAC AAG GAT GGC


 


TTC CTG TAC GTG TAC AAG GGC TAC CAG CCC ATC GAC


 


GTG GTG CGC GAT CTG CCC AGC GGC TTC AAC ACC CTG


 


AAG CCC ATC TTC AAG CTG CCC CTG GGC ATC AAC ATC


 


ACC AAC TTC CGC GCC ATC CTG ACC GCC TTC AGC CCC


 


GCC CAG GAC ATC TGG GGC ACC AGC GCC GCC GCC TAC


 


TTC GTG GGC TAC CTG AAG CCC ACC ACC TTC ATG CTG


 


AAG TAC GAT GAG AAC GGC ACC ATC ACC GAC GCC GTG


 


GAC TGC AGC CAG AAC CCC CTG GCC GAG CTG AAG TGC


 


AGC GTG AAG AGC TTC GAG ATC GAT AAG GGC ATC TAC


 


CAG ACC AGC AAC TTC CGC GTG GTG CCC AGC GGC GAC


 


GTG GTG CGC TTC CCC AAC ATC ACC AAC CTG TGT CCC


 


TTC GGC GAG GTG TTC AAC GCC ACC AAG TTC CCC AGC


 


GTG TAC GCC TGG GAG CGC AAG AAG ATC AGC AAC TGC


 


GTG GCC GAC TAC AGC GTG CTG TAC AAC AGC ACC TTC


 


TTC AGC ACC TTC AAG TGC TAC GGC GTG AGC GCC ACC


 


AAG CTG AAC GAT CTG TGC TTC AGC AAC GTG TAC GCC


 


GAC AGC TTC GTG GTG AAG GGC GAT GAT GTG CGC CAG


 


ATC GCC CCC GGC CAG ACC GGC GTG ATC GCC GAT TAC


 


AAC TAC AAG CTG CCC GAC GAC TTC ATG GGC TGC GTG


 


CTG GCC TGG AAC ACC CGC AAC ATC GAC GCC ACC AGC


 


ACC GGC AAC TAC AAC TAC AAG TAC CGC TAC CTG CGC


 


CAT GGC AAG CTG CGC CCC TTC GAG CGC GAT ATC AGC


 


AAC GTG CCC TTC AGC CCC GAT GGC AAG CCC TGC ACC


 


CCC CCC GCC CTG AAC TGT TAC TGG CCC CTG AAC GAC


 


TAC GGC TTC TAC ACC ACC ACC GGC ATC GGC TAC CAG


 


CCC TAC CGC GTG GTG GTG CTG AGC TTC GAG CTG CTG


 


AAC GCC CCC GCC ACC GTG TGC GGC CCC AAG CTG AGC


 


ACC GAC CTG ATC AAG AAC CAG TGC GTG AAC TTC AAC


 


TTC AAC GGC CTG ACC GGC ACC GGC GTG CTG ACC CCC


 


AGC AGC AAG CGC TTC CAG CCC TTC CAG CAG TTC GGC


 


CGC GAT GTG AGC GAC TTC ACC GAT AGC GTG CGC GAC


 


CCC AAG ACC AGC GAG ATC CTG GAT ATC AGC CCC TGC


 


AGC TTC GGC GGC GTG AGC GTG ATC ACC CCC GGC ACC


 


AAC GCC AGC AGC GAG GTG GCC GTG CTG TAC CAG GAT


 


GTG AAC TGT ACC GAT GTG AGC ACC GCC ATC CAC GCC


 


GAT CAG CTG ACC CCC GCC TGG CGC ATC TAC AGC ACC


 


GGC AAC AAC GTG TTC CAG ACC CAG GCC GGC TGC CTG


 


ATC GGC GCC GAG CAT GTG GAC ACC AGC TAC GAG TGT


 


GAC ATC CCC ATC GGC GCC GGC ATC TGT GCC AGC TAC


 


CAC ACC GTG AGC CTG CTG CGC AGC ACC AGC CAG AAG


 


AGC ATC GTG GCC TAC ACC ATG AGC CTG GGC GCC GAT


 


AGC AGC ATC GCC TAC AGC AAC AAC ACC ATC GCC ATC


 


CCC ACC AAC TTC AGC ATC AGC ATC ACC ACC GAG GTG


 


ATG CCC GTG AGC ATG GCC AAG ACC AGC GTG GAC TGC


 


AAC ATG TAC ATC TGC GGC GAT AGC ACC GAG TGC GCC


 


AAC CTG CTG CTG CAG TAC GGC AGC TTC TGC ACC CAG


 


CTG AAC CGC GCC CTG AGC GGC ATC GCC GCC GAG CAG


 


GAT CGC AAC ACC CGC GAG GTG TTC GCC CAG GTG AAG


 


CAG ATG TAC AAG ACC CCC ACC CTG AAG TAC TTC GGC


 


GGC TTC AAC TTC AGC CAG ATC CTG CCC GAT CCC CTG


 


AAG CCC ACC AAG CGC AGC TTC ATC GAG GAT CTG CTG


 


TTC AAC AAG GTG ACC CTG GCC GAT GCC GGC TTC ATG


 


AAG CAG TAC GGC GAG TGC CTG GGC GAT ATC AAC GCC


 


CGC GAT CTG ATC TGC GCC CAG AAG TTC AAC GGC CTG


 


ACC GTG CTG CCC CCC CTG CTG ACC GAC GAC ATG ATC


 


GCC GCC TAC ACC GCC GCC CTG GTG AGC GGC ACC GCC


 


ACC GCC GGC TGG ACC TTC GGC GCC GGC GCC GCC CTG


 


CAG ATC CCC TTC GCC ATG CAG ATG GCC TAC CGC TTC


 


AAC GGC ATC GGC GTG ACC CAG AAC GTG CTG TAC GAG


 


AAC CAG AAG CAG ATC GCC AAC CAG TTC AAC AAG GCC


 


ATC AGC CAG ATC CAG GAG AGC CTG ACC ACC ACC AGC


 


ACC GCC CTG GGC AAG CTG CAG GAC GTG GTG AAC CAG


 


AAC GCC CAG GCC CTG AAC ACC CTG GTG AAG CAG CTG


 


AGC AGC AAC TTC GGC GCC ATC AGC AGC GTG CTG AAC


 


GAC ATC CTG AGC CGC CTG GAT AAG GTG GAG GCC GAG


 


GTG CAG ATC GAT CGC CTG ATC ACC GGC CGC CTG CAG


 


AGC CTG CAG ACC TAC GTG ACC CAG CAG CTG ATC CGC


 


GCC GCC GAG ATC CGC GCC AGC GCC AAC CTG GCC GCC


 


ACC AAG ATG AGC GAG TGC GTG CTG GGC CAG AGC AAG


 


CGC GTG GAT TTC TGC GGC AAG GGC TAC CAC CTG ATG


 


AGC TTC CCC CAG GCC GCC CCC CAT GGC GTG GTG TTC


 


CTG CAC GTG ACC TAC GTG CCC AGC CAG GAG CGC AAC


 


TTC ACC ACC GCC CCC GCC ATC TGC CAC GAG GGC AAG


 


GCC TAC TTC CCC CGC GAG GGC GTG TTC GTG TTC AAC


 


GGC ACC AGC TGG TTC ATC ACC CAG CGC AAC TTC TTC


 


AGC CCC CAG ATC ATC ACC ACC GAT AAC ACC TTC GTG


 


AGC GGC AAC TGC GAT GTG GTG ATC GGC ATC ATC AAC


 


AAC ACC GTG TAC GAT CCC CTG CAG CCC GAG CTG GAC


 


AGC TTC AAG GAG GAG CTG GAT AAG TAC TTC AAG AAC


 


CAC ACC AGC CCC GAC GTG GAT CTG GGC GAT ATC AGC


 


GGC ATC AAC GCC AGC GTG GTG AAC ATC CAG AAG GAG


 


ATC GAT CGC CTG AAC GAG GTG GCC AAG AAC CTG AAC


 


GAG AGC CTG ATC GAC CTG CAG GAG CTG GGC AAG TAC


 


GAG CAG TAC ATC AAG TGG CCC TGG






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:4 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:4 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:4, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:4 is shown in Table 10.









TABLE 10






AMINO
Number in



ACID
SEQ ID NO: 4

















A
Ala
38


R
Arg
23


C
Cys
20


G
Gly
44


H
His
9


I
Ile
38


L
Leu
46


K
Lys
31


M
Met
8


F
Phe
53


P
Pro
37


S
Ser
56


T
Thr
58


W
Trp
6


Y
Tyr
35


V
Val
53


N
Asn
46


D
Asp
44


Q
Gln
21


E
Glu
17









Using the amino acid composition shown in Table 10, a human codon-optimized coding region which encodes SEQ ID NO:4 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:4 as follows: the 53 phenylalanine codons are TTC, the 46 leucine codons are CTG, the 38 isoleucine codons are ATC, the 8 methionine codons are ATG, the 53 valine codons are GTG, the 56 serine codons are AGC, the 37 proline codons are CCC, the 58 threonine codons are ACC, the 38 alanine codons are GCC, the 35 tyrosine codons are TAC, the 9 histidine codons are CAC, the 21 glutamine codons are CAG, the 46 asparagine codons are AAC, the 31 lysine codons are AAG, the 44 aspartic acid codons are GAC, the 17 glutamic acid codons are GAG, the 20 cysteine codons are TGC; the 6 tryptophan codons are TGG, the 23 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 44 glycine codons are GGC. The codon-optimized S1 coding region designed by this method is presented herein as SEQ I) NO:27.








ATGTTCATCTTCCTGCTGTTCCTGACCCTGACCAGCGGCAGCGACCTGGA



 


CAGATGCACCACCTTCGACGACGTGCAGGCCCCCAACTACACCCAGCACA


 


CCAGCAGCATGAGAGGCGTGTACTACCCCGACGAGATCTTCAGAAGCGAC


 


ACCCTGTACCTGACCCAGGACCTGTTCCTGCCCTTCTACAGCAACGTGAC


 


CGGCTTCCACACCATCAACCACACCTTCGGCAACCCCGTGATCCCCTTCA


 


AGGACGGCATCTACTTCGCCGCCACCGAGAAGAGCAACGTGGTGAGAGGC


 


TGGGTGTTCGGCAGCACCATGAACAACAAGAGCCAGAGCGTGATCATCAT


 


CAACAACAGCACCAACGTGGTGATCAGAGCCTGCAACTTCGAGCTGTGCG


 


ACAACCCCTTCTTCGCCGTGAGCAAGCCCATGGGCACCCAGACCCACACC


 


ATGATCTTCGACAACGCCTTCAACTGCACCTTCGAGTACATCAGCGACGC


 


CTTCAGCCTGGACGTGAGCGAGAAGAGCGGCAACTTCAAGCACCTGAGAG


 


AGTTCGTGTTCAAGAACAAGGACGGCTTCCTGTACGTGTACAAGGGCTAC


 


CAGCCCATCGACGTGGTGAGAGACCTGCCCAGCGGCTTCAACACCCTGAA


 


GCCCATCTTCAAGCTGCCCCTGGGCATCAACATCACCAACTTCAGAGCCA


 


TCCTGACCGCCTTCAGCCCCGCCCAGGACATCTGGGGCACCAGCGCCGCC


 


GCCTACTTCGTGGGCTACCTGAAGCCCACCACCTTCATGCTGAAGTACGA


 


CGAGAACGGCACCATCACCGACGCCGTGGACTGCAGCCAGAACCCCCTGG


 


CCGAGCTGAAGTGCAGCGTGAAGAGCTTCGAGATCGACAAGGGCATCTAC


 


CAGACCAGCAACTTCAGAGTGGTGCCCAGCGGCGACGTGGTGAGATTCCC


 


CAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAAGT


 


TCCCCAGCGTGTACGCCTGGGAGAGAAAGAAGATCAGCAACTGCGTGGCC


 


GACTACAGCGTGCTGTACAACAGCACCTTCTTCAGCACCTTCAAGTGCTA


 


CGGCGTGAGCGCCACCAAGCTGAACGACCTGTGCTTCAGCAACGTGTACG


 


CCGACAGCTTCGTGGTGAAGGGCGACGACGTGAGACAGATCGCCCCCGGC


 


CAGACCGGCGTGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCAT


 


GGGCTGCGTGCTGGCCTGGAACACCAGAAACATCGACGCCACCAGCACCG


 


GCAACTACAACTACAAGTACAGATACCTGAGACACGGCAAGCTGAGACCC


 


TTCGAGAGAGACATCAGCAACGTGCCCTTCAGCCCCGACGGCAAGCCCTG


 


CACCCCCCCCGCCCTGAACTGCTACTGGCCCCTGAACGACTACGGCTTCT


 


ACACCACCACCGGCATCGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGC


 


TTCGAGCTGCTGAACGCCCCCGCCACCGTGTGCGGCCCCAAGCTGAGCAC


 


CGACCTGATCAAGAACCAGTGCGTGAACTTCAACTTCAACGGCCTGACCG


 


GCACCGGCGTGCTGACCCCCAGCAGCAAGAGATTCCAGCCCTTCCAGCAG


 


TTCGGCAGAGACGTGAGCGACTTCACCGACAGCGTGAGAGACCCCAAGAC


 


CAGCGAGATCCTGGACATCAGCCCCTGCAGCTTCGGCGGCGTGAGCGTGA


 


TCACCCCCGGCACCAACGCCAGCAGCGAGGTGGCCGTGCTGTACCAGGAC


 


GTGAACTGCACCGACGTGAGCACCGCCATCCACGCCGACCAGCTGACCCC


 


CGCCTGGAGAATCTACAGCACCGGCAACAACGTGTTCCAGACCCAGGCCG


 


GCTGCCTGATCGGCGCCGAGCACGTGGACACCAGCTACGAGTGCGACATC


 


CCCATCGGCGCCGGCATCTGCGCCAGCTACCACACCGTGAGCCTGCTGAG


 


AAGCACCAGCCAGAAGAGCATCGTGGCCTACACCATGAGCCTGGGCGCC






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:4 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:4 as follows: about 24 of the 53 phenylalanine codons are TTT, and about 29 of the phenylalanine codons are TTC; about 3 of the 46 leucine codons are TTA, about 6 of the leucine codons are TTG, about 6 of the leucine codons are CTT, about 9 of the leucine codons are CTC, about 4 of the leucine codons are CTA, and about 18 of the leucine codons are CTG; about 13 of the 38 isoleucine codons are ATT, about 18 of the isoleucine codons are ATC, and about 7 of the isoleucine codons are ATA; the 8 methionine codons are ATG; about 10 of the 53 valine codons are GTT, about 13 of the valine codons are GTC, about 5 of the valine codons are GTA, and about 25 of the valine codons are GTG; about 10 of the 56 serine codons are TCT, about 12 of the serine codons are TCC, about 8 of the serine codons are TCA, about 3 of the serine codons are TCG, about 9 of the serine codons are AGT, and about 14 of the serine codons are AGC; about 10 of the 37 proline codons are CCT, about 12 of the proline codons are CCC, about 11 of the proline codons are CCA, and about 4 of the proline codons are CCG; about 14 of the 58 threonine codons are ACT, about 21 of the threonine codons are ACC, about 16 of the threonine codons are ACA, and about 7 of the threonine codons are ACG; about 10 of the 38 alanine codons are GCT, about 15 of the alanine codons are GCC, about 9 of the alanine codons are GCA, and about 4 of the alanine codons are GCG; about 15 of the 35 tyrosine codons are TAT and about 20 of the tyrosine codons are TAC; about 4 of the 9 histidine codons are CAT and about 5 of the histidine codons are CAC; about 5 of the 21 glutamine codons are CAA and about 16 of the glutamine codons are CAG; about 21 of the 46 asparagine codons are AAT and about 25 of the asparagine codons are AAC; about 13 of the 31 lysine codons are AAA and about 18 of the lysine codons are AAG; about 20 of the 44 aspartic acid codons are GAT and about 24 of the aspartic acid codons are GAC; about 7 of the 17 glutamic acid codons are GAA and about 10 of the glutamic acid codons are GAG; about 9 of the 20 cysteine codons are TGT and about 11 of the cysteine codons are TGC; the 6 tryptophan codons are TGG; about 2 of the 23 arginine codons are CGT, about 4 of the arginine codons are CGC, about 3 of the arginine codons are CGA, about 5 of the arginine codons are CGG, about 4 of the arginine codons are AGA, and about 5 of the arginine codons are AGG; and about 7 of the 44 glycine codons are GGT, about 15 of the glycine codons are GGC, about 11 of the glycine codons are GGA, and about 11 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:4, optimized according to codon usage in humans is presented herein as SEQ ID NO:26.








ATG TTT ATC TTT TTG CTG TTT CTC ACA TTA ACT TCG



 


GGG TCT GAC CTG GAC CGG TGC ACC ACA TTC GAT GAC


 


GTC CAA GCC CCC AAC TAC ACT CAG CAT ACA TCT AGC


 


ATG CGC GGC GTG TAC TAC CCA GAT GAG ATC TTT AGG


 


TCC GAC ACC CTT TAT CTG ACC CAG GAC CTT TTT CTT


 


CCT TTC TAC TCT AAT GTA ACT GGG TTC CAT ACC ATC


 


AAC CAT ACC TTT GGC AAC CCA GTG ATT CCA TTT AAG


 


GAT GGT ATT TAC TTC GCC GCG ACC GAG AAA TCA AAT


 


GTT GTG CGC GGC TGG GTT TTC GGC TCC ACC ATG AAC


 


AAT AAG AGT CAG TCC GTA ATT ATC ATT AAC AAT AGT


 


ACA AAC GTG GTG ATC AGG GCA TGT AAT TTT GAA TTG


 


TGC GAC AAC CCT TTC TTC GCT GTA AGC AAA CCC ATG


 


GGG ACG CAG ACT CAC ACG ATG ATC TTC GAT AAC GCT


 


TTC AAT TGC ACG TTT GAG TAC ATA TCC GAT GCC TTT


 


TCT CTA GAT GTG TCC GAA AAA TCA GGG AAT TTT AAG


 


CAC CTG AGA GAG TTC GTC TTT AAG AAC AAG GAC GGT


 


TTC TTG TAC GTG TAC AAG GGA TAC CAG CCG ATC GAC


 


GTG GTG CGG GAC CTA CCC AGC GGA TTC AAC ACC CTC


 


AAG CCC ATT TTT AAG CTC CCA CTG GGT ATC AAT ATA


 


ACT AAC TTC AGA GCC ATT CTC ACA GCT TTC TCT CCA


 


GCT CAG GAT ATT TGG GGG ACT AGT GCG GCA GCT TAT


 


TTC GTG GGA TAC CTT AAG CCC ACA ACC TTC ATG TTG


 


AAA TAC GAT GAG AAC GGA ACC ATA ACT GAC GCA GTT


 


GAC TGC TCA CAG AAC CCC CTC GCA GAG TTG AAA TGC


 


TCA GTT AAA TCC TTT GAG ATC GAC AAG GGT ATT TAC


 


CAG ACC AGT AAC TTT AGA GTC GTG CCG TCA GGC GAC


 


GTC GTG AGG TTT CCT AAC ATC ACA AAT CTA TGT CCT


 


TTC GGA GAA GTG TTC AAT GCC ACA AAG TTC CCC AGC


 


GTG TAC GCC TGG GAG CGA AAA AAG ATA TCT AAC TGC


 


GTC GCA GAC TAC AGC GTA CTG TAT AAC AGC ACT TTT


 


TTC AGC ACC TTT AAG TGT TAT GGG GTG TCA GCA ACA


 


AAA CTG AAC GAT CTC TGC TTT TCA AAC GTT TAT GCC


 


GAT TCC TTC GTT GTC AAG GGA GAC GAT GTC CGT CAA


 


ATT GCT CCC GGG CAA ACT GGC GTT ATC GCT GAC TAT


 


AAC TAT AAA CTG CCA GAC GAT TTT ATG GGG TGT GTC


 


CTC GCA TGG AAT ACG CGC AAC ATC GAT GCG ACC TCT


 


ACC GGA AAC TAC AAC TAT AAA TAT AGG TAT CTT CGG


 


CAC GGG AAA TTA CGG CCG TTC GAG CGA GAT ATT TCG


 


AAC GTG CCT TTC AGT CCC GAT GGA AAA CCA TGT ACT


 


CCT CCA GCC CTC AAT TGT TAC TGG CCA TTG AAT GAC


 


TAC GGG TTC TAC ACG ACA ACT GGA ATA GGC TAT CAG


 


CCT TAT CGT GTC GTC GTT CTT TCT TTC GAA CTG CTG


 


AAT GCT CCC GCC ACG GTG TGC GGT CCA AAA CTC AGC


 


ACC GAC CTG ATC AAG AAT CAG TGC GTG AAT TTC AAT


 


TTC AAC GGC CTG ACA GGC ACA GGC GTT CTG ACC CCA


 


AGC TCC AAG CGC TTC CAG CCC TTC CAG CAA TTT GGC


 


AGG GAT GTG TCC GAC TTT ACC GAT TCA GTG CGA GAT


 


CCC AAG ACC AGT GAA ATA CTA GAC ATT TCT CCG TGT


 


AGC TTT GGC GGC GTG TCT GTC ATT ACT CCT GGG ACG


 


AAT GCC TCG AGC GAG GTG GCG GTG TTA TAT CAG GAC


 


GTT AAT TGT ACA GAC GTC AGT ACC GCC ATA CAT GCT


 


GAT CAG CTG ACT CCT GCA TGG AGA ATC TAC TCC ACA


 


GGA AAT AAT GTG TTT CAG ACA CAA GCA GGT TGC CTG


 


ATC GGA GCC GAA CAC GTC GAC ACC AGC TAC GAA TGT


 


GAT ATC CCT ATC GGT GCC GGC ATC TGC GCT AGT TAT


 


CAC ACA GTA AGC CTG CTG CGG AGC ACC AGT CAG AAG


 


TCC ATT GTG GCC TAT ACT ATG TCC CTG GGC GCC






Another representative codon-optimized coding region encoding SEQ ID NO:4 is presented herein as SEQ ID NO:45.








ATG TTC ATC TTC CTG CTG TTT CTG ACA CTG ACT TCT



 


GGC TCA GAT CTG GAT AGA TGC ACT ACC TTT GAC GAT


 


GTA CAG GCC CCC AAC TAC ACT CAG CAC ACA TCG TCC


 


ATG CGA GGC GTG TAT TAC CCC GAC GAG ATC TTC AGA


 


AGT GAC ACT CTG TAC CTG ACA CAG GAC CTG TTC CTG


 


CCC TTT TAC TCT AAC GTG ACT GGC TTT CAC ACT ATC


 


AAC CAT ACC TTC GGC AAC CCC GTA ATC CCC TTC AAG


 


GAT GGC ATC TAT TTT GCC GCC ACC GAG AAG TCC AAC


 


GTG GTG AGG GGC TGG GTC TTC GGC AGT ACG ATG AAC


 


AAC AAG TCT CAG TCC GTG ATA ATC ATA AAC AAC AGT


 


ACT AAC GTG GTT ATA AGA GCC TGC AAC TTC GAG CTG


 


TGC GAC AAC CCC TTC TTC GCC GTG TCC AAG CCC ATG


 


GGC ACA CAG ACC CAC ACC ATG ATA TTC GAC AAC GCC


 


TTT AAC TGT ACT TTC GAG TAT ATA AGC GAT GCC TTC


 


AGT CTG GAT GTT TCT GAG AAG TCA GGC AAC TTT AAG


 


CAT CTG AGA GAG TTC GTA TTC AAG AAC AAG GAC GGC


 


TTT CTG TAT GTT TAT AAG GGC TAC CAG CCC ATA GAT


 


GTC GTG CGG GAT CTG CCC AGC GGC TTC AAC ACA CTG


 


AAG CCC ATT TTT AAG CTG CCC CTG GGC ATC AAC ATA


 


ACC AAC TTT AGA GCC ATC CTG ACT GCC TTT AGC CCC


 


GCC CAG GAT ATA TGG GGC ACT AGC GCC GCC GCC TAT


 


TTC GTC GGC TAC CTG AAG CCC ACC ACA TTC ATG CTG


 


AAG TAC GAT AGA AAC GGC ACA ATT ACG GAT GCC GTA


 


GAT TGC AGT CAG AAC CCC CTG GCC GAG CTG AAG TGC


 


AGT GTG AAG TCT TTC GAG ATC GAC AAG GGC ATA TAC


 


CAG ACT TCT AAC TTT CGG GTG GTT CCC AGC GGC GAC


 


GTT GTT AGG TTT CCC AAC ATC ACC AAC CTG TGC CCC


 


TTC GGC GAG GTG TTT AAC GCC ACA AAG TTC CCC TCC


 


GTA TAT GCC TGG GAG AGG AAG AAG ATT TCG AAC TGC


 


GTG GCC GAC TAT AGC GTC CTG TAC AAC TCT ACA TTC


 


TTT TCT ACA TTC AAG TGC TAC GGC GTC AGT GCC ACT


 


AAG CTG AAC GAC CTG TGC TTC AGC AAC GTG TAT GCC


 


GAC TCA TTT GTA GTT AAG GGC GAT GAT GTG AGA CAG


 


ATT GCC CCC GGC CAG ACA GGC GTG ATC GCC GAT TAT


 


AAC TAT AAG CTG CCC GAC GAT TTC ATG GGC TGC GTT


 


CTG GCC TGG AAC ACA AGG AAC ATC GAT GCC ACT AGC


 


ACT GGC AAC TAC AAC TAC AAG TAC AGG TAT CTG AGA


 


CAC GGC AAG CTG AGG CCC TTC GAG CGA GAT ATC AGT


 


AAC GTA CCC TTC AGT CCC GAC GGC AAG CCC TGC ACT


 


CCC CCC GCC CTG AAC TGC TAT TGG CCC CTG AAC GAC


 


TAC GGC TTT TAT ACC ACT ACA GGC ATC GGC TAC CAG


 


CCC TAC AGG GTT GTG GTG CTG AGC TTC GAG CTG CTG


 


AAC GCC CCC GCC ACT GTT TGC GGC CCC AAG CTG TCA


 


ACG GAT CTG ATC AAG AAC CAG TGC GTA AAC TTT AAC


 


TTT AAC GGC CTG ACA GGC ACA GGC GTC CTG ACT CCC


 


TCT AGT AAG AGA TTC CAG CCC TTT CAG CAG TTC GGC


 


CGC GAC GTC AGC GAT TTT ACG GAT AGT GTG AGA GAT


 


CCC AAG ACC AGC GAG ATC CTG GAC ATT AGT CCC TGT


 


TCT TTC GGC GGC GTG TCT GTC ATA ACG CCC GGC ACG


 


AAC GCC TCT TCT GAG GTC GCC GTT CTG TAC CAG GAC


 


GTC AAC TGT ACA GAC GTC TCC ACA GCC ATA CAC GCC


 


GAT CAG CTG ACT CCC GCC TGG AGA ATT TAC TCT ACC


 


GGC AAC AAC GTC TTC CAG ACC CAG GCC GGC TGC CTG


 


ATC GGC GCC GAG CAT GTG GAT ACT TCC TAC GAG TGC


 


GAC ATA CCC ATC GGC GCC GGC ATT TGC GCC TCG TAC


 


CAT ACC GTG TCT CTG CTG AGA TCT ACC TCT CAG AAG


 


AGT ATC GTT GCC TAC ACT ATG TCC CTG GGC GCC






A representative codon-optimized coding region encoding SEQ ID NO:4 according to the “standardized optimization” method is presented herein as SEQ ID NO: 68.








ATG TTC ATC TTC CTG CTG TTC CTG ACC CTG ACC AGC



 


GGC AGC GAT CTG GAC CGC TGC ACC ACC TTC GAC GAT


 


GTG CAG GCC CCC AAC TAC ACC CAG CAC ACC AGC AGC


 


ATG CGC GGC GTG TAC TAC CCC GAT GAG ATC TTC CGC


 


AGC GAT ACC CTG TAC CTG ACC CAG GAT CTG TTC CTG


 


CCC TTC TAC AGC AAC GTG ACC GGC TTC CAT ACC ATC


 


AAC CAC ACC TTC GGC AAC CCC GTG ATC CCC TTC AAG


 


GAT GGC ATC TAC TTC GCC GCC ACC GAG AAG AGC AAC


 


GTG GTG CGC GGC TGG GTG TTC GGC AGC ACC ATG AAC


 


AAC AAG AGC CAG AGC GTG ATC ATC ATC AAC AAC AGC


 


ACC AAC GTG GTG ATC CGC GCC TGC AAC TTC GAG CTG


 


TGC GAC AAC CCC TTC TTC GCC GTG AGC AAG CCC ATG


 


GGC ACC CAG ACC CAC ACC ATG ATC TTC GAC AAC GCC


 


TTC AAC TGC ACC TTC GAG TAC ATC AGC GAT GCC TTC


 


AGC CTG GAC GTG AGC GAG AAG AGC GGC AAC TTC AAG


 


CAT CTG CGC GAG TTC GTG TTC AAG AAC AAG GAT GGC


 


TTC CTG TAC GTG TAC AAG GGC TAC CAG CCC ATC GAC


 


GTG GTG CGC GAC CTG CCC AGC GGC TTC AAC ACC CTG


 


AAG CCC ATC TTC AAG CTG CCC CTG GGC ATC AAC ATC


 


ACC AAC TTC CGC GCC ATC CTG ACC GCC TTC AGC CCC


 


GCC CAG GAT ATC TGG GGC ACC AGC GCC GCC GCC TAC


 


TTC GTG GGC TAC CTG AAG CCC ACC ACC TTC ATG CTG


 


AAG TAC GAC GAG AAC GGC ACC ATC ACC GAT GCC GTG


 


GAT TGC AGC CAG AAC CCC CTG GCC GAG CTG AAG TGC


 


AGC GTG AAG AGC TTC GAG ATC GAT AAG GGC ATC TAC


 


CAG ACC AGC AAC TTC CGC GTG GTG CCC AGC GGC GAC


 


GTG GTG CGC TTC CCC AAC ATC ACC AAC CTG TGC CCC


 


TTC GGC GAG GTG TTC AAC GCC ACC AAG TTC CCC AGC


 


GTG TAC GCC TGG GAG CGC AAG AAG ATC AGC AAC TGC


 


GTG GCC GAT TAC AGC GTG CTG TAC AAC AGC ACC TTC


 


TTC AGC ACC TTC AAG TGC TAC GGC GTG AGC GCC ACC


 


AAG CTG AAC GAC CTG TGC TTC AGC AAC GTG TAC GCC


 


GAC AGC TTC GTG GTG AAG GGC GAC GAC GTG CGC CAG


 


ATC GCC CCC GGC CAG ACC GGC GTG ATC GCC GAT TAC


 


AAC TAC AAG CTG CCC GAT GAC TTC ATG GGC TGC GTG


 


CTG GCC TGG AAC ACC CGC AAC ATC GAT GCC ACC AGC


 


ACC GGC AAC TAC AAC TAC AAG TAC CGC TAC CTG CGC


 


CAC GGC AAG CTG CGC CCC TTC GAG CGC GAT ATC AGC


 


AAC GTG CCC TTC AGC CCC GAT GGC AAG CCC TGC ACC


 


CCC CCC GCC CTG AAC TGT TAC TGG CCC CTG AAC GAT


 


TAC GGC TTC TAC ACC ACC ACC GGC ATC GGC TAC CAG


 


CCC TAC CGC GTG GTG GTG CTG AGC TTC GAG CTG CTG


 


AAC GCC CCC GCC ACC GTG TGC GGC CCC AAG CTG AGC


 


ACC GAC CTG ATC AAA AAC CAG TGC GTG AAC TTC AAC


 


TTC AAC GGC CTG ACC GGC ACC GGC GTG CTG ACC CCC


 


AGC AGC AAG CGC TTC CAG CCC TTC CAG CAG TTC GGC


 


CGC GAC GTG AGC GAC TTC ACC GAC AGC GTG CGC GAT


 


CCC AAG ACC AGC GAG ATC CTG GAT ATC AGC CCC TGC


 


AGC TTC GGC GGC GTG AGC GTG ATC ACC CCC GGC ACC


 


AAC GCC AGC AGC GAG GTG GCC GTG CTG TAC CAG GAC


 


GTG AAC TGC ACC GAT GTG AGC ACC GCC ATC CAC GCC


 


GAT CAG CTG ACC CCC GCC TGG CGC ATC TAC AGC ACC


 


GGC AAC AAC GTG TTC CAG ACC CAG GCC GGC TGT CTG


 


ATC GGC GCC GAG CAT GTG GAC ACC AGC TAC GAG TGT


 


GAT ATC CCC ATC GGC GCC GGC ATC TGC GCC AGC TAC


 


CAT ACC GTG AGC CTG CTG CGC AGC ACC AGC CAG AAG


 


AGC ATC GTG GCC TAC ACC ATG AGC CTG GGC GCC






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:6 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:6 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:6, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:6 is shown in Table 11.










TABLE 11








Number in



AMINO ACID
SEQ ID NO: 6




















A
Ala
43



R
Arg
16



C
Cys
10



G
Gly
30



H
His
5



I
Ile
36



L
Leu
46



K
Lys
25



M
Met
10



F
Phe
28



P
Pro
19



S
Ser
35



T
Thr
38



W
Trp
4



Y
Tyr
17



V
Val
33



N
Asn
35



D
Asp
26



Q
Gln
34



E
Glu
23










Using the amino acid composition shown in Table 11, a human codon-optimized coding region which encodes SEQ ID NO:6 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:6 as follows: the 28 phenylalanine codons are TTC, the 46 leucine codons are CTG, the 36 isoleucine codons are ATC, the 10 methionine codons are ATG, the 33 valine codons are GTG, the 35 serine codons are AGC, the 19 proline codons are CCC, the 38 threonine codons are ACC, the 43 alanine codons are GCC, the 17 tyrosine codons are TAC, the 5 histidine codons are CAC, the 34 glutamine codons are CAG, the 35 asparagine codons are AAC, the 25 lysine codons are AAG, the 26 aspartic acid codons are GAC, the 23 glutamic acid codons are GAG, the 10 cysteine codons are TGC, the 4 tryptophan codon is TGG, the 16 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 30 glycine codons are GGC. The codon-optimized coding region designed by this method is presented herein as SEQ ID NO:29.








GAC AGC AGC ATC GCC TAC AGC AAC AAC ACC ATC GCC



 


ATC CCC ACC AAC TTC AGC ATC AGC ATC ACC ACC GAG


 


GTG ATG CCC GTG AGC ATG GCC AAG ACC AGC GTG GAC


 


TGC AAC ATG TAC ATC TGC GGC GAC AGC ACC GAG TGC


 


GCC AAC CTG CTG CTG CAG TAC GGC AGC TTC TGC ACC


 


CAG CTG AAC CGG GCC CTG AGC GGC ATC GCC GCC GAG


 


CAG GAC CGG AAC ACC CGG GAG GTG TTC GCC CAG GTG


 


AAG CAG ATG TAC AAG ACC CCC ACC CTG AAG TAC TTC


 


GGC GGC TTC AAC TTC AGC CAG ATC CTG CCC GAC CCC


 


CTG AAG CCC ACC AAG CGG AGC TTC ATC GAG GAC CTG


 


CTG TTC AAC AAG GTG ACC CTG GCC GAC GCC GGC TTC


 


ATG AAG CAG TAC GGC GAG TGC CTG GGC GAC ATC AAC


 


GCC CGG GAC CTG ATC TGC GCC CAG AAG TTC AAC GGC


 


CTG ACC GTG CTG CCC CCC CTG CTG ACC GAC GAC ATG


 


ATC GCC GCC TAC ACC GCC GCC CTG GTG AGC GGC ACC


 


GCC ACC GCC GGC TGG ACC TTC GGC GCC GGC GCC GCC


 


CTG CAG ATC CCC TTC GCC ATG CAG ATG GCC TAC CGG


 


TTC AAC GGC ATC GGC GTG ACC CAG AAC GTG CTG TAC


 


GAG AAC CAG AAG CAG ATC GCC AAC CAG TTC AAC AAG


 


GCC ATC AGC CAG ATC CAG GAG AGC CTG ACC ACC ACC


 


AGC ACC GCC CTG GGC AAG CTG CAG GAC GTG GTG AAC


 


CAG AAC GCC CAG GCC CTG AAC ACC CTG GTG AAG CAG


 


CTG AGC AGC AAC TTC GGC GCC ATC AGC AGC GTG CTG


 


AAC GAC ATC CTG AGC CGG CTG GAC AAG GTG GAG GCC


 


GAG GTG CAG ATC GAC CGG CTG ATC ACC GGC CGG CTG


 


CAG AGC CTG CAG ACC TAC GTG ACC CAG CAG CTG ATC


 


CGG GCC GCC GAG ATC CGG GCC AGC GCC AAC CTG GCC


 


GCC ACC AAG ATG AGC GAG TGC GTG CTG GGC CAG AGC


 


AAG CGG GTG GAC TTC TGC GGC AAG GGC TAC CAC CTG


 


ATG AGC TTC CCC CAG GCC GCC CCC CAC GGC GTG GTG


 


TTC CTG CAC GTG ACC TAC GTG CCC AGC CAG GAG CGG


 


AAC TTC ACC ACC GCC CCC GCC ATC TGC CAC GAG GGC


 


AAG GCC TAC TTC CCC CGG GAG GGC GTG TTC GTG TTC


 


AAC GGC ACC AGC TGG TTC ATC ACC CAG CGG AAC TTC


 


TTC AGC CCC CAG ATC ATC ACC ACC GAC AAC ACC TTC


 


GTG AGC GGC AAC TGC GAC GTG GTG ATC GGC ATC ATC


 


AAC AAC ACC GTG TAC GAC CCC CTG CAG CCC GAG CTG


 


GAC AGC TTC AAG GAG GAG CTG GAC AAG TAC TTC AAG


 


AAC CAC ACC AGC CCC GAC GTG GAC CTG GGC GAC ATC


 


AGC GGC ATC AAC GCC AGC GTG GTG AAC ATC CAG AAG


 


GAG ATC GAC CGG CTG AAC GAG GTG GCC AAG AAC CTG


 


AAC GAG AGC CTG ATC GAC CTG CAG GAG CTG GGC AAG


 


TAC GAG CAG TAC ATC AAG TGG CCC TGG






A codon-optimized coding region encoding SEQ ID NO:56 designed by this method is presented herein as SEQ ID NO:64.








ATG GAC AGC AGC ATC GCC TAC AGC AAC AAC ACC ATC



 


GCC ATC CCC ACC AAC TTC AGC ATC AGC ATC ACC ACC


 


GAG GTG ATG CCC GTG AGC ATG GCC AAG ACC AGC GTG


 


GAC TGC AAC ATG TAC ATC TGC GGC GAC AGC ACC GAG


 


TGC GCC AAC CTG CTG CTG CAG TAC GGC AGC TTC TGC


 


ACC CAG CTG AAC CGG GCC CTG AGC GGC ATC GCC GCC


 


GAG CAG GAC CGG AAC ACC CGG GAG GTG TTC GCC CAG


 


GTG AAG CAG ATG TAC AAG ACC CCC ACC CTG AAG TAC


 


TTC GGC GGC TTC AAC TTC AGC CAG ATC CTG CCC GAC


 


CCC CTG AAG CCC ACC AAG CGG AGC TTC ATC GAG GAC


 


CTG CTG TTC AAC AAG GTG ACC CTG GCC GAC GCC GGC


 


TTC ATG AAG CAG TAC GGC GAG TGC CTG GGC GAC ATC


 


AAC GCC CGG GAC CTG ATC TGC GCC CAG AAG TTC AAC


 


GGC CTG ACC GTG CTG CCC CCC CTG CTG ACC GAC GAC


 


ATG ATC GCC GCC TAC ACC GCC GCC CTG GTG AGC GGC


 


ACC GCC ACC GCC GGC TGG ACC TTC GGC GCC GGC GCC


 


GCC CTG CAG ATC CCC TTC GCC ATG CAG ATG GCC TAC


 


CGG TTC AAC GGC ATC GGC GTG ACC CAG AAC GTG CTG


 


TAC GAG AAC CAG AAG CAG ATC GCC AAC CAG TTC AAC


 


AAG GCC ATC AGC CAG ATC CAG GAG AGC CTG ACC ACC


 


ACC AGC ACC GCC CTG GGC AAG CTG CAG GAC GTG GTG


 


AAC CAG AAC GCC CAG GCC CTG AAC ACC CTG GTG AAG


 


CAG CTG AGC AGC AAC TTC GGC GCC ATC AGC AGC GTG


 


CTG AAC GAC ATC CTG AGC CGG CTG GAC AAG GTG GAG


 


GCC GAG GTG CAG ATC GAC CGG CTG ATC ACC GGC CGG


 


CTG CAG AGC CTG CAG ACC TAC GTG ACC CAG CAG CTG


 


ATC CGG GCC GCC GAG ATC CGG GCC AGC GCC AAC CTG


 


GCC GCC ACC AAG ATG AGC GAG TGC GTG CTG GGC CAG


 


AGC AAG CGG GTG GAC TTC TGC GGC AAG GGC TAC CAC


 


CTG ATG AGC TTC CCC CAG GCC GCC CCC CAC GGC GTG


 


GTG TTC CTG CAC GTG ACC TAC GTG CCC AGC CAG GAG


 


CGG AAC TTC ACC ACC GCC CCC GCC ATC TGC CAC GAG


 


GGC AAG GCC TAC TTC CCC CGG GAG GGC GTG TTC GTG


 


TTC AAC GGC ACC AGC TGG TTC ATC ACC CAG CGG AAC


 


TTC TTC AGC CCC CAG ATC ATC ACC ACC GAC AAC ACC


 


TTC GTG AGC GGC AAC TGC GAC GTG GTG ATC GGC ATC


 


ATC AAC AAC ACC GTG TAC GAC CCC CTG CAG CCC GAG


 


CTG GAC AGC TTC AAG GAG GAG CTG GAC AAG TAC TTC


 


AAG AAC CAC ACC AGC CCC GAC GTG GAC CTG GGC GAC


 


ATC AGC GGC ATC AAC GCC AGC GTG GTG AAC ATC CAG


 


AAG GAG ATC GAC CGG CTG AAC GAG GTG GCC AAG AAC


 


CTG AAC GAG AGC CTG ATC GAC CTG CAG GAG CTG GGC


 


AAG TAC GAG CAG TAC ATC AAG TGG CCC TGG






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:6 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:6 as follows: about 13 of the 28 phenylalanine codons are TTT, and about 15 of the phenylalanine codons are TTC; about 3 of the 46 leucine codons are TTA, about 6 of the leucine codons are TTG, about 6 of the leucine codons are CTT, about 9 of the leucine codons are CTC, about 4 of the leucine codons are CTA, and about 18 of the leucine codons are CTG; about 13 of the 36 isoleucine codons are ATT, about 17 of the isoleucine codons are ATC, and about 6 of the isoleucine codons are ATA; the 10 methionine codons are ATG; about 6 of the 33 valine codons are GTT, about 15 of the valine codons are GTG, about 4 of the valine codons are GTA, and about 8 of the valine codons are GTC; about 6 of the 35 serine codons are TCT, about 8 of the serine codons are TCC, about 5 of the serine codons are TCA, about 2 of the serine codons are TCG, about 6 of the serine codons are AGT, and about 8 of the serine codons are AGC; about 5 of the 19 proline codons are CCT, about 6 of the proline codons are CCC, about 6 of the proline codons are CCA, and about 2 of the proline codons are CCG; about 9 of the 38 threonine codons are ACT, about 14 of the threonine codons are ACC, about 11 of the threonine codons are ACA, and about 4 of the threonine codons are ACG; about 11 of the 43 alanine codons are GCT, about 17 of the alanine codons are GCC, about 10 of the alanine codons are GCA, and about 5 of the alanine codons are GCG; about 7 of the 17 tyrosine codons are TAT and about 10 of the tyrosine codons are TAC; about 2 of the 5 histidine codons are CAT and about 3 of the histidine codons are CAC; about 9 of the 34 glutamine codons are CAA and about 25 of the glutarnine codons are CAG; about 16 of the 35 asparagine codons are AAT and about 19 of the asparagine codons are AAC; about 11 of the 25 lysine codons are AAA and about 14 of the lysine codons are AAG; about 12 of the 26 aspartic acid codons are GAT and about 14 of the aspartic acid codons are GAC; about 10 of the 23 glutamic acid codons are GAA and about 13 of the glutarnic acid codons are GAG; about 5 of the 10 cysteine codons are TGT and about 5 of the cysteine codons are TGC; the 4 tryptophan codons are TGG; about 1 of the 16 arginine codons is CGT, about 3 of the arginine codons are CGC, about 2 of the arginine codons are CGA, about 3 of the arginine codons are CGG, about 4 of the arginine codons are AGA, and about 3 of the arginine codons are AGG; and about 5 of the 30 glycine codons are GGT, about 10 of the glycine codons are GGC, about 8 of the glycine codons are GGA, and about 7 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:6, optimized according to codon usage in humans is presented herein as SEQ ID NO:28.








GAC AGT TCA ATC GCC TAT TCG AAC AAC ACT ATA GCA



 


ATC CCA ACA AAT TTT TCA ATT TCT ATA ACA ACA GAG


 


GTG ATG CCA GTG TCC ATG GCA AAG ACT AGC GTA GAC


 


TGC AAT ATG TAC ATC TGC GGA GAT TCT ACA GAA TGT


 


GCA AAC TTG CTG CTA CAG TAT GGA TCG TTC TGT ACC


 


CAG CTC AAC CGG GCG CTG AGC GGC ATT GCT GCC GAA


 


CAG GAT CGC AAT ACG AGA GAG GTG TTT GCT CAA GTG


 


AAA CAA ATG TAT AAG ACC CCA ACA TTG AAA TAC TTC


 


GGT GGA TTC AAT TTC AGT CAG ATT CTG CCA GAC CCA


 


CTC AAA CCC ACC AAG AGG AGC TTT ATT GAA GAT CTT


 


CTG TTC AAC AAA GTT ACC TTG GCC GAC GCT GGG TTT


 


ATG AAG CAA TAC GGT GAG TGC CTG GGC GAC ATT AAC


 


GCA CGA GAC CTG ATC TGC GCC CAG AAG TTT AAC GGG


 


CTC ACG GTT TTA CCG CCA CTG CTG ACT GAT GAT ATG


 


ATT GCC GCT TAC ACT GCG GCC CTT GTG AGT GGT ACC


 


GCA ACT GCT GGC TGG ACG TTT GGC GCT GGG GCG GCC


 


TTA CAG ATC CCT TTT GCC ATG CAG ATG GCC TAC AGG


 


TTC AAT GGA ATT GGT GTC ACT CAG AAT GTC CTG TAC


 


GAG AAC CAG AAA CAG ATC GCC AAC CAG TTC AAT AAA


 


GCT ATT TCA CAG ATT CAG GAA TCA CTT ACC ACA ACT


 


TCC ACG GCA CTC GGT AAA CTG CAG GAC GTG GTG AAT


 


CAG AAC GCT CAG GCA CTA AAT ACA CTC GTC AAG CAA


 


CTG AGT TCC AAT TTC GGG GCC ATA TCT AGC GTA TTG


 


AAC GAC ATC CTC AGT CGG CTC GAC AAA GTG GAG GCC


 


GAA GTC CAA ATA GAC CGT CTT ATC ACA GGC AGA CTA


 


CAG TCA TTG CAG ACC TAC GTT ACC CAG CAG TTG ATC


 


CGC GCC GCT GAG ATA CGA GCC TCC GCC AAT CTG GCC


 


GCT ACC AAA ATG TCT GAG TGT GTG CTC GGA CAA AGT


 


AAG CGG GTG GAT TTT TGC GGC AAG GGC TAT CAC CTC


 


ATG TCC TTC CCT CAA GCA GCA CCC CAC GGA GTC GTT


 


TTT CTG CAT GTG ACA TAC GTG CCT AGC CAG GAG AGA


 


AAC TTT ACC ACT GCG CCT GCC ATT TGT CAT GAA GGC


 


AAA GCT TAT TTT CCC CGC GAG GGG GTG TTC GTT TTC


 


AAC GGA ACT AGC TGG TTT ATC ACA CAA AGG AAT TTC


 


TTC TCC CCC CAG ATC ATC ACC ACC GAC AAC ACC TTT


 


GTC TCT GGA AAC TGT GAC GTC GTT ATA GGC ATC ATC


 


AAT AAT ACA GTA TAC GAT CCC CTG CAG CCC GAA CTT


 


GAC TCT TTC AAG GAG GAA CTA GAT AAG TAC TTC AAG


 


AAT CAC ACC AGC CCG GAT GTA GAT TTA GGG GAT ATT


 


AGC GGG ATT AAC GCA TCC GTG GTC AAC ATC CAA AAA


 


GAG ATT GAC AGA CTG AAC GAA GTG GCG AAG AAC CTG


 


AAT GAG TCC CTG ATC GAT CTT CAG GAG CTG GGC AAG


 


TAT GAA CAG TAT ATC AAG TGG CCT TGG






A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:56, optimized according to codon usage in humans is presented herein as SEQ ID NO:65.








ATG GAC AGT TCA ATC GCC TAT TCG AAC AAC ACT ATA



 


GCA ATC CCA ACA AAT TTT TCA ATT TCT ATA ACA ACA


 


GAG GTG ATG CCA GTG TCC ATG GCA AAG ACT AGC GTA


 


GAC TGC AAT ATG TAC ATC TGC GGA GAT TCT ACA GAA


 


TGT GCA AAC TTG CTG CTA CAG TAT GGA TCG TTC TGT


 


ACC CAG CTC AAC CGG GCG CTG AGC GGC ATT GCT GCC


 


GAA CAG GAT CGC AAT ACG AGA GAG GTG TTT GCT CAA


 


GTG AAA CAA ATG TAT AAG ACC CCA ACA TTG AAA TAC


 


TTC GGT GGA TTC AAT TTC AGT CAG ATT CTG CCA GAC


 


CCA CTC AAA CCC ACC AAG AGG AGC TTT ATT GAA GAT


 


CTT CTG TTC AAC AAA GTT ACC TTG GCC GAC GCT GGG


 


TTT ATG AAG CAA TAC GGT GAG TGC CTG GGC GAC ATT


 


AAC GCA CGA GAC CTG ATC TGC GCC CAG AAG TTT AAC


 


GGG CTC ACG GTT TTA CCG CCA CTG CTG ACT GAT GAT


 


ATG ATT GCC GCT TAC ACT GCG GCC CTT GTG AGT GGT


 


ACC GCA ACT GCT GGC TGG ACG TTT GGC GCT GGG GCG


 


GCC TTA CAG ATC CCT TTT GCC ATG CAG ATG GCC TAC


 


AGG TTC AAT GGA ATT GGT GTC ACT CAG AAT GTC CTG


 


TAC GAG AAC CAG AAA CAG ATC GCC AAC CAG TTC AAT


 


AAA GCT ATT TCA CAG ATT CAG GAA TCA CTT ACC ACA


 


ACT TCC ACG GCA CTC GGT AAA CTG CAG GAC GTG GTG


 


AAT CAG AAC GCT CAG GCA CTA AAT ACA CTC GTC AAG


 


CAA CTG AGT TCC AAT TTC GGG GCC ATA TCT AGC GTA


 


TTG AAC GAC ATC CTC AGT CGG CTC GAC AAA GTG GAG


 


GCC GAA GTC CAA ATA GAC CGT CTT ATC ACA GGC AGA


 


CTA CAG TCA TTG CAG ACC TAC GTT ACC CAG CAG TTG


 


ATC CGC GCC GCT GAG ATA CGA GCC TCC GCC AAT CTG


 


GCC GCT ACC AAA ATG TCT GAG TGT GTG CTC GGA CAA


 


AGT AAG CGG GTG GAT TTT TGC GGC AAG GGC TAT CAC


 


CTC ATG TCC TTC CCT CAA GCA GCA CCC CAC GGA GTC


 


GTT TTT CTG CAT GTG ACA TAC GTG CCT AGC CAG GAG


 


AGA AAC TTT ACC ACT GCG CCT GCC ATT TGT CAT GAA


 


GGC AAA GCT TAT TTT CCC CGC GAG GGG GTG TTC GTT


 


TTC AAC GGA ACT AGC TGG TTT ATC ACA CAA AGG AAT


 


TTC TTC TCC CCC CAG ATC ATC ACC ACC GAC AAC ACC


 


TTT GTC TCT GGA AAC TGT GAC GTC GTT ATA GGC ATC


 


ATC AAT AAT ACA GTA TAC GAT CCC CTG CAG CCC GAA


 


CTT GAC TCT TTC AAG GAG GAA CTA GAT AAG TAC TTC


 


AAG AAT CAC ACC AGC CCG GAT GTA GAT TTA GGG GAT


 


ATT AGC GGG ATT AAC GCA TCC GTG GTC AAC ATC CAA


 


AAA GAG ATT GAC AGA CTG AAC GAA GTG GCG AAG AAC


 


CTG AAT GAG TCC CTG ATC GAT CTT CAG GAG CTG GGC


 


AAG TAT GAA CAG TAT ATC AAG TGG CCT TGG






Another representative codon-optimized coding region encoding SEQ ID NO:6 is presented herein as SEQ ID NO:46.








GAT AGC AGC ATA GCC TAC TCA AAC AAC ACG ATC GCC



 


ATC CCC ACA AAC TTT TCC ATT TCC ATA ACT ACC GAG


 


GTG ATG CCC GTG AGC ATG GCC AAG ACA TCG GTA GAT


 


TGC AAC ATG TAC ATC TGT GGC GAT TCT ACA GAG TGT


 


GCC AAC CTG CTG CTG CAG TAC GGC TCT TTC TGC ACG


 


CAG CTG AAC AGG GCC CTG TCT GGC ATC GCC GCC GAG


 


CAG GAT CGG AAC ACA CGG GAG GTT TTC GCC CAG GTA


 


AAG CAG ATG TAT AAG ACG CCC ACT CTG AAG TAC TTC


 


GGC GGC TTC AAC TTC TCT CAG ATA CTG CCC GAC CCC


 


CTG AAG CCC ACT AAG AGG TCT TTT ATC GAG GAT CTG


 


CTG TTC AAC AAG GTT ACC CTG GCC GAT GCC GGC TTT


 


ATG AAG CAG TAT GGC GAG TGC CTG GGC GAC ATC AAC


 


GCC AGA GAT CTG ATA TGC GCC CAG AAG TTC AAC GGC


 


CTG ACT GTG CTG CCC CCC CTG CTG ACT GAC GAC ATG


 


ATC GCC GCC TAT ACC GCC GCC CTG GTG AGT GGC ACA


 


GCC ACT GCC GGC TGG ACA TTC GGC GCC GGC GCC GCC


 


CTG CAG ATC CCC TTC GCC ATG CAG ATG GCC TAC AGA


 


TTT AAC GGC ATT GGC GTC ACT CAG AAC GTC CTG TAT


 


GAG AAC CAG AAG CAG ATC GCC AAC CAG TTT AAC AAG


 


GCC ATA AGC CAG ATC CAG GAG TCA CTG ACA ACG ACA


 


AGT ACC GCC CTG GGC AAG CTG CAG GAT GTA GTG AAC


 


CAG AAC GCC CAG GCC CTG AAC ACT CTG GTT AAG CAG


 


CTG TCT AGC AAC TTC GGC GCC ATC AGT AGT GTT CTG


 


AAC GAT ATT CTG TCT AGG CTG GAC AAG GTC GAG GCC


 


GAG GTG CAG ATT GAT CGC CTG ATT ACC GGC AGA CTG


 


CAG AGT CTG CAG ACT TAT GTA ACT CAG CAG CTG ATC


 


AGA GCC GCC GAG ATT CGA GCC TCC GCC AAC CTG GCC


 


GCC ACA AAG ATG TCT GAG TGC GTC CTG GGC CAG AGT


 


AAG AGG GTT GAC TTC TGC GGC AAG GGC TAT CAT CTG


 


ATG TCT TTT CCC CAG GCC GCC CCC CAC GGC GTC GTG


 


TTC CTG CAC GTA ACT TAC GTG CCC AGT CAG GAG AGA


 


AAC TTT ACC ACT GCC CCC GCC ATC TGC CAC GAG GGC


 


AAG GCC TAC TTC CCC AGA GAG GGC GTG TTT GTG TTC


 


AAC GGC ACA TCT TGG TTC ATC ACC CAG AGG AAC TTT


 


TTC AGC CCC CAG ATC ATA ACA ACT GAC AAC ACT TTC


 


GTT TCG GGC AAC TGC GAC GTA GTG ATC GGC ATA ATA


 


AAC AAC ACC GTG TAC GAT CCC CTG CAG CCC GAG CTG


 


GAC AGC TTT AAG GAG GAG CTG GAC AAG TAC TTT AAG


 


AAC CAT ACC TCA CCC GAT GTG GAC CTG GGC GAC ATT


 


TCT GGC ATA AAC GCC TCC GTC GTC AAC ATC CAG AAG


 


GAG ATA GAT AGA CTG AAC GAG GTT GCG AAG AAC CTG


 


AAC GAG TCC CTG ATC GAT CTG CAG GAG CTG GGC AAG


 


TAC GAG CAG TAT ATA AAG TGG CCC TGG






Another representative codon-optimized coding region encoding SEQ ID NO:56 is presented herein as SEQ ID NO:66.








ATG GAT AGC AGC ATA GCC TAC TCA AAC AAC ACG ATC



 


GCC ATC CCC ACA AAC TTT TCC ATT TCC ATA ACT ACC


 


GAG GTG ATG CCC GTG AGC ATG GCC AAG ACA TCG GTA


 


GAT TGC AAC ATG TAC ATC TGT GGC GAT TCT ACA GAG


 


TGT GCC AAC CTG CTG CTG CAG TAC GGC TCT TTC TGC


 


ACG CAG CTG AAC AGG GCC CTG TCT GGC ATC GCC GCC


 


GAG CAG GAT CGG AAC ACA CGG GAG GTT TTC GCC CAG


 


GTA AAG CAG ATG TAT AAG ACG CCC ACT CTG AAG TAC


 


TTC GGC GGC TTC AAC TTC TCT CAG ATA CTG CCC GAC


 


CCC CTG AAG CCC ACT AAG AGG TCT TTT ATC GAG GAT


 


CTG CTG TTC AAC AAG GTT ACC CTG GCC GAT GCC GGC


 


TTT ATG AAG CAG TAT GGC GAG TGC CTG GGC GAC ATC


 


AAC GCC AGA GAT CTG ATA TGC GCC CAG AAG TTC AAC


 


GGC CTG ACT GTG CTG CCC CCC CTG CTG ACT GAC GAC


 


ATG ATC GCC GCC TAT ACC GCC GCC CTG GTG AGT GGC


 


ACA GCC ACT GCC GGC TGG ACA TTC GGC GCC GGC GCC


 


GCC CTG CAG ATC CCC TTC GCC ATG CAG ATG GCC TAC


 


AGA TTT AAC GGC ATT GGC GTC ACT CAG AAC GTC CTG


 


TAT GAG AAC CAG AAG CAG ATC GCC AAC CAG TTT AAC


 


AAG GCC ATA AGC CAG ATC CAG GAG TCA CTG ACA ACG


 


ACA AGT ACC GCC CTG GGC AAG CTG CAG GAT GTA GTG


 


AAC CAG AAC GCC CAG GCC CTG AAC ACT CTG GTT AAG


 


CAG CTG TCT AGC AAC TTC GGC GCC ATC AGT AGT GTT


 


CTG AAC GAT ATT CTG TCT AGG CTG GAC AAG GTC GAG


 


GCC GAG GTG CAG ATT GAT CGC CTG ATT ACC GGC AGA


 


CTG CAG AGT CTG CAG ACT TAT GTA ACT CAG CAG CTG


 


ATC AGA GCC GCC GAG ATT CGA GCC TCC GCC AAC CTG


 


GCC GCC ACA AAG ATG TCT GAG TGC GTC CTG GGC CAG


 


AGT AAG AGG GTT GAC TTC TGC GGC AAG GGC TAT CAT


 


CTG ATG TCT TTT CCC CAG GCC GCC CCC CAC GGC GTC


 


GTG TTC CTG CAC GTA ACT TAC GTG CCC AGT CAG GAG


 


AGA AAC TTT ACC ACT GCC CCC GCC ATC TGC CAC GAG


 


GGC AAG GCC TAC TTC CCC AGA GAG GGC GTG TTT GTG


 


TTC AAC GGC ACA TCT TGG TTC ATC ACC CAG AGG AAC


 


TTT TTC AGC CCC CAG ATC ATA ACA ACT GAC AAC ACT


 


TTC GTT TCG GGC AAC TGC GAC GTA GTG ATC GGC ATA


 


ATA AAC AAC ACC GTG TAC GAT CCC CTG CAG CCC GAG


 


CTG GAC AGC TTT AAG GAG GAG CTG GAC AAG TAC TTT


 


AAG AAC CAT ACC TCA CCC GAT GTG GAC CTG GGC GAC


 


ATT TCT GGC ATA AAC GCC TCC GTC GTC AAC ATC CAG


 


AAG GAG ATA GAT AGA CTG AAC GAG GTT GCC AAG AAC


 


CTG AAC GAG TCC CTG ATC GAT CTG CAG GAG CTG GGC


 


AAG TAC GAG CAG TAT ATA AAG TGG CCC TGG






In certain embodiments, a codon-optimized coding region encoding the full-length SARS-CoV spike protein (SEQ ID NO:23) is optimized according to any plant, animal, or microbial species, including humans. A codon-optimized coding region encoding SEQ ID NO:23 was first established using the “uniform” optimization protocol described above. However, certain additional adjustments to the sequence were carried out in order to eliminate, for example, newly opened reading frames being created on the opposite strand, splice acceptors, stretches of identical bases, or unwanted restriction enzyme sites. Making such adjustments is well within the capabilities of a person of ordinary skill in the art.
A codon-optimized coding region encoding SEQ ID NO:23 is conveniently synthesized as smaller fragments, which are then spliced together using restriction enzyme sites engineered into the sequence fragments. Examples of fragments of codon-optimized coding regions encoding SEQ ID NO:23 are as follows.
SEQ ID NO:57 has the following sequence:








GTCGACATGGTTATCTTTCTGCTGTTCCTCACCCTCACCAGCGGCAGCGA



 


TCTGGATAGGTGCACCACCTTCGACGACGTGCAGGCCCCCAACTACACCC


 


AGCACACCAGCAGCATGAGGGGCGTGTACTACCCCGACGAGATTTTCAGA


 


AGCGACACCCTGTACCTCACCCAGGACCTGTTCCTGCCCTTCTACAGCAA


 


CGTGACCGGCTTCCACACCATCAACCACACCTTCGGCAACCCCGTGATCC


 


CTTTCAAGGACGGCATCTACTTCGCCGCCACCGAGAAGAGCAATGTGGTG


 


CGGGGCTGGGTGTTCGGCAGCACCATGAACAACAAGAGCCAGAGCGTGAT


 


CATCATCAACAACAGCACCAACGTGGTGATCCGGGCCTGCAATTTCGAGC


 


TGTGCGACAACCCTTTCTTCGCCGTGTCCAAACCTATGGGCACCCAGACC


 


CACACCATGATCTTCGACAACGCCTTCAACTGCACCTTCGAGTACATCAG


 


CGACGCCTTCAGCCTGGATGTGAGCGAGAAGAGCGGCAACTTCAAGCACC


 


TGCGGGAGTTCGTGTTCAAGAACAAGGACGGCTTCCTGTACGTGTACAAG


 


GGCTACCAGCCCATCGACGTGGTGAGAGACCTGCCCAGCGGCTTCAACAC


 


CCTGAAGCCCATCTTCAAGCTGCCCCTGGGCATCAACATCACCAACTTCC


 


GGGCCATCCTCACCGCCTTTAGCCCTGCCCAGGATATCTGGGGCACCAGC


 


GCCGCTGCCTACTTCGTGGGCTACCTGAAGCCTACCACCTTCATGCTGAA


 


GTACGACGAGAACGGCACCATCACCGATGCCGTGGACTGCAGCCAGAACC


 


CCCTGGCCGAGCTGAAGTGCAGCGTGAAGAGCTTCGAGATCGACAAGGGC


 


ATCTACCAGACCAGCAACTTCAGAGTGGTGCCTAGCGGCGATGTGGTGAG


 


GTTCCCCAATATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCA


 


CCAAGTTCCCTAGCGTGTACGCCTGGGAGCGGAAGAAGATCAGCAACTGC


 


GTGGCCGATTACAGCGTGCTGTACAACTCCACCTTCTTCAGCACCTTCAA


 


GTGCTACGGCGTGAGCGCCACCAAGCTGAACGACCTGTGCTTCAGCAACG


 


TGTACGCCGACTCATTCGTGGTGAAGGGCGACGACGTGAGACAGATCGCC


 


CCTGGCCAGACCGGCGTGATCGCCGACTACAACTACAAGCTT






Nucleotides 7 to 1242 of SEQ ID NO:57 encode amino acids 1 to 412 of SEQ ID NO:23, with the exception that amino acid 2 (Phenylalanine, (F)) of SEQ ID NO:23 is replaced with valine (V). The translation product of nucleotides 7 to 1242 of SEQ ID NO:57 is presented herein as SEQ ID NO:58.








MVIFLLFLTLTSGSDLDRCTTFDDVQALPNYTQHTSSMRGVYYPDEIFRS



 


DTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVR


 


GWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTH


 


TMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKG


 


YQPIDVVRDLPSGFNTLKLPIFKLPLGINITNFRAILTAFSPAQDIWGTS


 


AAAYFVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKG


 


IYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNC


 


VADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIA


 


PGQTGVIADYNYKL






Nucleotides 1 to 6 of SEQ ID NO:57, GTCGAC, is a recognition site for the restriction enzyme Sal I. Nucleotides 1237 to 1242 of SEQ ID NO:57, AAGCTT, is a recognition site for the restriction enzyme Hind III.
SEQ ID NO:59 has the following sequence:








AAGCTTCCCGACGACTTCATGGGCTGCGTGCTGGCCTGGAACACCAGAAA



 


CATCGACGCCACCTCCACCGGCAACTACAATTACAAGTACCGCTACCTGA


 


GGCACGGCAAGCTGAGACCCTTCGAGCGGGACATCTCCAACGTGCCCTTC


 


AGCCCCGACGGCAAGCCCTGCACCCCCCCTGCCCTGAACTGCTACTGGCC


 


CCTGAACGACTACGGCTTCTACACCACCACCGGCATCGGCTATCAGCCCT


 


ACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGAACGCCCCTGCCACCGTG


 


TGCGGCCCCAAGCTGAGCACCGACCTCATCAAGAACCAGTGCGTGAACTT


 


CAACTTCAACGGCCTCACCGGCACCGGCGTGCTCACCCCCAGCAGCAAGA


 


GATTCCAGCCCTTCCAGCAGTTCGGCAGGGACGTGAGCGATTTCACCGAC


 


AGCGTGAGGGATCCTAAGACCAGCGAGATCCTGGACATCAGCCCTTGCAG


 


CTTCGGCGGCGTGTCCGTGATCACCCCCGGCACCAACGCCAGCAGCGAGG


 


TGGCCGTGCTGTACCAGGACGTGAACTGCACCGACGTGAGCACCGCCATC


 


CACGCCGACCAGCTCACCCCCGCCTGGAGAATCTACAGCACCGGCAACAA


 


CGTGTTCCAGACCCAGGCCGGCTGCCTCATCGGCGCCGAGCACGTGGACA


 


CCAGCTACGAGTGCGACATCCCCATCGGAGCCGGCATCTGCGCCAGCTAC


 


CACACCGTGAGCCTGCTGAGAAGCACCAGCCAGAAGAGCATCGTGGCCTA


 


CACCATGAGCCTGGGCGCCGACAGCAGCATCGCCTACAGCAACAACACCA


 


TCGCCATCCCCACCAACTTCAGCATCTCCATCACCACCGAGGTGATGCCC


 


GTGAGCATGGCCAAGACCAGCGTGGATTGCAACATGTACATCTGCGGCGA


 


CAGCACCGAGTGCGCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCC


 


AGCTGAACAGAGCCCTGAGCGGCATTGCCGCCGAGCAGGACAGAAACACC


 


AGGGAGGTGTTCGCCCAGGTGAAGCAGATGTATAAGACCCCCACCCTGAA


 


GTACTTCGGCGGGTTCAACTTCAGCCAGATCCTGCCCGATCCTCTGAAGC


 


CCACCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAGGTGACCCTG


 


GCCGACGCCGGCTTTATGAAGCAGTACGGCGAGTGCCTGGGCGATATCAA


 


CGCCAGGGACCTCATCTGCGCCCAGAAGTTCAACGGCTTGACCGTGCTGC


 


CCCCTCTGCTCACCGATGATATGATCGCCGCCTATACAGCCGCCCTGGTG


 


TCAGGCACCGCCACCGCCGGCTGGACCTTTGGCGCCGGAGCCGCCCTGCA


 


GATCCCCTTCGCCATGCAGATGGCCTACCGGT






Nucleotides 1 to 1431 of SEQ ID NO:59 encode amino acids 411 to 887 of SEQ ID NO:23. Nucleotides 1 to 6 of SEQ ID NO:59, AAGCTT, is a recognition site for the restriction enzyrne Hind III. Nucleotides 1237 to 1242 of SEQ ID NO:59, ACCGGT, is a recognition site for the restriction enzymes Age I and PinA I.
SEQ ID NO:60 has the following sequence:








ACCGGTTCAATGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAG



 


AAGCAGATCGCCAACCAGTTCAATAAGGCCATCTCCCAGATCCAGGAGAG


 


CCTCACCACCACAAGCACCGCCCTGGGCAAGCTGCAGGACGTGGTGAACC


 


AGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTC


 


GGCGCCATCAGCAGCGTGCTGAACGACATCCTGAGCAGGCTGGATAAGGT


 


GGAGGCCGAGGTGCAGATCGACAGACTCATCACCGGCAGACTGCAGAGCC


 


TGCAGACCTACGTGACCCAGCAGCTCATCAGAGCCGCCGAGATCAGAGCC


 


AGCGCCAATCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTGGGCCAGAG


 


CAAGAGAGTGGACTTCTGCGGCAAGGGCTATCACCTCATGAGCTTCCCTC


 


AGGCCGCTCCCCACGGCGTGGTGTTCCTGCACGTGACCTACGTGCCTAGC


 


CAGGAGAGGAATTTCACCACCGCCCCAGCCATCTGCCACGAGGGCAAGGC


 


CTACTTCCCCAGAGAGGGCGTGTTCGTGTTTAACGGCACCAGCTGGTTCA


 


TCACCCAGCGGAACTTCTTCAGCCCCCAGATCATCACCACAGACAACACC


 


TTCGTGTCCGGCAATTGCGACGTGGTCATCGGCATCATCAAATAACACCG


 


TGTACGACCCCCTGCAGCCCGAGCTGGATAGCTTCAAGGAGGAGCTGGAC


 


AAGTACTTCAAGAACCACACCTCCCCCGACGTGGACCTGGGCGACATCAG


 


CGGCATCAATGCCAGCGTGGTGAACATCCAGAAGGAGATCGACCGGCTGA


 


ACGAGGTGGCCAAGAACCTGAACGAGAGCCTCATCGACCTGCAGGAGCTG


 


GGAAAGTACGAGCAGTACATCAAGTGGCCCTGGTACGTGTGGCTGGGCTT


 


CATCGCCGGCCTCATCGCCATCGTGATGGTGACCATCCTGCTGTGCTGCA


 


TGACCAGCTGCTGCTCCTGCCTGAAGGGCGCCTGCAGCTGTGGCAGCTGC


 


TGCAAGTTCGACGAGGACGACTCAGAGCCCGTGCTGAAGGGCGTGAAGCT


 


GCACTACACCTGAAGATCT






Nucleotides 3 to 1109 of SEQ ID NO:60 encode amino acids 887 to 1255 of SEQ ID NO:23. Nucleotides 1 to 6 of SEQ ID NO:60, ACCGGT, is a recognition site for the restriction enzymes Age I and PinA I. Nucleotides 1113 to 1118 of SEQ ID NO:59, AGATCT, is a recognition site for the restriction enzyme Bgl II.
SEQ ID NOs 57, 59, and 60 are then spliced together using the restriction enzyme sites described above to produce a codon-optimized coding region encoding SEQ ID NO:23 in its entirety, with the exception that amino acid 2 (Phenylalanine, (F)) of SEQ ID NO:23 is replaced with valine (V). The spliced sequence is presented herein as SEQ ID NO:61.








GTCGACATGGTTATCTTTCTGCTGTTCCTCACCCTCACCAGCGGCAGCGA



 


TCTGGATAGGTGCACCACCTTCGACGACGTGCAGGCCCCCAACTACACCC


 


AGCACACCAGCAGCATGAGGGGCGTGTACTACCCCGACGAGATTTTCAGA


 


AGCGACACCCTGTACCTCACCCAGGACCTGTTCCTGCCCTTCTACAGCAA


 


CGTGACCGGCTTCCACACCATCAACCACACCTTCGGCAACCCCGTGATCC


 


CTTTCAAGGACGGCATCTACTTCGCCGCCACCGAGAAGAGCAATGTGGTG


 


CGGGGCTGGGTGTTCGGCAGCACCATGAACAACAAGAGCCAGAGCGTGAT


 


CATCATCAACAACAGCACCAACGTGGTGATCCGGGCCTGCAATTTCGAGC


 


TGTGCGACAACCCTTTCTTCGCCGTGTCCAAACCTATGGGCACCCAGACC


 


CACACCATGATCTTCGACAACGCCTTCAACTGCACCTTCGAGTACATCAG


 


CGACGCCTTCAGCCTGGATGTGAGCGAGAAGAGCGGCAACTTCAAGCACC


 


TGCGGGAGTTCGTGTTCAAGAACAAGGACGGCTTCCTGTACGTGTACAAG


 


GGCTACCAGCCCATCGACGTGGTGAGAGACCTGCCCAGCGGCTTCAACAC


 


CCTGAAGCCCATCTTCAAGCTGCCCCTGGGCATCAACATCACCAACTTCC


 


GGGCCATCCTCACCGCCTTTAGCCCTGCCCAGGATATCTGGGGCACCAGC


 


GCCGCTGCCTACTTCGTGGGCTACCTGAAGCCTACCACCTTCATGCTGAA


 


GTACGACGAGAACGGCACCATCACCGATGCCGTGGACTGCAGCCAGAACC


 


CCCTGGCCGAGCTGAAGTGCAGCGTGAAGAGCTTCGAGATCGACAAGGGC


 


ATCTACCAGACCAGCAACTTCAGAGTGGTGCCTAGCGGCGATGTGGTGAG


 


GTTCCCCAATATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCA


 


CCAAGTTCCCTAGCGTGTACGCCTGGGAGCGGAAGAAGATCAGCAACTGC


 


GTGGCCGATTACAGCGTGCTGTACAACTCCACCTTCTTCAGCACCTTCAA


 


GTGCTACGGCGTGAGCGCCACCAAGCTGAACGACCTGTGCTTCAGCAACG


 


TGTACGCCGACTCATTCGTGGTGAAGGGCGACGACGTGAGACAGATCGCC


 


CCTGGCCAGACCGGCGTGATCGCCGACTACAACTACAAGCTTCCCGACGA


 


CTTCATGGGCTGCGTGCTGGCCTGGAACACCAGAAACATCGACGCCACCT


 


CCACCGGCAACTACAATTACAAGTACCGCTACCTGAGGCACGGCAAGCTG


 


AGACCCTTCGAGCGGGACATCTCCAACGTGCCCTTCAGCCCCGACGGCAA


 


GCCCTGCACCCCCCCTGCCCTGAACTGCTACTGGCCCCTGAACGACTACG


 


GCTTCTACACCACCACCGGCATCGGCTATCAGCCCTACAGAGTGGTGGTG


 


CTGAGCTTCGAGCTGCTGAACGCCCCTGCCACCGTGTGCGGCCCCAAGCT


 


GAGCACCGACCTCATCAAGAACCAGTGCGTGAACTTCAACTTCAACGGCC


 


TCACCGGCACCGGCGTGCTCACCCCCAGCAGCAAGAGATTCCAGCCCTTC


 


CAGCAGTTCGGCAGGGACGTGAGCGATTTCACCGACAGCGTGAGGGATCC


 


TAAGACCAGCGAGATCCTGGACATCAGCCCTTGCAGCTTCGGCGGCGTGT


 


CCGTGATCACCCCCGGCACCAACGCCAGCAGCGAGGTGGCCGTGCTGTAC


 


CAGGACGTGAACTGCACCGACGTGAGCACCGCCATCCACGCCGACCAGCT


 


CACCCCCGCCTGGAGAATCTACAGCACCGGCAACAACGTGTTCCAGACCC


 


AGGCCGGCTGCCTCATCGGCGCCGAGCACGTGGACACCAGCTACGAGTGC


 


GACATCCCCATCGGAGCCGGCATCTGCGCCAGCTACCACACCGTGAGCCT


 


GCTGAGAAGCACCAGCCAGAAGAGCATCGTGGCCTACACCATGAGCCTGG


 


GCGCCGACAGCAGCATCGCCTACAGCAACAACACCATCGCCATCCCCACC


 


AACTTCAGCATCTCCATCACCACCGAGGTGATGCCCGTGAGCATGGCCAA


 


GACCAGCGTGGATTGCAACATGTACATCTGCGGCGACAGCACCGAGTGCG


 


CCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAACAGAGCC


 


CTGAGCGGCATTGCCGCCGAGCAGGACAGAAACACCAGGGAGGTGTTCGC


 


CCAGGTGAAGCAGATGTATAAGACCCCCACCCTGAAGTACTTCGGCGGGT


 


TCAACTTCAGCCAGATCCTGCCCGATCCTCTGAAGCCCACCAAGCGGAGC


 


TTCATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTT


 


TATGAAGCAGTACGGCGAGTGCCTGGGCGATATCAACGCCAGGGACCTCA


 


TCTGCGCCCAGAAGTTCAACGGCTTGACCGTGCTGCCCCCTCTGCTCACC


 


GATGATATGATCGCCGCCTATACAGCCGCCCTGGTGTCAGGCACCGCCAC


 


CGCCGGCTGGACCTTTGGCGCCGGAGCCGCCCTGCAGATCCCCTTCGCCA


 


TGCAGATGGCCTACCGGTTCAATGGCATCGGCGTGACCCAGAACGTGCTG


 


TACGAGAACCAGAAGCAGATCGCCAACCAGTTCAATAAGGCCATCTCCCA


 


GATCCAGGAGAGCCTCACCACCACAAGCACCGCCCTGGGCAAGCTGCAGG


 


ACGTGGTGAACCAGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTG


 


AGCAGCAACTTCGGCGCCATCAGCAGCGTGCTGAACGACATCCTGAGCAG


 


GCTGGATAAGGTGGAGGCCGAGGTGCAGATCGACAGACTCATCACCGGCA


 


GACTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTCATCAGAGCCGCC


 


GAGATCAGAGCCAGCGCCAATCTGUCCGCCACCAAGATGAGCGAGTGCGT


 


GCTGGGCCAGAGCAAGAGAGTGGACTTCTGCGGCAAGGGCTATCACCTCA


 


TGAGCTTCCCTCAGGCCGCTCCCCACGGCGTGGTGTTCCTGCACGTGACC


 


TACGTGCCTAGCCAGGAGAGGAATTTCACCACCGCCCCAGCCATCTGCCA


 


CGAGGGCAAGGCCTACTTCCCCAGAGAGGGCGTGTTCGTGTTTAACGGCA


 


CCAGCTGCTTCATCACCCAGCGGAACTTCTTCAGCCCCCAGATCATCACC


 


ACAGACAACACCTTCGTGTCCGGCAATTGCGACGTGGTCATCGGCATCAT


 


CAATAACACCGTGTACGACCCCCTGCAGCCCGAGCTGGATAGCTTCAAGG


 


AGGAGCTGGACAAGTACTTCAAGAACCACACCTCCCCCGACGTGGACCTG


 


GGCGACATCAGCGGCATCAATGCCAGCGTGGTGAACATCCAGAAGGAGAT


 


CGACCGGCTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCTCATCGACC


 


TGCAGGAGCTGGGAAAGTACGAGCAGTACATCAAGTGGCCCTGGTACGTG


 


TGGCTGGGCTTCATCGCCGGCCTCATCGCCATCGTGATGGTGACCATCCT


 


GCTGTGCTGCATGACCAGCTGCTGCTCCTGCCTGAAGGGCGCCTGCAGCT


 


GTGGCAGCTGCTGCAAGTTCGACGAGGACGACTCAGAGCCCGTGCTGAAG


 


GGCGTGAAGCTGCACTACACCTGAAGATCT






The translation product of nucleotides 7 to 3771 of SEQ ID NO:61 is presented herein as SEQ ID NO:62








MVIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSD



 


TLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFKDGIYFAATEKSNVVRG


 


WVFGSTMNNKSQSVIIINNSTNVVRACNFELCDNPFFAVSKPMGTQTHTM


 


IFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQ


 


PIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAA


 


YFVGYLKPTTFMLKYDENGTTTDAVDCSQNPLAELKCSVKSFEIDKGIYQ


 


TSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVAD


 


YSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQ


 


TGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPF


 


ERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSF


 


ELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQF


 


GRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDV


 


NCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIP


 


IGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFS


 


ISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSG


 


IAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIE


 


DLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDM


 


IAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYEN


 


QKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSN


 


FGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIR


 


ASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVP


 


SQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDN


 


TFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI


 


SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLG


 


FIAGLIALIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGV


 


KLHYT






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:8 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:8 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:8, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:8 is shown in Table 12.










TABLE 12








Number in



AMINO ACID
SEQ ID NO: 8




















A
Ala
84



R
Arg
41



C
Cys
33



G
Gly
77



H
His
14



I
Ile
73



L
Leu
92



K
Lys
57



M
Met
19



F
Phe
79



P
Pro
57



S
Ser
93



T
Thr
94



W
Trp
10



Y
Tyr
52



V
Val
89



N
Asn
81



D
Asp
71



Q
Gln
55



E
Glu
40










Using the amino acid composition shown in Table 12, a human codon-optimized coding region which encodes SEQ ID NO:8 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:8 as follows: the 79 phenylalanine codons are TTC, the 92 leucine codons are CTG, the 73 isoleucine codons are ATC, the 19 methionine codons are ATG, the 89 valine codons are GTG, the 93 serine codons are AGC, the 57 proline codons are CCC, the 94 threonine codons are ACC, the 84 alanine codons are GCC, the 52 tyrosine codons are TAC, the 14 histidine codons are CAC, the 55 glutamine codons are CAG, the 81 asparagine codons are AAC, the 57 lysine codons are AAG, the 71 aspartic acid codons are GAC, the 40 glutamic acid codons are GAG, the 33 cysteine codons are TGC, the 10 tryptophan codon is TGG, the 41 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 77 glycine codons are GGC. The codon-optimized coding region designed by this method is presented herein as SEQ ID NO:31.








ATG GAC GCC ATG AAG CGG GGC CTG TGC TGC GTG CTG



 


CTG CTG TGC GGC GCC GTG TTC GTG AGC CCC AGC GCC


 


CGG GGC AGC GGC AGC GAC CTG GAC CGG TGC ACC ACC


 


TTC GAC GAC GTG CAG GCC CCC AAC TAC ACC CAG CAC


 


ACC AGC AGC ATG CGG GGC GTG TAC TAC CCC GAC GAG


 


ATC TTC CGG AGC GAC ACC CTG TAC CTG ACC CAG GAC


 


CTG TTC CTG CCC TTC TAC AGC AAC GTG ACC GGC TTC


 


CAC ACC ATC AAC CAC ACC TTC GGC AAC CCC GTG ATC


 


CCC TTC AAG GAC GGC ATC TAC TTC GCC GCC ACC GAG


 


AAG AGC AAC GTG GTG CGG GGC TGG GTG TTC GGC AGC


 


ACC ATG AAC AAC AAG AGC CAG AGC GTG ATC ATC ATC


 


AAC AAC AGC ACC AAC GTG GTG ATC CGG GCC TGC AAC


 


TTC GAG CTG TGC GAC AAC CCC TTC TTC GCC GTG AGC


 


AAG CCC ATG GGC ACC CAG ACC CAC ACC ATG ATC TTC


 


GAC AAC GCC TTC AAC TGC ACC TTC GAG TAC ATC AGC


 


GAC GCC TTC AGC CTG GAC GTG AGC GAG AAG AGC GGC


 


AAC TTC AAG CAC CTG CGG GAG TTC GTG TTC AAG AAC


 


AAC GAC GGC TTC CTG TAC GTG TAC AAG GGC TAC CAG


 


CCC ATC GAC GTG GTG CGG GAC CTG CCC AGC GGC TTC


 


AAC ACC CTG AAG CCC ATC TTC AAG CTG CCC CTG GGC


 


ATC AAC ATC ACC AAC TTC CGG GCC ATC CTG ACC GCC


 


TTC AGC CCC GCC CAG GAC ATC TGG GGC ACC AGC GCC


 


GCC GCC TAC TTC GTG GGC TAC CTG AAG CCC ACC ACC


 


TTC ATG CTG AAG TAC GAC GAG AAC GGC ACC ATC ACC


 


GAC GCC GTG GAC TGC AGC CAG AAC CCC CTG GCC GAG


 


CTG AAG TGC AGC GTG AAG AGC TTC GAG ATC GAC AAG


 


GGC ATC TAC CAG ACC AGC AAC TTC CGG GTG GTG CCC


 


AGC GGC GAC GTG GTG CGG TTC CCC AAC ATC ACC AAC


 


CTG TGC CCC TTC GGC GAG GTG TTC AAC GCC ACC AAG


 


TTC CCC AGC GTG TAC GCC TGG GAG CGG AAG AAG ATC


 


AGC AAC TGC GTG GCC GAC TAC AGC GTG CTG TAC AAC


 


AGC ACC TTC TTC AGC ACC TTC AAG TGC TAC GGC GTG


 


AGC GCC ACC AAG CTG AAC GAC CTG TGC TTC AGC AAC


 


GTG TAC GCC GAC AGC TTC GTG GTG AAG GGC GAC GAC


 


GTG CGG CAG ATC GCC CCC GGC CAG ACC GGC GTG ATC


 


GCC GAC TAC AAC TAC AAG CTG CCC GAC GAC TTC ATG


 


GGC TGC GTG CTG GCC TGG AAC ACC CGG AAC ATC GAC


 


GCC ACC AGC ACC GGC AAC TAC AAC TAC AAG TAC CGG


 


TAC CTG CGG CAC GGC AAG CTG CGG CCC TTC GAG CGG


 


GAC ATC AGC AAC GTG CCC TTC AGC CCC GAC GGC AAG


 


CCC TGC ACC CCC CCC GCC CTG AAC TGC TAC TGG CCC


 


CTG AAC GAC TAC GGC TTC TAC ACC ACC ACC GGC ATC


 


GGC TAC CAG CCC TAC CGG GTG GTG GTG CTG AGC TTC


 


GAG CTG CTG AAC GCC CCC GCC ACC GTG TGC GGC CCC


 


AAG CTG AGC ACC GAC CTG ATC AAG AAC CAG TGC GTG


 


AAC TTC AAC TTC AAC GGC CTG ACC GGC ACC GGC GTG


 


CTG ACC CCC AGC AGC AAG CGG TTC CAG CCC TTC CAG


 


CAG TTC GGC CGG GAC GTG AGC GAC TTC ACC GAC AGC


 


GTG CGG GAC CCC AAG ACC AGC GAG ATC CTG GAC ATC


 


AGC CCC TGC AGC TTC GGC GGC GTG AGC GTG ATC ACC


 


CCC GGC ACC AAC GCC AGC AGC GAG GTG GCC GTG CTG


 


TAC CAG GAC GTG AAC TGC ACC GAC GTG AGC ACC GCC


 


ATC CAC GCC GAC CAG CTG ACC CCC GCC TGG CGG ATC


 


TAC AGC ACC GGC AAC AAC GTG TTC CAG ACC CAG GCC


 


GGC TGC CTG ATC GGC GCC GAG CAC GTG GAC ACC AGC


 


TAC GAG TGC GAC ATC CCC ATC GGC GCC GGC ATC TGC


 


GCC AGC TAC CAC ACC GTG AGC CTG CTG CGG AGC ACC


 


AGC CAG AAG AGC ATC GTG GCC TAC ACC ATG AGC CTG


 


GGC GCC GAC AGC AGC ATC GCC TAC AGC AAC AAC ACC


 


ATC GCC ATC CCC ACC AAC TTC AGC ATC AGC ATC ACC


 


ACC GAG GTG ATG CCC GTG AGC ATG GCC AAG ACC AGC


 


GTG GAC TGC AAC ATG TAC ATC TGC GGC GAC AGC ACC


 


GAG TGC GCC AAC CTG CTG CTG CAG TAC GGC AGC TTC


 


TGC ACC CAG CTG AAC CGG GCC CTG AGC GGC ATC GCC


 


GCC GAG CAG GAC CGG AAC ACC CGG GAG GTG TTC GCC


 


CAG GTG AAG CAG ATG TAC AAG ACC CCC ACC CTG AAG


 


TAC TTC GGC GGC TTC AAC TTC AGC CAG ATC CTG CCC


 


GAC CCC CTG AAG CCC ACC AAG CGG AGC TTC ATC GAG


 


GAC CTG CTG TTC AAC AAG GTG ACC CTG GCC GAC GCC


 


GGC TTC ATG AAG CAG TAC GGC GAG TGC CTG GGC GAC


 


ATC AAC GCC CGG GAC CTG ATC TGC GCC CAG AAG TTC


 


AAC GGC CTG ACC GTG CTG CCC CCC CTG CTG ACC GAC


 


GAC ATG ATC GCC GCC TAC ACC GCC GCC CTG GTG AGC


 


GGC ACC GCC ACC GCC GGC TGG ACC TTC GGC GCC GGC


 


GCC GCC CTG CAG ATC CCC TTC GCC ATG CAG ATG GCC


 


TAC CGG TTC AAC GGC ATC GGC GTG ACC CAG AAC GTG


 


CTG TAC GAG AAC CAG AAG CAG ATC GCC AAC CAG TTC


 


AAC AAG GCC ATC AGC CAG ATC CAG GAG AGC CTG ACC


 


ACC ACC AGC ACC GCC CTG GGC AAG CTG CAG GAC GTG


 


GTG AAC CAG AAC GCC CAG GCC CTG AAC ACC CTG GTG


 


AAG CAG CTG AGC AGC AAC TTC GGC GCC ATC AGC AGC


 


GTG CTG AAC GAC ATC CTG AGC CGG CTG GAC AAG GTG


 


GAG GCC GAG GTG CAG ATC GAC CGG CTG ATC ACC GGC


 


CGG CTG CAG AGC CTG CAG ACC TAC GTG ACC CAG CAG


 


CTG ATC CGG GCC GCC GAG ATC CGG GCC AGC GCC AAC


 


CTG GCC GCC ACC AAG ATG AGC GAG TGC GTG CTG GGC


 


CAG AGC AAG CGG GTG GAC TTC TGC GGC AAG GGC TAC


 


CAC CTG ATG AGC TTC CCC CAG GCC GCC CCC CAC GGC


 


GTG GTG TTC CTG CAC GTG ACC TAC GTG CCC AGC CAG


 


GAG CGG AAC TTC ACC ACC GCC CCC GCC ATC TGC CAC


 


GAG GGC AAG GCC TAC TTC CCC CGG GAG GGC GTG TTC


 


GTG TTC AAC GGC ACC AGC TGG TTC ATC ACC CAG CGG


 


AAC TTC TTC AGC CCC CAG ATC ATC ACC ACC GAC AAC


 


ACC TTC GTG AGC GGC AAC TGC GAC GTG GTG ATC GGC


 


ATC ATC AAC AAC ACC GTG TAC GAC CCC CTG CAG CCC


 


GAG CTG GAC AGC TTC AAG GAG GAG CTG GAC AAG TAC


 


TTC AAG AAC CAC ACC AGC CCC GAC GTG GAC CTG GGC


 


GAC ATC AGC GGC ATC AAC GCC AGC GTG GTG AAC ATC


 


CAG AAG GAG ATC GAC CGG CTG AAC GAG GTG GCC AAG


 


AAC CTG AAC GAG AGC CTG ATC GAC CTG CAG GAG CTG


 


GGC AAG TAC GAG CAG TAC ATC AAG TGG CCC TGG






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:8 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:8 as follows: about 36 of the 79 phenylalanine codons are TTT, and about 43 of the phenylalanine codons are TTC; about 7 of the 92 leucine codons are TTA, about 12 of the leucine codons are TTG, about 12 of the leucine codons are CTT, about 18 of the leucine codons are CTC, about 7 of the leucine codons are CTA, and about 36 of the leucine codons are CTG; about 26 of the 73 isoleucine codons are ATT, about 35 of the isoleucine codons are ATC, and about 12 of the isoleucine codons are ATA; the 19 methionine codons are ATG; about 16 of the 89 valine codons are GTT, about 41 of the valine codons are GTG, about 11 of the valine codons are GTA, and about 21 of the valine codons are GTC; about 17 of the 93 serine codons are TCT, about 20 of the serine codons are TCC, about 14 of the serine codons are TCA, about 5 of the serine codons are TCG, about 15 of the serine codons are AGT, and about 22 of the serine codons are AGC; about 16 of the 57 proline codons are CCT, about 19 of the proline codons are CCC, about 16 of the proline codons are CCA, and about 6 of the proline codons are CCG; about 23 of the 94 threonine codons are ACT, about 34 of the threonine codons are ACC, about 26 of the threonine codons are ACA, and about 11 of the threonine codons are ACG; about 22 of the 84 alanine codons are GCT, about 34 of the alanine codons are GCC, about 19 of the alanine codons are GCA, and about 9 of the alanine codons are GCG; about 23 of the 52 tyrosine codons are TAT and about 29 of the tyrosine codons are TAC; about 6 of the 14 histidine codons are CAT and about 8 of the histidine codons are CAC; about 14 of the 55 glutamine codons are CAA and about 41 of the glutamine codons are CAG; about 37 of the 81 asparagine codons are AAT and about 44 of the asparagine codons are AAC; about 24 of the 57 lysine codons are AAA and about 33 of the lysine codons are AAG; about 33 of the 71 aspartic acid codons are GAT and about 38 of the aspartic acid codons are GAC; about 17 of the 40 glutamic acid codons are GAA and about 23 of the glutamic acid codons are GAG; about 15 of the 33 cysteine codons are TGT and about 18 of the cysteine codons are TGC; the 10 tryptophan codons are TGG; about 3 of the 41 arginine codons are CGT, about 8 of the arginine codons are CGC, about 5 of the arginine codons are CGA, about 8 of the arginine codons are CGG, about 9 of the arginine codons are AGA, and about 8 of the arginine codons are AGG; and about 13 of the 77 glycine codons are GGT, about 26 of the glycine codons are GGC, about 19 of the glycine codons are GGA, and about 19 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:8, optimized according to codon usage in humans is presented herein as SEQ ID NO:30.








ATG GAT GCA ATG AAG CGG GGC CTG TGC TGC GTG CTC



 


CTG CTC TGC GGG GCG GTG TTT GTG AGC CCC AGT GCC


 


AGA GGT AGC GGC AGC GAT TTG GAT AGG TGC ACC ACA


 


TTT GAT GAC GTG CAG GCT CCC AAT TAC ACC CAG CAC


 


ACC AGT TCT ATG AGA GGA GTA TAC TAC CCT GAC GAG


 


ATC TTC CGC AGT GAT ACC CTA TAT TTA ACA CAA GAT


 


TTA TTC TTA CCC TTC TAC TCC AAC GTC ACA GGG TTT


 


CAC ACC ATC AAC CAC ACC TTC GGC AAC CCC GTG ATC


 


CCG TTT AAA GAT GGC ATT TAT TTC GCA GCC ACA GAG


 


AAG TCG AAT GTA GTG CGG GGT TGG GTG TTT GGA TCA


 


ACA ATG AAT AAT AAA TCT CAG TCC GTG ATC ATT ATT


 


AAC AAC TCT ACG AAT GTG GTT ATA CGA GCC TGT AAT


 


TTC GAG TTA TGC GAT AAT CCA TTT TTC GCG GTC AGT


 


AAA CCA ATG GGC ACT CAG ACC CAT ACG ATG ATT TTC


 


GAT AAC GCA TTC AAT TGT ACG TTT GAA TAC ATT TCT


 


GAT GCT TTT TCA CTC GAC GTT TCA GAA AAG TCT GGG


 


AAC TTC AAG CAT TTA AGA GAG TTC GTC TTT AAA AAT


 


AAA GAC GGG TTC CTG TAC GTG TAT AAA GGA TAC CAG


 


CCT ATC GAC GTG GTG CGG GAC CTG CCA AGC GGT TTT


 


AAT ACC CTG AAG CCC ATC TTT AAG CTG CCC CTG GGA


 


ATC AAT ATT ACA AAC TTC AGG GCT ATC CTC ACC GCT


 


TTT AGC CCA GCT CAG GAC ATA TGG GGA ACC TCC GCC


 


GCC GCC TAC TTC GTC GGA TAT TTG AAA CCA ACC ACA


 


TTC ATG CTG AAG TAT GAC GAA AAT GGG ACG ATT ACC


 


GAC GCC GTA GAC TGT AGT CAG AAC CCT TTG GCG GAG


 


TTG AAG TGC TCA GTC AAG AGC TTT GAG ATC GAC AAG


 


GGA ATT TAT CAA ACT AGC AAC TTC AGG GTG GTG CCC


 


TCC GGA GAT GTA GTT CGC TTC CCC AAC ATC ACC AAC


 


CTG TGC CCG TTC GGT GAG GTG TTT AAT GCA ACT AAA


 


TTC CCC TCA GTG TAT GCC TGG GAA AGA AAG AAA ATT


 


AGC AAC TGT GTT GCC GAT TAC AGC GTC CTT TAT AAC


 


TCA ACA TTC TTC TCT ACC TTT AAG TGC TAT GGT GTG


 


TCC GCC ACT AAG TTG AAC GAC CTC TGC TTT AGT AAC


 


GTG TAC GCT GAT TCC TTC GTG GTG AAA GGG GAT GAC


 


GTG CGT CAG ATT GCA CCG GGC CAG ACC GGA GTA ATC


 


GCC GAT TAC AAT TAC AAG TTG CCT GAC GAC TTC ATG


 


GGC TGC GTT CTA GCA TGG AAT ACC CGC AAC ATA GAT


 


GCC ACC TCA ACG GGG AAC TAC AAC TAC AAG TAC AGA


 


TAT CTG AGA CAC GGT AAG CTG CGG CCT TTT GAG CGG


 


GAT ATC TCC AAT GTG CCT TTT AGC CCC GAT GGC AAA


 


CCA TGC ACC CCA CCT GCC CTG AAT TGT TAT TGG CCT


 


TTG AAC GAT TAT GGA TTC TAC ACT ACC ACT GGG ATC


 


GGT TAT CAA CCC TAC CGG GTC GTC GTC CTG AGT TTT


 


GAA CTC TTG AAC GCG CCT GCA ACA GTC TGC GGA CCC


 


AAG CTG TCG ACA GAC CTT ATC AAG AAT CAG TGT GTG


 


AAC TTT AAC TTC AAT GGG CTC ACC GGT ACC GGT GTT


 


CTG ACT CCA TCT AGT AAG CGA TTT CAA CCA TTC CAA


 


CAG TTC GGC CGT GAC GTT TCC GAT TTT ACG GAT TCG


 


GTG CGT GAT CCA AAA ACA TCA GAG ATC CTT GAC ATA


 


TCG CCG TGT TCT TTT GGA GGC GTG TCT GTG ATT ACA


 


CCA GGC ACT AAT GCT AGT AGC GAA GTC GCT GTA CTA


 


TAC CAG GAC GTG AAC TGC ACC GAC GTG AGC ACG GCA


 


ATC CAC GCT GAC CAG CTG ACC CCC GCC TGG CGC ATC


 


TAC AGT ACA GGC AAT AAC GTC TTT CAG ACC CAG GCC


 


GGC TGT CTG ATT GGG GCT GAG CAC GTC GAC ACT TCC


 


TAT GAA TGT GAT ATT CCC ATC GGC GCT GGA ATT TGT


 


GCT AGC TAT CAC ACA GTC TCC CTT TTA AGA TCA ACC


 


AGC CAG AAA TCT ATT GTG GCT TAC ACA ATG TCT CTC


 


GGC GCA GAC TCA TCA ATT GCC TAT AGC AAC AAT ACC


 


ATT GCA ATC CCT ACC AAT TTT AGT ATA TCC ATA ACC


 


ACC GAG GTG ATG CCC GTG TCT ATG GCG AAA ACT TCC


 


GTC GAT TGC AAC ATG TAT ATC TGC GGG GAC TCC ACA


 


GAA TGC GCC AAC CTG CTT CTG CAG TAT GGA AGC TTC


 


TGT ACT CAA CTC AAC CGC GCA TTG TCT GGG ATT GCC


 


GCC GAG CAG GAT AGG AAT ACT AGA GAG GTG TTC GCT


 


CAG GTT AAA CAA ATG TAC AAG ACA CCG ACA CTT AAG


 


TAC TTC GGA GGT TTT AAC TTT TCC CAG ATA CTC CCT


 


GAC CCT CTA AAG CCT ACT AAA CGC AGT TTC ATC GAG


 


GAT CTC CTG TTT AAT AAG GTG ACA CTC GCC GAT GCT


 


GGC TTC ATG AAA CAA TAC GGA GAA TGC CTG GGA GAC


 


ATT AAC GCC AGA GAC CTG ATC TGT GCC CAG AAG TTC


 


AAC GGT CTG ACA GTA CTT CCT CCC CTT CTG ACG GAC


 


GAC ATG ATT GCT GCA TAC ACA GCC GCC CTA GTT AGC


 


GGC ACA GCC ACA GCT GGG TGG ACC TTT GGC GCT GGC


 


GCA GCG TTG CAG ATT CCA TTC GCG ATG CAG ATG GCT


 


TAC CGA TTT AAC GGG ATC GGC GTG ACT CAG AAT GTT


 


TTG TAT GAG AAC CAG AAA CAG ATC GCT AAT CAG TTT


 


AAC AAG GCA ATC AGC CAG ATA CAA GAA TCT CTG ACT


 


ACC ACA AGC ACC GCT CTG GGA AAA CTG CAG GAC GTG


 


GTG AAT CAG AAT GCA CAG GCC CTC AAC ACG CTC GTG


 


AAG CAG CTT AGT TCC AAT TTC GGG GCC ATC TCC TCC


 


GTT TTA AAT GAT ATC CTG AGT CGC CTG GAC AAG GTC


 


GAG GCC GAA GTT CAG ATC GAC CGC CTG ATC ACA GGG


 


AGG CTA CAA TCA TTG CAG ACT TAC GTG ACT CAG CAG


 


CTC ATA AGG GCT GCA GAG ATT AGG GCC TCT GCA AAC


 


CTT GCC GCG ACC AAG ATG TCC GAG TGT GTT CTC GGT


 


CAG TCC AAA CGG GTT GAC TTT TGT GGC AAA GGC TAC


 


CAT CTG ATG AGC TTC CCC CAG GCC GCA CCC CAT GGC


 


GTA GTC TTT CTG CAC GTA ACT TAT GTG CCA TCC CAA


 


GAA AGG AAC TTC ACT ACG GCG CCA GCC ATA TGC CAT


 


GAA GGT AAA GCA TAT TTC CCT CGA GAA GGG GTA TTT


 


GTT TTC AAC GGG ACT AGC TGG TTT ATT ACG CAG CGG


 


AAT TTC TTC TCA CCA CAA ATC ATC ACT ACT GAT AAC


 


ACA TTC GTC AGC GGC AAT TGT GAC GTC GTC ATT GGA


 


ATT ATA AAC AAC ACT GTG TAC GAT CCT CTG CAG CCG


 


GAA CTG GAT TCT TTT AAG GAG GAG CTC GAC AAG TAC


 


TTC AAA AAC CAT ACC TCG CCC GAC GTG GAC CTA GGC


 


GAT ATC TCT GGG ATT AAT GCC TCA GTA GTC AAC ATC


 


CAG AAG GAG ATA GAC CGA CTT AAT GAG GTT GCC AAG


 


AAT CTG AAT GAG AGT CTC ATC GAT CTG CAA GAA CTT


 


GGC AAG TAT GAA CAA TAT ATC AAA TGG CCA TGG






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:10 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:10 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:10, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:10 is shown in Table 13.









TABLE 13






AMINO
Number in



ACID
SEQ ID NO: 10

















A
Ala
41


R
Arg
25


C
Cys
23


G
Gly
47


H
His
9


I
Ile
37


L
Leu
46


K
Lys
32


M
Met
9


F
Phe
51


P
Pro
38


S
Ser
58


T
Thr
56


W
Trp
6


Y
Tyr
35


V
Val
56


N
Asn
46


D
Asp
45


Q
Gln
21


E
Glu
17









Using the amino acid composition shown in Table 13, a human codon-optimized coding region which encodes SEQ ID NO:10 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:10 as follows: the 51 phenylalanine codons are TTC, the 46 leucine codons are CTG, the 37 isoleucine codons are ATC, the 9 methionine codons are ATG, the 56 valine codons are GTG, the 58 serine codons are AGC, the 38 proline codons are CCC, the 56 threonine codons are ACC, the 41 alanine codons are GCC, the 35 tyrosine codons are TAC, the 9 histidine codons are CAC, the 21 glutamine codons are CAG, the 46 asparagine codons are AAC, the 32 lysine codons are AAG, the 45 aspartic acid codons are GAC, the 17 glutamic acid codons are GAG, the 23 cysteine codons are TGC, the 6 tryptophan codons are TGG, the 25 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 47 glycine codons are GGC. The codon-optimized coding region designed by this method is presented herein as SEQ ID NO:33.








ATG GAC GCC ATG AAG CGG GGC CTG TGC TGC GTG CTG



 


CTG CTG TGC GGC GCC GTG TTC GTG AGC CCC AGC GCC


 


CGG GGC AGC GGC AGC GAC CTG GAC CGG TGC ACC ACC


 


TTC GAC GAC GTG CAG GCC CCC AAC TAC ACC CAG CAC


 


ACC AGC AGC ATG CGG GGC GTG TAC TAC CCC GAC GAG


 


ATC TTC CGG AGC GAC ACC CTG TAC CTG ACC CAG GAC


 


CTG TTC CTG CCC TTC TAC AGC AAC GTG ACC GGC TTC


 


CAC ACC ATC AAC CAC ACC TTC GGC AAC CCC GTG ATC


 


CCC TTC AAG GAC GGC ATC TAC TTC GCC GCC ACC GAG


 


AAG AGC AAC GTG GTG CGG GGC TGG GTG TTC GGC AGC


 


ACC ATG AAC AAC AAG AGC CAG AGC GTG ATC ATC ATC


 


AAC AAC AGC ACC AAC GTG GTG ATC CGG GCC TGC AAC


 


TTC GAG CTG TGC GAC AAC CCC TTC TTC GCC GTG AGC


 


AAG CCC ATG GGC ACC CAG ACC CAC ACC ATG ATC TTC


 


GAC AAC GCC TTC AAC TGC ACC TTC GAG TAC ATC AGC


 


GAC GCC TTC AGC CTG GAC GTG AGC GAG AAG AGC GGC


 


AAC TTC AAG CAC CTG CGG GAG TTC GTG TTC AAG AAC


 


AAG GAC GGC TTC CTG TAC GTG TAC AAG GGC TAC CAG


 


CCC ATC GAC GTG GTG CGG GAC CTG CCC AGC GGC TTC


 


AAC ACC CTG AAG CCC ATC TTC AAG CTG CCC CTG GGC


 


ATC AAC ATC ACC AAC TTC CGG GCC ATC CTG ACC GCC


 


TTC AGC CCC GCC CAG GAC ATC TGG GGC ACC AGC GCC


 


GCC GCC TAC TTC GTG GGC TAC CTG AAG CCC ACC ACC


 


TTC ATG CTG AAG TAC GAC GAG AAC GGC ACC ATC ACC


 


GAC GCC GTG GAC TGC AGC CAG AAC CCC CTG GCC GAG


 


CTG AAG TGC AGC GTG AAG AGC TTC GAG ATC GAC AAG


 


GGC ATC TAC CAG ACC AGC AAC TTC CGG GTG GTG CCC


 


AGC GGC GAC GTG GTG CGG TTC CCC AAC ATC ACC AAC


 


CTG TGC CCC TTC GGC GAG GTG TTC AAC GCC ACC AAG


 


TTC CCC AGC GTG TAC GCC TGG GAG CGG AAG AAG ATC


 


AGC AAC TGC GTG GCC GAC TAC AGC GTG CTG TAC AAC


 


AGC ACC TTC TTC AGC ACC TTC AAG TGC TAC GGC GTG


 


AGC GCC ACC AAG CTG AAC GAC CTG TGC TTC AGC AAC


 


GTG TAC GCC GAC AGC TTC GTG GTG AAG GGC GAC GAC


 


GTG CGG CAG ATC GCC CCC GGC CAG ACC GGC GTG ATC


 


GCC GAC TAC AAC TAC AAG CTG CCC GAC GAC TTC ATG


 


GGC TGC GTG CTG GCC TGG AAC ACC CGG AAC ATC GAC


 


GCC ACC AGC ACC GGC AAC TAC AAC TAC AAG TAC CGG


 


TAC CTG CGG CAC GGC AAG CTG CGG CCC TTC GAG CGG


 


GAC ATC AGC AAC GTG CCC TTC AGC CCC GAC GGC AAG


 


CCC TGC ACC CCC CCC GCC CTG AAC TGC TAC TGG CCC


 


CTG AAC GAC TAC GGC TTC TAC ACC ACC ACC GGC ATC


 


GGC TAC CAG CCC TAC CGG GTG GTG GTG CTG AGC TTC


 


GAG CTG CTG AAC GCC CCC GCC ACC GTG TGC GGC CCC


 


AAG CTG AGC ACC GAC CTG ATC AAG AAC CAG TGC GTG


 


AAC TTC AAC TTC AAC GGC CTG ACC GGC ACC GGC GTG


 


CTG ACC CCC AGC AGC AAG CGG TTC CAG CCC TTC CAG


 


CAG TTC GGC CGG GAC GTG AGC GAC TTC ACC GAC AGC


 


GTG CGG GAC CCC AAG ACC AGC GAG ATC CTG GAC ATC


 


AGC CCC TGC AGC TTC GGC GGC GTG AGC GTG ATC ACC


 


CCC GGC ACC AAC GCC AGC AGC GAG GTG GCC GTG CTG


 


TAC CAG GAC GTG AAC TGC ACC GAC GTG AGC ACC GCC


 


ATC CAC GCC GAC CAG CTG ACC CCC GCC TGG CGG ATC


 


TAC AGC ACC GGC AAC AAC GTG TTC CAG ACC CAG GCC


 


GGC TGC CTG ATC GGC GCC GAG CAC GTG GAC ACC AGC


 


TAC GAG TGC GAC ATC CCC ATC GGC GCC GGC ATC TGC


 


GCC AGC TAC CAC ACC GTG AGC CTG CTG CGG AGC ACC


 


AGC CAG AAG AGC ATC GTG GCC TAC ACC ATG AGC CTG


 


GGC






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:10 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:10 as follows: about 23 of the 51 phenylalanine codons are TTT, and about 28 of the phenylalanine codons are TTC; about 3 of the 46 leucine codons are TTA, about 6 of the leucine codons are TTG, about 6 of the leucine codons are CTT, about 9 of the leucine codons are CTC, about 4 of the leucine codons are CTA, and about 18 of the leucine codons are CTG; about 13 of the 37 isoleucine codons are ATT, about 18 of the isoleucine codons are ATC, and about 6 of the isoleucine codons are ATA; the 9 methionine codons are ATG; about 10 of the 56 valine codons are GTT, about 26 of the valine codons are GTG, about 7 of the valine codons are GTA, and about 13 of the valine codons are GTC; about 11 of the 58 serine codons are TCT, about 13 of the serine codons are TCC, about 9 of the serine codons are TCA, about 3 of the serine codons are TCG, about 8 of the serine codons are AGT, and about 14 of the serine codons are AGC; about 11 of the 38 proline codons are CCT, about 13 of the proline codons are CCC, about 10 of the proline codons are CCA, and about 4 of the proline codons are CCG; about 14 of the 56 threonine codons are ACT, about 20 of the threonine codons are ACC, about 16 of the threonine codons are ACA, and about 6 of the threonine codons are ACG; about 11 of the 41 alanine codons are GCT, about 16 of the alanine codons are GCC, about 10 of the alanine codons are GCA, and about 4 of the alanine codons are GCG; about 15 of the 35 tyrosine codons are TAT and about 20 of the tyrosine codons are TAC; about 4 of the 9 histidine codons are CAT and about 5 of the histidine codons are CAC; about 5 of the 21 glutamine codons are CAA and about 16 of the glutarnine codons are CAG; about 21 of the 46 asparagine codons are AAT and about 25 of the asparagine codons are AAC; about 14 of the 32 lysine codons are AAA and about 18 of the lysine codons are AAG; about 21 of the 45 aspartic acid codons are GAT and about 24 of the aspartic acid codons are GAC; about 7 of the 17 glutamic acid codons are GAA and about 10 of the glutarnic acid codons are GAG; about 10 of the 23 cysteine codons are TGT and about 13 of the cysteine codons are TGC; the 6 tryptophan codons are TGG; about 2 of the 25 arginine codons are CGT, about 5 of the arginine codons are CGC, about 3 of the arginine codons are CGA, about 5 of the arginine codons are CGG, about 5 of the arginine codons are AGA, and about 5 of the arginine codons are AGG; and about 8 of the 47 glycine codons are GGT, about 16 of the glycine codons are GGC, about 11 of the glycine codons are GGA, and about 12 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO: 10, optimized according to codon usage in humans is presented herein as SEQ ID NO:32.








ATG GAC GCC ATG AAG CGA GGA CTG TGC TGC GTT TTG



 


TTG CTG TGC GGC GCA GTT TTT GTC AGT CCA TCC GCC


 


CGG GGG TCG GGA TCT GAC CTA GAT AGA TGC ACG ACC


 


TTC GAT GAC GTG CAG GCA CCA AAT TAC ACC CAA CAT


 


ACT TCA TCC ATG CGC GGC GTT TAC TAC CCC GAC GAA


 


ATC TTC CGG AGT GAC ACC CTG TAT CTG ACT CAG GAC


 


CTG TTT CTG CCC TTC TAC AGC AAT GTG ACA GGC TTT


 


CAC ACC ATT AAC CAT ACC TTC GGG AAT CCA GTA ATC


 


CCT TTT AAG GAT GGG ATT TAC TTT GCT GCT ACT GAG


 


AAA AGT AAT GTT GTC AGG GGG TGG GTT TTT GGC TCA


 


ACA ATG AAC AAT AAG TCT CAG AGT GTC ATC ATC ATT


 


AAC AAT TCT ACC AAT GTA GTC ATC AGA GCA TGC AAC


 


TTC GAG CTC TGT GAT AAC CCT TTC TTT GCT GTG TCT


 


AAG CCC ATG GGC ACT CAA ACA CAT ACC ATG ATC TTC


 


GAC AAT GCG TTC AAT TGT ACC TTT GAG TAT ATA TCA


 


GAC GCC TTC AGC CTA GAC GTC TCG GAA AAG TCC GGA


 


AAC TTT AAA CAC CTG CGG GAA TTC GTG TTT AAG AAC


 


AAA GAT GGA TTT TTG TAC GTA TAC AAG GGT TAT CAG


 


CCT ATC GAT GTC GTG CGT GAT CTG CCC TCC GGC TTC


 


AAC ACC CTG AAG CCT ATA TTC AAA CTA CCC CTA GGG


 


ATC AAC ATC ACC AAT TTT AGG GCA ATA CTT ACG GCA


 


TTT TCC CCA GCC CAG GAC ATC TGG GGA ACT TCC GCC


 


GCT GCC TAC TTT GTG GGC TAT CTC AAG CCT ACT ACT


 


TTC ATG CTT AAG TAT GAT GAG AAT GGC ACA ATC ACG


 


GAT GCA GTG GAT TGC TCG CAG AAT CCA CTT GCT GAG


 


CTG AAA TGC TCC GTA AAG AGC TTC GAA ATT GAT AAA


 


GGA ATC TAT CAG ACC AGC AAC TTC CGG GTC GTG CCC


 


TCT GGC GAC GTT GTC CGG TTC CCC AAC ATC ACC AAC


 


CTC TGC CCA TTC GGC GAG GTG TTC AAC GCT ACA AAA


 


TTC CCA AGT GTC TAC GCC TGG GAG AGG AAA AAG ATC


 


TCT AAT TGT GTG GCA GAT TAT TCC GTG TTA TAC AAC


 


AGC ACA TTC TTC TCA ACG TTC AAG TGT TAT GGC GTG


 


AGC GCC ACC AAG CTT AAC GAC CTC TGC TTC TCC AAT


 


GTA TAC GCT GAC TCT TTT GTG GTT AAG GGA GAC GAT


 


GTG CGA CAG ATC GCC CCG GGG CAA ACC GGA GTG ATT


 


GCG GAC TAC AAC TAT AAA CTG CCC GAC GAT TTC ATG


 


GGT TGT GTG CTT GCT TGG AAT ACG AGG AAC ATT GAC


 


GCA ACG AGC ACC GGG AAC TAT AAT TAC AAA TAT CGT


 


TAC CTG CGC CAT GGG AAA CTC AGA CCT TTT GAA CGA


 


GAT ATT AGC AAC GTC CCT TTC TCA CCG GAT GGG AAG


 


CCC TGT ACC CCA CCT GCC CTG AAC TGC TAT TGG CCT


 


CTC AAC GAC TAC GGC TTC TAC ACT ACC ACA GGG ATC


 


GGG TAC CAG CCC TAT CGC GTG GTG GTT CTC TCC TTT


 


GAA CTC CTT AAT GCT CCC GCG ACT GTG TGT GGG CCG


 


AAG TTG AGT ACT GAC TTA ATA AAA AAT CAA TGC GTA


 


AAC TTT AAC TTT AAT GGC TTG ACA GGT ACA GGT GTG


 


CTC ACA CCG AGT AGC AAA AGG TTC CAG CCA TTT CAG


 


CAA TTT GGC AGA GAT GTG TCT GAC TTT ACA GAC AGC


 


GTG CGC GAT CCT AAG ACT TCT GAG ATT TTA GAC ATC


 


TCA CCT TGT TCC TTT GGA GGA GTG AGC GTG ATA ACT


 


CCC GGT ACC AAC GCC TCA TCC GAA GTG GCT GTC CTG


 


TAT CAG GAC GTT AAT TGC ACC GAT GTC TCT ACA GCC


 


ATT CAC GCC GAT CAG CTG ACA CCA GCT TGG CGC ATC


 


TAC AGT ACC GGT AAC AAT GTT TTC CAG ACT CAG GCC


 


GGT TGT CTG ATT GGC GCC GAG CAC GTC GAC ACA TCT


 


TAC GAG TGC GAT ATT CCC ATA GGT GCC GGC ATT TGT


 


GCG AGC TAC CAC ACT GTA TCA CTG CTG AGA AGC ACA


 


AGC CAG AAA TCA ATT GTG GCA TAC ACA ATG TCC TTG


 


GGA GCA






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:12 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:12 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:12, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:12 is shown in Table 14.









TABLE 14






AMINO
Number in



ACID
SEQ ID NO: 12

















A
Ala
46


R
Arg
18


C
Cys
13


G
Gly
34


H
His
5


I
Ile
36


L
Leu
50


K
Lys
26


M
Met
12


F
Phe
29


P
Pro
20


S
Ser
38


T
Thr
38


W
Trp
4


Y
Tyr
17


V
Val
36


N
Asn
35


D
Asp
27


Q
Gln
34


E
Glu
23









Using the amino acid composition shown in Table 14, a human codon-optimized coding region which encodes SEQ ID NO:12 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:12 as follows: the 29 phenylalanine codons are TTC, the 50 leucine codons are CTG, the 36 isoleucine codons are ATC, the 12 methionine codons are ATG, the 36 valine codons are GTG, the 38 serine codons are AGC, the 20 proline codons are CCC, the 38 threonine codons are ACC, the 46 alanine codons are GCC, the 17 tyrosine codons are TAC, the 5 histidine codons are CAC, the 34 glutamine codons are CAG, the 35 asparagine codons are AAC, the 26 lysine codons are AAG, the 35 aspartic acid codons are GAC, the 23 glutamic acid codons are GAG, the 13 cysteine codons are TGC, the 4 tryptophan codon is TGG, the 18 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 34 glycine codons are GGC. The codon-optimized coding region designed by this method is presented herein as SEQ ID NO:35.








ATG GAC GCC ATG AAG CGG GGC CTG TGC TGC GTG CTG



 


CTG CTG TGC GGC GCC GTG TTC GTG AGC CCC AGC GCC


 


CGG GGC AGC GGC GAC AGC AGC ATC GCC TAC AGC AAC


 


AAC ACC ATC GCC ATC CCC ACC AAC TTC AGC ATC AGC


 


ATC ACC ACC GAG GTG ATG CCC GTG AGC ATG GCC AAG


 


ACC AGC GTG GAC TGC AAC ATG TAC ATC TGC GGC GAC


 


AGC ACC GAG TGC GCC AAC CTG CTG CTG CAG TAC GGC


 


AGC TTC TGC ACC CAG CTG AAC CGG GCC CTG AGC GGC


 


ATC GCC GCC GAG CAG GAC CGG AAC ACC CGG GAG GTG


 


TTC GCC CAG GTG AAG CAG ATG TAC AAG ACC CCC ACC


 


CTG AAG TAC TTC GGC GGC TTC AAC TTC AGC CAG ATC


 


CTG CCC GAC CCC CTG AAG CCC ACC AAG CGG AGC TTC


 


ATC GAG GAC CTG CTG TTC AAC AAG GTG ACC CTG GCC


 


GAC GCC GGC TTC ATG AAG CAG TAC GGC GAG TGC CTG


 


GGC GAC ATC AAC GCC CGG GAC CTG ATC TGC GCC CAG


 


AAG TTC AAC GGC CTG ACC GTG CTG CCC CCC CTG CTG


 


ACC GAC GAC ATG ATC GCC GCC TAC ACC GCC GCC CTG


 


GTG AGC GGC ACC GCC ACC GCC GGC TGG ACC TTC GGC


 


GCC GGC GCC GCC CTG CAG ATC CCC TTC GCC ATG CAG


 


ATG GCC TAC CGG TTC AAC GGC ATC GGC GTG ACC CAG


 


AAC GTG CTG TAC GAG AAC CAG AAG CAG ATC GCC AAC


 


CAG TTC AAC AAG GCC ATC AGC CAG ATC CAG GAG AGC


 


CTG ACC ACC ACC AGC ACC GCC CTG GGC AAG CTG CAG


 


GAC GTG GTG AAC CAG AAC GCC CAG GCC CTG AAC ACC


 


CTG GTG AAG CAG CTG AGC AGC AAC TTC GGC GCC ATC


 


AGC AGC GTG CTG AAC GAC ATC CTG AGC CGG CTG GAC


 


AAG GTG GAG GCC GAG GTG CAG ATC GAC CGG CTG ATC


 


ACC GGC CGG CTG CAG AGC CTG CAG ACC TAC GTG ACC


 


CAG CAG CTG ATC CGG GCC GCC GAG ATC CGG GCC AGC


 


GCC AAC CTG GCC GCC ACC AAG ATG AGC GAG TGC GTG


 


CTG GGC CAG AGC AAG CGG GTG GAC TTC TGC GGC AAG


 


GGC TAC CAC CTG ATG AGC TTC CCC CAG GCC GCC CCC


 


CAC GGC GTG GTG TTC CTG CAC GTG ACC TAC GTG CCC


 


AGC CAG GAG CGG AAC TTC ACC ACC GCC CCC GCC ATC


 


TGC CAC GAG GGC AAG GCC TAC TTC CCC CGG GAG GGC


 


GTG TTC GTG TTC AAC GGC ACC AGC TGG TTC ATC ACC


 


CAG CGG AAC TTC TTC AGC CCC CAG ATC ATC ACC ACC


 


GAC AAC ACC TTC GTG AGC GGC AAC TGC GAC GTG GTG


 


ATC GGC ATC ATC AAC AAC ACC GTG TAC GAC CCC CTG


 


CAG CCC GAG CTG GAC AGC TTC AAG GAG GAG CTG GAC


 


AAG TAC TTC AAG AAC CAC ACC AGC CCC GAC GTG GAC


 


CTG GGC GAC ATC AGC GGC ATC AAC GCC AGC GTG GTG


 


AAC ATC CAG AAG GAG ATC GAC CGG CTG AAC GAG GTG


 


GCC AAG AAC CTG AAC GAG AGC CTG ATC GAC CTG CAG


 


GAG CTG GGC AAG TAC GAG CAG TAC ATC AAG TGG CCC


 


TGG






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:12 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:12 as follows: about 13 of the 29 phenylalanine codons are TTT, and about 16 of the phenylalanine codons are TTC; about 4 of the 50 leucine codons are TTA, about 6 of the leucine codons are TTG, about 6 of the leucine codons are CTT, about 10 of the leucine codons are CTC, about 4 of the leucine codons are CTA, and about 20 of the leucine codons are CTG; about 13 of the 36 isoleucine codons are ATT, about 17 of the isoleucine codons are ATC, and about 6 of the isoleucine codons are ATA; the 12 methionine codons are ATG; about 6 of the 36 valine codons are GTT, about 9 of the valine codons are GTG, about 4 of the valine codons are GTA, and about 17 of the valine codons are GTG; about 7 of the 38 serine codons are TCT, about 8 of the serine codons are TCC, about 6 of the serine codons are TCA, about 2 of the serine codons are TCG, about 6 of the serine codons are AGT, and about 9 of the serine codons are AGC; about 6 of the 20 proline codons are CCT, about 7 of the proline codons are CCC, about 5 of the proline codons are CCA, and about 2 of the proline codons are CCG; about 9 of the 38 threonine codons are ACT, about 14 of the threonine codons are ACC, about 11 of the threonine codons are ACA, and about 4 of the threonine codons are ACG; about 12 of the 46 alanine codons are GCT, about 19 of the alanine codons are GCC, about 10 of the alanine codons are GCA, and about 5 of the alanine codons are GCG; about 7 of the 17 tyrosine codons are TAT and about 10 of the tyrosine codons are TAC; about 2 of the 5 histidine codons are CAT and about 3 of the histidine codons are CAC; about 9 of the 34 glutamine codons are CAA and about 25 of the glutamine codons are CAG; about 16 of the 35 asparagine codons are AAT and about 19 of the asparagine codons are AAC; about 11 of the 26 lysine codons are AAA and about 15 of the lysine codons are AAG; about 12 of the 27 aspartic acid codons are GAT and about 15 of the aspartic acid codons are GAC; about 16 of the 23 glutamic acid codons are GAA and about 13 of the glutamic acid codons are GAG; about 6 of the 13 cysteine codons are TGT and about 7 of the cysteine codons are TGC; the 4 tryptophan codons are TGG; about 1 of the 18 arginine codons are CGT, about 3 of the arginine codons are CGC, about 2 of the arginine codons are CGA, about 4 of the arginine codons are CGG, about 4 of the arginine codons are AGA, and about 4 of the arginine codons are AGG; and about 6 of the 34 glycine codons are GGT, about 12 of the glycine codons are GGC, about 8 of the glycine codons are GGA, and about 8 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:12, optimized according to codon usage in humans is presented herein as SEQ ID NO:34.








ATG GAT GCA ATG AAA AGA GGC CTG TGT TGT GTT CTG



 


CTG CTG TGT GGG GCG GTA TTT GTG AGT CCC TCT GCC


 


AGG GGA AGC GGC GAC AGC AGT ATA GCC TAC TCA AAC


 


AAT ACC ATC GCC ATT CCT ACA AAT TTT TCC ATC TCA


 


ATC ACG ACG GAA GTC ATG CCA GTT AGC ATG GCC AAA


 


ACC TCT GTC GAC TGC AAC ATG TAC ATC TGC GGA GAC


 


TCT ACT GAG TGC GCA AAC CTG CTC TTG CAG TAT GGC


 


TCG TTT TGC ACC CAG TTG AAT CGG GCC CTC AGT GGC


 


ATT GCC GCA GAA CAA GAT CGG AAT ACC AGG GAG GTC


 


TTC GCG CAA GTC AAG CAG ATG TAC AAA ACC CCT ACA


 


CTC AAA TAC TTC GGG GGG TTC AAC TTT AGC CAA ATC


 


CTG CCA GAC CCC CTC AAG CCT ACT AAG CGC AGT TTT


 


ATC GAA GAC TTA CTC TTT AAT AAG GTG ACA TTA GCT


 


GAT GCC GGA TTC ATG AAG CAG TAC GGA GAG TGC CTG


 


GGG GAT ATC AAC GCG CGG GAC CTA ATC TGT GCC CAG


 


AAG TTC AAC GGT CTG ACA GTG CTT CCG CCT CTC CTG


 


ACC GAT GAT ATG ATC GCA GCT TAC ACC GCC GCA CTG


 


GTT AGT GGT ACG GCC ACA GCA GGC TGG ACC TTC GGT


 


GCC GGT GCT GCC CTG CAA ATC CCA TTC GCG ATG CAG


 


ATG GCA TAC AGA TTT AAC GGC ATT GGA GTC ACC CAG


 


AAT GTC CTA TAC GAG AAC CAG AAG CAA ATC GCT AAC


 


CAG TTC AAC AAA GCC ATA TCC CAG ATT CAG GAG TCC


 


CTT ACT ACA ACC AGT ACT GCT TTA GGT AAA CTG CAA


 


GAT GTA GTG AAC CAG AAC GCT CAG GCC TTA AAT ACC


 


CTT GTT AAA CAG CTA TCC TCA AAC TTT GGG GCT ATC


 


TCC TCC GTG CTC AAC GAT ATC CTG AGC CGC CTC GAT


 


AAG GTG GAA GCG GAG GTC CAG ATC GAT AGA CTT ATT


 


ACA GGC AGG CTT CAG TCT CTC CAG ACC TAT GTC ACA


 


CAA CAG CTC ATT CGT GCT GCA GAG ATC CGC GCT TCC


 


GCC AAC TTG GCT GCA ACA AAG ATG TCT GAA TGT GTG


 


CTG GGA CAG AGC AAG AGA GTG GAC TTT TGT GGG AAA


 


GGC TAT CAC TTG ATG AGC TTC CCC CAG GCC GCC CCC


 


CAT GGA GTG GTA TTC CTA CAC GTG ACG TAC GTT CCA


 


TCT CAA GAA CGA AAT TTC ACC ACC GCA CCT GCC ATT


 


TGC CAC GAA GGG AAG GCT TAT TTC CCT CGA GAG GGC


 


GTG TTC GTT TTT AAC GGG ACT TCA TGG TTT ATA ACT


 


CAA AGG AAT TTC TTC TCG CCC CAG ATA ATT ACA ACA


 


GAC AAC ACT TTT GTG AGC GGC AAT TGC GAC GTG GTC


 


ATA GGT ATT ATT AAT AAT ACT GTG TAT GAC CCG CTG


 


CAG CCC GAA CTG GAC AGC TTT AAA GAG GAG CTG GAC


 


AAA TAC TTC AAG AAT CAT ACT TCA CCC GAC GTG GAT


 


CTG GGC GAC ATA TCC GGA ATC AAT GCC TCT GTG GTA


 


AAC ATT CAG AAG GAG ATC GAT CGG CTG AAC GAA GTG


 


GCT AAG AAT CTG AAT GAA TCA TTG ATT GAC CTT CAG


 


GAG TTG GGC AAG TAT GAG CAG TAT ATT AAA TGG CCA


 


TGG






Another representative codon-optimized coding region encoding SEQ ID NO:12 is presented herein as SEQ ID NO:47.








ATG GAT GCC ATG AAG CGA GGC CTG TGT TGC GTA CTG



 


CTG CTG TGC GGC GCC GTG TTT GTG AGC CCC AGC GCC


 


CGG GGC AGT GGC GAC AGC AGC ATC GCC TAT TCG AAC


 


AAC ACT ATT GCC ATA CCC ACA AAC TTC TCT ATA TCT


 


ATA ACT ACG GAG GTG ATG CCC GTG TCT ATG GCC AAG


 


ACT AGT GTA GAC TGC AAC ATG TAC ATC TGC GGC GAC


 


TCT ACT GAG TGC GCC AAC CTG CTG CTG CAG TAT GGC


 


TCT TTC TGC ACC CAG CTG AAC AGA GCC CTG AGT GGC


 


ATC GCC GCC GAG CAG GAC CGG AAC ACA AGA GAG GTT


 


TTC GCC CAG GTA AAG CAG ATG TAC AAG ACC CCC ACT


 


CTG AAG TAT TTT GGC GGC TTC AAC TTC TCT CAG ATC


 


CTG CCC GAT CCC CTG AAG CCC ACC AAG AGG TCT TTC


 


ATC GAG GAC CTG CTG TTC AAC AAG GTC ACT CTG GCC


 


GAT GCC GGC TTC ATG AAG CAG TAC GGC GAG TGC CTG


 


GGC GAC ATT AAC GCC CGC GAC CTG ATC TGT GCC CAG


 


AAG TTT AAC GGC CTG ACG GTC CTG CCC CCC CTG CTG


 


ACA GAT GAT ATG ATC GCC GCC TAC ACT GCC GCC CTG


 


GTC TCT GGC ACC GCC ACC GCC GGC TGG ACT TTC GGC


 


GCC GGC GCC GCC CTG CAG ATC CCC TTC GCC ATG CAG


 


ATG GCC TAT AGA TTT AAC GGC ATA GGC GTA ACT CAG


 


AAC GTC CTG TAC GAG AAC CAG AAG CAG ATC GCC AAC


 


CAG TTT AAC AAG GCC ATC TCC CAG ATT CAG GAG AGC


 


CTG ACA ACC ACT AGC ACT GCC CTG GGC AAG CTG CAG


 


GAC GTG GTG AAC CAG AAC GCC CAG GCC CTG AAC ACA


 


CTG GTT AAG CAG CTG AGT TCT AAC TTT GGC GCC ATA


 


TCC TCG GTG CTG AAC GAC ATA CTG TCA AGG CTG GAC


 


AAG GTC GAG GCC GAG GTT CAG ATA GAT AGA CTG ATC


 


ACA GGC AGA CTG CAG AGC CTG CAG ACC TAC GTT ACA


 


CAG CAG CTG ATC AGA GCC GCC GAG ATC AGA GCC TCA


 


GCC AAC CTG GCC GCC ACG AAG ATG TCT GAG TGC GTC


 


CTG GGC CAG TCT AAG AGA GTC GAT TTC TGC GGC AAG


 


GGC TAC CAC CTG ATG AGT TTC CCC CAG GCC GCC CCC


 


CAT GGC GTT GTA TTC CTG CAT GTG ACA TAT GTT CCC


 


TCC CAG GAG AGG AAC TTT ACC ACG GCC CCC GCC ATC


 


TGC CAC GAG GGC AAG GCC TAC TTC CCC AGA GAG GGC


 


GTG TTC GTT TTT AAC GGC ACT AGC TGG TTT ATT ACC


 


CAG AGG AAC TTC TTC TCC CCC CAG ATT ATA ACA ACA


 


GAT AAC ACT TTC GTG TCC GGC AAC TGC GAT GTT GTG


 


ATA GGC ATC ATT AAC AAC ACA GTG TAC GAT CCC CTG


 


CAG CCC GAG CTG GAT AGT TTT AAG GAG GAG CTG GAC


 


AAG TAT TTT AAG AAC CAC ACT TCC CCC GAT GTA GAC


 


CTG GGC GAT ATC AGT GGC ATA AAC GCC AGT GTC GTG


 


AAC ATA CAG AAG GAG ATC GAT AGG CTG AAC GAG GTG


 


GCC AAG AAC CTG AAC GAG TCA CTG ATC GAT CTG CAG


 


GAG CTG GGC AAG TAC GAG CAG TAT ATT AAG TGG CCC






A representative codon-optimized coding region encoding SEQ ID NO:12 according to the “standardized optimization” method is presented herein as SEQ ID NO: 69.








ATG GAT GCC ATG AAG CGC GGC CTG TGC TGT GTG CTG



 


CTG CTG TGT GGC GCC GTG TTC GTG AGC CCC AGC GCC


 


CGC GGC AGC GGC GAT AGC AGC ATC GCC TAC AGC AAC


 


AAC ACC ATC GCC ATC CCC ACC AAC TTC AGC ATC AGC


 


ATC ACC ACC GAG GTG ATG CCC GTG AGC ATG GCC AAG


 


ACC AGC GTG GAT TGC AAC ATG TAC ATC TGC GGC GAC


 


AGC ACC GAG TGC GCC AAC CTG CTG CTG CAG TAC GGC


 


AGC TTC TGC ACC CAG CTG AAC CGC GCC CTG AGC GGC


 


ATC GCC GCC GAG CAG GAC CGC AAC ACC CGC GAG GTG


 


TTC GCC CAG GTG AAG CAG ATG TAC AAG ACC CCC ACC


 


CTG AAG TAC TTC GGC GGC TTC AAC TTC AGC CAG ATC


 


CTG CCC GAC CCC CTG AAG CCC ACC AAG CGC AGC TTC


 


ATC GAG GAT CTG CTG TTC AAC AAG GTG ACC CTG GCC


 


GAC GCC GGC TTC ATG AAG CAG TAC GGC GAG TGC CTG


 


GGC GAC ATC AAC GCC CGC GAC CTG ATC TGC GCC GAC


 


AAG TTC AAC GGC CTG ACC GTG CTG CCC CCC CTG CTG


 


ACC GAT GAC ATG ATC GCC GCC TAC ACC GCC GCC CTG


 


GTG AGC GGC ACC GCC ACC GCC GGC TGG ACC TTC GGC


 


GCC GGC GCC GCC CTG CAG ATC CCC TTC GCC ATG CAG


 


ATG GCC TAC CGC TTC AAC GGC ATC GGC GTG ACC CAG


 


AAC GTG CTG TAC GAG AAC CAG AAG CAG ATC GCC AAC


 


CAG TTC AAC AAG GCC ATC AGC CAG ATC CAG GAG AGC


 


CTG ACC ACC ACC AGC ACC GCC CTG GGC AAG CTG CAG


 


GAT GTG GTG AAC CAG AAC GCC CAG GCC CTG AAC ACC


 


CTG GTG AAG CAG CTG AGC AGC AAC TTC GGC GCC ATC


 


AGC AGC GTG CTG AAC GAT ATC CTG AGC CGC CTG GAT


 


AAG GTG GAG GCC GAG GTG CAG ATC GAC CGC CTG ATC


 


ACC GGC CGC CTG CAG AGC CTG CAG ACC TAC GTG ACC


 


CAG CAG CTG ATC CGC GCC GCC GAG ATC CGC GCC AGC


 


GCC AAC CTG GCC GCC ACC AAG ATG AGC GAG TGC GTG


 


CTG GGC CAG AGC AAG CGC GTG GAT TTC TGC GGC AAG


 


GGC TAC CAC CTG ATG AGC TTC CCC CAG GCC GCC CCC


 


CAC GGC GTG GTG TTC CTG CAT GTG ACC TAC GTG CCC


 


AGC CAG GAG CGC AAC TTC ACC ACC GCC CCC GCC ATC


 


TGC CAC GAG GGC AAG GCC TAC TTC CCC CGC GAG GGC


 


GTG TTC GTG TTC AAC GGC ACC AGC TGG TTC ATC ACC


 


CAG CGC AAC TTC TTC AGC CCC CAG ATC ATC ACC ACC


 


GAC AAC ACC TTC GTG AGC GGC AAC TGC GAC GTG GTG


 


ATC GGC ATC ATC AAC AAC ACC GTG TAC GAT CCC CTG


 


CAG CCC GAG CTG GAT AGC TTC AAG GAG GAG CTG GAC


 


AAG TAC TTC AAG AAC CAT ACC AGC CCC GAT GTG GAT


 


CTG GGC GAC ATC AGC GGC ATC AAC GCC AGC GTG GTG


 


AAC ATC CAG AAG GAG ATC GAT CGC CTG AAC GAG GTG


 


GCC AAG AAC CTG AAC GAG AGC CTG ATC GAT CTG CAG


 


GAG CTG GGC AAG TAC GAG CAG TAC ATC AAG TGG CCC


 


TGG






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:14 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:14 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:14, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:14 is shown in Table 15.










TABLE 15








Number in



AMINO ACID
SEQ ID NO: 14




















A
Ala
34



R
Arg
31



C
Cys
0



G
Gly
45



H
His
5



I
Ile
11



L
Leu
26



K
Lys
29



M
Met
7



F
Phe
13



P
Pro
31



S
Ser
35



T
Thr
33



W
Trp
5



Y
Tyr
11



V
Val
11



N
Asn
25



D
Asp
22



Q
Gln
34



E
Glu
14










Using the amino acid composition shown in Table 15, a human codon-optimized coding region which encodes SEQ ID NO:14 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:14 as follows: the 13 phenylalanine codons are TTC, the 26 leucine codons are CTG, the 11 isoleucine codons are ATC, the 7 methionine codons are ATG, the 11 valine codons are GTG, the 35 serine codons are AGC, the 31 proline codons are CCC, the 33 threonine codons are ACC, the 34 alanine codons are GCC, the 11 tyrosine codons are TAC, the 5 histidine codons are CAC, the 34 glutamine codons are CAG, the 25 asparagine codons are AAC, the 29 lysine codons are AAG, the 22 aspartic acid codons are GAC, the 14 glutamic acid codons are GAG, the 5 tryptophan codons are TGG, the 31 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 45 glycine codons are GGC. The codon-optimized N coding region designed by this method is presented herein as SEQ ID NO:37.








ATGAGCGACAACGGCCCCCAGAGCAACCAGAGAAGCGCCCGCAGAATCAC



 


CTTCGGCGGCCCCACCGACAGCACCGACAACAACCAGAACGGCGGCAGAA


 


ACGGCGCCAGACCCAAGCAGAGAAGACCCCAGGGCCTGCCCAACAACACC


 


GCCAGCTGGTTCACCGCCCTGACCCAGCACGGCAAGGAGGAGCTGAGATT


 


CCCCAGAGGCCAGGGCGTGCCCATCAACACCAACAGCGGCCCCGACGACC


 


AGATCGGCTACTACAGAAGAGCCACCAGAAGAGTGAGAGGCGGCGACGGC


 


AAGATGAAGGAGCTGAGCCCCAGATGGTACTTCTACTACCTGGGCACCGG


 


CCCCGAGGCCAGCCTGCCCTACGGCGCCAACAAGGAGGGCATCGTGTGGG


 


TGGCCACCGAGGGCGCCCTGAACACCCCCAAGGACCACATCGGCACCAGA


 


AACCCCAACAACAACGCCGCCACCGTGCTGCAGCTGCCCCAGGGCACCAC


 


CCTGCCCAAGGGCTTCTACGCCGAGGGCAGCAGAGGCGGCAGCCAGGCCA


 


GCAGCAGAAGCAGCAGCAGAAGCAGAGGCAACAGCAGAAACAGCACCCCC


 


GGCAGCAGCAGAGGCAACAGCCCCGCCAGAATGGCCAGCGGCGGCGGCGA


 


GACCGCCCTGGCCCTGCTGCTGCTGGACAGACTGAACCAGCTGGAGAGCA


 


AGGTGAGCGGCAAGGGCCAGCAGCAGCAGGGCCAGACCGTGACCAAGAAG


 


AGCGCCGCCGAGGCCAGCAAGAAGCCCAGACAGAAGAGAACCGCCACCAA


 


GCAGTACAACGTGACCCAGGCCTTCGGCAGAAGAGGCCCCGAGCAGACCC


 


AGGGCAACTTCGGCGACCAGGACCTGATCAGACAGGGCACCGACTACAAG


 


CACTGGCCCCAGATCGCCCAGTTCGCCCCCAGCGCCAGCGCCTTCTTCGG


 


CATGAGCAGAATCGGCATGGAGGTGACCCCCAGCGGCACCTGGCTGACCT


 


ACCACGGCGCCATCAAGCTGGACGACAAGGACCCCCAGTTCAAGGACAAC


 


GTGATCCTGCTGAACAAGCACATCGACGCCTACAAGACCTTCCCCCCCAC


 


CGAGCCCAAGAAGGACAAGAAGAAGAAGACCGACGAGGCCCAGCCCCTGC


 


CCCAGAGACAGAAGAAGCAGCCCACCGTGACCCTGCTGCCCGCCGCCGAC


 


ATGGACGACTTCAGCAGACAGCTGCAGAACAGCATGAGCGGCGCCAGCGC


 


CGACAGCACCCAGGCC






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:14 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:14 as follows: about 4 of the 13 phenylalanine codons are TTT, and about 9 of the phenylalanine codons are TTC; about 1 of the 26 leucine codons are TTA, about 6 of the leucine codons are TTG, about 7 of the leucine codons are CTT, about 3 of the leucine codons are CTC, about 5 of the leucine codons are CTA, and about 4 of the leucine codons are CTG; about 7 of the 11 isoleucine codons are ATT, about 3 of the isoleucine codons are ATC, and about 1 of the isoleucine codons are ATA; the 7 methionine codons are ATG; about 4 of the 11 valine codons are GTT, about 4 of the valine codons are GTC, about 1 of the valine codons is GTA, and about 2 of the valine codons are GTG; about 10 of the 35 serine codons are TCT, about 3 of the serine codons are TCC, about 9 of the serine codons are TCA, about 1 of the serine codons is TCG, about 7 of the serine codons are AGT, and about 5 of the serine codons are AGC; about 10 of the 31 proline codons are CCT, about 9 of the proline codons are CCC, about 10 of the proline codons are CCA, and about 2 of the proline codons are CCG; about 17 of the 33 threonine codons are ACT, about 5 of the threonine codons are ACC, about 11 of the threonine codons are ACA, and about 0 of the threonine codons is ACG; about 14 of the 34 alanine codons are GCT, about 8 of the alanine codons are GCC, about 9 of the alanine codons are GCA, and about 3 of the alanine codons are GCG; about 2 of the 11 tyrosine codons are TAT and about 9 of the tyrosine codons are TAC; about 3 of the 5 histidine codons are CAT and about 2 of the histidine codons are CAC; about 24 of the 34 glutamine codons are CAA and about 10 of the glutamine codons are CAG; about 16 of the 25 asparagine codons are AAT and about 9 of the asparagine codons are AAC; about 20 of the 29 lysine codons are AAA and about 9 of the lysine codons are AAG; about 10 of the 22 aspartic acid codons are GAT and about 12 of the aspartic acid codons are GAC; about 7 of the 14 glutamic acid codons are GAA and about 7 of the glutamic acid codons are GAG; the 5 tryptophan codons are TGG; about 5 of the 31 arginine codons are CGT, about 8 of the arginine codons are CGC, about 6 of the arginine codons are CGA, about 0 of the arginine codons are CGG, about 10 of the arginine codons are AGA, and about 2 of the arginine codons are AGG; and about 10 of the 45 glycine codons are GGT, about 16 of the glycine codons are GGC, about 16 of the glycine codons are GGA, and about 3 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:14, optimized according to codon usage in humans is presented herein as SEQ ID NO:36.








ATG TCC GAT AAT GGT CCC CAG TCT AAC CAG AGG TCG



 


GCG CCA AGA ATC ACA TTC GGG GGC CCA ACA GAC AGT


 


ACC GAT AAC AAC CAG AAC GGC GGA AGA AAC GGG GCC


 


AGG CCC AAG CAG CGG AGA CCT CAG GGA TTA CCA AAT


 


AAT ACC GCA AGC TGG TTC ACA GCC CTG ACC CAG CAT


 


GGA AAA GAG GAA CTG AGA TTC CCT AGA GGA CAA GGG


 


GTG CCT ATT AAT ACT AAT AGC GGG CCT GAC GAT CAA


 


ATT GGC TAT TAT CGA CGT GCG ACT CGC CGT GTT AGA


 


GGG GGG GAC GGG AAG ATG AAG GAG CTT AGC CCA CGC


 


TGG TAC TTT TAC TAT CTG GGA ACC GGA CCT GAA GCT


 


AGT CTG CCC TAC GGC GCT AAC AAG GAG GGA ATA GTA


 


TGG GTC GCC ACG GAA GGT GCG TTG AAT ACT CCG AAA


 


GAT CAC ATC GGC ACC AGA AAT CCT AAC AAT AAC GCC


 


GCA ACC GTG CTA CAA TTA CCC CAG GGA ACT ACT CTG


 


CCG AAG GGG TTC TAT GCG GAG GGA AGC CGC GGC GGC


 


TCA CAA GCC AGT TCA CGC TCC AGC TCC CGG TCG AGG


 


GGT AAT TCC CGA AAC AGC ACC CCG GGA TCA TCT AGG


 


GGA AAC TCT CCC GCC CGG ATG GCC TCA GGC GGC GGC


 


GAA ACA GCT CTG GCT CTG CTA TTG CTG GAC CGG CTC


 


AAC CAG CTC GAG TCC AAA GTC TCT GGT AAA GGT CAG


 


CAG CAG CAG GGT CAA ACA GTG ACC AAA AAA AGT GCA


 


GCC GAG GCC AGC AAG AAA CCA CGC CAG AAA CGT ACG


 


GCC ACA AAG CAA TAC AAT GTG ACC CAA GCC TTT GGA


 


AGG CGG GGG CCC GAA CAG ACA CAG GGC AAT TTC GGC


 


GAT CAA GAT TTG ATA CGA CAG GGC ACT GAC TAC AAA


 


CAC TGG CCG CAG ATC GCT CAG TTT GCA CCT AGC GCC


 


TCC GCT TTC TTT GGC ATG AGT CGG ATT GGC ATG GAG


 


GTG ACA CCA TCA GGT ACT TGG TTA ACG TAC CAC GGG


 


GCA ATC AAA CTT GAT GAT AAA GAT CCC CAG TTT AAG


 


GAC AAC GTT ATC CTC CTG AAT AAG CAT ATT GAC GCC


 


TAT AAG ACC TTC CCC CCA ACC GAA CCA AAG AAG GAC


 


AAG AAG AAG AAG ACA GAC GAG GCA CAG CCT CTC CCC


 


CAG AGG CAG AAA AAG CAG CCT ACT GTC ACC CTT CTG


 


CCC GCT GCA GAC ATG GAT GAC TTT TCC CGC CAA CTC


 


CAG AAC TCT ATG AGT GGG GCT TCC GCT GAC TCT ACG


 


CAG GCC TGA






Another representative codon-optimized coding region encoding SEQ ID NO:14 is presented herein as SEQ ID NO:63. SEQ ID NO:14 is encoded by nucleotides 7 to 1275 of SEQ ID NO:63.








GTCGACATGAGCGACAACGGCCCCCAGAGCAACCAGAGAAGCGCCCCCAG



 


AATCACCTTTGGCGGCCCTACCGACAGCACCGACAACAACCAGAACGGCG


 


GCAGAAACGGCGCCAGACCCAAGCAGAGGAGACCCCAGGGCCTGCCCAAC


 


AACACCGCCAGCTGGTTCACCGCCCTCACCCAGCACGGCAAGGAGGAGCT


 


GAGATTCCCCAGAGGCCAGGGCGTGCCCATCAATACCAACAGCGGCCCAG


 


ACGATCAGATCGGCTACTACCGGAGGGCCACCAGAAGAGTGAGAGGCGGC


 


GACGGCAAGATGAAGGAGCTGAGCCCCCGGTGGTACTTCTACTACCTGGG


 


CACCGGCCCTGAGGCCAGCCTGCCCTACGGCGCCAACAAGGAGGGCATCG


 


TGTGGGTGGCCACCGAGGGCGCCCTGAATACCCCCAAGGACCACATCGGC


 


ACCAGGAACCCCAACAACAATGCCGCCACCGTGCTGCAGCTGCCCCAGGG


 


CACCACCCTGCCCAAGGGCTTCTACGCCGAGGGCAGCAGAGGCGGCAGCC


 


AGGCCAGCAGCAGAAGCAGCAGCAGGAGCAGGGGCAACAGCAGAAATAGC


 


ACCCCCGGCAGCAGCAGAGGAAATTCACCCGCCAGAATGGCCAGCGGCGG


 


AGGCGAGACCGCCCTGGCCCTGCTGCTCCTGGACAGGCTGAATCAGCTGG


 


AGAGCAAGGTGAGCGGCAAGGGCCAGCAACAGCAGGGACAGACCGTGACC


 


AAGAAGTCTGCCGCCGAGGCCAGCAAGAAGCCCAGGCAGAAGAGAACCGC


 


CACCAAGCAGTACAATGTGACCCAGGCCTTCGGCAGAAGAGGCCCCGAGC


 


AGACCCAGGGCAATTTCGGCGACCAGGACCTCATCAGACAGGGCACCGAC


 


TACAAGCACTGGCCTCAGATCGCCCAGTTCGCCCCCAGCGCCAGCGCCTT


 


CTTCGGCATGAGCCGGATCGGCATGGAGGTGACCCCCAGCGGCACCTGGC


 


TCACCTACCACGGCGCCATCAAGCTGGACGACAAGGACCCCCAGTTCAAG


 


GACAACGTGATCCTGCTGAACAAGCACATCGACGCCTACAAGACCTTCCC


 


ACCCACCGAGCCCAAGAAGGACAAGAAGAAGAAAACCGACGAGGCCCAGC


 


CCCTGCCCCAGAGACAGAAGAAGCAGCCCACCGTGACCCTGCTGCCTGCC


 


GCCGACATGGACGACTTCAGCCGCCAGCTGCAGAATAGCATGAGCGGCGC


 


CTCTGCCGATTCAACCCAGGCCTGAAGATCT






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:16 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:16 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:16, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:16 is shown in Table 16.










TABLE 16








Number in



AMINO ACID
SEQ ID NO: 16




















A
Ala
33



R
Arg
31



C
Cys
0



G
Gly
45



H
His
5



I
Ile
11



L
Leu
26



K
Lys
22



M
Met
7



F
Phe
12



P
Pro
28



S
Ser
35



T
Thr
30



W
Trp
5



Y
Tyr
11



V
Val
11



N
Asn
25



D
Asp
20



Q
Gln
33



E
Glu
12










Using the amino acid composition shown in Table 16, a human codon-optimized coding region which encodes SEQ ID NO:16 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:16 as follows: the 12 phenylalanine codons are TTC, the 26 leucine codons are CTG, the 11 isoleucine codons are ATC, the 7 methionine codons are ATG, the 11 valine codons are GTG, the 35 serine codons are AGC, the 28 proline codons are CCC, the 30 threonine codons are ACC, the 33 alanine codons are GCC, the 11 tyrosine codons are TAC, the 5 histidine codons are CAC, the 33 glutamine codons are CAG, the 25 asparagine codons are AAC, the 22 lysine codons are AAG, the 20 aspartic acid codons are GAC, the 12 glutamic acid codons are GAG, the 5 tryptophan codons are TGG, the 31 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 45 glycine codons are GGC. The codon-optimized N (minus NLS) coding region designed by this method is presented herein as SEQ ID NO:39.








ATGAGCGACAACGGCCCCCAGAGCAACCAGAGAAGCGCCCCCAGAATCAC



 


CTTCGGCGGCCCCACCGACAGCACCGACAACAACCAGAACGGCGGCAGAA


 


ACGGCGCCAGACCCAAGCAGAGAAGACCCCAGGGCCTGCCCAACAACACC


 


GCCAGCTGGTTCACCGCCCTGACCCAGCACGGCAAGGAGGAGCTGAGATT


 


CCCCAGAGGCCAGGGCGTGCCCATCAACACCAACAGCGGCCCCGACGACC


 


AGATCGGCTACTACAGAAGAGCCACCAGAAGAGTGAGAGGCGGCGACGGC


 


AAGATGAAGGAGCTGAGCCCCAGATGGTACTTCTACTACCTGGGCACCGG


 


CCCCGAGGCCAGCCTGCCCTACGGCGCCAACAAGGAGGGCATCGTGTGGG


 


TGGCCACCGAGGGCGCCCTGAACACCCCCAAGGACCACATCGGCACCAGA


 


AACCCCAACAACAACGCCGCCACCGTGCTGCAGCTGCCCCAGGGCACCAC


 


CCTGCCCAAGGGCTTCTACGCCGAGGGCAGCAGAGGCGGCAGCCAGGCCA


 


GCAGCAGAAGCAGCAGCAGAAGCAGAGGCAACAGCAGAAACAGCACCCCC


 


GGCAGCAGCAGAGGCAACAGCCCCGCCAGAATGGCCAGCGGCGGCGGCGA


 


GACCGCCCTGGCCCTGCTGCTGCTGGACAGACTGAACCAGCTGGAGAGCA


 


AGGTGAGCGGCAAGGGCCAGCAGCAGCAGGGCCAGACCGTGACCAAGAAG


 


AGCGCCGCCGAGGCCAGCAAGAAGCCCAGACAGAAGAGAACCGCCACCAA


 


GCAGTACAACGTGACCCAGGCCTTCGGCAGAAGAGGCCCCGAGCAGACCC


 


AGGGCAACTTCGGCGACCAGGACCTGATCAGACAGGGCACCGACTACAAG


 


CACTGGCCCCAGATCGCCCAGTTCGCCCCCAGCGCCAGCGCCTTCTTCGG


 


CATGAGCAGAATCGGCATGGAGGTGACCCCCAGCGGCACCTGGCTGACCT


 


ACCACGGCGCCATCAAGCTGGACGACAAGGACCCCCAGTTCAAGGACAAC


 


GTGATCCTGCTGAACAAGCACATCGACGCCTACCCCCTGCCCCAGAGACA


 


GAAGAAGCAGCCCACCGTGACCCTGCTGCCCGCCGCCGACATGGACGACT


 


TCAGCAGACAGCTGCAGAACAGCATGAGCGGCGCCAGCGCCGACAGCACC


 


CAGGCC






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:16 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:16 as follows: about 5 of the 12 phenylalanine codons are TTT, and about 7 of the phenylalanine codons are TTC; about 3 of the 26 leucine codons are TTA, about 3 of the leucine codons are TTG, about 3 of the leucine codons are CTT, about 5 of the leucine codons are CTC, about 2 of the leucine codons are CTA, and about 10 of the leucine codons are CTG; about 4 of the 11 isoleucine codons are ATT, about 5 of the isoleucine codons are ATC, and about 2 of the isoleucine codons are ATA; the 7 methionine codons are ATG; about 2 of the 11 valine codons are GTT, about 3 of the valine codons are GTC, about 1 of the valine codons is GTA, and about 5 of the valine codons are GTG; about 6 of the 35 serine codons are TCT, about 8 of the serine codons are TCC, about 5 of the serine codons are TCA, about 2 of the serine codons are TCG, about 6 of the serine codons are AGT, and about 8 of the serine codons are AGC; about 8 of the 28 proline codons are CCT, about 9 of the proline codons are CCC, about 8 of the proline codons are CCA, and about 3 of the proline codons are CCG; about 7 of the 30 threonine codons are ACT, about 11 of the threonine codons are ACC, about 9 of the threonine codons are ACA, and about 3 of the threonine codons are ACG; about 9 of the 33 alanine codons are GCT, about 13 of the alanine codons are GCC, about 7 of the alanine codons are GCA, and about 4 of the alanine codons are GCG; about 5 of the 11 tyrosine codons are TAT and about 6 of the tyrosine codons are TAC; about 2 of the 5 histidine codons are CAT and about 3 of the histidine codons are CAC; about 9 of the 33 glutamine codons are CAA and about 24 of the glutarnine codons are CAG; about 12 of the 25 asparagine codons are AAT and about 13 of the asparagine codons are AAC; about 9 of the 22 lysine codons are AAA and about 13 of the lysine codons are AAG; about 9 of the 20 aspartic acid codons are GAT and about 11 of the aspartic acid codons are GAC; about 5 of the 12 glutamic acid codons are GAA and about 7 of the glutamic acid codons are GAG; the 5 tryptophan codons are TGG; about 3 of the 31 arginine codons are CGT, about 6 of the arginine codons are CGC, about 3 of the arginine codons are CGA, about 6 of the arginine codons are CGG, about 7 of the arginine codons are AGA, and about 6 of the arginine codons are AGG; and about 7 of the 45 glycine codons are GGT, about 15 of the glycine codons are GGC, about 12 of the glycine codons are GGA, and about 11 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:16, optimized according to codon usage in humans is presented herein as SEQ ID NO:38.








ATG AGT GAT AAT GGC CCC CAG TCT AAC CAG AGG AGC



 


GCA CCG CGG ATC ACG TTC GGT GGC CCA ACC GAC TCA


 


ACA GAC AAT AAT CAG AAC GGA GGA CGC AAT GGT GCA


 


CGT CCT AAG CAG AGA CGC CCC CAA GGG CTG CCT AAT


 


AAT ACA GCA AGT TGG TTT ACC GCA CTC ACA CAA CAT


 


GGA AAG GAA GAG TTG CGG TTC CCC CGC GGC CAG GGC


 


GTG CCC ATC AAC ACA AAT AGC GGA CCC GAC GAT CAG


 


ATC GGA TAT TAC CGA AGA GCT ACA AGG AGA GTT CGC


 


GGC GGG GAT GGC AAG ATG AAG GAG CTA TCA CCA CGA


 


TGG TAC TTC TAT TAC CTC GGG ACA GGC CCA GAG GCC


 


TCG CTA CCA TAC GGG GCC AAC AAG GAG GGT ATT GTC


 


TGG GTC GCT ACC GAA GGG GCC CTG AAT ACA CCT AAA


 


GAC CAC ATA GGT ACC AGA AAT CCC AAC AAT AAC GCC


 


GCG ACC GTG TTA CAG CTT CCT CAG GGA ACG ACC CTT


 


CCA AAA GGG TTT TAC GCC GAA GGA TCT CGG GGA GGG


 


TCA CAG GCT AGC TCC CGT AGC TCC TCA AGG TCC AGG


 


GGG AAT TCT AGA AAC AGT ACA CCC GGC TCT AGC CGT


 


GGT AAC TCC CCA GCT CGC ATG GCA TCC GGC GGA GGG


 


GAA ACC GCT CTG GCT CTG CTC CTG TTA GAT CGG TTG


 


AAC CAA CTG GAA TCG AAG GTA TCC GGA AAG GGA CAG


 


CAG CAG CAA GGC CAG ACT GTG ACT AAG AAG TCC GCG


 


GCC GAG GCC AGT AAG AAA CCC CGC CAG AAA CGA ACT


 


GCC ACC AAA CAG TAT AAT GTG ACA CAG GCC TTC GGC


 


AGA CGG GGT CCA GAG CAG ACC CAA GGC AAC TTC GGG


 


GAT CAG GAC CTG ATT CGG CAG GGT ACC GAC TAT AAG


 


CAC TGG CCG CAA ATT GCT CAG TTT GCT CCC AGT GCG


 


AGT GCC TTC TTC GGC ATG TCT AGG ATC GGG ATG GAG


 


GTT ACT CCT AGC GGC ACT TGG CTT ACT TAT CAC GGA


 


GCC ATC AAA CTC GAT GAT AAG GAC CCA CAG TTT AAG


 


GAT AAC GTG ATT CTG CTG AAC AAA CAT ATA GAC GCG


 


TAC CCT CTC CCG CAA AGG CAG AAA AAA CAG CCT ACC


 


GTC ACG TTA CTG CCT GCC GCA GAC ATG GAC GAC TTT


 


TCT AGA CAG TTG CAA AAC AGC ATG TCA GGC GCA TCC


 


GCC GAT AGC ACT CAA GCT TGA






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:19 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:19 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:19, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:19 is shown in Table 17.










TABLE 17








Number in



AMINO ACID
SEQ ID NO: 19




















A
Ala
19



R
Arg
15



C
Cys
3



G
Gly
15



H
His
3



I
Ile
18



L
Leu
31



K
Lys
6



M
Met
7



F
Phe
11



P
Pro
6



S
Ser
11



T
Thr
13



W
Trp
7



Y
Tyr
9



V
Val
16



N
Asn
13



D
Asp
6



Q
Gln
5



E
Glu
7










Using the amino acid composition shown in Table 17, a human codon-optimized coding region which encodes SEQ ID NO:19 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:19 as follows: the 11 phenylalanine codons are TTC, the 31 leucine codons are CTG, the 18 isoleucine codons are ATC, the 7 methionine codons are ATG, the 16 valine codons are GTG, the 11 serine codons are AGC, the 6 proline codons are CCC, the 13 threonine codons are ACC, the 19 alanine codons are GCC, the 19 tyrosine codons are TAC, the 3 histidine codons are CAC, the 5 glutamine codons are CAG, the 13 asparagine codons are AAC, the 6 lysine codons are AAG, the 6 aspartic acid codons are GAC, the 7 glutamic acid codons are GAG, the 3 cysteine codons are TGC, the 7 tryptophan codons are TGG, the 15 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 43 glycine codons are GGC. The codon-optimized M coding region designed by this method is presented herein as SEQ ID NO:41.








ATGGCCGACAACGGCACCATCACCGTGGAGGAGCTGAAGCAGCTGCTGGA



 


GCAGTGGAACCTGGTGATCGGCTTCCTGTTCCTGGCCTGGATCATGCTGC


 


TGCAGTTCGCCTACAGCAACAGAAACAGATTCCTGTACATCATCAAGCTG


 


GTGTTCCTGTGGCTGCTGTGGCCCGTGACCCTGGCCTGCTTCGTGCTGGC


 


CGCCGTGTACAGAATCAACTGGGTGACCGGCGGCATCGCCATCGCCATGG


 


CCTGCATCGTGGGCCTGATGTGGCTGAGCTACTTCGTGGCCAGCTTCAGA


 


CTGTTCGCCAGAACCAGAAGCATGTGGAGCTTCAACCCCGAGACCAACAT


 


CCTGCTGAACGTGCCCCTGAGAGGCACCATCGTGACCAGACCCCTGATGG


 


AGAGCGAGCTGGTGATCGGCGCCGTGATCATCAGAGGCCACCTGAGAATG


 


GCCGGCCACCCCCTGGGCAGATGCGACATCAAGGACCTGCCCAAGGAGAT


 


CACCGTGGCCACCAGCAGAACCCTGAGCTACTACAAGCTGGGCGCCAGCC


 


AGAGAGTGGGCACCGACAGCGGCTTCGCCGCCTACAACAGATACAGAATC


 


GGCAACTACAAGCTGAACACCGACCACGCCGGCAGCAACGACAACATCGC


 


CCTGCTGGTGCAG






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:19 can be designed by the “full optimization” method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:19 as follows: about 5 of the 11 phenylalanine codons are TTT, and about 6 of the phenylalanine codons are TTC; about 3 of the 31 leucine codons are TTA, about 4 of the leucine codons are TTG, about 4 of the leucine codons are CTT, about 6 of the leucine codons are CTC, about 2 of the leucine codons are CTA, and about 12 of the leucine codons are CTG; about 6 of the 18 isoleucine codons are ATT, about 9 of the isoleucine codons are ATC, and about 3 of the isoleucine codons are ATA; the 7 methionine codons are ATG; about 3 of the 16 valine codons are GTT, about 4 of the valine codons are GTC, about 2 of the valine codons are GTA, and about 7 of the valine codons are GTG; about 2 of the 11 serine codons are TCT, about 2 of the serine codons are TCC, about 2 of the serine codons are TCA, about 1 of the serine codons is TCG, about 1 of the serine codons is AGT, and about 3 of the serine codons are AGC; about 2 of the 6 proline codons are CCT, about 2 of the proline codons are CCC, about 1 of the proline codons is CCA, and about 1 of the proline codons is CCG; about 3 of the 13 threonine codons are ACT, about 5 of the threonine codons are ACC, about 4 of the threonine codons are ACA, and about 1 of the threonine codons is ACG; about 5 of the 19 alanine codons are GCT, about 8 of the alanine codons are GCC, about 4 of the alanine codons are GCA, and about 2 of the alanine codons are GCG; about 4 of the 9 tyrosine codons are TAT and about 5 of the tyrosine codons are TAC; about 1 of the 3 histidine codons is CAT and about 2 of the histidine codons are CAC; about 1 of the 5 glutamine codons is CAA and about 4 of the glutamine codons are CAG; about 6 of the 13 asparagine codons are AAT and about 7 of the asparagine codons are AAC; about 3 of the 6 lysine codons are AAA and about 3 of the lysine codons are AAG; about 3 of the 6 aspartic acid codons are GAT and about 3 of the aspartic acid codons are GAC; about 3 of the 7 glutamic acid codons are GAA and about 4 of the glutamic acid codons are GAG; about 1 of the 3 cysteine codons is TGT and about 2 of the cysteine codons are TGC; the 7 tryptophan codons are TGG; about 1 of the 15 arginine codons is CGT, about 3 of the arginine codons are CGC, about 2 of the arginine codons are CGA, about 3 of the arginine codons are CGG, about 3 of the arginine codons are AGA, and about 3 of the arginine codons are AGG; and about 2 of the 15 glycine codons are GGT, about 5 of the glycine codons are GGC, about 4 of the glycine codons are GGA, and about 4 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative “fully optimized” codon-optimized coding region encoding SEQ ID NO:19, optimized according to codon usage in humans is presented herein as SEQ ID NO:40.








ATG GCT GAC AAC GGC ACC ATA ACC GTC GAG GAG CTT



 


AAA CAG TTA TTA GAA CAA TGG AAC TTG GTG ATA GGA


 


TTC CTC TTT CTG GCA TGG ATC ATG TTG CTT CAG TTC


 


GCC TAT TCT AAC CGC AAT AGG TTT TTG TAC ATT ATC


 


AAG CTG GTC TTC CTT TGG CTG CTC TGG CCC GTA ACA


 


CTA GCC TGT TTT GTT TTG GCG GCC GTG TAT CGG ATC


 


AAT TGG GTG ACA GGT GGC ATT GCT ATT GCG ATG GCT


 


TGC ATC GTG GGG CTG ATG TGG CTG TCG TAT TTC GTT


 


GCC TCA TTC CGG CTG TTT GCC CGA ACA AGG AGT ATG


 


TGG TCT TTT AAC CCC GAG ACC AAT ATT CTG CTC AAT


 


GTG CCT TTA CGC GGC ACT ATC GTG ACC CGG CCT CTA


 


ATG GAA TCC GAG CTG GTA ATT GGC GCA GTC ATC ATA


 


AGG GGG CAC CTC AGA ATG GCC GGG CAC CCA CTT GGG


 


AGA TGC GAC ATC AAG GAT CTG CCG AAG GAA ATT ACT


 


GTT GCA ACT TCA CGA ACG CTG AGC TAT TAC AAA CTG


 


GGA GCT AGC CAG AGA GTG GGT ACC GAC TCC GGC TTC


 


GCT GCC TAC AAC CGC TAC CGT ATC GGA AAT TAC AAA


 


CTC AAC ACA GAT CAT GCA GGA AGC AAT GAT AAC ATC


 


GCC CTC CTG GTC CAG TGA






In certain embodiments described herein, a codon-optimized coding region encoding SEQ ID NO:21 is optimized according to codon usage in humans (Homo sapiens). Alternatively, a codon-optimized coding region encoding SEQ ID NO:21 may be optimized according to codon usage in any plant, animal, or microbial species. Codon-optimized coding regions encoding SEQ ID NO:21, optimized according to codon usage in humans are designed as follows. The amino acid composition of SEQ ID NO:21 is shown in Table 18.










TABLE 18








Number in



AMINO ACID
SEQ ID NO: 21




















A
Ala
4



R
Arg
2



C
Cys
3



G
Gly
2



H
His
0



I
Ile
3



L
Leu
14



K
Lys
2



M
Met
1



F
Phe
4



P
Pro
2



S
Ser
7



T
Thr
5



W
Trp
0



Y
Tyr
4



V
Val
14



N
Asn
5



D
Asp
1



Q
Gln
0



E
Glu
3










Using the amino acid composition shown in Table 18, a human codon-optimized coding region which encodes SEQ ID NO:21 can be designed by any of the methods discussed herein. For “uniform” optimization, each amino acid is assigned the most frequent codon used in the human genome for that amino acid. According to this method, codons are assigned to the coding region encoding SEQ ID NO:21 as follows: the 4 phenylalanine codons are TTC, the 14 leucine codons are CTG, the 18 isoleucine codons are 3, the 1 methionine codon is ATG, the 14 valine codons are GTG, the 7 serine codons are AGC, the 2 proline codons are CCC, the 5 threonine codons are ACC, the 4 alanine codons are GCC, the 4 tyrosine codons are TAC, the 5 asparagine codons are AAC, the 2 lysine codons are AAG, the 1 aspartic acid codon is GAC, the 3 glutamic acid codons are GAG, the 3 cysteine codons are TGC, the 1 tryptophan codon is TGG, the 2 arginine codons are CGG, AGA, or AGG (the frequencies of usage of these three codons in the human genome are not significantly different), and the 2 glycine codons are GGC. The codon-optimized E coding region designed by this method is presented herein as SEQ ID NO:43.








ATG TAC AGC TTC GTG AGC GAG GAG ACC GGC ACC CTG



 


ATC GTG AAC AGC GTG CTG CTG TTC CTG GCC TTC GTG


 


GTG TTC CTG CTG GTG ACC CTG GCC ATC CTG ACC GCC


 


CTG CGG CTG TGC GCC TAC TGC TGC AAC ATC GTG AAC


 


GTG AGC CTG GTG AAG CCC ACC GTG TAC GTG TAC AGC


 


CGG GTG AAG AAC CTG AAC AGC AGC GAG GGC GTG CCC


 


GAC CTG CTG GTG TGA






Alternatively, a human codon-optimized coding region which encodes SEQ ID NO:21 can be designed by an optimization method, where each amino acid is assigned codons based on the frequency of usage in the human genome. These frequencies are shown in Table 4 above. Using this latter method, codons are assigned to the coding region encoding SEQ ID NO:21 as follows: about 1 of the 4 phenylalanine codons are TTT, and about 3 of the phenylalanine codons are TTC; about 2 of the 14 leucine codons are TTA, about 2 of the leucine codons are TTG, about 6 of the leucine codons are CTT, about 0 of the leucine codons are CTC, about 2 of the leucine codons are CTA, and about 2 of the leucine codons are CTG; about 1 of the 3 isoleucine codons are ATT, about 1 of the isoleucine codons are ATC, and about 1 of the isoleucine codons are ATA; the 1 methionine codons are ATG; about 6 of the 14 valine codons are GTT, about 3 of the valine codons are GTC, about 3 of the valine codons are GTA, and about 2 of the valine codons are GTG; about 2 of the 7 serine codons are TCT, about 0 of the serine codons are TCC, about 1 of the serine codons are TCA, about 2 of the serine codons is TCG, about 1 of the serine codons is AGT, and about 1 of the serine codons are AGC; about 1 of the 2 proline codons are CCT, about 0 of the proline codons are CCC, about 1 of the proline codons is CCA, and about 0 of the proline codons is CCG; about 1 of the 5 threonine codons are ACT, about 0 of the threonine codons are ACC, about 2 of the threonine codons are ACA, and about 2 of the threonine codons is ACG; about 1 of the 4 alanine codons are GCT, about 1 of the alanine codons are GCC, about 0 of the alanine codons are GCA, and about 2 of the alanine codons are GCG; about 0 of the 4 tyrosine codons are TAT and about 4 of the tyrosine codons are TAC; about 3 of the 5 asparagine codons are AAT and about 2 of the asparagine codons are AAC; about 2 of the 2 lysine codons are AAA and about 0 of the lysine codons are AAG; about 1 of the 1 aspartic acid codons are GAT and about 0 of the aspartic acid codons are GAC; about 3 of the 3 glutamic acid codons are GAA and about 0 of the glutamic acid codons are GAG; about 1 of the 3 cysteine codons is TGT and about 2 of the cysteine codons are TGC; about 1 of the 2 arginine codons is CGT, about 0 of the arginine codons are CGC, about 1 of the arginine codons are CGA, about 0 of the arginine codons are CGG, about 0 of the arginine codons are AGA, and about 0 of the arginine codons are AGG; and about 1 of the 2 glycine codons are GGT, about 0 of the glycine codons are GGC, about 1 of the glycine codons are GGA, and about 0 of the glycine codons are GGG.
As described above, the term “about” means that the number of amino acids encoded by a certain codon may be one more or one less than the number given. It would be understood by those of ordinary skill in the art that the total number of any amino acid in the polypeptide sequence must remain constant, therefore, if there is one “more” of one codon encoding a give amino acid, there would have to be one “less” of another codon encoding that same amino acid.
A representative fully codon-optimized coding region encoding SEQ ID NO:21, optimized according to codon usage in humans is presented herein as SEQ ID NO:42.








ATG TAC AGC TTT GTG TCT GAA GAA ACA GGA ACG TTG



 


ATA GTT AAT AGT GTT TTG CTT TTC TTA GCG TTC GTA


 


GTC TTC CTT CTT GTC ACA CTT GCC ATT TTA ACT GCG


 


CTT CGT CTA TGC GCT TAC TGT TGC AAT ATC GTA AAC


 


GTG TCG CTT GTT AAA CCA ACG GTT TAC GTA TAC TCG


 


CGA GTT AAA AAC CTG AAT TCT TCA GAA GGT GTT CCT


 


GAT CTG CTA GTC TAA






Another representative codon-optimized coding region encoding SEQ ID NO:21 is presented herein as SEQ ID NO:48.








ATG TAT AGT TTT GTG AGT GAG GAG ACG GGC ACC CTG



 


ATT GTC AAC TCA GTG CTG CTG TTC CTG GCC TTT GTT


 


GTC TTC CTG CTG GTA ACT CTG GCC ATC CTG ACT GCC


 


CTG AGA CTG TGC GCC TAC TGC TGC AAC ATC GTG AAC


 


GTC TCT CTG GTA AAG CCC ACA GTT TAC GTG TAT TCT


 


AGG GTG AAG AAC CTG AAC TCC AGC GAG GGC GTT CCC


 


GAT CTG CTG GTA TGA






Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence using the “uniform optimization,” “full optimization,” “minimal optimization,” or other optimization methods, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the “EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG—Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences. For example, the “backtranslation” function found on the Entelechon website at www.entelechon.com/eng/backtranslation.html (visited Jul. 9, 2002), and the “backtranseq” function available on the Institute of Pasteur website at bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Oct. 15, 2002). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.
A number of options are available for synthesizing codon-optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.
The codon-optimized coding regions can be versions encoding any gene products from any strain, derivative, or variant of SARS-CoV, or fragments, variants, or derivatives of such gene products. For example, nucleic acid fragments of codon-optimized coding regions encoding the S, N, E or M polypeptides, or fragments, variants or derivatives thereof. Codon-optimized coding regions encoding other SARS-CoV polypeptides or fragments, variants, or derivatives thereof (e.g., those encoding certain predicted open reading frames in the SARS-CoV genome), are included within the present invention. Additional, non-codon-optimized polynucleotides encoding SARS-CoV polypeptides or other polypeptides may be included as well.
Compositions and Methods
In certain embodiments, the present invention is directed to compositions and methods of raising a detectable immune in a vertebrate by administering in vivo, into a tissue of a vertebrate, one or more polynucleotides comprising at least one wild-type coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof, and/or at least one codon-optimized coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof. In addition, the present invention is directed to compositions and methods of raising a detectable immune response in a vertebrate by administering to the vertebrate a composition comprising one or more polynucleotides as described herein, and at least one isolated SARS-CoV component, or isolated polypeptide. The SARS-CoV component may be inactivated virus, attenuated virus, a viral vector expressing an isolated SARS-CoV polypeptide, or a SARS-CoV virus protein, fragment, variant or derivative thereof.
The polynucleotides comprising at least one coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof, and/or at least one codon-optimized coding region encoding a SARS-CoV polypeptide may be administered either prior to, at the same time (simultaneously), or subsequent to the administration of the SARS-CoV component, or isolated polypeptide.
The SARS-CoV component, or isolated polypeptide in combination with polynucleotides comprising at least one coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof, and/or at least one codon-optimized coding region encoding a SARS-CoV polypeptide compositions may be referred to as “combinatorial polynucleotide vaccine compositions” or “single formulation heterologous prime-boost vaccine compositions.”
The isolated SARS-CoV polypeptides of the invention may be in any form, and are generated using techniques well known in the art. Examples include isolated SARS-CoV proteins produced recombinantly, isolated SARS-CoV proteins directly purified from their natural milieu, recombinant (non-SARS-COV) virus vectors expressing an isolated SARS-CoV protein, or proteins delivered in the form of an inactivated SARS-CoV vaccine, such as conventional vaccines.
When utilized, an isolated SARS-CoV component, or polypeptide or fragment, variant or derivative thereof is administered in an immunologically effective amount. Canine coronavirus, known to infect swine, turkeys, mice, calves, dogs, cats, rodents, avians and humans, may be administered as a live viral vector vaccine at a dose rate per dog of 105-108 pfu, or as a typical subunit vaccine at 10 ug-1 mg of polypeptide, according to U.S. Pat. No. 5,661,006, incorporated by reference herein in its entirety. Similarly, Bovine coronavirus is administered to animals in an antigen vaccine composition at dose of about 1 to about 100 micrograms of subunit antigen, according to U.S. Pat. No. 5,369,026, incorporated by reference herein in its entirety. The effective amount of SARS-CoV component or isolated polypeptide, and polynucleotides as described herein are determinable by one of ordinary skill in the art based upon several factors, including the antigen being expressed, the age and weight of the subject, and the precise condition requiring treatment and its severity, and route of administration.
In the instant invention, the combination of conventional antigen vaccine compositions with the polynucleotides comprising at least one coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof, and/or at least one codon-optimized coding region encoding a SARS-CoV polypeptide compositions provides for therapeutically beneficial effects at dose sparing concentrations. For example, immunological responses sufficient for a therapeutically beneficial effect in patients predetermined for an approved commercial product, such as for the typical animal coronavirus products described above, may be attained by using less of the product when supplemented or enhanced with the appropriate amount of polynucleotides comprising at least one coding region encoding a SARS-CoV or codon-optimized nucleic acid. Thus, dose sparing is contemplated by administration of conventional coronavirus vaccines administered in combination with the nucleic acids of the invention.
In particular, the dose of an antigen SARS-CoV vaccine may be reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% when administered in combination with the nucleic acid compositions of the invention.
Similarly, a desirable level of an immunological response afforded by a DNA-based pharmaceutical alone may be attained with less DNA by including an aliquot of antigen SARS-CoV vaccine. Further, using a combination of conventional and DNA-based pharmaceuticals may allow both materials to be used in lesser amounts, while still affording the desired level of immune response arising from administration of either component alone in higher amounts (e.g., one may use less of either immunological product when they are used in combination). This may be manifest not only by using lower amounts of materials being delivered at any time, but also to leads to reducing the number of administrations in a vaccination regime (e.g., 2 versus 3 or 4 injections), and/or to reducing the kinetics of the immunological response (e.g., desired response levels are attained in 3 weeks instead of 6 weeks after immunization).
In particular, the dose of DNA-based pharmaceuticals, may be reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% when administered in combination with antigen SARS-CoV vaccines.
Determining the precise amounts of DNA based pharmaceutical and SARS-CoV antigen is based on a number of factors as described above, and is readily determined by one of ordinary skill in the art.
In addition to dose sparing, the claimed combinatorial compositions provide for a broadening of the immune response and/or enhanced beneficial immune responses. Such broadened or enhanced immune responses are achieved by: adding DNA to enhance cellular responses to a conventional vaccine; adding a conventional vaccine to a DNA pharmaceutical to enhance humoral response; using a combination that induces additional epitopes (both humoral and/or cellular) to be recognized and/or responded to in a more desirable way (epitope broadening); employing a DNA-conventional vaccine combination designed for a particular desired spectrum of immunological responses; and/or obtaining a desirable spectrum by using higher amounts of either component. The broadened immune response is measurable by one of ordinary skill in the art by standard immunological assays specific for the desirable response spectrum.
Both broadening and dose sparing may be obtained simultaneously.
In addition, the present invention is directed to compositions and methods of raising a detectable immune response in a vertebrate by administering to the vertebrate a composition comprising one or more SARS-CoV polynucleotides as described herein. The compositions of the invention may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 polynucleotides, as described herein, encoding different SARS-CoV polypeptides or fragments, variants or derivatives thereof in the same composition.
The coding regions encoding SARS-CoV polypeptides or fragments, variants, or derivatives thereof may be codon optimized for a particular vertebrate. Codon optimization is carried out by the methods described herein; for example, in certain embodiments codon-optimized coding regions encoding polypeptides of SARS-CoV, or nucleic acid fragments of such coding regions encoding fragments, variants, or derivatives thereof are optimized according to the codon usage of the particular vertebrate. The polynucleotides of the invention are incorporated into the cells of the vertebrate in vivo, and an immunologically effective amount of a SARS-CoV polypeptide or a fragment, variant, or derivative thereof is produced in vivo. The coding regions encoding a SARS-CoV polypeptide or a fragment, variant, or derivative thereof may be codon optimized for mammals, e.g., humans, apes, monkeys (e.g., owl, squirrel, cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), orangutans, baboons, gibbons, and chimpanzees, dogs, wolves, cats, lions, and tigers, horses, donkeys, zebras, cows, pigs, sheep, deer, giraffes, bears, rabbits, mice, ferrets, seals, whales; birds, e.g., ducks, geese, terns, shearwaters, gulls, turkeys, chickens, quail, pheasants, geese, starlings and budgerigars; or other vertebrates.
In particular, the present invention relates to codon-optimized coding regions encoding polypeptides of SARS-CoV, or fragments, variants, or derivatives thereof, or nucleic acid fragments of such coding regions or fragments, variants, or derivatives thereof, which have been optimized according to human codon usage. For example, human codon-optimized coding regions encoding polypeptides of SARS-CoV, or fragments, variants, or derivatives thereof are prepared by substituting one or more codons preferred for use in human genes for the codons naturally used in the DNA sequence encoding the SARS-CoV polypeptide or a fragment, variant, or derivative thereof. Also provided are polynucleotides, vectors, and other expression constructs comprising wild-type coding regions or codon-optimized coding regions encoding polypeptides of SARS-CoV, or nucleic acid fragments of such wild-type coding regions or codon-optimized coding regions including variants, or derivatives thereof. Also provided are pharmaceutical compositions comprising polynucleotides, vectors, and other expression constructs comprising wild-type coding regions or codon-optimized coding regions encoding polypeptides of SARS-CoV, or nucleic acid fragments of such coding regions encoding variants, or derivatives thereof; and various methods of using such polynucleotides, vectors and other expression constructs. Coding regions encoding SARS-CoV polypeptides may be uniformly optimized, fully optimized, or minimally optimized, or otherwise optimized, as described herein.
The present invention is further directed towards polynucleotides comprising coding regions or codon-optimized coding regions encoding polypeptides of SARS-CoV antigens, for example, (predicted ORF's), optionally in conjunction with other antigens. The invention is also directed to polynucleotides comprising nucleic acid fragments or codon-optimized nucleic acid fragments encoding fragments, variants and derivatives of these polypeptides.
In certain embodiments, the present invention provides an isolated polynucleotide comprising a nucleic acid fragment, where the nucleic acid fragment is a fragment of a coding region or a codon optimized coding region encoding a polypeptide at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a SARS-CoV polypeptide, e.g., S, N, E or M, and where the nucleic acid fragment is a variant of a coding region or a codon optimized coding region encoding an SARS-CoV polypeptide, e.g., S, N, E or M. The human codon-optimized coding region can be optimized for any vertebrate species and by any of the methods described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining, Penalty=30 Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
Isolated SARS-CoV Polypeptides
The present invention is further drawn to compositions which include at least one polynucleotide comprising one or more nucleic acid fragments, where each nucleic acid fragment is a fragment of a coding region or a codon-optimized coding region operably encoding an SARS-CoV polypeptide or fragment, variant, or derivative thereof; together with and one or more isolated SARS-CoV, components, polypeptides or fragments, variants or derivatives thereof, i.e., “combinatorial polynucleotide vaccine compositions” or “single formulation heterologous prime-boost vaccine compositions.” The isolated SARS-CoV polypeptides of the invention may be in any form, and are generated using techniques well known in the art. Examples include isolated SARS-CoV proteins produced recombinantly, isolated SARS-CoV proteins directly purified from their natural milieu, and recombinant (non-SARS-CoV) virus vectors expressing an isolated SARS-CoV protein.
Similarly, the isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof to be delivered (either a recombinant protein, a purified subunit, or viral vector expressing an isolated SARS-CoV polypeptide) may be any isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof, including but not limited to the S, S1, S2, N, E or M proteins or fragments, variants or derivatives thereof. Fragments include, but are not limited to the soluble portion of the S protein and the S1 and S2 domains of the S protein. In certain embodiments, a derivative protein may be a fusion protein. It should be noted that any isolated SARS-CoV polypeptide or fragment, variant, or derivative thereof described herein may be combined in a composition with any polynucleotide comprising a nucleic acid fragment, where the nucleic acid fragment is a fragment of a coding region or a codon-optimized coding region operably encoding a SARS-CoV polypeptide or fragment, variant, or derivative thereof. The proteins may be different, the same, or may be combined in any combination of one or more isolated SARS-CoV proteins and one or more polynucleotides.
In certain embodiments, the isolated SARS-CoV polypeptides, or fragments, derivatives or variants thereof may be fused to or conjugated to a second isolated SARS-CoV polypeptide, or fragment, derivative or variant thereof, or may be fused to other heterologous proteins, including for example, hepatitis B proteins including, but not limited to the hepatitis B core antigen (HBcAg), or those derived from diphtheria or tetanus. The second isolated SARS-CoV polypeptide or other heterologous protein may act as a “carrier” that potentiates the immunogenicity of the SARS-CoV polypeptide or a fragment, variant, or derivative thereof to which it is attached. Hepatitis B virus proteins and fragments and variants thereof useful as carriers within the scope of the invention are disclosed in U.S. Pat. Nos. 6,231,864 and 5,143,726, incorporated by reference in their entireties. Polynucleotides comprising coding regions encoding said fused or conjugated proteins are also within the scope of the invention.
Methods and Administration
The present invention also provides methods for delivering a SARS-CoV polypeptide or a fragment, variant, or derivative thereof to a human, which comprise administering to a human one or more of the polynucleotide compositions described herein such that upon administration of polynucleotide compositions such as those described herein, a SARS-CoV polypeptide or a fragment, variant, or derivative thereof is expressed in human cells, in an amount sufficient to generate an immune response to SARS-CoV; or administering the SARS-CoV polypeptide or a fragment, variant, or derivative thereof itself to the human in an amount sufficient to generate an immune response.
The present invention further provides methods for delivering a SARS-CoV polypeptide or a fragment, variant, or derivative thereof to a human, which comprise administering to a vertebrate one or more of the compositions described herein; such that upon administration of compositions such as those described herein, an immune response is generated in the vertebrate.
The term “vertebrate” is intended to encompass a singular “vertebrate” as well as plural “vertebrates” and comprises mammals and birds, as well as fish, reptiles, and amphibians.
The term “mammal” is intended to encompass a singular “mammal” and plural “mammals,” and includes, but is not limited to humans; primates such as apes, monkeys (e.g., owl, squirrel, cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), orangutans, baboons, gibbons, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equines such as horses, donkeys, and zebras, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; ursids such as bears; and others such as rabbits, mice, ferrets, seals, whales. In particular, the mammal can be a human subject, a food animal or a companion animal.
The term “bird” is intended to encompass a singular “bird” and plural “birds,” and includes, but is not limited to feral water birds such as ducks, geese, terns, shearwaters, and gulls; as well as domestic avian species such as turkeys, chickens, quail, pheasants, geese, and ducks. The term “bird” also encompasses passerine birds such as starlings and budgerigars.
The present invention further provides a method for generating, enhancing or modulating an immune response to SARS-CoV comprising administering to a vertebrate one or more of the compositions described herein. In this method, the compositions may include one or more isolated polynucleotides comprising at least one nucleic acid fragment where the nucleic acid fragment is a fragment of a coding region or a codon-optimized coding region encoding an SARS-CoV polypeptide, or a fragment, variant, or derivative thereof. In another embodiment, the compositions may include multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) polynucleotides as described herein, such polynucleotides encoding different SARS CoV polypeptides in the same composition.
In another embodiment, the compositions may include both a polynucleotide as described above; and also an isolated SARS-CoV polypeptide, or a fragment, variant, or derivative thereof, wherein the protein is provided as a recombinant protein, in particular, a fusion protein, a purified subunit, viral vector expressing the protein, or inactivated virus. Thus, the latter compositions include both a polynucleotide encoding a SARS-CoV polypeptide or a fragment, variant, or derivative thereof and an isolated SARS-CoV polypeptide or a fragment, variant, or derivative thereof. The SARS-CoV polypeptide or a fragment, variant, or derivative thereof encoded by the polynucleotide of the compositions need not be the same as the isolated SARS-CoV polypeptide or a fragment, variant, or derivative thereof of the compositions. Compositions to be used according to this method may be univalent, bivalent, trivalent or multivalent.
The polynucleotides of the compositions may comprise a fragment of a coding region or a human (or other vertebrate) codon-optimized coding region encoding a protein of SARS-CoV, or a fragment, variant, or derivative thereof. The polynucleotides are incorporated into the cells of the vertebrate in vivo, and an antigenic amount of the SARS-CoV polypeptide, or fragment, variant, or derivative thereof, is produced in vivo. Upon administration of the composition according to this method, the SARS-CoV polypeptide or a fragment, variant, or derivative thereof is expressed in the vertebrate in an amount sufficient to elicit an immune response. Such an immune response might be used, for example, to generate antibodies to the SARS-CoV for use in diagnostic assays or as laboratory reagents, or as therapeutic or preventative vaccines as described herein.
The present invention further provides a method for generating, enhancing, or modulating a protective and/or therapeutic immune response to SARS-CoV in a vertebrate, comprising administering to a vertebrate in need of therapeutic and/or preventative immunity one or more of the compositions described herein. In this method, the compositions include one or more polynucleotides comprising at least one nucleic acid fragment, where the nucleic acid fragment is a fragment of a wild-type coding region or a codon-optimized coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof. In a further embodiment, the composition used in this method includes both an isolated polynucleotide comprising at least one nucleic acid fragment, where the nucleic acid fragment is a fragment of a wild-type coding region or a codon-optimized coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof; and at least one isolated SARS-CoV polypeptide, or a fragment, variant, or derivative thereof. Thus, the latter composition includes both an isolated polynucleotide encoding a SARS-CoV polypeptide or a fragment, variant, or derivative thereof and an isolated SARS-CoV polypeptide or a fragment, variant, or derivative thereof, for example, a recombinant protein, a purified subunit, or viral vector expressing the protein. Upon administration of the composition according to this method, the SARS-CoV polypeptide or a fragment, variant, or derivative thereof is expressed in the vertebrate in a therapeutically or prophylactically effective amount.
In certain embodiments, the polynucleotide or polypeptide compositions of the present invention may be administered to a vertebrate where the vertebrate is used as an in vivo model to observe the effects of individual or multiple SARS-CoV polypeptides in vivo. This approach would not only eliminate the species specific barrier to studying SARS-CoV, but would allow for the study of the immunopathology of SARS-CoV polypeptides as well as SARS-CoV polypeptide specific effects with out using infectious SARS-CoV virus. An in vivo vertebrate model of SARS infection would be useful, for example, in developing treatments for one or more aspects of SARS infection by mimicking those aspects of infection without the potential hazards associated with handling the infectious virus
As used herein, an “immune response” refers to the ability of a vertebrate to elicit an immune reaction to a composition delivered to that vertebrate. Examples of immune responses include an antibody response or a cellular, e.g., T-cell, response. One or more compositions of the present invention may be used to prevent SARS-CoV infection in vertebrates, e.g., as a prophylactic or prevenative vaccine (also sometimes referred to in the art as a “protective” vaccine), to establish or enhance immunity to SARS-CoV in a healthy individual prior to exposure to SARS-CoV or contraction of Severe Acute Respiratory Syndrome (SARS), thus preventing the syndrome or reducing the severity of SARS symptoms. As used herein, “a detectable immune response” refers to an immunogenic response to the polynucleotides and polypeptides of the present invention, which can be measured or observed by standard protocols. These protocols include, but are not limited to, immunoblot analysis (western), fluorescence-activated cell sorting (FACS), immunoprecipitation analysis, ELISA, cytolytic T-cell response, ELISPOT, and chromium release assay. An immune response may also be “detected” through challenge of immunized animals with virulent SARS-CoV, either before or after vaccination. ELISA assays are performed as described by Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989). Cytolytic T-cell responses are measured as described in Hartikka et al. “Vaxfectin Enhances the Humoral Response to Plasmid DNA-encoded Antigens.” Vaccine 19: 1911-1923 (2001), which is hereby incorporated in its entirety by reference. Standard ELISPOT technology is used for the CD4+ and CD8+ T-cell assays as described in Example 6A. Standard chromium release assays are used to measure specific cytotoxic T lymphocyte (CTL) activity against the various SARS-CoV antigens.
As mentioned above, compositions of the present invention may be used both to prevent SARS-CoV infection, and also to therapeutically treat SARS-CoV infection. In individuals already exposed to SARS-CoV, or already suffering from SARS, the present invention is used to further stimulate the immune system of the vertebrate, thus reducing or eliminating the symptoms associated with that disease or disorder. As defined herein, “treatment ” refers to the use of one or more compositions of the present invention to prevent, cure, retard, or reduce the severity of SARS symptoms in a vertebrate, and/or result in no worsening of SARS over a specified period of time in a vertebrate which has already been exposed to SARS-CoV and is thus in need of therapy. The term “prevention” refers to the use of one or more compositions of the present invention to generate immunity in a vertebrate which has not yet been exposed to a particular strain of SARS-CoV, thereby preventing or reducing disease symptoms if the vertebrate is later exposed to the particular strain of SARS-CoV. The methods of the present invention therefore may be referred to as therapeutic vaccination or preventative or prophylactic vaccination. It is not required that any composition of the present invention provide total immunity to SARS-CoV or totally cure or eliminate all SARS symptoms. As used herein, a “vertebrate in need of therapeutic and/or preventative immunity” refers to an individual for whom it is desirable to treat, i.e., to prevent, cure, retard, or reduce the severity of SARS symptoms, and/or result in no worsening of SARS over a specified period of time. Vertebrates to treat and/or vaccinate include humans, apes, monkeys (e.g., owl, squirrel, cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), orangutans, baboons, gibbons, and chimpanzees, dogs, wolves, cats, lions, and tigers, horses, donkeys, zebras, cows, pigs, sheep, deer, giraffes, bears, rabbits, mice, ferrets, seals, whales, ducks, geese, terns, shearwaters, gulls, turkeys, chickens, quail, pheasants, geese, starlings and budgerigars.
One or more compositions of the present invention are utilized in a “prime boost” regimen. An example of a “prime boost” regimen may be found in Yang, Z. et al. J. Virol. 77:799-803 (2002). In these embodiments, one or more polynucleotide vaccine compositions of the present invention are delivered to a vertebrate, thereby priming the immune response of the vertebrate to SARS-CoV, and then a second immunogenic composition is utilized as a boost vaccination. One or more compositions of the present invention are used to prime immunity, and then a second immunogenic composition, e.g., a recombinant viral vaccine or vaccines, a different polynucleotide vaccine, or one or more purified subunit isolated SARS-CoV polypeptides or fragments, variants or derivatives thereof is used to boost the anti-SARS-CoV immune response.
In one embodiment, a priming composition and a boosting composition are delivered to a vertebrate in separate doses and vaccinations. For example, a single composition may comprise one or more polynucleotides encoding SARS-CoV protein(s), fragment(s), variant(s), or derivative(s) thereof and/or one or more isolated SARS-CoV polypeptide(s) or fragment(s), variant(s), or derivative(s) thereof as the priming component. The polynucleotides encoding the SARS-CoV polypeptides fragments, variants, or derivatives thereof may be contained in a single plasmid or viral vector or in multiple plasmids or viral vectors. At least one polynucleotide encoding a SARS-CoV protein and/or one or more SARS-CoV isolated polypeptide can serve as the boosting component. In this embodiment, the compositions of the priming component and the compositions of the boosting component may be contained in separate vials. In one example, the boosting component is administered approximately 1 to 6 months after administration of the priming component.
In one embodiment, a priming composition and a boosting composition are combined in a single composition or single formulation. For example, a single composition may comprise an isolated SARS-CoV polypeptide or a fragment, variant, or derivative thereof as the priming component and a polynucleotide encoding an SARS-CoV protein as the boosting component. In this embodiment, the compositions may be contained in a single vial where the priming component and boosting component are mixed together. In general, because the peak levels of expression of protein from the polynucleotide does not occur until later (e.g., 7-10 days) after administration, the polynucleotide component may provide a boost to the isolated protein component. Compositions comprising both a priming component and a boosting component are referred to herein as “combinatorial vaccine compositions” or “single formulation heterologous prime-boost vaccine compositions.” In addition, the priming composition may be administered before the boosting composition, or even after the boosting composition, if the boosting composition is expected to take longer to act.
In another embodiment, the priming composition may be administered simultaneously with the boosting composition, but in separate formulations where the priming component and the boosting component are separated.
The terms “priming” or “primary” and “boost” or “boosting” as used herein may refer to the initial and subsequent immunizations, respectively, i.e., in accordance with the definitions these terms normally have in immunology. However, in certain embodiments, e.g., where the priming component and boosting component are in a single formulation, initial and subsequent immunizations may not be necessary as both the “prime” and the “boost” compositions are administered simultaneously.
In certain embodiments, one or more compositions of the present invention are delivered to a vertebrate by methods described herein, thereby achieving an effective therapeutic and/or an effective preventative immune response. More specifically, the compositions of the present invention may be administered to any tissue of a vertebrate, including, but not limited to, muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue, e.g., myocardium, endocardium, and pericardium, lymph tissue, blood tissue, bone tissue, pancreas tissue, kidney tissue, gall bladder tissue, stomach tissue, intestinal tissue, testicular tissue, ovarian tissue, uterine tissue, vaginal tissue, rectal tissue, nervous system tissue, eye tissue, glandular tissue, tongue tissue, and connective tissue, e.g., cartilage.
Furthermore, the compositions of the present invention may be administered to any internal cavity of a vertebrate, including, but not limited to, the lungs, the mouth, the nasal cavity, the stomach, the peritoneal cavity, the intestine, any heart chamber, veins, arteries, capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity, the rectal cavity, joint cavities, ventricles in brain, spinal canal in spinal cord, the ocular cavities, the lumen of a duct of a salivary gland or a liver. When the compositions of the present invention are administered to the lumen of a duct of a salivary gland or liver, the desired polypeptide is expressed in the salivary gland and the liver such that the polypeptide is delivered into the blood stream of the vertebrate from each of the salivary gland or the liver. Certain modes for administration to secretory organs of a gastrointestinal system using the salivary gland, liver and pancreas to release a desired polypeptide into the bloodstream are disclosed in U.S. Pat. Nos. 5,837,693 and 6,004,944, both of which are incorporated herein by reference in their entireties.
In certain embodiments, the compositions are administered to muscle, either skeletal muscle or cardiac muscle, or to lung tissue. Specific, but non-limiting modes for administration to lung tissue are disclosed in Wheeler, C. J., et al., Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996), which is incorporated herein by reference in its entirety.
According to the disclosed methods, compositions of the present invention can be administered by intramuscular (i.m.), subcutaneous (s.c.), or intrapulmonary routes. Other suitable routes of administration include, but are not limited to intratracheal, transdermal, intraocular, intranasal, inhalation, intracavity, intravenous (i.v.), intraductal (e.g., into the pancreas) and intraparenchymal (i.e., into any tissue) administration. Transdermal delivery includes, but is not limited to intradermal (e.g., into the dermis or epidermis), transdermal (e.g., percutaneous) and transmucosal administration (i.e., into or through skin or mucosal tissue). Intracavity administration includes, but is not limited to administration into oral, vaginal, rectal, nasal, peritoneal, or intestinal cavities as well as, intrathecal (i.e., into spinal canal), intraventricular (i.e., into the brain ventricles or the heart ventricles), inraatrial (i.e., into the heart atrium) and sub arachnoid (i.e., into the sub arachnoid spaces of the brain) administration.
Any mode of administration can be used so long as the mode results in the expression of the desired peptide or protein, in the desired tissue, in an amount sufficient to generate an immune response to SARS-CoV and/or to generate a prophylactically or therapeutically effective immune response to SARS-CoV in a vertebrate in need of such response. Administration means of the present invention include needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., “gene guns” or pneumatic “needleless” injectors) Med-E-Jet (Vahlsing, H., et al., J. Immunol. Methods 171:11-22 (1994)), Pigjet (Schrijver, R., et al., Vaccine 15: 1908-1916 (1997)), Biojector (Davis, H., et al., Vaccine 12: 1503-1509 (1994); Gramzinski, R., et al., Mol. Med. 4: 109-118 (1998)), AdvantaJet (Linmayer, I., et al., Diabetes Care 9:294-297 (1986)), Medi-jector (Martins, J., and Roedl, E. J. Occup. Med. 21:821-824 (1979)), gelfoam sponge depots, other commercially available depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, use of polynucleotide coated suture (Qin, Y., et al., Life Sciences 65: 2193-2203 (1999)) or topical applications during surgery. Certain modes of administration are intramuscular needle-based injection and pulmonary application via catheter infusion. Energy-assisted plasmid delivery (EAPD) methods may also be employed to administer the compositions of the invention. One such method involves the application of brief electrical pulses to injected tissues, a procedure commonly known as electroporation. See generally Mir, L. M. et al., Proc. Natl. Acad. Sci USA 96:4262-7 (1999); Hartikka, J. et al., Mol. Ther. 4:407-15 (2001); Mathiesen, I., Gene Ther. 6:508-14(1999); Rizzuto G. et al., Hum. Gen. Ther. 11:1891-900 (2000). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
Determining an effective amount of one or more compositions of the present invention depends upon a number of factors including, for example, the antigen being expressed or administered directly, (e.g., S, N, E or M, or fragments, variants, or derivatives thereof), the age and weight of the subject, the precise condition requiring treatment and its severity, and the route of administration. Based on the above factors, determining the precise amount, number of doses, and timing of doses are within the ordinary skill in the art and will be readily determined by the attending physician or veterinarian.
Compositions of the present invention may include various salts, excipients, delivery vehicles and/or auxiliary agents as are disclosed, e.g., in U.S. Patent Application Publication 2002/0019358, published Feb. 14, 2002, which is incorporated herein by reference in its entirety.
Furthermore, compositions of the present invention may include one or more transfection facilitating compounds that facilitate delivery of polynucleotides to the interior of a cell, and/or to a desired location within a cell. As used herein, the terms “transfection facilitating compound,” “transfection facilitating agent,” and “transfection facilitating material” are synonymous, and may be used interchangeably. It should be noted that certain transfection facilitating compounds may also be “adjuvants” as described infra, i.e., in addition to facilitating delivery of polynucleotides to the interior of a cell, the compound acts to alter or increase the immune response to the antigen encoded by that polynucleotide. Examples of the transfection facilitating compounds include, but are not limited to inorganic materials such as calcium phosphate, alum (aluminum sulfate), and gold particles (e.g., “powder” type delivery vehicles); peptides that are, for example, cationic, intercell targeting (for selective delivery to certain cell types), intracell targeting (for nuclear localization or endosomal escape), and ampipathic (helix forming or pore forming); proteins that are, for example, basic (e.g., positively charged) such as histones, targeting (e.g., asialoprotein), viral (e.g., Sendai virus coat protein), and pore-forming; lipids that are, for example, cationic (e.g., DMRIE, DOSPA, DC-Chol), basic (e.g., steryl amine), neutral (e.g., cholesterol), anionic (e.g., phosphatidyl serine), and zwitterionic (e.g., DOPE, DOPC); and polymers such as dendrimers, star-polymers, “homogenous” poly-amino acids (e.g., poly-lysine, poly-arginine), “heterogeneous” poly-amino acids (e.g., mixtures of lysine & glycine), co-polymers, polyvinylpyrrolidinone (PVP), poloxamers (e.g., CRL 1005) and polyethylene glycol (PEG). A transfection facilitating material can be used alone or in combination with one or more other transfection facilitating materials. Two or more transfection facilitating materials can be combined by chemical bonding (e.g., covalent and ionic such as in lipidated polylysine, PEGylated polylysine) (Toncheva, et al., Biochim. Biophys. Acta 1380(3):354-368 (1988)), mechanical mixing (e.g., free moving materials in liquid or solid phase such as “polylysine+cationic lipids”) (Gao and Huang, Biochemistry 35:1027-1036 (1996); Trubetskoy, et al., Biochem. Biophys. Acta 1131:311-313 (1992)), and aggregation (e.g., co-precipitation, gel forming such as in cationic lipids+poly-lactide, and polylysine+gelatin).
One category of transfection facilitating materials is cationic lipids. Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide (DOGS) and dipalmitoyl-phophatidylethanolamine-5-carboxyspermylamide (DPPES). Cationic cholesterol derivatives are also useful, including {3β-[N-N′,N′-dimethylamino)ethane]-carbomoyl}-cholesterol (DC-Chol). Dimethyldioctdecyl-ammonium bromide (DDAB), N-(3-aminopropyl)-N,N-(bis-(2-tetradecyloxyethyl))-N-methyl-ammonium bromide (PA-DEMO), N-(3-aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methyl-ammonium bromide (PA-DELO), N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium bromide (PA-TELO), and N1-(3-aminopropyl)((2-dodecyloxy)ethyl)-N2-(2-dodecyloxy)ethyl-1-piperazinaminium bromide (GA-LOE-BP) can also be employed in the present invention.
Non-diether cationic lipids, such as DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORI diester), 1-O-oleyl-2-oleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium (DORI ester/ether), and their salts promote in vivo gene delivery. In some embodiments, cationic lipids comprise groups attached via a heteroatom attached to the quaternary ammonium moiety in the head group. A glycyl spacer can connect the linker to the hydroxyl group.
Specific, but non-limiting cationic lipids for use in certain embodiments of the present invention include DMRIE ((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide), GAP-DMORIE ((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium bromide), and GAP-DLRIE ((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanaminium bromide).
Other specific but non-limiting cationic surfactants for use in certain embodiments of the present invention include Bn-DHRIE, DhxRIE, DhxRIE-OAc, DhxRIE-OBz and Pr-DOctRIE-OAc. These lipids are disclosed in U.S. patent application No. 60/435,303. In another aspect of the present invention, the cationic surfactant is Pr-DOctRIE-OAc.
Other cationic lipids include (±)-N,N-dimethyl-N-[2-(sperminecarboxamido) ethyl]-2,3-bis(dioleyloxy)-1-propaniminium pentahydrochloride (DOSPA), (±)-N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaniminium bromide (β-aminoethyl-DMRIE or βAE-DMRIE) (Wheeler, et al., Biochim. Biophys. Acta 1280:1-11 (1996), and (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996)), which have been developed from DMRIE.
Other examples of DMRIE-derived cationic lipids that are useful for the present invention are (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-1-propanaminium bromide (GAP-DDRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), (±)-N-((N′′-methyl)-N′-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GMU-DMRIE), (±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (DLRIE), and (±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis-([Z]-9-octadecenyIoxy)propyl-1- propaniminium bromide (HP-DORIE).
In the embodiments where the immunogenic composition comprises a cationic lipid, the cationic lipid may be mixed with one or more co-lipids. For purposes of definition, the term “co-lipid” refers to any hydrophobic material which may be combined with the cationic lipid component and includes amphipathic lipids, such as phospholipids, and neutral lipids, such as cholesterol. Cationic lipids and co-lipids may be mixed or combined in a number of ways to produce a variety of non-covalently bonded macroscopic structures, including, for example, liposomes, multilamellar vesicles, unilamellar vesicles, micelles, and simple films. One non-limiting class of co-lipids are the zwitterionic phospholipids, which include the phosphatidylethanolamines and the phosphatidylcholines. Examples of phosphatidylethanolamines, include DOPE, DMPE and DPyPE. In certain embodiments, the co-lipid is DPyPE, which comprises two phytanoyl substituents incorporated into the diacylphosphatidylethanolamine skeleton.
In other embodiments, the co-lipid is DOPE, CAS name 1,2-diolyeoyl-sn-glycero-3-phosphoethanolamine.
When a composition of the present invention comprises a cationic lipid and co-lipid, the cationic lipid:co-lipid molar ratio may be from about 9:1 to about 1:9, from about 4:1 to about 1:4, from about 2:1 to about 1:2, or about 1:1.
In order to maximize homogeneity, the cationic lipid and co-lipid components may be dissolved in a solvent such as chloroform, followed by evaporation of the cationic lipid/co-lipid solution under vacuum to dryness as a film on the inner surface of a glass vessel (e.g., a Rotovap round-bottomed flask). Upon suspension in an aqueous solvent, the amphipathic lipid component molecules self-assemble into homogenous lipid vesicles. These lipid vesicles may subsequently be processed to have a selected mean diameter of uniform size prior to complexing with, for example, a polynucleotide or a codon-optimized polynucleotide of the present invention, according to methods known to those skilled in the art. For example, the sonication of a lipid solution is described in Felgner et al., Proc. Natl. Acad. Sci. USA 8:,7413-7417 (1987) and in U.S. Pat. No. 5,264,618, the disclosures of which are incorporated herein by reference.
In those embodiments where the composition includes a cationic lipid, polynucleotides of the present invention are complexed with lipids by mixing, for example, a plasmid in aqueous solution and a solution of cationic lipid:co-lipid as prepared herein are mixed. The concentration of each of the constituent solutions can be adjusted prior to mixing such that the desired final plasmid/cationic lipid:co-lipid ratio and the desired plasmid final concentration will be obtained upon mixing the two solutions. The cationic lipid:co-lipid mixtures are suitably prepared by hydrating a thin film of the mixed lipid materials in an appropriate volume of aqueous solvent by vortex mixing at ambient temperatures for about 1 minute. The thin films are prepared by admixing chloroform solutions of the individual components to afford a desired molar solute ratio followed by aliquoting the desired volume of the solutions into a suitable container. The solvent is removed by evaporation, first with a stream of dry, inert gas (e.g., argon) followed by high vacuum treatment.
Other hydrophobic and amphiphilic additives, such as, for example, sterols, fatty acids, gangliosides, glycolipids, lipopeptides, liposaccharides, neobees, niosomes, prostaglandins and sphingolipids, may also be included in compositions of the present invention. In such compositions, these additives may be included in an amount between about 0.1 mol % and about 99.9 mol % (relative to total lipid), about 1-50 mol %, or about 2-25 mol %.
Additional embodiments of the present invention are drawn to compositions comprising an auxiliary agent which is administered before, after, or concurrently with the polynucleotide. As used herein, an “auxiliary agent” is a substance included in a composition for its ability to enhance, relative to a composition which is identical except for the inclusion of the auxiliary agent, the entry of polynucleotides into vertebrate cells in vivo, and/or the in vivo expression of polypeptides encoded by such polynucleotides. Certain auxiliary agents may, in addition to enhancing entry of polynucleotides into cells, enhance an immune response to an immunogen encoded by the polynucleotide. Auxiliary agents of the present invention include nonionic, anionic, cationic, or zwitterionic surfactants or detergents, with nonionic surfactants or detergents being preferred, chelators, DNase inhibitors, poloxamers, agents that aggregate or condense nucleic acids, emulsifying or solubilizing agents, wetting agents, gel-forming agents, and buffers.
Auxiliary agents for use in compositions of the present invention include, but are not limited to non-ionic detergents and surfactants IGEPAL CA 630®, NONIDET NP-40, Nonidet® P40, Tween-20®, Tween-80TM, Pluronic® F68 (ave. MW: 8400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 80%), Pluronic F770® (ave. MW: 6600; approx. MW of hydrophobe, 2100; approx. wt. % of hydrophile, 70%), Pluronic P65® (ave. MW: 3400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 50%), Triton X-100TM, and Triton X-114TM; the anionic detergent sodium dodecyl sulfate (SDS); the sugar stachyose; the condensing agent DMSO; and the chelator/DNAse inhibitor EDTA, CRL 1005 (12 kDa, 5% POE), and BAK (Benzalkonium chloride 50% solution, available from Ruger Chemical Co. Inc.). In certain specific embodiments, the auxiliary agent is DMSO, Nonidet P40, Pluronic F68® (ave. MW: 8400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 80%), Pluronic F77® (ave. MW: 6600; approx. MW of hydrophobe, 2100; approx. wt. % of hydrophile, 70%), Pluronic P65® (ave. MW: 3400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 50%), Pluronic L64® (ave. MW: 2900; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 40%), and Pluronic F108® (ave. MW: 14600; approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 80%). See, e.g., U.S. Patent Application Publication No. 2002/0019358, published Feb. 14, 2002, which is incorporated herein by reference in its entirety.
Certain compositions of the present invention may further include one or more adjuvants before, after, or concurrently with the polynucleotide. The term “adjuvant” refers to any material having the ability to (1) alter or increase the immune response to a particular antigen or (2) increase or aid an effect of a pharmacological agent. It should be noted, with respect to polynucleotide vaccines, that an “adjuvant,” may be a transfection facilitating material. Similarly, certain “transfection facilitating materials” described supra, may also be an “adjuvant.” An adjuvant may be used with a composition comprising a polynucleotide of the present invention. In a prime-boost regimen, as described herein, an adjuvant may be used with either the priming immunization, the booster immunization, or both. Suitable adjuvants include, but are not limited to, cytokines and growth factors; bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viruses and virally-derived materials, poisons, venoms, imidazoquiniline compounds, poloxamers, and cationic lipids.
A great variety of materials have been shown to have adjuvant activity through a variety of mechanisms. Any compound which may increase the expression, antigenicity or immunogenicity of the polypeptide is a potential adjuvant. The present invention provides an assay to screen for improved immune responses to potential adjuvants. Potential adjuvants which may be screened for their ability to enhance the immune response according to the present invention include, but are not limited to: inert carriers, such as alum, bentonite, latex, and acrylic particles; pluronic block polymers, such as TiterMax® (block copolymer CRL-8941, squalene (a metabolizable oil) and a microparticulate silica stabilizer), depot formers, such as Freunds adjuvant, surface active materials, such as saponin, lysolecithin, retinal, Quil A, liposomes, and pluronic polymer formulations; macrophage stimulators, such as bacterial lipopolysaccharide; alternate pathway complement activators, such as insulin, zymosan, endotoxin, and levamisole; and non-ionic surfactants, such as poloxamers, poly(oxyethylene)-poly(oxypropylene) tri-block copolymers. Also included as adjuvants are transfection-facilitating materials, such as those described above.
Poloxamers which may be screened for their ability to enhance the immune response according to the present invention include, but are not limited to, commercially available poloxamers such as Pluronic® surfactants, which are block copolymers of propylene oxide and ethylene oxide in which the propylene oxide block is sandwiched between two ethylene oxide blocks. Examples of Pluronic® surfactants include Pluronic® L121 (ave. MW: 4400; approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 10%), Pluronic® L101 (ave. MW: 3800; approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 10%), Pluronic® L81 (ave. MW: 2750; approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile, 10%), Pluronic® L61 (ave. MW: 2000; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 10%), Pluronic® L31 (ave. MW: 1100; approx. MW of hydrophobe, 900; approx. wt. % of hydrophile, 10%), Pluronic® L122 (ave. MW: 5000; approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 20%), Pluronic® L92 (ave. MW: 3650; approx. MW of hydrophobe, 2700; approx. wt. % of hydrophile, 20%), Pluronic® L72 (ave. MW: 2750; approx. MW of hydrophobe, 2100; approx. wt. % of hydrophile, 20%), Pluronic® L62 (ave. MW: 2500; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 20%), Pluronic® L42 (ave. MW: 1630; approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile, 20%), Pluronic® L63 (ave. MW: 2650; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 30%), Pluronic® L43 (ave. MW: 1850; approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile, 30%), Pluronic® L64 (ave. MW: 2900; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 40%), Pluronic® L44 (ave. MW: 2200; approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile, 40%), Pluronic® L35 (ave. MW: 1900; approx. MW of hydrophobe, 900; approx. wt. % of hydrophile, 50%), Pluronic® P123 (ave. MW: 5750; approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 30%), Pluronic® P103 (ave. MW: 4950; approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 30%), Pluronic® P104 (ave. MW: 5900; approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 40%), Pluronic® P84 (ave. MW: 4200; approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile, 40%), Pluronic® P105 (ave. MW: 6500; approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 50%), Pluronic® P85 (ave. MW: 4600; approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile, 50%), Pluronic® P75 (ave. MW: 4150; approx. MW of hydrophobe, 2100; approx. wt. % of hydrophile, 50%), Pluronic® P65 (ave. MW: 3400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 50%), Pluronic® F127 (ave. MW: 12600; approx. MW of hydrophobe, 3600; approx. wt. % of hydrophile, 70%), Pluronic® F98 (ave. MW: 13000; approx. MW of hydrophobe, 2700; approx. wt. % of hydrophile, 80%), Pluronic® F87 (ave. MW: 7700; approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile, 70%), Pluronic® F77 (ave. MW: 6600; approx. MW of hydrophobe, 2100; approx. wt. % of hydrophile, 70%), Pluronic® F108 (ave. MW: 14600; approx. MW of hydrophobe, 3000; approx. wt. % of hydrophile, 80%), Pluronic® F98 (ave. MW: 13000; approx. MW of hydrophobe, 2700; approx. wt. % of hydrophile, 80%), Pluronic® F88 (ave. MW: 11400; approx. MW of hydrophobe, 2400; approx. wt. % of hydrophile, 80%), Pluronic® F68 (ave. MW: 8400; approx. MW of hydrophobe, 1800; approx. wt. % of hydrophile, 80%), Pluronic® F38 (ave. MW: 4700; approx. MW of hydrophobe, 900; approx. wt. % of hydrophile, 80%).
Reverse poloxamers which may be screened for their ability to enhance the immune response according to the present invention include, but are not limited to Pluronic® R 31R1 (ave. MW: 3250; approx. MW of hydrophobe, 3100; approx. wt. % of hydrophile, 10%), Pluronic® R 25R1 (aye. MW: 2700; approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 10%), Pluronic® R 17R1 (ave. MW: 1900; approx. MW of hydrophobe, 1700; approx. wt. % of hydrophile, 10%), Pluronic® R 31R2 (ave. MW: 3300; approx. MW of hydrophobe, 3100; approx. wt. % of hydrophile, 20%), Pluronic® R 25R2 (ave. MW: 3100; approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 20%), Pluronic® R 17R2 (ave. MW: 2150; approx. MW of hydrophobe, 1700; approx. wt. % of hydrophile, 20%), Pluronic® R 12R3 (ave. MW: 1800; approx. MW of hydrophobe, 1200; approx. wt. % of hydrophile, 30%), Pluronic® R 31R4 (ave. MW: 4150; approx. MW of hydrophobe, 3100; approx. wt. % of hydrophile, 40%), Pluronic® R 25R4 (ave. MW: 3600; approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 40%), Pluronic® R 22R4 (ave. MW: 3350; approx. MW of hydrophobe, 2200; approx. wt. % of hydrophile, 40%), Pluronic® R 17R4 (ave. MW: 3650; approx. MW of hydrophobe, 1700; approx. wt. % of hydrophile, 40%), Pluronic® R 25R5 (ave. MW: 4320; approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 50%), Pluronic® R 10R5 (ave. MW: 1950; approx. MW of hydrophobe, 1000; approx. wt. % of hydrophile, 50%), Pluronic® R 25R8 (ave. MW: 8550; approx. MW of hydrophobe, 2500; approx. wt. % of hydrophile, 80%), Pluronic® R 17R8 (ave. MW: 7000; approx. MW of hydrophobe, 1700; approx. wt. % of hydrophile, 80%), and Pluronic® R 10R8 (ave. MW: 4550; approx. MW of hydrophobe, 1000; approx. wt. % of hydrophile, 80%).
Other commercially available poloxamers which may be screened for their ability to enhance the immune response according to the present invention include compounds that are block copolymer of polyethylene and polypropylene glycol such as Synperonic® L121 (ave. MW: 4400), Synperonic® L122 (ave. MW: 5000), Synperonic® P104 (ave. MW: 5850), Synperonic® P105 (ave. MW: 6500), Synperonic® P123 (ave. MW: 5750), Synperonic® P85 (ave. MW: 4600) and Synperonic® P94 (ave. MW: 4600), in which L indicates that the surfactants are liquids, P that they are pastes, the first digit is a measure of the molecular weight of the polypropylene portion of the surfactant and the last digit of the number, multiplied by 10, gives the percent ethylene oxide content of the surfactant; and compounds that are nonylphenyl polyethylene glycol such as Synperonic® NP10 (nonylphenol ethoxylated surfactant—10% solution), Synperonic® NP30 (condensate of 1 mole of nonylphenol with 30 moles of ethylene oxide) and Synperonic® NP5 (condensate of 1 mole of nonylphenol with 5.5 moles of naphthalene oxide).
Other poloxamers which may be screened for their ability to enhance the immune response according to the present invention include: (a) a polyether block copolymer comprising an A-type segment and a B-type segment, wherein the A-type segment comprises a linear polymeric segment of relatively hydrophilic character, the repeating units of which contribute an average Hansch-Leo fragmental constant of about −0.4 or less and have molecular weight contributions between about 30 and about 500, wherein the B-type segment comprises a linear polymeric segment of relatively hydrophobic character, the repeating units of which contribute an average Hansch-Leo fragmental constant of about −0.4 or more and have molecular weight contributions between about 30 and about 500, wherein at least about 80% of the linkages joining the repeating units for each of the polymeric segments comprise an ether linkage; (b) a block copolymer having a polyether segment and a polycation segment, wherein the polyether segment comprises at least an A-type block, and the polycation segment comprises a plurality of cationic repeating units; and (c) a polyether-polycation copolymer comprising a polymer, a polyether segment and a polycationic segment comprising a plurality of cationic repeating units of formula —NH—R0, wherein R0 is a straight chain aliphatic group of 2 to 6 carbon atoms, which may be substituted, wherein said polyether segments comprise at least one of an A-type of B-type segment. See U.S. Pat. No. 5,656,611, by Kabonov, et al., which is incorporated herein by reference in its entirety. Other poloxamers of interest include CRL1005 (12 kDa, 5% POE), CRL8300 (11 kDa, 5% POE), CRL2690 (12 kDa, 10% POE), CRL4505 (15 kDa, 5% POE) and CRL1415 (9 kDa, 10% POE).
Other auxiliary agents which may be screened for their ability to enhance the immune response according to the present invention include, but are not limited to Acacia (gum arabic); the poloxyethylene ether R—O—(C2H4O)x—H (BRIJ®), e.g., polyethylene glycol dodecyl ether (BRIJ® 35, x=23), polyethylene glycol dodecyl ether (BRIJ® 30, x=4), polyethylene glycol hexadecyl ether (BRIJ® 52 x=2), polyethylene glycol hexadecyl ether (BRIJ® 56, x=10), polyethylene glycol hexadecyl ether (BRIJ® 58P, x=20), polyethylene glycol octadecyl ether (BRIJ® 72, x=2), polyethylene glycol octadecyl ether (BRIJ® 76, x=10), polyethylene glycol octadecyl ether (BRIJ® 78P, x=20), polyethylene glycol oleyl ether (BRIJ® 92V, x=2), and polyoxyl 10 oleyl ether (BRIJ® 97, x=10); poly-D-glucosamine (chitosan); chlorbutanol; cholesterol; diethanolamine; digitonin; dimethylsulfoxide (DMSO), ethylenediamine tetraacetic acid (EDTA); glyceryl monosterate; lanolin alcohols; mono- and di-glycerides; monoethanolamine; nonylphenol polyoxyethylene ether (NP-40®); octylphenoxypolyethoxyethanol (NONIDET NP-40 from Amresco); ethyl phenol poly (ethylene glycol ether)n, n=11 (Nonidet® P40 from Roche); octyl phenol ethylene oxide condensate with about 9 ethylene oxide units (nonidet P40); IGEPAL CA 630® ((octyl phenoxy) polyethoxyethanol; structurally same as NONIDET NP-40); oleic acid; oleyl alcohol; polyethylene glycol 8000; polyoxyl 20 cetostearyl ether; polyoxyl 35 castor oil; polyoxyl 40 hydrogenated castor oil; polyoxyl 40 stearate; polyoxyethylene sorbitan monolaurate (polysorbate 20, or TWEEN-20®; polyoxyethylene sorbitan monooleate (polysorbate 80, or TWEEN-80®); propylene glycol diacetate; propylene glycol monstearate; protamine sulfate; proteolytic enzymes; sodium dodecyl sulfate (SDS); sodium monolaurate; sodium stearate; sorbitan derivatives (SPAN®), e.g., sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate (SPAN® 65), sorbitan monooleate (SPAN® 80), and sorbitan trioleate (SPAN® 85); 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa-hexaene (squalene); stachyose; stearic acid; sucrose; surfactin (lipopeptide antibiotic from Bacillus subtilis); dodecylpoly(ethyleneglycolether)g (Thesit®) MW 582.9; octyl phenol ethylene oxide condensate with about 9-10 ethylene oxide units (Triton X-100TM); octyl phenol ethylene oxide condensate with about 7-8 ethylene oxide units (Triton X-114TM); tris(2-hydroxyethyl)amine (trolamine); and emulsifying wax.
In certain adjuvant compositions, the adjuvant is a cytokine. A composition of the present invention can comprise one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines, or a polynucleotide encoding one or more cytokines, chemokines, or compounds that induce the production of cytokines and chemokines. Examples include, but are not limited to granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), interferon omega (IFNω), interferon tau (IFNθ), interferon gamma inducing factor I (IGIF), transforming growth factor beta (TGF-β), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), Leishmania elongation initiating factor (LEIF), and Flt-3 ligand.
In certain compositions of the present invention, the polynucleotide construct may be complexed with an adjuvant composition comprising (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium bromide (GAP-DMORIE). The composition may also comprise one or more co-lipids, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or 1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE). An adjuvant composition comprising; GAP-DMORIE and DPyPE at a 1:1 molar ratio is referred to herein as VaxfectinTM. See, e.g., PCT Publication No. WO 00/57917, which is incorporated herein by reference in its entirety.
In other embodiments, the polynucleotide itself may function as an adjuvant as is the case when the polynucleotides of the invention are derived, in whole or in part, from bacterial DNA. Bacterial DNA containing motifs of unmethylated CpG-dinucleotides (CpG-DNA) triggers innate immune cells in vertebrates through a pattern recognition receptor (including toll receptors such as TLR 9) and thus possesses potent immunostimulatory effects on macrophages, dendritic cells and B-lymphocytes. See, e.g., Wagner, H., Curr. Opin. Microbiol. 5:62-69 (2002); Jung, J. et al., J. Immunol. 169: 2368-73 (2002); see also Klinman, D. M. et al., Proc. Natl Acad. Sci. U.S.A. 93:2879-83 (1996). Methods of using unmethylated CpG-dinucleotides as adjuvants are described in, for example, U.S. Pat. Nos. 6,207,646, 6,406,705, and 6,429,199, the disclosures of which are herein incorporated by reference.
The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated protection. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th2 response into a primarily cellular, or Th1 response.
In certain embodiments, the compositions of the present invention may be administered in the absence of one or more transfection facilitating materials or auxiliary agents. It has been shown that, surprisingly, the cells of living vertebrates are capable of taking up and expressing polynucleotides that have been injected in vivo, even in the absence of any agent to facilitate transfection. Cohen, J., Science 259: 1691-1692; Felgner, P., Scientific American 276: 102-106 (1997). These references are hereby incorporated by reference in their entireties. Thus, by way of non-limiting examples, nucleic acid molecules and/or polynucleotides of the present invention (e.g., plasmid DNA, mRNA, linear DNA, or oligonucleotides) may be administered in the absence of any one of, or any combination of more than one of the following transfection facilitating materials or auxiliary agents as described herein: inorganic materials including but not limited to calcium phosphate, alum, and/or gold particles; peptides including, but not limited to cationic peptides, amphipathic peptides, intercell targeting peptides, and/or intracell targetting peptides; proteins, including, but not limited to basic (i.e., positively-charged) proteins, targeting proteins, viral proteins, and/or pore-forming proteins; lipids, including but not limited to cationic lipids, anionic lipids, basic lipids, neutral lipids, and/or zwitterionic lipids; polymers including but not limited to dendrimers, star-polymers, “homogeneous” poly-amino acids, “heterogenous” poly-amino acids, co-polymers, PVP, poloxamers, and/or PEG; surfactants, including but not limited to anionic surfactants, cationic surfactants, and zwitterionic surfactants; detergents, including but not limited to anionic detergents, cationic detergents, and zwitterionic detergents; chelators, including but not limited to EDTA; DNase inhibitors; condensing agents including, but not limited to DMSO; emulsifying or solublizing agents; gel-forming agents; buffers, and/or adjuvants.
Nucleic acid molecules and/or polynucleotides of the present invention, e.g., plasmid DNA, mRNA, linear DNA or oligonucleotides, may be solubilized in any of various buffers. Suitable buffers include, for example, phosphate buffered saline (PBS), normal saline, Tris buffer, and sodium phosphate (e.g., 150 mM sodium phosphate). Insoluble polynucleotides may be solubilized in a weak acid or weak base, and then diluted to the desired volume with a buffer. The pH of the buffer may be adjusted as appropriate. In addition, a pharmaceutically acceptable additive can be used to provide an appropriate osmolarity. Such additives are within the purview of one skilled in the art. For aqueous compositions used in vivo, sterile pyrogen-free water can be used. Such formulations will contain an effective amount of a polynucleotide together with a suitable amount of an aqueous solution in order to prepare pharmaceutically acceptable compositions suitable for administration to a human.
Compositions of the present invention can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., Mack Publishing Co., Easton, Pa. (1980), and Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), both of which are incorporated herein by reference in their entireties. Although the composition may be administered as an aqueous solution, it can also be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art. In addition, the composition may contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives.
Passive Immunotherapy
Antibody therapy can be subdivided into two principally different activities: (i) passive immunotherapy using intact non-labeled antibodies or labeled antibodies and (ii) active immunotherapy using anti-idiotypes for re-establishment of network balance in autoimmunity
In passive immunotherapy, naked antibodies are administered to neutralize an antigen or to direct effector functions to targeted membrane associated antigens. Neutralization would be of a lymphokine, a hormone, or an anaphylatoxin, i.e., C5a. Effector functions include complement fixation, macrophage activation and recruitment, and antibody-dependent cell-mediated cytotoxicity (ADCC). Naked antibodies have been used to treat leukemia (Ritz, S.F. et al Blood, 58:141-152 (1981)) and antibodies to GD2 have been used in treatments of neuroblastomas (Schulz et al. Cancer Res. 44:5914 (1984)) and melanomas (Irie et al., Proc. Natl. Acad. Sci. 83: 8694 (1986) One major advantage of passive antibody immunization is that it provides immediate immunity that can last for weeks and possibly months. Casadevall, A. “Passive Antibody Administration (Immediate Immunity) as a Specific Defense against Biological Weapons.” Emerging Infectious Diseases. 8:833-841(2002).
The invention also provides for antibodies specifically reactive with SARS Co-V polypeptides which have been produced from an immune response elicited by the administration, to a vertebrate, of polynucleotide and polypeptides of the present invention. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A vertebrate such as a mouse, a hamster, a rabbit, a horse, a human, or non-human primate can be immunized with an immunogenic form of a SARS Co-V polypeptide or polynucleotide, of the present invention, encoding an immunogenic form of a SARS-CoV polypeptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of the SARS-CoV polypeptide can be administered in the presence of adjuvant and as part of compositions described herein. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
The antibodies of the invention are immunospecific for antigenic determinants of the SARS-CoV polypeptides of the invention, e.g., antigenic determinants of a polypeptide of the invention or a closely related human or non-human mammalian homolog (e.g., 90% homologous and at least about 95% homologous). In an alternative embodiment of the invention, the SARS Co-V antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80% percent homologous to a sequence of the invention. By “not substantially cross react,” is meant that the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, less than 5 percent, or less than 1 percent, of the binding affinity for a protein of the invention. In an alternative embodiment, there is no cross-reactivity between viral and mammalian antigens.
In one embodiment, purified monoclonal antibodies or polyclonal antibodies containing the variable heavy and light sequences are used as therapeutic and prophylactic agents to treat or prevent SARS-CoV infection by passive antibody therapy. In general, this will comprise administering a therapeutically or prophylactically effective amount of the monoclonal or polyclonal antibodies to a susceptible vertebrate or one exhibiting SARS Co-V infection. A dosage effective amount will range from about 50 to 20,000 μg/Kg, and from about 100 to 5000 μg/Kg. However, suitable dosages will vary dependening on factors such as the condition of the treated host, weight, etc. Suitable effective dosages may be determined by those skilled in the art.
In an alternative embodiment, purified antibodies and the polynucleotides or polypeptides of the present invention are administered simultaneously (at the same time) or subsequent to the administration of the isolated antibodies, thereby providing both immediate and long lasting protection.
The monoclonal or polyclonal antibodies may be administered by any mode of administration suitable for administering antibodies. Typically, the subject antibodies will be administered by injection, e.g., intravenous, intramuscular, or intraperitoneal injection (as described previously), or aerosol. Aerosol administration is particularly preferred if the subjects treated comprise newborn infants.
Formulation of antibodies in pharmaceutically acceptable form may be effected by known methods, using known pharmaceutical carriers and excipients. Suitable carriers and excipients include by way of non-limiting example buffered saline, and bovine serum albumin.
Any polynucleotides or polypeptides, as described herein, can be used to produce the isolated antibodies of the invention. For example, SARS-CoV proteins S, N, M, and E, fragments, variants and derivatives thereof, are purified as described in Example 2. The purified protein then serves as an antigen for producing SARS-CoV specific monoclonal and polyclonal antibodies.
Any vertebrate can serve as a host for antibody production. Preferred hosts include, but are not limited to human, non-human primate, mouse, rabbit, horse, goat, donkey, cow, sheep, chickens, cat, dog. Alternatively, antibodies can be produced by cultivation ex vivo of lymphocytes from primed donors stimulated with CD40 resulting in expansion of human B cells Banchereau et al., Science 251:70 (1991); Zhani et al., J. Immunol. 144:2955-2960, (1990); Tohma et al., J. Immunol. 146:2544-2552 (1991). Furthermore, an extra in vitro booster step can be used to obtain a higher yield of antibodies prior to immortalization of the cells. See Chaudhuri et al., Cancer Supplement 73: 1098-1104 (1994); Steenbakkers et al. Hum. Antibod. Hybridomas 4: 166-173 (1993); Ferrarro et al., Hum. Antibod. Hybridomas 4:80-85 (1993); Kwekkeboom et al., Immunol. Methods 160:117-127 (1993), which are herein incorporated by reference.
An alternative to human primed donors, is to “recreate” or mimic splenic conditions in an immunocompromised animal host, such as the “Severe Combined Immune Deficient” (SCID) mouse. Human lymphocytes are readily adopted by the SCID mouse (hu-SCID) and produce high levels of immunoglobulins Mosier et al, Nature 335:256 (1988); McCune et al, Science 241:1632-1639 (1988). Moreover, if the donor used for reconstitution has been exposed to a particular antigen, a strong secondary response to the same antigen can be elicited in such mice. Duchosal et al. Nature 355:258-262 (1992).
The term “antibody” as used herein is intended to include fragments thereof which are also specifically reactive with SARS-CoV polypeptides. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example. F(ab′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. The antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-SARS-CoV portion.
Both monoclonal and polyclonal antibodies (Ab) directed against SARS-CoV polypeptides or SARS-CoV polypeptide variants, and antibody fragments such as Fab′ and F(ab′) 2, can be used to block the action of SARS-CoV polypeptides and allow the study of the role of a particular SARS-CoV polypeptide of the invention in the infectious life cycle of the virus and in pathogenesis.
Moreover, the antibodies possess utility as immunoprobes for diagnosis of SARS Co-V infection. This generally comprises taking a sample, e.g., respiratory fluid, of a person suspected of having SARS-CoV infection and incubating the sample with the subject human monoclonal antibodies to detect the presence of SARS-CoV infected cells. This involves directly or indirectly labeling the subject human antibodies with a reporter molecule which provides for detection of human monoclonal antibody SARS-CoV immune complexes. Examples of known labels include by way of non-limiting example enzymes, e.g.,. β-lactamase, luciferase, and radiolabels. Methods for effecting immunodetection of antigens using monoclonal antibodies are well known in the art.
The following examples are included for purposes of illustration only and are not intended to limit the scope of the present invention, which is defined by the appended claims. All references cited in the Examples are incorporated herein by reference in their entireties.
EXAMPLES
Materials and Methods
The following materials and methods apply generally to all the examples disclosed herein. Specific materials and methods are disclosed in each example, as necessary.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology (including PCR), vaccinology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989).
Gene Construction
Constructs of the present invention are constructed based on the sequence information provided herein or in the art utilizing standard molecular biology techniques, including, but not limited to the following. First, a series complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the construct are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends. The single-stranded ends of each pair of oligonucleotides are designed to anneal with a single-stranded end of an adjacent oligonucleotide duplex. Several adjacent oligonucleotide pairs prepared in this manner are allowed to anneal, and approximately five to six adjacent oligonucleotide duplex fragments are then allowed to anneal together via the cohesive single stranded ends. This series of annealed oligonucleotide duplex fragments is then ligated together and cloned into a suitable plasmid, such as the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Constructs prepared in this manner, comprising 5 to 6 adjacent 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence of the construct is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. The oligonucleotides and primers referred to herein can easily be designed by a person of skill in the art based on the sequence information provided herein and in the art, and such can be synthesized by any of a number of commercial nucleotide providers, for example Retrogen, San Diego, Calif.
Plasmid Vector
Constructs of the present invention can be inserted, for example, into eukaryotic expression vectors VR1012 or VR10551. These vectors are built on a modified pUC18 background (see Yanisch-Perron, C., et al. Gene 33:103-119 (1985)), and contain a kanamycin resistance gene, the human cytomegalovirus immediate early promoter/enhancer and intron A, and the bovine growth hormone transcription termination signal, and a polylinker for inserting foreign genes. See Hartikka, J., et al., Hum. Gene Ther. 7:1205-1217 (1996). However, other standard commercially available eukaryotic expression vectors may be used in the present invention, including, but not limited to: plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and plasmid pCI (available from Promega, Madison, Wis.).
An optimized backbone plasmid, termed VR-10551 has minor changes from the VR-1012 backbone described above. The VR-10551 vector is derived from and similar to VR-1012 in that it uses the human cytomegalovirus immediate early (hCMV-IE) gene enhancer/promoter and 5′untranslated region (UTR), including the hCMV-IE Intron A. The changes from the VR-1012 to the VR-10551 include some modifications to the multiple cloning site, and a modified rabbit ∃globin 3′untranslated region/polyadenylation signal sequence/transcriptional terminator has been substituted for the same functional domain derived from the bovine growth hormone gene.
Plasmid DNA Purification
Plasmid DNA may be transformed into competent cells of an appropriate Escherichia coli strain (including but not limited to the DH5α strain) and highly purified covalently closed circular plasmid DNA may be isolated by a modified lysis procedure (Horn, N. A., et al., Hum. Gene Ther. 6:565-573 (1995)) followed by standard double CsCl-ethidium bromide gradient ultracentrifugation (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989)). Alternatively, plasmid DNAs are purified using Giga columns from Qiagen (Valencia, Calif.) according to the kit instructions. All plasmid preparations are free of detectable chromosomal DNA, RNA and protein impurities based on gel analysis and the bicinchoninic protein assay (Pierce Chem. Co., Rockford Ill.). Endotoxin levels are measured using Limulus Amebocyte Lysate assay (LAL, Associates of Cape Cod, Falmouth, Mass.) in Endotoxin Units/mg of plasmid DNA. The spectrophotometric A260/A280 ratios of the DNA solutions are also determined. Plasmids are ethanol precipitated and resuspended in an appropriate solution, e.g., 150 mM sodium phosphate (for other appropriate excipients and auxiliary agents, see U.S. Patent Application Publication 20020019358, published Feb. 14, 2002). DNA is stored at −20EC until use. DNA is diluted by mixing it with 300 mM salt solutions and by adding appropriate amount of USP water to obtain 1 mg/ml plasmid DNA in the desired salt at the desired molar concentration.
Injections of Plasmid DNA
The quadriceps muscles of restrained awake mice (e.g., female 6-12 week old BALB/c mice from Harlan Sprague Dawley, Indianapolis, Ind.) are injected bilaterally with 50 μg of DNA in 50 μl solution (100 μg in 100 μl total per mouse) using a disposable plastic insulin syringe and 28G 1⁄2 needle (Becton-Dickinson, Franklin Lakes, N.J., Cat. No. 329430) fitted with a plastic collar cut from a micropipette tip, as previously described (Hartikka, J., et al., Hum. Gene Ther. 7:1205-1217 (1996).
Animal care will comply with the “Guide for the Use and Care of Laboratory Animals,” Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press, Washington, D.C., 1996 as well as with Vical's Institutional Animal Care and Use Committee.
Example 1
Construction of Expression Vectors
Plasmid constructs comprising the native coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg are constructed as follows. The S, S1, S2, N, M, or E genes from SARS-CoV Urbani or other strains (e.g., CUKH-Su10, TOR2 and BJ01) are isolated from viral RNA by RT PCR, or prepared by direct synthesis if the wildtype sequence is known, by standard methods and are inserted into the vector VR-10551 via standard restriction sites, by standard methods.
Plasmid constructs comprising human codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, are prepared as follows. The codon-optimized coding regions are generated using the full, minimal, uniform, or other codon optimization methods described herein. The coding regions or codon optimized coding regions are constructed using standard PCR methods described herein, or are ordered commercially. The coding regions or codon-optimized coding regions are inserted into the vector VR-10551 via standard restriction sites, by standard methods.
Examples of constructs to be made are listed in Table 19.










TABLE 19





Gene
Strain
Backbone
Wild type/Codon optimized


















S
Urbani
10551
Wild type


S
Urbani
10551
Codon optimized


S1
Urbani
1012
Wild type


S1
Urbani
10551
Codon optimized


S2
Urbani
10551
Wild type


S2
Urbani
10551
Codon optimized


N
Urbani
10551
Wild type


N
Urbani
10551
Codon optimized


M
Urbani
10551
Wild type


M
Urbani
10551
Codon optimized


E
Urbani
10551
Wild type


E
Urbani
10551
Codon optimized









Plasmids constructed as above are propagated in Escherichia coli and purified by the alkaline lysis method (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 2 (1989)). CsCl-banded DNA are ethanol precipitated and resuspended in 0.9% saline to a final concentration of 2 mg/ml for injection. Alternately, plasmids are purified using any of a variety of commercial kits, or by other known procedures involving differential precipitation and/or chromatographic purification.
Expression is tested by formulating each of the plasmids in DMRIE/DOPE and transfecting cell lines including, but not limited to VM92 cells, fungal cells, including yeast cells such as Saccharomyces spp. cells; insect cells such as Drosophila S2, Spodoptera Sf9 or Sf21 cells and Trichoplusa High-Five cells; other animal cells (particularly mammalian cells and human cells) such as MDCK, CV1, 3T3, CPAE, A10, Sp2/0-Ag14, PC12, CHO, COS, VERO, HeLa, Bowes melanoma cells, SW-13, NCI-H295, RT4, HT-1376, UM-UC-3, IM-9, KG-1, R54;11, A-172, U-87MG, BT-20, MCF-7, SK-BR-3, ChaGo K-1, CCD-14Br, CaSki, ME-180, FHC, HT-29, Caco-2, SW480, HuTu8O, Tera 1, NTERA-2, AN3 CA, KLE, RL95-2, Caki-1, ACHN, 769 P, CCRF-CEM, Hut 78, MOLT 4, HL-60, Hep-3B, HepG2, SK-HEP1, A-549, NCI-H146, NCI-H82, NCI-H82, SK-LU-1, WI-38, MRC-5, HLF-a, CCD-19Lu, C39, Hs294T, SK-MEL5, COLO 829, U266B1, RPMI 2650, BeWo, JEG-3, JAR, SW 1353, MeKam, and SCC-4; and higher plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art.
The supernatants are collected and the protein production tested by Western blot or ELISA. The relative expression of the wild type and codon optimized constructs are compared.
In addition to plasmids encoding single SARS-CoV proteins, single plasmids which contain a portion of a SARS-CoV coding region are constructed according to standard methods. For example, portions of a SARS-CoV coding region that is too large to be contained in a single plasmid may be inserted into two or more plasmids. Also, single plasmids which contain two or more SARS-CoV coding regions are constructed according to standard methods. For example, a polycistronic construct, where two or more SARS-CoV coding regions are transcribed as a single transcript in eukaryotic cells may be constructed by separating the various coding regions with IRES sequences (Jang et al. “A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation.” J. Virol. 62: 2636-43 (1988); Jang et al. “Cap-independent Translation of Picornavirus RNAs: Structure and Function of the Internal Ribosomal Entry Site.” Enzyme 44:292-309(1990)).
Alternatively, two or more coding regions may be inserted into a single plasmid, each with their own promoter sequence.
Example 2
In Vitro Expression of SARS-CoV Subunit Proteins
Expression of SARS-CoV Nucleocapsid (N) and Spike (S) constructs were tested in vitro by transfection of a mouse melanoma cell line (VM92). The following expression constructs were transfected individually into VM92 cells and cultured for a period of time. All SARS-CoV sequences described below, were cloned into the VR1012 expression vector. The VR9208 expression plasmid contains a nucleotide sequence encoding the SARS-CoV S1 domain which was codon-optimized according to the full optimization method described herein and is disclosed in SEQ ID NO:50. The VR9204 expression plasmid contains a nucleotide sequence encoding a fragment of the SARS-CoV S1 which corresponds to amino acids 1-417 of the SARS-CoV S1 protein. The coding sequence in VR9204 was also codon optimized according to the full optimization method described herein.

    
    
        VR9219—expressing full-length SARS-CoV N protein
        VR9208—expressing SARS-CoV S1 domain of the S protein (amino acids 1-683 of the S protein)
        VR9204—expressing a fragment of the SARS-CoV S1 domain (amino acids 1-417 of the S1 domain)
        VR9209—expressing SARS-CoV S2 domain of the S protein
        VR9210—expressing SARS-CoV secreted S protein
    
    


Both cell extracts and cell culture medium supernatants were analyzed by Western blot. The presence of the SARS-CoV N protein and S proteins were detected using commercial rabbit polyclonal antibodies which reconginze the N protein from SARS-CoV strain Urbani (IMG-543; Imgenex, San Diego, Calif.) and the S proteins from SARS-CoV strain Urbani (IMG-557, 542 and 541; Imgenex, Diego, Calif.). Western blot results are summarized below:
In both the supemantant and cell lystates from cells transfected with the VR9219 plasmid, protein bands of a molecular weight of between 37 and 50 kDa (as estimated by a protein molecular weight standard) were detectable. The SARS-CoV N protein has an expected molecule weight of 46 kDa. This result is consistent with efficient expression of the SARS-CoV N antigen.
The supernantant and cell lysates from cells transfected with four different SARS-CoV S antigen constructs were individually analyzed for the presence of the S antigen. The results are summarized below.
A protein band of 85-110 kDa (as estimated by a protein molecular weight standard) was detected by Western blot in both the lysate and supernatant of cells transfected with the VR9204 plasmid (S1 domain—fragment).
A protein band of about 150 kDa (as estimated by a protein molecular weight standard) was detected by Western blot in both the lysate and supernatant of cells transfected with the VR9208 plasmid (S1 domain).
A protein band of approximately 111 kDa (as estimated by a protein molecular weight standard) was detected by Western blot in both the lysate and supernatant of cells transfected with the VR9209 plasmid (S2 domain).
A protein band of about 190 kDa (as estimated by a protein molecular weight standard) was detected by Western blot in both the lysate and supernatant of cells transfected with the VR9210 plasmid (secreted S).
These results are consistent with efficient expression and secretion of SARS-CoV Spike protein. Due to the presence of glycosylation sites in the S protein, the molecular weight is difficult to acurrately predict.
Example 3
Preparation of SARS-CoV Subunit Proteins
Recombinantly prepared SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, for use as subunit proteins in the various combination therapies and compositions described herein, are prepared using the following procedure.
Eukaryotic cells transfected with expression plasmids such as those described in Example 1 are used to express SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg. Alternatively, a baculovirus system can be used wherein insect cells such as, but not limited to, Sf9, Sf21, or D.Mel-2 cells are infected with recombinant baculoviruses which can express SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg. Other in vitro expression systems may be used, and are well known to those of ordinary skill in the art. For baculovirus expression of non-secreted forms of these proteins, cells which are infected with recombinant baculoviruses capable of expressing SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, are collected by knocking and scraping cells off the bottom of the flask in which they are grown. Cells infected with baculoviruses for 24 or 48 hours are less easy to detach from flask and may lyse, thus care must be taken with their removal. Eukaryotic cells which are transfected, either transiently or permanently, with expression plasmids encoding non-secreted forms of SARS-CoV proteins are gently scraped of the bottom of the flasks in which they are grown. Flasks containing the cells are then rinsed with PBS and the cells are transferred to 250 ml conical tubes. The tubes are spun at 1000 rpm in J-6 centrifuge (300×g) for about 5-10 minutes. The cell pellets are washed two times with PBS and then resuspended in about 10-20 ml of PBS in order to count. The cells are fmally resuspended at a concentration of about 2×107 cells/ml in RSB (10 mM Tris pH=7.5, 1.5 mM MgCl2, 10 mM KCl).
At this point either a total cell lysate is prepared, or cytoplasmic and nuclear fractions are separated. Approximately 106 infected cells are used per lane of a standard SDS-PAGE mini-protein gel for gel analysis purposes. When separating cytoplasmic and nuclear fractions, 10% NP40 is added to the cells for a final concentration of 0.5%. The cell-NP40 mixture is vortexed and placed on ice for 10 minutes, vortexing occasionally. After ice incubation, the cells are spun at 1500 rpm in a J-6 centrifuge (600×1) for 10 minutes. The supemantant is removed, which is the cytoplasmic fraction. The remaining pellet, containing the nuclei, is washed two times with buffer C (20 mM HEPES pH=7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) to remove cytoplasmic proteins. The nuclei are resuspended in buffer C to 5×107 nuclei/ml. The nuclei are vortexed vigorously to break up particles and an aliquot is removed for the mini-protein gel, which is the nuclei fraction.
Whole cell lysates are prepared by simply resuspending the requisite number of cells in gel sample buffer.
For gel analysis, a small amount (about 106 nuclear equivalents) of the nuclear pellet is resuspended directly in gel sample buffer and run with equivalent amounts of whole cells, cytoplasm, and nuclei. Those fractions containing the SARS-CoV protein of interest are detected by Western blot analysis as described herein.
Following analysis as described above, larger quantities of crude subunit proteins are prepared from batch cell cultures by protein purification methods well known by those of ordinary skill in the art, e.g., the use of HPLC.
Secreted versions of SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg are isolated from cell culture supernatants using various protein purification methods well known to those of ordinary skill in the art.
Example 4
Preparation of Vaccine Formulations
Plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are formulated with the poloxamer CRL 1005 and BAK (Benzalkonium chloride 50% solution, available from Ruger Chemical Co. Inc.) by the following methods. Specific final concentrations of each component of the formulae are described in the following methods, but for any of these methods, the concentrations of each component may be varied by basic stoichiometric calculations known by those of ordinary skill in the art to make a final solution having the desired concentrations.
For example, the concentration of CRL 1005 is adjusted depending on, for example, transfection efficiency, expression efficiency, or imunogenicity, to achieve a final concentration of between about 1 mg/ml to about 75 mg/ml, for example, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6.5 mg/ml, about 7 mg/ml, about 7.5 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, or about 75 mg/ml of CRL 1005.
Similarly, the concentration of DNA is adjusted depending on many factors, including the amount of a formulation to be delivered, the age and weight of the subject, the delivery method and route and the immunogenicity of the antigen being delivered. In general, formulations of the present invention are adjusted to have a final concentration from about 1 ng/ml to about 30 mg/ml of plasmid (or other polynucleotide). For example, a formulation of the present invention may have a final concentration of about 1 ng/ml, about 5 ng/ml, about 10 ng/ml, about 50 ng/ml, about 100 ng/ml, about 500 ng/ml, about 1 μg/ml, about 5 μg/ml, about 10 μg/ml, about 50 μg/ml, about 200 μg/ml, about 400 μg/ml, about 600 μg/ml, about 800 μg/ml, about 1 mg/ml, about 2 mg/ml, about 2.5, about 3 mg/ml, about 3.5, about 4 mg/ml, about 4.5, about 5 mg/ml, about 5.5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 20 mg/ml, or about 30 mg/ml of a plasmid.
Certain formulations of the present invention include a cocktail of plasmids (see, e.g., Example 1 supra) of the present invention, e.g., comprising coding regions encoding SARS-CoV proteins, for example SARS-CoV S, S1, S2, N, M, or E and optionally, plasmids encoding immunity enhancing proteins, e.g., cytokines. Various plasmids desired in a cocktail are combined together in PBS or other diluent prior to the addition to the other ingredients. Furthermore, plasmids may be present in a cocktail at equal proportions, or the ratios may be adjusted based on, for example, relative expression levels of the antigens or the relative immunogenicity of the encoded antigens. Thus, various plasmids in the cocktail may be present in equal proportions, or up to twice or three times as much of one plasmid may be included relative to other plasmids in the cocktail.
Additionally, the concentration of BAK may be adjusted depending on, for example, a desired particle size and improved stability. Indeed, in certain embodiments, formulations of the present invention include CRL 1005 and DNA, but are free of BAK. In general BAK-containing formulations of the present invention are adjusted to have a final concentration of BAK from about 0.05 mM to about 0.5 mM. For example, a formulation of the present invention may have a final BAK concentration of about 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM.
The total volume of the formulations produced by the methods below may be scaled up or down, by choosing apparatus of proportional size. Finally, in carrying out any of the methods described below, the three components of the formulation, BAK, CRL 1005, and plasmid DNA, may be added in any order. In each of these methods described below the term “cloud point” refers to the point in a temperature shift, or other titration, at which a clear solution becomes cloudy, ie., when a component dissolved in a solution begins to precipitate out of solution.
Thermal Cycling of a Pre-Mixed Formulation
This example describes the preparation of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a total volume of 3.6 ml. The ingredients are combined together at a temperature below the cloud point and then the formulation is thermally cycled to room temperature (above the cloud point) several times, according to the protocol outlined in FIG. 2.
A 1.28 mM solution of BAK is prepared in PBS, 846 μl of the solution is placed into a 15 ml round bottom flask fitted with a magnetic stirring bar, and the solution is stirred with moderate speed, in an ice bath on top of a stirrer/hotplate (hotplate off) for 10 minutes. CRL 1005 (27 μl) is then added using a 100 μl positive displacement pipette and the solution is stirred for a further 60 minutes on ice. Plasmids comprising coding regions or codon-optimized coding regions encoding SARS-CoV proteins, for example, S, S1, S2, N, M, or E, as described herein, and optionally, additional plasmids comprising codon-optimized or non-codon-optimized coding regions encoding, e.g., additional SARS-CoV proteins, and or other proteins, e.g., cytokines, are mixed together at desired proportions in PBS to achieve 6.4 mg/ml total DNA. This plasmid cocktail is added dropwise, slowly, to the stirring solution over 1 min using a 5 ml pipette. The solution at this point (on ice) is clear since it is below the cloud point of the poloxamer and is further stirred on ice for 15 min. The ice bath is then removed, and the solution is stirred at ambient temperature for 15 minutes to produce a cloudy solution as the poloxamer passes through the cloud point.
The flask is then placed back into the ice bath and stirred for a further 15 minutes to produce a clear solution as the mixture is cooled below the poloxamer cloud point. The ice bath is again removed and the solution stirred at ambient temperature for a further 15 minutes. Stirring for 15 minutes above and below the cloud point (total of 30 minutes), is defined as one thermal cycle. The mixture is cycled six more times. The resulting formulation may be used immediately, or may be placed in a glass vial, cooled below the cloud point, and frozen at −80° C. for use at a later time.
Thermal Cycling, Dilution and Filtration of a Pre-mixed Formulation, Using Increased Concentrations of CRL 1005
This example describes the preparation of a formulation comprising 0.3 mM BAK, 34 mg/ml or 50 mg/ml CRL 1005, and 2.5 mg/ml of DNA in a final volume of 4.0 ml. The ingredients are combined together at a temperature below the cloud point, then the formulation is thermally cycled to room temperature (above the cloud point) several times, diluted, and filtered according to the protocol outlined in FIG. 3.
Plasmids comprising wild-type or codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, and or other proteins, e.g., cytokines, are mixed together at desired proportions in PBS to achieve 6.4 mg/ml total DNA. This plasmid cocktail is placed into the 15 ml round bottom flask fitted with a magnetic stirring bar, and for the formulation containing 50 mg/ml CRL 1005, 3.13 ml of a solution containing about 3.2 mg/ml of e.g., S1 encoding plasmid and about 3.2 mg/ml S2 encoding plasmid (about 6.4 mg/ml total DNA) is placed into the 15 ml round bottom flask fitted with a magnetic stirring bar, and the solutions are stirred with moderate speed, in an ice bath on top of a stirrer/hotplate (hotplate off) for 10 minutes. CRL 1005 (136 μl for 34 mg/ml final concentration, and 100 μl for 50 mg/ml final concentration) is then added using a 200 μl positive displacement pipette and the solution is stirred for a further 30 minutes on ice. Solutions of 1.6 mM and 1.8 mM BAK are prepared in PBS, and 739 μl of 1.6 mM and 675 μl of 1.8 mM are then added dropwise, slowly, to the stirring poloxamer solutions with concentrations of 34 mg/ml or 50 mg/ml mixtures, respectively, over 1 min using a 1 ml pipette. The solutions at this point are clear since they are below the cloud point of the poloxamer and are stirred on ice for 30 min. The ice baths are then removed; the solutions stirred at ambient temperature for 15 minutes to produce cloudy solutions as the poloxamer passes through the cloud point.
The flasks are then placed back into the ice baths and stirred for a further 15 minutes to produce clear solutions as the mixtures cooled below the poloxamer cloud point. The ice baths are again removed and the solutions stirred for a further 15 minutes. Stirring for 15 minutes above and below the cloud point (total of 30 minutes), is defined as one thermal cycle. The mixtures are cycled two more times.
In the meantime, two Steriflip® 50 ml disposable vacuum filtration devices, each with a 0.22 μm Millipore Express® membrane (available from Millipore, cat # SCGP00525) are placed in an ice bucket, with a vacuum line attached and left for 1 hour to allow the devices to equilibrate to the temperature of the ice. The poloxamer formulations are then diluted to 2.5 mg/ml DNA with PBS and filtered under vacuum.
The resulting formulations may be used immediately, or may be transferred to glass vials, cooled below the cloud point, and frozen at −80° C. for use at a later time.
A Simplified Method Without Thermal Cycling
This example describes a simplified preparation of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005, and 5 mg/ml of DNA in a total volume of 2.0 ml. The ingredients are combined together at a temperature below the cloud point and then the formulation is simply filtered and then used or stored, according to the protocol outlined in FIG. 4.
A 0.77 mM solution of BAK is prepared in PBS, and 780 μl of the solution is placed into a 15 ml round bottom flask fitted with a magnetic stirring bar, and the solution is stirred with moderate speed, in an ice bath on top of a stirrer/hotplate (hotplate off) for 15 minutes. CRL 1005 (15 μl) is then added using a 100 μl positive displacement pipette and the solution is stirred for a further 60 minutes on ice. Plasmids comprising coding regions or codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, and or other proteins, e.g., cytokines, are mixed together at desired proportions in PBS to achieve a final concentration of about 8.3 mg/ml total DNA. This plasmid cocktail is added dropwise, slowly, to the stirring solution over 1 min using a 5 ml pipette. The solution at this point (on ice) is clear since it is below the cloud point of the poloxamer and is further stirred on ice for 15 min.
In the meantime, one Steriflip® 50 ml disposable vacuum filtration device, with a 0.22 μm Millipore Express® membrane (available from Millipore, cat # SCGP00525) is placed in an ice bucket, with a vacuum line attached and left for 1 hour to allow the device to equilibrate to the temperature of the ice. The poloxamer formulation is then filtered under vacuum, below the cloud point and then allowed to warm above the cloud point. The resulting formulations may be used immediately, or may be transferred to glass vials, cooled below the cloud point and then frozen at −80° C. for use at a later time.
Example 5
Animal Immunizations
The immunogenicity of the various SARS-CoV expression products encoded polynucleotides and codon-optimized polynucleotides described herein are initially evaluated based on each plasmid's ability to mount an immune response in vivo. Plasmids are tested individually and in combinations by injecting single constructs as well as multiple constructs. Immunizations are initially carried out in animals, such as mice, rabbits, goats, sheep, domestic cats, non-human primates, or other suitable animal, by intramuscular (IM) injections. Serum is collected from immunized animals, and the antigen specific antibody response is quantified by ELISA assay using purified immobilized antigen proteins in a protein—immunized subject antibody—anti-species antibody type assay, according to standard protocols. The tests of immunogenicity further include measuring antibody titer, neutralizing antibody titer, T-cell proliferation, T-cell secretion of cytokines, and cytolytic T cell responses. Correlation to protective levels of the immune responses in humans are made according to methods well known by those of ordinary skill in the art. See above.
A. DNA Formulations
Plasmid DNA is formulated with a poloxamer by any of the methods described in Example 3. Alternatively, plasmid DNA is prepared as described above and dissolved at a concentration of about 0.1 mg/ml to about 10 mg/ml, preferably about 1 mg/ml, in PBS with or without transfection-facilitating cationic lipids, e.g., DMRIE/DOPE at a 4:1 DNA:lipid mass ratio. Alternative DNA formulations include 150 mM sodium phosphate instead of PBS, adjuvants, e.g., VaxfectinTM at a 4:1 DNA: VaxfectinTM mass ratio, mono-phosphoryl lipid A (detoxified endotoxin) from S. minnesota (MPL) and trehalosedicorynomycolateAF (TDM), in 2% oil (squalene)-Tween 80-water (MPL+TDM, available from Sigma/Aldrich, St. Louis, Mo., (catalog # M6536)), a solubilized mono-phosphoryl lipid A formulation (AF, available from Corixa), or (±)-N-(3-Acetoxypropyl)-N,N-dimethyl-2,3-bis(octyloxy)-1-propanaminium chloride (compound # VC1240) (see Shriver, J. W. et al., Nature 415:331-335 (2002), and P.C.T. Publication No. WO 02/00844 A2, each of which is incorporated herein by reference in its entirety).
B. Animal Immunizations
Plasmid constructs comprising codon-optimized or non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are injected into BALB/c mice as single plasmids or as cocktails of two or more plasmids, as either DNA in PBS or formulated with the poloxamer-based delivery system: 2 mg/ml DNA, 3 mg/ml CRL 1005, and 0.1 mM BAK. Groups of 10 mice are immunized three times, at biweekly intervals, and serum is obtained to determine antibody titers to each of the antigens. Groups are also included in which mice are immunized with a trivalent preparation, containing each of three plasmid constructs expressing any of the SARS Co-V polypeptides, e.g., soluble, extracellular S1, M, and N polypeptides, in equal mass.
An example of an immunization schedule is as follows:













Day −3
Pre-bleed


Day 0
Plasmid injections, intramuscular, bilateral in rectus femoris,



5-50 μg/leg


Day 20
Serum Collection


Day 21
Plasmid injections, intramuscular, bilateral in rectus femoris,



5-50 μg/leg


Day 48
Serum Collection


Day 49
Plasmid injections, intramuscular, bilateral in rectus femoris,



5-50 μg/leg


Day 59
Serum collection









Serum antibody titers, at the various time points are determined by ELISA, using as the antigen SARS-CoV protein preparations including, but not limited to, purified recombinant proteins, transfection supernatants and lysates from mammalian or insect cells transfected with the various plasmids described herein, or live, inactivated, or lysed SARS-CoV virus.
C. Immunization of Mice with Vaccine Formulations Using a VAXFECTINTM Adjuvant
VAXFECTINTM (a 1:1 molar ratio of the cationic lipid VC1052 and the neutral co-lipid DPyPE) is a synthetic cationic lipid formulation which has shown promise for its ability to enhance antibody titers against an antigen when administered with DNA encoding the antigen intramuscularly to mice. See Hartikka et al. “Vaxfectin Enhances the Humoral Response to Plasmid DNA-encoded Antigens.” Vaccine 19: 1911-1923 (2001).
In mice, intramuscular injection of VAXFECTINTM formulated with, for example, DNA encoding the IAV NP protein increased antibody titers to NP up to 20-fold to levels that could not be reached with DNA alone. In rabbits, complexing DNA with VAXFECTINTM enhanced antibody titers up to 50-fold. Thus, VAXFECTINTM shows promise as a delivery system and as an adjuvant in a DNA vaccine.
VaxfectinTMmixtures are prepared by mixing chloroform solutions of VC1052 cationic lipid with chloroform solutions of DpyPE neutral co-lipid. Dried films are prepared in 2 ml sterile glass vials by evaporating the chloroform under a stream of nitrogen, and placing the vials under vacuum overnight to remove solvent traces. Each vial contains 1.5 μmole each of VC1052 and DPyPE. Liposomes are prepared by adding sterile water followed by vortexing. The resulting liposome solution is mixed with DNA at a phosphate mole:cationic lipid mole ratio of 4:1.
Plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are mixed together at desired proportions in PBS to achieve a final concentration of at 1.0 mg/ml. The plasmid cocktail, as well as the controls, are formulated with VAXFECTINTM. Groups of 5 Balb/c female mice are injected bilaterally in the rectus femoris muscle with 50 μl of DNA solution (100 μl total/mouse), on days 1 and 21 and 49 with each formulation. Mice are bled for serum on days 0 (prebleed), 20 (bleed 1), and 41 (bleed 2), and 62 (bleed 3), and up to 40 weeks post-injection. Antibody titers to the various SARS CoV proteins encoded by the plasmid DNAs are measured by ELISA as described elsewhere herein.
Cytolytic T-cell responses are measured as described in Hartikka et al. “Vaxfectin Enhances the Humoral Response to Plasmid DNA-encoded Antigens.” Vaccine 19: 1911-1923 (2001) and is incorporated herein in its entirety by reference. Standard ELISPOT technology is used for the CD4+ and CD8+ T-cell assays as described in Example 6, part A.
D. Production of SARS-CoV Antisera in Animals
Plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are prepared according to the immunization scheme described above and injected into a suitable animal for generating polyclonal antibodies. Serum is collected and the antibody titered as above.
Monoclonal antibodies are also produced using hybridoma technology. Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur. J. Immunol. 6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976); Hammerling, et al., in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981), pp. 563-681, each of which is incorporated herein by reference in its entirety. In general, such procedures involve immunizing an animal (preferably a mouse) as described above. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (Sp2/0), available from the American Type Culture Collection, Rockville, Md. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al., Gastroenterology 80:225-232 (1981), incorporated herein by reference in its entirety. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the various SARS-CoV proteins.
Alternatively, additional antibodies capable of binding to SARS-CoV proteins described herein may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, various SARS-CoV-specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the SARS-CoV protein-specific antibody can be blocked by the cognate SARS-CoV protein. Such antibodies comprise anti-idiotypic antibodies to the SARS-CoV protein-specific antibody and can be used to immunize an animal to induce formation of further SARS-CoV-specific antibodies.
It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, SARS-CoV polypeptide binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
It may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques 4:214 (1986); Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi, et al., EP 171496; Morrison, et al., EP 173494; Neuberger, et al., WO 8601533; Robinson, et al., WO 8702671; Boulianne, et al., Nature 312:643 (1984); Neuberger, et al., Nature 314:268 (1985).
These antibodies are used, for example, in diagnostic assays, as a research reagent, or to further immunize animals to generate SARS-CoV-specific anti-idiotypic antibodies. Non-limiting examples of uses for anti-SARS-CoV antibodies include use in Western blots, ELISA (competitive, sandwich, and direct), immunofluorescence, immunoelectron microscopy, radioimmunoassay, immunoprecipitation, agglutination assays, immunodiffision, immunoelectrophoresis, and epitope mapping. Weir, D. Ed. Handbook of Experimental Immunology, 4th ed. Vols. I and II, Blackwell Scientific Publications (1986).
Example 6
Mouse and Rabbit Immunogenicity Studies to SARS-CoV Antigens
Balb/c mice were injected intramuscularly bilaterally with 100 μg of SARS-CoV antigen expressing plasmid. VR9204, VR9208, VR9209, VR9210, VR9219 plasmids were formulated in PBS and DMRIE:DOPE at a 4:1 DNA:lipid mass ratio.
New Zealand white rabbits were injected intramuscularly bilaterally with 1 mg of SARS-CoV antigen expressing plasmid (VR9219 (N antigen) or VR9204 (S1 fragment antigen), formulated with DMRIE:DOPE, on days 1, 28 and 56. Rabbit sera anti-antigen titers were determined by ELISA assay. The ELISA assay was performed according to standard protocols. ELISA plates used in the assay were coated with cell culture supernatants, from cells transfected with the a SARS-CoV antigen plasmid. Sera from rabbits which had been injected with the corresponding plasmid was then applied to the plates. Bound rabbit antibodies were detected using an alkaline phosphatase-modified donkey anti-rabbit IgG monoclonal antibody (Jackson Immuno Research; Cat No. 711-055-152). Bound antibodies were detected by standard colorimetric method after 2.5 hours of incubation with chromogenic substrates. Optical Density was determined at a wavelength of 405 nm. The results of the ELISA assay are summarized below.
Data shown in Table 20 demonstrate the presence of anti-nucleocapsid antibodies at day 21 in rabbits injected with plasmid VR9219 expressing full-length SARS-CoV nucleocapsid antigen. The antibody titers reach a plateau at day 42 (1:400 dilution).
In another experiment, rabbits were injected with plasmid VR9204, which expresses a fragment of the SARS-CoV Spike S1 domain. ELISA plates were coated with in vitro-produced full length-secreted Spike protein from cells transfected with plasmid VR9210. Antibodies IMG-542 and IMG-557, which recognize amino acids 288-303 and 1124-1140 of the SARS-CoV spike protein respectively (available from lmgenex, San Diego, Calif.), were used as positive controls in the ELISA assay. An ELISA plate coated with supernatant from VR1012-transfected VM92 cells was used as a negative control in the ELISA assay. The data shown in Table 20 demonstrate the presence of anti-Spike antibodies at days 42 and 50 after injection.







TABLE 20







Anti-SARS CoV Antigen Titers (Rabbits)










Nucleocapsid
S1 fragment



Plamsid - VR9219
Plasmid - VR9204



 1/400 sera dilution
 1/200 sera dilution















Day 21
0.92
0.22



Day 42
3.9
0.74



Day 50
NA
0.51



Day 80
4
NA



Pre-bleed
0.13
0.19



IMG-542
NA
0.44



IMG-557
NA
2.41



VR1012
0.15
0.21










Example 7
Mucosal Vaccination and Electrically Assisted Plasmid Delivery
A. Mucosal DNA Vaccination
Plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, (100 μg/50 μl total DNA) are delivered to BALB/c mice at 0, 2 and 4 weeks via i.m., intranasal (i.n.), intravenous (i.v.), intravaginal (i.vag.), intrarectal (i.r.) or oral routes. The DNA is delivered unformulated, formulated with the cationic lipids DMRIE/DOPE (DD) or GAP-DLRIE/DOPE (GD), or formulatated with a poloxamer as described in Example 3. As endpoints, serum IgG titers against the various SARS-CoV antigens are measured by ELISA and splenic T-cell responses are measured by antigen-specific production of IFN-gamma and IL-4 in ELISPOT assays. Standard chromium release assays are used to measure specific cytotoxic T lymphocyte (CTL) activity against the various SARS-CoV antigens. In addition, IgG and IgA responses against the various SARS-CoV antigens are analyzed by ELISA of vaginal washes.
B. Electrically-Assisted Plasmid Delivery
In vivo gene delivery may be enhanced through the application of brief electrical pulses to injected tissues, a procedure referred to herein as electrically-assisted plasmid delivery. See, e.g., Aihara, H. & Miyazaki, J. Nat. Biotechnol. 16:867-70 (1998); Mir, L. M. et al., Proc. Natl Acad. Sci. USA 96:4262-67 (1999); Hartikka, J. et al., Mol. Ther. 4:407-15 (2001); and Mir, L. M. et al.; Rizzuto, G. et al., Hum Gene Ther 11:1891-900 (2000); Widera, G. et al, J. of Immuno. 164: 4635-4640 (2000). The use of electrical pulses for cell electropermeabilization has been used to introduce foreign DNA into prokaryotic and eukaryotic cells in vitro. Cell permeabilization can also be achieved locally, in vivo, using electrodes and optimal electrical parameters that are compatible with cell survival.
The electroporation procedure can be performed with various electroporation devices. These devices include external plate type electrodes or invasive needle/rod electrodes and can possess two electrodes or multiple electrodes placed in an array. Distances between the plate or needle electrodes can vary depending upon the number of electrodes, size of target area and treatment subject.
The TriGrid needle array, used in examples described herein, is a three electrode array comprising three elongate electrodes in the approximate shape of a geometric triangle. Needle arrays may include single, double, three, four, five, six or more needles arranged in various array formations. The electrodes are connected through conductive cables to a high voltage switching device that is connected to a power supply.
The electrode array is placed into the muscle tissue, around the site of nucleic acid injection, to a depth of approximately 3 mm to 3 cm. The depth of insertion varies depending upon the target tissue and the size of the patient receiving electroporation. After injection of foreign nucleic acid, such as plasmid DNA, and a period of time sufficient for distribution of the nucleic acid, square wave electrical pulses are applied to the tissue. The amplitude of each pulse ranges from about 100 volts to about 1500 volts, e.g., about 100 volts, about 200 volts, about 300 volts, about 400 volts, about 500 volts, about 600 volts, about 700 volts, about 800 volts, about 900 volts, about 1000 volts, about 1100 volts, about 1200 volts, about 1300 volts, about 1400 volts, or about 1500 volts or about 1-1.5 kV/cm, based on the spacing between electrodes. Each pulse has a duration of about 1 μs to about 1000 μs, e.g., about 1 μs, about 10 μs, about 50 μs, about 100 μs, about 200 μs, about 300 μs, about 400 μs, about 500 μs, about 600 μs, about 700 μs, about 800 μs, about 900 μs, or about 1000 μs, and a pulse frequency on the order of about 1-10 Hz. The polarity of the pulses may be reversed during the electroporation procedure by switching the connectors to the pulse generator. Pulses are repeated multiple times. The electroporation parameters (e.g., voltage amplitude, duration of pulse, number of pulses, depth of electrode insertion and frequency) will vary based on target tissue type, number of electrodes used and distance of electrode spacing, as would be understood by one of ordinary skill in the art.
Immediately after completion of the pulse regimen, subjects receiving electroporation can be optionally treated with membrane stabilizing agents to prolong cell membrane permeability as a result of the electroporation.
Examples of membrane stabilizing agents include, but are not limited to, steroids (e.g., dexamethasone, methylprednisone and progesterone), angiotensin II and vitamin E. A single dose of dexamethasone, approximately 0.1 mg per kilogram of body weight, should be sufficient to achieve a beneficial affect.
EAPD techniques such as electroporation can also be used for plasmids contained in liposome formulations. The liposome—plasmid suspension is administered to the animal or patient and the site of injection is treated with a safe but effective electrical field generated, for example, by a TriGrid needle array. The electroporation may aid in plasmid delivery to the cell by destabilizing the liposome bilayer so that membrane fusion between the liposome and the target cellular structure occurs. Electroporation may also aid in plasmid delivery to the cell by triggering the release of the plasmid, in high concentrations, from the liposome at the surface of the target cell so that the plasmid is driven across the cell membrane by a concentration gradient via the pores created in the cell membrane as a result of the electroporation.
Female BALB/c mice aged 8-10 weeks are anesthetized with inhalant isoflurane and maintained under anesthesia for the duration of the electroporation procedure. The legs are shaved prior to treatment. Plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are administered to BALB/c mice (n=10) via unilateral injection in the quadriceps with 25 μg total of a plasmid DNA per mouse using an 0.3 cc insulin syringe and a 26 gauge, 1⁄2 length needle fitted with a plastic collar to regulate injection depth. Approximately one minute after injection, electrodes are applied. Modified caliper electrodes are used to apply the electrical pulse. See Hartikka J. et al. Mol Ther 188:407-415 (2001). The caliper electrode plates are coated with conductivity gel and applied to the sides of the injected muscle before closing to a gap of 3 mm for administration of pulses. EAPD is applied using a square pulse type at 1-10 Hz with a field strength of 100-500 V/cm, 1-10 pulses, of 10-100 ms each.
Mice are vaccinated±EAPD at 0, 2 and 4 weeks. As endpoints, serum IgG titers against the various SARS-CoV antigens are measured by ELISA and splenic T-cell responses are measured by antigen-specific production of IFN-gamma and IL-4 in ELISPOT assays. Standard chromium release assays are used to measure specific cytotoxic T lymphocyte (CTL) activity against the various SARS-CoV antigens.
Rabbits (n=3) are given bilateral injections in the quadriceps muscle with plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector. The implantation area is shaved and the TriGrid electrode array is implanted into the target region of the muscle. 3.0 mg of plasmid DNA is administered per dose through the injection port of the electrode array. An injection collar is used to control the depth of injection. Electroporation begins approximately one minute after injection of the plasmid DNA is complete. Electroporation is administered with a TriGrid needle array, with eletrodes evenly spaced 7 mm apart, using an Ichor TGP-2 pulse generator. The array is inserted into the target muscle to a depth of about 1 to 2 cm. 4-8 pulses are administered. Each pulse has a duration of about 50-100 μs, an amplitude of about 1-1.2 kV/cm and a pulse frequency of 1 Hz. The injection and electroporation may be repeated.
Sera are collected from vaccinated rabbits at various time points. As endpoints, serum IgG titers against the various SARS-CoV antigens are measured by ELISA and PBMC T-cell proliferative responses are measured by antigen-specific production of IFN-gamma and IL-4 in ELISPOT assays or by quantification of intracellular cytokine staining. Standard chromium release assays are used to measure specific cytotoxic T lymphocyte (CTL) activity against the various SARS-CoV antigens.
To test the effect of electroporation on therapeutic protein expression in non-human primates, male or female rhesus monkeys are given either 2 or 6 EAPD-assisted i.m. injections of plasmid constructs comprising codon-optimized and/or non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, (0.1 to 10 mg DNA total per animal). Target muscle groups include, but are not limited to, bilateral rectus fermoris, cranial tibialis, biceps, gastrocenemius or deltoid muscles. The target area is shaved and a needle array, comprising between 4 and 10 electrodes, spaced between 0.5-1.5 cm apart, is implanted into the target muscle. Once injections are complete, a sequence of brief electrical pulses is applied to the electrodes implanted in the target muscle using an Ichor TGP-2 pulse generator. The pulses have an amplitude of approximately 120-200V. The pulse sequence is completed within one second. During this time, the target muscle may make brief contractions or twitches. The injection and electroporation may be repeated.
Sera are collected from vaccinated monkeys at various time points. As endpoints, serum IgG titers against the various SARS-CoV antigens are measured by ELISA and PBMC T-cell proliferative responses are measured by antigen-specific production of IFN-gamma and IL-4 in ELISPOT assays or by quantification of intracellular cytokine staining Standard chromium release assays are used to measure specific cytotoxic T lymphocyte (CTL) activity against the various SARS-CoV antigens.
Example 8
Combinatorial DNA Vaccine Using Heterologous Prime-Boost Vaccination
This Example describes vaccination with a combinatorial formulation including one or more polynucleotides comprising at least one codon-optimized or non-codon optimized coding regions encoding a SARS-CoV protein or fragment, variant, or derivative thereof prepared with an adjuvant and/or transfection facilitating agent; and also an isolated SARS-CoV protein or fragment, variant, or derivative thereof. Thus, antigen is provided in two forms. The exogenous isolated protein stimulates antigen specific antibody and CD4+ T-cell responses, while the polynucleotide-encoded protein, produced as a result of cellular uptake and expression of the coding region, stimulates a CD8+ T-cell response. Unlike conventional “prime-boost” vaccination strategies, this approach provides different forms of antigen in the same formulation. Because antigen expression from the DNA vaccine doesn't peak until 7-10 days after injection, the DNA vaccine provides a boost for the protein component. Furthermore, the formulation takes advantage of the immunostimulatory properties of the bacterial plasmid DNA.
A. Formulation Determinations for SARS-CoV proteins
This example mainly describes this procedure using an S2 subunit protein; however, the methods described herein are applicable to any SARS-CoV subunit protein combined with any polynucleotide vaccine formulation. For example any polynucleotide comprising a codon-optimized or non-codon-optimized coding region encoding any SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg may be combined with any subunit SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg. Because only a small amount of protein is needed in this method, it is conceivable that the approach could be used to reduce the dose of other types of protein or antibody based vaccines, not described herein, when administered in combination with the polynucleotides and polypeptides of the present invention. The decreased dosing of other vaccines would allow for the increased availability of scarce or expensive vaccines. This feature would be particularly important for vaccines against pandemic SARS or biological warfare agents.
In this example, an injection dose of 10 μg SARS-CoV S protein, subunit 2 (S2) DNA per mouse, prepared essentially as described in Example 2 and in Ulmer, J. B., et al., Science 259:1745-49 (1993) and Ulmer, J. B. et al., J Virol. 72:5648-53 (1998) is pre-determined in dose response studies to induce T cell and antibody responses in the linear range of the dose response and results in a response rate of greater than 95% of mice injected. Each formulation, either a plasmid comprising a codon-optimized or non-codon-optimized coding region encoding S2 alone (“S2 DNA”), or S2 DNA+/−S2 protein formulated with Ribi I or the cationic lipids, DMRIE:DOPE or Vaxfectin TMis prepared in the recommended buffer for that vaccine modality. For injections with S2 DNA formulated with cationic lipid, the DNA is diluted in 2×PBS to 0.2 mg/ml+/−purified recombinant S2 protein (produced in baculovirus as described in Example 2) at 0.08 mg/ml. Each cationic lipid is reconstituted from a dried film by adding 1 ml of sterile water for injection (SWFI) to each vial and vortexing continuously for 2 min., then diluted with SWFI to a final concentration of 0.15 mM. Equal volumes of S2 DNA (+/−S2 protein) and cationic lipid are mixed to obtain a DNA to cationic lipid molar ratio of 4:1. For injections with DNA containing Ribi I adjuvant (Sigma), Ribi I is reconstituted with saline to twice the final concentration. Ribi I (2×) is mixed with an equal volume of S2 DNA at 0.2 mg/ml in saline+/−S2 protein at 0.08 mg/ml. For immunizations without cationic lipid or Ribi, S2 DNA is prepared in 150 mM sodium phosphate buffer, pH 7.2. For each experiment, groups of 9 BALB/c female mice at 7-9 weeks of age are injected with 50 μl of S2 DNA+/−S2 protein, cationic lipid or Ribi I. Injections are given bilaterally in each rectus femoris at day 0 and day 21. The mice are bled by OSP on day 20 and day 33 and serum titers of individual mice are measured.
S2 specific serum antibody titers are determined by indirect binding ELISA using 96 well ELISA plates coated overnight at 4° C. with purified recombinant S2 protein at 0.5 μg per well in BBS buffer pH 8.3. S2-coated wells are blocked with 1% bovine serum albumin in BBS for 1 h at room temperature. Two-fold serial dilutions of sera in blocking buffer are incubated for 2 h at room temperature and detected by incubating with alkaline phosphatase conjugated (AP) goat anti-mouse IgG-Fc (Jackson Immunoresearch, West Grove, Pa.) at 1:5000 for 2 h at room temperature. Color is developed with 1 mg/ml para-nitrophenyl phosphate (Calbiochem, La Jolla, Calif.) in 50 mM sodium bicarbonate buffer, pH 9.8 and 1 MM MgCl2 and the absorbance read at 405 nm. The titer is the reciprocal of the last dilution exhibiting an absorbance value 2 times that of pre-bleed samples.
Standard ELISPOT technology, used to identify the number of interferon gamma (IFN-γ) secreting cells after stimulation with specific antigen (spot forming cells per million splenocytes, expressed as SFU/million), is used for the CD4+ and CD8+ T-cell assays. For the screening assays, 3 mice from each group are sacrificed on day 34, 35, and 36. At the time of collection, spleens from each group are pooled, and single cell suspensions made in cell culture media using a dounce homogenizer. Red blood cells are lysed, and cells washed and counted. For the CD4+ and CD8+ assays, cells are serially diluted 3-fold, starting at 106 cells per well and transferred to 96 well ELISPOT plates pre-coated with anti-murine IFN-γ monoclonal antibody. Spleen cells are stimulated with the H-2Kd binding peptide, TYQRTRALV (SEQ ID NO: 55) at 1 μg/ml and recombinant murine IL-2 at 1 U/ml for the CD8+ assay and with purified recombinant S2 protein at 20 μg/ml for the CD4+ assay. Cells are stimulated for 20-24 hours at 37° C. in 5% CO2, then the cells are washed out and biotin labeled anti-IFN-γ monoclonal antibody added for a 2 hour incubation at room temperature. Plates are washed and horseradish peroxidase-labeled avidin is added. After a 1-hour incubation at room temperature, AEC substrate is added and “spots” developed for 15 min. Spots are counted using the Immunospot automated spot counter (C.T.L. Inc., Cleveland Ohio.). Thus, CD4+ and CD8+ responses are measured in three separate assays, using spleens collected on each of three consecutive days.
B. Determining Combinatorial Formulations with SARS-CoV Polynucleotide Constructs
Plasmid constructs comprising codon-optimized or non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are used in the prime-boost compositions described herein. For the prime-boost modalities, the same protein may be used for the boost, e.g., DNA encoding S2 with S2 protein, or a heterologous boost may be used, e.g., DNA encoding S2 with an M protein boost. Each formulation, the plasmid comprising a coding region for the SARS-CoV protein alone, or the plasmid comprising a coding region for the SARS-CoV protein plus the isolated protein, is formulated with Ribi I or the cationic lipids, DMRIE:DOPE or VaxfectinTM. The formulations are prepared in the recommended buffer for that vaccine modality. Exemplary formulations, using S2 as an example, are described herein. Other plasmid/protein formulations, including multivalent formulations, can be easily prepared by one of ordinary skill in the art by following this example. For injections with DNA formulated with cationic lipid, the DNA is diluted in 2×PBS to 0.2 mg/ml+/−purified recombinant SARS-CoV protein at 0.08 mg/ml. Each cationic lipid is reconstituted from a dried film by adding 1 ml of sterile water for injection (SWFI) to each vial and vortexing continuously for 2 min., then diluted with SWFI to a final concentration of 0.15 mM. Equal volumes of S2 DNA (+/−S2 protein) and cationic lipid are mixed to obtain a DNA to cationic lipid molar ratio of 4:1. For injections with DNA containing Ribi I adjuvant (Sigma), Ribi I is reconstituted with saline to twice the final concentration. Ribi I (2×) is mixed with an equal volume of S2 DNA at 0.2 mg/ml in saline+/−S2 protein at 0.08 mg/ml. For immunizations without cationic lipid or Ribi, S2 DNA is prepared in 150 mM sodium phosphate buffer, pH 7.2. For each experiment, groups of 9 BALB/c female mice at 7-9 weeks of age are injected with 50 .mu.l of S2 DNA+/−S2 protein, cationic lipid or Ribi I. The formulations are administered to BALB/c mice (n=10) via bilateral injection in each rectus femoris at day 0 and day 21.
The mice are bled on day 20 and day 33, and serum titers of individual mice to the various SARS-CoV antigens are measured. Serum antibody titers specific for the various SARS-CoV antigens are determined by ELISA. Standard ELISPOT technology, used to identify the number of interferon gamma (IFN-γ) secreting cells after stimulation with specific antigen (spot forming cells per million splenocytes, expressed as SFU/million), is used for the CD4+ and CD8+ T-cell assays using 3 mice from each group vaccinated as above, sacrificed on day 34, 35, and 36, post vaccination.
Example 9
Challenge in Non-Human Primates
The purpose of these studies is to evaluate three or more of the optimal plasmid DNA vaccine formulations for immunogenicity in non-human primates. Prelmimary challenge experiments may be carried out in toher suitable animal modes, for example birds as described below, or in domestic cats. Rhesus or cynomologus monkeys (6/group) are vaccinated with plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding SARS-CoV proteins, for example, SARS-CoV S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2 proteins, fusions thereof, or fragments, variants or derivatives of such proteins either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, intramuscularly 0.1 to 2 mg DNA combined with cationic lipid, and/or poloxamer and/or aluminum phosphate based or other adjuvants at 0, 1 and 4 months.
Blood is drawn twice at baseline and then again at the time of and two weeks following each vaccination, and then again 4 months following the last vaccination. At 2 weeks post-vaccination, plasma is analyzed for humoral response and PBMCs are monitored for cellular responses, by standard methods described herein. Animals are monitored for 4 months following the final vaccination to determine the durability of the immune response.
Animals are challenged within 2-4 weeks following the final vaccination. Animals are challenged intratracheally with the suitable dose of virus based on preliminary challege studies. Nasal swabs, pharyngeal swabs and lung lavages are collected at days 0, 2, 4, 6, 8 and 11 post-challenge and will be assayed for cell-free virus titers on monkey kidney cells. After challenge, animals are monitored for clinical symptoms, e.g., rectal temperature, body weight, leukocyte counts, and in addition, hematocrit and respiratory rate. Oropharyngeal swab samples are taken to allow determination of the length of viral shedding. Illness is scored using a variety of conventional illness scoring methods such as the system developed by Berendt & Hall (Infect Immun 16:476-479 (1977)), and will be analyzed by analysis of variance and the method of least significant difference.
Example 10
Challenge in Birds
In this example, various vaccine formulations of the present invention are tested in a chicken SARS-CoV model. For these studies a SARS-CoV is used for the challenge. Plasmid constructs comprising codon-optimized and non-codon-optimized coding regions encoding S, S1, S2, N, M, E, soluble S, soluble S1, soluble S2, soluble TPA-S, soluble TPA-S1, and soluble TPA-S2, as described herein, fusions; or alternatively, coding regions (either codon-optimized or non-codon optimized) encoding various SARS-CoV proteins or fragments, variants or derivatives, either alone or as fusions with a carrier protein, e.g., HBcAg, as well as various controls, e.g., empty vector, are formulated with cationic lipid, and/or poloxamer and/or aluminum phosphate based or other adjuvants. The vaccine formulations are delivered at a dose of about 1-10 μg, delivered IM into the defeathered breast area, at 0 and 1 month. The animals are bled for antibody results 3 weeks following the second vaccine. Antibody titers against the various SARS-CoV antigens are determined using techniques described in the literature. See, e.g., Kodihalli S. et al., Vaccine 18:2592-9 (2000). The birds are challenged intranasally with 0.1 mL containing 100 LD50 3 weeks post second vaccination. The birds are monitored daily for 10 days for disease symptoms, which include gasping, coughing and nasal discharge, wet eyes and swollen sinuses, reduced food consumption and weight loss. Tracheal and cloacal swabs are taken 4 days following challenge for virus titration.
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and any compositions or methods which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

## Claims

1. A composition comprising an isolated polynucleotide comprising a nucleic acid fragment which encodes amino acids 1-417 of a SARS-CoV-S1 polypeptide, and a carrier,
wherein said nucleic acid fragment is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 45, SEQ ID NO: 50 and SEQ ID NO: 68
wherein said composition, when administered to a vertebrate in a sufficient amount, elicits a detectable immune response against SARS CoV; and
wherein said composition further comprises a component of SARS-CoV selected from the group consisting of inactivated virus, attenuated virus, a viral vector expressing an isolated SARS-CoV virus polypeptide, and an isolated polypeptide from a SARS-CoV virus protein, fragment, variant or derivative thereof and/or one or more additional polynucleotides comprising at least one coding region encoding a SARS-CoV polypeptide, or a fragment, variant, or derivative thereof.

2. The composition of claim 1 wherein the isolated polynucleotide further comprises a heterologous nucleic acid.

3. The composition of claim 2, wherein said heterologous nucleic acid encodes polypeptide fused to said amino acids encoded by said nucleic acid fragment.

4. The composition of claim 3, wherein said heterologous polypeptide comprises a small self assembly polypeptide, and wherein said heterologous polypeptide self assembles into multimers.

5. The composition of claim 3, wherein said heterologous polypeptide is a secretory signal peptide.

6. The composition of claim 1, wherein the isolated polynucleotide is DNA, and wherein said nucleic acid fragment is operably associated with a promoter.

7. The composition of claim 1, wherein the isolated polynucleotide is messenger RNA (mRNA).

8. The composition of claim 1 comprising a vector comprising the isolated polynucleotide.

9. The composition of claim 8 wherein the vector is a plasmid.

10. The composition of claim 9, wherein said vector is VR9208.

11. The composition of claim 9, wherein said vector is VR9204.

12. The composition of claim 1, further comprising a component selected from the group consisting of an adjuvant and a transfection facilitating compound.

13. The composition of claim 12, wherein said adjuvant is selected from the group consisting of:
(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium bromide (GAP-DMORIE) and a neutral lipid;
a cytokine;
mono-phosphoryl lipid A and trehalosedicorynomycolateAF (MPL+TDM);
a solubilized mono-phosphoryl lipid A formulation; and
CRL1005/BAK.

14. The composition of claim 12, comprising the transfection facilitating compound (±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide) (DMRIE).

15. A method for raising a detectable immune response to a SARS-CoV polypeptide, comprising administering to a vertebrate the composition of claim 12 in an amount sufficient to elicit a detectable immune response to the encoded polypeptide.

16. A method for raising a detectable immune response to a SARS-CoV polypeptide, comprising administering to a vertebrate the composition of claim 1 in an amount sufficient to elicit a detectable immune response to the encoded polypeptide.