# Patent

## Title
Infectious DNA vaccines against chikungunya virus

## Abstract

Described herein are i-DNATM vectors and vaccines and methods for using the same. The i-DNATM generates live attenuated vaccines in eukaryotic cells in vitro or in vivo for pathogenic RNA viruses, particularly chikungunya virus (CHIKV). When iDNA is injected into the vaccine recipient, RNA of live attenuated virus is generated by in vivo transcription in the recipient's tissues. This initiates production of progeny attenuated viruses in the tissues of the vaccine recipient, as well as elicitation of an effective immune response protecting against wild-type, non-attenuated virus.

## Background
CROSS-REFERENCE TO PRIOR APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/519,948, filed Nov. 7, 2012, now allowed, which is a National Stage of PCT/US2011/000001, filed Jan. 3, 2011, and designating the United States (published in English on Jul. 7, 2011, as WO 2011/082388 A2; the title and abstract were also published in English), which claims priority of U.S. Provisional Patent Application No. 61/291,682, filed Dec. 31, 2009, each hereby expressly incorporated by reference in its entirety and each assigned to the assignee hereof.


GOVERNMENT INTERESTS
The U.S. Government provided the inventors with materials and/or reagents that may be related to the subject matter of this application. Accordingly, the U.S. government may have certain rights in the subject matter.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 19, 2015, is named 20009201-0031_SL.txt and is 110,235 bytes in size.
FIELD
Live attenuated and DNA vaccines against chikungunya virus and system and methods for making and administering such vaccines.
BACKGROUND
A variety of vaccines and systems and methods for making and administering the same have been suggested. However, such vaccines, systems and methods are not optimal.
SUMMARY
Described herein are vectors comprising DNA encoding an infectious RNA molecule and an RNA polymerase promoter, where the DNA encoding an infectious RNA molecule is operably linked to the RNA polymerase promoter and the infectious RNA molecules encodes a chikungunya virus (CHIKV). In certain embodiments, the CHIKV is non-pathogenic. Also described, are vaccines for chikungunya comprising the DNA described above, and methods for using the vaccines to immunize against a CHIKV. Also described, are homogenous clonally purified live attenuated CHIKV virus prepared from cultured cells transfected with the DNA described above, vaccines for chikungunya comprising the same, and methods for using the vaccines to immunize against a CHIKV virus.
This application also provides vectors comprising DNA encoding an infectious RNA molecule and a cytomegalovirus (CMV) RNA polymerase promoter, where the DNA encoding an infectious RNA molecule is operably linked to the CMV RNA polymerase promoter, the CMV RNA polymerase promoter is located from about 13 to about 17 (preferably 15 nucleotides as exemplified on FIGS. 3-7 and 9) nucleotide residues upstream of the 5′ end of said DNA encoding an infectious RNA molecule, and the infectious RNA molecule encodes an attenuated CHIKV virus. In certain embodiments, the CHIKV is a chimeric virus containing sequences from CHIKV as well as from another alphavirus. In certain embodiments, the DNA encoding the infectious RNA is modified to improve characteristics of said infectious RNA and of the described vector.
In an exemplary embodiment a vector is described comprising: (a) DNA encoding an infectious RNA molecule; and (b) an RNA polymerase promoter; wherein: (i) the DNA encoding an infectious RNA molecule is operably linked to the RNA polymerase promoter; and (ii) the infectious RNA molecule encodes a chikungunya virus (CHIKV). In some examples, the infectious RNA molecule encodes a non-pathogenic chikungunya virus. In some examples, the RNA polymerase promoter comprises a cytomegalovirus (CMV) RNA polymerase promoter, the CMV RNA polymerase promoter is located from about 13 to about 17 nucleotide residues upstream of the 5′ end of the DNA encoding an infectious RNA molecule, and the infectious RNA molecule encodes an attenuated CHIKV virus. In various examples, a vector can comprise the DNA sequence listed in FIGS. 3-7.
In some embodiments, the CHIKV is a chimeric virus containing sequences from CHIKV as well as from another alphavirus. The DNA encoding the infectious RNA may also be modified to improve characteristics of the infectious RNA and of the described vector.
A vaccine for chikungunya virus can comprise a therapeutically effective amount of such vectors. In other examples, a vaccine for chikungunya virus can comprise an attenuated CHIKV virus produced by isolating the CHIKV virus from cells transfected by the DNA vectors described herein. A homogeneous clonally purified live attenuated virus may be prepared from cultured cells transfected with the DNA vectors described herein. Such a preparation is particularly useful as a vaccine.
Alternatively, a vector may comprise (a) DNA encoding an infectious RNA molecule; and (b) an RNA polymerase promoter; wherein: (i) the DNA encoding an infectious RNA molecule is operably linked to the RNA polymerase promoter; and (ii) the infectious RNA molecule encodes an alphavirus and contains sequences from CHIKV as well as from another alphavirus. One example of such a vector comprises the sequence of FIG. 9.
The vectors and vaccines described herein are useful for providing a method for immunizing a mammal against a chikungunya virus comprising the step of administering the vaccine to a mammal, such as an animal or human.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a comparison of live attenuated vaccines, DNA vaccines, and the described CHIKV i-DNATM vaccine.
FIG. 2 illustrates an example CHIKV i-DNATM vaccine and CHIKV i-DNATM immunization. The full-length CHIKV cDNA is placed downstream from optimized promoter (Popt). In animal cells, for example cells of humans or mice, injected with i-DNATM, transcription from the promoter yields a full-length infectious genomic RNA capable of initiating productive replication of the CHIKV live attenuated virus particles and inducing specific immune responses.
FIGS. 3A-3E illustrate an example (nucleotide sequence) of i-DNATM that encodes the 181/25 live attenuated vaccine (SEQ ID NO: 1); wherein FIG. 3A shows the nucleotides of SEQ ID NO1 at relative positions 1-2520, FIG. 3B shows the nucleotides of SEQ ID NO1 at relative positions 2521-5220, FIG. 3C shows the nucleotides of SEQ ID NO1 at relative positions 5221-7920, FIG. 3D shows the nucleotides of SEQ ID NO1 at relative positions 7921-10620, FIG. 3E shows the nucleotides of SEQ ID NO1 at relative positions 10621-13251. The precise 5′ and 3′ ends of i-DNA-encoded functional genomic RNA of CHIKV are determined by optimized CMV promoter and ribozyme, respectively.
FIGS. 4A-4E illustrate an example (nucleotide sequence) of i-DNATM that encodes the 181/25 live attenuated vaccine without ribozyme sequences (SEQ ID NO: 2); wherein FIG. 4A shows the nucleotides of SEQ ID NO2 at relative positions 1-2580, FIG. 4B shows the nucleotides of SEQ ID NO2 at relative positions 2581-5280, FIG. 4C shows the nucleotides of SEQ ID NO2 at relative positions 5281-7980, FIG. 4D shows the nucleotides of SEQ ID NO2 at relative positions 7981-10680, FIG. 4E shows the nucleotides of SEQ ID NO2 at relative positions 10681-12923. The precise 5′ end of i-DNA-encoded CHIKV RNA is determined by optimized CMV promoter. The location of 3′ end of i-DNATM encoded CHIKV RNA is unknown but such RNA is capable of replicating and generating live CHIKV.
FIGS. 5A-5E illustrate an example (nucleotide sequence) of i-DNATM that encodes the 181/25 live attenuated vaccine with the duplicated 26S promoter (SEQ ID NO: 3); wherein FIG. 5A shows the nucleotides of SEQ ID NO3 at relative positions 1-2460, FIG. 5B shows the nucleotides of SEQ ID NO3 at relative positions 2461-5160, FIG. 5C shows the nucleotides of SEQ ID NO3 at relative positions 5161-7860, FIG. 5D shows the nucleotides of SEQ ID NO3 at relative positions 7861-10560, FIG. 5E shows the nucleotides of SEQ ID NO3 at relative positions 10561-13031. In this i-DNATM construct, capsid (C) and glycoproteins (GPs) of CHIKV are encoded from separate 26S promoters within CHIKV RNA.
FIGS. 6A-6E illustrate an example (nucleotide sequence) of i-DNATM that encodes the 181/25 live attenuated vaccine with the nuclear transport element at the 3′ terminus (SEQ ID NO: 4); wherein FIG. 6A shows the nucleotides of SEQ ID NO4 at relative positions 1-2460, FIG. 6B shows the nucleotides of SEQ ID NO4 at relative positions 2461-5160, FIG. 6C shows the nucleotides of SEQ ID NO4 at relative positions 5161-7860, FIG. 6D shows the nucleotides of SEQ ID NO4 at relative positions 7861-10560, FIG. 6E shows the nucleotides of SEQ ID NO4 at relative positions 10561-13163. In this i-DNATM construct, a nuclear transport element sequence is introduced prior to polyA. This sequence enhances transport of CHIKV RNA from the nucleus to the cytoplasm.
FIGS. 7A-7E illustrate an example (nucleotide sequence) of i-DNATM that encodes the chimeric vaccine comprising the TC-83 live attenuated vaccine (SEQ ID NO: 5); wherein FIG. 7A shows the nucleotides of SEQ ID NO5 at relative positions 1-2400, FIG. 7B shows the nucleotides of SEQ ID NO5 at relative positions 2401-5100, FIG. 7C shows the nucleotides of SEQ ID NO5 at relative positions 5101-7800, FIG. 7D shows the nucleotides of SEQ ID NO5 at relative positions 7801-10500, FIG. 7E shows the nucleotides of SEQ ID NO5 at relative positions 10501-12173. In this chimeric i-DNATM construct, its structural gene region is replaced with the structural gene region (C and GPs) of CHIKV 181/25 live attenuated vaccine.
FIG. 8 illustrates the CHIKV i-DNATM constructs from FIGS. 3-7. The genomic RNA from CHIKV is shown on the top. The i-DNATM plasmid contains a DNA fragment (dashed box) that encodes (i) optimized eukaryotic promoter Popt (preferably optimized CMV promoter) and (ii) the full-length cDNA of CHIKV functional RNA encoding elements necessary for replication of genomic CHIKV RNA and generation of live attenuated CHIKV in vitro or in vivo. The indicated nucleotide sequence domains can be used in any of the constructs. For example, ribozyme (FIG. 3(A-E)) can also be used in the constructs described in FIGS. 5-7 and 9.
FIGS. 9A-9E illustrate an example (nucleotide sequence) of chimeric CHIKV i-DNATM derived from live attenuated strain 181/25 that contains C-GPs polyprotein from TC-83 (SEQ ID NO: 6); wherein FIG. 9A shows the nucleotides of SEQ ID NO6 at relative positions 1-2460, FIG. 9B shows the nucleotides of SEQ ID NO6 at relative positions 2461-5160, FIG. 9C shows the nucleotides of SEQ ID NO6 at relative positions 5161-7860, FIG. 9D shows the nucleotides of SEQ ID NO6 at relative positions 7861-10560, FIG. 9E shows the nucleotides of SEQ ID NO6 at relative positions 10561-12944. This chimeric i-DNATM construct can be used as a vaccine against Venezuelan equine encephalitis virus (VEE). Structural polyproteins from the other alphaviruses also can be used in place of the TC-83 to develop CHIKV-based i-DNATM vaccines against the respective alphaviruses.
FIG. 10(A-C) illustrates example photomicrographs of Chinese hamster ovary (CHO) cells transfected with the Full-Length CHIKV i-DNATM, clone #10 (sequence of FIG. 4(A-E)), by immunofluorescence assay using specific anti-CHIKV antibody at 48 hr post transfection.
FIG. 11(A-B) illustrates (A) determination of the titer and plaque morphology of live attenuated virus derived from i-DNA, clone #10, FIG. 4(A-E); and, (B) plaques derived from 181/25 IND vaccine that was passed once in CHO cells. The i-DNATM-derived CHIKV Virus has a uniform plaque size as compared to “classic” IND Vaccine 181/25.
FIG. 12 illustrates sequence variation within 181/25 IND vaccine (SEQ ID NOs: 7-18). The vaccine was resuspended in saline and passed once in CHO cells. Viral RNA was isolated, cDNA was prepared by reverse transcription PCR and cloned into pCR2.1 vector. The sequences of cloned cDNA fragments were determined and compared to known CHIKV sequences from GenBank.



DETAILED DESCRIPTION
Chikungunya is a mosquito-borne disease caused by Chikungunya virus (CHIKV). CHIKV is a member of the Alphavirus genus in the family Togaviridae. The Alphavirus genus consists of 29 distinct species (along with O'nyong'nyong virus, Ross River virus, Sindbis virus, Semliki Forest virus, VEE and others) that either cause encephalitis, febrile illness with arthralgia, or are not known to cause disease in humans. Members of this genus are primarily vector-borne; nearly all of them are utilizing mosquitoes as their invertebrate vectors (Powers and Brault, 2009). As used herein, CHIKV includes chimeric viruses that contain sequences from CHIKV as well as from another alphavirus, such chimeric viruses preferably comprising at least 50% CHIKV sequences and/or an antigenic portion of CHIKV.
Like all alphaviruses, CHIKV has a genome consisting of a linear, positive sense, single-stranded RNA molecule of approximately 12 kb in length (Khan et al., 2002). The nonstructural proteins required for viral replication are encoded in the 5′ two thirds of the genome and are regulated from 49S promoter, while the structural genes are collinear with the 3′ one-third and utilize 26S internal promoter. The 5′ end of the genome has a 7-methylguanosine cap while the 3′ end is polyadenylated. There are also 3′ noncoding repeat sequence elements that generate predicted secondary structures (Khan et al., 2002).
CHIKV causes explosive outbreaks and significant morbidity in many countries. The virus is widely spread and can easily be imported into naïve regions due to travel from endemic areas. CHIKV is also responsible for numerous laboratory acquired infections. Different approaches have been used to develop CHIKV vaccine including formalin inactivated (Kitaoka, 1967), live attenuated (Levitt et al., 1986) and chimeric alphavirus vaccine (Wang et al., 2008). Various formulations of potential CHIKV vaccines have been examined and subjected to human clinical trials (reviewed by (Powers and Brault, 2009). However, no licensed vaccine is currently available against chikungunya.
Vaccines are needed to control CHIKV. Ideally, the vaccine should have high degree of safety, induce efficient immunity and protection, be genetically stable, and not require a “cold chain” from vaccine manufacturer to vaccine recipient. Reduced cost and simplicity of production are important, because the main reservoir of CHIKV and the majority of cases are located in the tropical countries with limited resources.
Live attenuated vaccines against viral diseases are attractive because of rapid onset of immunity and efficient protection. Successful application of live attenuated vaccines resulted in the control of infectious diseases caused by many RNA viruses such as poliomyelitis, measles, mumps, rabies, rubella, and yellow fever. Approximately 60% of vaccines licensed for use in the U.S. are live attenuated vaccines. Among four viral vaccines recently approved by the FDA, three represent live attenuated vaccines—against rotavirus, influenza A and B, and varicella-zoster virus. This indicates that a live attenuated platform continues to be very attractive for vaccine development.
However, safety is the major concern for live attenuated vaccines. A typical live attenuated vaccine represents a population of viruses containing multiple genetic variants, or quasispecies that have various characteristics including pathogenic potential. The quasispecies diversity of RNA viruses can be associated with pathogenicity. Additionally, live associated vaccines contain impurities and adventitious agents derived from the cells that are used for vaccine manufacturing. Such impurities/adventitious agents can be associated with allergic reactions and elevated reactogenicity in the vaccine recipients.
Relatively recently, DNA vaccines have become a popular vaccination platform. A typical DNA vaccine contains a vaccine-relevant gene downstream from strong eukaryotic promoter, such as cytomegalovirus (CMV) promoter. For vaccination, DNA vaccine is injected into the tissues of a vaccine recipient, where it penetrates through the cellular and nuclear membranes of permissive cells. In the nuclei of host cells, transcription from CMV promoter occurs and the transcribed mRNA migrates from the nucleus into the cytoplasm, where translation and expression of vaccine-relevant antigen takes place. Thus, antigen is generated directly in the tissues of the vaccine recipient, which results in the induction of immunity to the antigen of interest. The advantage of DNA vaccines are the simplicity, low cost of production, the genetic stability, high level of purity and no need for a cold chain. The disadvantages of DNA vaccines are that multiple booster vaccinations and high quantities of DNA are required to induce an immune response. The need for multiple boost and high quantities of DNA injected into the nuclei of many cells raises concern that DNA vaccines can integrate into the host DNA and cause insertional mutagenesis.
Described herein is a novel chikungunya vaccine that combines the advantages of both live attenuated virus and DNA vaccine platforms. Namely, an “infectious DNA” (i-DNATM) CHIKV vaccine is described that represents a DNA vaccine that generates a live attenuated vaccine against chikungunya in vivo. The difference between the traditional DNA vaccine and the i-DNATM vaccine described herein is that traditional DNA vaccine encodes a gene of interest, whereas i-DNATM vaccine encodes the entire functional genomic RNA of live attenuated CHIKV. When CHIKV i-DNATM is injected into vaccine recipient, it enters the nucleus and transcribes the entire infectious RNA of attenuated CHIKV, which initiates replication of live attenuated vaccine in the tissues in vivo and results in rapid induction of immunity to chikungunya.
This new platform for vaccination against CHIKV combines the advantages of conventional live attenuated and DNA vaccines (FIGS. 1 and 2). Like DNA vaccines, it is genetically stable, inexpensive and simple in manufacturing, and does not require a cold chain. Like live attenuated vaccine, it requires a single small dose to induce effective immunity.
While combining the advantages of both attenuated and DNA vaccine platforms, the CHIKV i-DNATM vaccine lacks the disadvantages of both. Unlike live attenuated vaccines, the i-DNATM is genetically stable and represents a homogenous, clonally purified and well-characterized DNA that can be easily purified to high levels of purity. Unlike conventional DNA vaccines, the CHIKV i-DNATM is capable of inducing effective immunity with a single vaccination, with no multiple boosts. Also, only a low dose of i-DNATM is needed. For example, a low dose of about 1 ng to about 1 μg, preferably about 10 ng to about 1 μg, and more preferably about 100 ng to about 1 μg of i-DNATM could be used. Further, when compared with a conventional DNA vaccine, one could use about 5 fold to about 100 fold less i-DNATM, more preferably about 10 fold to about 100 fold less i-DNATM, even more preferably about 25 fold to about 100 fold less i-DNATM and most preferably about 50 fold to about 100 fold less i-DNATM. The nucleus is only needed to generate the initial few copies of RNA genome, and after that, replication of live attenuated CHIKV virus occurs in the cell cytoplasm, thus drastically reducing the possibility of genetic mutagenesis of host DNA. For example, the use of the CHIKV i-DNATM could reduce the possibility of genetic mutagenesis of host DNA by at least 50%, preferably by at least about 70%, more preferably by at least about 80%, even more preferably by at least about 90% and most preferably by about 100%.
The i-DNATM can be utilized in various ways to create vaccines against CHIKV. For example, the i-DNATM can be introduced by electroporation or any other acceptable way known in the art into eukaryotic cells acceptable for vaccine production. The live attenuated CHIKV generated from an i-DNATM clone represents a homogenous virus population and contains a lower number of quasispecies thus representing an advantage over traditional live attenuated vaccines. For example, the virus population generated from an i-DNATM clone could contain at least about 50% less quasispecies, preferably at least about 70% less quasispecies, more preferably at least about 80% less quasispecies, even more preferably at least about 90% less quasispecies and most preferably about 100% less quasispecies than the number of quasispecies generated by a conventional vaccine. Such homeogenous live attenuated CHIKV generated from i-DNATM plasmid can be configured into a pharmaceutically acceptable formulation suitable for vaccine administration to people. Alternatively, the i-DNATM can be administered to people in a pharmaceutically acceptable way as shown, for example, in FIG. 2.
A similar system has been developed for flaviviruses, for example West Nile virus (for example, U.S. Pat. No. 7,459,163, incorporated herein by reference in its entirety). An i-DNATM vaccine for Venezuelan equine encephalitis (VEE) virus, another alphavirus is described in commonly-owned International Application No. PCT/US2009/004133, incorporated herein by reference in its entirety. However, until now, there has been no i-DNATM vaccine system for CHIKV. The difficulty is in the configuring CHIKV i-DNATM to combine several characteristics that are normally not present in a CHIKV virus during its normal life cycle.
In exemplary embodiments, CHIKV i-DNATM plasmid is efficiently grown as a high-copy plasmid in bacteria (which are not a normal host for CHIKV) and, therefore i-DNATM is preferably free of any strong secondary structures, cryptic origins of replication or open reading frames (ORFs) encoding toxic products or of any other known and unknown elements inhibiting growth of bacteria or synthesis of plasmid DNA.
In other exemplary embodiments, the genomic CHIKV RNA is efficiently transcribed in the host cell nuclei. Nucleoplasm in the nuclei normally represents a hostile environment (because of splicing and other RNA processing mechanisms) for RNAs that are evolved to replicate in the cytoplasm such as CHIKV RNA. After transcription, the resulting RNA is preferably able to avoid splicing machinery and also successfully migrate from the nucleus to the cytoplasm via nuclear pores, the process that is tightly controlled by the cellular proteins and factors. The RNA of cytoplasmic viruses such as CHIKV do not normally have elements that ensure synthesis and transport of intact full-length RNA in the nucleus. Therefore, the i-DNATM should preferably be free of cryptic splice sites or other elements precluding effective transcription and transport of RNA into the cytoplasm.
In other exemplary embodiments, in order to replicate in the cytoplasm and generate a live attenuated vaccine, the transcribed RNA should be functional and have authentic 5′ and 3′ ends capable of supporting RNA replication.
In further exemplary embodiments, in order to ensure synthesis and transport from the nucleus to cytoplasm of transcribed CHIKV RNA, certain sequences can be either removed or introduced into CHIKV i-DNATM.
Herein are described examples of several configurations of CHIKV i-DNATM that can be used to generate live attenuated CHIKV in vitro or in vivo and can be configured into pharmaceutically-acceptable chikungunya vaccines. The CHIKV i-DNATM vaccines described herein are also expected to protect against O'nyong'nyong virus, a related alphavirus. Accordingly, i-DNATM vaccines described herein could also be used in a method for immunizing a mammal against the O'nyong'nyong virus, which would include administering an exemplary vaccine to the mammal.
EXAMPLES
Example 1
Live attenuated vaccine candidate CHIK 181/25 (TSI-GSD-218) was generated from CHIKV strain 15561 and later successfully tested in phase II clinical trials (Edelman et al., 2000; Levitt et al., 1986). However, CHIKV 181/25 strain has common drawbacks with other live vaccines. For example, there is the risk of reversion to a virulent form, which has been shown previously (Parker, 1994). In addition to safety risks associated with the possibility of genetic reversion, other weaknesses include heterogeneity of virus population within the vaccine, presence of impurities and adventitious agents derived from cells substrate and during vaccine preparation; costly and inconvenient requirement for a cold chain delivery, difficulties in production of large amounts of live attenuated virus, and the requirement of constant quality control measures to maintain the attenuated genotype unchanged.
An i-DNATM molecule contains the full-length cDNA copy of the 181/25 genomic RNA under control of the CMV promoter (FIG. 3(A-E)). The distance between the promoter and the start of CHIKV cDNA (15±2 nucleotides according to our numbering, see nucleotide sequence on FIG. 3(A-E) for details) ensures generation of genomic CHIKV RNA with functional 5′ terminus capable of RNA amplification and replication. The polyA tail, ribozyme sequence and the transcription termination sequences ensure generation of CHIKV RNA with functional 3′ terminus. The entire DNA fragment shown on FIG. 3(A-E) includes (1) CMV promoter/enhancer sequences, (2) the full-length CHIKV cDNA, (3) polyA tail, (4) ribozyme, and (5) transcription termination sequences. Such DNA fragment can be cloned and propagated in E. coli as a part of plasmids known in the art including but not limited to pcDNA3.1, pCR2.1, pUC19 and others. Following transcription from i-DNATM shown in FIG. 3(A-E), the genomic RNA of live attenuated vaccine is generated in vivo, which initiates limited replication of live attenuated vaccine virus and generation of a protective immune response (FIG. 2).
In addition to mutations contained in the live attenuated virus strain 181/25 of CHIKV, other configurations of attenuating mutations can be used for the development of CHIKV i-DNATM vaccines. Additional sequence variations including deletions, insertions or substitutions can be used to improve characteristics of CHIKV i-DNATM
FIG. 4(A-E) shows the nucleotide sequence of a variant i-DNATM that is similar to that shown on FIG. 3(A-E) but containing no ribozyme sequences. Surprisingly, such an i-DNATM without a ribozyme is capable of transcribing the functional infectious CHIKV RNA and generating live attenuated CHIKV virus. FIG. 10(A-C) demonstrates that transfection of Chinese hamster ovary (CHO) cells with i-DNATM plasmid (clone #10, either in supercoiled form or linearized by using NotI enzyme) results in expression of CHIKV antigens in the majority of CHO cells suggesting that (i) no cryptic splicing sites or other restrictive elements were present in the i-DNATM or encoded RNA and (ii) that transfection results in live virus, which ensures effective spread of antigen expression in transfected CHO cells. Further, i-DNATM can accommodate additional sequences that can improve certain characteristics of i-DNATM vaccines. Examples of such i-DNATM sequences are shown on FIGS. 5 and 6.
Also, chimeric live attenuated alphavirus protecting against CHIKV and O'nyong'nyong virus can be made by placing CHIKV structural genes in place of another alphavirus structural genes. FIG. 7(A-E) shows an example of a nucleotide sequence, in which CHIKV structural genes derived from strain 181/25 are introduced into TC-83 live attenuated virus in place of the TC-83 structural genes. Again, such vaccine can either (1) represent homogenous virus generated from i-DNATM in vitro in a pharmaceutically acceptable way, or (2) represent the i-DNATM construct formulated in a pharmaceutically acceptable way for administration in vivo.
Alternatively, chimeric vaccines against alphaviruses can be made by introducing structural proteins from other alphaviruses into CHIKV i-DNA. FIG. 9(A-E) shows an example of a nucleotide sequence of a chimeric CHIKV i-DNATM containing the structural polyprotein from TC-83 virus in place of the structural polyprotein of CHIKV 181/25 virus. Such chimeric i-DNATM can be used for production of either homogenous virus vaccines or i-DNATM vaccines against VEE infections.
Example 2
Generation of live attenuated CHIKV in vitro using i-DNA. When CHIKV i-DNATM molecule (FIGS. 3-9) is introduced into cells in vitro, for example by transfection, the CHIKV viral RNA is generated in the cells. The resulting RNA is “infectious” and initiates production of the CHIKV live attenuated virus vaccine in the cells (FIG. 10(A-C)). The live attenuated CHIKV accumulates in the culture medium and can be harvested and the titer of live attenuated virus and plaque morphology can be determined by plaque assay (FIG. 11A). The live attenuated CHIKV can be formulated in a pharmaceutically acceptable way according to current state of the art.
Example 3
Vaccination in vivo with live attenuated CHIKV vaccine generated from i-DNATM in vitro. CHIKV virus vaccine can be harvested from cultured cells as described in Example 2 and used in a pharmaceutically acceptable formulation for vaccination of animals or people according to current state of the art. Administration can be by any route typically used for vaccination, including subcutaneous, intravenous, intramuscular, combinations thereof and the like. An advantage of vaccine that is generated from the i-DNATM is that it represents homogeneous progeny virus generated from the same, well-characterized, stable DNA.
FIG. 11(A-B) shows plaque size homogeneity for i-DNATM-derived virus (FIG. 11A) as compared to the more heterogenous plaque sizes in a “classic” 181/25 IND vaccine (FIG. 11B). Homogenous plaque size is expected to result in higher safety of i-DNA-derived live attenuated virus because large plaques in 181/25 IND vaccine (FIG. 11B) can indicate presence of revertants to virulent virus. Revertants to virulent virus are detected in the virus isolated from patients that experience adverse effects after vaccination with 181/25 IND vaccine (Genbank entry EF452494, note isolation source=“viremic vaccine recipient”).
FIG. 12 shows sequencing results of several clones generated from 181/25 IND vaccine by reverse transcription and PCR. Vaccine 181/25 was passed once in CHO cells, the RNA isolated and Reverse Transcription PCR (RT-PCR) conducted. The RT-PCR fragments were cloned and sequenced.
The cDNA fragments were sequenced and compared to the sequences of several virulent CHIKV strains from GenBank as well as to the original 181/25 sequence. Analysis revealed that several sequenced clones contain “reversion” mutations to the virulent virus. The result shown in FIG. 12 is that in the sequenced region, one out of seven clones has the same sequence as 181/25 IND vaccine, whereas others had nucleotide substitutions present in virulent isolates. Only clone 3.5_40 out of seven has an isoleucine (I) residue at amino acid 301, as in 181/25, whereas other six clones have the threonine (T) residue that is present in the virulent wild type CHIKV isolates and in the VR1 isolate from 181/25-vaccinated sick patient. Heterogeneity was also detected at position 314.
The vaccines described herein can provide higher safety and regulatory advantages. Because the same, clonally purified, i-DNATM can be used for the production of different vaccine lots, these vaccine will have greater uniformity and lot-to-lot consistency compared to current vaccines, which can accumulate mutations during virus passages.
Example 4
Vaccination in vivo by using i-DNATM vaccine. Alternatively, CHIKV i-DNATM (FIGS. 3-8) can be administered in a pharmaceutically-acceptable formulation into the vaccine recipient directly, for example intramuscularly or intravenously, as illustrated in FIG. 2. Direct i-DNATM administration to the vaccine recipient initiates production of CHIKV vaccine in the tissues of the patient in vivo, and provides successful vaccination against chikungunya. An additional advantage of i-DNATM immunization versus conventional live attenuated virus vaccine stems from the immunogenic characteristics of the i-DNATM itself. Bacterially produced DNA, including i-DNATM, contains unmethylated CpG motifs. These motifs activate toll-like receptor (TLR) signaling pathway, which results in induction of innate immunity and production of pro-inflammatory cytokines and type 1 interferons (IFN 1) shortly after DNA injection. Signaling through receptors induces robust cytokine response from myeloid DCs and IFN 1 production from plasmocytoid DCs as well as stimulates cross-presentation of exogenous antigens and CTL T responses. Thus, i-DNATM immunization leads to activation and maturation of DCs even before virus particles are released. Such pre-activated DCs enhance specific immune responses induced by newly synthesized live attenuated virus.
Example 5
i-DNATM vaccines with de-optimized codons. One vaccine, i-DNAC, contains the full-length cDNA copy of the 181/25 IND vaccine RNA genome but translational codons within the capsid gene are de-optimized via down-selected codons. Another vaccine, i-DNAE2-E1, also contains the full-length cDNA copy of the 181/25 IND vaccine RNA genome but translational codons within the E2-E1 gene region are similarly de-optimized. E1, E2 and C genes are selected based on immunogenicity of these antigens in mice and on the current knowledge about adaptive immune responses following CHIKV infection in humans. The de-optimized codons change only the nucleotide sequence and do not give rise to changes in the amino acid sequence. These silent mutations increase genetic stability and preserve the attenuated phenotype and are designed so that at least two independent genetic mutations are necessary for each codon to revert to a wild-type CHIKV codon.
The synthetic 181/25 fragment encompassing 3,771 base pairs of 181/25 structural gene region 7567-11313 (C-E1-E2) is synthesized biochemically (GenScript, Piscataway, N.J.). The recombinant 181/25 i-DNA, i-DNAC and i-DNAE2-E1 constructs are transfected into CHO-K1 cells in vitro. Samples of live attenuated viruses are collected from culture medium and cells at 6 hour intervals for 96 hours. The live CHIKV viruses produced via transfection of cells and phenotypic features of these recovered viruses are evaluated in vitro, examining kinetic parameters of replication in tissue culture, antigenic properties, genetic stability, and molecular heterogeneity by the following in vitro assays: (1) plaque assay and phenotype; (2) virus growth curves; (3) western blot; (4) immunofluorescence; (5) at least 10 passages in E. coli; (6) reverse transcription PCR, and (7) DNA sequencing of the entire structural region of at least 120 plaque isolates derived from each virus including 181/25 control (to assess population heterogeneity and genetic stability in mammalian cells). The ability of new CHIKV vaccine antigens to react with human antisera from recent CHIKV clinical cases is evaluated via ELISA.
Example 6
Animal model testing of i-DNATM vaccine. Production of plasmid i-DNATM from E. coli is done using established methods for production of the bacterial cell bank, fermentation, harvest/lysis of the biomass, and downstream DNA purification. This process results in a sterile DNA product with about 95% supercoiled DNA and an A260/A280 ratio of about 1.9, as well as minimal residual endotoxin, RNA, genomic DNA, and protein impurities. Quality control includes (1) endotoxin testing, (2) agarose gel, (3) SDS-PAGE; (4) restriction enzyme analysis and (5) DNA sequencing. A maximum of 100 ng of i-DNATM is injected intramuscularly (i.m.) into: (1) newborn ICR mice; (2) 14-day-old CD-1 mice; (3) aged (3-month-old) CD-1 mice; and (4) immunosuppressed hamsters.
Alternatively, i-DNATM is delivered into the quadriceps muscles by in vivo electroporation in a total volume of 0.1 ml using the BTX ECM600 with 2-needle Array Electrode (BTX/Harvard Apparatus, Holliston, Mass.). The PolyPlus InVivo-Jet PEI transfection reagent (PolyPlus, Illkirch, France) can be used as another transfection method. Safety and immunogenicity parameters include (1) viremia; (2) morbidity; (3) cytokine profiles; (4) CHIKV ELISA; (5) virus-specific neutralizing antibody responses; (6) cell-mediated immunity; and (7) anti-DNATM antibody.
For the ICR and CD-1 mice, sixty animals from each strain-are divided into 5 groups (12 mice per group): (1) 181/25 i-DNA; (2) i-DNAC; (3) i-DNAE2-E1; (4) 181/25 virus (positive control); and (5) mock-vaccinated (negative control). Each i-DNATM plasmid is injected as a single 100 ng dose in 20-40μ i.m. Control animals are injected with 105 PFU of 181/25 virus vaccine. Blood samples are taken every 2-7 days for viremia and serology. At days 0, 2, 4, 8, and 16 after immunization, 3 mice from each group are euthanized and bled by cardiac puncture. TLR9 activation by i-DNATM is expected to trigger pro-inflammatory cytokine production at 12-16 h, shortly before the release of the infectious virus, thus enhancing adaptive immune responses. Innate immunity is tested by cytokine ELISA at different time points after i-DNATM immunization by using reagents for IFN-γ, IL-1α/β, TNF-α, MCP-1, IL-4, IL-6 and IL-12p40 (BD Biosciences, San Jose, Calif.). INF-α and INF-β is measured by ELISA (PBL Biomed Labs, NJ). The levels of biological active IFN are determined using an EMCV-L929 bioassay (Daffis et al., 2007). Specific immune responses are measured by IgG ELISA, plaque reduction neutralization assay (PRNT), and by IFN-γ ELISPOT assay using pools of 15-mer peptides overlapping by 11 amino acids (Mimotopes, Melbourne, Australia) (Muthumani et al., 2008). Briefly, ELISPOT plates are coated with anti-IFN-γ Ab and incubated for 12 h at 40 C. Plates are washed and blocked with 1% BSA. After washing, 25×104 splenocytes are added to wells in triplets and stimulated overnight by incubation with specific E1-, E2-, and C-derived peptide pools at 37° C. After stimulation, the cells are washed and incubated with biotinylated anti-mouse IFN-γ (R&D Systems). Tissue samples (liver, spleen, lymph nodes, lung, kidney, and brain) are homogenized in PBS with 1% FBS and used for plaque assay or RNA extraction with Trizol.
Cyclophosphamide (CYP) treated Syrian golden hamsters (Mesocricetus auratus) (Harlan Sprague Dawley) are used to evaluate safety and immunogenicity of vaccines in the immunocompromised conditions (Mateo et al., 2007). A total of 26 hamsters are used in this study. The CYP-treated animals are divided into six groups. Three groups receive the original and modified i-DNAs. The three control groups include a group that receives the 181/25 vaccine; a control group that receives the CYP treatment but no vaccine, and a control group that receives no CYP and no vaccine (Vertebrate Animals). The i-DNATM constructs are injected i.m. as a single dose (100 ng). The 181/25 control virus is injected in 100μ (105 PFU). Animals are examined daily for 8 weeks (end-point) for any evidence of adverse effects. Temperature and body weight is recorded. Animals are bled every 2 days for the 1st week, then every 7 days for virus detection and serology.
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        Each of the below references is incorporated herein by reference in its entirety.
    
    
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## Claims

1. A vaccine for chikungunya virus (CHIKV) comprising an attenuated CHIKV produced by isolating said CHIKV from cells transfected by a vector comprising:
(a) DNA encoding an infectious RNA molecule; and
(b) a eukaryotic RNA polymerase promoter;
wherein:
(i) the DNA encoding the infectious RNA molecule is operably linked to the eukaryotic RNA polymerase promoter; and
(ii) the infectious RNA molecule encodes a CHIKV.

2. The vaccine of claim 1, wherein the infectious RNA molecule encodes an attenuated CHIKV.

3. The vaccine of claim 2, wherein:
the eukaryotic RNA polymerase promoter comprises a cytomegalovirus (CMV) RNA polymerase promoter.

4. The vaccine of claim 2, wherein the CHIKV is a chimeric virus containing sequences from CHIKV as well as from another alphavirus.

5. The vaccine of claim 2, wherein the vector comprises sequences that allow transport of the transcribed infectious RNA molecule from nucleus to cytoplasm or that allow generation of transcribed infectious RNA molecule comprising functional 5′ and 3′ termini for replication and amplification.

6. A homogeneous clonally purified live attenuated virus population prepared from cells transfected with a vector comprising:
(a) DNA encoding an infectious RNA molecule; and
(b) a eukaryotic RNA polymerase promoter;
wherein:
(i) the DNA encoding the infectious RNA molecule is operably linked to the eukaryotic RNA polymerase promoter; and
(ii) the infectious RNA molecule encodes a CHIKV.

7. The virus population of claim 6, wherein the infectious RNA molecule encodes an attenuated CHIKV.

8. The virus population of claim 7, wherein:
the eukaryotic RNA polymerase promoter comprises a cytomegalovirus (CMV) RNA polymerase promoter.

9. The virus population of claim 7, wherein the CHIKV is a chimeric virus containing sequences from CHIKV as well as from another alphavirus.

10. The virus population of claim 7, wherein the vector comprises sequences that allow transport of the transcribed infectious RNA molecule from nucleus to cytoplasm or that allow generation of transcribed infectious RNA molecule comprising functional 5′ and 3′ termini for replication and amplification.

11. A vaccine comprising the virus population of claim 6.

12. A method of immunizing a mammal against a CHIKV, the method comprising a step of administering to the mammal an effective amount of the vaccine of claim 11.

13. A homogeneous clonally purified live attenuated virus comprising an infectious CHIKV RNA molecule operably linked to a eukaryotic RNA polymerase promoter.

14. A vaccine comprising the virus population of claim 7.

15. The vaccine of claim 3, wherein the CMV RNA polymerase promoter is located from about 13 to about 17 nucleotide residues upstream of a 5′ end of the DNA encoding the infectious RNA molecule.

16. The virus population of claim 8, wherein the CMV RNA polymerase promoter is located from about 13 to about 17 nucleotide residues upstream of a 5′ end of the DNA encoding the infectious RNA molecule.

17. A method of immunizing a mammal against CHIKV, the method comprising a step of administering to the mammal an effective amount of the vaccine of claim 1.

18. A method of immunizing a mammal against CHIKV, the method comprising a step of administering to the mammal an effective amount of the vaccine of claim 2.

19. A method of immunizing a mammal against CHIKV, the method comprising a step of administering to the mammal an effective amount of the vaccine of claim 14.