# Patent

## Title
Synthetic peptides and uses therefore

## Abstract

Aspects of the present invention are related to a synthetic polypeptide comprising a plurality of different segments from one or more parent polypeptides, linked together in such a way that is different from their linkage in the one or more parent polypeptides, in such way as to impede, abrogate or otherwise alter at least one function associated with the linkage in the one or more parent polypeptides, and in such a way that their product is not similar in function and/or structure to at least a portion of the one or more parent polypeptides. Additional aspects include a synthetic polynucleotide encoding the synthetic polypeptide, methods of production, a synthetic construct and methods of using the synthetic polypeptide, nucleotide construct and compositions comprising the above. Another aspect is directed towards a computer program product for designing these synthetic polypeptides and polynucleotides.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is the U.S. National Phase under 35 U.S.C. §371 of International Application No. PCT/AU01/00622, filed May 25, 2001, designating the United States and published in English, which claims priority to Australian Application No. PQ 7761, filed May 26, 2000, the disclosure of which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
THIS INVENTION relates generally to agents for modulating immune responses. More particularly, the present invention relates to a synthetic polypeptide comprising a plurality of different segments of a parent polypeptide, wherein the segments are linked to each other such that one or more functions of the parent polypeptide are impeded, abrogated or otherwise altered and such that the synthetic polypeptide, when introduced into a suitable host, can elicit an immune response against the parent polypeptide. The invention also relates to synthetic polynucleotides encoding the synthetic polypeptides and to synthetic constructs comprising these polynucleotides. The invention further relates to the use of the polypeptides and polynucleotides of the invention in compositions for modulating immune responses. The invention also extends to methods of using such compositions for prophylactic and/or therapeutic purposes.
Bibliographic details of various publications referred to in this specification are collected at the end of the description.
BACKGROUND OF THE INVENTION
The modem reductionist approach to vaccine and therapy development has been pursued for a number of decades and attempts to focus only on those parts of pathogens or of cancer proteins which are relevant to the immune system. To date the performance of this approach has been relatively poor considering the vigorous research carried out and the number of effective vaccines and therapies that it has produced. This approach is still being actively pursued, however, despite its poor performance because vaccines developed using this approach are often extremely safe and because only by completely understanding the immune system can new vaccine strategies be developed.
One area that has benefited greatly from research efforts is knowledge about how the adaptive immune system operates and more specifically how T and B cells learn to recognise specific parts of pathogens and cancers. T cells are mainly involved in cell-mediated immunity whereas B cells are involved in the generation of antibody-mediated immunity. The two most important types of T cells involved in adaptive cellular immunity are αβ CD8+ cytotoxic T lymphocytes (CTL) and CD4+ T helper lymphocytes. CTL are important mediators of cellular immunity against many viruses, tumours, some bacteria and some parasites because they are able to kill infected cells directly and secrete various factors which can have powerful effects on the spread of infectious organisms. CTLs recognise epitopes derived from foreign intracellular proteins, which are 8-10 amino acids long and which are presented by class I major histocompatibility complex (MHC) molecules (in humans called human lymphocyte antigens—HLAs) (Jardetzky et al., 1991; Fremont et al., 1992; Rotzschke et al., 1990). T helper cells enhance and regulate CTL responses and are necessary for the establishment of long-lived memory CTL. They also inhibit infectious organisms by secreting cytokines such as IFN-γ. T helper cells recognise epitopes derived mostly from extracellular proteins which are 12-25 amino acids long and which are presented by class II MHC molecules (Chicz et al., 1993; Newcomb et al., 1993). B cells, or more specifically the antibodies they secrete, are important mediators in the control and clearance of mostly extracellular organisms. Antibodies recognise mainly conformational determinants on the surface of organisms, for example, although sometimes they may recognise short linear determinants.
Despite significant advances towards understanding how T and linear B cell epitopes are processed and presented to the immune system, the full potential of epitope-based vaccines has not been fully exploited. The main reason for this is the large number of different T cell epitopes, which have to be included into such vaccines to cover the extreme HLA polymorphism in the human population. The human HLA diversity is one of the main reasons why whole pathogen vaccines frequently provide better population coverage than subunit or peptide-based vaccine strategies. There is a range of epitope-based strategies though which have tried to solve this problem, e.g. peptide blends, peptide conjugates and polyepitope vaccines (ie comprising strings of multiple epitopes) (Dyall et al., 1995; Thomson et al., 1996; Thomson et al., 1998; Thomson et al., 1998). These approaches however will always be sub optimal not only because of the slow pace of epitope characterisation but also, because it is virtually impossible for them to cover every existing HLA polymorphism in the population. A number of strategies have sought to avoid both problems by not identifying epitopes and instead incorporating larger amounts of sequence information e.g., approaches using whole genes or proteins and approaches that mix multiple protein or gene sequences together. The proteins used by these strategies however sometimes still function and therefore can compromise vaccine safety e.g. whole cancer proteins. Alternative strategies have tried to improve the safety of vaccines by fragmenting the genes and expressing them either separately or as complex mixtures e.g., library DNA immunisation or by ligating such fragments back together. These approaches are still sub-optimal because they are too complex, generate poor levels of immunity, cannot guarantee that all proteins no longer function and/or that all fragments are present, which compromises substantially complete immunological coverage.
The lack of a safe and efficient vaccine strategy that can provide substantially complete immunological coverage is an important problem, especially when trying to develop vaccines against rapidly mutating and persistent viruses such as HIV and hepatitis C virus, because partial population coverage could allow vaccine-resistant pathogens to re-emerge in the future. Human immunodeficiency virus (HIV) is an RNA lentivirus virus approximately 9 kb in length, which infects CD4+ T cells, causing T cell decline and AIDS typically 3-8 years after infection. It is currently the most serious human viral infection, evidenced by the number of people currently infected with HIV or who have died from AIDS, estimated by the World Health Organisation (WHO) and UNAIDS in their AIDS epidemic update (December 1999) to be 33.6 and 16.3 million people, respectively. The spread of HIV is also now increasing fastest in areas of the world where over half of the human population reside, hence an effective vaccine is desperately needed to curb the spread of this epidemic. Despite the urgency, an effective vaccine for HIV is still some way off because of delays in defining the correlates of immune protection, lack of a suitable animal model, existence of up to 8 different subtypes of HIV and a high HIV mutation rate.
A significant amount of research has been carried out to try and develop a vaccine capable of generating neutralising antibody responses that can protect against field isolates of HIV. Despite these efforts, it is now clear that the variability, instability and inaccessibility of critical determinants on the HIV envelope protein will make it extremely difficult and perhaps impossible to develop such a vaccine (Kwong et al., 1998). The limited ability of antibodies to block HIV infection is also supported by the observation that development of AIDS correlates primarily with a reduction in CTL responsiveness to HIV and not to altered antibody levels (Ogg et al., 1998). Hence CTL-mediated and not antibody-mediated responses appear to be critical for maintaining the asymptomatic state in vivo. There is also some evidence to suggest that pre-existing HIV-specific CTL responses can block the establishment of a latent HIV infection. This evidence comes from a number of cases where individuals have generated HIV-specific CTL responses without becoming infected and appear to be protected from establishing latent HIV infections despite repeated virus exposure (Rowland-Jones et al., 1995; Parmiani 1998). Taken together, these observations suggest that a vaccine capable of generating a broad range of strong CTL responses may be able to stop individuals from becoming latently infected with HIV or at least allow infected individuals to remain asymptomatic for life. Virtually all of the candidate HIV vaccines developed to date have been derived from subtype B HIV proteins (western world subtype) whereas the majority of the HIV infections worldwide are caused by subtypes A/E or C (E and A are similar except in the envelop protein)(referred to as developing world subtypes). Hence existing candidate vaccines may not be suitable for the more common HIV subtypes. Recently, there has been some evidence that B subtype vaccines may be partially effective against other common HIV subtypes (Rowland-Jones et al., 1998). Accordingly, the desirability of a vaccine still remains, whose effectiveness is substantially complete against all isolates of all strains of HIV.
SUMMARY OF THE INVENTION
The present invention is predicated in part on a novel strategy for enhancing the efficacy of an immunopotentiating composition. This strategy involves utilising the sequence information of a parent polypeptide to produce a synthetic polypeptide that comprises a plurality of different segments of the parent polypeptide, which are linked sequentially together in a different arrangement relative to that of the parent polypeptide. As a result of this change in relationship, the sequence of the linked segments in the synthetic polypeptide is different to a sequence contained within the parent polypeptide. As more fully described hereinafter, the present strategy is used advantageously to cause significant disruption to the structure and/or function of the parent polypeptide while minimising the destruction of potentially useful epitopes encoded by the parent polypeptide.
Thus, in one aspect of the present invention, there is provided a synthetic polypeptide comprising a plurality of different segments of at least one parent polypeptide, wherein the segments are linked together in a different relationship relative to their linkage in the at least one parent polypeptide.
In one embodiment, the synthetic polypeptide consists essentially of different segments of a single parent polypeptide.
In an alternate embodiment, the synthetic polypeptide consists essentially of different segments of a plurality of different parent polypeptides.
Suitably, said segments in said synthetic polypeptide are linked sequentially in a different order or arrangement relative to that of corresponding segments in said at least one parent polypeptide.
Preferably, at least one of said segments comprises partial sequence identity or homology to one or more other said segments. The sequence identity or homology is preferably contained at one or both ends of said at least one segment.
In another aspect, the invention resides in a synthetic polynucleotide encoding the synthetic polypeptide as broadly described above.
According to yet another aspect, the invention contemplates a synthetic construct comprising a said polynucleotide as broadly described above that is operably linked to a regulatory polynucleotide.
In a further aspect of the invention, there is provided a method for producing a synthetic polynucleotide as broadly described above, comprising:

    
    
        linking together in the same reading frame a plurality of nucleic acid sequences encoding different segments of at least one parent polypeptide to form a synthetic polynucleotide whose sequence encodes said segments linked together in a different relationship relative to their linkage in the at least one parent polypeptide.
    
    


Preferably, the method further comprises fragmenting the sequence of a respective parent polypeptide into fragments and linking said fragments together in a different relationship relative to their linkage in said parent polypeptide sequence. In a preferred embodiment of this type, the fragments are randomly linked together.
Suitably, the method further comprises reverse translating the sequence of a respective parent polypeptide or a segment thereof to provide a nucleic acid sequence encoding said parent polypeptide or said segment. In a preferred embodiment of this type, an amino acid of said parent polypeptide sequence is reverse translated to provide a codon, which has higher translational efficiency than other synonymous codons in a cell of interest. Suitably, an amino acid of said parent polypeptide sequence is reverse translated to provide a codon which, in the context of adjacent or local sequence elements, has a lower propensity of forming an undesirable sequence (e.g., a palindromic sequence or a duplicated sequence) that is refractory to the execution of a task (e.g., cloning or sequencing).
In another aspect, the invention encompasses a computer program product for designing the sequence of a synthetic polypeptide as broadly described above, comprising:

    
    
        code that receives as input the sequence of at least one parent polypeptide;
        code that fragments the sequence of a respective parent polypeptide into fragments;
        code that links together said fragments in a different relationship relative to their linkage in said parent polypeptide sequence; and
        a computer readable medium that stores the codes.
    
    


In yet another aspect, the invention provides a computer program product for designing the sequence of a synthetic polynucleotide as broadly described above, comprising:

    
    
        code that receives as input the sequence of at least one parent polypeptide;
        code that fragments the sequence of a respective parent polypeptide into fragments;
        code that reverse translates the sequence of a respective fragment to provide a nucleic acid sequence encoding said fragment;
        code that links together in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which said fragments are linked together in a different relationship relative to their linkage in the at least one parent polypeptide sequence; and
        a computer readable medium that stores the codes.
    
    


In still yet another aspect, the invention provides a computer for designing the sequence of a synthetic polypeptide as broadly described above, wherein said computer comprises:

    
    
        (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said machine-readable data comprise the sequence of at least one parent polypeptide;
        (b) a working memory for storing instructions for processing said machine-readable data;
        (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine readable data to provide said synthetic polypeptide sequence; and
        (d) an output hardware coupled to said central processing unit, for receiving said synthetic polypeptide sequence.
    
    


In a preferred embodiment, the processing of said machine readable data comprises fragmenting the sequence of a respective parent polypeptide into fragments and linking together said fragments in a different relationship relative to their linkage in the sequence of said parent polypeptide.
In still yet another aspect, the invention resides in a computer for designing the sequence of a synthetic polynucleotide as broadly described above, wherein said computer comprises:

    
    
        (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said machine-readable data comprise the sequence of at least one parent polypeptide;
        (b) a working memory for storing instructions for processing said machine-readable data;
        (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine readable data to provide said synthetic polynucleotide sequence; and
        (d) an output hardware coupled to said central processing unit, for receiving said synthetic polynucleotide sequence.
    
    


In a preferred embodiment, the processing of said machine readable data comprises fragmenting the sequence of a respective parent polypeptide into fragments, reverse translating the sequence of a respective fragment to provide a nucleic acid sequence encoding said fragment and linking together in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which said fragments are linked together in a different relationship relative to their linkage in the at least one parent polypeptide sequence.
According to another aspect, the invention contemplates a composition, comprising an immunopotentiating agent selected from the group consisting of a synthetic polypeptide as broadly described above, a synthetic polynucleotide as broadly described above and a synthetic construct as broadly described above, together with a pharmaceutically acceptable carrier.
The composition may optionally comprise an adjuvant.
In a further aspect, the invention encompasses a method for modulating an immune response, which response is preferably directed against a pathogen or a cancer, comprising administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the group consisting of a synthetic polypeptide as broadly described above, a synthetic polynucleotide as broadly described above and a synthetic construct as broadly described above, or a composition as broadly described above.
According to still a further aspect of the invention, there is provided a method for treatment and/or prophylaxis of a disease or condition, comprising administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the group consisting of a synthetic polypeptide as broadly described above, a synthetic polynucleotide as broadly described above and a synthetic construct as broadly described above, or a composition as broadly described above.
The invention also encompasses the use of the synthetic polypeptide, the synthetic polynucleotide and the synthetic construct as broadly described above in the study, and modulation of immune responses.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic representation showing the number of people living with AIDS in 1998 in various parts of the world and most prevalent HIV clades in these regions. Estimates generated by UNAIDS.
FIG. 2 is a graphical representation showing trends in the incidence of the common HIV clades and estimates for the future. Graph from the International Aids Vaccine Initiative (IAVI).
FIG. 3 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV gag [SEQ ID NO: 1] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV clade consensus sequences for the HIV gag protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 4 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV pol [SEQ ID NO: 2] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV lade consensus sequences for the HIV pol protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR98-485.
FIG. 5 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV vif [SEQ ID NO: 3] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV clade consensus sequences for the HIV vif protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR98-485.
FIG. 6 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV vpr [SEQ ID NO: 4] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV lade consensus sequences for the HIV vpr protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 7 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV tat [SEQ ID NO: 5] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV lade consensus sequences for the HIV tat protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 8 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV rev [SEQ ID NO: 6] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV lade consensus sequences for the HIV rev protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 9 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV vpu [SEQ ID NO: 7] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV lade consensus sequences for the HIV vpu protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 10 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV env [SEQ ID NO: 8] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV clade consensus sequences for the HIV env protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 11 is a diagrammatic representation showing overlapping segments of a parent polypeptide sequence for HIV nef [SEQ ID NO: 9] used for the construction of an embodiment of an HIV Savine. Also shown are the alignments of common HIV clade consensus sequences for the HIV nef protein from the HIV Molecular Immunology Database 1997, Editors Bette Korber, John Moore, Cristian Brander, Richard Koup, Barton Haynes and Bruce Walker. Publisher, Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N.Mex., Pub LAUR 98-485.
FIG. 12 is a diagrammatic representation depicting the systematic segmentation of the designed degenerate consensus sequences for each HIV protein and the reverse translation of each segment into a DNA sequence. Also shown is the number of segments used during random rearrangement and amino acids that were removed. Amino acids surrounded by an open square were removed from the design, because degenerate codons to cater for the desired amino acid combination required too many degenerate bases to comply with the incorporation of degenerate sequence rules outlined in the description of the invention herein. Amino acids surrounded by an open circle were removed only in the segment concerned mainly because they were coded for in an oligonucleotide overlap region. Amino acids marked with an asterisk were designed differently in one fragment compared to the corresponding overlap region (see tat gene)
FIG. 13 is a diagrammatic representation showing the first and second most frequently used codons in mammals used to reverse translate HIV protein segments. Also shown are all first and second most frequently used degenerate codons for two amino acids where only one base is varied. Codons used where more than one base was varied were worked out in each case by comparing all the codons for each amino acid. The IUPAC codes for degenerate bases are also shown.
FIG. 14 illustrates the construction plan for the HIV Savine showing the approximate sizes of the subcassettes, cassettes and full-length Savine cDNA and the restriction sites involved in joining them together. Also shown are the extra sequences added onto each subcassette during their design and a brief description of how the subcassettes, cassettes and full length cDNA were constructed and transferred into appropriate DNA plasmids. Description of full length construction: pA was cleaved with XhoI/SalI and cloned into XhoI arms of the B cassette; pAB was cleaved with XhoI and cloned into XhoI arms of the C cassette; full length construct is excisable with either XbaI/BamHI at the 5′ end or BglII at the 3′ end. Options for excising cassettes: A) XbaI/BamHI at the 5′ end, BglII/XhoI at the 3′ end; B) XbaI/BamHI at the 5′ end, BglII/SalI at the 3′ end; C) XbaI/BamHI at the 5′ end, BglII/SalI at the 3′ end. Cleaving plasmid vectors: pDNAVacc is cleavable with XbaI/XhoI (DNA vaccination); pBCB07 or pTK7.5 vectors are cleavable with BamHI/SalI (Recombinant Vaccinia); pAvipox vector pAF09 is cleavable with BamHI/SalI (Recombinant Avipox).
FIG. 15 shows the full length DNA (17253 bp) and protein sequence (5742 aas) of the HIV Savine construct. Fragment boundaries are shown, together with the position of each fragment in each designed HIV protein, fragment number (in brackets), spacer residues (two alanine residues) and which fragment the spacer was for (open boxes and arrows). The location of residual restriction site joining sequences corresponding to subcassette or cassette boundaries (shaded boxes) are also shown, along with start and stop codons, Kozak sequence, the location of the murine influenza virus CTL epitope sequence (near the 3′ end), important restriction sites at each end and the position of each degenerate amino acid (indicated by ‘X’).
FIG. 16 depicts the layout and position of oligonucleotides in the designed DNA sequence for subcassette A1. The sequences which anneal to the short amplification oligonucleotides are indicated by hatched boxes and the position of oligonucleotide overlap regions are dark shaded.
FIG. 17: Panel (a) depicts the stepwise asymmetric PCR of the two halves of subcassette A1 (lanes 2-5 and 7-9, respectively) and final splicing together by SOEing (lane 10). DNA standards in lane 1 are pUC18 digested with Sau3AI. Panel (b) shows the stepwise ligation-mediated joining and PCR amplification of each cassette as indicated. DNA standards in lane 1 are SPP1 cut with EcoRI.
FIG. 18: Panel (a) shows summary of the construction of the DNA vaccine plasmids that express one HIV Savine cassette. Panel (b) shows a summary of the construction of the plasmids used for marker rescue recombination to generate Vaccinia viruses expressing one HIV Savine cassette. Panel (c) shows a summary of the construction of the DNA vaccine plasmids which each express a version of the full-length HIV Savine cDNA
FIG. 19 shows restimulation of HIV specific polyclonal CTL responses from three HIV-infected patients by the HIV Savine constructs. PBMCs from three different patients were restimulated for 7 days by infection with Vaccinia virus pools expressing the HIV Savine cassettes: Pool 1 included VV-AC1 and VV-BC1; Pool 2 included VV-AC2, VV-BC2 and VV-CC2. The restimulated PBMCs were then mixed with autologous LCLs (effector to target ratio of 50:1), which were either uninfected or infected with either Vaccinia viruses expressing the HIV proteins gag (VV-gag), env (VV-env) or pol (VV-pol), VV-HIV Savine pools 1 (light bars) or 2 (dark bars) or a control Vaccinia virus (VV-Lac) and the amount of 51Cr released used to determine percent specific lysis. K562 cells were used to determine the level of NK cell-mediated killing in their stimulated culture.
FIG. 20 is a diagrammatic representation showing CD4+ proliferation of PBMCs from HIV-1 infected patients restimulated with either Pool1 or Pool2 of the HIV-1 Savine. Briefly PBMCs were stained with CFSE and culture for 6 days with or without VVs encoding either pooh or pool2 of the HIV-1 Savine. Restimulated Cells were then labelled with antibodies and analysed by FACS.
FIG. 21 is a graphical representation showing the CTL response in mice vaccinated with the HIV Savine. C57BL6 mice were immunised with the HIV-1 Savine DNA vaccine comprising the six plasmids described in FIG. 18a (100 μg total DNA was given as 50 μg/leg i.m.). One week later Poxviruses (1×107 pfu) comprising Pool 1 of the HIV-1 Savine were used to boost the immune responses. Three weeks later splenocytes from these mice were restimulated with VV-Pool 1 or VV-Pool 2 for 5 days and the resultant effectors used in a 51Cr release cytotoxicity assay against targets infected with CTRVV, VV-pools or VV expressing the natural antigens from HIV-1.
FIG. 22 shows immune responses of HIV Immune Macaques (vaccinated with recombinant FPV expressing gag-pol and challenged with HIV-1 2 years prior to experiment). Monkeys 1 and 2 were immunised once at day 0 with VV Savine pool 1 (Three VVs which together express the entire HIV Savine). Monkey 3 was immunised twice with FPV-gag-pol ie., Day 0 is 3 weeks after first FPV-gag-pol immunisation. A) IFN-γ detection by ELISPOT of whole blood (0.5 mL, venous blood heparin-anticoagulated) stimulated with Aldrithiol-2 inactivated whole HIV-1 (20 hours, 20 μg/mL). Plasma samples were then centrifuged (1000×g) and assayed in duplicate for antigen-specific IFN using capture ELISA. B) Flow cytometric detection of HIV-1 specific CD69+/CD8+ T cells. Freshly isolated PBMCs were stimulated with inactivated HIV-1 as above for 16 hours, washed and labelled with the antibodies. Cells were then analysed using a FACScaliburTM flow cytometer and data. analysed using Cell-Quest software. C) Flow cytometric detection of HIV-1 specific CD69+/CD4+ T cells carried out as in B).
FIG. 23 shows a diagram of a system used to carry out the instructions encoded by the storage medium of FIGS. 28 and 29.
FIG. 24 depicts a flow diagram showing an embodiment of a method for designing synthetic polynucleotide and synthetic polypeptides of the invention.
FIG. 25 shows an algorithm, which inter alia utilises the steps of the method shown in FIG. 24.
FIG. 26 shows an example of applying the algorithm of FIG. 25 to an input consensus polyprotein sequence of Hepatitis C1a to execute the segmentation of the polyprotein sequence, the rearrangement of the segments, the linkage of the rearranged segments and the outputting of synthetic polynucleotide and polypeptide sequences for the preparation of Savines for treating and/or preventing Hepatitis C infection.
FIG. 27 illustrates an example of applying the algorithm of FIG. 25 to input consensus melanocyte differentiation antigens (gp100, MART, TRP-1, Tyros, Trp-2, MC1R, MUC1F and MUC1R) and to consensus melanoma specific antigens (BAGE, GAGE-1, gp100In4, MAGE-1, MAGE-3, PRAME, TRP2IN2, NYNSO1a, NYNSO1b and LAGE1) to facilitate segmentation of those sequences, to rearrange the segments, to link the rearranged segments and to synthetic polynucleotide and polypeptide sequences for the preparation of Savines for treating and/or preventing melanoma.
FIG. 28 shows a cross section of a magnetic storage medium.
FIG. 29 shows a cross section of an optically readable data storage medium.
FIG. 30 shows six HIV Savine cassette sequences (A1 [SEQ ID NO: 393], A2 [SEQ ID NO: 399], B1[SEQ ID NO: 395], B2 [SEQ ID NO: 401], C1 [SEQ ID NO: 397] and C2 [SEQ ID NO: 403]). A1, B1 and C1 can be joined together using, for example, convenient restriction enzyme sites provided at the ends of each cassette to construct an embodiment of a full length HIV Savine [SEQ ID NO: 405]. A2, B2 and C2 can also be joined together to provide another embodiment of a full length HIV Savine with 350 aa mutations common in major HIV clades. The cassettes A/B/C can be joined into single constructs using specific restriction enzyme sites incorporated after the start codon or before the stop codon in the cassettes



BRIEF DESCRIPTION OF THE SEQUENCES: SUMMARY TABLE









TABLE A





SEQUENCE ID




NUMBER
SEQUENCE
Length


















SEQ ID NO: 1
GAG consensus polypeptide
499
aa


SEQ ID NO: 2
POL consensus polypeptide
995
aa


SEQ ID NO: 3
VIF consensus polypeptide
192
aa


SEQ ID NO: 4
VPR consensus polypeptide
96
aa


SEQ ID NO: 5
TAT consensus polypeptide
102
aa


SEQ ID NO: 6
REV consensus polypeptide
123
aa


SEQ ID NO: 7
VPU consensus polypeptide
81
aa


SEQ ID NO: 8
ENV consensus polypeptide
651
aa


SEQ ID NO: 9
NEF consensus polypeptide
206
aa


SEQ ID NO: 10
GAG segment 1
90
nts


SEQ ID NO: 11
Polypeptide encoded by SEQ ID NO: 10
30
aa


SEQ ID NO: 12
GAG segment 2
90
nts


SEQ ID NO: 13
Polypeptide encoded by SEQ ID NO: 12
30
aa


SEQ ID NO: 14
GAG segment 3
90
nts


SEQ ID NO: 15
Polypeptide encoded by SEQ ID NO: 14
30
aa


SEQ ID NO: 16
GAG segment 4
90
nts


SEQ ID NO: 17
Polypeptide encoded by SEQ ID NO: 16
30
aa


SEQ ID NO: 18
GAG segment 5
90
nts


SEQ ID NO: 19
Polypeptide encoded by SEQ ID NO: 18
30
aa


SEQ ID NO: 20
GAG segment 6
90
nts


SEQ ID NO: 21
Polypeptide encoded by SEQ ID NO: 20
30
aa


SEQ ID NO: 22
GAG segment 7
90
nts


SEQ ID NO: 23
Polypeptide encoded by SEQ ID NO: 22
30
aa


SEQ ID NO: 24
GAG segment 8
90
nts


SEQ ID NO: 25
Polypeptide encoded by SEQ ID NO: 24
30
aa


SEQ ID NO: 26
GAG segment 9
90
nts


SEQ ID NO: 27
Polypeptide encoded by SEQ ID NO: 26
30
aa


SEQ ID NO: 28
GAG segment 10
90
nts


SEQ ID NO: 29
Polypeptide encoded by SEQ ID NO: 28
30
aa


SEQ ID NO: 30
GAG segment 11
90
nts


SEQ ID NO: 31
Polypeptide encoded by SEQ ID NO: 30
30
aa


SEQ ID NO: 32
GAG segment 12
90
nts


SEQ ID NO: 33
Polypeptide encoded by SEQ ID NO: 32
30
aa


SEQ ID NO: 34
GAG segment 13
90
nts


SEQ ID NO: 35
Polypeptide encoded by SEQ ID NO: 34
30
aa


SEQ ID NO: 36
GAG segment 14
90
nts


SEQ ID NO: 37
Polypeptide encoded by SEQ ID NO: 36
30
aa


SEQ ID NO: 38
GAG segment 15
90
nts


SEQ ID NO: 39
Polypeptide encoded by SEQ ID NO: 38
30
aa


SEQ ID NO: 40
GAG segment 16
90
nts


SEQ ID NO: 41
Polypeptide encoded by SEQ ID NO: 40
30
aa


SEQ ID NO: 42
GAG segment 17
90
nts


SEQ ID NO: 43
Polypeptide encoded by SEQ ID NO: 42
30
aa


SEQ ID NO: 44
GAG segment 18
90
nts


SEQ ID NO: 45
Polypeptide encoded by SEQ ID NO: 44
30
aa


SEQ ID NO: 46
GAG segment 19
90
nts


SEQ ID NO: 47
Polypeptide encoded by SEQ ID NO: 46
30
aa


SEQ ID NO: 48
GAG segment 20
90
nts


SEQ ID NO: 49
Polypeptide encoded by SEQ ID NO: 48
30
aa


SEQ ID NO: 50
GAG segment 21
90
nts


SEQ ID NO: 51
Polypeptide encoded by SEQ ID NO: 50
30
aa


SEQ ID NO: 52
GAG segment 22
90
nts


SEQ ID NO: 53
Polypeptide encoded by SEQ ID NO: 52
30
aa


SEQ ID NO: 54
GAG segment 23
90
nts


SEQ ID NO: 55
Polypeptide encoded by SEQ ID NO: 54
30
aa


SEQ ID NO: 56
GAG segment 24
90
nts


SEQ ID NO: 57
Polypeptide encoded by SEQ ID NO: 56
30
aa


SEQ ID NO: 58
GAG segment 25
90
nts


SEQ ID NO: 59
Polypeptide encoded by SEQ ID NO: 58
30
aa


SEQ ID NO: 60
GAG segment 26
90
nts


SEQ ID NO: 61
Polypeptide encoded by SEQ ID NO: 60
30
aa


SEQ ID NO: 62
GAG segment 27
90
nts


SEQ ID NO: 63
Polypeptide encoded by SEQ ID NO: 62
30
aa


SEQ ID NO: 64
GAG segment 28
90
nts


SEQ ID NO: 65
Polypeptide encoded by SEQ ID NO: 64
30
aa


SEQ ID NO: 66
GAG segment 29
90
nts


SEQ ID NO: 67
Polypeptide encoded by SEQ ID NO: 66
30
aa


SEQ ID NO: 68
GAG segment 30
90
nts


SEQ ID NO: 69
Polypeptide encoded by SEQ ID NO: 68
30
aa


SEQ ID NO: 70
GAG segment 31
90
nts


SEQ ID NO: 71
Polypeptide encoded by SEQ ID NO: 70
30
aa


SEQ ID NO: 72
GAG segment 32
90
nts


SEQ ID NO: 73
Polypeptide encoded by SEQ ID NO: 72
30
aa


SEQ ID NO: 74
GAG segment 33
57
nts


SEQ ID NO: 75
Polypeptide encoded by SEQ ID NO: 74
19
aa


SEQ ID NO: 76
POL segment 1
90
nts


SEQ ID NO: 77
Polypeptide encoded by SEQ ID NO: 76
30
aa


SEQ ID NO: 78
POL segment 2
90
nts


SEQ ID NO: 79
Polypeptide encoded by SEQ ID NO: 78
30
aa


SEQ ID NO: 80
POL segment 3
90
nts


SEQ ID NO: 81
Polypeptide encoded by SEQ ID NO: 80
30
aa


SEQ ID NO: 82
POL segment 4
90
nts


SEQ ID NO: 83
Polypeptide encoded by SEQ ID NO: 82
30
aa


SEQ ID NO: 84
POL segment 5
90
nts


SEQ ID NO: 85
Polypeptide encoded by SEQ ID NO: 84
30
aa


SEQ ID NO: 86
POL segment 6
90
nts


SEQ ID NO: 87
Polypeptide encoded by SEQ ID NO: 86
30
aa


SEQ ID NO: 88
POL segment 7
90
nts


SEQ ID NO: 89
Polypeptide encoded by SEQ ID NO: 88
30
aa


SEQ ID NO: 90
POL segment 8
90
nts


SEQ ID NO: 91
Polypeptide encoded by SEQ ID NO: 90
30
aa


SEQ ID NO: 92
POL segment 9
90
nts


SEQ ID NO: 93
Polypeptide encoded by SEQ ID NO: 92
30
aa


SEQ ID NO: 94
POL segment 10
90
nts


SEQ ID NO: 95
Polypeptide encoded by SEQ ID NO: 94
30
aa


SEQ ID NO: 96
POL segment 11
90
nts


SEQ ID NO: 97
Polypeptide encoded by SEQ ID NO: 96
30
aa


SEQ ID NO: 98
POL segment 12
90
nts


SEQ ID NO: 99
Polypeptide encoded by SEQ ID NO: 98
30
aa


SEQ ID NO: 100
POL segment 13
90
nts


SEQ ID NO: 101
Polypeptide encoded by SEQ ID NO: 100
30
aa


SEQ ID NO: 102
POL segment 14
90
nts


SEQ ID NO: 103
Polypeptide encoded by SEQ ID NO: 102
30
aa


SEQ ID NO: 104
POL segment 15
90
nts


SEQ ID NO: 105
Polypeptide encoded by SEQ ID NO: 104
30
aa


SEQ ID NO: 106
POL segment 16
90
nts


SEQ ID NO: 107
Polypeptide encoded by SEQ ID NO: 106
30
aa


SEQ ID NO: 108
POL segment 17
90
nts


SEQ ID NO: 109
Polypeptide encoded by SEQ ID NO: 108
30
aa


SEQ ID NO: 110
POL segment 18
90
nts


SEQ ID NO: 111
Polypeptide encoded by SEQ ID NO: 110
30
aa


SEQ ID NO: 112
POL segment 19
90
nts


SEQ ID NO: 113
Polypeptide encoded by SEQ ID NO: 112
30
aa


SEQ ID NO: 114
POL segment 20
90
nts


SEQ ID NO: 115
Polypeptide encoded by SEQ ID NO: 114
30
aa


SEQ ID NO: 116
POL segment 21
90
nts


SEQ ID NO: 117
Polypeptide encoded by SEQ ID NO: 116
30
aa


SEQ ID NO: 118
POL segment 22
90
nts


SEQ ID NO: 119
Polypeptide encoded by SEQ ID NO: 118
30
aa


SEQ ID NO: 120
POL segment 23
90
nts


SEQ ID NO: 121
Polypeptide encoded by SEQ ID NO: 120
30
aa


SEQ ID NO: 122
POL segment 24
90
nts


SEQ ID NO: 123
Polypeptide encoded by SEQ ID NO: 122
30
aa


SEQ ID NO: 124
POL segment 25
90
nts


SEQ ID NO: 125
Polypeptide encoded by SEQ ID NO: 124
30
aa


SEQ ID NO: 126
POL segment 26
90
nts


SEQ ID NO: 127
Polypeptide encoded by SEQ ID NO: 126
30
aa


SEQ ID NO: 128
POL segment 27
90
nts


SEQ ID NO: 129
Polypeptide encoded by SEQ ID NO: 128
30
aa


SEQ ID NO: 130
POL segment 28
90
nts


SEQ ID NO: 131
Polypeptide encoded by SEQ ID NO: 130
30
aa


SEQ ID NO: 132
POL segment 29
90
nts


SEQ ID NO: 133
Polypeptide encoded by SEQ ID NO: 132
30
aa


SEQ ID NO: 134
POL segment 30
90
nts


SEQ ID NO: 135
Polypeptide encoded by SEQ ID NO: 134
30
aa


SEQ ID NO: 136
POL segment 31
90
nts


SEQ ID NO: 137
Polypeptide encoded by SEQ ID NO: 136
30
aa


SEQ ID NO: 138
POL segment 32
90
nts


SEQ ID NO: 139
Polypeptide encoded by SEQ ID NO: 138
30
aa


SEQ ID NO: 140
POL segment 33
90
nts


SEQ ID NO: 141
Polypeptide encoded by SEQ ID NO: 140
30
aa


SEQ ID NO: 142
POL segment 34
90
nts


SEQ ID NO: 143
Polypeptide encoded by SEQ ID NO: 142
30
aa


SEQ ID NO: 144
POL segment 35
90
nts


SEQ ID NO: 145
Polypeptide encoded by SEQ ID NO: 144
30
aa


SEQ ID NO: 146
POL segment 36
90
nts


SEQ ID NO: 147
Polypeptide encoded by SEQ ID NO: 146
30
aa


SEQ ID NO: 148
POL segment 37
90
nts


SEQ ID NO: 149
Polypeptide encoded by SEQ ID NO: 148
30
aa


SEQ ID NO: 150
POL segment 38
90
nts


SEQ ID NO: 151
Polypeptide encoded by SEQ ID NO: 150
30
aa


SEQ ID NO: 152
POL segment 39
90
nts


SEQ ID NO: 153
Polypeptide encoded by SEQ ID NO: 152
30
aa


SEQ ID NO: 154
POL segment 40
90
nts


SEQ ID NO: 155
Polypeptide encoded by SEQ ID NO: 154
30
aa


SEQ ID NO: 156
POL segment 41
90
nts


SEQ ID NO: 157
Polypeptide encoded by SEQ ID NO: 156
30
aa


SEQ ID NO: 158
POL segment 42
90
nts


SEQ ID NO: 159
Polypeptide encoded by SEQ ID NO: 158
30
aa


SEQ ID NO: 160
POL segment 43
90
nts


SEQ ID NO: 161
Polypeptide encoded by SEQ ID NO: 160
30
aa


SEQ ID NO: 162
POL segment 44
90
nts


SEQ ID NO: 163
Polypeptide encoded by SEQ ID NO: 162
30
aa


SEQ ID NO: 164
POL segment 45
90
nts


SEQ ID NO: 165
Polypeptide encoded by SEQ ID NO: 164
30
aa


SEQ ID NO: 166
POL segment 46
90
nts


SEQ ID NO: 167
Polypeptide encoded by SEQ ID NO: 166
30
aa


SEQ ID NO: 168
POL segment 47
90
nts


SEQ ID NO: 169
Polypeptide encoded by SEQ ID NO: 168
30
aa


SEQ ID NO: 170
POL segment 48
90
nts


SEQ ID NO: 171
Polypeptide encoded by SEQ ID NO: 170
30
aa


SEQ ID NO: 172
POL segment 49
90
nts


SEQ ID NO: 173
Polypeptide encoded by SEQ ID NO: 172
30
aa


SEQ ID NO: 174
POL segment 50
90
nts


SEQ ID NO: 175
Polypeptide encoded by SEQ ID NO: 174
30
aa


SEQ ID NO: 176
POL segment 51
90
nts


SEQ ID NO: 177
Polypeptide encoded by SEQ ID NO: 176
30
aa


SEQ ID NO: 178
POL segment 52
90
nts


SEQ ID NO: 179
Polypeptide encoded by SEQ ID NO: 178
30
aa


SEQ ID NO: 180
POL segment 53
90
nts


SEQ ID NO: 181
Polypeptide encoded by SEQ ID NO: 180
30
aa


SEQ ID NO: 182
POL segment 54
90
nts


SEQ ID NO: 183
Polypeptide encoded by SEQ ID NO: 182
30
aa


SEQ ID NO: 184
POL segment 55
90
nts


SEQ ID NO: 185
Polypeptide encoded by SEQ ID NO: 184
30
aa


SEQ ID NO: 186
POL segment 56
90
nts


SEQ ID NO: 187
Polypeptide encoded by SEQ ID NO: 186
30
aa


SEQ ID NO: 188
POL segment 57
90
nts


SEQ ID NO: 189
Polypeptide encoded by SEQ ID NO: 188
30
aa


SEQ ID NO: 190
POL segment 58
90
nts


SEQ ID NO: 191
Polypeptide encoded by SEQ ID NO: 190
30
aa


SEQ ID NO: 192
POL segment 59
90
nts


SEQ ID NO: 193
Polypeptide encoded by SEQ ID NO: 192
30
aa


SEQ ID NO: 194
POL segment 60
90
nts


SEQ ID NO: 195
Polypeptide encoded by SEQ ID NO: 194
30
aa


SEQ ID NO: 196
POL segment 61
90
nts


SEQ ID NO: 197
Polypeptide encoded by SEQ ID NO: 196
30
aa


SEQ ID NO: 198
POL segment 62
90
nts


SEQ ID NO: 199
Polypeptide encoded by SEQ ID NO: 198
30
aa


SEQ ID NO: 200
POL segment 63
90
nts


SEQ ID NO: 201
Polypeptide encoded by SEQ ID NO: 200
30
aa


SEQ ID NO: 202
POL segment 64
90
nts


SEQ ID NO: 203
Polypeptide encoded by SEQ ID NO: 202
30
aa


SEQ ID NO: 204
POL segment 65
90
nts


SEQ ID NO: 205
Polypeptide encoded by SEQ ID NO: 204
30
aa


SEQ ID NO: 206
POL segment 66
60
nts


SEQ ID NO: 207
Polypeptide encoded by SEQ ID NO: 206
20
aa


SEQ ID NO: 208
VIF segment 1
90
nts


SEQ ID NO: 209
Polypeptide encoded by SEQ ID NO: 208
30
aa


SEQ ID NO: 210
VIF segment 2
90
nts


SEQ ID NO: 211
Polypeptide encoded by SEQ ID NO: 210
30
aa


SEQ ID NO: 212
VIF segment 3
90
nts


SEQ ID NO: 213
Polypeptide encoded by SEQ ID NO: 212
30
aa


SEQ ID NO: 214
VIF segment 4
90
nts


SEQ ID NO: 215
Polypeptide encoded by SEQ ID NO: 214
30
aa


SEQ ID NO: 216
VIF segment 5
90
nts


SEQ ID NO: 217
Polypeptide encoded by SEQ ID NO: 216
30
aa


SEQ ID NO: 218
VIF segment 6
90
nts


SEQ ID NO: 219
Polypeptide encoded by SEQ ID NO: 218
30
aa


SEQ ID NO: 220
VIF segment 7
90
nts


SEQ ID NO: 221
Polypeptide encoded by SEQ ID NO: 220
30
aa


SEQ ID NO: 222
VIF segment 8
90
nts


SEQ ID NO: 223
Polypeptide encoded by SEQ ID NO: 222
30
aa


SEQ ID NO: 224
VIF segment 9
90
nts


SEQ ID NO: 225
Polypeptide encoded by SEQ ID NO: 224
30
aa


SEQ ID NO: 226
VIF segment 10
90
nts


SEQ ID NO: 227
Polypeptide encoded by SEQ ID NO: 226
30
aa


SEQ ID NO: 228
VIF segment 11
90
nts


SEQ ID NO: 229
Polypeptide encoded by SEQ ID NO: 228
30
aa


SEQ ID NO: 230
VIF segment 12
81
nts


SEQ ID NO: 231
Polypeptide encoded by SEQ ID NO: 230
27
aa


SEQ ID NO: 232
VPR segment 1
90
nts


SEQ ID NO: 233
Polypeptide encoded by SEQ ID NO: 232
30
aa


SEQ ID NO: 234
VPR segment 2
90
nts


SEQ ID NO: 235
Polypeptide encoded by SEQ ID NO: 234
30
aa


SEQ ID NO: 236
VPR segment 3
90
nts


SEQ ID NO: 237
Polypeptide encoded by SEQ ID NO: 236
30
aa


SEQ ID NO: 238
VPR segment 4
90
nts


SEQ ID NO: 239
Polypeptide encoded by SEQ ID NO: 238
30
aa


SEQ ID NO: 240
VPR segment 5
90
nts


SEQ ID NO: 241
Polypeptide encoded by SEQ ID NO: 240
30
aa


SEQ ID NO: 242
VPR segment 6
63
nts


SEQ ID NO: 243
Polypeptide encoded by SEQ ID NO: 242
21
aa


SEQ ID NO: 244
TAT segment 1
90
nts


SEQ ID NO: 245
Polypeptide encoded by SEQ ID NO: 244
30
aa


SEQ ID NO: 246
TAT segment 2
90
nts


SEQ ID NO: 247
Polypeptide encoded by SEQ ID NO: 246
30
aa


SEQ ID NO: 248
TAT segment 3
90
nts


SEQ ID NO: 249
Polypeptide encoded by SEQ ID NO: 248
30
aa


SEQ ID NO: 250
TAT segment 4
90
nts


SEQ ID NO: 251
Polypeptide encoded by SEQ ID NO: 250
30
aa


SEQ ID NO: 252
TAT segment 5
90
nts


SEQ ID NO: 253
Polypeptide encoded by SEQ ID NO: 252
30
aa


SEQ ID NO: 254
TAT segment 6
81
nts


SEQ ID NO: 255
Polypeptide encoded by SEQ ID NO: 254
27
aa


SEQ ID NO: 256
REV segment 1
90
nts


SEQ ID NO: 257
Polypeptide encoded by SEQ ID NO: 256
30
aa


SEQ ID NO: 258
REV segment 2
90
nts


SEQ ID NO: 259
Polypeptide encoded by SEQ ID NO: 258
30
aa


SEQ ID NO: 260
REV segment 3
90
nts


SEQ ID NO: 261
Polypeptide encoded by SEQ ID NO: 260
30
aa


SEQ ID NO: 262
REV segment 4
90
nts


SEQ ID NO: 263
Polypeptide encoded by SEQ ID NO: 262
30
aa


SEQ ID NO: 264
REV segment 5
90
nts


SEQ ID NO: 265
Polypeptide encoded by SEQ ID NO: 264
30
aa


SEQ ID NO: 266
REV segment 6
90
nts


SEQ ID NO: 267
Polypeptide encoded by SEQ ID NO: 266
30
aa


SEQ ID NO: 268
REV segment 7
90
nts


SEQ ID NO: 269
Polypeptide encoded by SEQ ID NO: 268
30
aa


SEQ ID NO: 270
REV segment 8
54
nts


SEQ ID NO: 271
Polypeptide encoded by SEQ ID NO: 270
18
aa


SEQ ID NO: 272
VPU segment 1
90
nts


SEQ ID NO: 273
Polypeptide encoded by SEQ ID NO: 272
30
aa


SEQ ID NO: 274
VPU segment 2
90
nts


SEQ ID NO: 275
Polypeptide encoded by SEQ ID NO: 274
30
aa


SEQ ID NO: 276
VPU segment 3
90
nts


SEQ ID NO: 277
Polypeptide encoded by SEQ ID NO: 276
30
aa


SEQ ID NO: 278
VPU segment 4
90
nts


SEQ ID NO: 279
Polypeptide encoded by SEQ ID NO: 278
30
aa


SEQ ID NO: 280
VPU segment 5
63
nts


SEQ ID NO: 281
Polypeptide encoded by SEQ ID NO: 280
21
aa


SEQ ID NO: 282
ENV segment 1
90
nts


SEQ ID NO: 283
Polypeptide encoded by SEQ ID NO: 282
30
aa


SEQ ID NO: 284
ENV segment 2
90
nts


SEQ ID NO: 285
Polypeptide encoded by SEQ ID NO: 284
30
aa


SEQ ID NO: 286
ENV segment 3
90
nts


SEQ ID NO: 287
Polypeptide encoded by SEQ ID NO: 286
30
aa


SEQ ID NO: 288
ENV segment 4
90
nts


SEQ ID NO: 289
Polypeptide encoded by SEQ ID NO: 288
30
aa


SEQ ID NO: 290
ENV segment 5
90
nts


SEQ ID NO: 291
Polypeptide encoded by SEQ ID NO: 290
30
aa


SEQ ID NO: 292
ENV segment 6
90
nts


SEQ ID NO: 293
Polypeptide encoded by SEQ ID NO: 292
30
aa


SEQ ID NO: 294
ENV segment 7
90
nts


SEQ ID NO: 295
Polypeptide encoded by SEQ ID NO: 294
30
aa


SEQ ID NO: 296
ENV segment 8
90
nts


SEQ ID NO: 297
Polypeptide encoded by SEQ ID NO: 296
30
aa


SEQ ID NO: 298
ENV segment 9
57
nts


SEQ ID NO: 299
Polypeptide encoded by SEQ ID NO: 298
19
aa


SEQ ID NO: 300
GAP A segment 1
90
nts


SEQ ID NO: 301
Polypeptide encoded by SEQ ID NO: 300
30
aa


SEQ ID NO: 302
GAP A segment 2
90
nts


SEQ ID NO: 303
Polypeptide encoded by SEQ ID NO: 302
30
aa


SEQ ID NO: 304
GAP A segment 3
90
nts


SEQ ID NO: 305
Polypeptide encoded by SEQ ID NO: 304
30
aa


SEQ ID NO: 306
GAP A segment 4
90
nts


SEQ ID NO: 307
Polypeptide encoded by SEQ ID NO: 306
30
aa


SEQ ID NO: 308
GAP A segment 5
90
nts


SEQ ID NO: 309
Polypeptide encoded by SEQ ID NO: 308
30
aa


SEQ ID NO: 310
GAP A segment 6
90
nts


SEQ ID NO: 311
Polypeptide encoded by SEQ ID NO: 310
30
aa


SEQ ID NO: 312
GAP A segment 7
75
nts


SEQ ID NO: 313
Polypeptide encoded by SEQ ID NO: 312
25
nts


SEQ ID NO: 314
GAP B segment 1
90
nts


SEQ ID NO: 315
Polypeptide encoded by SEQ ID NO: 314
30
aa


SEQ ID NO: 316
GAP B segment 2
90
nts


SEQ ID NO: 317
Polypeptide encoded by SEQ ID NO: 316
30
aa


SEQ ID NO: 318
GAP B segment 3
90
nts


SEQ ID NO: 319
Polypeptide encoded by SEQ ID NO: 318
30
aa


SEQ ID NO: 320
GAP B segment 4
90
nts


SEQ ID NO: 321
Polypeptide encoded by SEQ ID NO: 320
30
aa


SEQ ID NO: 322
GAP B segment 5
90
nts


SEQ ID NO: 323
Polypeptide encoded by SEQ ID NO: 322
30
aa


SEQ ID NO: 324
GAP B segment 6
90
nts


SEQ ID NO: 325
Polypeptide encoded by SEQ ID NO: 324
30
aa


SEQ ID NO: 326
GAP B segment 7
90
nts


SEQ ID NO: 327
Polypeptide encoded by SEQ ID NO: 326
30
aa


SEQ ID NO: 328
GAP B segment 8
90
nts


SEQ ID NO: 329
Polypeptide encoded by SEQ ID NO: 328
30
aa


SEQ ID NO: 330
GAP B segment 9
90
nts


SEQ ID NO: 331
Polypeptide encoded by SEQ ID NO: 330
30
aa


SEQ ID NO: 332
GAP B segment 10
90
nts


SEQ ID NO: 333
Polypeptide encoded by SEQ ID NO: 332
30
aa


SEQ ID NO: 334
GAP B segment 11
90
nts


SEQ ID NO: 335
Polypeptide encoded by SEQ ID NO: 334
30
aa


SEQ ID NO: 336
GAP B segment 12
90
nts


SEQ ID NO: 337
Polypeptide encoded by SEQ ID NO: 336
30
aa


SEQ ID NO: 338
GAP B segment 13
90
nts


SEQ ID NO: 339
Polypeptide encoded by SEQ ID NO: 338
30
aa


SEQ ID NO: 340
GAP B segment 14
90
nts


SEQ ID NO: 341
Polypeptide encoded by SEQ ID NO: 340
30
aa


SEQ ID NO: 342
GAP B segment 15
90
nts


SEQ ID NO: 343
Polypeptide encoded by SEQ ID NO: 342
30
aa


SEQ ID NO: 344
GAP B segment 16
90
nts


SEQ ID NO: 345
Polypeptide encoded by SEQ ID NO: 344
30
aa


SEQ ID NO: 346
GAP B segment 17
90
nts


SEQ ID NO: 347
Polypeptide encoded by SEQ ID NO: 346
30
aa


SEQ ID NO: 348
GAP B segment 18
90
nts


SEQ ID NO: 349
Polypeptide encoded by SEQ ID NO: 348
30
aa


SEQ ID NO: 350
GAP B segment 19
90
nts


SEQ ID NO: 351
Polypeptide encoded by SEQ ID NO: 350
30
aa


SEQ ID NO: 352
GAP B segment 20
90
nts


SEQ ID NO: 353
Polypeptide encoded by SEQ ID NO: 352
30
aa


SEQ ID NO: 354
GAP B segment 21
90
nts


SEQ ID NO: 355
Polypeptide encoded by SEQ ID NO: 354
30
aa


SEQ ID NO: 356
GAP B segment 22
90
nts


SEQ ID NO: 357
Polypeptide encoded by SEQ ID NO: 356
30
aa


SEQ ID NO: 358
GAP B segment 23
90
nts


SEQ ID NO: 359
Polypeptide encoded by SEQ ID NO: 358
30
aa


SEQ ID NO: 360
GAP B segment 24
90
nts


SEQ ID NO: 361
Polypeptide encoded by SEQ ID NO: 360
30
aa


SEQ ID NO: 362
GAP B segment 25
90
nts


SEQ ID NO: 363
Polypeptide encoded by SEQ ID NO: 362
30
aa


SEQ ID NO: 364
GAP B segment 26
66
nts


SEQ ID NO: 365
Polypeptide encoded by SEQ ID NO: 364
22
aa


SEQ ID NO: 366
NEF segment 1
90
nts


SEQ ID NO: 367
Polypeptide encoded by SEQ ID NO: 366
30
aa


SEQ ID NO: 368
NEF segment 2
90
nts


SEQ ID NO: 369
Polypeptide encoded by SEQ ID NO: 368
30
aa


SEQ ID NO: 370
NEF segment 3
90
nts


SEQ ID NO: 371
Polypeptide encoded by SEQ ID NO: 370
30
aa


SEQ ID NO: 372
NEF segment 4
90
nts


SEQ ID NO: 373
Polypeptide encoded by SEQ ID NO: 372
30
aa


SEQ ID NO: 374
NEF segment 5
90
nts


SEQ ID NO: 375
Polypeptide encoded by SEQ ID NO: 374
30
aa


SEQ ID NO: 376
NEF segment 6
90
nts


SEQ ID NO: 377
Polypeptide encoded by SEQ ID NO: 376
30
aa


SEQ ID NO: 378
NEF segment 7
90
nts


SEQ ID NO: 379
Polypeptide encoded by SEQ ID NO: 378
30
aa


SEQ ID NO: 380
NEF segment 8
90
nts


SEQ ID NO: 381
Polypeptide encoded by SEQ ID NO: 380
30
aa


SEQ ID NO: 382
NEF segment 9
90
nts


SEQ ID NO: 383
Polypeptide encoded by SEQ ID NO: 382
30
aa


SEQ ID NO: 384
NEF segment 10
90
nts


SEQ ID NO: 385
Polypeptide encoded by SEQ ID NO: 384
30
aa


SEQ ID NO: 386
NEF segment 11
90
nts


SEQ ID NO: 387
Polypeptide encoded by SEQ ID NO: 386
30
aa


SEQ ID NO: 388
NEF segment 12
90
nts


SEQ ID NO: 389
Polypeptide encoded by SEQ ID NO: 388
30
aa


SEQ ID NO: 390
NEF segment 13
78
nts


SEQ ID NO: 391
Polypeptide encoded by SEQ ID NO: 390
26
aa


SEQ ID NO: 392
HIV Cassette A1
5703
nts


SEQ ID NO: 393
Polypeptide encoded by SEQ ID NO: 392
1896
aa


SEQ ID NO: 394
HIV Cassette B1
5685
nts


SEQ ID NO: 395
Polypeptide encoded by SEQ ID NO: 394
1890
aa


SEQ ID NO: 396
HIV Cassette C1
5925
nts


SEQ ID NO: 397
Polypeptide encoded by SEQ ID NO: 396
1967
aa


SEQ ID NO: 398
HIV Cassette A2
5703
nts


SEQ ID NO: 399
Polypeptide encoded by SEQ ID NO: 398
1896
aa


SEQ ID NO: 400
HIV Cassette B2
5685
nts


SEQ ID NO: 401
Polypeptide encoded by SEQ ID NO: 400
1890
aa


SEQ ID NO: 402
HIV Cassette C2
5925
nts


SEQ ID NO: 403
Polypeptide encoded by SEQ ID NO: 402
1967
aa


SEQ ID NO: 404
HIV complete Savine
17244
nts


SEQ ID NO: 405
Polypeptide encoded by SEQ ID NO: 404
5747
aa


SEQ ID NO: 406
HepC1a consensus polyprotein sequence
3011
aa


SEQ ID NO: 407
HepC1a segment 1
90
nts


SEQ ID NO: 408
Polypeptide encoded by SEQ ID NO: 407
30
aa


SEQ ID NO: 409
HepC1a segment 2
90
nts


SEQ ID NO: 410
Polypeptide encoded by SEQ ID NO: 409
30
aa


SEQ ID NO: 411
HepC1a segment 3
90
nts


SEQ ID NO: 412
Polypeptide encoded by SEQ ID NO: 411
30
aa


SEQ ID NO: 413
HepC1a segment 4
90
nts


SEQ ID NO: 414
Polypeptide encoded by SEQ ID NO: 413
30
aa


SEQ ID NO: 415
HepC1a segment 5
90
nts


SEQ ID NO: 416
Polypeptide encoded by SEQ ID NO: 415
30
aa


SEQ ID NO: 417
HepC1a segment 6
90
nts


SEQ ID NO: 418
Polypeptide encoded by SEQ ID NO: 417
30
aa


SEQ ID NO: 419
HepC1a segment 7
90
nts


SEQ ID NO: 420
Polypeptide encoded by SEQ ID NO: 419
30
aa


SEQ ID NO: 421
HepC1a segment 8
90
nts


SEQ ID NO: 422
Polypeptide encoded by SEQ ID NO: 421
30
aa


SEQ ID NO: 423
HepC1a segment 9
90
nts


SEQ ID NO: 424
Polypeptide encoded by SEQ ID NO: 423
30
aa


SEQ ID NO: 425
HepC1a segment 10
90
nts


SEQ ID NO: 426
Polypeptide encoded by SEQ ID NO: 425
30
aa


SEQ ID NO: 427
HepC1a segment 11
90
nts


SEQ ID NO: 428
Polypeptide encoded by SEQ ID NO: 427
30
aa


SEQ ID NO: 429
HepC1a segment 12
90
nts


SEQ ID NO: 430
Polypeptide encoded by SEQ ID NO: 429
30
aa


SEQ ID NO: 431
HepC1a segment 13
90
nts


SEQ ID NO: 432
Polypeptide encoded by SEQ ID NO: 431
30
aa


SEQ ID NO: 433
HepC1a segment 14
90
nts


SEQ ID NO: 434
Polypeptide encoded by SEQ ID NO: 433
30
aa


SEQ ID NO: 435
HepC1a segment 15
90
nts


SEQ ID NO: 436
Polypeptide encoded by SEQ ID NO: 435
30
aa


SEQ ID NO: 437
HepC1a segment 16
90
nts


SEQ ID NO: 438
Polypeptide encoded by SEQ ID NO: 437
30
aa


SEQ ID NO: 439
HepC1a segment 17
90
nts


SEQ ID NO: 440
Polypeptide encoded by SEQ ID NO: 439
30
aa


SEQ ID NO: 441
HepC1a segment 18
90
nts


SEQ ID NO: 442
Polypeptide encoded by SEQ ID NO: 441
30
aa


SEQ ID NO: 443
HepC1a segment 19
90
nts


SEQ ID NO: 444
Polypeptide encoded by SEQ ID NO: 443
30
aa


SEQ ID NO: 445
HepC1a segment 20
90
nts


SEQ ID NO: 446
Polypeptide encoded by SEQ ID NO: 445
30
aa


SEQ ID NO: 447
HepC1a segment 21
90
nts


SEQ ID NO: 448
Polypeptide encoded by SEQ ID NO: 447
30
aa


SEQ ID NO: 449
HepC1a segment 22
90
nts


SEQ ID NO: 450
Polypeptide encoded by SEQ ID NO: 449
30
aa


SEQ ID NO: 451
HepC1a segment 23
90
nts


SEQ ID NO: 452
Polypeptide encoded by SEQ ID NO: 451
30
aa


SEQ ID NO: 453
HepC1a segment 24
90
nts


SEQ ID NO: 454
Polypeptide encoded by SEQ ID NO: 453
30
aa


SEQ ID NO: 455
HepC1a segment 25
90
nts


SEQ ID NO: 456
Polypeptide encoded by SEQ ID NO: 455
30
aa


SEQ ID NO: 457
HepC1a segment 26
90
nts


SEQ ID NO: 458
Polypeptide encoded by SEQ ID NO: 457
30
aa


SEQ ID NO: 459
HepC1a segment 27
90
nts


SEQ ID NO: 460
Polypeptide encoded by SEQ ID NO: 459
30
aa


SEQ ID NO: 461
HepC1a segment 28
90
nts


SEQ ID NO: 462
Polypeptide encoded by SEQ ID NO: 461
30
aa


SEQ ID NO: 463
HepC1a segment 29
90
nts


SEQ ID NO: 464
Polypeptide encoded by SEQ ID NO: 463
30
aa


SEQ ID NO: 465
HepC1a segment 30
90
nts


SEQ ID NO: 466
Polypeptide encoded by SEQ ID NO: 465
30
aa


SEQ ID NO: 467
HepC1a segment 31
90
nts


SEQ ID NO: 468
Polypeptide encoded by SEQ ID NO: 467
30
aa


SEQ ID NO: 469
HepC1a segment 32
90
nts


SEQ ID NO: 470
Polypeptide encoded by SEQ ID NO: 469
30
aa


SEQ ID NO: 471
HepC1a segment 33
90
nts


SEQ ID NO: 472
Polypeptide encoded by SEQ ID NO: 471
30
aa


SEQ ID NO: 473
HepC1a segment 34
90
nts


SEQ ID NO: 474
Polypeptide encoded by SEQ ID NO: 473
30
aa


SEQ ID NO: 475
HepC1a segment 35
90
nts


SEQ ID NO: 476
Polypeptide encoded by SEQ ID NO: 475
30
aa


SEQ ID NO: 477
HepC1a segment 36
90
nts


SEQ ID NO: 478
Polypeptide encoded by SEQ ID NO: 477
30
aa


SEQ ID NO: 479
HepC1a segment 37
90
nts


SEQ ID NO: 480
Polypeptide encoded by SEQ ID NO: 479
30
aa


SEQ ID NO: 481
HepC1a segment 38
90
nts


SEQ ID NO: 482
Polypeptide encoded by SEQ ID NO: 481
30
aa


SEQ ID NO: 483
HepC1a segment 39
90
nts


SEQ ID NO: 484
Polypeptide encoded by SEQ ID NO: 483
30
aa


SEQ ID NO: 485
HepC1a segment 40
90
nts


SEQ ID NO: 486
Polypeptide encoded by SEQ ID NO: 485
30
aa


SEQ ID NO: 487
HepC1a segment 41
90
nts


SEQ ID NO: 488
Polypeptide encoded by SEQ ID NO: 487
30
aa


SEQ ID NO: 489
HepC1a segment 42
90
nts


SEQ ID NO: 490
Polypeptide encoded by SEQ ID NO: 489
30
aa


SEQ ID NO: 491
HepC1a segment 43
90
nts


SEQ ID NO: 492
Polypeptide encoded by SEQ ID NO: 491
30
aa


SEQ ID NO: 493
HepC1a segment 44
90
nts


SEQ ID NO: 494
Polypeptide encoded by SEQ ID NO: 493
30
aa


SEQ ID NO: 495
HepC1a segment 45
90
nts


SEQ ID NO: 496
Polypeptide encoded by SEQ ID NO: 495
30
aa


SEQ ID NO: 497
HepC1a segment 46
90
nts


SEQ ID NO: 498
Polypeptide encoded by SEQ ID NO: 497
30
aa


SEQ ID NO: 499
HepC1a segment 47
90
nts


SEQ ID NO: 500
Polypeptide encoded by SEQ ID NO: 499
30
aa


SEQ ID NO: 501
HepC1a segment 48
90
nts


SEQ ID NO: 502
Polypeptide encoded by SEQ ID NO: 501
30
aa


SEQ ID NO: 503
HepC1a segment 49
90
nts


SEQ ID NO: 504
Polypeptide encoded by SEQ ID NO: 503
30
aa


SEQ ID NO: 505
HepC1a segment 50
90
nts


SEQ ID NO: 506
Polypeptide encoded by SEQ ID NO: 505
30
aa


SEQ ID NO: 507
HepC1a segment 51
90
nts


SEQ ID NO: 508
Polypeptide encoded by SEQ ID NO: 507
30
aa


SEQ ID NO: 509
HepC1a segment 52
90
nts


SEQ ID NO: 510
Polypeptide encoded by SEQ ID NO: 509
30
aa


SEQ ID NO: 511
HepC1a segment 53
90
nts


SEQ ID NO: 512
Polypeptide encoded by SEQ ID NO: 511
30
aa


SEQ ID NO: 513
HepC1a segment 54
90
nts


SEQ ID NO: 514
Polypeptide encoded by SEQ ID NO: 513
30
aa


SEQ ID NO: 515
HepC1a segment 55
90
nts


SEQ ID NO: 516
Polypeptide encoded by SEQ ID NO: 515
30
aa


SEQ ID NO: 517
HepC1a segment 56
90
nts


SEQ ID NO: 518
Polypeptide encoded by SEQ ID NO: 517
30
aa


SEQ ID NO: 519
HepC1a segment 57
90
nts


SEQ ID NO: 520
Polypeptide encoded by SEQ ID NO: 519
30
aa


SEQ ID NO: 521
HepC1a segment 58
90
nts


SEQ ID NO: 522
Polypeptide encoded by SEQ ID NO: 521
30
aa


SEQ ID NO: 523
HepC1a segment 59
90
nts


SEQ ID NO: 524
Polypeptide encoded by SEQ ID NO: 523
30
aa


SEQ ID NO: 525
HepC1a segment 60
90
nts


SEQ ID NO: 526
Polypeptide encoded by SEQ ID NO: 525
30
aa


SEQ ID NO: 527
HepC1a segment 61
90
nts


SEQ ID NO: 528
Polypeptide encoded by SEQ ID NO: 527
30
aa


SEQ ID NO: 529
HepC1a segment 62
90
nts


SEQ ID NO: 530
Polypeptide encoded by SEQ ID NO: 529
30
aa


SEQ ID NO: 531
HepC1a segment 63
90
nts


SEQ ID NO: 532
Polypeptide encoded by SEQ ID NO: 531
30
aa


SEQ ID NO: 533
HepC1a segment 64
90
nts


SEQ ID NO: 534
Polypeptide encoded by SEQ ID NO: 533
30
aa


SEQ ID NO: 535
HepC1a segment 65
90
nts


SEQ ID NO: 536
Polypeptide encoded by SEQ ID NO: 535
30
aa


SEQ ID NO: 537
HepC1a segment 66
90
nts


SEQ ID NO: 538
Polypeptide encoded by SEQ ID NO: 537
30
aa


SEQ ID NO: 539
HepC1a segment 67
90
nts


SEQ ID NO: 540
Polypeptide encoded by SEQ ID NO: 539
30
aa


SEQ ID NO: 541
HepC1a segment 68
90
nts


SEQ ID NO: 542
Polypeptide encoded by SEQ ID NO: 541
30
aa


SEQ ID NO: 543
HepC1a segment 69
90
nts


SEQ ID NO: 544
Polypeptide encoded by SEQ ID NO: 543
30
aa


SEQ ID NO: 545
HepC1a segment 70
90
nts


SEQ ID NO: 546
Polypeptide encoded by SEQ ID NO: 545
30
aa


SEQ ID NO: 547
HepC1a segment 71
90
nts


SEQ ID NO: 548
Polypeptide encoded by SEQ ID NO: 547
30
aa


SEQ ID NO: 549
HepC1a segment 72
90
nts


SEQ ID NO: 550
Polypeptide encoded by SEQ ID NO: 549
30
aa


SEQ ID NO: 551
HepC1a segment 73
90
nts


SEQ ID NO: 552
Polypeptide encoded by SEQ ID NO: 551
30
aa


SEQ ID NO: 553
HepC1a segment 74
90
nts


SEQ ID NO: 554
Polypeptide encoded by SEQ ID NO: 553
30
aa


SEQ ID NO: 555
HepC1a segment 75
90
nts


SEQ ID NO: 556
Polypeptide encoded by SEQ ID NO: 555
30
aa


SEQ ID NO: 557
HepC1a segment 76
90
nts


SEQ ID NO: 558
Polypeptide encoded by SEQ ID NO: 557
30
aa


SEQ ID NO: 559
HepC1a segment 77
90
nts


SEQ ID NO: 560
Polypeptide encoded by SEQ ID NO: 559
30
aa


SEQ ID NO: 561
HepC1a segment 78
90
nts


SEQ ID NO: 562
Polypeptide encoded by SEQ ID NO: 561
30
aa


SEQ ID NO: 563
HepC1a segment 79
90
nts


SEQ ID NO: 564
Polypeptide encoded by SEQ ID NO: 563
30
aa


SEQ ID NO: 565
HepC1a segment 80
90
nts


SEQ ID NO: 566
Polypeptide encoded by SEQ ID NO: 565
30
aa


SEQ ID NO: 567
HepC1a segment 81
90
nts


SEQ ID NO: 568
Polypeptide encoded by SEQ ID NO: 567
30
aa


SEQ ID NO: 569
HepC1a segment 82
90
nts


SEQ ID NO: 570
Polypeptide encoded by SEQ ID NO: 569
30
aa


SEQ ID NO: 571
HepC1a segment 83
90
nts


SEQ ID NO: 572
Polypeptide encoded by SEQ ID NO: 571
30
aa


SEQ ID NO: 573
HepC1a segment 84
90
nts


SEQ ID NO: 574
Polypeptide encoded by SEQ ID NO: 573
30
aa


SEQ ID NO: 575
HepC1a segment 85
90
nts


SEQ ID NO: 576
Polypeptide encoded by SEQ ID NO: 575
30
aa


SEQ ID NO: 577
HepC1a segment 86
90
nts


SEQ ID NO: 578
Polypeptide encoded by SEQ ID NO: 577
30
aa


SEQ ID NO: 579
HepC1a segment 87
90
nts


SEQ ID NO: 580
Polypeptide encoded by SEQ ID NO: 579
30
aa


SEQ ID NO: 581
HepC1a segment 88
90
nts


SEQ ID NO: 582
Polypeptide encoded by SEQ ID NO: 581
30
aa


SEQ ID NO: 583
HepC1a segment 89
90
nts


SEQ ID NO: 584
Polypeptide encoded by SEQ ID NO: 583
30
aa


SEQ ID NO: 585
HepC1a segment 90
90
nts


SEQ ID NO: 586
Polypeptide encoded by SEQ ID NO: 585
30
aa


SEQ ID NO: 587
HepC1a segment 91
90
nts


SEQ ID NO: 588
Polypeptide encoded by SEQ ID NO: 587
30
aa


SEQ ID NO: 589
HepC1a segment 92
90
nts


SEQ ID NO: 590
Polypeptide encoded by SEQ ID NO: 589
30
aa


SEQ ID NO: 591
HepC1a segment 93
90
nts


SEQ ID NO: 592
Polypeptide encoded by SEQ ID NO: 591
30
aa


SEQ ID NO: 593
HepC1a segment 94
90
nts


SEQ ID NO: 594
Polypeptide encoded by SEQ ID NO: 593
30
aa


SEQ ID NO: 595
HepC1a segment 95
90
nts


SEQ ID NO: 596
Polypeptide encoded by SEQ ID NO: 595
30
aa


SEQ ID NO: 597
HepC1a segment 96
90
nts


SEQ ID NO: 598
Polypeptide encoded by SEQ ID NO: 597
30
aa


SEQ ID NO: 599
HepC1a segment 97
90
nts


SEQ ID NO: 600
Polypeptide encoded by SEQ ID NO: 599
30
aa


SEQ ID NO: 601
HepC1a segment 98
90
nts


SEQ ID NO: 602
Polypeptide encoded by SEQ ID NO: 601
30
aa


SEQ ID NO: 603
HepC1a segment 99
90
nts


SEQ ID NO: 604
Polypeptide encoded by SEQ ID NO: 603
30
aa


SEQ ID NO: 605
HepC1a segment 100
90
nts


SEQ ID NO: 606
Polypeptide encoded by SEQ ID NO: 605
30
aa


SEQ ID NO: 607
HepC1a segment 101
90
nts


SEQ ID NO: 608
Polypeptide encoded by SEQ ID NO: 607
30
aa


SEQ ID NO: 609
HepC1a segment 102
90
nts


SEQ ID NO: 610
Polypeptide encoded by SEQ ID NO: 609
30
aa


SEQ ID NO: 611
HepC1a segment 103
90
nts


SEQ ID NO: 612
Polypeptide encoded by SEQ ID NO: 611
30
aa


SEQ ID NO: 613
HepC1a segment 104
90
nts


SEQ ID NO: 614
Polypeptide encoded by SEQ ID NO: 613
30
aa


SEQ ID NO: 615
HepC1a segment 105
90
nts


SEQ ID NO: 616
Polypeptide encoded by SEQ ID NO: 615
30
aa


SEQ ID NO: 617
HepC1a segment 106
90
nts


SEQ ID NO: 618
Polypeptide encoded by SEQ ID NO: 617
30
aa


SEQ ID NO: 619
HepC1a segment 107
90
nts


SEQ ID NO: 620
Polypeptide encoded by SEQ ID NO: 619
30
aa


SEQ ID NO: 621
HepC1a segment 108
90
nts


SEQ ID NO: 622
Polypeptide encoded by SEQ ID NO: 621
30
aa


SEQ ID NO: 623
HepC1a segment 109
90
nts


SEQ ID NO: 624
Polypeptide encoded by SEQ ID NO: 623
30
aa


SEQ ID NO: 625
HepC1a segment 110
90
nts


SEQ ID NO: 626
Polypeptide encoded by SEQ ID NO: 625
30
aa


SEQ ID NO: 627
HepC1a segment 111
90
nts


SEQ ID NO: 628
Polypeptide encoded by SEQ ID NO: 627
30
aa


SEQ ID NO: 629
HepC1a segment 112
90
nts


SEQ ID NO: 630
Polypeptide encoded by SEQ ID NO: 629
30
aa


SEQ ID NO: 631
HepC1a segment 113
90
nts


SEQ ID NO: 632
Polypeptide encoded by SEQ ID NO: 631
30
aa


SEQ ID NO: 633
HepC1a segment 114
90
nts


SEQ ID NO: 634
Polypeptide encoded by SEQ ID NO: 633
30
aa


SEQ ID NO: 635
HepC1a segment 115
90
nts


SEQ ID NO: 636
Polypeptide encoded by SEQ ID NO: 635
30
aa


SEQ ID NO: 637
HepC1a segment 116
90
nts


SEQ ID NO: 638
Polypeptide encoded by SEQ ID NO: 637
30
aa


SEQ ID NO: 639
HepC1a segment 117
90
nts


SEQ ID NO: 640
Polypeptide encoded by SEQ ID NO: 639
30
aa


SEQ ID NO: 641
HepC1a segment 118
90
nts


SEQ ID NO: 642
Polypeptide encoded by SEQ ID NO: 641
30
aa


SEQ ID NO: 643
HepC1a segment 119
90
nts


SEQ ID NO: 644
Polypeptide encoded by SEQ ID NO: 643
30
aa


SEQ ID NO: 645
HepC1a segment 120
90
nts


SEQ ID NO: 646
Polypeptide encoded by SEQ ID NO: 645
30
aa


SEQ ID NO: 647
HepC1a segment 121
90
nts


SEQ ID NO: 648
Polypeptide encoded by SEQ ID NO: 647
30
aa


SEQ ID NO: 649
HepC1a segment 122
90
nts


SEQ ID NO: 650
Polypeptide encoded by SEQ ID NO: 649
30
aa


SEQ ID NO: 651
HepC1a segment 123
90
nts


SEQ ID NO: 652
Polypeptide encoded by SEQ ID NO: 651
30
aa


SEQ ID NO: 653
HepC1a segment 124
90
nts


SEQ ID NO: 654
Polypeptide encoded by SEQ ID NO: 653
30
aa


SEQ ID NO: 655
HepC1a segment 125
90
nts


SEQ ID NO: 656
Polypeptide encoded by SEQ ID NO: 655
30
aa


SEQ ID NO: 657
HepC1a segment 126
90
nts


SEQ ID NO: 658
Polypeptide encoded by SEQ ID NO: 657
30
aa


SEQ ID NO: 659
HepC1a segment 127
90
nts


SEQ ID NO: 660
Polypeptide encoded by SEQ ID NO: 659
30
aa


SEQ ID NO: 661
HepC1a segment 128
90
nts


SEQ ID NO: 662
Polypeptide encoded by SEQ ID NO: 661
30
aa


SEQ ID NO: 663
HepC1a segment 129
90
nts


SEQ ID NO: 664
Polypeptide encoded by SEQ ID NO: 663
30
aa


SEQ ID NO: 665
HepC1a segment 130
90
nts


SEQ ID NO: 666
Polypeptide encoded by SEQ ID NO: 665
30
aa


SEQ ID NO: 667
HepC1a segment 131
90
nts


SEQ ID NO: 668
Polypeptide encoded by SEQ ID NO: 667
30
aa


SEQ ID NO: 669
HepC1a segment 132
90
nts


SEQ ID NO: 670
Polypeptide encoded by SEQ ID NO: 669
30
aa


SEQ ID NO: 671
HepC1a segment 133
90
nts


SEQ ID NO: 672
Polypeptide encoded by SEQ ID NO: 671
30
aa


SEQ ID NO: 673
HepC1a segment 134
90
nts


SEQ ID NO: 674
Polypeptide encoded by SEQ ID NO: 673
30
aa


SEQ ID NO: 675
HepC1a segment 135
90
nts


SEQ ID NO: 676
Polypeptide encoded by SEQ ID NO: 675
30
aa


SEQ ID NO: 677
HepC1a segment 136
90
nts


SEQ ID NO: 678
Polypeptide encoded by SEQ ID NO: 677
30
aa


SEQ ID NO: 679
HepC1a segment 137
90
nts


SEQ ID NO: 680
Polypeptide encoded by SEQ ID NO: 679
30
aa


SEQ ID NO: 681
HepC1a segment 138
90
nts


SEQ ID NO: 682
Polypeptide encoded by SEQ ID NO: 681
30
aa


SEQ ID NO: 683
HepC1a segment 139
90
nts


SEQ ID NO: 684
Polypeptide encoded by SEQ ID NO: 683
30
aa


SEQ ID NO: 685
HepC1a segment 140
90
nts


SEQ ID NO: 686
Polypeptide encoded by SEQ ID NO: 685
30
aa


SEQ ID NO: 687
HepC1a segment 141
90
nts


SEQ ID NO: 688
Polypeptide encoded by SEQ ID NO: 687
30
aa


SEQ ID NO: 689
HepC1a segment 142
90
nts


SEQ ID NO: 690
Polypeptide encoded by SEQ ID NO: 689
30
aa


SEQ ID NO: 691
HepC1a segment 143
90
nts


SEQ ID NO: 692
Polypeptide encoded by SEQ ID NO: 691
30
aa


SEQ ID NO: 693
HepC1a segment 144
90
nts


SEQ ID NO: 694
Polypeptide encoded by SEQ ID NO: 693
30
aa


SEQ ID NO: 695
HepC1a segment 145
90
nts


SEQ ID NO: 696
Polypeptide encoded by SEQ ID NO: 695
30
aa


SEQ ID NO: 697
HepC1a segment 146
90
nts


SEQ ID NO: 698
Polypeptide encoded by SEQ ID NO: 697
30
aa


SEQ ID NO: 699
HepC1a segment 147
90
nts


SEQ ID NO: 700
Polypeptide encoded by SEQ ID NO: 699
30
aa


SEQ ID NO: 701
HepC1a segment 148
90
nts


SEQ ID NO: 702
Polypeptide encoded by SEQ ID NO: 701
30
aa


SEQ ID NO: 703
HepC1a segment 149
90
nts


SEQ ID NO: 704
Polypeptide encoded by SEQ ID NO: 703
30
aa


SEQ ID NO: 705
HepC1a segment 150
90
nts


SEQ ID NO: 706
Polypeptide encoded by SEQ ID NO: 705
30
aa


SEQ ID NO: 707
HepC1a segment 151
90
nts


SEQ ID NO: 708
Polypeptide encoded by SEQ ID NO: 707
30
aa


SEQ ID NO: 709
HepC1a segment 152
90
nts


SEQ ID NO: 710
Polypeptide encoded by SEQ ID NO: 709
30
aa


SEQ ID NO: 711
HepC1a segment 153
90
nts


SEQ ID NO: 712
Polypeptide encoded by SEQ ID NO: 711
30
aa


SEQ ID NO: 713
HepC1a segment 154
90
nts


SEQ ID NO: 714
Polypeptide encoded by SEQ ID NO: 713
30
aa


SEQ ID NO: 715
HepC1a segment 155
90
nts


SEQ ID NO: 716
Polypeptide encoded by SEQ ID NO: 715
30
aa


SEQ ID NO: 717
HepC1a segment 156
90
nts


SEQ ID NO: 718
Polypeptide encoded by SEQ ID NO: 717
30
aa


SEQ ID NO: 719
HepC1a segment 157
90
nts


SEQ ID NO: 720
Polypeptide encoded by SEQ ID NO: 719
30
aa


SEQ ID NO: 721
HepC1a segment 158
90
nts


SEQ ID NO: 722
Polypeptide encoded by SEQ ID NO: 721
30
aa


SEQ ID NO: 723
HepC1a segment 159
90
nts


SEQ ID NO: 724
Polypeptide encoded by SEQ ID NO: 723
30
aa


SEQ ID NO: 725
HepC1a segment 160
90
nts


SEQ ID NO: 726
Polypeptide encoded by SEQ ID NO: 725
30
aa


SEQ ID NO: 727
HepC1a segment 161
90
nts


SEQ ID NO: 728
Polypeptide encoded by SEQ ID NO: 727
30
aa


SEQ ID NO: 729
HepC1a segment 162
90
nts


SEQ ID NO: 730
Polypeptide encoded by SEQ ID NO: 729
30
aa


SEQ ID NO: 731
HepC1a segment 163
90
nts


SEQ ID NO: 732
Polypeptide encoded by SEQ ID NO: 731
30
aa


SEQ ID NO: 733
HepC1a segment 164
90
nts


SEQ ID NO: 734
Polypeptide encoded by SEQ ID NO: 733
30
aa


SEQ ID NO: 735
HepC1a segment 165
90
nts


SEQ ID NO: 736
Polypeptide encoded by SEQ ID NO: 735
30
aa


SEQ ID NO: 737
HepC1a segment 166
90
nts


SEQ ID NO: 738
Polypeptide encoded by SEQ ID NO: 737
30
aa


SEQ ID NO: 739
HepC1a segment 167
90
nts


SEQ ID NO: 740
Polypeptide encoded by SEQ ID NO: 739
30
aa


SEQ ID NO: 741
HepC1a segment 168
90
nts


SEQ ID NO: 742
Polypeptide encoded by SEQ ID NO: 741
30
aa


SEQ ID NO: 743
HepC1a segment 169
90
nts


SEQ ID NO: 744
Polypeptide encoded by SEQ ID NO: 743
30
aa


SEQ ID NO: 745
HepC1a segment 170
90
nts


SEQ ID NO: 746
Polypeptide encoded by SEQ ID NO: 745
30
aa


SEQ ID NO: 747
HepC1a segment 171
90
nts


SEQ ID NO: 748
Polypeptide encoded by SEQ ID NO: 747
30
aa


SEQ ID NO: 749
HepC1a segment 172
90
nts


SEQ ID NO: 750
Polypeptide encoded by SEQ ID NO: 749
30
aa


SEQ ID NO: 751
HepC1a segment 173
90
nts


SEQ ID NO: 752
Polypeptide encoded by SEQ ID NO: 751
30
aa


SEQ ID NO: 753
HepC1a segment 174
90
nts


SEQ ID NO: 754
Polypeptide encoded by SEQ ID NO: 753
30
aa


SEQ ID NO: 755
HepC1a segment 175
90
nts


SEQ ID NO: 756
Polypeptide encoded by SEQ ID NO: 755
30
aa


SEQ ID NO: 757
HepC1a segment 176
90
nts


SEQ ID NO: 758
Polypeptide encoded by SEQ ID NO: 757
30
aa


SEQ ID NO: 759
HepC1a segment 177
90
nts


SEQ ID NO: 760
Polypeptide encoded by SEQ ID NO: 759
30
aa


SEQ ID NO: 761
HepC1a segment 178
90
nts


SEQ ID NO: 762
Polypeptide encoded by SEQ ID NO: 761
30
aa


SEQ ID NO: 763
HepC1a segment 179
90
nts


SEQ ID NO: 764
Polypeptide encoded by SEQ ID NO: 763
30
aa


SEQ ID NO: 765
HepC1a segment 180
90
nts


SEQ ID NO: 766
Polypeptide encoded by SEQ ID NO: 765
30
aa


SEQ ID NO: 767
HepC1a segment 181
90
nts


SEQ ID NO: 768
Polypeptide encoded by SEQ ID NO: 767
30
aa


SEQ ID NO: 769
HepC1a segment 182
90
nts


SEQ ID NO: 770
Polypeptide encoded by SEQ ID NO: 769
30
aa


SEQ ID NO: 771
HepC1a segment 183
90
nts


SEQ ID NO: 772
Polypeptide encoded by SEQ ID NO: 771
30
aa


SEQ ID NO: 773
HepC1a segment 184
90
nts


SEQ ID NO: 774
Polypeptide encoded by SEQ ID NO: 773
30
aa


SEQ ID NO: 775
HepC1a segment 185
90
nts


SEQ ID NO: 776
Polypeptide encoded by SEQ ID NO: 775
30
aa


SEQ ID NO: 777
HepC1a segment 186
90
nts


SEQ ID NO: 778
Polypeptide encoded by SEQ ID NO: 777
30
aa


SEQ ID NO: 779
HepC1a segment 187
90
nts


SEQ ID NO: 780
Polypeptide encoded by SEQ ID NO: 779
30
aa


SEQ ID NO: 781
HepC1a segment 188
90
nts


SEQ ID NO: 782
Polypeptide encoded by SEQ ID NO: 781
30
aa


SEQ ID NO: 783
HepC1a segment 189
90
nts


SEQ ID NO: 784
Polypeptide encoded by SEQ ID NO: 783
30
aa


SEQ ID NO: 785
HepC1a segment 190
90
nts


SEQ ID NO: 786
Polypeptide encoded by SEQ ID NO: 785
30
aa


SEQ ID NO: 787
HepC1a segment 191
90
nts


SEQ ID NO: 788
Polypeptide encoded by SEQ ID NO: 787
30
aa


SEQ ID NO: 789
HepC1a segment 192
90
nts


SEQ ID NO: 790
Polypeptide encoded by SEQ ID NO: 789
30
aa


SEQ ID NO: 791
HepC1a segment 193
90
nts


SEQ ID NO: 792
Polypeptide encoded by SEQ ID NO: 791
30
aa


SEQ ID NO: 793
HepC1a segment 194
90
nts


SEQ ID NO: 794
Polypeptide encoded by SEQ ID NO: 793
30
aa


SEQ ID NO: 795
HepC1a segment 195
90
nts


SEQ ID NO: 796
Polypeptide encoded by SEQ ID NO: 795
30
aa


SEQ ID NO: 797
HepC1a segment 196
90
nts


SEQ ID NO: 798
Polypeptide encoded by SEQ ID NO: 797
30
aa


SEQ ID NO: 799
HepC1a segment 197
90
nts


SEQ ID NO: 800
Polypeptide encoded by SEQ ID NO: 799
30
aa


SEQ ID NO: 801
HepC1a segment 198
90
nts


SEQ ID NO: 802
Polypeptide encoded by SEQ ID NO: 801
30
aa


SEQ ID NO: 803
HepC1a segment 199
90
nts


SEQ ID NO: 804
Polypeptide encoded by SEQ ID NO: 803
30
aa


SEQ ID NO: 805
HepC1a segment 200
90
nts


SEQ ID NO: 806
Polypeptide encoded by SEQ ID NO: 805
30
aa


SEQ ID NO: 807
HepC1a segment 201
45
nts


SEQ ID NO: 808
Polypeptide encoded by SEQ ID NO: 807
15
aa


SEQ ID NO: 809
HepC1a scrambled
17955
nts


SEQ ID NO: 810
Polypeptide encoded by SEQ ID NO: 809
5985
aa


SEQ ID NO: 811
HepC Cassette A
6065
nts


SEQ ID NO: 812
Polypeptide encoded by SEQ ID NO: 811
2011
aa


SEQ ID NO: 813
HepC Cassette B
6069
nts


SEQ ID NO: 814
Polypeptide encoded by SEQ ID NO: 813
2010
aa


SEQ ID NO: 815
HepC Cassette C
6030
nts


SEQ ID NO: 816
Polypeptide encoded by SEQ ID NO: 815
1997
aa


SEQ ID NO: 817
gp100 consensus polypeptide
661
aa


SEQ ID NO: 818
MART consensus polypeptide
118
aa


SEQ ID NO: 819
TRP-1 consensus polypeptide
248
aa


SEQ ID NO: 820
Tyros consensus polypeptide
529
aa


SEQ ID NO: 821
TRP2 consensus polypeptide
519
aa


SEQ ID NO: 822
MC1R consensus polypeptide
317
aa


SEQ ID NO: 823
MUC1F consensus polypeptide
125
aa


SEQ ID NO: 824
MUC1R consensus polypeptide
312
aa


SEQ ID NO: 825
BAGE consensus polypeptide
43
aa


SEQ ID NO: 826
GAGE-1 consensus polypeptide
138
aa


SEQ ID NO: 827
gp1001n4 consensus polypeptide
51
aa


SEQ ID NO: 828
MAGE-1 consensus polypeptide
309
aa


SEQ ID NO: 829
MAGE-3 consensus polypeptide
314
aa


SEQ ID NO: 830
PRAME consensus polypeptide
509
aa


SEQ ID NO: 831
TRP21N2 consensus polypeptide
54
aa


SEQ ID NO: 832
NYNSO1a consensus polypeptide
180
aa


SEQ ID NO: 833
NYNSO1b consensus polypeptide
58
aa


SEQ ID NO: 834
LAGE1 consensus polypeptide
180
aa


SEQ ID NO: 835
gp100 segment 1
90
nts


SEQ ID NO: 836
Polypeptide encoded by SEQ ID NO: 835
30
aa


SEQ ID NO: 837
gp100 segment 2
90
nts


SEQ ID NO: 838
Polypeptide encoded by SEQ ID NO: 837
30
aa


SEQ ID NO: 839
gp100 segment 3
90
nts


SEQ ID NO: 840
Polypeptide encoded by SEQ ID NO: 839
30
aa


SEQ ID NO: 841
gp100 segment 4
90
nts


SEQ ID NO: 842
Polypeptide encoded by SEQ ID NO: 841
30
aa


SEQ ID NO: 843
gp100 segment 5
90
nts


SEQ ID NO: 844
Polypeptide encoded by SEQ ID NO: 843
30
aa


SEQ ID NO: 845
gp100 segment 6
90
nts


SEQ ID NO: 846
Polypeptide encoded by SEQ ID NO: 845
30
aa


SEQ ID NO: 847
gp100 segment 7
90
nts


SEQ ID NO: 848
Polypeptide encoded by SEQ ID NO: 847
30
aa


SEQ ID NO: 849
gp100 segment 8
90
nts


SEQ ID NO: 850
Polypeptide encoded by SEQ ID NO: 849
30
aa


SEQ ID NO: 851
gp100 segment 9
90
nts


SEQ ID NO: 852
Polypeptide encoded by SEQ ID NO: 851
30
aa


SEQ ID NO: 853
gp100 segment 10
90
nts


SEQ ID NO: 854
Polypeptide encoded by SEQ ID NO: 853
30
aa


SEQ ID NO: 855
gp100 segment 11
90
nts


SEQ ID NO: 856
Polypeptide encoded by SEQ ID NO: 855
30
aa


SEQ ID NO: 857
gp100 segment 12
90
nts


SEQ ID NO: 858
Polypeptide encoded by SEQ ID NO: 857
30
aa


SEQ ID NO: 859
gp100 segment 13
90
nts


SEQ ID NO: 860
Polypeptide encoded by SEQ ID NO: 859
30
aa


SEQ ID NO: 861
gp100 segment 14
90
nts


SEQ ID NO: 862
Polypeptide encoded by SEQ ID NO: 861
30
aa


SEQ ID NO: 863
gp100 segment 15
90
nts


SEQ ID NO: 864
Polypeptide encoded by SEQ ID NO: 863
30
aa


SEQ ID NO: 865
gp100 segment 16
90
nts


SEQ ID NO: 866
Polypeptide encoded by SEQ ID NO: 865
30
aa


SEQ ID NO: 867
gp100 segment 17
90
nts


SEQ ID NO: 868
Polypeptide encoded by SEQ ID NO: 867
30
aa


SEQ ID NO: 869
gp100 segment 18
90
nts


SEQ ID NO: 870
Polypeptide encoded by SEQ ID NO: 869
30
aa


SEQ ID NO: 871
gp100 segment 19
90
nts


SEQ ID NO: 872
Polypeptide encoded by SEQ ID NO: 871
30
aa


SEQ ID NO: 873
gp100 segment 20
90
nts


SEQ ID NO: 874
Polypeptide encoded by SEQ ID NO: 873
30
aa


SEQ ID NO: 875
gp100 segment 21
90
nts


SEQ ID NO: 876
Polypeptide encoded by SEQ ID NO: 875
30
aa


SEQ ID NO: 877
gp100 segment 22
90
nts


SEQ ID NO: 878
Polypeptide encoded by SEQ ID NO: 877
30
aa


SEQ ID NO: 879
gp100 segment 23
90
nts


SEQ ID NO: 880
Polypeptide encoded by SEQ ID NO: 879
30
aa


SEQ ID NO: 881
gp100 segment 24
90
nts


SEQ ID NO: 882
Polypeptide encoded by SEQ ID NO: 881
30
aa


SEQ ID NO: 883
gp100 segment 25
90
nts


SEQ ID NO: 884
Polypeptide encoded by SEQ ID NO: 883
30
aa


SEQ ID NO: 885
gp100 segment 26
90
nts


SEQ ID NO: 886
Polypeptide encoded by SEQ ID NO: 885
30
aa


SEQ ID NO: 887
gp100 segment 27
90
nts


SEQ ID NO: 888
Polypeptide encoded by SEQ ID NO: 887
30
aa


SEQ ID NO: 889
gp100 segment 28
90
nts


SEQ ID NO: 890
Polypeptide encoded by SEQ ID NO: 889
30
aa


SEQ ID NO: 891
gp100 segment 29
90
nts


SEQ ID NO: 892
Polypeptide encoded by SEQ ID NO: 891
30
aa


SEQ ID NO: 893
gp100 segment 30
90
nts


SEQ ID NO: 894
Polypeptide encoded by SEQ ID NO: 893
30
aa


SEQ ID NO: 895
gp100 segment 31
90
nts


SEQ ID NO: 896
Polypeptide encoded by SEQ ID NO: 895
30
aa


SEQ ID NO: 897
gp100 segment 32
90
nts


SEQ ID NO: 898
Polypeptide encoded by SEQ ID NO: 897
30
aa


SEQ ID NO: 899
gp100 segment 33
90
nts


SEQ ID NO: 900
Polypeptide encoded by SEQ ID NO: 899
30
aa


SEQ ID NO: 901
gp100 segment 34
90
nts


SEQ ID NO: 902
Polypeptide encoded by SEQ ID NO: 901
30
aa


SEQ ID NO: 903
gp100 segment 35
90
nts


SEQ ID NO: 904
Polypeptide encoded by SEQ ID NO: 903
30
aa


SEQ ID NO: 905
gp100 segment 36
90
nts


SEQ ID NO: 906
Polypeptide encoded by SEQ ID NO: 905
30
aa


SEQ ID NO: 907
gp100 segment 37
90
nts


SEQ ID NO: 908
Polypeptide encoded by SEQ ID NO: 907
30
aa


SEQ ID NO: 909
gp100 segment 38
90
nts


SEQ ID NO: 910
Polypeptide encoded by SEQ ID NO: 909
30
aa


SEQ ID NO: 911
gp100 segment 39
90
nts


SEQ ID NO: 912
Polypeptide encoded by SEQ ID NO: 911
30
aa


SEQ ID NO: 913
gp100 segment 40
90
nts


SEQ ID NO: 914
Polypeptide encoded by SEQ ID NO: 913
30
aa


SEQ ID NO: 915
gp100 segment 41
90
nts


SEQ ID NO: 916
Polypeptide encoded by SEQ ID NO: 915
30
aa


SEQ ID NO: 917
gp100 segment 42
90
nts


SEQ ID NO: 918
Polypeptide encoded by SEQ ID NO: 917
30
aa


SEQ ID NO: 919
gp100 segment 43
90
nts


SEQ ID NO: 920
Polypeptide encoded by SEQ ID NO: 919
30
aa


SEQ ID NO: 921
gp100 segment 44
60
nts


SEQ ID NO: 922
Polypeptide encoded by SEQ ID NO: 921
20
aa


SEQ ID NO: 923
MART segment 1
90
nts


SEQ ID NO: 924
Polypeptide encoded by SEQ ID NO: 923
30
aa


SEQ ID NO: 925
MART segment 2
90
nts


SEQ ID NO: 926
Polypeptide encoded by SEQ ID NO: 925
30
aa


SEQ ID NO: 927
MART segment 3
90
nts


SEQ ID NO: 928
Polypeptide encoded by SEQ ID NO: 927
30
aa


SEQ ID NO: 929
MART segment 4
90
nts


SEQ ID NO: 930
Polypeptide encoded by SEQ ID NO: 929
30
aa


SEQ ID NO: 931
MART segment 5
90
nts


SEQ ID NO: 932
Polypeptide encoded by SEQ ID NO: 931
30
aa


SEQ ID NO: 933
MART segment 6
90
nts


SEQ ID NO: 934
Polypeptide encoded by SEQ ID NO: 933
30
aa


SEQ ID NO: 935
MART segment 7
90
nts


SEQ ID NO: 936
Polypeptide encoded by SEQ ID NO: 935
30
aa


SEQ ID NO: 937
MART segment 8
51
nts


SEQ ID NO: 938
Polypeptide encoded by SEQ ID NO: 937
17
aa


SEQ ID NO: 939
trp-1 segment 1
90
nts


SEQ ID NO: 940
Polypeptide encoded by SEQ ID NO: 939
30
aa


SEQ ID NO: 941
trp-1 segment 2
90
nts


SEQ ID NO: 942
Polypeptide encoded by SEQ ID NO: 941
30
aa


SEQ ID NO: 943
trp-1 segment 3
90
nts


SEQ ID NO: 944
Polypeptide encoded by SEQ ID NO: 943
30
aa


SEQ ID NO: 945
trp-1 segment 4
90
nts


SEQ ID NO: 946
Polypeptide encoded by SEQ ID NO: 945
30
aa


SEQ ID NO: 947
trp-1 segment 5
90
nts


SEQ ID NO: 948
Polypeptide encoded by SEQ ID NO: 947
30
aa


SEQ ID NO: 949
trp-1 segment 6
90
nts


SEQ ID NO: 950
Polypeptide encoded by SEQ ID NO: 949
30
aa


SEQ ID NO: 951
trp-1 segment 7
90
nts


SEQ ID NO: 952
Polypeptide encoded by SEQ ID NO: 951
30
aa


SEQ ID NO: 953
trp-1 segment 8
90
nts


SEQ ID NO: 954
Polypeptide encoded by SEQ ID NO: 953
30
aa


SEQ ID NO: 955
trp-1 segment 9
90
nts


SEQ ID NO: 956
Polypeptide encoded by SEQ ID NO: 955
30
aa


SEQ ID NO: 957
trp-1 segment 10
90
nts


SEQ ID NO: 958
Polypeptide encoded by SEQ ID NO: 957
30
aa


SEQ ID NO: 959
trp-1 segment 11
90
nts


SEQ ID NO: 960
Polypeptide encoded by SEQ ID NO: 959
30
aa


SEQ ID NO: 961
trp-1 segment 12
90
nts


SEQ ID NO: 962
Polypeptide encoded by SEQ ID NO: 961
30
aa


SEQ ID NO: 963
trp-1 segment 13
90
nts


SEQ ID NO: 964
Polypeptide encoded by SEQ ID NO: 963
30
aa


SEQ ID NO: 965
trp-1 segment 14
90
nts


SEQ ID NO: 966
Polypeptide encoded by SEQ ID NO: 965
30
aa


SEQ ID NO: 967
trp-1 segment 15
90
nts


SEQ ID NO: 968
Polypeptide encoded by SEQ ID NO: 967
30
aa


SEQ ID NO: 969
trp-1 segment 16
81
nts


SEQ ID NO: 970
Polypeptide encoded by SEQ ID NO: 969
27
aa


SEQ ID NO: 971
tyros segment 1
90
nts


SEQ ID NO: 972
Polypeptide encoded by SEQ ID NO: 971
30
aa


SEQ ID NO: 973
tyros segment 2
90
nts


SEQ ID NO: 974
Polypeptide encoded by SEQ ID NO: 973
30
aa


SEQ ID NO: 975
tyros segment 3
90
nts


SEQ ID NO: 976
Polypeptide encoded by SEQ ID NO: 975
30
aa


SEQ ID NO: 977
tyros segment 4
90
nts


SEQ ID NO: 978
Polypeptide encoded by SEQ ID NO: 977
30
aa


SEQ ID NO: 979
tyros segment 5
90
nts


SEQ ID NO: 980
Polypeptide encoded by SEQ ID NO: 979
30
aa


SEQ ID NO: 981
tyros segment 6
90
nts


SEQ ID NO: 982
Polypeptide encoded by SEQ ID NO: 981
30
aa


SEQ ID NO: 983
tyros segment 7
90
nts


SEQ ID NO: 984
Polypeptide encoded by SEQ ID NO: 983
30
aa


SEQ ID NO: 985
tyros segment 8
90
nts


SEQ ID NO: 986
Polypeptide encoded by SEQ ID NO: 985
30
aa


SEQ ID NO: 987
tyros segment 9
90
nts


SEQ ID NO: 988
Polypeptide encoded by SEQ ID NO: 987
30
aa


SEQ ID NO: 989
tyros segment 10
90
nts


SEQ ID NO: 990
Polypeptide encoded by SEQ ID NO: 989
30
aa


SEQ ID NO: 991
tyros segment 11
90
nts


SEQ ID NO: 992
Polypeptide encoded by SEQ ID NO: 991
30
aa


SEQ ID NO: 993
tyros segment 12
90
nts


SEQ ID NO: 994
Polypeptide encoded by SEQ ID NO: 993
30
aa


SEQ ID NO: 995
tyros segment 13
90
nts


SEQ ID NO: 996
Polypeptide encoded by SEQ ID NO: 995
30
aa


SEQ ID NO: 997
tyros segment 14
90
nts


SEQ ID NO: 998
Polypeptide encoded by SEQ ID NO: 997
30
aa


SEQ ID NO: 999
tyros segment 15
90
nts


SEQ ID NO: 1000
Polypeptide encoded by SEQ ID NO: 999
30
aa


SEQ ID NO: 1001
tyros segment 16
90
nts


SEQ ID NO: 1002
Polypeptide encoded by SEQ ID NO: 1001
30
aa


SEQ ID NO: 1003
tyros segment 17
90
nts


SEQ ID NO: 1004
Polypeptide encoded by SEQ ID NO: 1003
30
aa


SEQ ID NO: 1005
tyros segment 18
90
nts


SEQ ID NO: 1006
Polypeptide encoded by SEQ ID NO: 1005
30
aa


SEQ ID NO: 1007
tyros segment 19
90
nts


SEQ ID NO: 1008
Polypeptide encoded by SEQ ID NO: 1007
30
aa


SEQ ID NO: 1009
tyros segment 20
90
nts


SEQ ID NO: 1010
Polypeptide encoded by SEQ ID NO: 1009
30
aa


SEQ ID NO: 1011
tyros segment 21
90
nts


SEQ ID NO: 1012
Polypeptide encoded by SEQ ID NO: 1011
30
aa


SEQ ID NO: 1013
tyros segment 22
90
nts


SEQ ID NO: 1014
Polypeptide encoded by SEQ ID NO: 1013
30
aa


SEQ ID NO: 1015
tyros segment 23
90
nts


SEQ ID NO: 1016
Polypeptide encoded by SEQ ID NO: 1015
30
aa


SEQ ID NO: 1017
tyros segment 24
90
nts


SEQ ID NO: 1018
Polypeptide encoded by SEQ ID NO: 1017
30
aa


SEQ ID NO: 1019
tyros segment 25
90
nts


SEQ ID NO: 1020
Polypeptide encoded by SEQ ID NO: 1019
30
aa


SEQ ID NO: 1021
tyros segment 26
90
nts


SEQ ID NO: 1022
Polypeptide encoded by SEQ ID NO: 1021
30
aa


SEQ ID NO: 1023
tyros segment 27
90
nts


SEQ ID NO: 1024
Polypeptide encoded by SEQ ID NO: 1023
30
aa


SEQ ID NO: 1025
tyros segment 28
90
nts


SEQ ID NO: 1026
Polypeptide encoded by SEQ ID NO: 1025
30
aa


SEQ ID NO: 1027
tyros segment 29
90
nts


SEQ ID NO: 1028
Polypeptide encoded by SEQ ID NO: 1027
30
aa


SEQ ID NO: 1029
tyros segment 30
90
nts


SEQ ID NO: 1030
Polypeptide encoded by SEQ ID NO: 1029
30
aa


SEQ ID NO: 1031
tyros segment 31
90
nts


SEQ ID NO: 1032
Polypeptide encoded by SEQ ID NO: 1031
30
aa


SEQ ID NO: 1033
tyros segment 32
90
nts


SEQ ID NO: 1034
Polypeptide encoded by SEQ ID NO: 1033
30
aa


SEQ ID NO: 1035
tyros segment 33
90
nts


SEQ ID NO: 1036
Polypeptide encoded by SEQ ID NO: 1035
30
aa


SEQ ID NO: 1037
tyros segment 34
90
nts


SEQ ID NO: 1038
Polypeptide encoded by SEQ ID NO: 1037
30
aa


SEQ ID NO: 1039
tyros segment 35
69
nts


SEQ ID NO: 1040
Polypeptide encoded by SEQ ID NO: 1039
23
aa


SEQ ID NO: 1041
trp2 segment 1
90
nts


SEQ ID NO: 1042
Polypeptide encoded by SEQ ID NO: 1041
30
aa


SEQ ID NO: 1043
trp2 segment 2
90
nts


SEQ ID NO: 1044
Polypeptide encoded by SEQ ID NO: 1043
30
aa


SEQ ID NO: 1045
trp2 segment 3
90
nts


SEQ ID NO: 1046
Polypeptide encoded by SEQ ID NO: 1045
30
aa


SEQ ID NO: 1047
trp2 segment 4
90
nts


SEQ ID NO: 1048
Polypeptide encoded by SEQ ID NO: 1047
30
aa


SEQ ID NO: 1049
trp2 segment 5
90
nts


SEQ ID NO: 1050
Polypeptide encoded by SEQ ID NO: 1049
30
aa


SEQ ID NO: 1051
trp2 segment 6
90
nts


SEQ ID NO: 1052
Polypeptide encoded by SEQ ID NO: 1051
30
aa


SEQ ID NO: 1053
trp2 segment 7
90
nts


SEQ ID NO: 1054
Polypeptide encoded by SEQ ID NO: 1053
30
aa


SEQ ID NO: 1055
trp2 segment 8
90
nts


SEQ ID NO: 1056
Polypeptide encoded by SEQ ID NO: 1055
30
aa


SEQ ID NO: 1057
trp2 segment 9
90
nts


SEQ ID NO: 1058
Polypeptide encoded by SEQ ID NO: 1057
30
aa


SEQ ID NO: 1059
trp2 segment 10
90
nts


SEQ ID NO: 1060
Polypeptide encoded by SEQ ID NO: 1059
30
aa


SEQ ID NO: 1061
trp2 segment 11
90
nts


SEQ ID NO: 1062
Polypeptide encoded by SEQ ID NO: 1061
30
aa


SEQ ID NO: 1063
trp2 segment 12
90
nts


SEQ ID NO: 1064
Polypeptide encoded by SEQ ID NO: 1063
30
aa


SEQ ID NO: 1065
trp2 segment 13
90
nts


SEQ ID NO: 1066
Polypeptide encoded by SEQ ID NO: 1065
30
aa


SEQ ID NO: 1067
trp2 segment 14
90
nts


SEQ ID NO: 1068
Polypeptide encoded by SEQ ID NO: 1067
30
aa


SEQ ID NO: 1069
trp2 segment 15
90
nts


SEQ ID NO: 1070
Polypeptide encoded by SEQ ID NO: 1069
30
aa


SEQ ID NO: 1071
trp2 segment 16
90
nts


SEQ ID NO: 1072
Polypeptide encoded by SEQ ID NO: 1071
30
aa


SEQ ID NO: 1073
trp2 segment 17
90
nts


SEQ ID NO: 1074
Polypeptide encoded by SEQ ID NO: 1073
30
aa


SEQ ID NO: 1075
trp2 segment 18
90
nts


SEQ ID NO: 1076
Polypeptide encoded by SEQ ID NO: 1075
30
aa


SEQ ID NO: 1077
trp2 segment 19
90
nts


SEQ ID NO: 1078
Polypeptide encoded by SEQ ID NO: 1077
30
aa


SEQ ID NO: 1079
trp2 segment 20
90
nts


SEQ ID NO: 1080
Polypeptide encoded by SEQ ID NO: 1079
30
aa


SEQ ID NO: 1081
trp2 segment 21
90
nts


SEQ ID NO: 1082
Polypeptide encoded by SEQ ID NO: 1081
30
aa


SEQ ID NO: 1083
trp2 segment 22
90
nts


SEQ ID NO: 1084
Polypeptide encoded by SEQ ID NO: 1083
30
aa


SEQ ID NO: 1085
trp2 segment 23
90
nts


SEQ ID NO: 1086
Polypeptide encoded by SEQ ID NO: 1085
30
aa


SEQ ID NO: 1087
trp2 segment 24
90
nts


SEQ ID NO: 1088
Polypeptide encoded by SEQ ID NO: 1087
30
aa


SEQ ID NO: 1089
trp2 segment 25
90
nts


SEQ ID NO: 1090
Polypeptide encoded by SEQ ID NO: 1089
30
aa


SEQ ID NO: 1091
trp2 segment 26
90
nts


SEQ ID NO: 1092
Polypeptide encoded by SEQ ID NO: 1091
30
aa


SEQ ID NO: 1093
trp2 segment 27
90
nts


SEQ ID NO: 1094
Polypeptide encoded by SEQ ID NO: 1093
30
aa


SEQ ID NO: 1095
trp2 segment 28
90
nts


SEQ ID NO: 1096
Polypeptide encoded by SEQ ID NO: 1095
30
aa


SEQ ID NO: 1097
trp2 segment 29
90
nts


SEQ ID NO: 1098
Polypeptide encoded by SEQ ID NO: 1097
30
aa


SEQ ID NO: 1099
trp2 segment 30
90
nts


SEQ ID NO: 1100
Polypeptide encoded by SEQ ID NO: 1099
30
aa


SEQ ID NO: 1101
trp2 segment 31
90
nts


SEQ ID NO: 1102
Polypeptide encoded by SEQ ID NO: 1101
30
aa


SEQ ID NO: 1103
trp2 segment 32
90
nts


SEQ ID NO: 1104
Polypeptide encoded by SEQ ID NO: 1103
30
aa


SEQ ID NO: 1105
trp2 segment 33
90
nts


SEQ ID NO: 1106
Polypeptide encoded by SEQ ID NO: 1105
30
aa


SEQ ID NO: 1107
trp2 segment 34
84
nts


SEQ ID NO: 1108
Polypeptide encoded by SEQ ID NO: 1107
28
aa


SEQ ID NO: 1109
MC1R segment 1
90
nts


SEQ ID NO: 1110
Polypeptide encoded by SEQ ID NO: 1109
30
aa


SEQ ID NO: 1111
MC1R segment 2
90
nts


SEQ ID NO: 1112
Polypeptide encoded by SEQ ID NO: 1111
30
aa


SEQ ID NO: 1113
MC1R segment 3
90
nts


SEQ ID NO: 1114
Polypeptide encoded by SEQ ID NO: 1113
30
aa


SEQ ID NO: 1115
MC1R segment 4
90
nts


SEQ ID NO: 1116
Polypeptide encoded by SEQ ID NO: 1115
30
aa


SEQ ID NO: 1117
MC1R segment 5
90
nts


SEQ ID NO: 1118
Polypeptide encoded by SEQ ID NO: 1117
30
aa


SEQ ID NO: 1119
MC1R segment 6
90
nts


SEQ ID NO: 1120
Polypeptide encoded by SEQ ID NO: 1119
30
aa


SEQ ID NO: 1121
MC1R segment 7
90
nts


SEQ ID NO: 1122
Polypeptide encoded by SEQ ID NO: 1121
30
aa


SEQ ID NO: 1123
MC1R segment 8
90
nts


SEQ ID NO: 1124
Polypeptide encoded by SEQ ID NO: 1123
30
aa


SEQ ID NO: 1125
MC1R segment 9
90
nts


SEQ ID NO: 1126
Polypeptide encoded by SEQ ID NO: 1125
30
aa


SEQ ID NO: 1127
MC1R segment 10
90
nts


SEQ ID NO: 1128
Polypeptide encoded by SEQ ID NO: 1127
30
aa


SEQ ID NO: 1129
MC1R segment 11
90
nts


SEQ ID NO: 1130
Polypeptide encoded by SEQ ID NO: 1129
30
aa


SEQ ID NO: 1131
MC1R segment 12
90
nts


SEQ ID NO: 1132
Polypeptide encoded by SEQ ID NO: 1131
30
aa


SEQ ID NO: 1133
MC1R segment 13
90
nts


SEQ ID NO: 1134
Polypeptide encoded by SEQ ID NO: 1133
30
aa


SEQ ID NO: 1135
MC1R segment 14
90
nts


SEQ ID NO: 1136
Polypeptide encoded by SEQ ID NO: 1135
30
aa


SEQ ID NO: 1137
MC1R segment 15
90
nts


SEQ ID NO: 1138
Polypeptide encoded by SEQ ID NO: 1137
30
aa


SEQ ID NO: 1139
MC1R segment 16
90
nts


SEQ ID NO: 1140
Polypeptide encoded by SEQ ID NO: 1139
30
aa


SEQ ID NO: 1141
MC1R segment 17
90
nts


SEQ ID NO: 1142
Polypeptide encoded by SEQ ID NO: 1141
30
aa


SEQ ID NO: 1143
MC1R segment 18
90
nts


SEQ ID NO: 1144
Polypeptide encoded by SEQ ID NO: 1143
30
aa


SEQ ID NO: 1145
MC1R segment 19
90
nts


SEQ ID NO: 1146
Polypeptide encoded by SEQ ID NO: 1145
30
aa


SEQ ID NO: 1147
MC1R segment 20
90
nts


SEQ ID NO: 1148
Polypeptide encoded by SEQ ID NO: 1147
30
aa


SEQ ID NO: 1149
MC1R segment 21
63
nts


SEQ ID NO: 1150
Polypeptide encoded by SEQ ID NO: 1149
21
aa


SEQ ID NO: 1151
MUC1F segment 1
90
nts


SEQ ID NO: 1152
Polypeptide encoded by SEQ ID NO: 1151
30
aa


SEQ ID NO: 1153
MUC1F segment 2
90
nts


SEQ ID NO: 1154
Polypeptide encoded by SEQ ID NO: 1153
30
aa


SEQ ID NO: 1155
MUC1F segment 3
90
nts


SEQ ID NO: 1156
Polypeptide encoded by SEQ ID NO: 1155
30
aa


SEQ ID NO: 1157
MUC1F segment 4
90
nts


SEQ ID NO: 1158
Polypeptide encoded by SEQ ID NO: 1157
30
aa


SEQ ID NO: 1159
MUC1F segment 5
90
nts


SEQ ID NO: 1160
Polypeptide encoded by SEQ ID NO: 1159
30
aa


SEQ ID NO: 1161
MUC1F segment 6
90
nts


SEQ ID NO: 1162
Polypeptide encoded by SEQ ID NO: 1161
30
aa


SEQ ID NO: 1163
MUC1F segment 7
90
nts


SEQ ID NO: 1164
Polypeptide encoded by SEQ ID NO: 1163
30
aa


SEQ ID NO: 1165
MUC1F segment 8
72
nts


SEQ ID NO: 1166
Polypeptide encoded by SEQ ID NO: 1165
24
aa


SEQ ID NO: 1167
MUC1R segment 1
90
nts


SEQ ID NO: 1168
Polypeptide encoded by SEQ ID NO: 1167
30
aa


SEQ ID NO: 1169
MUC1R segment 2
90
nts


SEQ ID NO: 1170
Polypeptide encoded by SEQ ID NO: 1169
30
aa


SEQ ID NO: 1171
MUC1R segment 3
90
nts


SEQ ID NO: 1172
Polypeptide encoded by SEQ ID NO: 1171
30
aa


SEQ ID NO: 1173
MUC1R segment 4
90
nts


SEQ ID NO: 1174
Polypeptide encoded by SEQ ID NO: 1173
30
aa


SEQ ID NO: 1175
MUC1R segment 5
90
nts


SEQ ID NO: 1176
Polypeptide encoded by SEQ ID NO: 1175
30
aa


SEQ ID NO: 1177
MUC1R segment 6
90
nts


SEQ ID NO: 1178
Polypeptide encoded by SEQ ID NO: 1177
30
aa


SEQ ID NO: 1179
MUC1R segment 7
90
nts


SEQ ID NO: 1180
Polypeptide encoded by SEQ ID NO: 1179
30
aa


SEQ ID NO: 1181
MUC1R segment 8
90
nts


SEQ ID NO: 1182
Polypeptide encoded by SEQ ID NO: 1181
30
aa


SEQ ID NO: 1183
MUC1R segment 9
90
nts


SEQ ID NO: 1184
Polypeptide encoded by SEQ ID NO: 1183
30
aa


SEQ ID NO: 1185
MUC1R segment 10
90
nts


SEQ ID NO: 1186
Polypeptide encoded by SEQ ID NO: 1185
30
aa


SEQ ID NO: 1187
MUC1R segment 11
90
nts


SEQ ID NO: 1188
Polypeptide encoded by SEQ ID NO: 1187
30
aa


SEQ ID NO: 1189
MUC1R segment 12
90
nts


SEQ ID NO: 1190
Polypeptide encoded by SEQ ID NO: 1189
30
aa


SEQ ID NO: 1191
MUC1R segment 13
90
nts


SEQ ID NO: 1192
Polypeptide encoded by SEQ ID NO: 1191
30
aa


SEQ ID NO: 1193
MUC1R segment 14
90
nts


SEQ ID NO: 1194
Polypeptide encoded by SEQ ID NO: 1193
30
aa


SEQ ID NO: 1195
MUC1R segment 15
90
nts


SEQ ID NO: 1196
Polypeptide encoded by SEQ ID NO: 1195
30
aa


SEQ ID NO: 1197
MUC1R segment 16
90
nts


SEQ ID NO: 1198
Polypeptide encoded by SEQ ID NO: 1197
30
aa


SEQ ID NO: 1199
MUC1R segment 17
90
nts


SEQ ID NO: 1200
Polypeptide encoded by SEQ ID NO: 1199
30
aa


SEQ ID NO: 1201
MUC1R segment 18
90
nts


SEQ ID NO: 1202
Polypeptide encoded by SEQ ID NO: 1201
30
aa


SEQ ID NO: 1203
MUC1R segment 19
90
nts


SEQ ID NO: 1204
Polypeptide encoded by SEQ ID NO: 1203
30
aa


SEQ ID NO: 1205
MUC1R segment 20
90
nts


SEQ ID NO: 1206
Polypeptide encoded by SEQ ID NO: 1205
30
aa


SEQ ID NO: 1207
MUC1R segment 21
48
nts


SEQ ID NO: 1208
Polypeptide encoded by SEQ ID NO: 1207
16
aa


SEQ ID NO: 1209
Differentiation Savine
16638
nts


SEQ ID NO: 1210
Polypeptide encoded by SEQ ID NO: 1209
5546
aa


SEQ ID NO: 1211
BAGE segment 1
90
nts


SEQ ID NO: 1212
Polypeptide encoded by SEQ ID NO: 1211
30
aa


SEQ ID NO: 1213
BAGE segment 2
90
nts


SEQ ID NO: 1214
Polypeptide encoded by SEQ ID NO: 1213
30
aa


SEQ ID NO: 1215
BAGE segment 3
51
nts


SEQ ID NO: 1216
Polypeptide encoded by SEQ ID NO: 1215
17
aa


SEQ ID NO: 1217
GAGE-1 segment 1
90
nts


SEQ ID NO: 1218
Polypeptide encoded by SEQ ID NO: 1217
30
aa


SEQ ID NO: 1219
GAGE-1 segment 2
90
nts


SEQ ID NO: 1220
Polypeptide encoded by SEQ ID NO: 1219
30
aa


SEQ ID NO: 1221
GAGE-1 segment 3
90
nts


SEQ ID NO: 1222
Polypeptide encoded by SEQ ID NO: 1221
30
aa


SEQ ID NO: 1223
GAGE-1 segment 4
90
nts


SEQ ID NO: 1224
Polypeptide encoded by SEQ ID NO: 1223
30
aa


SEQ ID NO: 1225
GAGE-1 segment 5
90
nts


SEQ ID NO: 1226
Polypeptide encoded by SEQ ID NO: 1225
30
aa


SEQ ID NO: 1227
GAGE-1 segment 6
90
nts


SEQ ID NO: 1228
Polypeptide encoded by SEQ ID NO: 1227
30
aa


SEQ ID NO: 1229
GAGE-1 segment 7
90
nts


SEQ ID NO: 1230
Polypeptide encoded by SEQ ID NO: 1229
30
aa


SEQ ID NO: 1231
GAGE-1 segment 8
90
nts


SEQ ID NO: 1232
Polypeptide encoded by SEQ ID NO: 1231
30
aa


SEQ ID NO: 1233
GAGE-1 segment 9
66
nts


SEQ ID NO: 1234
Polypeptide encoded by SEQ ID NO: 1233
22
aa


SEQ ID NO: 1235
gp1001n4 segment 1
90
nts


SEQ ID NO: 1236
Polypeptide encoded by SEQ ID NO: 1235
30
aa


SEQ ID NO: 1237
gp1001n4 segment 2
90
nts


SEQ ID NO: 1238
Polypeptide encoded by SEQ ID NO: 1237
30
aa


SEQ ID NO: 1239
gp1001n4 segment 3
75
nts


SEQ ID NO: 1240
Polypeptide encoded by SEQ ID NO: 1239
25
aa


SEQ ID NO: 1241
MAGE-1 segment 1
90
nts


SEQ ID NO: 1242
Polypeptide encoded by SEQ ID NO: 1241
30
aa


SEQ ID NO: 1243
MAGE-1 segment 2
90
nts


SEQ ID NO: 1244
Polypeptide encoded by SEQ ID NO: 1243
30
aa


SEQ ID NO: 1245
MAGE-1 segment 3
90
nts


SEQ ID NO: 1246
Polypeptide encoded by SEQ ID NO: 1245
30
aa


SEQ ID NO: 1247
MAGE-1 segment 4
90
nts


SEQ ID NO: 1248
Polypeptide encoded by SEQ ID NO: 1247
30
aa


SEQ ID NO: 1249
MAGE-1 segment 5
90
nts


SEQ ID NO: 1250
Polypeptide encoded by SEQ ID NO: 1249
30
aa


SEQ ID NO: 1251
MAGE-1 segment 6
90
nts


SEQ ID NO: 1252
Polypeptide encoded by SEQ ID NO: 1251
30
aa


SEQ ID NO: 1253
MAGE-1 segment 7
90
nts


SEQ ID NO: 1254
Polypeptide encoded by SEQ ID NO: 1253
30
aa


SEQ ID NO: 1255
MAGE-1 segment 8
90
nts


SEQ ID NO: 1256
Polypeptide encoded by SEQ ID NO: 1255
30
aa


SEQ ID NO: 1257
MAGE-1 segment 9
90
nts


SEQ ID NO: 1258
Polypeptide encoded by SEQ ID NO: 1257
30
aa


SEQ ID NO: 1259
MAGE-1 segment 10
90
nts


SEQ ID NO: 1260
Polypeptide encoded by SEQ ID NO: 1259
30
aa


SEQ ID NO: 1261
MAGE-1 segment 11
90
nts


SEQ ID NO: 1262
Polypeptide encoded by SEQ ID NO: 1261
30
aa


SEQ ID NO: 1263
MAGE-1 segment 12
90
nts


SEQ ID NO: 1264
Polypeptide encoded by SEQ ID NO: 1263
30
aa


SEQ ID NO: 1265
MAGE-1 segment 13
90
nts


SEQ ID NO: 1266
Polypeptide encoded by SEQ ID NO: 1265
30
aa


SEQ ID NO: 1267
MAGE-1 segment 14
90
nts


SEQ ID NO: 1268
Polypeptide encoded by SEQ ID NO: 1267
30
aa


SEQ ID NO: 1269
MAGE-1 segment 15
90
nts


SEQ ID NO: 1270
Polypeptide encoded by SEQ ID NO: 1269
30
aa


SEQ ID NO: 1271
MAGE-1 segment 16
90
nts


SEQ ID NO: 1272
Polypeptide encoded by SEQ ID NO: 1271
30
aa


SEQ ID NO: 1273
MAGE-1 segment 17
90
nts


SEQ ID NO: 1274
Polypeptide encoded by SEQ ID NO: 1273
30
aa


SEQ ID NO: 1275
MAGE-1 segment 18
90
nts


SEQ ID NO: 1276
Polypeptide encoded by SEQ ID NO: 1275
30
aa


SEQ ID NO: 1277
MAGE-1 segment 19
90
nts


SEQ ID NO: 1278
Polypeptide encoded by SEQ ID NO: 1277
30
aa


SEQ ID NO: 1279
MAGE-1 segment 20
84
nts


SEQ ID NO: 1280
Polypeptide encoded by SEQ ID NO: 1279
28
aa


SEQ ID NO: 1281
MAGE-3 segment 1
90
nts


SEQ ID NO: 1282
Polypeptide encoded by SEQ ID NO: 1281
30
aa


SEQ ID NO: 1283
MAGE-3 segment 2
90
nts


SEQ ID NO: 1284
Polypeptide encoded by SEQ ID NO: 1283
30
aa


SEQ ID NO: 1285
MAGE-3 segment 3
90
nts


SEQ ID NO: 1286
Polypeptide encoded by SEQ ID NO: 1285
30
aa


SEQ ID NO: 1287
MAGE-3 segment 4
90
nts


SEQ ID NO: 1288
Polypeptide encoded by SEQ ID NO: 1287
30
aa


SEQ ID NO: 1289
MAGE-3 segment 5
90
nts


SEQ ID NO: 1290
Polypeptide encoded by SEQ ID NO: 1289
30
aa


SEQ ID NO: 1291
MAGE-3 segment 6
90
nts


SEQ ID NO: 1292
Polypeptide encoded by SEQ ID NO: 1291
30
aa


SEQ ID NO: 1293
MAGE-3 segment 7
90
nts


SEQ ID NO: 1294
Polypeptide encoded by SEQ ID NO: 1293
30
aa


SEQ ID NO: 1295
MAGE-3 segment 8
90
nts


SEQ ID NO: 1296
Polypeptide encoded by SEQ ID NO: 1295
30
aa


SEQ ID NO: 1297
MAGE-3 segment 9
90
nts


SEQ ID NO: 1298
Polypeptide encoded by SEQ ID NO: 1297
30
aa


SEQ ID NO: 1299
MAGE-3 segment 10
90
nts


SEQ ID NO: 1300
Polypeptide encoded by SEQ ID NO: 1299
30
aa


SEQ ID NO: 1301
MAGE-3 segment 11
90
nts


SEQ ID NO: 1302
Polypeptide encoded by SEQ ID NO: 1301
30
aa


SEQ ID NO: 1303
MAGE-3 segment 12
90
nts


SEQ ID NO: 1304
Polypeptide encoded by SEQ ID NO: 1303
30
aa


SEQ ID NO: 1305
MAGE-3 segment 13
90
nts


SEQ ID NO: 1306
Polypeptide encoded by SEQ ID NO: 1305
30
aa


SEQ ID NO: 1307
MAGE-3 segment 14
90
nts


SEQ ID NO: 1308
Polypeptide encoded by SEQ ID NO: 1307
30
aa


SEQ ID NO: 1309
MAGE-3 segment 15
90
nts


SEQ ID NO: 1310
Polypeptide encoded by SEQ ID NO: 1309
30
aa


SEQ ID NO: 1311
MAGE-3 segment 16
90
nts


SEQ ID NO: 1312
Polypeptide encoded by SEQ ID NO: 1311
30
aa


SEQ ID NO: 1313
MAGE-3 segment 17
90
nts


SEQ ID NO: 1314
Polypeptide encoded by SEQ ID NO: 1313
30
aa


SEQ ID NO: 1315
MAGE-3 segment 18
90
nts


SEQ ID NO: 1316
Polypeptide encoded by SEQ ID NO: 1315
30
aa


SEQ ID NO: 1317
MAGE-3 segment 19
90
nts


SEQ ID NO: 1318
Polypeptide encoded by SEQ ID NO: 1317
30
aa


SEQ ID NO: 1319
MAGE-3 segment 20
90
nts


SEQ ID NO: 1320
Polypeptide encoded by SEQ ID NO: 1319
30
aa


SEQ ID NO: 1321
MAGE-3 segment 21
54
nts


SEQ ID NO: 1322
Polypeptide encoded by SEQ ID NO: 1321
18
aa


SEQ ID NO: 1323
PRAME segment 1
90
nts


SEQ ID NO: 1324
Polypeptide encoded by SEQ ID NO: 1323
30
aa


SEQ ID NO: 1325
PRAME segment 2
90
nts


SEQ ID NO: 1326
Polypeptide encoded by SEQ ID NO: 1325
30
aa


SEQ ID NO: 1327
PRAME segment 3
90
nts


SEQ ID NO: 1328
Polypeptide encoded by SEQ ID NO: 1327
30
aa


SEQ ID NO: 1329
PRAME segment 4
90
nts


SEQ ID NO: 1330
Polypeptide encoded by SEQ ID NO: 1329
30
aa


SEQ ID NO: 1331
PRAME segment 5
90
nts


SEQ ID NO: 1332
Polypeptide encoded by SEQ ID NO: 1331
30
aa


SEQ ID NO: 1333
PRAME segment 6
90
nts


SEQ ID NO: 1334
Polypeptide encoded by SEQ ID NO: 1333
30
aa


SEQ ID NO: 1335
PRAME segment 7
90
nts


SEQ ID NO: 1336
Polypeptide encoded by SEQ ID NO: 1335
30
aa


SEQ ID NO: 1337
PRAME segment 8
90
nts


SEQ ID NO: 1338
Polypeptide encoded by SEQ ID NO: 1337
30
aa


SEQ ID NO: 1339
PRAME segment 9
90
nts


SEQ ID NO: 1340
Polypeptide encoded by SEQ ID NO: 1339
30
aa


SEQ ID NO: 1341
PRAME segment 10
90
nts


SEQ ID NO: 1342
Polypeptide encoded by SEQ ID NO: 1341
30
aa


SEQ ID NO: 1343
PRAME segment 11
90
nts


SEQ ID NO: 1344
Polypeptide encoded by SEQ ID NO: 1343
30
aa


SEQ ID NO: 1345
PRAME segment 12
90
nts


SEQ ID NO: 1346
Polypeptide encoded by SEQ ID NO: 1345
30
aa


SEQ ID NO: 1347
PRAME segment 13
90
nts


SEQ ID NO: 1348
Polypeptide encoded by SEQ ID NO: 1347
30
aa


SEQ ID NO: 1349
PRAME segment 14
90
nts


SEQ ID NO: 1350
Polypeptide encoded by SEQ ID NO: 1349
30
aa


SEQ ID NO: 1351
PRAME segment 15
90
nts


SEQ ID NO: 1352
Polypeptide encoded by SEQ ID NO: 1351
30
aa


SEQ ID NO: 1353
PRAME segment 16
90
nts


SEQ ID NO: 1354
Polypeptide encoded by SEQ ID NO: 1353
30
aa


SEQ ID NO: 1355
PRAME segment 17
90
nts


SEQ ID NO: 1356
Polypeptide encoded by SEQ ID NO: 1355
30
aa


SEQ ID NO: 1357
PRAME segment 18
90
nts


SEQ ID NO: 1358
Polypeptide encoded by SEQ ID NO: 1357
30
aa


SEQ ID NO: 1359
PRAME segment 19
90
nts


SEQ ID NO: 1360
Polypeptide encoded by SEQ ID NO: 1359
30
aa


SEQ ID NO: 1361
PRAME segment 20
90
nts


SEQ ID NO: 1362
Polypeptide encoded by SEQ ID NO: 1361
30
aa


SEQ ID NO: 1363
PRAME segment 21
90
nts


SEQ ID NO: 1364
Polypeptide encoded by SEQ ID NO: 1363
30
aa


SEQ ID NO: 1365
PRAME segment 22
90
nts


SEQ ID NO: 1366
Polypeptide encoded by SEQ ID NO: 1365
30
aa


SEQ ID NO: 1367
PRAME segment 23
90
nts


SEQ ID NO: 1368
Polypeptide encoded by SEQ ID NO: 1367
30
aa


SEQ ID NO: 1369
PRAME segment 24
90
nts


SEQ ID NO: 1370
Polypeptide encoded by SEQ ID NO: 1369
30
aa


SEQ ID NO: 1371
PRAME segment 25
90
nts


SEQ ID NO: 1372
Polypeptide encoded by SEQ ID NO: 1371
30
aa


SEQ ID NO: 1373
PRAME segment 26
90
nts


SEQ ID NO: 1374
Polypeptide encoded by SEQ ID NO: 1373
30
aa


SEQ ID NO: 1375
PRAME segment 27
90
nts


SEQ ID NO: 1376
Polypeptide encoded by SEQ ID NO: 1375
30
aa


SEQ ID NO: 1377
PRAME segment 28
90
nts


SEQ ID NO: 1378
Polypeptide encoded by SEQ ID NO: 1377
30
aa


SEQ ID NO: 1379
PRAME segment 29
90
nts


SEQ ID NO: 1380
Polypeptide encoded by SEQ ID NO: 1379
30
aa


SEQ ID NO: 1381
PRAME segment 30
90
nts


SEQ ID NO: 1382
Polypeptide encoded by SEQ ID NO: 1381
30
aa


SEQ ID NO: 1383
PRAME segment 31
90
nts


SEQ ID NO: 1384
Polypeptide encoded by SEQ ID NO: 1383
30
aa


SEQ ID NO: 1385
PRAME segment 32
90
nts


SEQ ID NO: 1386
Polypeptide encoded by SEQ ID NO: 1385
30
aa


SEQ ID NO: 1387
PRAME segment 33
90
nts


SEQ ID NO: 1388
Polypeptide encoded by SEQ ID NO: 1387
30
aa


SEQ ID NO: 1389
PRAME segment 34
54
nts


SEQ ID NO: 1390
Polypeptide encoded by SEQ ID NO: 1389
18
aa


SEQ ID NO: 1391
TRP21N2 segment 1
90
nts


SEQ ID NO: 1392
Polypeptide encoded by SEQ ID NO: 1391
30
aa


SEQ ID NO: 1393
TRP21N2 segment 2
90
nts


SEQ ID NO: 1394
Polypeptide encoded by SEQ ID NO: 1393
30
aa


SEQ ID NO: 1395
TRP21N2 segment 3
84
nts


SEQ ID NO: 1396
Polypeptide encoded by SEQ ID NO: 1395
28
aa


SEQ ID NO: 1397
NYNSO1a segment 1
90
nts


SEQ ID NO: 1398
Polypeptide encoded by SEQ ID NO: 1397
30
aa


SEQ ID NO: 1399
NYNSO1a segment 2
90
nts


SEQ ID NO: 1400
Polypeptide encoded by SEQ ID NO: 1399
30
aa


SEQ ID NO: 1401
NYNSO1a segment 3
90
nts


SEQ ID NO: 1402
Polypeptide encoded by SEQ ID NO: 1401
30
aa


SEQ ID NO: 1403
NYNSO1a segment 4
90
nts


SEQ ID NO: 1404
Polypeptide encoded by SEQ ID NO: 1403
30
aa


SEQ ID NO: 1405
NYNSO1a segment 5
90
nts


SEQ ID NO: 1406
Polypeptide encoded by SEQ ID NO: 1405
30
aa


SEQ ID NO: 1407
NYNSO1a segment 6
90
nts


SEQ ID NO: 1408
Polypeptide encoded by SEQ ID NO: 1407
30
aa


SEQ ID NO: 1409
NYNSO1a segment 7
90
nts


SEQ ID NO: 1410
Polypeptide encoded by SEQ ID NO: 1409
30
aa


SEQ ID NO: 1411
NYNSO1a segment 8
90
nts


SEQ ID NO: 1412
Polypeptide encoded by SEQ ID NO: 1411
30
aa


SEQ ID NO: 1413
NYNSO1a segment 9
90
nts


SEQ ID NO: 1414
Polypeptide encoded by SEQ ID NO: 1413
30
aa


SEQ ID NO: 1415
NYNSO1a segment 10
90
nts


SEQ ID NO: 1416
Polypeptide encoded by SEQ ID NO: 1415
30
aa


SEQ ID NO: 1417
NYNSO1a segment 11
90
nts


SEQ ID NO: 1418
Polypeptide encoded by SEQ ID NO: 1417
30
aa


SEQ ID NO: 1419
NYNSO1a segment 12
57
nts


SEQ ID NO: 1420
Polypeptide encoded by SEQ ID NO: 1419
19
aa


SEQ ID NO: 1421
NYNSO1b segment 1
90
nts


SEQ ID NO: 1422
Polypeptide encoded by SEQ ID NO: 1421
30
aa


SEQ ID NO: 1423
NYNSO1b segment 2
90
nts


SEQ ID NO: 1424
Polypeptide encoded by SEQ ID NO: 1423
30
aa


SEQ ID NO: 1425
NYNSO1b segment 3
90
nts


SEQ ID NO: 1426
Polypeptide encoded by SEQ ID NO: 1425
30
aa


SEQ ID NO: 1427
NYNSO1b segment 4
51
nts


SEQ ID NO: 1428
Polypeptide encoded by SEQ ID NO: 1427


SEQ ID NO: 1429
LAGE1 segment 1
90
nts


SEQ ID NO: 1430
Polypeptide encoded by SEQ ID NO: 1429
30
aa


SEQ ID NO: 1431
LAGE1 segment 2
90
nts


SEQ ID NO: 1432
Polypeptide encoded by SEQ ID NO: 1431
30
aa


SEQ ID NO: 1433
LAGE1 segment 3
90
nts


SEQ ID NO: 1434
Polypeptide encoded by SEQ ID NO: 1433
30
aa


SEQ ID NO: 1435
LAGE1 segment 4
90
nts


SEQ ID NO: 1436
Polypeptide encoded by SEQ ID NO: 1435
30
aa


SEQ ID NO: 1437
LAGE1 segment 5
90
nts


SEQ ID NO: 1438
Polypeptide encoded by SEQ ID NO: 1437
30
aa


SEQ ID NO: 1439
LAGE1 segment 6
90
nts


SEQ ID NO: 1440
Polypeptide encoded by SEQ ID NO: 1439
30
aa


SEQ ID NO: 1441
LAGE1 segment 7
90
nts


SEQ ID NO: 1442
Polypeptide encoded by SEQ ID NO: 1441
30
aa


SEQ ID NO: 1443
LAGE1 segment 8
90
nts


SEQ ID NO: 1444
Polypeptide encoded by SEQ ID NO: 1443
30
aa


SEQ ID NO: 1445
LAGE1 segment 9
90
nts


SEQ ID NO: 1446
Polypeptide encoded by SEQ ID NO: 1445
30
aa


SEQ ID NO: 1447
LAGE1 segment 10
90
nts


SEQ ID NO: 1448
Polypeptide encoded by SEQ ID NO: 1447
30
aa


SEQ ID NO: 1449
LAGE1 segment 11
90
nts


SEQ ID NO: 1450
Polypeptide encoded by SEQ ID NO: 1449
30
aa


SEQ ID NO: 1451
LAGE1 segment 12
57
nts


SEQ ID NO: 1452
Polypeptide encoded by SEQ ID NO: 1451
19
aa


SEQ ID NO: 1453
Melanoma cancer specific Savine
10623
nts


SEQ ID NO: 1454
Polypeptide encoded by SEQ ID NO: 1453
3541
aa


SEQ ID NO: 1455
FIG. 16 A1S1 99mer
99
nts


SEQ ID NO: 1456
FIG. 16 A1S2 100mer
100
nts


SEQ ID NO: 1457
FIG. 16 A1S3 100mer
100
nts


SEQ ID NO: 1458
FIG. 16 A1S4 100mer
100
nts


SEQ ID NO: 1459
FIG. 16 A1S5 100mer
100
nts


SEQ ID NO: 1460
FIG. 16 A1S6 99mer
99
nts


SEQ ID NO: 1461
FIG. 16 A1S7 97mer
99
nts


SEQ ID NO: 1462
FIG. 16 A1S8 100mer
100
nts


SEQ ID NO: 1463
FIG. 16 A1S9 100mer
100
nts


SEQ ID NO: 1464
FIG. 16 A1S10 75mer
76
nts


SEQ ID NO: 1465
FIG. 16 A1F 20mer
20
nts


SEQ ID NO: 1466
FIG. 16 A1R 20mer
20
nts


SEQ ID NO: 1467
Amino acid sequence of immunostimulatory domain of an
16
aa



invasin protein from Yersinia spp.









DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein, the term “about” refers to a quantity, level, value, dimension, size, or amount that varies by as much as 30%, preferably by as much as 20%, and more preferably by as much as 10% to a reference quantity, level, value, dimension, size, or amount.
By “antigen-binding molecule” is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
The term “clade” as used herein refers to a hypothetical species of an organism and its descendants or a monophyletic group of organisms. Clades carry a definition, based on ancestry, and a diagnosis, based on synapomorphies. It should be noted that diagnoses of clades could change while definitions do not.
Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
By “expression vector” is meant any autonomous genetic element capable of directing the synthesis of a protein encoded by the vector. Such expression vectors are known by practitioners in the art.
As used herein, the term “function” refers to a biological, enzymatic, or therapeutic function.
“Homology” refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table B infra. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
To enhance an immune response (“immunoenhancement”), as is well-known in the art, means to increase an animal's capacity to respond to foreign or disease-specific antigens (e.g., cancer antigens) i.e., those cells primed to attack such antigens are increased in number, activity, and ability to detect and destroy the those antigens. Strength of immune response is measured by standard tests including: direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (see, e.g., Provinciali M. et al (1992, J. Immunol. Meth. 155: 19-24), cell proliferation assays (see, e.g., Vollenweider, I. and Groseurth, P. J. (1992, J. Immunol. Meth. 149: 133-135), immunoassays of immune cells and subsets (see, e.g. Loeffler, D. A., et al. (1992, Cytom. 13: 169-174); Rivoltini, L.; et al. (1992. Can. Immunol. Immunother. 34: 241-251); or skin tests for cell-mediated immunity (see, e.g., Chang, A. E. et al (1993, Cancer Res. 53: 1043-1050). Any statistically significant increase in strength of immune response as measured by the foregoing tests is considered “enhanced immune response” “immunoenhancement” or “immunopotentiation” as used herein. Enhanced immune response is also indicated by physical manifestations such as fever and inflammation, as well as healing of systemic and local infections, and reduction of symptoms in disease, i.e., decrease in tumour size, alleviation of symptoms of a disease or condition including, but not restricted to, leprosy, tuberculosis, malaria, naphthous ulcers, herpetic and papillomatous warts, gingivitis, artherosclerosis, the concomitants of AIDS such as Kaposi's sarcoma, bronchial infections, and the like. Such physical manifestations also define “enhanced immune response” “immunoenhancement” or “immunopotentiation” as used herein.
Reference herein to “immuno-interactive” includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state.
By “modulating” is meant increasing or decreasing, either directly or indirectly, an immune response against a target antigen of a member selected from the group consisting of a cancer and an organism, preferably a pathogenic organism.
By “natural gene” is meant a gene that naturally encodes a protein.
The term “natural polypeptide” as used herein refers to a polypeptide that exists in nature.
By “obtained from” is meant that a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source of the host. For example, the extract can be obtained from a tissue or a biological fluid isolated directly from the host.
The term “oligonucleotide” as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term “oligonucleotide” typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term “polynucleotide” or “nucleic acid” is typically used for large oligonucleotides.
By “operably linked” is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.
The term “parent polypeptide” as used herein typically refers to a polypeptide encoded by a natural gene. However, it is possible that the parent polypeptide corresponds to a protein that is not naturally-occurring but has been engineered using recombinant techniques. In this instance, a polynucleotide encoding the parent polypeptide may comprise different but synonymous codons relative to a natural gene encoding the same polypeptide. Alternatively, the parent polypeptide may not correspond to a natural polypeptide sequence. For example, the parent polypeptide may comprise one or more consensus sequences common to a plurality of polypeptides.
The term “patient” refers to patients of human or other mammal and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that “patient” does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes).
By “pharmaceutically-acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to a mammal.
“Polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
The term “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.
By “primer” is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerising agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerisation agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotide residues, although it can contain fewer nucleotide residues. Primers can be large polynucleotides, such as from about 35 nucleotides to several kilobases or more. Primers can be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridise and serve as a site for the initiation of synthesis. By “substantially complementary”, it is meant that the primer is sufficiently complementary to hybridise with a target polynucleotide. Preferably, the primer contains no mismatches with the template to which it is designed to hybridise but this is not essential. For example, non-complementary nucleotide residues can be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non-complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridise therewith and thereby form a template for synthesis of the extension product of the primer.
“Probe” refers to a molecule that binds to a specific sequence or sub-sequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the “target polynucleotide”, through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridisation conditions. Probes can be labelled directly or indirectly.
By “recombinant polypeptide” is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 50 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.
The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software.
The term “synthetic polynucleotide” as used herein refers to a polynucleotide formed in vitro by the manipulation of a polynucleotide into a form not normally found in nature. For example, the synthetic polynucleotide can be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory polynucleotide operably linked to the polynucleotide.
The term “synonymous codon” as used herein refers to a codon having a different nucleotide sequence than another codon but encoding the same amino acid as that other codon.
By “translational efficiency” is meant the efficiency of a cell's protein synthesis machinery to incorporate the amino acid encoded by a codon into a nascent polypeptide chain. This efficiency can be evidenced, for example, by the rate at which the cell is able to synthesise the polypeptide from an RNA template comprising the codon, or by the amount of the polypeptide synthesised from such a template.
By “vector” is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is preferably a viral or viral-derived vector, which is operably functional in animal and preferably mammalian cells. Such vector may be derived from a poxvirus, an adenovirus or yeast. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptII gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
2. Synthetic Polypeptides
The inventors have surprisingly discovered that the structure of a parent polypeptide can be disrupted sufficiently to impede, abrogate or otherwise alter at least one function of the parent polypeptide, while simultaneously minimising the destruction of potentially useful epitopes that are present in the parent polypeptide, by fusing, coupling or otherwise linking together different segments of the parent polypeptide in a different relationship relative to their linkage in the parent polypeptide. As a result of this change in relationship, the sequence of the linked segments in the resulting synthetic polypeptide is different to a sequence contained within the parent polypeptide. The synthetic polypeptides of the invention are useful as immunopotentiating agents, and are referred to elsewhere in the specification as scrambled antigen vaccines, super attenuated vaccines or “Savines”.
Thus, the invention broadly resides in a synthetic polypeptide comprising a plurality of different segments of at least one parent polypeptide, wherein said segments are linked together in a different relationship relative to their linkage in the at least one parent polypeptide.
It is preferable but not essential that the segments in said synthetic polypeptide are linked sequentially in a different order or arrangement relative to that of corresponding segments in said at least one parent polypeptide. For example, in the case of a parent polypeptide that comprises four contiguous or overlapping segments A-B-C-D, these segments may be linked in 23 other possible orders to form a synthetic polypeptide. These orders may be selected from the group consisting of: A-B-D-C, A-C-B-D, A-C-D-B, A-D-B-C, A-D-C-B, B-A-C-D, B-A-D-C, B-C-A-D, B-C-D-A, B-D-A-C, B-D-C-A, C-A-B-D, C-A-D-B, C-B-A-D, C-B-D-A, C-D-A-B, C-D-B-A, D-A-B-C, D-A-C-B, D-B-A-C, D-B-C-A, D-C-A-B, and D-C-B-A. Although the rearrangement of the segments is preferably random, it is especially preferable to exclude or otherwise minimize rearrangements that result in complete or partial reassembly of the parent sequence (e.g., ADBC, BACD, DABC). It will be appreciated, however, that the probability of such complete or partial reassembly diminishes as the number of segments for rearrangement increases.
The order of the segments is suitably shuffled, reordered or otherwise rearranged relative to the order in which they exist in the parent polypeptide so that the structure of the polypeptide is disrupted sufficiently to impede, abrogate or otherwise alter at least one function associated with the parent polypeptide. Preferably, the segments of the parent polypeptide are randomly rearranged in the synthetic polypeptide.
The parent polypeptide is suitably a polypeptide that is associated with a disease or condition. For example, the parent polypeptide may be a polypeptide expressed by a pathogenic organism or a cancer. Alternatively, the parent polypeptide can be a self peptide related to an autoimmune disease including, but are not limited to, diseases such as diabetes (e.g., juvenile diabetes), multiple sclerosis, rheumatoid arthritis, myasthenia gravis, atopic dermatitis, and psoriasis and ankylosing spondylitis. Accordingly, the synthetic molecules of the present invention may also have utility for the induction of tolerance in a subject afflicted with an autoimmune disease or condition or with an allergy or other condition to which tolerance is desired. For example tolerance may be induced by contacting an immature dendritic cell of the individual to be treated with a synthetic polypeptide of the invention or by expressing in an immature dendritic cell a synthetic polynucleotide of the invention. Tolerance may also be induced against antigens causing allergic responses (e.g., asthma, hay fever). In this case, the parent polypeptide is suitably an allergenic protein including, but not restricted to, house-dust-mite allergenic proteins as for example described by Thomas and Smith (1998, Allergy, 53(9): 821-832).
The pathogenic organism includes, but is not restricted to, yeast, a virus, a bacterium, and a parasite. Any natural host of the pathogenic organism is contemplated by the present invention and includes, but is not limited to, mammals, avians and fish. In a preferred embodiment, the pathogenic organism is a virus, which may be an RNA virus or a DNA virus. Preferably, the RNA virus is Human Immunodeficiency Virus (HIV), Poliovirus, and Influenza virus, Rous sarcoma virus, or a Flavivirus such as Japanese encephalitis virus. In a preferred embodiment, the RNA virus is a Hepatitis virus including, but not limited to, Hepatitis strains A, B and C. Suitably, the DNA virus is a Herpesvirus including, but not limited to, Herpes simplex virus, Epstein-Barr virus, Cytomegalovirus and Parvovirus. In a preferred embodiment, the virus is HIV and the parent polypeptide is suitably selected from env, gag, pol, vif, vpr, tat, rev, vpu and nef, or combination thereof. In an alternate preferred embodiment, the virus is Hepatitis C1a virus and the parent polypeptide is the Hepatitis C1a virus polyprotein.
In another embodiment, the pathogenic organism is a bacterium, which includes, but is not restricted to, Neisseria species, Meningococcal species, Haemophilus species Salmonella species, Streptococcal species, Legionella species and Mycobacterium species.
In yet another embodiment, the pathogenic organism is a parasite, which includes, but is not restricted to, Plasmodium species, Schistosoma species, Leishmania species, Trypanosoma species, Toxoplasma species and Giardia species.
Any cancer or tumour is contemplated by the present invention. For example, the cancer or tumour includes, but is not restricted to, melanoma, lung cancer, breast cancer, cervical cancer, prostate cancer, colon cancer, pancreatic cancer, stomach cancer, bladder cancer, kidney cancer, post transplant lymphoproliferative disease (PTLD), Hodgkin's Lymphoma and the like. Preferably, the cancer or tumour relates to melanoma. In a preferred embodiment of this type, the parent polypeptide is a melanocyte differentiation antigen which is suitably selected from gp100, MART, TRP-1, Tyros, TRP2, MC1R, MUC1F, MUC1R or a combination thereof In an alternate preferred embodiment of this type, the parent polypeptide is a melanoma-specific antigen which is suitably selected from BAGE, GAGE-1, gp100In4, MAGE-1, MAGE-3, PRAME, TRP2IN2, NYNSO1a, NYNSO1b, LAGE1 or a combination thereof.
In a preferred embodiment, the segments are selected on the basis of size. A segment according to the invention may be of any suitable size that can be utilised to elicit an immune response against an antigen encoded by the parent polypeptide. A number of factors can influence the choice of segment size. For example, the size of a segment should be preferably chosen such that it includes, or corresponds to the size of, T cell epitopes and their processing requirement. Practitioners in the art will recognise that class I-restricted T cell epitopes can be between 8 and 10 amino acids in length and if placed next to unnatural flanking residues, such epitopes can generally require 2 to 3 natural flanking amino acids to ensure that they are efficiently processed and presented. Class II-restricted T cell epitopes can range between 12 and 25 amino acids in length and may not require natural flanking residues for efficient proteolytic processing although it is believed that natural flanking residues may play a role. Another important feature of class II-restricted epitopes is that they generally contain a core of 9-10 amino acids in the middle which bind specifically to class II MHC molecules with flanking sequences either side of this core stabilising binding by associating with conserved structures on either side of class II MHC antigens in a sequence independent manner (Brown et al., 1993). Thus the functional region of class II-restricted epitopes is typically less than 15 amino acids long. The size of linear B cell epitopes and the factors effecting their processing, like class II-restricted epitopes, are quite variable although such epitopes are frequently smaller in size than 15 amino acids. From the foregoing, it is preferable, but not essential, that the size of the segment is at least 4 amino acids, preferably at least 7 amino acids, snore preferably at least 12 amino acids, more preferably at least 20 amino acids and more preferably at least 30 amino acids. Suitably, the size of the segment is less than 2000 amino acids, more preferably less than 1000 amino acids, more preferably less than 500 amino acids, more preferably less than 200 amino acids, more preferably less than 100 amino acids, more preferably less than 80 amino acids and even more preferably less than 60 amino acids and still even more preferably less than 40 amino acids. In this regard, it is preferable that the size of the segments is as small as possible so that the synthetic polypeptide adopts a functionally different structure relative to the structure of the parent polypeptide. It is also preferable that the size of the segments is large enough to minimise loss of T cell epitopes. In an especially preferred embodiment, the size of the segment is about 30 amino acids.
An optional spacer may be utilised to space adjacent segments relative to each other. Accordingly, an optional spacer may be interposed between some or all of the segments. The spacer suitably alters proteolytic processing and/or presentation of adjacent segment(s). In a preferred embodiment of this type, the spacer promotes or otherwise enhances proteolytic processing and/or presentation of adjacent segment(s). Preferably, the spacer comprises at least one amino acid. The at least one amino acid is suitably a neutral amino acid. The neutral amino acid is preferably alanine. Alternatively, the at least one amino acid is cysteine.
In a preferred embodiment, segments are selected such that they have partial sequence identity or homology with one or more other segments. Suitably, at one or both ends of a respective segment there is comprised at least 4 contiguous amino acids, preferably at least 7 contiguous amino acids, more preferably at least 10 contiguous amino acids, more preferably at least 15 contiguous amino acids and even more preferably at least 20 contiguous amino acids that are identical to, or homologous with, an amino acid sequence contained within one or more other of said segments. Preferably, at the or each end of a respective segment there is comprised less than 500 contiguous amino acids, more preferably less than 200 contiguous amino acids, more preferably less than 100 contiguous amino acids, more preferably less than 50 contiguous amino acids, more preferably less than 40 contiguous amino acids, and even more preferably less than 30 contiguous amino acids that are identical to, or homologous with, an amino acid sequence contained within one or more other of said segments. Such sequence overlap (also referred to elsewhere in the specification as “overlapping fragments” or “overlapping segments”) is preferable to ensure potential epitopes at segment boundaries are not lost and to ensure that epitopes at or near segment boundaries are processed efficiently if placed beside or near amino acids that inhibit processing. Preferably, the segment size is about twice the size of the overlap.
In a preferred embodiment, when segments have partial sequence homology therebetween, the homologous sequences suitably comprise conserved and/or non-conserved amino acid differences. Exemplary conservative substitutions are listed in the following table.










TABLE B







Original
Exemplary



Residue
Substitutions









Ala
Ser



Arg
Lys



Asn
Gln, His



Asp
Glu



Cys
Ser



Gln
Asn



Glu
Asp



Gly
Pro



His
Asn, Gln



Ile
Leu, Val



Leu
Ile, Val



Lys
Arg, Gln, Glu



Met
Leu, Ile,



Phe
Met, Leu, Tyr



Ser
Thr



Thr
Ser



Trp
Tyr



Tyr
Trp, Phe



Val
Ile, Leu










Conserved or non-conserved differences may correspond to polymorphisms in corresponding parent polypeptides. Polymorphic polypeptides are expressed by various pathogenic organisms and cancers. For example, the polymorphic polypeptides may be expressed by different viral strains or clades or by cancers in different individuals.
Sequence overlap between respective segments is preferable to minimise destruction of any epitope sequences that may result from any shuffling or rearrangement of the segments relative to their existing order in the parent polypeptide. If overlapping segments as described above are employed to form a synthetic polypeptide, it may not be necessary to change the order in which those segments are linked together relative to the order in which corresponding segments are normally present in the parent polypeptide. In this regard, such overlapping segments when linked together in the synthetic polypeptide can adopt a different structure relative to the structure of the parent polypeptide, wherein the different structure does not provide for one or more functions associated with the parent polypeptide. For example, in the case of four segments A-B-C-D each spanning 30 contiguous amino acids of the parent polypeptide and having a 10-amino acid overlapping sequence with one or more adjacent segments, the synthetic polypeptide will have duplicated 10-amino acid sequences bridging segments A-B, B-C and C-D. The presence of these duplicated sequences may be sufficient to render a different structure and to abrogate or alter function relative to the parent polypeptide.
In a preferred embodiment, segment size is about 30 amino acids and sequence overlap at one or both ends of a respective segment is about 15 amino acids. However, it will be understood that other suitable segment sizes and sequence overlap sizes are contemplated by the present invention, which can be readily ascertained by persons of skill in the art.
It is preferable but not necessary to utilise all the segments of the parent polypeptide in the construction of the synthetic polypeptide. Suitably, at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80% and still even more preferably at least 90% of the parent polypeptide sequence is used in the construction of the synthetic polypeptide. However, it will be understood that the more sequence information from a parent polypeptide that is utilised to construct the synthetic polypeptide, the greater the population coverage will be of the synthetic polypeptide as an immunogen. Preferably, no sequence information from the parent polypeptide is excluded (e.g., because of an apparent lack of immunological epitopes).
Persons of skill in the art will appreciate that when preparing a synthetic polypeptide against a pathogenic organism (e.g., a virus) or a cancer, it may be preferable to use sequence information from a plurality of different polypeptides expressed by the organism or the cancer. Accordingly, in a preferred embodiment, segments from a plurality of different polypeptides are linked together to form a synthetic polypeptide according to the invention. It is preferable in this respect to utilise as many parent polypeptides as possible from, or in relation to, a particular source in the construction of the synthetic polypeptide. The source of parent polypeptides includes, but is not limited to, a pathogenic organism and a cancer. Suitably, at least about 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80% and still even more preferably at least 90% of the parent polypeptides expressed by the source is used in the construction of the synthetic polypeptide. Preferably, parent polypeptides from a virus include, but are not restricted to, latent polypeptides, regulatory polypeptides or polypeptides expressed early during their replication cycle. Suitably, parent polypeptides from a parasite or bacterium include, but are not restricted to, secretory polypeptides and polypeptides expressed on the surface of the parasite or bacteria. It is preferred that parent polypeptides from a cancer or tumour are cancer specific polypeptides.
Suitably, hypervariable sequences within the parent polypeptide are excluded from the construction of the synthetic polypeptide.
The synthetic polypeptides of the inventions may be prepared by any suitable procedure known to those of skill in the art. For example, the polypeptide may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (1989, Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford) and in Roberge et at (1995, Science 269: 202). Syntheses may employ, for example, either t-butyloxycarbonyl (t-Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) chemistries (see Chapter 9.1, of Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE, John Wiley & Sons, Inc. 1995-1997; Stewart and Young, 1984, Solid Phase Peptide Synthesis, 2nd ed. Pierce Chemical Co., Rockford, Ill.; and Atherton and Shephard, supra).
Alternatively, the polypeptides may be prepared by a procedure including the steps of:

    
    
        (a) preparing a synthetic construct including a synthetic polynucleotide encoding a synthetic polypeptide wherein said synthetic polynucleotide is operably linked to a regulatory polynucleotide, wherein said synthetic polypeptide comprises a plurality of different segments of a parent polypeptide, wherein said segments are linked together in a different relationship relative to their linkage in the parent polypeptide;
        (b) introducing the synthetic construct into a suitable host cell;
        (c) culturing the host cell to express the synthetic polypeptide from said synthetic construct; and
        (d) isolating the synthetic polypeptide.
    
    


The synthetic construct is preferably in the form of an expression vector. For example, the expression vector can be a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. Typically, the regulatory polynucleotide may include, but is not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. The regulatory polynucleotide will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory polynucleotides are known in the art for a variety of host cells.
In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
The expression vector may also include a fusion partner (typically provided by the expression vector) so that the synthetic polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner. The main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide. In order to express said fusion polypeptide, it is necessary to ligate a polynucleotide according to the invention into the expression vector so that the translational reading frames of the fusion partner and the polynucleotide coincide.
Well known examples of fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS6), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively. Many such matrices are available in “kit” form, such as the QIAexpressTM system (Qiagen) useful with (HIS6) fusion partners and the Pharmacia GST purification system. In a preferred embodiment, the recombinant polynucleotide is expressed in the commercial vector pFLAGTM.
Another fusion partner well known in the art is green fluorescent protein (GFP). This fusion partner serves as a fluorescent “tag” which allows the fusion polypeptide of the invention to be identified by fluorescence microscopy or by flow cytometry. The GFP tag is useful when assessing subcellular localisation of a fusion polypeptide of the invention, or for isolating cells which express a fusion polypeptide of the invention. Flow cytometric methods such as fluorescence activated cell sorting (FACS) are particularly useful in this latter application. Preferably, the fusion partners also have protease cleavage sites, such as for Factor Xa, Thrombin and inteins (protein introns), which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom. The liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation. Fusion partners according to the invention also include within their scope “epitope tags”, which are usually short peptide sequences for which a specific antibody is available. Well known examples of epitope tags for which specific monoclonal antibodies are readily available include c-Myc, influenza virus, haemagglutinin and FLAG tags. Alternatively, a fusion partner may be provided to promote other forms of immunity. For example, the fusion partner may be an antigen-binding molecule that is immuno-interactive with a conformational epitope on a target antigen or to a post-translational modification of a target antigen (e.g., an antigen-binding molecule that is immuno-interactive with a glycosylated target antigen).
The step of introducing the synthetic construct into the host cell may be effected by any suitable method including transfection, and transformation, the choice of which will be dependent on the host cell employed. Such methods are well known to those of skill in the art.
Synthetic polypeptides of the invention may be produced by culturing a host cell transformed with the synthetic construct. The conditions appropriate for protein expression will vary with the choice of expression vector and the host cell. This is easily ascertained by one skilled in the art through routine experimentation.
Suitable host cells for expression may be prokaryotic or eukaryotic. One preferred host cell for expression of a polypeptide according to the invention is a bacterium. The bacterium used may be Escherichia coli. Alternatively, the host cell may be an insect cell such as, for example, SF9 cells that may be utilised with a baculovirus expression system.
The synthetic polypeptide may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
The amino acids of the synthetic polypeptide can be any non-naturally occurring or any naturally occurring amino acid. Examples of unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated by the present invention is shown in TABLE C.








TABLE C





Non-conventional Amino Acid
Non-conventional Amino Acid







α-aminobutyric acid
L-N-methylalanine


α-amino-α-methylbutyrate
L-N-methylarginine


aminocyclopropane-carboxylate
L-N-methylasparagine


aminoisobutyric acid
L-N-methylaspartic acid


aminonorbornyl-carboxylate
L-N-methylcysteine


cyclohexylalanine
L-N-methylglutamine


cyclopentylalanine
L-N-methylglutamic acid


L-N-methylisoleucine
L-N-methylhistidine


D-alanine
L-N-methylleucine


D-arginine
L-N-methyllysine


D-aspartic acid
L-N-methylmethionine


D-cysteine
L-N-methylnorleucine


D-glutamate
L-N-methylnorvaline


D-glutamic acid
L-N-methylornithine


D-histidine
L-N-methylphenylalanine


D-isoleucine
L-N-methylproline


D-leucine
L-N-methlylserine


D-lysine
L-N-methylthreonine


D-methionine
L-N-methyltryptophan


D-ornithine
L-N-methyltyrosine


D-phenylalanine
L-N-methylvaline


D-proline
L-N-methylethylglycine


D-serine
L-N-methyl-t-butylglycine


D-threonine
L-norleucine


D-tryptophan
L-norvaline


D-tyrosine
α-methyl-aminoisobutyrate


D-valin
α-methyl-γ-aminobutyrate


D-α-methylalanine
α-methylcyclohexylalanine


D-α-methylarginine
α-methylcylcopentylalanine


D-α-methylasparagine
α-methyl-α-napthylalanine


D-α-methylaspartate
α-methylpenicillamine


D-α-methylcysteine
N-(4-aminobutyl)glycine


D-α-methylglutamine
N-(2-aminoethyl)glycine


D-α-methylhistidine
N-(3-aminopropyl)glycine


D-α-methylisoleucine
N-amino-α-methylbutyrate


D-α-methylleucine
α-napthylalanine


D-α-methyllysine
N-benzylglycine


D-α-methylmethionine
N-(2-carbamylediyl)glycine


D-α-methylornithiine
N-(carbamylmethyl)glycine


D-α-methylphenylalanine
N-(2-carboxyethyl)glycine


D-α-methylproline
N-(carboxymethyl)glycine


D-α-methylserine
N-cyclobutylglycine


D-α-methylthreonine
N-cycloheptylglycine


D-α-methyltryptophan
N-cyclohexylglycine


D-α-methyltyrosine
N-cyclodecylglycine


L-α-methylleucine
L-α-methyllysine


L-α-methylmethionine
L-α-methylnorleucine


L-α-methylnorvatine
L-α-methylornithine


L-α-methylphenylalanine
L-α-methylproline


L-α-methylserine
L-α-methylthreonine


L-α-methyltryptophan
L-α-methyltyrosine


L-α-methylvaline
L-α-methylhomophenylalanine


N-(N-(2,2-diphenylethyl
N-(N-(3,3-diphenylpropyl


carbamylmethyl)glycine
carbamylmethyl)glycine


1-carboxy-1-(2,2-diphenyl-ethyl


amino)cyclopropane









The invention also contemplates modifying the synthetic polypeptides of the invention using ordinary molecular biological techniques so as to alter their resistance to proteolytic degradation or to optimise solubility properties or to render them more suitable as an immunogenic agent.
3. Preparation of Synthetic Polynucleotides of the Invention
The invention contemplates synthetic polynucleotides encoding the synthetic polypeptides as for example described in Section 2 supra. Polynucleotides encoding segments of a parent polypeptide can be produced by any suitable technique. For example, such polynucleotides can be synthesised de novo using readily available machinery. Sequential synthesis of DNA is described, for example, in U.S. Pat. No 4,293,652. Instead of de novo synthesis, recombinant techniques may be employed including use of restriction endonucleases to cleave a polynucleotide encoding at least a segment of the parent polypeptide and use of ligases to ligate together in frame a plurality of cleaved polynucleotides encoding different segments of the parent polypeptide. Suitable recombinant techniques are described for example in the relevant sections of Ausubel, et al. (supra) and of Sambrook, et al., (supra) which are incorporated herein by reference. Preferably, the synthetic polynucleotide is constructed using splicing by overlapping extension (SOEing) as for example described by Horton et al. (1990, Biotechniques 8(5): 528-535; 1995, Mol Biotechnol. 3(2): 93-99; and 1997, Methods Mol Biol. 67: 141-149). However, it should be noted that the present invention is not dependent on, and not directed to, any one particular technique for constructing the synthetic construct.
Various modifications to the synthetic polynucleotides may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
The invention therefore contemplates a method of producing a synthetic polynucleotide as broadly described above, comprising linking together in the same reading frame at least two nucleic acid sequences encoding different segments of a parent polypeptide to form a synthetic polynucleotide, which encodes a synthetic polypeptide according to the invention. Suitably, nucleic acid sequences encoding at least 10 segments, preferably at least 20 segments, more preferably at least 40 segments and more preferably at least 100 segments of a parent polypeptide are employed to produce the synthetic polynucleotide.
Preferably, the method further comprises selecting segments of the parent polypeptide, reverse translating the selected segments and preparing nucleic acid sequences encoding the selected segments. It is preferred that the method further comprises randomly linking the nucleic acid sequences together to form the synthetic polynucleotide. The nucleic acid sequences may be oligonucleotides or polynucleotides.
Suitably, segments are selected on the basis of size. Additionally, or in the alternative, segments are selected such that they have partial sequence identity or homology (i.e., sequence overlap) with one or more other segments. A number of factors can influence segment size and sequence overlap as mentioned above. In the case of sequence overlap, large amounts of duplicated nucleic acid sequences can sometimes result in sections of nucleic acid being lost during nucleic acid amplification (e.g., polymerase chain reaction, PCR) of such sequences, recombinant plasmid propagation in a bacterial host or during amplification of recombinant viruses containing such sequences. Accordingly, in a preferred embodiment, nucleic acid sequences encoding segments having sequence identity or homology with one or more other encoded segments are not linked together in an arrangement in which the identical or homologous sequences are contiguous. Also, it is preferable that different codons are used to encode a specific amino acid in a duplicated region. In this context, an amino acid of a parent polypeptide sequence is preferably reverse translated to provide a codon which, in the context of adjacent or local sequence elements, has a lower propensity of forming an undesirable sequence (e.g., a duplicated sequence or a palindromic sequence) that is refractory to the execution of a task (e.g., cloning or sequencing). Alternatively, segments may be selected such that they contain a carboxyl terminal leucine residue or such that reverse translated sequences encoding the segments contain restriction enzyme sites for convenient splicing of the reverse translated sequences.
The method optionally further comprises linking a spacer oligonucleotide encoding at least one spacer residue between segment-encoding nucleic acids. Such spacer residue(s) may be advantageous in ensuring that epitopes within the segments are processed and presented efficiently. Preferably, the spacer oligonucleotide encodes 2 to 3 spacer residues. The spacer residue is suitably a neutral amino acid, which is preferably alanine.
Optionally, the method further comprises linking in the same reading frame as other segment-containing nucleic acid sequences at least one variant nucleic acid sequence which encodes a variant segment having a homologous but not identical amino acid sequence relative to other encoded segments. Suitably, the variant segment comprises conserved and/or non-conserved amino acid differences relative to one or more other encoded segments. Such differences may correspond to polymorphisms as discussed above. In a preferred embodiment, degenerate bases are designed or built in to the at least one variant nucleic acid sequence to give rise to all desired homologous sequences.
When a large number of polymorphisms is intended to be covered, it is preferred that multiple synthetic polynucleotides are constructed rather than a single synthetic polynucleotide, which encodes all variant segments. For example, if there is less than 85% homology between polymorphic polypeptides, then it is preferred that more than one synthetic polynucleotide is constructed.
Preferably, the method further comprises optimising the codon composition of the synthetic polynucleotide such that it is translated efficiently by a host cell. In this regard, it is well known that the translational efficiency of different codons varies between organisms and that such differences in codon usage can be utilized to enhance the level of protein expression in a particular organism. In this regard, reference may be made to Seed et al. (International Application Publication No WO 96/09378) who disclose the replacement of existing codons in a parent polynucleotide with synonymous codons to enhance expression of viral polypeptides in mammalian host cells. Preferably, the first or second most frequently used codons are employed for codon optimization.
Preferably, gene splicing by overlap extension or “gene SOEing” (supra) is employed for the construction of the synthetic polynucleotide which is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5′-ends of the primers, any pair of PCR products can be made to share a common sequence at one end. Under PCR conditions, the common sequence allows strands from two different fragments to hybridise to one another, forming an overlap. Extehsion of this overlap by DNA polymerase yields a recombinant molecule. However, a problem with long synthetic constructs is that mutations generally incorporate into amplified products during synthesis. In this instance, it is preferred that resolvase treatment is employed at various steps of the synthesis. Resolvases are bacteriophage-encoded endonucleases which recognise disruptions or mispairing of double stranded DNA and are primarily used by bacteriophages to resolve Holliday junctions (Mizuuchi, 1982; Youil et al., 1995). For example, T7 endonuclease I can be employed in synthetic DNA constructions to recognise mutations and cleave corrupted dsDNA. The mutated DNA strands are then hybridised to non-mutant or correct DNA sequences, which results in a mispairing of DNA bases. The mispaired bases are recognized by the resolvase, which then cleaves the DNA nearby leaving only correctly hybridised sequences intact. Preferably a thermostable resolvase enzyme is employed during splicing or amplification so that errors are not incorporated in downstream synthesis products.
Synthetic polynucleotides according to the invention can be operably linked to a regulatory polynucleotide in the form a synthetic construct as for example described in Section 2 supra. Synthetic constructs of the invention have utility inter alia as nucleic acid vaccines. The choice of regulatory polynucleotide and synthetic construct will depend on the intended host.
Exemplary expression vectors for expression of a synthetic polypeptide according to the invention include, but are not restricted to, modified Ankara Vaccinia virus as for example described by Allen et al. (2000, J. Immunol. 164(9): 4968-4978), fowlpox virus as for example described by Boyle and Coupar (1988, Virus Res. 10: 343-356) and the herpes simplex amplicons described for example by Fong et al. in U.S. Pat. No. 6,051,428. Alternatively, Adenovirus and Epstein-Barr virus vectors, which are preferably capable of accepting large amounts of DNA or RNA sequence information, can be used.
Preferred promoter sequences that can be utilized for expression of synthetic polypeptides include the P7.5 or PE/L promoters as for example disclosed by Kumar and Boyle. (1990, Virology 179:151-158), CMV and RSV promoters.
The synthetic construct optionally further includes a nucleic acid sequence encoding an immunostimulatory molecule. The immunostimulatory molecule may be fusion partner of the synthetic polypeptide. Alternatively, the immunostimulatory molecule may be translated separately from the synthetic polypeptide. Preferably, the immunostimulatory molecule comprises a general immunostimulatory peptide sequence. For example, the immunostimulatory peptide sequence may comprise a domain of an invasin protein (Inv) from the bacteria Yersinia spp as for example disclosed by Brett et al. (1993, Eur. J. Immunol. 23: 1608-1614). This immune stimulatory property results from the capability of this invasin domain to interact with the β1 integrin molecules present on T cells, particularly activated immune or memory T cells. A preferred embodiment of the invasin domain (Inv) for linkage to a synthetic polypeptide has been previously described in U.S. Pat. No. 5,759,551. The said Inv domain has the sequence: Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe [SEQ ID NO; 1467] or is an immune stimulatory homologue thereof from the corresponding region in another Yersinia species invasin protein. Such homologues thus may contain substitutions, deletions or insertions of amino acid residues to accommodate strain to strain variation, provided that the homologues retain immune stimulatory properties. The general immunostimulatory sequence may optionally be linked to the synthetic polypeptide by a spacer sequence.
In an alternate embodiment, the immunostimulatory molecule may comprise an immunostimulatory membrane or soluble molecule, which is suitably a T cell co-stimulatory molecule. Preferably, the T cell co-stimulatory molecule is a B7 molecule or a biologically active fragment thereof, or a variant or derivative of these. The B7 molecule includes, but is not restricted to, B7-1 and B7-2. Preferably, the B7 molecule is B7-1. Alternatively, the T cell co-stimulatory molecule may be an ICAM molecule such as ICAM-1 and ICAM-2.
In another embodiment, the immunostimulatory molecule can be a cytokine, which includes, but is not restricted to, an interleukin, a lymphokine, tumour necrosis factor and an interferon. Alternatively, the immunostimulatory molecule may comprise an immunomodulatory oligonucleotide as for example disclosed by Krieg in U.S. Pat. No. 6,008,200.
Suitably, the size of the synthetic polynucleotide does not exceed the ability of host cells to transcribe, translate or proteolytically process and present epitopes to the immune system. Practitioners in the art will also recognise that the size of the synthetic polynucleotide can impact on the capacity of an expression vector to express the synthetic polynucleotide in a host cell. In this connection, it is known that the efficacy of DNA vaccination reduces with expression vectors greater that 20-kb. In such situations it is preferred that a larger number of smaller synthetic constructs is utilized rather than a single large synthetic construct.
4. Immunopotentiating Compositions
The invention also contemplates a composition, comprising an immunopotentiating agent selected from the group consisting of a synthetic polypeptide as described in Section 2, and a synthetic polynucleotide or a synthetic construct as described in Section 3, together with a pharmaceutically acceptable carrier. One or more immunopotentiating agents can be used as actives in the preparation of immunopotentiating compositions. Such preparation uses routine methods known to persons skilled in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 1983A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. For example, the effectiveness of an adjuvant may be determined by measuring the amount of antibodies resulting from the administration of the composition, wherein those antibodies are directed against one or more antigens presented by the treated cells of the composition.
The immunopotentiating agents may be formulated into a composition as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic basis such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic basis as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
If desired, devices or compositions containing the immunopotentiating agents suitable for sustained or intermittent release could be, in effect, implanted in the body or topically applied thereto for the relatively slow release of such materials into the body.
The compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
Administration of the gene therapy construct to said mammal, preferably a human, may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor. An example of the latter approach would be stem cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the vector comprising the Sox18 nucleic acid. The stem cells are cultured for a period and then transferred to the mammal being treated.
With regard to nucleic acid based compositions, all modes of delivery of such compositions are contemplated by the present invention. Delivery of these compositions to cells or tissues of an animal may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g., lipofectin or lipofectamine), electroporation, calcium phosphate or DEAE-dextran-mediated transfection, for example. In an alternate embodiment, a synthetic construct may be used as a therapeutic or prophylactic composition in the form of a “naked DNA” composition as is known in the art. A discussion of suitable delivery methods may be found in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al.; John Wiley & Sons Inc., 1997 Edition) or on the Internet site DNAvaccine.com. The compositions may be administered by intradermal (e.g., using panjetTM delivery) or intramuscular routes.
The step of introducing the synthetic polynucleotide into a target cell will differ depending on the intended use and species, and can involve one or more of non-viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R.C., (1993 Science 260 926-932) which is hereby incorporated by reference. Such methods can include, for example:

    A. Local application of the synthetic polynucleotide by injection (Wolff et al., 1990, Science 247 1465-1468, which is hereby incorporated by reference), surgical implantation, instillation or any other means. This method can also be used in combination with local application by injection, surgical implantation, instillation or any other means, of cells responsive to the protein encoded by the synthetic polynucleotide so as to increase the effectiveness of that treatment. This method can also be used in combination with local application by injection, surgical implantation, instillation or any other means, of another factor or factors required for the activity of said protein.
    B. General systemic delivery by injection of DNA, (Calabretta et al., 1993, Cancer Treat. Rev. 19 169-179, which is incorporated herein by reference), or RNA, alone or in combination with liposomes (Zhu et al., 1993, Science 261 209-212, which is incorporated herein by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is incorporated herein by reference) or any other mediator of delivery. Improved targeting might be achieved by linking the synthetic polynucleotide to a targeting molecule (the so-called “magic bullet” approach employing, for example, an antibody), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the protein encoding said synthetic polynucleotide , or of cells responsive to said protein.
    C. Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate: Chen et al., 1987, Mole. Cell Biochem. 7 2745-2752, or of cationic lipids and polyamines: Rose et al., 1991, BioTech. 10 520-525, which articles are incorporated herein by reference), infection, injection, electroporation (Shigekawa et al., 1988, BioTech. 6 742-751, which is incorporated herein by reference) or any other way so as to increase the expression of said synthetic polynucleotide in those cells. The modification can be mediated by plasmid, bacteriophage, cosmid, viral (such as adenoviral or retroviral; Mulligan, 1993, Science 260 926-932; Miller, 1992, Nature 357 455-460; Salmons et al., 1993, Hum. Gen. Ther. 4 129-141, which articles are incorporated herein by reference) or other vectors, or other agents of modification such as liposomes (Zhu et al., 1993, Science 261 209-212, which is incorporated herein by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is incorporated herein by reference), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al., 1991, Science 254 1507-1512 and by Dhawan et al., 1991, Science 254 1509-1512, which articles are incorporated herein by reference. Treated cells can be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.


Also encapsulated by the present invention is a method for treatment and/or prophylaxis of a disease or condition, comprising administering to a patient in need of such treatment a therapeutically effective amount of a composition as broadly described above. The disease or condition may be caused by a pathogenic organism or a cancer as for example described above.
In a preferred embodiment, the immunopotentiating composition of the invention is suitable for treatment of, or prophylaxis against, a cancer. Cancers which could be suitably treated in accordance with the practices of this invention include cancers of the lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone liver, oesophagus, brain, testicle, uterus, melanoma and the various leukemias and lymphomas.
In an alternate embodiment, the immunopotentiating composition is suitable for treatment of, or prophylaxis against, a viral, bacterial or parasitic infection. Viral infections contemplated by the present invention include, but are not restricted to, infections caused by HIV, Hepatitis, Influenza, Japanese encephalitis virus, Epstein-Barr virus and respiratory syncytial virus. Bacterial infections include, but are not restricted to, those caused by Neisseria species, Meningococcal species, Haemophilus species Salmonella species, Streptococcal species, Legionella species and Mycobacterium species. Parasitic infections encompassed by the invention include, but are not restricted to, those caused by Plasmodium species, Schistosoma species, Leishmania species, Trypanosoma species, Toxoplasma species and Giardia species.
The above compositions or vaccines may be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective to alleviate patients from the disease or condition or as is prophylactically effective to prevent incidence of the disease or condition in the patient. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over time such as a reduction or cessation of blood loss. The quantity of the composition or vaccine to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the composition or vaccine for administration will depend on the judgement of the practitioner. In determining the effective amount of the composition or vaccine to be administered in the treatment of a disease or condition, the physician may evaluate the progression of the disease or condition over time. In any event, those of skill in the art may readily determine suitable dosages of the composition or vaccine of the invention.
In a preferred embodiment, DNA-based immunopotentiating agent (e.g., 100 μg) is delivered intradermally into a patient at day 1 and at week 8 to prime the patient. A recombinant poxvirus (e.g., at 107 pfu/mL) from which substantially the same immunopotentiating agent can be expressed is then delivered intradermally as a booster at weeks 16 and 24, respectively.
The effectiveness of the immunisation may be assessed using any suitable technique. For example, CTL lysis assays may be employed using stimulated splenocytes or peripheral blood mononuclear cells (PBMC) on peptide coated or recombinant virus infected cells using 51Cr labelled target cells. Such assays can be performed using for example primate, mouse or human cells (Allen et al., 2000, J. Immunol. 164(9): 4968-4978 also Woodberry et al., infra). Alternatively, the efficacy of the immunisation may be monitored using one or more techniques including, but not limited to, HLA class I Tetramer staining—of both fresh and stimulated PBMCs (see for example Allen et al., supra), proliferation assays (Allen et al., supra), ElispotTM Assays and intracellular INF-gamma staining (Allen et al., supra), ELISA Assays—for linear B cell responses; and Western blots of cell sample expressing the synthetic polynucleotides.
5. Computer Related Embodiments
The design or construction of a synthetic polypeptide sequence or a synthetic polynucleotide sequence according to the invention is suitably facilitated with the assistance of a computer programmed with software, which inter alia fragments a parent sequence into fragments, and which links those fragments together in a different relationship relative to their linkage in the parent sequence. The ready use of a parent sequence for the construction of a desired synthetic molecule according to the invention requires that it be stored in a computer-readable format. Thus, in accordance with the present invention, sequence data relating to a parent molecule (e.g. a parent polypeptide) is stored in a machine-readable storage medium, which is capable of processing the data to fragment the sequence of the parent molecule into fragments and to link together the fragments in a different relationship relative to their linkage in the parent molecule.
Therefore, another embodiment of the present invention provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data which, when used by a machine programmed with instructions for using said data, fragments a parent sequence into fragments, and links those fragments together in a different relationship relative to their linkage in the parent sequence. In a preferred embodiment of this type, a machine-readable data storage medium is provided that is capable of reverse translating the sequence of a respective fragment to provide a nucleic acid sequence encoding the fragment and to link together in the same reading frame each of the nucleic acid sequences to provide a polynucleotide sequence that codes for a polypeptide sequence in which said fragments are linked together in a different relationship relative to their linkage in a parent polypeptide sequence.
In another embodiment, the invention encompasses a computer for designing the sequence of a synthetic polypeptide and/or a synthetic polynucleotide of the invention, wherein the computer comprises wherein said computer comprises: (a) a machine readable data storage medium comprising a data storage material encoded with machine readable data, wherein said machine readable data comprises the sequence of a parent polypeptide; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine-readable data into said synthetic polypeptide sequence and/or said synthetic polynucleotide; and (d) an output hardware coupled to said central processing unit, for receiving said synthetic polypeptide sequence and/or said synthetic polynucleotide.
In yet another embodiment, the invention contemplates a computer program product for designing the sequence of a synthetic polynucleotide of the invention, comprising code that receives as input the sequence of a parent polypeptide, code that fragments the sequence of the parent polypeptide into fragments, code that reverse translates the sequence of a respective fragment to provide a nucleic acid sequence encoding the fragment, code that links together in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which said fragments are linked together in a different relationship relative to their linkage in the parent polypeptide sequence, and a computer readable medium that stores the codes.
A version of these embodiments is presented in FIG. 23, which shows a system 10 including a computer 11 comprising a central processing unit (“CPU”) 20, a working memory 22 which may be, e.g., RAM (random-access memory) or “core” memory, mass storage memory 24 (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals 26, one or more keyboards 28, one or more input lines 30, and one or more output lines 40, all of which are interconnected by a conventional bidirectional system bus 50.
Input hardware 36, coupled to computer 11 by input lines 30, may be implemented in a variety of ways. For example, machine-readable data of this invention may be inputted via the use of a modem or modems 32 connected by a telephone line or dedicated data line 34. Alternatively or additionally, the input hardware 36 may comprise CD. Alternatively, ROM drives or disk drives 24 in conjunction with display terminal 26, keyboard 28 may also be used as an input device.
Output hardware 46, coupled to computer 11 by output lines 40, may similarly be implemented by conventional devices. By way of example, output hardware 46 may include CRT display terminal 26 for displaying a synthetic polynucleotide sequence or a synthetic polypeptide sequence as described herein. Output hardware might also include a printer 42, so that hard copy output may be produced, or a disk drive 24, to store system output for later use.
In operation, CPU 20 coordinates the use of the various input and output devices 36,46 coordinates data accesses from mass storage 24 and accesses to and from working memory 22, and determines the sequence of data processing steps. A number of programs may be used to process the machine readable data of this invention. Exemplary programs may use for example the steps outlined in the flow diagram illustrated in FIG. 24. Broadly, these steps include (1) inputting at least one parent polypeptide sequence; (2) optionally adding to alanine spacers at the ends of each polypeptide sequence; (3) fragmenting the polypeptide sequences into fragments (e.g., 30 amino acids long), which are preferably overlapping (e.g., by 15 amino acids); (4) reverse translating the fragment to provide a nucleic acid sequence for each fragment and preferably using for the reverse translation first and second most translationally efficient codons for a cell type, wherein the codons are preferably alternated out of frame with each other in the overlaps of consecutive fragments; (5) randomly rearranging the fragments; (6) checking whether rearranged fragments recreate at least a portion of a parent polypeptide sequence; (7) repeating randomly rearranging the fragments when rearranged fragments recreate said at least a portion; or otherwise (8) linking the rearranged fragments together to produce a synthetic polypeptide sequence and/or a synthetic polynucleotide sequence; and (9) outputting said synthetic polypeptide sequence and/or a synthetic polynucleotide sequence. An example of an algorithm which uses inter alia the aforementioned steps is shown in FIG. 25. By way of example, this algorithm has been used for the design of synthetic polynucleotides and synthetic polypeptides according to the present invention for Hepatitis C1a and for melanoma, as illustrated in FIGS. 26 and 27.
FIG. 28 shows a cross section of a magnetic data storage medium 100 which can be encoded with machine readable data, or set of instructions, for designing a synthetic molecule of the invention, which can be carried out by a system such as system 10 of FIG. 23. Medium 100 can be a conventional floppy diskette or hard disk, having a suitable substrate 101, which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides, containing magnetic domains (not visible) whose polarity or orientation can be altered magnetically. Medium 100 may also have an opening (not shown) for receiving the spindle of a disk drive or other data storage device 24. The magnetic domains of coating 102 of medium 100 are polarised or oriented so as to encode in manner which may be conventional, machine readable data such as that described herein, for execution by a system such as system 10 of FIG. 23.
FIG. 29 shows a cross section of an optically readable data storage medium 110 which also can be encoded with such a machine-readable data, or set of instructions, for designing a synthetic molecule of the invention, which can be carried out by a system such as system 10 of FIG. 23. Medium 110 can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk, which is optically readable and magneto-optically writable. Medium 100 preferably has a suitable substrate 111, which may be conventional, and a suitable coating 112, which may be conventional, usually of one side of substrate 111.
In the case of CD-ROM, as is well known, coating 112 is reflective and is impressed with a plurality of pits 113 to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of coating 112. A protective coating 114, which preferably is substantially transparent, is provided on top of coating 112.
In the case of a magneto-optical disk, as is well known, coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown). The orientation of the domains can be read by measuring the polarisation of laser light reflected from coating 112. The arrangement of the domains encodes the data as described above.
In order that the invention may be readily understood and put into practical effect, particular preferred non-limiting embodiments will now be described as follows.
EXAMPLES
Example 1
Preparation of an HIV Savine
Experimental Protocol
Plasmids
The plasmid pDNAVacc is ampicillin resistant and contains an expression cassette comprising a CMV promoter and enhancer, a synthetic intron, a multiple cloning site (MCS) and a SV40poly A signal sequence (Thomson et al., 1998). The plasmid pTK7.5 and contains a selection cassette, a pox virus 7.5 early/late promoter and a MCS flanked on either side by Vaccinia virus TK gene sequences.
Recombinant Vaccinia Viruses
Recombinant Vaccinia viruses expressing the gag, env (IIB) and pol (LAI) genes of HIV-1 were used as previously described and denoted VV-GAG, VV-POL, VV-ENV (Woodberry et al., 1999; Kent et al., 1998).
Marker Rescue Recombination
Recombinant Vaccinia viruses containing Savine constructs were generated by marker rescue recombination, using protocols described previously (Boyle et al., 1985). Plaque purified viruses were tested for the TK phenotype and for the appropriate genome arrangement by Southern blot and PCR.
Oligonucleotides
Oligonucleotides 50 nmol scale and desalted were purchased from Life Technologies. Short oligonucleotides were resuspended in 100 μL of water, their concentration determined, then diluted to 20 μM for use in PCR or sequencing reactions. Long oligonucleotides for splicing reactions were denatured for 5 minutes at 94° C. in 20 μL of formamide loading buffer then 0.5 μL gel purified on a 6% polyacrylamide gel. Gel slices containing full-length oligonucleotides were visualised with ethidium bromide, excised, placed in EppendorfTM tubes, combined with 200 μL of water before being crushed using the plunger of a 1 mL syringe. Before being used in splicing reactions the crushed gel was resuspended in an appropriate volume of buffer and 1-2 μL of the resuspendate used directly in the splicing reactions.
Sequencing
Sequencing was performed using Dye terminator sequencing reactions and analyzed by the Biomedical Resource Facility at the John Curtin School of Medical Research using an ABI automated sequencer.
Restimulation of Lymphocytes from HIV Infected Patients
Two pools of recombinant Vaccinia viruses containing VV-AC1+VV-BC1 (Pool 1) or VV-AC2+VV-BC2+VV-CC2 (Pool 2) were used to restimulate lymphocytes from the blood samples of HIV-infected patients. Briefly CTL lines were generated from HIV-infected donor PBMC. A fifth of the total PBMC were infected with either Pool 1 or Pool 2 Vaccinia viruses then added back to the original cell suspension. The infected cell suspension was then cultured with IL-7 for 1 week.
CTL Assays
Restimulated PBMCs were used as effectors in a standard 51Cr-release CTL assay. Targets were autologous EBV-transformed lymphoblastoid cell lines (LCLs) infected with the following viruses: Pool 1, Pool 2, VV-GAG, VV-POL or VV-ENV. Assay controls included uninfected targets, targets infected with VV-lacZ (virus control) and K562 cells.
Results
HIV Savine Design
A main goal of the Savine strategy is to include as much protein sequence information from a pathogen or cancer as possible in such a way that potential T cell epitopes remain intact and so that the vaccine or therapy is extremely safe. An HIV Savine is described herein not only to compare this strategy to other strategies but also, to produce an HIV vaccine that would provide the maximum possible population coverage as well as catering for the major HIV clades.
A number of design criteria was first determined to exploit the many advantages of using a synthetic approach. One advantage is that it is possible to use consensus protein sequences to design these vaccines. Using consensus sequences for a highly variable virus like HIV should provide better vaccine coverage because individual viral isolate sequences may have lost epitopes which induce CTL against the majority of other viral isolates. Thus, using the-consensus sequences of each HIV lade rather than individual isolate sequences should provide better vaccine coverage. Taking this one step further, a consensus sequence that covers all HIV clades should theoretically provide better coverage than using just the consensus sequences for individual clades. Before designing such a sequence however, it was decided that a more appropriate and focussed HIV vaccine might be constructed if the various clades were first ranked according to their relative importance. To establish such a ranking the following issues were considered, current prevalence of each lade, the rate at which each lade is increasing and the capacity of various regions of the world to cope with the HIV pandemic (FIGS. 1 and 2). These criteria produced the following ranking, Clade E≧clade A>clade C>clade B>cldade D>other clades. Clades E and A were considered to almost equal since they are very similar except in their envelope protein sequences, which differ considerably.
Another advantage of synthesising a designed sequence is that it is possible to incorporate degenerate sequences into their design. In the case of HIV, this means that more than one amino acid can be included at various positions to improve the ability of the vaccine to cater for the various HIV clades and isolates. Coverage is improved because mutations in different HIV clades and also in individual isolate sequences, while mostly destroying specific T cell epitopes, can result in the formation of new potentially useful epitopes nearby (Goulder et al., 1997). Incorporating degenerate amino acid sequences, however, also means that more than one construct must be made and mixed together. The number of constructs required depends on the frequency with which mutations are incorporated into the design. While this approach requires the construction of additional constructs, these constructs can be prepared from the same set of degenerate long oligonucleotides, significantly reducing the cost of providing such considerable interclade coverage.
A set of degeneracy rules was developed for the incorporation of amino acid mutations into the design which meant that a maximum of eight constructs would be required so that theoretically all combinations were present, as follows: 1) Two amino acids at three positions (or less) within any group of nine amino acids (i.e., present in a CTL epitope); 2) Three amino acids at one position and two at another (or not) within any group of nine amino acids; 3) Four amino acids at one position and two at another (or not) within any group of nine amino acids. The reason why these rules were applied to nine amino acids (the average CTL epitope size) and not to larger stretches of amino acid sequence to cater for class II restricted epitopes, is because class II-restricted epitopes generally have a core sequence of nine amino acids in the middle which bind specifically to class II MHC molecules with the extra flanking sequences stabilising binding, by associating with either side of class II MHC antigens in a largely sequence independent manner (Brown et al., 1993).
Using the HIV clade ranking described above, the amino acid degeneracy rules and in some situations the similarity between amino acids, a degenerate consensus protein sequence was designed for each HIV protein using the consensus protein sequences for Molecular Immunology Database, 1997). It is important to note that in some situations the order with which each of the above design criteria was applied was altered. Each time this was done the primary goal however was to increase the ability of the Savine to cater for interclade differences. Two isolate sequences, GenBank accession U51189 and U46016, for lade E and lade C, respectively, were used when a consensus sequence for some HIV proteins from these two clades was unavailable (Gao et al., 1996; Salminen et al., 1996). The design of a consensus sequence for the hypervariable regions of the HIV envelope protein and in some cases between these regions (hypervariable regions 1-2 and 3-5) was difficult and so these regions were excluded from the vaccine design.
Once a degenerate consensus sequence was designed for each HIV protein, an approach was then determined for incorporating all the protein sequences safely into the vaccine. One convenient approach to ensure that a vaccine will be safe is to systematically fragment and randomly rearrange the protein sequences together thus abrogating or otherwise altering their structure and function. The protein sequences still have to be immunologically functional however, meaning that the process used to fragment the sequences should not destroy potential epitopes. To decide on the best approach for systematically fragmenting protein sequences, the main criteria used was the size of T epitopes and their processing requirements. Class I-restricted T cell epitopes are 8-10 amino acids long and generally require 2-3 natural flanking amino acids to ensure their efficient processing and presentation if placed next to unnatural flanking residues (Del Val et al., 1991; Thomson et al., 1995). Class II-restricted T cell epitopes range between 12-25 amino acids long and do appear to require natural flanking residues for processing however, it is difficult to rule out a role for natural flanking residues in all cases due to the complexity of their processing pathways (Thomson et al., 1998). Also class II-restricted epitopes despite being larger than CTL epitopes generally have a core sequence of 9-10 amino acids, which binds to MHC molecules in a sequence specific fashion. Thus, based on current knowledge, it was decided that an advantageous approach was to overlap the fragments by at least 15 amino acids to ensure that potential epitopes which might lie across fragment boundaries are not lost and to ensure that CTL epitopes near fragment boundaries, that are placed beside or near inhibitory amino acids in adjacent fragments, are processed efficiently. In deciding the optimal fragment size, the main criteria used were that size had to be small enough to cause the maximum disruption to the structure and function of proteins but large enough to cover the sequence information as efficiently as possible without any further unnecessary duplication. Based on these criteria the fragments would be twice the overlap size, in this case 30 amino acids long.
The designed degenerate protein sequences were then separated into fragments 30 amino acid long and overlapping by fifteen amino acids. Two alanine amino acids were also added to the start and end of the first and last fragment for each protein or envelop protein segment to ensure these fragments were not placed directly adjacent to amino acids capable of blocking epitope processing (Del Val et al., 1991). The next step was to reverse translate each protein sequence back into DNA. Duplicating DNA sequences was avoided when constructing DNA sequences encoding a tandem repeat of identical or homologous amino acid sequences to maximise expression of the Savine. In this regard, the first and second most commonly used mammalian codons (shown in FIG. 12) were assigned to amino acids in these repeat regions, wherein a first codon was used to encode an amino acid in one of the repeated sequences and wherein a second but synonymous codon was used for the other repeated sequence (e.g., see the gag HIV protein in FIG. 13). To cater for the designed amino acid mutations more than one base was assigned to some positions using the IUPAC DNA codes without exceeding more than three base variations (eight possible combinations) in any group of 27 bases (FIG. 12). Where a particular combination of amino acids could not be incorporated, because too many degenerate bases would be required, some or all of the amino acid degeneracy was removed according to the protein consensus design rules outlined above. Also the degenerate codons were checked to determine if they could encode a stop codon, if stop codons could not be avoided then the amino acid degeneracy was also simplified again according to the protein consensus design rules outlined above.
The designed DNA segments were then scrambled randomly and joined to create twenty-two subcassettes approximately 840 bp in size. Extra DNA sequences incorporating sites for one of the cohesive restriction enzymes XbaI, SpeI, AvrII or NheI and 3 additional base pairs (to cater for premature Taq polymerase termination) were then added to each end of each subcassette (FIG. 14). Some of these extra DNA sequences also contained, the cohesive restriction sites for SalI or XhoI, Kozak signal sequences and start or stop codons to enable the subcassettes to be joined and expressed either as three large cassettes or one full length protein (FIGS. 14 and 15).
In designing the HIV Savine one issue that required investigation was whether such a large DNA molecule would be fully expressed and Whether epitopes encoded near the end of the molecule would be efficiently presented to the immune system. The inventors also wished to show that mixing two or more degenerate Savine constructs together could induce T cell responses that recognise mutated sequences. To examine both issues DNA coding for a degenerate murine influenza nucleoprotein CTL epitope, NP365-373, which differs by two amino acids at positions 71 and 72 in influenza strain A/PR/8/34 compared to the A/NT/60/68 strain and restricted by H2-Db, was inserted before the last stop codon at the end of the HIV Savine design (FIG. 15). An important and unusual characteristic of both of these naturally occurring NP365-373 sequences, which enabled the present inventors to examine the effectiveness of incorporating mutated sequences, is that they generate CTL responses which do not cross react with the alternate sequence (Townsend et. al., 1986). This is an unusual characteristic because epitopes not destroyed by mutation usually induce CTL responses that cross-react.
Up to ten long oligonucleotides up to 100 bases long and two short amplification oligonucleotides were synthesized to enable construction of each subcassette (Life Technologies). In designing each oligonucleotide the 3′ end and in most cases also the 5′ end had to be either a ‘c’ or a ‘g’ to ensure efficient extension during PCR splicing. The overlap region for each long oligonucleotide was designed to be at least 16 bp with approximately 50% G/C content. Also oligonucleotide overlaps were not placed where degenerate DNA bases coded for degenerate amino acids to avoid splicing difficulties later. Where this was too difficult some degenerate bases were removed according to the protein consensus design rules outlined above and indicated in FIG. 12 FIG. 16 shows an example of the oligonucleotides design for each subcassette.
Construction of the HIV Savine
Five of each group of ten designed oligonucleotides were spliced together using stepwise asymmetric PCR (Sandhu et al., 1992) and Splicing by Overlap Extension (SOEing) (FIG. 17a). Each subcassette was then PCR amplified, cloned into pBluescriptTM II KS- using BamHI/EcoRI and 16 individual clones sequenced. Mutations, deletions and insertions were present in the large majority of the clones for each subcassette, despite acrylamide gel purification of the long oligonucleotides. In order to construct a fictional Savine with minimal mutations, two clones for each subcassette with no insertions or deletions and hence a complete open reading frame and with minimal numbers of non-designed mutations, were selected from the sixteen available. The subcassettes were then excised from their plasmids and joined by stepwise PCR-amplified ligation using the polymerase blend ElongaseTM (Life Technology), T4 DNA ligase and the cohesive restriction enzymes XbaI/SpeI/AvrII/NheI, to generate two copies of cassettes A, B and C as outlined in FIG. 14 and shown in FIG. 17b. Predicted sequences for these cassettes are shown in FIG. 30. Each cassette was then reamplifled by PCR with ElongaseTM, cloned into pBluescriptTM II KS- and 3 of the resulting plasmid clones sequenced using 12 of the 36 sequencing primers designed to cover the full length construct. Clones with minimal or no further mutations were selected for transfer into plasmids for DNA vaccination or used to make recombinant poxviruses. A summary of the number of designed and non-designed mutations in each Savine construct is presented in Table 1.







TABLE 1







Summary of mutations









Number of mutations















Expected
Actual



Construct
No. aas
Designed
in 2 clones
in 2 clones
Non-designed















Cassette A
1896
249
124
107
 5 (AC1),







 8 (AC2)


Cassette B
1184
260
130
124
11 (BC1),







 4 (BC2)


Cassette C
1969
276
138
121
10 (CC1),







14 (CC2)


Full length
5742
785
392
352
26 (FL1),







26 (FL2)









Summary of the mutations present in the two full-length clones constructed as determined by sequencing. Includes the number of mutations designed, expected and actually present in the 2 clones and the number of non-designed mutations in each cassette and full-length clone.
HIV Savine DNA vaccines and Recombinant Vaccinia viruses
To test the immunological effectiveness of the HIV Savine constructs the cassette sequences were transferred into DNA vaccine and poxvirus vectors. These vectors when used either separately in immunological assays described below or together in a ‘prime-boost ’ protocol which has been shown previously to generate strong T cell responses in vivo (Kent et al., 1997).
DNA Vaccination plasmids were constructed by excising the cassettes from the selected plasmid clones with XbaI/XhoI (cassette A) or XbaI/SalI (cassettes B and C) and ligating them into pDNAVacc cut with XbaI/XhoI to create pDVAC1, pDVAC2, pDVBC1, pDVBC2, pDVCC1, pDVCC2, respectively (FIG. 18a). These plasmids were then further modified by cloning into their XbaI site a DNA fragment excised using XbaI/AvrII from pTUMERA2 and encoding a synthetic endoplasmic reticulum (ER) signal sequence from the Adenovirus E1A protein (Persson et al., 1980) (FIG. 18a). ER signal sequences have been shown previously to enhance the presentation of both CTL and T helper epitopes in vivo (Ishioka, G.Y., 1999; Thomson et al., 1998). The plasmids pDVERAC1, pDVERBC1, pDVERCC1 andpDVERAC2, pDVERBC2, pDVERCC2 were then mixed together to create, plasmid pool 1 and pool 2 respectively. Each plasmid pool collectively encodes one copy of the designed full-length HIV Savine.
Plasmids to generate recombinant Vaccinia viruses which express HIV Savine sequences were constructed by excising the various HIV Savine cassettes from the selected plasmid clones using BamHI/XhoI (cassette A) or BamHI/SalI (cassettes B and C) and cloned into the marker rescue plasmid, pTK7.5, cleaved with BamHI/SalI. These pTK7.5-derived plasmids were then used to generate recombinant Vaccinia viruses by marker rescue recombination using established protocols (Boyle et al., 1985) to generate VV-AC1, VV-AC2, VV-BC1, VV-BC2, VV-CC1 and VV-CC2 (FIG. 18b).
Two further DNA vaccine plasmids were constructed each encoding a version of the full length HIV Savine (FIG. 18c). Briefly, the two versions of cassette B were excised with )XhoI and cloned into the corresponding selected plasmid clones containing cassette A sequences that were cut with XhoI/SalI to generate pBSAB 1 and pBSAB2 respectively. The joined A/B cassettes in pBSAB 1 and pBSAB2 were excised with XbaI/XhoI and cloned into pDVCC1 and pDVCC2, respectively, and cleaved with XbaI/XhoI to generate pDVFL1 and pDVFL2. These were then further modified to contain an ER signal sequence using the same cloning strategy as outlined in FIG. 18a. 
Restimulation of HIV specific lymphocytes from HIV infected patients
The present inventors examined the capacity of the HIV Savine to restimulate HIV-specific polyclonal CTL responses from HIV-infected patients. PBMCs from three different patients were restimulated in vitro with two HIV Savine Vaccinia virus pools (Pool 1 included VV-AC1 andVV-BC1; Pool 2 included VV-AC2, VV-BC2 and VV-CC2) then used in CTL lysis assays against LCLs infected either with one of the Savine Vaccinia virus pools or Vaccinia viruses which express gag, env or pol. FIG. 19 clearly shows, that in all three assays, both HIV Savine viral pools restimulated HIV-specific CTL responses which could recognise targets expressing whole natural HIV antigens and not targets which were uninfected or infected with the control Vaccinia virus. Furthermore, in all three cases, both pools restimulated responses that recognized all three natural HIV antigens. This result suggests that the combined Savine constructs will provide broader immunological coverage than single antigen based vaccine approaches. The level of lysis in each case of targets infected with Savine viral pools was significantly higher than the lysis recorded for any other infected target. This probably reflects the combined CTL responses to gag, pol, and env plus other HIV antigens not analyzed here but whose sequences are also incorporated into the Savine constructs.
CTL recognition of each HIV antigen is largely controlled by each patient's HLA background hence the pattern of CTL lysis for whole HIV antigens is different in each patient. Interestingly, this CTL lysis pattern did not change when the second Savine Vaccinia virus pool was used for CTL restimulation. In these assays, therefore, the inventors were unable to demonstrate clear differences between pools 1 and 2, despite pool 1 lacking a Vaccinia virus expressing cassette CC1 and despite the many amino acid differences between the A and B cassettes in each pool (see table 1).
From the foregoing, the present inventors have developed a novel vaccine/therapeutic strategy. In one embodiment, pathogen or cancer protein sequences are systemically fragmented, reverse translated back into DNA, rearranged randomly then joined back together. The designed synthetic DNA sequence is then constructed using long oligonucleotides and can be transferred into a range of delivery vectors. The vaccine vectors used here were DNA vaccine plasmids and recombinant poxvirus vectors which have been previously shown to elicit strong T cell responses when used together in a ‘prime-boost’ protocol (Kent et al., 1997). An important advantage of scrambled antigen vaccines or ‘Savines’ is that the amount of starting sequence information for the design can be easily expanded to include the majority of the protein sequences from a pathogen or for cancer, thereby providing the maximum possible vaccine or therapy coverage for a given population.
An embodiment of the systematic fragmentation approach described herein was based on the size and processing requirements for T cell epitopes and was designed to cause maximal disruption to the structure and function of protein sequences. This fragmentation approach ensures that the maximum possible range of T cell epitopes will be present from any incorporated protein sequence without the protein being functional and able to compromise vaccine safety
Another important advantage of Savines is that consensus protein sequences can be used for their design. This feature is only applicable when the design needs to cater for pathogen or cancer antigens whose sequence varies considerably. HIV is a highly mutagenic virus, hence this feature was utilized extensively to design a vaccine which has the potential to cover not only field isolates of HIV but also the major HIV clades involved in the current HIV pandemic. To construct the HIV Savine, one set of losynthesizedleotides was synthesized, which included degenerate bases in such a way that 8 constructs are theoretically required for the vaccine to contain all combinations in any stretch of 9 amino acids. The inventors believe that this approach can be improved for the following reasons: 1) While degenerate bases should be theoretically equally represented, in practice some degenerate bases were biased towards one base or the other, leading to a lower than expected frequency of the designed mutations in the two full length HIV Savines which were constructed (see Table 1). 2) Only sequence combinations actually present in the HIV lade consensus sequences are required to get full clade coverage, hence the number of full length constructs needed could be reduced. To reduce the number of constructs however, separate sets of long oligonucleotides would have to be synthesized, significantly increasing the cost, time and effort required to generate a vaccine capable of such considerable vaccine coverage.
A significant problem during the construction of the HIV Savine synthetic DNA sequence was the incorporation of non-designed mutations. The most serious types of mutations were insertions, deletions or those giving rise to stop codons, all of which change the frame of the synthesized sequences and/or caused premature truncation of the Savine proteins. These types of mutation were removed during construction of the HIV Savines by sequencing multiple clones after subcassette and cassette construction and selecting functional clones. The major source of these non-designed mutations was in the long oligonucleotides used for Savine synthesis, despite their gel purification. This problem could be reduced by making the initial subcassettes smaller thereby reducing the possibility of corrupted oligonucleotides being incorporated into each subcassette clone. The second major cause of non-designed mutations was the large number of PCR cycles required for the PCR and ligation-mediated joining of the subcassettes. Including extra sequencing and clone selection steps during the subcassette joining process should help to reduce the frequency of non-designed mutations in future constructs. Finally, another method that could help reduce the frequency of such mutations at all stages is to use resolvase treatment. Resolvases are bacteriophage-encoded endonucleases which recognise disruptions to double stranded DNA and are primarily used by bacteriophages to resolve Holliday junctions (Mizuuchi, 1982; Youil et al., 1995). T7 endonuclease I has already been used by the present inventors in synthetic DNA constructions to recognise mutations and cleave corrupted dsDNA to allow gel purification of correct sequences. Cleavage of corrupted sequences occurs because after a simple denaturing and hybridization step mutated DNA hybridizes to correct DNA sequences and results in a mispairing of DNA bases which is able to be recognized by the resolvase. This method resulted in a 50% reduction in the frequency of errors. Further optimization of this method and the use of a thermostable version of this type of enzyme could further reduce the frequency of errors during long Savine construction.
Two pools of Vaccinia viruses expressing Savine cassettes were both shown to restimulate HIV-specific responses from three different patients infected with B lade HIV viruses. These results provide a clear indication that the HIV Savine should provide broad coverage of the population because each patient had a different HLA pattern yet both pools were able to restimulate HIV-specific CTL responses in all three patients against all three natural HIV proteins tested. Also, both pools were shown to restimulate virtually identical CTL patterns in all three patients. This result was unexpected because some responses should have been lost or gained due to the amino acid differences between the two pools and because Pool 1 is only capable of expressing 2⁄3 of the full length HIV Savine. There are two suggested reasons why the pattern of CTL lysis was not altered between the two viral pools. Firstly, the sequences in the Savine constructs are nearly all duplicated because the fragment sequences overlap. Hence the loss of a third of the Savine may not have excluded sufficient T cell epitopes for differences to be detected in only three patient samples against only three HIV proteins. Secondly, while mutations often destroy T cell epitopes, if they remain functional, then the CTL they generate frequently can recognise alternate epitope sequences. Taken together this finding indirectly suggests that combining only two Savine constructs may provide robust multiclade coverage. Further experiments are being carried out to directly examine the capacity of the HIV Savine to stimulate CTL generated by different strains of HIV virus. The capacity of the two HIV-1 Savine Vaccinia vector pools to stimulate CD4+ T cell HIV-1 specific responses from infected patients was also tested (FIG. 20). Both patients showed significant proliferation of CD4+ T cells although both pools did not show consistent patterns suggesting that the two pools may provide wider vaccine coverage than using either pool independently.
The present inventors have generated a novel vaccine strategy, which has been used to generate what the inventors believe to be the most effective HIV candidate vaccine to date. The inventors have used this vaccine to immunize naive mice. FIG. 21 shows conclusively that the HIV-1 Savine described above can generate a Gag and Nef CTL response in naive mice. It should be noted, however, that the Nef CTL epitope appeared to exist only in Pool 1 since it was not restimulated by Pool 2. This is further proof of the utility of combining HIV-1 Savine Pool 1 and Pool 2 components together to provide broader vaccine coverage.
The HIV-1 Savine Vaccinia vectors have also been used to restimulate in vivo HIV-1 responses in pre-immune M. nemestrina monkeys. These experiments (FIG. 22) showed, by INF-γ ELISPOT and CD69 expression on both CD4 and CD8 T cells, that the ability of the HIV-1 SAVINE to restimulate HIV-1 specific responses in vivo is equivalent or perhaps better than another HIV-1 candidate vaccine.
This is a generic strategy able to be applied to many other human infections or cancers where T-cell responses are considered to be important for protection or recovery. With this in mind the inventors have begun constructing Savines for melanoma, cervical cancer and Hepatitis C. In the case of melanoma, the majority of the currently identified melanoma antigens have been divided into two groups, one containing antigens associated with melanoma and one containing differentiation antigens from melanocytes, which are often upregulated in melanomas. Two Savine constructs are presently being constructed to cater for these two groups. The reason for making the distinction is that treatment of melanoma might first proceed using the Savine that incorporates fragments of melanoma specific antigens only. If this Savine fails to control some metastases then the less specific Savine containing the melanocyte-specific antigens can then be used. It is important to point out that other cancers also express many of the antigens specific to melanomas e.g., testicular and breast cancers. Hence the melanoma specific Savine may have therapeutic benefits for other cancers.
A small Savine is also being constructed for cervical cancer. This Savine will contain two antigens, E6 and E7, from two strains of human papilloma virus (HPV), HPV-16 and HPV-18, directly linked with causing the majority of cervical cancers worldwide. There is a large number of sequence differences in these two antigens between the two strains which would normally require two Savines to be constructed. However since this Savine is small, the antigen fragments from both strains are being scrambled together. While it is normally better for the Savine approach to include all or a majority of the antigens from a virus, in this case only E6 and E7 are expressed during viral latency or in cervical carcinomas. Hence in the interests of simplicity, the rest of the HPV genome will not be included although all HPV antigens would be desirable in a Savine against genital warts.
Two Savines have also been constructed for two strains of hepatitis C, a major cause of liver disease in the world. Hepatitis C is similar to HIV in the requirements for a vaccine or therapeutic. However, the major hepatitis C strains share significantly lower homology, 69-79%, with one another than do the various HIV clades. To cater for this the inventors have decided to construct two separate constructs to cater for the two major strains present in Australia, types 1a and 3a, which together cause approximately 80-95% of hepatitis C infections in this country. Both constructs will be approximately the same size as the HIV Savine but will be blended together into a single vaccine or therapy.
Overall it is believed that the Savine vaccine strategy is a generic technology likely to be applied to a wide range of human diseases. It is also believed that because it is not necessary to characterize each antigen, this technology will be actively applied to animal vaccines as well where research into vaccines or therapies is often inhibited by the lack of specific reagents, modest research budgets and poor returns on animal vaccines.
Example 2
Hepatitis C Savine
Synthetic immunomodulatory molecules have also been designed for treating Hepatitis C. In one example, the algorithm of FIG. 25 was applied to a consensus polyprotein sequence of Hepatitis C1a to facilitate its segmentation into overlapping segments (30 aa segments overlapping by 15aa), the rearrangement of these segments into a scrambled order and the output of Savine nucleic acid and amino acid sequences, as shown in FIG. 26. Exemplary DNA cassettes (A, B and C) are also shown in FIG. 26, which contain suitable restriction enzyme sites at their ends to facilitate their joining into a single expressible open reading frame.
Example 3
Melanonia Savine
The algorithm of FIG. 25 was also applied to melanocyte differentiation antigens (gp100, MART, TRP-1, Tyros, Trp-2, MC1R, MUC1F and MUC1R) and to melanoma specific antigens (BAGE, GAGE-1, gp100In4, MAGE-1, MAGE-3, PRAME, TRP2IN2, NYNSO1a, NYNSO1b and LAGE1), as shown in FIG. 27, to provide separate Savine nucleic acid and amino acid sequences for treating or preventing melanoma.
Example 4
Resolvase Repair Experiment
A resolvase can be used advantageously to repair errors in polynucleotides. The following procedure outlines resolvase repair of a synthetic 340 bp fragment in which DNA errors were common.
Method
The 340 bp fragment was PCR amplified and gel purified on a 4% agarose gel. After spin purifying, 10 ul of the eluate corresponding to approximately 100 ng was subjected to the resolvase repair treatment. The rest of the DNA sample was stored for later cloning as the untreated control.
2 μL of 10×PCR buffer, 2 μL of 20 MM MgCl2 and 6 μL of MilliQTM water (MQW) and Taq DNA polymerase were added to the 10 μL DNA sample. The mixture was subjected to the following thermal profile; 95° C. for 5 min, 65° C. for 30 min, cooled and held at 37° C. Five μL of 10×T7 endonuclease I buffer, 8 μL of 1/50 μL of T7endoI enzyme stock and 17 μL of MQW were added, mixed and incubated for 30 min. Loading buffer was added to the sample and the sample was electrophoresed on a 4% agarose gel. A faint band corresponding to the full length fragment was excised and subjected to 15 further cycles of PCR. The amplified fragment was agarose gel purified and, along with the untreated DNA sample, cloned into pBluescript. Eleven plasmid clones for each DNA sample were sequenced and the number and type of errors compared (see table) Buffers were as follows:
10×T7Endonuclease Buffer
2.5 ml 1M TRIS pH7.8, 0.5 ml 1M MgCl2, 25 μL 1 M DTT, 50 μL 10 mg/mL BSA, 2 mL MQW made up to a total of 5 mL.
T7 Endonuclease I Stock
Concentrated sample of enzyme prepared by, and obtained from, Jeff Babon (St Vincent's Hospital) was diluted 1/50 using the following dilution buffer: 50 μL 1 M TRIS pH7.8, 0.1 μL 1M EDTA pH8, 5 μL 100 mM glutathione, 50 μL 10 mg/mL BSA, 2.3 mL MQW, 2.5 mL glycerol made up to a total of 5 mL.
Results
The results are summarized in Tables 2 and 3.







TABLE 2







Total Errors










Untreated
Resolvase treated







A/T to G/C = 6
A/T to G/C = 1



G/C to A/T = 12
G/C to A/T = 7



A/T to deletion = 1
A/T to deletion = 1



G/C to deletion = 6
G/C to deletion = 3

















TABLE 3







Clone Summary










Untreated
Resolvase treated







6/11 contained deletions
3/11 contained deletions



9/11 contained mutations
7/11 contained mutations



2/11 correct
3/11 correct










DISCUSSION/CONCLUSION
While overall the number of correct clones obtained was not significantly different, there was a significant difference in the level of errors. This reduction in errors becomes more significant as greater numbers of long oligonucleotides are joined into the one construct i.e., increasing the difference between untreated versus treated samples in the chance of obtaining a correct clone. It is believed that combining another resolvase such as T4 endonuclease VII may further enhance repair or increase the bias against errors.
Importantly, this experiment was not optimized e.g., by using proofreading PCR enzymes or optimized conditions. Finally if the repair reaction is carried out during normal PCR, for example, by including a thermostable resolvase, it is believed that amplification of already damaged long oligonucleotides, and the normal accumulation of PCR induced errors, even using error reading polymerases during PCR, could be reduced significantly. The repair of damaged long oligonucleotides is particularly important for synthesis of long DNA fragment such as in Savines because, while the rate of long oligonucleotide damage is typically <5%, after joining 10 oligonucleotides, the error rate approaches 50%. This is true even using the best proofreading PCR enzymes because these enzymes do not verify the sequence integrity using correct oligonucleotide templates that exist as a significant majority (95%) in a joining reaction.
The disclosure of every patent, patent application, and publication cited herein is incorporated herein by reference in its entirety.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application
Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.
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## Claims

1. A synthetic polypeptide comprising at least three different segments of an HIV polypeptide, wherein the size of each segment is from about 20 to about 60 amino acids, wherein the segments are linked together in the same polypeptide chain but in a different relationship relative to their linkage in said HIV polypeptide, wherein the linkage of the segments impedes, abrogates or otherwise alters at least one function associated with said HIV polypeptide, wherein the sequence of any plurality of segments so linked is different from a sequence contained within said HIV polypeptide, wherein the sequence of at least one of said segments overlaps with the sequence of at least one other of said segments, wherein the sequence overlap is at least 4 contiguous amino acids in length, and wherein the synthetic polypeptide comprises at least 30% of the sequence of said HIV polypeptide.

2. The synthetic polypeptide of claim 1, further comprising a segment from at least one other polypeptide.

3. The synthetic polypeptide of claim 1, further comprising different segments from at least one other polypeptide, wherein the segments are linked together in a different relationship relative to their linkage in the at least one other polypeptide to impede, abrogate or otherwise alter at least one function associated with the at least one other polypeptide, and wherein the sequence of any plurality of segments so linked is different from a sequence contained within said at least one other polypeptide.

4. The synthetic polypeptide of claim 1, wherein the segments in said synthetic polypeptide are linked sequentially in a different order or arrangement relative to their linkage in said HIV polypeptide.

5. The synthetic polypeptide of claim 1, wherein the segments in said synthetic polypeptide are randomly rearranged relative to their linkage in said HIV polypeptide.

6. The synthetic polypeptide of claim 1, wherein the size of an individual segment is from about 20 to about 30 amino acids.

7. The synthetic polypeptide of claim 6, wherein the size of an individual segment is about 30 amino acids.

8. The synthetic polypeptide of claim 1, wherein the sequence overlap is contained at one or both ends of an individual segment.

9. The synthetic polypeptide of claim 1, wherein an optional spacer is interposed between some or all of the segments.

10. The synthetic polypeptide of claim 9, wherein the spacer alters proteolytic processing and/or presentation of adjacent segment(s).

11. The synthetic polypeptide of claim 10, wherein the spacer comprises at least one alanine residue.

12. The synthetic polypeptide of claim 10, wherein the spacer comprises at least one alanine residue.

13. The synthetic polypeptide of claim 1, wherein the at least one parent polypeptide is selected from the group consisting of env, gag, pol, vif, vpr, tat, rev, vpu and nef, or a combination thereof.

14. A method for producing the synthetic polynucleotide according to claim 1, said method comprising:
linking together in the same reading frame nucleic acid sequences encoding the plurality of different segments; and
expressing the nucleotide sequences to produce said synthetic polypeptide.

15. The method of claim 14, further comprising segmenting the sequence of an individual parent polypeptide into segments and linking said segments together in a different relationship relative to their linkage in the individual parent polypeptide sequence.

16. The method of claim 15, wherein the segments are randomly linked together.

17. The method of claim 14, further comprising reverse translating the sequence of an individual parent polypeptide or a segment thereof to provide a nucleic acid sequence encoding said parent polypeptide or said segment.

18. The method of claim 17, wherein an amino acid of an individual parent polypeptide sequence is reverse translated to provide a codon, which has higher translational efficiency than other synonymous codons in a cell of interest.

19. The method of claim 17, wherein an amino acid of an individual parent polypeptide sequence is reverse translated to provide a codon which, in the context of adjacent or local sequence elements, has a lower propensity of forming an undesirable sequence that is refractory to the execution of a task selected from the group consisting of cloning and sequencing.

20. The method of claim 17, wherein an amino acid of an individual parent polypeptide sequence is reverse translated to provide a codon which, in the context of adjacent or local sequence elements, has a lower propensity of forming an undesirable sequence selected from a palindromic sequence or a duplicated sequence, which is refractory to the execution of a task selected from the group consisting of cloning or sequencing.

21. The method of claim 14, further comprising linking a spacer oligonucleotide encoding at least one spacer residue between segment-encoding nucleic acids.

22. The method of claim 21, wherein spacer oligonucleotide encodes 2 to 3 spacer residues.

23. A composition comprising the synthetic polypeptide of claim 1, and a pharmaceutically acceptable carrier.

24. The composition of claim 23, further comprising an adjuvant.

25. A method for inducing an immune response comprising administering to a patient in need thereof an effective amount of an immunopotentiating agent selected from the group consisting of:
a) the synthetic polypeptide of claim 1,
b) a synthetic polynucleotide encoding the synthetic polypeptide of a) above,
c) a synthetic construct comprising a synthetic polynucleotide encoding the synthetic polypeptide of b) above wherein the synthetic polynucleotide is operably linked to a regulatory polynucleotide, and
d) a composition wherein said composition comprises an immunopotentiating agent selected from the group consisting of a) above, b) above and c) above, and a pharmaceutically acceptable carrier.

26. The method of claim 24, wherein the spacer residue is a neutral amino acid.

27. The method of claim 24, wherein the space residue is alanine.

28. The method of claim 25, wherein the immune response is directed against HIV.

29. The synthetic polypeptide of claim 1, wherein the overlap is about 15 amino acids.

30. The synthetic polypeptide of claim 1, wherein said segments are from HIV gag or env.

31. The synthetic peptide of claim 1, wherein said segments have a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 281, 283, 285, 287, 289, 291, 293, 295, 297, and 299.

32. The synthetic polypeptide of claim 1, wherein the sequence overlap is at least 7 contiguous amino acids in length.

33. The synthetic polypeptide of claim 1, wherein the sequence overlap is at least 10 contiguous amino acids in length.

34. The synthetic polypeptide of claim 1, wherein the sequence overlap is at least 15 contiguous amino acids in length.

35. The synthetic polypeptide of claim 1, wherein the sequence overlap is at least 20 contiguous amino acids in length.