AU2001259957B2 - Synthetic peptides and uses therefore - Google Patents

Synthetic peptides and uses therefore Download PDF

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AU2001259957B2
AU2001259957B2 AU2001259957A AU2001259957A AU2001259957B2 AU 2001259957 B2 AU2001259957 B2 AU 2001259957B2 AU 2001259957 A AU2001259957 A AU 2001259957A AU 2001259957 A AU2001259957 A AU 2001259957A AU 2001259957 B2 AU2001259957 B2 AU 2001259957B2
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polypeptide
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Ian Alistair Ramshaw
Scott Anthony Thomson
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Savine Therapeutics Pty Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Description

WO 01/90197 PCT/AU01/00622 Synthetic Peptides And Uses Therefore.
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 cellmediated immunity whereas B cells are involved in the generation of antibody-mediated immunity. The two most important types ofT cells involved in adaptive cellular immunity WO 01/90197 PCT/AU01/00622 -2are ap 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-y. 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 detenninants 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 epitopebased 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 epitopebased strategies though which have tried to solve this problem, 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 approaches using whole genes or proteins and approaches that mix multiple protein or gene sequences together. The proteins used by these strategies WO 01/90197 PCT/AU01/00622 -3however sometimes still function and therefore can compromise vaccine safety 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 irmnunisation 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 reemerge 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 (Decempber 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 WO 01/90197 PCT/AU01/00622 -4in 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; Panniani 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.
PCT/AU 1/00622 Received 07 February 2002 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 from one or more parent polypeptides, wherein the segments are linked together in a different relationship relative to their linkage in the or each parent polypeptide to impede, abrogate or otherwise alter at least one function associated therewith and wherein any pair of segments so linked does not result in a product whose structure and/or function is substantially similar to at least a portion of the or each parent polypeptide.
In one embodiment, the synthetic polypeptide consists essentially of different segments from a single parent polypeptide.
In an alternate embodiment, the synthetic polypeptide consists essentially of different segments from 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 the or each 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.
AMENDED SHEET
IPEA/AU
PCT/AUO 1/00622 Received 07 February 2002 -6- 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 nucleic acid sequences encoding a plurality of different segments from one or more parent polypeptides to form a synthetic polynucleotide whose sequence encodes said segments linked together in a different relationship relative to their linkage in the or each parent polypeptide to impede, abrogate or otherwise alter at least one function associated therewith and wherein any pair of segments so linked does not result in a product whose structure and/or function is substantially similar to at least a portion of the or each parent polypeptide.
Preferably, the method further comprises 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. In a preferred embodiment of this type, the segments are randomly linked together.
Suitably, the method further comprises 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. 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 a palindromic sequence or a duplicated sequence) that is refractory to the execution of a task cloning or sequencing).
AMENDED SHEET PCT/AU01/00622 Received 07 February 2002 -7- 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 the or each parent polypeptide; code that segments the sequence of the or each parent polypeptide into segments; code that links together said segments in a different relationship relative to their linkage in the or each parent polypeptide sequence to impede, abrogate or otherwise alter at least one function associated therewith and wherein any pair of segments so linked does not result in a product whose structure and/or function is substantially similar to at least a portion of the or each parent polypeptide; 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 segments the sequence of a respective parent polypeptide into segments; code that reverse translates the sequence of an individual segment to provide a nucleic acid sequence encoding said segment; code that receives as input the sequence of the or each parent polypeptide; code that segments the sequence of the or each parent polypeptide into segments; code that reverse translates the sequence of an individual segment to provide a nucleic acid sequence encoding said individual segment; 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 segments are linked together in a different relationship relative to their linkage in the or each parent polypeptide sequence to impede, abrogate or otherwise alter at least one function associated therewith and wherein any pair of segments so linked does not result in a product whose structure and/or function is substantially similar to at least a portion of the or each parent polypeptide; and AMENDED SHEET PCT/AUO 1/00622 Received 07 February 2002 -8a 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 machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said machine-readable data comprise the sequence of the or each parent polypeptide; a working memory for storing instructions for processing said machine-readable data; a central-processing unit coupled to said working memory and to said machinereadable data storage medium for processing said machine readable data to provide said synthetic polypeptide sequence, wherein said processing comprises segmenting the sequence of the or each parent polypeptide into segments and linking together said segments in a different relationship relative to their linkage in the or each parent polypeptide to impede, abrogate or otherwise alter at least one function associated therewith and wherein any pair of segments so linked does not result in a product whose structure and/or function is substantially similar'to at least a portion of the or each parent polypeptide; and an output hardware coupled to said central processing unit, for receiving said synthetic polypeptide sequence.
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 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; a working memory tor storing instructions for processing said machine-readable data; a central-processing unit coupled to said working memory and to said machinereadable data storage medium for processing said machine readable data to provide said AMEN ,ED SHEET 1PEE~DTi AU E 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 to 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.
According to a first aspect of the present invention, there is provided a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, wherein the segments in the synthetic polypeptide are linked sequentially in a different order or arrangement relative to their linkage in the parent polypeptide to impede or abrogate at least one function associated therewith and wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide.
According to a second aspect of the present invention, there is provided a synthetic polynucleotide encoding the synthetic polypeptide of the first aspect.
According to a third aspect of the present invention, there is provided a method for producing a synthetic polynucleotide, the method comprising: linking together in the same reading frame nucleic acid sequences encoding a plurality of different segments from a parent polypeptide to form synthetic polynucleotide whose sequence encodes the segments linked sequentially in a different order or arrangement relative to their linkage in the parent polypeptide to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide.
[R:\LI BZ\Vaughan\Proscutions\752598AU]752598A Uconsistories.RL2.doc:VN B 9a According to a fourth aspect of the present invention, there is provided a synthetic construct comprising the synthetic polynucleotide encoding a synthetic polypeptide as defined in the first aspect operably linked to a regulatory polynucleotide.
According to a fifth aspect of the present invention, there is provided an immunopotentiating composition, comprising an immunopotentiating agent selected from the synthetic polypeptide of the first aspect, the synthetic polynucleotide of the second aspect or the synthetic construct of the fourth aspect, together with a pharmaceutically acceptable carrier.
According to a sixth aspect of the present invention, there is provided use of an 0o immunopotentiating agent selected from the synthetic polypeptide of the first aspect, the synthetic polynucleotide of the second aspect, the synthetic construct of the fourth aspect or the composition of the fifth aspect in the manufacture of a medicament for modulating an immune response.
According to a seventh aspect of the present invention, there is provided a computer program product for designing the sequence of a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, the program product comprising: code that receives as input the sequence of the parent polypeptide; code that segments the sequence of the parent polypeptide into segments; code that links sequentially the segments in a different order or arrangement relative to their linkages in the parent polypeptide sequence to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide sequence; and a computer readable medium that stores the codes.
According to an eighth aspect of the present invention, there is provided a computer program product for designing the sequence of a synthetic polynucleotide that encodes a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, the computer program product comprising: code that receives as input the sequence of the parent polypeptide; code that segments the sequence of the parent polypeptide into segments; code that reverse translates the sequence of an individual segment to provide a nucleic acid sequence encoding the individual segment; code that links sequentially in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which the segments are linked sequentially in a different order or arrangement relative R:\II BZ\Vaughan\Prosecutions\752598AU]752598AU-consistories.RL2doc:VNB 9b to their linkage in the parent polypeptide sequence to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide sequence; and a computer readable medium that stores the codes.
According to a ninth aspect of the present invention, there is provided a computer for designing the sequence of a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, the computer comprising: a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein the machine-readable data comprise the sequence of the parent polypeptide; a working memory for storing instructions for processing the machinereadable data; a central-processing unit that processes the machine readable data coupled to the working memory and to the machine-readable data storage medium for processing the machine readable data to provide the synthetic polypeptide sequence, wherein the processing comprises segmenting the sequence of the parent polypeptide into segments and linking sequentially the segments in a different order or arrangement relative to their linkage in the parent polypeptide to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide; and an output hardware coupled to the central processing unit, for receiving the synthetic polypeptide sequence.
According to a tenth aspect of the present invention, there is provided a computer for designing the sequence of a synthetic polynucleotide that encodes a synthetic polypeptide comprising a plurality of different segment of one or more parent polypeptides, the computer comprising: a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein the machine-readable data comprise the sequence of the parent polypeptide; a working memory for storing instructions for processing the machinereadable data; a central-processing unit that processes the machine readable data coupled to the working memory and to the machine-readable data storage medium for processing the machine readable data to provide the synthetic polynucleotide sequence, wherein the processing comprises segmenting the sequence of the parent polypeptide into segments, RLI BZ\Vaughan\Prosecutions\752598AU]752598AU-consistories.RL2doc:VN3 9c I reverse translating the sequence of the segments to provide for each segment a Snucleic acid sequence encoding the segment and linking sequentially in the same reading Sframe each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which the segments are linked sequentially in a different order or arrangement relative to their linkages in the parent polypeptide to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide; and an output hardware coupled to the central processing unit, for receiving the t synthetic polynucleotide sequence.
According to an eleventh aspect of the present invention, there is provided a Smethod for modulating an immune response directed against a pathogen or a cancer, wherein said method comprises administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the synthetic polypeptide of the first aspect, the synthetic polynucleotide of the second aspect, the synthetic construct of the fourth aspect or the composition of the fifth aspect.
According to a twelfth aspect of the present invention, there is provided a method for treatment and/or prophylaxis of a disease or condition, wherein said method comprises administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the synthetic polypeptide of the first aspect, the synthetic polynucleotide of the second aspect, the synthetic construct of the fourth aspect or the composition of the fifth aspect.
IR:\I-IBZ\Vaughan\Prosecutions\752598AU]752598AU-consistories. RL2.doc:VNB WO 01/90197 PCT/AU01/00622 BRIEF DESCRIPTION OF THE DRAWINGS Figure 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.
Figure 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).
Figure 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, New Mexico, Pub LAUR 98-485.
Figure 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 clade 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, New Mexico, Pub LAUR98-485.
Figure 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, New Mexico, Pub LAUR98-485.
WO 01/90197 PCT/AU01/00622 -11- Figure 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 clade 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, New Mexico, Pub LAUR 98-485.
Figure 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 clade 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, New Mexico, Pub LAUR 98-485.
Figure 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 clade 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, New Mexico, Pub LAUR 98-485.
Figure 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 clade 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, New Mexico, Pub LAUR 98-485.
Figure 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 PCT/AU01/00622 Received 07 February 2002 -12consensus 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, New Mexico, Pub LAUR 98-485.
Figure 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, New Mexico, Pub LAUR 98-485.
Figure 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 segment compared to the corresponding overlap region (see tat gene) Figure 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.
Figure 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 AMEdDEFD SHEET PAiALAU PCT/AU01/00622 Received 07 February 2002 -13added 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 Xhol and cloned into Xhol arms of the C cassette; full length construct is excisable with either XbalBamHI at the 5' end or BglII at the 3' end. Options for excising cassettes: A) XbalBamHI at the 5' end, BgllIXhoI at the 3' end; B) XbaIlBamHI at the 5' end, BglIIWSalI at the 3' end; C) XbaI/BamHI at the 5' end, BglllSall at the 3' end. Cleaving plasmid vectors: pDNAVacc is cleavable with XbaIIXhoI (DNA vaccination); pBCB07 or pTK7.5 vectors are cleavable with BamHVSalI (Recombinant Vaccinia); pAvipox vector pAF09 is cleavable with BamHI/SaI (Recombinant Avipox).
Figure 15 shows the full length DNA (17253 bp) and protein sequence (5742 aas) of the HIV Savine construct. Segment boundaries are shown, together with the position of each segment in each designed HIV protein, segment number (in brackets), spacer residues (two alanine residues) and which segment 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 Figure 16 depicts the layout and position of oligonucleotides in the designed DNA sequence for subcassette Al. The sequences which anneal to the short amplification oligonucleotides are indicated by hatched boxes and the position of oligonucleotide overlap regions are dark shaded.
Figure 17: Panel depicts the stepwise asymmetric PCR of the two halves of subcassette Al (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 shows the stepwise ligation-mediated joining and PCR amplification of each cassette as indicated.
DNA standards in lane 1 are SPP1 cut with EcoRI.
Figure 18: Panel shows summary of the construction of the DNA vaccine plasmids that express one HIV Savine cassette. Panel shows a summary of the AMENDED SHEET
IPRAIAU
WO 01/90197 PCT/AU01/00622 -14construction of the plasmids used for marker rescue recombination to generate Vaccinia viruses expressing one HIV Savine cassette. Panel shows a summary of the construction of the DNA vaccine plasmids which each express a version of the full-length HIV Savine cDNA Figure 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 andVV-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 (VVpol), VV- HIV Savine pools 1 (light bars) or 2 (dark bars) or a control Vaccinia virus (VV- Lac) and the amount of 5 Cr released used to determine percent specific lysis. K562 cells were used to determine the level of NK cell-mediated killing in their stimulated culture.
Figure 20 is a diagrammatic representation showing CD4+ proliferation of PBMCs from HIV-1 infected patients restimulated with either Pooll or Pool2 of the HIV-1 Savine. Briefly PBMCs were stained with CFSE and culture for 6 days with or without VVs encoding either pooll or pool2 of the HIV-1 Savine. Restimulated Cells were then labelled with antibodies and analysed by FACS.
Figure 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 Figure 18a (100 /g total DNA was given as 50 /ig/leg One week later Poxviruses (lx10 7 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 5Cr release cytotoxicity assay against targets infected with CTRVV, VV-pools or VV expressing the natural antigens from HIV-1.
Figure 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 WO 01/90197 PCT/AU01/00622 twice with FPV-gag-pol Day 0 is 3 weeks after first FPV-gag-pol immunisation. A) IFN-y detection by ELISPOT of whole blood (0.5 mL, venous blood heparinanticoagulated) stimulated with Aldrithiol-2 inactivated whole HIV-1 (20 hours, Plasma samples were then centrifuged (1000xg) 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).
Figure 23 shows a diagram of a system used to carry out the instructions encoded by the storage medium of Figures 28 and 29.
Figure 24 depicts a flow diagram showing an embodiment of a method for designing synthetic polynucleotide and synthetic polypeptides of the invention.
Figure 25 shows an algorithm, which inter alia utilises the steps of the method shown in Figure 24.
Figure 26 shows an example of applying the algorithm of Figure 25 to an input consensus polyprotein sequence of Hepatitis C la 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.
Figure 27 illustrates an example of applying the algorithm of Figure 25 to input consensus melanocyte differentiation antigens (gpl00, MART, TRP-1, Tyros, Trp-2, MC1R, MUC1F and MUC1R) and to consensus melanoma specific antigens (BAGE, GAGE-1, gpl00In4, MAGE-1, MAGE-3, PRAME, TRP21N2, NYNSOla, NYNSOlb 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.
Figure 28 shows a cross section of a magnetic storage medium.
Figure 29 shows a cross section of an optically readable data storage medium.
WO 01/90197 PCT/AU01/00622 -16- Figure 30 shows six HIIV Savine cassette sequences (Al [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]). Al, 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 WO 01/90197 PCT/AU01/00622 -17- BRIEF DESCRIPTION OF THE SEQUENCES: SUMMARY TABLE TABLE A SEQUENCE ID SEQUENCE LENGTH Y^^iIJEBv SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 GAG consensus polypeptide POL consensus polypeptide VIF consensus polypeptide VPR consensus polypeptide TAT consensus polypeptide REV consensus polypeptide VPU consensus polypeptide ENV consensus polypeptide NEF consensus polypeptide GAG segment 1 Polypeptide encoded by SEQ ID NO: 10 GAG segment 2 Polypeptide encoded by SEQ ID NO: 12 GAG segment 3 Polypeptide encoded by SEQ ID NO: 14 GAG segment 4 Polypeptide encoded by SEQ ID NO: 16 GAG segment 5 Polypeptide encoded by SEQ ID NO: 18 GAG segment 6 Polypeptide encoded by SEQ ID NO: 20 GAG segment 7 499 aa 995 aa 192 aa 96 aa 102 aa 123 aa 81 aa 651 aa 206 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 WO 0190197PCT/AU01/00622 18s- SEQUENCE ID SE QUENTCE LENG(F SEQ ED NO: 23 SEQ ID NO: 24 SIEQ ID NO: 25 SBQ ID NO: 26 SEQ ED NO: 27 SEQ DD NO: 28 SEQ DD NO: 29 SEQ ED NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 3 8 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NSO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 SEQ ED NO: 45 SEQ IID NO: 46 Polypeptide encoded by SEQ ID NO: 22 GAG segment 8 Polypeptide encoded by SEQ ED NO: 24 GAG segment 9 Polypeptide encoded by SEQ lID NO: 26 GAG segment 10 Polypeptide encoded by SEQ ID NO: 28 GAG segment 11 Polyp eptide encoded by SEQ liD NO: 30 GAG segment 12 Polypeptide encoded by SEQ ID NO: 32 GAG segment 13 Polypeptide encoded by SEQ DD NO: 34 GAG segment 14 Polyp eptide encoded by SEQ ID NO: 36 GAG segment 15 Polypeptide, encoded by SEQ ID NO: 38 GAG segment 16 Polypeptide encoded by SEQ ED NO: 40 GAG segment 17 Polypeptide encoded by SEQ ID NO: 42 GAG segment 18 Polypeptide encoded by SEQ ID NO: 44 GAG segment 19 30 aa 90 nts 30 an 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -19- SEQUEN'CE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 59 SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 68 SEQ ID NO: 69 SEQ ID NO: 70 Polypeptide encoded by SEQ ID NO: 46 GAG segment 20 Polypeptide encoded by SEQ ID NO: 48 GAG segment 21 Polypeptide encoded by SEQ ID NO: 50 GAG segment 22 Polypeptide encoded by SEQ ID NO: 52 GAG segment 23 Polypeptide encoded by SEQ ID NO: 54 GAG segment 24 Polypeptide encoded by SEQ ID NO: 56 GAG segment 25 Polypeptide encoded by SEQ ID NO: 58 GAG segment 26 Polypeptide encoded by SEQ ID NO: 60 GAG segment 27 Polypeptide encoded by SEQ ID NO: 62 GAG segment 28 Polypeptide encoded by SEQ ID NO: 64 GAG segment 29 Polypeptide encoded by SEQ ID NO: 66 GAG segment 30 Polypeptide encoded by SEQ ID NO: 68 GAG segment 31 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 SSEQUENE D SEUENCE LE .NGTH SEQ ID NO: 71 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 74 SEQ ID NO: 75 SEQ ID NO: 76 SEQ ID NO: 77 SEQ ID NO: 78 SEQ ID NO: 79 SEQ ID NO: 80 SEQ ID NO: 81 SEQ ID NO: 82 SEQ ID NO: 83 SEQ ID NO: 84 SEQ ID NO: 85 SEQ ID NO: 86 SEQ ID NO: 87 SEQ ID NO: 88 SEQ ID NO: 89 SEQ ID NO: 90 SEQ ID NO: 91 SEQ ID NO: 92 SEQ ID NO: 93 SEQ ID NO: 94 Polypeptide encoded by SEQ ID NO: 70 GAG segment 32 Polypeptide encoded by SEQ ID NO: 72 GAG segment 33 Polypeptide encoded by SEQ ID NO: 74 POL segment 1 Polypeptide encoded by SEQ ID NO: 76 POL segment 2 Polypeptide encoded by SEQ ID NO: 78 POL segment 3 Polypeptide encoded by SEQ ID NO: 80 POL segment 4 Polypeptide encoded by SEQ ID NO: 82 POL segment 5 Polypeptide encoded by SEQ ID NO: 84 POL segment 6 Polypeptide encoded by SEQ ID NO: 86 POL segment 7 Polypeptide encoded by SEQ ID NO: 88 POL segment 8 Polypeptide encoded by SEQ ID NO: 90 POL segment 9 Polypeptide encoded by SEQ ID NO: 92 POL segment 10 30 aa 90 nts 30 aa 57 nts 19 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 WO 0190197PCT/AU01/00622 -21- NEQUMBE ID STQUENCEjLNGJ SEQ DD NO: 95 SEQ ID NO: 96 SEQ ID NO: 97 SEQ ID NO: 98 SEQ 11D NO: 99 SIEQ ID NO: 100 SIEQ 1DNO: 10 1 SEQ ID NO: 102 SEQ ID NO: 103 SEQ ID NO: 104 SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 107 SEQ ID NO: 108 SEQ IDNO0: 109 SEQ lID NO: 110 SEQIDNO: 111 SEQ ID NO: 112 SEQ D NO: 113 SEQ ED NO: 114 SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 117 SEQ ID NO: 118 Polypeptide encoded by SEQ ID NO: 94 POL segment I11 Polypeptide encoded by SEQ DD NO: 96 POL segment 12 Polypeptide encoded by SEQ ED NO: 98 POL segment 13 Polypeptide encoded by SEQ ID NO: 100 POL segment 14 Polypeptide encoded by SEQ lID NO: 102 POL segment 15 Polypeptide encoded by SEQ HiD NO: 104 POL segment 16 Polypeptide encoded by SEQ EiD NO: 106 POL segment 17 Polypeptide encoded by SEQ ID NO: 108 POL segment 18 Polypeptide encoded by SEQ I1D NO: 110 POL segment 19 Polypeptide encoded by SEQ ID NO: 112 POL segment 20 Polypeptide encoded by SEQ ID NO: 114 POL segment 21 Polypeptide, encoded by SEQ IDD NO: 116 POL segment 22 30 aa 90 uts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -22- SEQUENCE ID SEQUENACE LENGTH N_ __ER I I SEQ ID NO: 119 SEQ ID NO: 120 SEQ ID NO: 121 SEQ ID NO: 122 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO: 126 SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO: 129 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132 SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 SEQ ID NO: 137 SEQ ID NO: 138 SEQ ID NO: 139 SEQ ID NO: 140 SEQ ID NO: 141 SEQ ID NO: 142 Polypeptide encoded by SEQ ID NO: 118 POL segment 23 Polypeptide encoded by SEQ ID NO: 120 POL segment 24 Polypeptide encoded by SEQ ID NO: 122 POL segment 25 Polypeptide encoded by SEQ ID NO: 124 POL segment 26 Polypeptide encoded by SEQ ID NO: 126 POL segment 27 Polypeptide encoded by SEQ ID NO: 128 POL segment 28 Polypeptide encoded by SEQ ID NO: 130 POL segment 29 Polypeptide encoded by SEQ ID NO: 132 POL segment 30 Polypeptide encoded by SEQ ID NO: 134 POL segment 31 Polypeptide encoded by SEQ ID NO: 136 POL segment 32 Polypeptide encoded by SEQ ID NO: 138 POL segment 33 Polypeptide encoded by SEQ ID NO: 140 POL segment 34 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -23- SEQUENCE ID SEQUENCE LENGTH SEQ ID NO: 143 SEQ ID NO: 144 SEQ ID NO: 145 SEQ ID NO: 146 SEQ ID NO: 147 SEQ ID NO: 148 SEQ ID NO: 149 SEQ ID NO: 150 SEQ ID NO: 151 SEQ ID NO: 152 SEQ ID NO: 153 SEQ ID NO: 154 SEQ ID NO: 155 SEQ ID NO: 156 SEQ ID NO: 157 SEQ ID NO: 158 SEQ IDNO: 159 SEQ ID NO: 160 SEQ ID NO: 161 SEQ ID NO: 162 SEQ ID NO: 163 SEQ ID NO: 164 SEQ ID NO: 165 SEQ ID NO: 166 Polypeptide encoded by SEQ ID NO: 142 POL segment 35 Polypeptide encoded by SEQ ID NO: 144 POL segment 36 Polypeptide encoded by SEQ ID NO: 146 POL segment 37 Polypeptide encoded by SEQ ID NO: 148 POL segment 38 Polypeptide encoded by SEQ ID NO: 150 POL segment 39 Polypeptide encoded by SEQ ID NO: 152 POL segment 40 Polypeptide encoded by SEQ ID NO: 154 POL segment 41 Polypeptide encoded by SEQ ID NO: 156 POL segment 42 Polypeptide encoded by SEQ ID NO: 158 POL segment 43 Polypeptide encoded by SEQ ID NO: 160 POL segment 44 Polypeptide encoded by SEQ ID NO: 162 POL segment 45 Polypeptide encoded by SEQ ID NO: 164 POL segment 46 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -24- SEQUENCE ID IEEC T.1 NUM:BER' ,SUN
LN
SEQ ID NO: 167 SEQ ID NO: 168 SEQ ID NO: 169 SEQ ID NO: 170 SEQ ID NO: 171 SEQ ID NO: 172 SEQ ID NO: 173 SEQ ID NO: 174 SEQ ID NO: 175 SEQ ID NO: 176 SEQ ID NO: 177 SEQ ID NO: 178 SEQ ID NO: 179 SEQ ID NO: 180 SEQ ID NO: 181 SEQ ID NO: 182 SEQ ID NO: 183 SEQ ID NO: 184 SEQ ID NO: 185 SEQ ID NO: 186 SEQ ID NO: 187 SEQ ID NO: 188 SEQ ID NO: 189 SEQ ID NO: 190 Polypeptide encoded by SEQ ID NO: 166 POL segment 47 Polypeptide encoded by SEQ ID NO: 168 POL segment 48 Polypeptide encoded by SEQ ID NO: 170 POL segment 49 Polypeptide encoded by SEQ ID NO: 172 POL segment 50 Polypeptide encoded by SEQ ID NO: 174 POL segment 51 Polypeptide encoded by SEQ ID NO: 176 POL segment 52 Polypeptide encoded by SEQ ID NO: 178 POL segment 53 Polypeptide encoded by SEQ ID NO: 180 POL segment 54 Polypeptide encoded by SEQ ID NO: 182 POL segment 55 Polypeptide encoded by SEQ ID NO: 184 POL segment 56 Polypeptide encoded by SEQ ID NO: 186 POL segment 57 Polypeptide encoded by SEQ ID NO: 188 POL segment 58 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts U I WO 01/90197 PCT/AU01/00622 SEQUENCE ID SEQUENCE r LENGTH NUMBER I SEQ ID NO: 191 SEQ ID NO: 192 SEQ ID NO: 193 SEQ ID NO: 194 SEQ ID NO: 195 SEQ ID NO: 196 SEQ ID NO: 197 SEQ ID NO: 198 SEQ ID NO: 199 SEQ ID NO: 200 SEQ ID NO: 201 SEQ ID NO: 202 SEQ ID NO: 203 SEQ ID NO: 204 SEQ ID NO: 205 SEQ ID NO: 206 SEQ ID NO: 207 SEQ ID NO: 208 SEQ ID NO: 209 SEQ ID NO: 210 SEQ ID NO: 211 SEQ ID NO: 212 SEQ ID NO: 213 SEQ ID NO: 214 Polypeptide encoded by SEQ ID NO: 190 POL segment 59 Polypeptide encoded by SEQ ID NO: 192 POL segment 60 Polypeptide encoded by SEQ ID NO: 194 POL segment 61 Polypeptide encoded by SEQ ID NO: 196 POL segment 62 Polypeptide encoded by SEQ ID NO: 198 POL segment 63 Polypeptide encoded by SEQ ID NO: 200 POL segment 64 Polypeptide encoded by SEQ ID NO: 202 POL segment 65 Polypeptide encoded by SEQ ID NO: 204 POL segment 66 Polypeptide encoded by SEQ ID NO: 206 VIF segment 1 Polypeptide encoded by SEQ ID NO: 208 VIF segment 2 Polypeptide encoded by SEQ ID NO: 210 VIF segment 3 Polypeptide encoded by SEQ ID NO: 212 VIF segment 4 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 60 nts 20 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -26- SEQENCE ID SEQUENCE- LENGTH
NUMBER
SEQ ID NO: 215 SEQ ID NO: 216 SEQ ID NO: 217 SEQ ID NO: 218 SEQ ID NO: 219 SEQ ID NO: 220 SEQ ID NO: 221 SEQ ID NO: 222 SEQ ID NO: 223 SEQ ID NO: 224 SEQ ID NO: 225 SEQ ID NO: 226 SEQ ID NO: 227 SEQ ID NO: 228 SEQ ID NO: 229 SEQ ID NO: 230 SEQ ID NO: 231 SEQ ID NO: 232 SEQ ID NO: 233 SEQ ID NO: 234 SEQ ID NO: 235 SEQ ID NO: 236 SEQ ID NO: 237 SEQ ID NO: 238 Polypeptide encoded by SEQ ID NO: 214 VIF segment 5 Polypeptide encoded by SEQ ID NO: 216 VIF segment 6 Polypeptide encoded by SEQ ID NO: 218 VIF segment 7 Polypeptide encoded by SEQ ID NO: 220 VIF segment 8 Polypeptide encoded by SEQ ID NO: 222 VIF segment 9 Polypeptide encoded by SEQ ID NO: 224 VIF segment 10 Polypeptide encoded by SEQ ID NO: 226 VIF segment 11 Polypeptide encoded by SEQ ID NO: 228 VIF segment 12 Polypeptide encoded by SEQ ID NO: 230 VPR segment 1 Polypeptide encoded by SEQ ID NO: 232 VPR segment 2 Polypeptide encoded by SEQ ID NO: 234 VPR segment 3 Polypeptide encoded by SEQ ID NO: 236 VPR segment 4 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 81 nts 27 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -27 SEQUENCE ID SEQUENCE LENGT 'NiA SEQ ID NO: 239 SEQ ID NO: 240 SEQ ID NO: 241 SEQ ID NO: 242 SEQ ID NO: 243 SEQ ID NO: 244 SEQ ID NO: 245 SEQ ID NO: 246 SEQ ID NO: 247 SEQ ID NO: 248 SEQ ID NO: 249 SEQ ID NO: 250 SEQ ID NO: 251 SEQ ID NO: 252 SEQ ID NO: 253 SEQ ID NO: 254 SEQ ID NO: 255 SEQ ID NO: 256 SEQ ID NO: 257 SEQ ID NO: 258 SEQ ID NO: 259 SEQ ID NO: 260 SEQ ID NO: 261 SEQ ID NO: 262 Polypeptide encoded by SEQ ID NO: 238 VPR segment 5 Polypeptide encoded by SEQ ID NO: 240 VPR segment 6 Polypeptide encoded by SEQ ID NO: 242 TAT segment 1 Polypeptide encoded by SEQ ID NO: 244 TAT segment 2 Polypeptide encoded by SEQ ID NO: 246 TAT segment 3 Polypeptide encoded by SEQ ID NO: 248 TAT segment 4 Polypeptide encoded by SEQ ID NO: 250 TAT segment 5 Polypeptide encoded by SEQ ID NO: 252 TAT segment 6 Polypeptide encoded by SEQ ID NO: 254 REV segment 1 Polypeptide encoded by SEQ ID NO: 256 REV segment 2 Polypeptide encoded by SEQ ID NO: 258 REV segment 3 Polypeptide encoded by SEQ ID NO: 260 REV segment 4 30 aa 90 nts 30 aa 63 nts 21 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 81 nts 27 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts j I WO 01/90197 WO 0190197PCT/AU01/00622 -28 SEQUENCE ID SEQ UENCE LXT
N/UMBERP
SEQ ID NO: 263 SEQ ID NO: 264 SEQ ID NO: 265 SEQ ID NO: 266 SEQ ED NO: 267 SEQ ED NO: 268 SEQ ID NO: 269 SEQ EID NO: 270 SEQ ID NO: 271 SEQ DD NO: 272 SEQ ID NO: 273 SEQ ID NO: 274 SEQ ED NO: 275 SEQ ID NO: 276 SEQ ID NO: 277 SEQ ID NO: 278 SEQ ID NO: 279 SEQ ID NO: 280 SEQ H) NO: 281 SEQ H) NO: 282 SEQ ID NO: 283 SEQ ID NO: 284 SEQ ED NO: 285 SEQ ID NO: 286 Polypeptide encoded by SEQ ID NO: 262 REV segment 5 Polypeptide encoded by SEQ ED NO: 264 REV segment 6 Polyp eptide encoded by SEQ ID NO: 266 REV segment 7 Polypeptide encoded by SEQ ID) NO: 268 REV segment 8 Polyp eptide encoded by SEQ ID NO: 270 VPU segment 1 Polypeptide, encoded by SEQ H) NO: 272 VPU segment 2 Polyp eptide encoded by SEQ ID NO: 274 VPU segment 3 Polypeptide encoded by SEQ lID NO: 276 VPU segment 4 Polypeptide encoded by SEQ IID NO: 278 VPU segment 5 Polypeptide encoded by SEQ ED NO: 280 ENV segment 1 Polypeptide encoded by SEQ ED NO: 282 ENV segment 2 Polyp eptide encoded by SEQ ID NO: 284 ENV segment 3 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 54 nts 18 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 63 nts 21 aa 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -29- SEUMERCE ID SEQUENCE LENGTH NUMiER |M SEQ ID NO: 287 SEQ ID NO: 288 SEQ ID NO: 289 SEQ ID NO: 290 SEQ ID NO: 291 SEQ ID NO: 292 SEQ ID NO: 293 SEQ ID NO: 294 SEQ ID NO: 295 SEQ ID NO: 296 SEQ ID NO: 297 SEQ ID NO: 298 SEQ ID NO: 299 SEQ ID NO: 300 SEQ ID NO: 301 SEQ ID NO: 302 SEQ ID NO: 303 SEQ ID NO: 304 SEQ ID NO: 305 SEQ ID NO: 306 SEQ ID NO: 307 SEQ ID NO: 308 SEQ ID NO: 309 SEQ ID NO: 310 Polypeptide encoded by SEQ ID NO: 286 ENV segment 4 Polypeptide encoded by SEQ ID NO: 288 ENV segment 5 Polypeptide encoded by SEQ ID NO: 290 ENV segment 6 Polypeptide encoded by SEQ ID NO: 292 ENV segment 7 Polypeptide encoded by SEQ ID NO: 294 ENV segment 8 Polypeptide encoded by SEQ ID NO: 296 ENV segment 9 Polypeptide encoded by SEQ ID NO: 298 GAP A segment 1 Polypeptide encoded by SEQ ID NO: 300 GAP A segment 2 Polypeptide encoded by SEQ ID NO: 302 GAP A segment 3 Polypeptide encoded by SEQ ID NO: 304 GAP A segment 4 Polypeptide encoded by SEQ ID NO: 306 GAP A segment 5 Polypeptide encoded by SEQ ID NO: 308 GAP A segment 6 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 57 nts 19 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 SEQUENCE ID SEQUENCE LENGTH NUMBER SEQ ID NO: 311 SEQ ID NO: 312 SEQ ID NO: 313 SEQ ID NO: 314 SEQ ID NO: 315 SEQ ID NO: 316 SEQ ID NO: 317 SEQ ID NO: 318 SEQ ID NO: 319 SEQ ID NO: 320 SEQ ID NO: 321 SEQ ID NO: 322 SEQ ID NO: 323 SEQ ID NO: 324 SEQ ID NO: 325 SEQ ID NO: 326 SEQ ID NO: 327 SEQ ID NO: 328 SEQ ID NO: 329 SEQ ID NO: 330 SEQ ID NO: 331 SEQ ID NO: 332 SEQ ID NO: 333 Polypeptide encoded by SEQ ID NO: 310 GAP A segment 7 Polypeptide encoded by SEQ ID NO: 312 GAP B segment 1 Polypeptide encoded by SEQ ID NO: 314 GAP B segment 2 Polypeptide encoded by SEQ ID NO: 316 GAP B segment 3 Polypeptide encoded by SEQ ID NO: 318 GAP B segment 4 Polypeptide encoded by SEQ ID NO: 320 GAP B segment 5 Polypeptide encoded by SEQ ID NO: 322 GAP B segment 6 Polypeptide encoded by SEQ ID NO: 324 GAP B segment 7 Polypeptide encoded by SEQ ID NO: 326 GAP B segment 8 Polypeptide encoded by SEQ ID NO: 328 GAP B segment 9 Polypeptide encoded by SEQ ID NO: 330 GAP B segment 10 Polypeptide encoded by SEQ ID NO: 332 GAP B segment 11 30 aa 75 nts 25 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts SEQ ID NO: 334 WO 01/90197 PCT/AU01/00622 -31- SEQUENCE ID SEQUENCE LE NGTH
'NUMBER
SEQ ID NO: 335 SEQ ID NO: 336 SEQ ID NO: 337 SEQ ID NO: 338 SEQ ID NO: 339 SEQ ID NO: 340 SEQ ID NO: 341 SEQ ID NO: 342 SEQ ID NO: 343 SEQ ID NO: 344 SEQ ID NO: 345 SEQ ID NO: 346 SEQ ID NO: 347 SEQ ID NO: 348 SEQ ID NO: 349 SEQ ID NO: 350 SEQ ID NO: 351 SEQ ID NO: 352 SEQ ID NO: 353 SEQ ID NO: 354 SEQ ID NO: 355 SEQ ID NO: 356 SEQ ID NO: 357 SEQ ID NO: 358 Polypeptide encoded by SEQ ID NO: 334 GAP B segment 12 Polypeptide encoded by SEQ ID NO: 336 GAP B segment 13 Polypeptide encoded by SEQ ID NO: 338 GAP B segment 14 Polypeptide encoded by SEQ ID NO: 340 GAP B segment 15 Polypeptide encoded by SEQ ID NO: 342 GAP B segment 16 Polypeptide encoded by SEQ ID NO: 344 GAP B segment 17 Polypeptide encoded by SEQ ID NO: 346 GAP B segment 18 Polypeptide encoded by SEQ ID NO: 348 GAP B segment 19 Polypeptide encoded by SEQ ID NO: 350 GAP B segment 20 Polypeptide encoded by SEQ ID NO: 352 GAP B segment 21 Polypeptide encoded by SEQ ID NO: 354 GAP B segment 22 Polypeptide encoded by SEQ ID NO: 356 GAP B segment 23 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -32- SEQUE ID NO: 359
NUMBER
SEQ ID NO: 360 SEQ ID NO: 35961 SEQ ID NO: 360 SEQ ID NO: 361 SEQ ID NO: 362 SEQ ID NO: 363 SEQ ID NO: 364 SEQ ID NO: 365 SEQ ID NO: 366 SEQ ID NO: 367 SEQ ID NO: 36870 SEQ ID NO: 36971 SEQ ID NO: 370 SEQ ID NO: 371 SEQ ID NO: 372 SEQ ID NO: 373 SEQ ID NO: 374 SEQ ID NO: 375 SEQ ID NO: 376 SEQ ID NO: 377 SEQ ID NO: 378 SEQ ID NO: 37981 SEQ ID NO: 380 SEQ ID NO: 381 SEQ ID NO: 382 EQUENCE LENGTH Polypeptide encoded by SEQ ID NO: 358 GAP B segment 24 Polypeptide encoded by SEQ ID NO: 360 GAP B segment 25 Polypeptide encoded by SEQ ID NO: 362 GAP B segment 26 Polypeptide encoded by SEQ ID NO: 364 NEF segment 1 Polypeptide encoded by SEQ ID NO: 366 NEF segment 2 Polypeptide encoded by SEQ ID NO: 368 NEF segment 3 Polypeptide encoded by SEQ ID NO: 370 NEF segment 4 Polypeptide encoded by SEQ ID NO: 372 NEF segment 5 Polypeptide encoded by SEQ ID NO: 374 NEF segment 6 Polypeptide encoded by SEQ ID NO: 376 NEF segment 7 Polypeptide encoded by SEQ ID NO: 378 NEF segment 8 Polypeptide encoded by SEQ ID NO: 380 NEF segment 9 30 aa 90 nts 30 aa 90 nts 30 aa 66 nts 22 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts WO 01/90197 PCT/AU01/00622 -33- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 383 SEQ ID NO: 384 SEQ ID NO: 385 SEQ ID NO: 386 SEQ ID NO: 387 SEQ ID NO: 388 SEQ ID NO: 389 SEQ ID NO: 390 SEQ ID NO: 391 SEQ ID NO: 392 SEQ ID NO: 393 SEQ ID NO: 394 SEQ ID NO: 395 SEQ ID NO: 396 SEQ ID NO: 397 SEQ ID NO: 398 SEQ ID NO: 399 SEQ ID NO: 400 SEQ ID NO: 401 SEQ ID NO: 402 SEQ ID NO: 403 SEQ ID NO: 404 SEQ ID NO: 405 SEQ ID NO: 406 Polypeptide encoded by SEQ ID NO: 382 NEF segment 10 Polypeptide encoded by SEQ ID NO: 384 NEF segment 11 Polypeptide encoded by SEQ ID NO: 386 NEF segment 12 Polypeptide encoded by SEQ ID NO: 388 NEF segment 13 Polypeptide encoded by SEQ ID NO: 390 HIV Cassette Al Polypeptide encoded by SEQ ID NO:392 HIV Cassette B1 Polypeptide encoded by SEQ ID NO: 394 HIV Cassette C1 Polypeptide encoded by SEQ ID NO: 396 HIV Cassette A2 Polypeptide encoded by SEQ ID NO: 398 HIV Cassette B2 Polypeptide encoded by SEQ ID NO: 400 HIV Cassette C2 Polypeptide encoded by SEQ ID NO: 402 HIV complete Savine Polypeptide encoded by SEQ ID NO: 404 HepCla consensus polyprotein sequence 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 78 nts 26 aa 5703 nts 1896 aa 5685 nts 1890 aa 5925 nts 1967 aa 5703 nts 1896 aa 5685 nts 1890 aa 5925 nts 1967 aa 17244 nts 5747 aa 3011 aa WO 01/90197 PCT/AU01/00622 -34- SEQUENCEID :SEQUENCE LENGTH 1 l ~to ffi^ l l SEQ ID NO: 407 SEQ ID NO: 408 SEQ ID NO: 409 SEQ ID NO: 410 SEQ ID NO: 411 SEQ ID NO: 412 SEQ ID NO: 413 SEQ ID NO: 414 SEQ ID NO: 415 SEQ ID NO: 416 SEQ ID NO: 417 SEQ ID NO: 418 SEQ ID NO: 419 SEQ ID NO: 420 SEQ ID NO: 421 SEQ ID NO: 422 SEQ ID NO: 423 SEQ ID NO: 424 SEQ ID NO: 425 SEQ ID NO: 426 SEQ ID NO: 427 SEQ ID NO: 428 SEQ ID NO: 429 SEQ ID NO: 430 HepCla segment 1 Polypeptide encoded by SEQ ID NO: 407 HepC 1 a segment 2 Polypeptide encoded by SEQ ID NO: 409 HepCla segment 3 Polypeptide encoded by SEQ ID NO: 411 HepCla segment 4 Polypeptide encoded by SEQ ID NO: 413 HepCla segment 5 Polypeptide encoded by SEQ ID NO: 415 HepCla segment 6 Polypeptide encoded by SEQ ID NO: 417 HepCla segment 7 Polypeptide encoded by SEQ ID NO: 419 HepCla segment 8 Polypeptide encoded by SEQ ID NO: 421 HepCla segment 9 Polypeptide encoded by SEQ ID NO: 423 HepCla segment 10 Polypeptide encoded by SEQ ID NO: 425 HepCla segment 11 Polypeptide encoded by SEQ ID NO: 427 HepCla segment 12 Polypeptide encoded by SEQ ID NO: 429 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 35 NUMERl SEQUrENCELI 27( f SEQ IID NO: 43 1 SEQ ID NO: 432 SEQ ID NO: 433 SEQ ED NO: 434 SEQ 1ID NO: 435 SEQ ID NO: 436 SEQ ID NO: 437 SEQ ]D NO: 438 S EQ ID NO: 439 SEQ ID NO: 440 SEQ ID NO: 441 SEQ ID NO: 442 SEQ ED NO: 443 SEQ IID NO: 444 SEQ IT)NO: 445 SEQ IIDNO: 446 SEQED NO: 447 SEQ ID NO: 448 SEQ ]D NO: 449 SEQ 1ID NO: 450 SEQ ID NO: 451 SEQ ID NO: 452 SEQ ID NO: 453 SEQ ID NO: 454
I
HepCla segment 13 Polypeptide encoded by SEQ ID NO: 431 HepClIa segment 14 Polypeptide encoded by SEQ H)D NO: 433 HepClIa segment 15 Polypeptide encoded by SEQ ID NO: 435 HepCl1a segmient 16 Polypeptide encoded by SEQ ID NO: 437 HepCla segment 17 Polypeptide encoded by SEQ ID NO: 439 HepCla segment 18 Polypeptide encoded by SEQ ED NO: 441 HepCl asegnment 19 Polypeptide encoded by SEQ ID NO: 443 HepCla segment 20 Polypeptide encoded by SEQ ID NO: 445 HepCla segment 21 Polypeptide encoded by SEQ ID NO: 447 HepCl1a segment 22 Polypeptide encoded by SEQ ID NO: 449 HepCl a segment 23 Polypeptide encoded by SEQ ID NO: 451 HepC 1 a segment 24 Polypeptide encoded by SEQ ID NO: 453 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 ats 30 aa 90 nts 30 aa 30 aa 90 nts 30 aa 90 nts 30 aa 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 -36- SYEQUEACE ID SEQA~UErcELf GTI NUMBER jLNT SEQ ID NO: 455 SEQ lED NO: 456 SEQ ED NO: 457 SEQBD NO: 458 SEQ TD NO: 459 SEQ IDNO: 460 SEQ ID NO: 461 SEQ ID NO: 462 SEQ ID NO: 463 SEQ TD NO: 464 SEQ ID NO: 465 SEQ ID NO: 466 SEQ ID NQ, 467 SEQ ID NO: 468 SEQ ID NO: 469 SEQ ID NO: 470 SEQ ID NO: 471 SEQ ID NO: 472 SEQ ID NO: 473 SEQ IID NO: 474 SEQ ED NO: 475 SEQ ID NO: 476 SEQ ID NO: 477 SEQ ID NO: 478 HepClIa segment 25 Polypeptide encoded by SEQ IID NO: 455 HepCl1a segment 26 Polypeptide encoded by SEQ ID NO: 457 IHepCla segment 27 Polypeptide encoded by SEQ ED NO: 459 HepClIa segment 28 Polypeptide encoded by SEQ ID NO: 461 HepCla segment 29 Polypeptide encoded by SEQ IID NO: 463 HepCl a segment 30 Polypeptide encoded by SEQ ID NO: 465 HepCla segment 31 Polypeptide encoded by SEQ ED NO: 467 HepCla segment 32 Polypeptide encoded by SEQ ID NO: 469 HepCla segment 33 Polypeptide encoded by SEQ ID NO: 471 HepCla segment 34 Polypeptide encoded by SEQ liD NO: 473 HepCl a segmnent 35 Polypeptide encoded by SEQ lID NO: 475 lHepClIa segment 36 Polypeptide encoded by SEQ ID NO: 477 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -37- SEQUENCE ID
NUMBER'
SEQ ID NO: 479 SEQ ID NO: 480 SEQ ID NO: 481 SEQ ID NO: 482 SEQ ID NO: 483 SEQ ID NO: 484 SEQ ID NO: 485 SEQ ID NO: 486 SEQ ID NO: 487 SEQ ID NO: 488 SEQ ID NO: 489 SEQ ID NO: 490 SEQ ID NO: 491 SEQ ID NO: 492 SEQ ID NO: 493 SEQ ID NO: 494 SEQ ID NO: 495 SEQ ID NO: 496 SEQ ID NO: 497 SEQ ID NO: 498 SEQ ID NO: 499 SEQ ID NO: 500 SEQ ID NO: 501 SEQ ID NO: 502 SEQUENCE LENGTH HepCla segment 37 Polypeptide encoded by SEQ ID NO: 479 HepCla segment 38 Polypeptide encoded by SEQ ID NO: 481 HepCla segment 39 Polypeptide encoded by SEQ ID NO: 483 HepCla segment 40 Polypeptide encoded by SEQ ID NO: 485 HepCla segment 41 Polypeptide encoded by SEQ ID NO: 487 HepCla segment 42 Polypeptide encoded by SEQ ID NO: 489 HepCla segment 43 Polypeptide encoded by SEQ ID NO: 491 HepCla segment 44 Polypeptide encoded by SEQ ID NO: 493 HepCla segment 45 Polypeptide encoded by SEQ ID NO: 495 HepCla segment 46 Polypeptide encoded by SEQ ID NO: 497 HepCla segment 47 Polypeptide encoded by SEQ ID NO: 499 HepCla segment 48 Polypeptide encoded by SEQ ID NO: 501 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 38 SEQ()UENACE. ID SEQUENCE LEAGTYH
NUMBER
SEQ IID NO: 503 SEQ ED NO: 504 SEQ EID NO: 505 SEQ DED NO: 506 SEQ ID NO: 507 SEQ ED NO: 508 SEQ ID NO: 509 SEQ IUD NO: 510 SEQ ID NO: 511 SEQ IiDNO- 512 SEQ ID NO: 513 SEQ IDNO: 514 SEQ ID NO: 515 SEQ ID NO: 516 SEQ ID NO: 517 SEQ ID NO: 518 SEQ ID NO: 519 SEQ ID NO: 520 SEQ ID NO: 521 SEQ IDNO: 522 SEQ ID NO: 523 SEQ ID NO: 524 SEQ ID NO: 525 SEQ ID NO: 526 HepCla segment 49 Polypeptide encoded by SEQ ID NO: 503 HepCla segment 50 Polypeptide encoded by SEQ ID NO: 505 HepCla segment 51 Polypeptide encoded by SEQ ID NO: 507 HepCl1a segment 52 Polypeptide encoded by SEQ ID NO: 509 HepClIa segment 53 Polyp eptide encoded by SEQ ID NO: 511 HepClIa segment 54 Polypeptide encoded by SEQ ID NO: 513 HepCla segment 55 Polypeptide'encoded by SEQ ED NO: 515 lHepCla segment 56 Polypeptide encoded by SEQ ED NO: 517 HepCla segment 57 Polypeptide encoded by SEQ ED NO: 519 lHepCla segment 58 Polypeptide encoded by SEQ ED NO: 521 HepCla segment 59 Polypeptide encoded by SEQ ID) NO: 523 HcpCla segment 60 Polypeptide encoded by SEQ ID NO: 525 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -39- SEQUENCEID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 527 SEQ ID NO: 528 SEQ ID NO: 529 SEQ ID NO: 530 SEQ ID NO: 531 SEQ ID NO: 532 SEQ ID NO: 533 SEQ ID NO: 534 SEQ ID NO: 535 SEQ ID NO: 536 SEQ ID NO: 537 SEQ ID NO: 538 SEQ ID NO: 539 SEQ ID NO: 540 SEQ ID NO: 541 SEQ ID NO: 542 SEQ ID NO: 543 SEQ ID NO: 544 SEQ ID NO: 545 SEQ ID NO: 546 SEQ ID NO: 547 SEQ ID NO: 548 SEQ ID NO: 549 SEQ ID NO: 550 HepCla segment 61 Polypeptide encoded by SEQ ID NO: 527 HepCla segment 62 Polypeptide encoded by SEQ ID NO: 529 HepCla segment 63 Polypeptide encoded by SEQ ID NO: 531 HepCla segment 64 Polypeptide encoded by SEQ ID NO: 533 HepCla segment 65 Polypeptide encoded by SEQ ID NO: 535 HepCla segment 66 Polypeptide encoded by SEQ ID NO: 537 HepCla segment 67 Polypeptide encoded by SEQ ID NO: 539 HepCla segment 68 Polypeptide encoded by SEQ ID NO: 541 HepCla segment 69 Polypeptide encoded by SEQ ID NO: 543 HepCla segment 70 Polypeptide encoded by SEQ ID NO:545 HepCla segment 71 Polypeptide encoded by SEQ ID NO: 547 HepCla segment 72 Polypeptide encoded by SEQ ID NO: 549 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 SEQUjENCEID SEQUENCE, LENlGTH SEQED NO: 551 SEQ ID NO: 552 SEQ ID NO: 553 SEQ DD NO: 554 SEQ IID NO: 555 SEQ ID NO: 556 SEQ TD NO: 557 SEQ ID NO: 558 SEQ ID NO: 559 SEQ ID NO: 560 SEQ EiD NO: 561 SEQ TD NO: 562 SEQ ID NO: 563 SEQ ID NO: 564 SEQ ID NO: 565 SEQ ID NO: 566 SEQ ID NO: 567 SEQED NO: 568 SEQ IOD NO: 569 SEQ IOD NO: 570 SEQ ID NO: 571 SEQ ID NO: 572 SEQ H) NO: 573 SEQ ID NO: 574 HepClIa segment 73 Polypeptide encoded by SEQ 1D NO: 551 IlepCl a segment 74 Polypeptide encoded by SEQ ID NO: 553 llepCla segment 75 Polypeptide encoded by SEQ ID NO: 555 HepClta segment 76 Polypeptide encoded by SEQ ID NO: 557 HepCl1a segment 77 Polypeptide encoded by SEQ lID NO: 559 HepCla segment 78 Polypeptide encoded by SEQ ID NO: 561 HepCl1a segment 79 Polypeptide encoded by SEQ ED NO: 563 }{epC Ia segment 8 0 Polypeptide encoded by SEQ ED NO: 565 HepClIa segment 81 Polypeptide encoded by SEQ ED NO: 567 HepCla segment 82 Polypeptide encoded by SEQ TD NO: 569 HepCla segment 83 Polypeptide encoded by SEQ ID NO: 571 HepCla segment 84 Polypeptide encoded by SEQ ID NO: 573 90 nts 30 aa 90 nts 30 aa 30 aa 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts -30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 30 aa L J WO 01/90197 WO 0190197PCT/AU01/00622 -41- SEQ UENCE ID SEQUENCE LENGCTH SEQ ID NO: 575 SEQ DD NO: 576 SEQ MD NO: 577 SEQ BD NO: 578 SEQ IID NO: 579 SEQ ID NO: 580 SEQ ID NO: 581 SEQ ID NO: 582 SEQ ID NO: 583 SEQ ID NO: 584 SEQ ID NO: 585 SEQ ID NO: 586 SEQ ID NO: 587 SEQ ED NO: 5 88 SEQ ]ID NO: 589 SEQ EIDNO: 590 SEQBD NO: 591 SEQ ID NO: 592 SEQ ID NO: 593 SEQ ID NO: 594 SEQ ID NO: 595 SEQ ID NO: 596 SEQ ID NO: 597 SEQ ID NO: 598 HepC Ia segment 85 Polypeptide encoded by SEQ ID NO: 575 HepCl a segment 86 Polypeptide, encoded by SEQ ED NO: 577 HepCla segment 87 Polypeptide encoded by SEQ ID NO: 579 HepCla segment 88 Polypeptide encoded by SEQ ID NO: 581 1{epCla segment 89 Polypeptide encoded by SEQ ID NO: 583 HepClIa segment 90 Polypeptide encoded by SEQ ID NO: 585 IHepCl1a segment 91 Polypeptide encoded by SEQ lID NO: 587 HepClIa segment 92 Polypeptide encoded by SEQ ID NO: 589 HepCl a segment 93 Polypeptide, encoded by SEQ EID NO: 591 HepCla segment 94 Polypeptide encoded by SEQ ID NO: 593 HepCla segment 95 Polypeptide encoded by SEQ ID NO: 595 HepClIa segment 96 Polypeptide encoded by SEQ ID NO: 597 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 uts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa I WO 01/90197 PCT/AU01/00622 -42- SEQUENCEI D SEQUENCE LENGTT NUMBER
LEGT
SEQ ID NO: 599 SEQ ID NO: 600 SEQ ID NO: 601 SEQ ID NO: 602 SEQ ID NO: 603 SEQ ID NO: 604 SEQ ID NO: 605 SEQ ID NO: 606 SEQ ID NO: 607 SEQ ID NO: 608 SEQ ID NO: 609 SEQ ID NO: 610 SEQ ID NO: 611 SEQ ID NO: 612 SEQ ID NO: 613 SEQ ID NO: 614 SEQ ID NO: 615 SEQ ID NO: 616 SEQ ID NO: 617 SEQ IDNO: 618 SEQ ID NO: 619 SEQ ID NO: 620 SEQ ID NO: 621 SEQ ID NO: 622 HepCla segment 97 Polypeptide encoded by SEQ ID NO: 599 HepCla segment 98 Polypeptide encoded by SEQ ID.NO: 601 HepCla segment 99 Polypeptide encoded by SEQ ID NO: 603 HepCla segment 100 Polypeptide encoded by SEQ ID NO: 605 HepCla segment 101 Polypeptide encoded by SEQ ID NO: 607 HepCla segment 102 Polypeptide encoded by SEQ ID NO: 609 HepCla segment 103 Polypeptide encoded by SEQ ID NO: 611 HepCla segment 104 Polypeptide encoded by SEQ ID NO: 613 HepCla segment 105 Polypeptide encoded by SEQ ID NO: 615 HepCla segment 106 Polypeptide encoded by SEQ ID NO: 617 HepCla segment 107 Polypeptide encoded by SEQ ID NO: 619 HepCla segment 108 Polypeptide encoded by SEQ ID NO: 621 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 43 SEQ UENCE ID, SEQUENrCE LEN'GTH, NUM4BER SEQ IID NO: 623 SEQ DD NO: 624 SEQ ID NO: 625 SEQ ID NO: 626 SEQ ID NO: 627 SEQ ID NO: 628 SEQ ID NO: 629 SEQID NO: 630 SEQ lID NO: 631 SEQ ID NO: 632 SEQ ID NO: 633 SEQ ID NO: 634 SEQ I1DNO: 635 SEQ 1D NO: 636 SEQ ID NO: 637 SEQ ID NO: 638 SEQBD NO: 639 SEQID NO: 640 SEQ ID NO: 641 SEQ ID NO: 642 SEQ ID NO: 643 SEQ ID NO: 644 SEQ I1D NO: 645 SEQ ID NO: 646 HepCl1a segment 109 Polypeptide encoded by SEQ ID NO: 623 HepCl1a segment 110 Polypeptide encoded by SEQ TD NO: 625 HepCla segmnent Ill1' Polypeptide encoded by SEQ ID NO: 627 HepClIa segment 112 Polypeptide encoded by SEQ ID NO: 629 HepClIa segment 113 Polypeptide encoded by SEQ ID NO: 631 HepCla segment 114 Polypeptide encoded by SEQ I0D NO: 633 HepCla segment 115 Polypeptide encoded by SEQ I1D NO: 635 HepClIa segment 116 Polypeptide encoded by SEQ ID NO: 637 HepCla segment 117 Polypeptide encoded by SEQ ID NO: 639 HepCl1a segment 118 Polypeptide encoded by SEQ lID NO: 641 HepCl1a segment 119 Polypeptide encoded by SEQ ID NO: 643 HepCla segment 120 Polypeptide encoded by SEQ lID NO: 645 90 nts 30 aa 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -44- SEQUENCE ID SEQUENCE LENGTH: 1
NUMBERI
SEQ ID NO: 647 SEQ ID NO: 648 SEQ ID NO: 649 SEQ ID NO: 650 SEQ ID NO: 651 SEQ ID NO: 652 SEQ ID NO: 653 SEQ ID NO: 654 SEQ ID NO: 655 SEQ ID NO: 656 SEQ ID NO: 657 SEQ ID NO: 658 SEQ ID NO: 659 SEQ ID NO: 660 SEQ ID NO: 661 SEQ ID NO: 662 SEQ ID NO: 663 SEQ ID NO: 664 SEQ ID NO: 665 SEQ ID NO: 666 SEQ ID NO: 667 SEQ ID NO: 668 SEQ ID NO: 669 SEQ ID NO: 670 HepCla segment 121 Polypeptide encoded by SEQ ID NO: 647 HepCla segment 122 Polypeptide encoded by SEQ ID NO: 649 HepCla segment 123 Polypeptide encoded by SEQ ID NO: 651 HepCla segment 124 Polypeptide encoded by SEQ ID NO: 653 HepCla segment 125 Polypeptide encoded by SEQ ID NO: 655 HepCla segment 126 Polypeptide encoded by SEQ ID NO: 657 HepCla segment 127 Polypeptide encoded by SEQ ID NO: 659 HepCla segment 128 Polypeptide encoded by SEQ ID NO: 661 HepCla segment 129 Polypeptide encoded by SEQ ID NO: 663 HepCla segment 130 Polypeptide encoded by SEQ ID NO: 665 HepCla segment 131 Polypeptide encoded by SEQ ID NO: 667 HepCla segment 132 Polypeptide encoded by SEQ ID NO: 669 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa -J WO 01/90197 PCT/AU01/00622 SEQUENCE ID SEQUENCE LENGTH
NUMBER
1 V^C.M \f l SEQ ID NO: 671 SEQ ID NO: 672 SEQ ID NO: 673 SEQ ID NO: 674 SEQ ID NO: 675 SEQ ID NO: 676 SEQ ID NO: 677 SEQ ID NO: 678 SEQ ID NO: 679 SEQ ID NO: 680 SEQ ID NO: 681 SEQ ID NO: 682 SEQ ID NO: 683 SEQ ID NO: 684 SEQ ID NO: 685 SEQ ID NO: 686 SEQ ID NO: 687 SEQ ID NO: 688 SEQ ID NO: 689 SEQ ID NO: 690 SEQ ID NO: 691 SEQ ID NO: 692 SEQ ID NO: 693 SEQ ID NO: 694 HepCla segment 133 Polypeptide encoded by SEQ ID NO: 671 HepCla segment 134 Polypeptide encoded by SEQ ID NO: 673 HepCla segment 135 Polypeptide encoded by SEQ ID NO: 675 HepCla segment 136 Polypeptide encoded by SEQ ID NO: 677 HepCla segment 137 Polypeptide encoded by SEQ ID NO: 679 HepCla segment 138 Polypeptide encoded by SEQ ID NO: 681 HepCla segment 139 Polypeptide encoded by SEQ ID NO: 683 HepCla segment 140 Polypeptide encoded by SEQ ID NO: 685 HepCla segment 141 Polypeptide encoded by SEQ ID NO: 687 HepCla segment 142 Polypeptide encoded by SEQ ID NO: 689 HepCla segment 143 Polypeptide encoded by SEQ ID NO: 691 HepCla segment 144 Polypeptide encoded by SEQ ID NO: 693 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 -46 SEQ UETCE ID SEQUENCE LENGLH NUMBER J SEQ ED NO: 695 SEQ lID NO: 696 SEQ lID NO: 697 SEQ IID NO: 698 SEQ IID NO: 699 SEQ ID NO: 700 SEQ ID NO: 701 SEQ ID NO: 702 SEQ ID NO: 703 SEQ ID NO: 704 SEQ H) NO: 705 SEQ IIDNO: 706 SEQ ID NO: 707 SEQ ID NO: 708 SEQ ID NO: 709 SEQ TD NO: 710 SEQ ED NO: 711 SEQ ED NO: 712 SEQ IDNO: 713 SEQ ID NO: 714 SEQ ID NO: 715 SEQ ID NO: 716 SEQ ID NO: 717 SEQ ID NO: 718 HepCl a segment 145 Polypeptide encoded by SEQ ED NO: 695 HepClIa segment 146 Polypeptide encoded by SEQ ID NO: 697 HepCla segment 147 Polyp eptide encoded by SEQ ID NO: 699 HepCl1a segment 148 Polypeptide encoded by SEQ ID NO: 701 HapCl1a segment 149 Polypeptide encoded by SEQ ID NO: 703 IlepCla segment 150 Polyp eptide encoded by SEQ IID NO: 705 HepCl a segment 151 Polypeptide encoded by SEQ ED NO: 707 HepCl a segment 152 Polypeptide encoded by SEQ ID NO: 709 HepCla segment 153 Polypeptide encoded by SEQ ED NO: 711 HepCla segment 154 Polypeptide encoded by SEQ I) NO: 713 flepCla segment 155 Polypeptide encoded by SEQ ID NO: 715 HepCla segment 156 Polypeptide encoded by SEQ ID NO: 717 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 9D nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -47- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 719 SEQ ID NO: 720 SEQ ID NO: 721 SEQ ID NO: 722 SEQ ID NO: 723 SEQ ID NO: 724 SEQ ID NO: 725 SEQ ID NO: 726 SEQ ID NO: 727 SEQ ID NO: 728 SEQ ID NO: 729 SEQ ID NO: 730 SEQ ID NO: 731 SEQ ID NO: 732 SEQ ID NO: 733 SEQ ID NO: 734 SEQ ID NO: 735 SEQ ID NO: 736 SEQ ID NO: 737 SEQ ID NO: 738 SEQ ID NO: 739 SEQ ID NO: 740 SEQ ID NO: 741 SEQ ID NO: 742 HepCla segment 157 Polypeptide encoded by SEQ ID NO: 719 HepCla segment 158 Polypeptide encoded by SEQ ID NO: 721 HepCla segment 159 Polypeptide encoded by SEQ ID NO: 723 HepCla segment 160 Polypeptide encoded by SEQ ID NO: 725 HepCla segment 161 Polypeptide encoded by SEQ ID NO: 727 HepCla segment 162 Polypeptide encoded by SEQ ID NO: 729 HepCla segment 163 Polypeptide encoded by SEQ ID NO: 731 HepCla segment 164 Polypeptide encoded by SEQ ID NO: 733 HepCla segment 165 Polypeptide encoded by SEQ ID NO: 735 HepCla segment 166 Polypeptide encoded by SEQ ID NO: 737 HepCla segment 167 Polypeptide encoded by SEQ ID NO: 739 HepCla segment 168 Polypeptide encoded by SEQ ID NO: 741 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -48- SEQUENCEID 1 SEQUENCE LENGTH
NUMBER
SEQ ID NO: 743 SEQ ID NO: 744 SEQ ID NO: 745 SEQ ID NO: 746 SEQ ID NO: 747 SEQ ID NO: 748 SEQ ID NO: 749 SEQ ID NO: 750 SEQ ID NO: 751 SEQ ID NO: 752 SEQ ID NO: 753 SEQ ID NO: 754 SEQ ID NO: 755 SEQ ID NO: 756 SEQ ID NO: 757 SEQ ID NO: 758 SEQ ID NO: 759 SEQ ID NO: 760 SEQ ID NO: 761 SEQ ID NO: 762 SEQ ID NO: 763 SEQ ID NO: 764 SEQ ID NO: 765 SEQ ID NO: 766 1 HepCla segment 169 Polypeptide encoded by SEQ ID NO: 743 HepCla segment 170 Polypeptide encoded by SEQ ID NO: 745 HepCla segment 171 Polypeptide encoded by SEQ ID NO: 747 HepCla segment 172 Polypeptide encoded by SEQ ID NO: 749 HepCla segment 173 Polypeptide encoded by SEQ ID NO: 751 HepCla segment 174 Polypeptide encoded by SEQ ID NO: 753 HepCla segment 175 Polypeptide encoded by SEQ ID NO: 755 HepCla segment 176 Polypeptide encoded by SEQ ID NO: 757 HepCla segment 177 Polypeptide encoded by SEQ ID NO: 759 HepCla segment 178 Polypeptide encoded by SEQ ID NO: 761 HepCla segment 179 Polypeptide encoded by SEQ ID NO: 763 HepCla segment 180 Polypeptide encoded by SEQ ID NO: 765 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -49- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 767 SEQ ID NO: 768 SEQ ID NO: 769 SEQ ID NO: 770 SEQ ID NO: 771 SEQ ID NO: 772 SEQ ID NO: 773 SEQ ID NO: 774 SEQ ID NO: 775 SEQ ID NO: 776 SEQ ID NO: 777 SEQ ID NO: 778 SEQ ID NO: 779 SEQ ID NO: 780 SEQ ID NO: 781 SEQ ID NO: 782 SEQ ID NO: 783 SEQ ID NO: 784 SEQ ID NO: 785 SEQ ID NO: 786 SEQ ID NO: 787 SEQ ID NO: 788 SEQ ID NO: 789 SEQ ID NO: 790 HepCla segment 181 Polypeptide encoded by SEQ ID NO: 767 HepCla segment 182 Polypeptide encoded by SEQ ID NO: 769 HepCla segment 183 Polypeptide encoded by SEQ ID NO: 771 HepCla segment 184 Polypeptide encoded by SEQ ID NO: 773 HepCla segment 185 Polypeptide encoded by SEQ ID NO: 775 HepCla segment 186 Polypeptide encoded by SEQ ID NO: 777 HepCla segment 187 Polypeptide encoded by SEQ ID NO: 779 HepCla segment 188 Polypeptide encoded by SEQ ID NO: 781 HepCla segment 189 Polypeptide encoded by SEQ ID NO: 783 HepCla segment 190 Polypeptide encoded by SEQ ID NO: 785 HepCla segment 191 Polypeptide encoded by SEQ ID NO: 787 HepCla segment 192 Polypeptide encoded by SEQ ID NO: 789 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 SEQ0[EATCE ID' S- EUECE LENGTH N -U-4BRER! SEQ DD NO: 791 SEQ 11D NO: 792 SEQ ID NO: 793 SEQ ED NO: 794 SEQ ID NO: 795 SEQ ID NO: 796 SEQ ID NO: 797 SEQ ID NO: 798 SEQ ID NO: 799 SEQ ID NO: 800 SEQ ID NO: 801 SEQ ID NO: 802 SEQ IDNO: 803 SEQ IIDNO: 804 SEQ ID NO: 805 SEQ ID NO: 806 SEQ ID NO: 807 SEQ ID NO: 808 SEQID NO: 809 SEQ ID NO-- 810 SEQ ED NO: 8 11 SEQ ID NO: 8 12 SEQ ID NO: 813 SEQ ID NO: 814 HepClIa segment 193 Polypeptide encoded by SEQ ID NO: 791 HepCla segment 194 Polypeptide encoded by SEQ ID NO: 793 HepCl1a segment 195 Polypeptide encoded by SEQ ID NO: 795 HepClIa segment 196 Polypeptide encoded by SEQ ID NO: 797 IHepClIa segment 197 Polypeptide encoded by SEQ I7D NO: 799 llepClIa segment 198 Polypeptide encoded by SEQ ID NO: 801 HepC Ia segment 199 Polyp eptide encoded by SEQ ED NO: 803 HepCla segment 200 Polypeptide encoded by SEQ ED NO: 805 HepCla segment 201 Polypeptide encoded by SEQ ED NO: 807 HepC 1 a scrambled Polypeptide encoded by SEQ ID NO: 809 flepC Cassette A Polypeptide encoded by SEQ ED NO: 811 HepC Cassette B Polypeptide encoded by SEQ ID NO: 813 90 nts 30 aa.
90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 45 nts 15 aa 17955 nts 5985 aa 6065 nts 2011 aa 6069 nts 2010 aa.
L I WO 01/90197 WO 0190197PCT/AU01/00622 -51 SEQ0UENCE-ID SEQUENCE LENGTH SEQ DD NO: 815 SEQ ID NO: 816 SEQ H) NO: 817 SEQ ID NO: 818 SEQ ID NO: 819 SEQ ID NO: 820 SEQ ID NO: 821 SEQ ID NO: 822 SEQ ID NO: 823 SEQ ID NO: 824 SEQ IDJ NO: 825 SEQ ID NO: 826 SEQ ID NO: 827 SEQ ID NO: 828 SEQ ID NO: 829 SEQ ID NO: 830 SEQBD NO: 831 SEQ ID NO: 832 SEQ ID NO: 833 SEQ ID NO: 834 SEQ BD NO: 835 SEQ DD NO: 836 SEQ IDNO: 837 SEQ ID NO: 838 HepC Cassette C Polypeptide encoded by SEQ BD NO: 815 gplOO consensus polypeptide MART consensus polyp eptide TRP-1 consensus polypeptide Tyros consensus polypeptide TRP2 consensus polypeptide, MC1R consensus polypeptide MUClF consensus polypeptide MUC1R consensus polypeptide BAGE consensus polypeptide GAGE- I consensus polypeptide gp 1 001n4 consensus polyp eptide MAGE-1 consensus polypeptide, MAGE-3 consensus polyp eptide, PRAVE consensus polypeptide TRP2IN2 consensus polypeptide NYNSO0 1 a consensus polypeptide NYNSOib consensus polypeptide LAGE1 consensus polypeptide, gpIOO segment 1 Polypeptide encoded by SEQ ED NO: 835 gp 100 segment 2 Polypeptide encoded by SEQ liD NO: 837 6030 nts 1997 aa 661 aa 118 aa 248 aa 529 aa 519 aa 317 aa 125 aa 312 aa.
43 aa 138 aa 51 aa.
309 aa 314 aa 509 aa 54 aa 180 aa 58 aa 180 aa 90 nts 30 aa 90 uts 30 aa .1~ WO 01/90197 WO 0190197PCT/AU01/00622 52 SEQ UENTCE ID SEQUETTNCIE LEN"GTH SEQ BID NO: 839 SEQ DD NO: 840 SEQ JID NO: 841 SEQ ID NO: 842 SEQ ID NO: 843 SEQ ID NO: 844 SEQ lID NO: 845 SEQ ID NO: 846 SEQ ID NO: 847 SEQ ID NO: 848 SEQ IIDNO: 849 SEQ TD NO: 850 SEQ ID NO: 851 SEQ ED NO: 852 SEQ ID NO: 853 SEQ ID NO: 854 SEQID NO: 855 SEQ ID NO: 856 SEQ ID NO: 857 SEQ ID NO: 858 SEQ ID NO: 859 SEQ ID NO: 860 SEQ ID NO: 861 SEQ lID NO: 862 gplOO segment 3 Polyp eptide encoded by SEQ ID NO: 839 gplOO segment 4 Polypeptide encoded by SEQ ID NO: 841 gplOO segment 5 Polypeptide encoded by SEQ lID NO: 843 gpl100 segment 6 IPolypeptide encoded by SEQ ID NO: 845 gplOO segment 7 Polypeptide encoded by SEQ MD NO: 847 gplOO segment 8 Polypeptide encoded by SEQ ED NO: 849 gp, 10 segment 9 Polypeptide encoded by SEQ ED NO: 851 gplOO segment 10 Polypeptide encoded by SEQ ID NO: 853 gplOO segment 11 Polypeptide encoded by SEQ ID NO: 855 gplOO segment 12 Polypeptide encoded by SEQ lID NO: 857 gplOO segmientl13 Polyp eptide encoded by SEQ ID NO: 859 gplOO segment 14 Polyp eptide encoded by SEQ ID NO: 8 61 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -53- SE9QUENCE ID SEQUENCE LENGTI:
NUMBER
SEQ ID NO: 863 SEQ ID NO: 864 SEQ ID NO: 865 SEQ ID NO: 866 SEQ ID NO: 867 SEQ ID NO: 868 SEQ ID NO: 869 SEQ ID NO: 870 SEQ ID NO: 871 SEQ ID NO: 872 SEQ ID NO: 873 SEQ ID NO: 874 SEQ ID NO: 875 SEQ ID NO: 876 SEQ ID NO: 877 SEQ ID NO: 878 SEQ ID NO: 879 SEQ ID NO: 880 SEQ ID NO: 881 SEQ ID NO: 882 SEQ ID NO: 883 SEQ ID NO: 884 SEQ ID NO: 885 SEQ ID NO: 886 gpl00 segment 15 Polypeptide encoded by SEQ ID NO: 863 gplOO segment 16 Polypeptide encoded by SEQ ID NO: 865 gpl00 segment 17 Polypeptide encoded by SEQ ID NO: 867 gpl00 segment 18 Polypeptide encoded by SEQ ID NO: 869 gpl00 segment 19 Polypeptide encoded by SEQ ID NO: 871 gp100 segment 20 Polypeptide encoded by SEQ ID NO: 873 gp100 segment 21 Polypeptide encoded by SEQ ID NO: 875 gp100 segment 22 Polypeptide encoded by SEQ ID NO: 877 gpl00 segment 23 Polypeptide encoded by SEQ ID NO: 879 gplOO segment 24 Polypeptide encoded by SEQ ID NO: 881 gpl00 segment 25 Polypeptide encoded by SEQ ID NO: 883 gpl00 segment 26 Polypeptide encoded by SEQ ID NO: 885 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 -54- SEQ UENiCE ID 1SEQUENCE LENG-TH
NUMBER
SEQ IID NO: 887 SEQ IID NO: 888 SEQ ID NO: 889 SEQID NO: 890 SEQ ID NO: 891 SEQ H) NO: 892 SEQ ED NO: 893 SEQ ID NO: 894 SEQ ID NO: 895 SEQ ID NO: 896 SEQ IDNO: 897 SEQ ID NO: 898 SEQ IIDNO: 899 SEQ H)NO: 900 SEQ ID NO: 901 SEQ IIDNO: 902 SEQ ID NO: 903 SEQ ED NO: 904 SEQ D NO: 905 SEQ ID NO: 906 SEQ ID NO: 907 SEQ ID NO: 908 SEQ ID NO: 909 SEQ ID NO: 910 gplOO segment 27 Polypeptide encoded by SEQ ED NO: 887 gplOO segment 28 Polypeptide encoded by SEQ ID NO: 889 gpl100 segment 29 Polypeptide encoded by SEQ ID NO: 891 gpl1OO segment 30 Polypeptide encoded by SEQ ID NO: 893 gpl1OO segment 31 Polypeptide encoded by SEQ ID NO: 895 gpl1OO segment 32 Polypeptide encoded by SEQ ID NO: 897 gpllOO segment 33 Polypeptide encoded by SEQ ED NO: 899 gplOO segment 34 Polypeptide encoded by SEQ ID NO: 901 gpIOO segment 35 Polypeptide encoded by SEQ ED NO: 903 gpIOO segment 36 Polypeptide encoded by SEQ ED NO: 905 gplOO segment 37 Polypeptide, encoded by SEQ WD NO: 907 gplOO segment 38 Polyp eptide, encoded by SEQ ID NO: 909 90 nts 90 nts 30 aa 90 nts 90 nts 90 nts 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 30 aa 90 nts 90 nts 30 aa WO 01/90197 PCT/AU01/00622 SEQUENCE ID SEQUENCE LENTH
NUMBER
SEQ ID NO: 911 SEQ ID NO: 912 SEQ ID NO: 913 SEQ ID NO: 914 SEQ ID NO: 915 SEQ ID NO: 916 SEQ ID NO: 917 SEQ ID NO: 918 SEQ ID NO: 919 SEQ ID NO: 920 SEQ ID NO: 921 SEQ ID NO: 922 SEQ ID NO: 923 SEQ ID NO: 924 SEQ ID NO: 925 SEQ ID NO: 926 SEQ ID NO: 927 SEQ ID NO: 928 SEQ ID NO: 929 SEQ ID NO: 930 SEQ ID NO: 931 SEQ ID NO: 932 SEQ ID NO: 933 SEQ ID NO: 934 gpl00 segment 39 Polypeptide encoded by SEQ ID NO: 911 gpl00 segment 40 Polypeptide encoded by SEQ ID NO: 913 gpl00 segment 41 Polypeptide encoded by SEQ ID NO: 915 gpl00 segment 42 Polypeptide encoded by SEQ ID NO: 917 gp100 segment 43 Polypeptide encoded by SEQ ID NO: 919 gplOO segment 44 Polypeptide encoded by SEQ ID NO: 921 MART segment 1 Polypeptide encoded by SEQ ID NO: 923 .MART segment 2 Polypeptide encoded by SEQ ID NO: 925 MART segment 3 Polypeptide encoded by SEQ ID NO: 927 MART segment 4 Polypeptide encoded by SEQ ID NO: 929 MART segment 5 Polypeptide encoded by SEQ ID NO: 931 MART segment 6 Polypeptide encoded by SEQ ID NO: 933 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 20 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 -56- SE QUENCE ii) SEQUENCE 1 LE NG TH- NUMBR I SEQ DD NO: 935 SEQ JID NO: 936 SEQ ID NO: 937 SEQ ID NO: 938 SEQ ID NO: 939 SEQ ID NO: 940 SEQ ID NO: 941 SEQ H) NO: 942 SEQ H) NO: 943 SEQ ID NO: 944 SEQID)NO: 945 SEQ ID NO: 946 SEQ ID NO: 947 SEQ JID NO: 948 SEQ TD NO: 949 SEQ ED NO: 950 SEQ ED NO: 951 SEQ ID NO: 952 SEQ ID NO: 953 SEQ ID NO: 954 SEQ ID NO: 955 SEQ ID NO: 956 SEQ ID NO: 957 SEQ IID NO: 958 MART segment 7 Polypeptide encoded by SEQ NO: 935 MAkRT segment 8 Polypeptide encoded by SEQ ]ID NO: 937 trp-1 segment 1 Polypeptide encoded by SEQ ID NO: 939 trp- 1 segment 2 Polypeptide encoded by SEQ ID NO: 941 ft-p-i segment 3 Polypeptide encoded by SEQ ID NO: 943 trp- 1 segment 4 Polypeptide encoded by SEQ IID NO: 945 trp-1 segment 5 Polypeptide encoded by SEQ ID NO: 947 trp-1I segment 6 Polypeptide encoded by SEQ ID NO: 949 trp-1 segment 7 Polyp eptide encoded by SEQ I1) NO: 951 ftp-i segment 8 Polypeptide encoded by SEQ ID NO: 953 ftp-i segment 9 Polyp eptide encoded by SEQ ID iNO: 955 trp-i segment 10 Polypeptide encoded by SEQ ID NO: 957 90 nts 30 aa 5 1 nts 17 aa 30 aa 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -57- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 959 SEQ ID NO: 960 SEQ ID NO: 961 SEQ ID NO: 962 SEQ ID NO: 963 SEQ ID NO: 964 SEQ ID NO: 965 SEQ ID NO: 966 SEQ ID NO: 967 SEQ ID NO: 968 SEQ ID NO: 969 SEQ ID NO: 970 SEQ ID NO: 971 SEQ ID NO: 972 SEQ ID NO: 973 SEQ ID NO: 974 SEQ ID NO: 975 SEQ ID NO: 976 SEQ ID NO: 977 SEQ ID NO: 978 SEQ ID NO: 979 SEQ ID NO: 980 SEQ ID NO: 981 SEQ ID NO: 982 trp-1 segment 11 Polypeptide encoded by SEQ ID NO: 959 trp-1 segment 12 Polypeptide encoded by SEQ ID NO: 961 trp-1 segment 13 Polypeptide encoded by SEQ ID NO: 963 trp-i segment 14 Polypeptide encoded by SEQ ID NO: 965 trp-1 segment 15 Polypeptide encoded by SEQ ID NO: 967 trp-1 segment 16 Polypeptide encoded by SEQ ID NO: 969 tyros segment 1 Polypeptide encoded by SEQ ID NO: 971 tyros segment 2 Polypeptide encoded by SEQ ID NO: 973 tyros segment 3 Polypeptide encoded by SEQ ID NO: 975 tyros segment 4 Polypeptide encoded by SEQ ID NO: 977 tyros segment 5 Polypeptide encoded by SEQ ID NO: 979 tyros segment 6 Polypeptide encoded by SEQ ID NO: 981 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 81 nts 27 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -58- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 983 SEQ ID NO: 984 SEQ ID NO: 985 SEQ ID NO: 986 SEQ ID NO: 987 SEQ ID NO: 988 SEQ ID NO: 989 SEQ ID NO: 990 SEQ ID NO: 991 SEQ ID NO: 992 SEQ ID NO: 993 SEQ ID NO: 994 SEQ ID NO: 995 SEQ ID NO: 996 SEQ ID NO: 997 SEQ ID NO: 998 SEQ ID NO: 999 SEQ ID NO: 1000 SEQ ID NO: 1001 SEQ ID NO: 1002 SEQ ID NO: 1003 SEQ ID NO: 1004 SEQ ID NO: 1005 SEQ ID NO: 1006 tyros segment 7 Polypeptide encoded by SEQ ID NO: 983 tyros segment 8 Polypeptide encoded by SEQ ID NO: 985 tyros segment 9 Polypeptide encoded by SEQ ID NO: 987 tyros segment 10 Polypeptide encoded by SEQ ID NO: 989 tyros segment 11 Polypeptide encoded by SEQ ID NO: 991 tyros segment 12 Polypeptide encoded by SEQ ID NO: 993 tyros segment 13 Polypeptide encoded by SEQ ID NO: 995 tyros segment 14 Polypeptide encoded by SEQ ID NO: 997 tyros segment 15 Polypeptide encoded by SEQ ID NO: 999 tyros segment 16 Polypeptide encoded by SEQ ID NO: 1001 tyros segment 17 Polypeptide encoded by SEQ ID NO: 1003 tyros segment 18 Polypeptide encoded by SEQ ID NO: 1005 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa I WO 01/90197 PCT/AU01/00622 -59- SEQUENCE ID SEQUENCE LENGTH SEQ ID NO: 1007 SEQ ]D NO: 1008 SEQ ID NO: 1009 SEQ ID NO: 1010 SEQ ID NO: 1011 SEQ ID NO: 1012 SEQ ID NO: 1013 SEQ ID NO: 1014 SEQ ID NO: 1015 SEQ ID NO: 1016 SEQ ID NO: 1017 SEQ IDNO: 1018 SEQ ID NO: 1019 SEQ ID NO: 1020 SEQ ID NO: 1021 SEQ ID NO: 1022 SEQ ID NO: 1023 SEQ ID NO: 1024 SEQ ID NO: 1025 SEQ ID NO: 1026 SEQ ID NO: 1027 SEQ ID NO: 1028 SEQ ID NO: 1029 SEQ ID NO: 1030 tyros segment 19 Polypeptide encoded by SEQ ID NO: 1007 tyros segment 20 Polypeptide encoded by SEQ ID NO: 1009 tyros segment 21 Polypeptide encoded by SEQ ID NO: 1011 tyros segment 22 Polypeptide encoded by SEQ ID NO: 1013 tyros segment 23 Polypeptide encoded by SEQ ID NO: 1015 tyros segment 24 Polypeptide encoded by SEQ ID NO: 1017 tyros segment 25 Polypeptide encoded by SEQ ID NO: 1019 tyros segment 26 Polypeptide encoded by SEQ ID NO: 1021 tyros segment 27 Polypeptide encoded by SEQ ID NO: 1023 tyros segment 28 Polypeptide encoded by SEQ ID NO: 1025 tyros segment 29 Polypeptide encoded by SEQ ID NO: 1027 tyros segment 30 Polypeptide encoded by SEQ ID NO: 1029 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 SEQUENCE'l IDEun 1C LENGTH- NUMWBER SQEC SEQ ID NO: 1031 SEQ ID NO: 1032 SEQ 11) NO: 1033 SEQ ID NO: 1034 SEQ ID NO: 1035 SEQ IED NO: 1036 SEQ ID NO: 103 7 SEQED NO: 103 8 SEQID NO: 1039 SEQ ID NO: 1040 SEQ ID NO: 1041 SEQ ID NO: 1042 SEQ ID NO: 1043.
SEQ ID NO: 1044 SEQ ED NO: 1045 SEQ ID NO: 1046 SEQ ID NO: 1047 SEQ ID NO: 1048 SEQ ID NO: 1049 SEQ ID NO: 1050 SEQ ID NO: 1051 SEQ ID NO: 1052 SEQ lID NO: 1053 ~SEQ lID NO: 1054 tyros segment 31 Polyp eptide encoded by SEQ ID NO: 1031 tyros segment 32 Polypeptide encoded by SEQ ED NO: 1033 tyros segment 33 Polypeptide encoded by SEQ HD NO: 103 5 tyros segment 34 Polypeptide encoded by SEQ EiD NO: 1037 tyros segment 35 Polypeptide encoded by SEQ ED NO: 1039 trp2 segmnent 1 Polypeptide encoded by SEQ ED NO: 1041 trp2 segment 2 Polypeptide encoded by SEQ ED NO: 1043 frp2 segment 3 Polypeptide encoded by SEQ ID NO: 1045 trp2 segment 4 Polyp eptide encoded by SEQ ID NO: 1047 trp2 segment 5 Polypeptide encoded by SEQ ID NO: 1049 trp2 segment 6 Polypeptide encoded by SEQ ID NO: 1051 trp2 segment 7 Polypeptide encoded by SEQ ID NO: 1053 90 nts 90 nts 90 nts 30 aa 90 nts 30 aa 69 nts 23 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 30 aa WO 01/90197 PCT/AU01/00622 -61- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 1055 SEQ ID NO: 1056 SEQ ID NO: 1057 SEQ ID NO: 1058 SEQ ID NO: 1059 SEQ ID NO: 1060 SEQ ID NO: 1061 SEQ ID NO: 1062 SEQ ID NO: 1063 SEQ ID NO: 1064 SEQ ID NO: 1065 SEQ ID NO: 1066 SEQ ID NO: 1067 SEQ ID NO: 1068 SEQ ID NO: 1069 SEQ ID NO: 1070 SEQ ID NO: 1071 SEQ ID NO: 1072 SEQ ID NO: 1073 SEQ ID NO: 1074 SEQ ID NO: 1075 SEQ ID NO: 1076 SEQ ID NO: 1077 SEQ ID NO: 1078 trp2 segment 8 Polypeptide encoded by SEQ ID NO: 1055 trp2 segment 9 Polypeptide encoded by SEQ ID NO: 1057 trp2 segment 10 Polypeptide encoded by SEQ ID NO: 1059 trp2 segment 11 Polypeptide encoded by SEQ ID NO: 1061 trp2 segment 12 Polypeptide encoded by SEQ ID NO: 1063 trp2 segment 13 Polypeptide encoded by SEQ ID NO: 1065 trp2 segment 14 Polypeptide encoded by SEQ ID NO: 1067 trp2 segment 15 Polypeptide encoded by SEQ ID NO: 1069 trp2 segment 16 Polypeptide encoded by SEQ ID NO: 1071 trp2 segment 17 Polypeptide encoded by SEQ ID NO: 1073 trp2 segment 18 Polypeptide encoded by SEQ ID NO: 1075 trp2 segment 19 Polypeptide encoded by SEQ ID NO: 1077 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -62- SEQUENCE ID SEQUENCE LENGTH fNUMBER'.
SEQ ID NO: 1079 SEQ ID NO: 1080 SEQ ID NO: 1081 SEQ ID NO: 1082 SEQ ID NO: 1083 SEQ ID NO: 1084 SEQ ID NO: 1085 SEQ ID NO: 1086 SEQ ID NO: 1087 SEQ ID NO: 1088 SEQ ID NO: 1089 SEQ ID NO: 1090 SEQ ID NO: 1091 SEQ ID NO: 1092 SEQ ID NO: 1093 SEQ ID NO: 1094 SEQ ID NO: 1095 SEQ ID NO: 1096 SEQ ID NO: 1097 SEQ ID NO: 1098 SEQ ID NO: 1099 SEQ ID NO: 1100 SEQ ID NO: 1101 SEQ ID NO: 1102 trp2 segment 20 Polypeptide encoded by SEQ ID NO: 1079 trp2 segment 21 Polypeptide encoded by SEQ ID NO: 1081 trp2 segment 22 Polypeptide encoded by SEQ ID NO: 1083 trp2 segment 23 Polypeptide encoded by SEQ ID NO: 1085 trp2 segment 24 Polypeptide encoded by SEQ ID NO: 1087 trp2 segment 25 Polypeptide encoded by SEQ ID NO: 1089 trp2 segment 26 Polypeptide encoded by SEQ ID NO: 1091 trp2 segment 27 Polypeptide encoded by SEQ ID NO: 1093 trp2 segment 28 Polypeptide encoded by SEQ ID NO: 1095 trp2 segment 29 Polypeptide encoded by SEQ ID NO: 1097 trp2 segment 30 Polypeptide encoded by SEQ ID NO: 1099 trp2 segment 31 Polypeptide encoded by SEQ ID NO: 1101 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 WO 0190197PCT/AU01/00622 63 SEQU( ENCE LD SEQUt ENCE LENG-TH- N UMBER SEQ ID NO: 1103 SEQIED NO: 1104 SEQ ED NO: 1105 SEQ ID NO: 1106 SEQ ID NO: 1107 SEQ ID NO: 1108 SEQ DD NO: 1109' SEQ ID NO: 1110 SEQ EID NO: 1111 SEQ H) NO: 1112 SEQ ID NO: 1113 SEQID NO: 1114 SEQ IDNO: 1115 SEQ ID NO: 1116 SEQ TD NO: 1117 SEQID NO: 1118 SEQ ED NO: 1119 SEQ ID NO: 1120 SEQ ID NO: 1121 SEQ ID NO: 1122 SEQ ID NO: 1123 SEQ ID NO: 1124 SEQ ID NO: 1125 SEQ ID NO. 1126 trp2 segment 32 Polypeptide encoded by SEQ ID NO: 1103 trp2 segment 33 Polyp eptide encoded by SEQ ED NO: 1105 trp2 segment 34 Polyp eptide encoded by SEQ ID NO: 1107 MC1R segment 1 Polypeptide encoded by SEQ lID NO: 1109 MCIR segment 2 Polyp eptide encoded by SEQ ED NO: 1111I MC1R segment 3 Polypeptide encoded by SEQ ID NO: 1113 MC1R segment 4 Polypeptide encoded by SEQ ID NO: 1115 MC1R segment 5 Polypeptide encoded by SEQ lID NO: 1117 MC1R segment 6 Polypeptide encoded by SEQ ED NO: 1119 MC1R segment 7 Polypeptide encoded by SEQ EID NO: 1121 MCIR segment 8 Polypeptide encoded by SEQ ID NO: 1123 MC1R segment 9 Polyp eptide encoded by SEQ ID NO: 1125 90 nts 90 nts 84 nts 28 aa 90 nts 30 aa 90 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -64- SE Q UENCE ID 'SEQ UENCE LENG TH
NUM*BER
SEQ ID NO: 1127 SEQ ID NO: 1128 SEQ ID NO: 1129 SEQ ID NO: 1130 SEQ ID NO: 1131 SEQ ID NO: 1132 SEQ ID NO: 1133 SEQ ID NO: 1134 SEQ ID NO: 1135 SEQ ID NO: 1136 SEQ ID NO: 1137 SEQ ID NO: 1138 SEQ ID NO: 1139 SEQ ID NO: 1140 SEQ ID NO: 1141 SEQ ID NO: 1142 SEQ ID NO: 1143 SEQ ID NO: 1144 SEQ ID NO: 1145 SEQ ID NO: 1146 SEQ ID NO: 1147 SEQ ID NO: 1148 SEQ ID NO: 1149 SEQ ID NO: 1150 MC1R segment 10 Polypeptide encoded by SEQ ID NO: 1127 MC1R segment 11 Polypeptide encoded by SEQ ID NO: 1129 MC1R segment 12 Polypeptide encoded by SEQ ID NO: 1131 MC1R segment 13 Polypeptide encoded by SEQ ID NO: 1133 MC1R segment 14 Polypeptide encoded by SEQ ID NO: 1135 MC1R segment 15 Polypeptide encoded by SEQ ID NO: 1137 MC1R segment 16 Polypeptide encoded by SEQ ID NO: 1139 MC1R segment 17 Polypeptide encoded by SEQ ID NO: 1141 MC1R segment 18 Polypeptide encoded by SEQ ID NO: 1143 MC1R segment 19 Polypeptide encoded by SEQ ID NO: 1145 MC1R segment 20 Polypeptide encoded by SEQ ID NO: 1147 MC1R segment 21 Polypeptide encoded by SEQ ID NO: 1149 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 63 nts 21 aa WO 01/90197 WO 0190197PCT/AU01/00622 65 SEQUENCEJD SEIQUEICE' LENGTH1 SEQ IDNO: 1151 SEQ lID NO: 1152 SEQ ID NO: 1153 SEQ ID NO: 1154 SEQ 11) NO: 1155 SEQ lID NO: 1156 SEQ DD NO: 1157 SEQ DD NO: 1158 SEQIDNO: 1159 SEQ 1ID NO: 1160 SEQ ID NO: 1161 SEQ ID NO: 1162 SEQ H) NO: 1163 SEQ ID NO: 1164 SEQ TD NO: 1165 SEQ HD NO: 1166 SEQ ID NO: 1167 SEQ ID NO: 1168 SEQ ID NO: 1169 SEQ ID NO: 1170 SEQ ID NO: 1171 SEQ ID NO: 1172 SEQ H) NO: 1173 SEQ ED NO: 1174 MUCiF segment 1 Polypeptide, encoded by SEQ ID NO: 1151 MUC1IF segment 2 Polypeptide encoded by SEQ DD NO: 1153 MLTC1F segment 3 Polypeptide encoded by SEQ ID NO: 1155 WLC IF segment 4 Polypeptide encoded by SEQ ID NO: 1157 M-UGIF segment 5 Polyp eptide encoded by SEQ liD NO: 1159 MUC IF segment 6 Polypeptide, encoded by SEQ ID NO: 1161 IvIIC1F segment 7 Polypeptide encoded by SEQ 11D NO: 1163 MIUCIF segment 8 Polypeptide encoded by SEQ ID NO: 1165 AMCR segment 1 Polypeptide encoded by SEQ ED NO: 1167 MUCiR segment 2 Polypeptide encoded by SEQ ED NO: 1169 MIUCIR segment 3 Polypeptide encoded by SEQ I1D NO: 1171 M[JC1R segment 4 Polypeptide encoded by SEQ 1ID NO: 1173 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 72 nts 24 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -66- 'SEQUENCE ID SEQUENCE LENGH
NUMBER
SEQ ID NO: 1175 SEQ ID NO: 1176 SEQ ID NO: 1177 SEQ ID NO: 1178 SEQ ID NO: 1179 SEQ ID NO: 1180 SEQ ID NO: 1181 SEQ ID NO: 1182 SEQ ID NO: 1183 SEQ ID NO: 1184 SEQ ID NO: 1185 SEQ ID NO: 1186 SEQ ID NO: 1187 SEQ ID NO: 1188 SEQ ID NO: 1189 SEQ ID NO: 1190 SEQ ID NO: 1191 SEQ ID NO: 1192 SEQ ID NO: 1193 SEQ ID NO: 1194 SEQ ID NO: 1195 SEQ ID NO: 1196 SEQ ID NO: 1197 MUCIR segment 5 Polypeptide encoded by SEQ ID NO: 1175 MUC1R segment 6 Polypeptide encoded by SEQ ID NO: 1177 MUC1R segment 7 Polypeptide encoded by SEQ ID NO: 1179 MUC1R segment 8 Polypeptide encoded by SEQ ID NO: 1181 MUC1R segment 9 Polypeptide encoded by SEQ ID NO: 1183 MUCI1R segment 10 Polypeptide encoded by SEQ ID NO: 1185 MUCIR segment It Polypeptide encoded by SEQ ID NO: 1187 MUC1R segment 12 Polypeptide encoded by SEQ ID NO: 1189 MUC1R segment 13 Polypeptide encoded by SEQ ID NO: 1191 MUC1R segment 14 Polypeptide encoded by SEQ ID NO: 1193 MUCIR segment 15 Polypeptide encoded by SEQ ID NO: 1195 MUC1R segment 16 Polypeptide encoded by SEQ ID NO: 1197 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts SEQ H) NO: 1198 30 aa WO 01/90197 PCT/AU01/00622 -67- SEQUENCE ID SEQUENCE LE NGTH M 1 l l SEQ ID NO: 1199 SEQ ID NO: 1200 SEQ ID NO: 1201 SEQ ID NO: 1202 SEQ ID NO: 1203 SEQ ID NO: 1204 SEQ ID NO: 1205 SEQ ID NO: 1206 SEQ ID NO: 1207 SEQ ID NO: 1208 SEQ ID NO: 1209 SEQ ID NO: 1210 SEQ ID NO: 1211 SEQ ID NO: 1212 SEQ ID NO: 1213 SEQ ID NO: 1214 SEQ ID NO: 1215 SEQ ID NO: 1216 SEQ ID NO: 1217 SEQ ID NO: 1218 SEQ ID NO: 1219 SEQ ID NO: 1220 SEQ ID NO: 1221 SEQ ID NO: 1222 MUC1R segment 17 Polypeptide encoded by SEQ ID NO: 1199 MUC1R segment 18 Polypeptide encoded by SEQ ID NO: 1201 MUC1R segment 19 Polypeptide encoded by SEQ ID NO: 1203 MUC1R segment 20 Polypeptide encoded by SEQ ID NO: 1205 MUC1R segment 21 Polypeptide encoded by SEQ ID NO: 1207 Differentiation Savine Polypeptide encoded by SEQ ID NO: 1209 BAGE segment 1 Polypeptide encoded by SEQ ID NO: 1211 BAGE segment 2 Polypeptide encoded by SEQ ID NO: 1213 BAGE segment 3 Polypeptide encoded by SEQ ID NO: 1215 GAGE-1 segment 1 Polypeptide encoded by SEQ ID NO: 1217 GAGE-1 segment 2 Polypeptide encoded by SEQ ID NO: 1219 GAGE-1 segment 3 Polypeptide encoded by SEQ ID NO: 1221 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 48 nts 16 aa 16638 nts 5546 aa 90 nts 30 aa 90 nts 30 aa 51 nts 17 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -68- SEQUEATCEID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 1223 SEQ ID NO: 1224 SEQ ID NO: 1225 SEQ ID NO: 1226 SEQ ID NO: 1227 SEQ ID NO: 1228 SEQ ID NO: 1229 SEQ ID NO: 1230 SEQ ID NO: 1231 SEQ ID NO: 1232 SEQ ID NO: 1233 SEQ ID NO: 1234 SEQ ID NO: 1235 SEQ ID NO: 1236 SEQ ID NO: 1237 SEQ ID NO: 1238 SEQ ID NO: 1239 SEQ ID NO: 1240 SEQ ID NO: 1241 SEQ ID NO: 1242 SEQ ID NO: 1243 SEQ ID NO: 1244 SEQ ID NO: 1245 SEQ ID NO: 1246 GAGE-1 segment 4 Polypeptide encoded by SEQ ID NO: 1223 GAGE-1 segment 5 Polypeptide encoded by SEQ ID NO: 1225 GAGE-1 segment 6 Polypeptide encoded by SEQ ID NO: 1227 GAGE-1 segment 7 Polypeptide encoded by SEQ ID NO: 1229 GAGE-1 segment 8 Polypeptide encoded by SEQ ID NO: 1231 GAGE-1 segment 9 Polypeptide encoded by SEQ ID NO: 1233 gpl001n4 segment 1 Polypeptide encoded by SEQ ID NO: 1235 gpl001n4 segment 2 Polypeptide encoded by SEQ ID NO: 1237 gpl001n4 segment 3 Polypeptide encoded by SEQ ID NO: 1239 MAGE-1 segment 1 Polypeptide encoded by SEQ ID NO: 1241 MAGE-1 segment 2 Polypeptide encoded by SEQ ID NO: 1243 MAGE-1 segment 3 Polypeptide encoded by SEQ ID NO: 1245 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 66 nts 22 aa 90 nts 30 aa 90 nts 30 aa 75 nts 25 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -69- SEQUENCEID SEQUENCE LEINFGTH
NUMBER
SEQ ID NO: 1247 SEQ ID NO: 1248 SEQ ID NO: 1249 SEQ ID NO: 1250 SEQ ID NO: 1251 SEQ ID NO: 1252 SEQ ID NO: 1253 SEQ ID NO: 1254 SEQ ID NO: 1255 SEQ ID NO: 1256 SEQ ID NO: 1257 SEQ ID NO: 1258 SEQ ID NO: 1259 SEQ ID NO: 1260 SEQ ID NO: 1261 SEQ ID NO: 1262 SEQ ID NO: 1263 SEQ ID NO: 1264 SEQ ID NO: 1265 SEQ ID NO: 1266 SEQ ID NO: 1267 SEQ ID NO: 1268 SEQ ID NO: 1269 SEQ ID NO: 1270 MAGE-1 segment 4 Polypeptide encoded by SEQ ID NO: 1247 MAGE-1 segment 5 Polypeptide encoded by SEQ ID NO: 1249 MAGE-1 segment 6 Polypeptide encoded by SEQ ID NO: 1251 MAGE-1 segment 7 Polypeptide encoded by SEQ ID NO: 1253 MAGE-1 segment 8 Polypeptide encoded by SEQ ID NO: 1255 MAGE-1 segment 9 Polypeptide encoded by SEQ ID NO: 1257 MAGE-1 segment 10 Polypeptide encoded by SEQ ID NO: 1259 MAGE-1 segment 11 Polypeptide encoded by SEQ ID NO: 1261 MAGE-1 segment 12 Polypeptide encoded by SEQ ID NO: 1263 MAGE-1 segment 13 Polypeptide encoded by SEQ ID NO: 1265 MAGE-1 segment 14 Polypeptide encoded by SEQ ID NO: 1267 MAGE-1 segment 15 Polypeptide encoded by SEQ ID NO: 1269 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 SEQUENCE ID SEQUENCE
NUMBER
SEQ ID NO: 1271 SEQ ID NO: 1272 SEQ ID NO: 1273 SEQ ID NO: 1274 SEQ ID NO: 1275 SEQ ID NO: 1276 SEQ ID NO: 1277 SEQ ID NO: 1278 SEQ ID NO: 1279 SEQ ID NO: 1280 SEQ ID NO: 1281 SEQ ID NO: 1282 SEQ ID NO: 1283 SEQ ID NO: 1284 SEQ ID NO: 1285 SEQ ID NO: 1286 SEQ ID NO: 1287 SEQ ID NO: 1288 SEQ ID NO: 1289 SEQ ID NO: 1290 SEQ ID NO: 1291 SEQ ID NO: 1292 SEQ ID NO: 1293 SEQ ID NO: 1294 MAGE-1 segment 16 Polypeptide encoded by SEQ ID NO: 1271 MAGE-1 segment 17 Polypeptide encoded by SEQ ID NO: 1273 MAGE-1 segment 18 Polypeptide encoded by SEQ ID NO: 1275 MAGE-1 segment 19 Polypeptide encoded by SEQ ID NO: 1277' MAGE-1 segment 20 Polypeptide encoded by SEQ ID NO: 1279 MAGE-3 segment 1 Polypeptide encoded by SEQ ID NO: 1281 MAGE-3 segment 2 Polypeptide encoded by SEQ ID NO: 1283 MAGE-3 segment 3 Polypeptide encoded by SEQ ID NO: 1285 MAGE-3 segment 4 Polypeptide encoded by SEQ ID NO: 1287 MAGE-3 segment 5 Polypeptide encoded by SEQ ID NO: 1289 MAGE-3 segment 6 Polypeptide encoded by SEQ ID NO: 1291 MAGE-3 segment 7 Polypeptide encoded by SEQ ID NO: 1293 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 84 nts 28 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -71- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 1295 SEQ ID NO: 1296 SEQ ID NO: 1297 SEQ ID NO: 1298 SEQ ID NO: 1299 SEQ ID NO: 1300 SEQ ID NO: 1301 SEQ ID NO: 1302 SEQ ID NO: 1303 SEQ ID NO: 1304 SEQ ID NO: 1305 SEQ ID NO: 1306 SEQ ID NO: 1307 SEQ ID NO: 1308 SEQ ID NO: 1309 SEQ ID NO: 1310 SEQ TD NO: 1311 SEQ ID NO: 1312 SEQ ID NO: 1313 SEQ ID NO: 1314 SEQ ID NO: 1315 SEQ ID NO: 1316 SEQ ID NO: 1317 SEQ ID NO: 1318 MAGE-3 segment 8 Polypeptide encoded by SEQ ID NO: 1295 MAGE-3 segment 9 Polypeptide encoded by SEQ ID NO: 1297 MAGE-3 segment 10 Polypeptide encoded by SEQ ID NO: 1299 MAGE-3 segment 11 Polypeptide encoded by SEQ ID NO: 1301 MAGE-3 segment 12 Polypeptide encoded by SEQ ID NO: 1303 MAGE-3 segment 13 Polypeptide encoded by SEQ ID NO: 1305 MAGE-3 segment 14 Polypeptide encoded by SEQ ID NO: 1307 MAGE-3 segment 15 Polypeptide encoded by SEQ ID NO: 1309 MAGE-3 segment 16 Polypeptide encoded by SEQ ID NO: 1311 MAGE-3 segment 17 Polypeptide encoded by SEQ ID NO: 1313 MAGE-3 segment 18 Polypeptide encoded by SEQ ID NO: 1315 MAGE-3 segment 19 Polypeptide encoded by SEQ ID NO: 1317 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -72- SEQUENCE ID SEQ UENE LENGTH
NUMBER
SEQ ID NO: 1319 SEQ ID NO: 1320 SEQ ID NO: 1321 SEQ ID NO: 1322 SEQ ID NO: 1323 SEQ ID NO: 1324 SEQ ID NO: 1325 SEQ ID NO: 1326 SEQ ID NO: 1327 SEQ ID NO: 1328 SEQ ID NO: 1329 SEQ ID NO: 1330 SEQ ID NO: 1331 SEQ ID NO: 1332 SEQ ID NO: 1333 SEQ ID NO: 1334 SEQ ID NO: 1335 SEQ ID NO: 1336 SEQ IDNO: 1337 SEQ ID NO: 1338 SEQ ID NO: 1339 SEQ ID NO: 1340 SEQ ID NO: 1341 SEQ ID NO: 1342 MAGE-3 segment 20 Polypeptide encoded by SEQ ID NO: 1319 MAGE-3 segment 21 Polypeptide encoded by SEQ ID NO: 1321 PRAME segment 1 Polypeptide encoded by SEQ ID NO: 1323 PRAME segment 2 Polypeptide encoded by SEQ ID NO: 1325 PRAME segment 3 Polypeptide encoded by SEQ ID NO: 1327 FRAME segment 4 Polypeptide encoded by SEQ ID NO: 1329 PRAME segment 5 Polypeptide encoded by SEQ ID NO: 1331 PRAME segment 6 Polypeptide encoded by SEQ ID NO: 1333 PRAME segment 7 Polypeptide encoded by SEQ ID NO: 1335 PRAME segment 8 Polypeptide encoded by SEQ ID NO: 1337 PRAME segment 9 Polypeptide encoded by SEQ ID NO: 1339 PRAME segment 10 Polypeptide encoded by SEQ ID NO: 1341 90 nts 30 aa 54 nts 18 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -73- SEQUENCE ID SEQUENCE LENGTH
]TUMIBLR
SEQ ID NO: 1343 SEQ ID NO: 1344 SEQ ID NO: 1345 SEQ ID NO: 1346 SEQ ID NO: 1347 SEQ ID NO: 1348 SEQ ID NO: 1349 SEQ ID NO: 1350 SEQ ID NO: 1351 SEQ ID NO: 1352 SEQ ID NO: 1353 SEQ ID NO: 1354 SEQ ID NO: 1355 SEQ ID NO: 1356 SEQ ID NO: 1357 SEQ ID NO: 1358 SEQ ID NO: 1359 SEQ ID NO: 1360 SEQ ID NO: 1361 SEQ ID NO: 1362 SEQ ID NO: 1363 SEQ ID NO: 1364 SEQ ID NO: 1365 SEQ ID NO: 1366 FRAME segment 11 Polypeptide encoded by SEQ ID NO: 1343 PRAME segment 12 Polypeptide encoded by SEQ ID NO: 1345 FRAME segment 13 Polypeptide encoded by SEQ ID NO: 1347 FRAME segment 14 Polypeptide encoded by SEQ ID NO: 1349 FRAME segment 15 Polypeptide encoded by SEQ ID NO: 1351 FRAME segment 16.
Polypeptide encoded by SEQ ID NO: 1353 FRAME segment 17 Polypeptide encoded by SEQ ID NO: 1355 PRAME segment 18 Polypeptide encoded by SEQ ID NO: 1357 PRAME segment 19 Polypeptide encoded by SEQ ID NO: 1359 PRAME segment 20 Polypeptide encoded by SEQ ID NO: 1361 FRAME segment 21 Polypeptide encoded by SEQ ID NO: 1363 FRAME segment 22 Polypeptide encoded by SEQ ID NO: 1365 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -74- SEQUENC ID SEQUENCE L ENGTH
NUMBER
1 1 SEQ ID NO: 1367 SEQ ID NO: 1368 SEQ ID NO: 1369 SEQ ID NO: 1370 SEQ ID NO: 1371 SEQ ID NO: 1372 SEQ ID NO: 1373 SEQ ID NO: 1374 SEQ ID NO: 1375 SEQ ID NO: 1376 SEQ ID NO: 1377 SEQ ID NO: 1378 SEQ ID NO: 1379 SEQ ID NO: 1380 SEQ ID NO: 1381 SEQ ID NO: 1382 SEQ ID NO: 1383 SEQ ID NO: 1384 SEQ ID NO: 1385 SEQ ID NO: 1386 SEQ ID NO: 1387 SEQ ID NO: 1388 SEQ ID NO: 1389 SEQ ID NO: 1390 PRAME segment 23 Polypeptide encoded by SEQ ID NO: 1367 PRAME segment 24 Polypeptide encoded by SEQ ID NO: 1369 PRAME segment 25 Polypeptide encoded by SEQ ID NO: 1371 PRAME segment 26 Polypeptide encoded by SEQ ID NO: 1373 PRAME segment 27 Polypeptide encoded by SEQ ID NO: 1375 PRAME segment 28 Polypeptide encoded by SEQ ID NO: 1377 PRAME segment 29 Polypeptide encoded by SEQ ID NO: 1379 PRAME segment 30 Polypeptide encoded by SEQ ID NO: 1381 PRAME segment 31 Polypeptide encoded by SEQ ID NO: 1383 PRAME segment 32 Polypeptide encoded by SEQ ID NO: 1385 PRAME segment 33 Polypeptide encoded by SEQ ID NO: 1387 PRAME segment 34 Polypeptide encoded by SEQ ID NO: 1389 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 54 nts 18 aa WO 01/90197 PCT/AU01/00622 SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 1391 SEQ ID NO: 1392 SEQ ID NO: 1393 SEQ ID NO: 1394 SEQ ID NO: 1395 SEQ ID NO: 1396 SEQ ID NO: 1397 SEQ ID NO: 1398 SEQ ID NO: 1399 SEQ ID NO: 1400 SEQ ID NO: 1401 SEQ ID NO: 1402 SEQ ID NO: 1403 SEQ ID NO: 1404 SEQ ID NO: 1405 SEQ ID NO: 1406 SEQ ID NO: 1407 SEQ ID NO: 1408 SEQ ID NO: 1409 SEQ ID NO: 1410 SEQ ID NO: 1411 SEQ ID NO: 1412 SEQ ID NO: 1413 SEQ ID NO: 1414 TRP21N2 segment 1 Polypeptide encoded by SEQ ID NO: 1391 TRP21N2 segment 2 Polypeptide encoded by SEQ ID NO: 1393 TRP21N2 segment 3 Polypeptide encoded by SEQ ID NO: 1395 NYNSO1a segment 1 Polypeptide encoded by SEQ ID NO: 1397 NYNSOla segment 2 Polypeptide encoded by SEQ ID NO: 1399 NYNSOla segment 3 Polypeptide encoded by SEQ ID NO: 1401 NYNSOla segment 4 Polypeptide encoded by SEQ ID NO: 1403 NYNSOla segment 5 Polypeptide encoded by SEQ ID NO: 1405 NYNSOla segment 6 Polypeptide encoded by SEQ ID NO: 1407 NYNSOla segment 7 Polypeptide encoded by SEQ ID NO: 1409 NYNSOla segment 8 Polypeptide encoded by SEQ ID NO: 1411 NYNSOla segment 9 Polypeptide encoded by SEQ ID NO: 1413 90 nts 30 aa 90 nts 30 aa 84 nts 28 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -76- SEQUENCE ID SEQUENCE LENGTH
NUMBER
SEQ ID NO: 1415 SEQ ID NO: 1416 SEQ ID NO: 1417 SEQ ID NO: 1418 SEQ ID NO: 1419 SEQ ID NO: 1420 SEQ ID NO: 1421 SEQ ID NO: 1422 SEQ ID NO: 1423 SEQ ID NO: 1424 SEQ ID NO: 1425 SEQ ID NO: 1426 SEQ ID NO: 1427 SEQ ID NO: 1428 SEQ ID NO: 1429 SEQ ID NO: 1430 SEQ ID NO: 1431 SEQ ID NO: 1432 SEQ ID NO: 1433 SEQ ID NO: 1434 SEQ ID NO: 1435 SEQ ID NO: 1436 SEQ ID NO: 1437 SEQ ID NO: 1438 NYNSOla segment 10 Polypeptide encoded by SEQ ID NO: 1415 NYNSOla segment 11 Polypeptide encoded by SEQ ID NO: 1417 NYNSO1a segment 12 Polypeptide encoded by SEQ ID NO: 1419 NYNSOib segment 1 Polypeptide encoded by SEQ ID NO: 1421 NYNSOib segment 2 Polypeptide encoded by SEQ ID NO: 1423 NYNSOib segment 3 Polypeptide encoded by SEQ ID NO: 1425 NYNSOib segment 4 Polypeptide encoded by SEQ ID NO: 1427 LAGE1 segment 1 Polypeptide encoded by SEQ ID NO: 1429 LAGE1 segment 2 Polypeptide encoded by SEQ ID NO: 1431 LAGE1 segment 3 Polypeptide encoded by SEQ ID NO: 1433 LAGE1 segment 4 Polypeptide encoded by SEQ ID NO: 1435 LAGEl segment 5 Polypeptide encoded by SEQ ID NO: 1437 90 nts 30 aa 90 nts 30 aa 57 nts 19 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 51 nts 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa WO 01/90197 PCT/AU01/00622 -77- SEQUENCE ID :SEQUENE ENGTH
NUMBER_
SEQ ID NO: 1439 SEQ ID NO: 1440 SEQ ID NO: 1441 SEQ ID NO: 1442 SEQ ID NO: 1443 SEQ ID NO: 1444 SEQ ID NO: 1445 SEQ ID NO: 1446 SEQ ID NO: 1447 SEQ ID NO: 1448 SEQ ID NO: 1449 SEQ ID NO: 1450 SEQ ID NO: 1451 SEQ ID NO: 1452 SEQ ID NO: 1453 SEQ ID NO: 1454 SEQ ID NO: 1455 SEQ ID NO: 1456 SEQ ID NO: 1457 SEQ ID NO: 1458 SEQ ID NO: 1459 SEQ ID NO: 1460 SEQ ID NO: 1461 SEQ ID NO: 1462 LAGE1 segment 6 Polypeptide encoded by SEQ ID NO: 1439 LAGE1 segment 7 Polypeptide encoded by SEQ ID NO: 1441 LAGE1 segment 8 Polypeptide encoded by SEQ ID NO: 1443 LAGE1 segment 9 Polypeptide encoded by SEQ ID NO: 1445 LAGE1 segment 10 Polypeptide encoded by SEQ ID NO: 1447 LAGE1 segment 11 Polypeptide encoded by SEQ ID NO: 1449 LAGE1 segment 12 Polypeptide encoded by SEQ ID NO: 1451 Melanoma cancer specific Savine Polypeptide encoded by SEQ ID NO: 1453 Figure 16 A1S1 99mer Figure 16 A1S2 100mer Figure 16 A1S3 100mer Figure 16 A1S4 100mer Figure 16 AIS5 100mer Figure 16 A1S6 99mer Figure 16 A1S7 97mer Figure 16 A1S8 100mer 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 90 nts 30 aa 57 nts 19 aa 10623 nts 3541 aa 99 nts 100 nts 100 nts 100 nts 100 nts 99 nts 99 nts 100 nts WO 01/90197 PCT/AU01/00622 -78- SEQENE 1 ~SEQUENCE LA'NG-TH4 NMB ER? SEQ DD NO: 1463 Figure 16 Al1S9 lO0mer 100 nits SEQ DD NO: 1464 Figure 16 Ai1Sl 10l5mer 76 nts SEQ D NO: 1465 Figure 16 AIF 20mer 20 nts SEQ MD NO: 1466 Figure 16 AIR 20mer 20 nts SEQ H) NO: 1467 Amino acid sequence of inimunostimulatory 16 aa domain of an invasin protein from Yersinia spp.
WO 01/90197 PCT/AU01/00622 -79- DETAILED DESCRIPTION OF THE INVENTION 1. Definitions The articles and "an are used herein to refer to one or to more than one 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.
WO 01/90197 PCT/AU01/00622 "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 cancer antigens) 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, Provinciali M. et al (1992, J. Immunol. Meth. 155: 19-24), cell proliferation assays (see, Vollenweider, I. and Groseurth, P. J. (1992, J. Immunol.
Meth. 149: 133-135), immunoassays of immune cells and subsets (see, Loeffler, D.
et al. (1992, Cytom. 13: 169-174); Rivoltini, et al. (1992, Can. Immunol.
Immunother. 34: 241-251); or skin tests for cell-mediated immunity (see, 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, 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.
WO 01/90197 PCT/AU01/00622 -81 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 polynuclcotide is transcribed and the polypeptide is translated.
WO 01/90197 PCT/AU01/00622 82- 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 ofpolypeptides.
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 sheep, cows, horses, donkeys, pigs), laboratory test animals rabbits, mice, rats, guinea pigs, hamsters), companion animals cats, dogs) and captive wild animals 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, eRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 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 WO 01/90197 PCT/AU01/00622 -83 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, noncomplementary 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, 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 WO 01/90197 PCT/AU01/00622 84units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise a sequence only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and 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 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 Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment 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 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 A, T, C, G, I) or the identical amino acid residue Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, 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 the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present WO 01/90197 PCT/AU01/00622 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, California, 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, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, 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 WO 01/90197 PCT/AU01/00622 -86into 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 nptllI gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
PCT/AU01/00622 Received 07 February 2002 -87- 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 from one or more parent polypeptides, wherein the segments are linked together in a different relationship relative to their linkage in the or each parent polypeptide to impede, abrogate or otherwise alter at least one function associated therewith and wherein any pair of segments so linked does not result in a product whose structure and/or function is substantially similar to at least a portion of the or each 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 three 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 minimise rearrangements that result in complete or partial reassembly of the parent sequence 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.
AMENDED SHEET JPA4JAu PCT/AU01/00622 Received 07 February 2002 -87A 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 AMENDED SHEET IP2A4U WO 01/90197 PCT/AU01/00622 -88function 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 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 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 Cla virus and the parent polypeptide is the Hepatitis Cla virus polyprotein.
WO 01/90197 PCT/AU01/00622 89- 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 gpl00, MART, TRP-1, Tyros, TRP2, MC1R, MUC1F, MUCIR 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, gpl00In4, MAGE-1, MAGE-3, PRAME, TRP2IN2, NYNSOla, NYNSOlb, 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 WO 01/90197 PCT/AU01/00622 stabilising binding by associating with conserved structures on either side of class II MHC antigens in a sequence independent manner (Brown et 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 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, nore preferably at least 12 amino acids, more preferably at least 20 amino acids and more preferably at least 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 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 WO 01/90197 PCT/AU01/00622 -91end 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 nonconserved amino acid differences. Exemplary conservative substitutions are listed in the following table.
TABLE B Original Residue Rxinplaty Substitutons Ala Ser Arg Lys Asn Gin, His Asp Glu Cys Ser Gin Asn Glu Asp Gly Pro His Asn, Gin Ile Leu, Val Leu Ile, Val WO 01/90197 PCT/AU01/00622 -92- Original Residue Exerplaiy Substitutrions, Lys Arg, Gin, 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 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.
WO 01/90197 PCT/AU01/00622 -93- 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 preferably at least 40%, more preferably at least 50%, even more preferably at least 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 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 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 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 WO 01/90197 PCT/AU01/00622 -94the 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 al (1995, Science 269: 202). Syntheses may employ, for example, either t-butyloxycarbonyl (t-Boc) or 9fluorenylmethyloxycarbonyl (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: 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; introducing the synthetic construct into a suitable host cell; culturing the host cell to express the synthetic polypeptide from said synthetic construct; and 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 WO 01/90197 PCT/AU01/00622 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 (HIS 6 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 QIAexpress T M system (Qiagen) useful with
(HIS
6 fusion partners and the Pharmacia GST purification system. In a preferred embodiment, the recombinant polynucleotide is expressed in the commercial vector pFLAGT.
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, WO 01/90197 PCT/AU01/00622 -96or 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 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 WO 01/90197 WO 0190197PCT/AU01/00622 97 Sections 16 and 17; Ausubel et CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley Sons, ic. 1994-1998), in particular Chapters 10 and 16; and Coligan et al., CUIRRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley Sons, Ic. 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, 6aminohexanoic acid, 4-ami-no-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6methyiheptanoic acid, t-butylglyciine, 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 TAB3LE C.
TABLE C j~o-co~vetianal (11nillf, acid }71 6norcelioLnfl an Z1ino acid ci-aminobutyric acid ou-aniino-ou-methylbutyrate aminocyclopropane-carhoxylate aminoisobutyric acid aminonorbornyl-carboxylate cyclohexylalanine cyclopentylalamine L-N-methylisoleucine D-alanine D-arginine D-aspartic acid D-cysteine D-glutainate D-glutamic acid L-N-methylalanine L--N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-N-methylhistidine L-N-methylleucine L-N-methyllysine L-N-methylmnethionine L-N-methylnorleucine L-N-methylnorvaline L-N-rnethylornithine WO 01/90197 WO 0190197PCT/AU01/00622 -98- Non-conventional amnino acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-ornithine D-plienylalanine D-proline D-serine D-threonine D-tryptophan D-tyrosine D-valine D-a-inethylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-oa-methylcysteine D-a-methylglutamine D-a-inethylhistidine D-ca-methylisoleucine D-a-rnethylleucine D-a-naethyllysine D-a-mietlhylrethionine D-a-rnethylornithiine Mon con veional amino acid L-N-methylphenylalanine L-N-methylproline L-N-medlylserine L-N-methylthreoriine L-N-methyltryptophan L-N-methyltyrosine L-N--methylvaline L-N-methylethylglycine L-N-rnethyl-t-butylglycine L-norleucine L-norvaline ci-mthyl-aminoisobutyrate ou-methy-y-aminobutyrate c-methylcyclohexylalanine a-methylcylcopentylalanine oa-methy1-a-napthylalanine a-methylpenicillamine N-(4-aminobutyl)glycine N-(2-aminoethyl)glycine N-(3-aminopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbaniylediyl)glycine >-(carbamylmethyl)glycine WO 01/90197 WO 0190197PCT/AU01/00622 Non7-convenitionaI amlino acid1 99 1Non7-conventOnal amino acid D-a-methylphenylalanine D-a-metliylproline D-c-methylserine D-c-methylthreoniine D-a-methyltryptophan D-a-methyltyrosine L-ca-methylleucine L-a-methylmnethionine L-a-methylnorvatine L-ai-methylphenylalanine L-a-methylserine L-a-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl carbamylmethyl)glycine 1 -carboxy-l1-{2,2-diphenyl-ethyl amino)cyclopropano N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine L-a~-methy11ysine L-a-methylnorleucine L-c-methylomithine L-c-methylproline L-a-methylthreonine L-u-methyltyrosine L-N-mnethylhomophenylalanine N-(N-(3,3-diphenylpropyl carbamytrmethyl)glycine The invention also contemplates modifying the synthetic po1ypeptides 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 polynutieeotides 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.
WO 01/90197 PCT/AU01/00622 100- Sequential synthesis of DNA is described, for example, in U.S. Patent 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 528-535; 1995, Mol Biotechnol. 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 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.
WO 01/90197 PCT/AU01/00622 -101- 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 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 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 a duplicated sequence or a palindromic sequence) that is refractory to the execution of a task 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 WO 01/90197 PCT/AU01/00622 -102above. 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 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 utilised 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 optimisation.
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 ofrecombining 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 WO 01/90197 PCT/AU01/00622 103non-mutant or correct DNA sequences, which results in a mispairing of DNA bases. The mispaired bases are recognised 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. Patent 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 utilised 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 (Lnv) 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 fl 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 WO 01/90197 PCT/AU01/00622 -104in 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 costimulatory 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. Patent 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 utilised 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 WO 01/90197 PCT/AU01/00622 -105described 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), Nacetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), Nacetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( '-2'-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-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.
WO 01/90197 PCT/AU01/00622 -106- 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 inc 1 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 Oral formulations include such normally employed excipients as, for example, pharmaceutical grades ofmamnitol, 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 Soxl8 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 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 using panjet T M delivery) or intramuscular routes.
WO 01/90197 PCT/AU01/00622 -107- 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, (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 WO 01/90197 PCT/AU01/00622 -108expression 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 WO 01/90197 PCT/AU01/00622 -109infections 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 100 is delivered intradermally into a patient at day I and at week 8 to prime the patient. A recombinant poxvirus at 10 7 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 5'Cr 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). Alteratively, 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 Alien et al., supra), proliferation assays (Allen et al., supra), ElispotTM Assays and intracellular INF- PCT/AUO 1/00622 Received 07 February 2002 -110gamma staining (Allen et al., supra), ELISA Assays for linear B cell responses; and Western blots of cell sample expressing the synthetic polynucleotides.
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 segments a parent sequence into segments, and which links those segments 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 a parent polypeptide) is stored in a machine-readable storage medium, which is capable of processing the data to segment the sequence of the parent molecule into segments and to link together the segments in a different relationship relative to their linkage in the parent molecule.
Therefore, another embodiment of the present invention provides a machinereadable 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, segments a parent sequence into segments, and links those segments 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 segment to provide a nucleic acid sequence encoding the segment 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 segments 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 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; a working memory for storing instructions for processing said machine-readable data; AMENDED SHEET IPEA. tU PCT/AUO 1/00622 Received 07 February 2002 -111a 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 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 segments the sequence of the parent polypeptide into segments, code that reverse translates the sequence of a respective segment to provide a nucleic acid sequence encoding the segment, 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 segments 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 Figure 23, which shows a system including a computer 11 comprising a central processing unit 20, a working memory 22 which may be, 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 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 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 AMENDED SHEET i P FA 1A U PCT/AU01/00622 Received 07 February 2002 -112printer 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 Figure 24.
Broadly, these steps include inputting at least one parent polypeptide sequence; (2) optionally adding to alanine spacers at the ends of each polypeptide sequence; (3) segmenting the polypeptide sequences into segments 30 amino acids long), which are preferably overlapping by 15 amino acids); reverse translating the segment to provide a nucleic acid sequence for each segment 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 segments; randomly rearranging the segments; checking whether rearranged segments recreate at least a portion of a parent polypeptide sequence; (7) repeating randomly rearranging the segments when rearranged segments recreate said at least a portion; or otherwise linking the rearranged segments 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 Figure 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 C la and for melanoma, as illustrated in Figures 26 and 27.
Figure 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 Figure 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 AMEN DED SHEE IPEA,Ai WO 01/90197 PCT/AU01/00622 -113magnetic 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 Figure 23.
Figure 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 Figure 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.
WO 01/90197 PCT/AU01/00622 -114-
EXAMPLES
EXAMPLE 1 Preparation of an HIVSavine 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 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 L of water, their concentration determined, then diluted to 20 gM for use in PCR or sequencing reactions.
Long oligonucleotides for splicing reactions were denatured for 5 minutes at 94 0 C in 20 gL of formamide loading buffer then 0.5 gL gel purified on a 6% polyacrylamide gel.
WO 01/90197 PCT/AU01/00622 -115- Gel slices containing full-length oligonucleotides were visualised with ethidium bromide, excised, placed in Eppendorf r M tubes, combined with 200 pL 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 pL 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 HIVinfected 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 s5Cr-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 WO 01/90197 PCT/AU01/00622 -116an 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 clade 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 clade, the rate at which each clade is increasing and the capacity of various regions of the world to cope with the HIV pandemic (Figures 1 and These criteria produced the following ranking, Clade E clade A clade C clade B clade 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.
PCT/AUO 1/00622 Received 07 February 2002 -117- 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 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 each HIV clade compiled by the Los Alamos HIV sequence database (Figures 3-11) (HIV 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 clade E and clade 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 segment 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 segment the AMENDED SHEET iPp.AU PCT/AUO 1/00622 Received 07 February 2002 -118sequences should not destroy potential epitopes. To decide on the best approach for systematically segmenting 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 1-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 segments by at least 15 amino acids to ensure that potential epitopes which might lie across segment boundaries are not lost and to ensure that CTL epitopes near segment boundaries, that are placed beside or near inhibitory amino acids in adjacent segments, are processed efficiently. In deciding the optimal segment 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 segments would be twice the overlap size, in this case 30 amino acids long.
The designed degenerate protein sequences were then separated into segments 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 segment for each protein or envelop protein segment to ensure these segments 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 (sh-)wn in Figure 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 see the gag HIV protein in Figure 13). To cater AMENDED SHEET
!PE!,AU
WO 01/90197 PCT/AU01/00622 -119for 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 (Figure 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, Spel, AvrII or NheI and 3 additional base pairs (to cater for premature Taq polymerase termination) were then added to each end of each subcassette (Figure 14). Some of these extra DNA sequences also contained, the cohesive restriction sites for Sail 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 (Figures 14 and 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/60/68strain and restricted by H2-Db, was inserted before the last stop codon at the end of the HIV Savine design (Figure 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.
WO 01/90197 PCT/AU01/00622 -120- Up to ten long oligonucleotides up to 100 bases long and two short amplification oligonucleotides were synthesised to enable construction of each subcassette (Life Technologies). In designing each oligonucleotide the 3' end and in most cases also the end had to be either a or a 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 Figure 12. Figure 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) (Figure 17a). Each subcassette was then PCR amplified, cloned into pBluescriptTM II KS- using BamHUIEcoRI 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 functional 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 Xba/SpeI/AvrINheI, to generate two copies of cassettes A, B and C as outlined in Figure 14 and shown in Figure 17b. Predicted sequences for these cassettes are shown in Figure 30. Each cassette was then reamplified by PCR with Elongase T M cloned into pBluescript T 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.
WO 01/90197 PCT/AU01/00622 -121- TABLE 1 Summary of mutations Number of mutations Constricti No. aas Designed Expected Actual in2 Non-designed in 2 clones clones 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.
HIVSavine 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 'primeboost' 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 XbaIlXhol (cassette A) or XbaISalI (cassettes B and C) and ligating them into pDNAVacc cut with Xbal/Xhol to create pDVAC1, pDVAC2, pDVBC1, pDVBC2, pDVCC1, pDVCC2, respectively (Figure 18a). These plasmids were then further modified by cloning into their XbaI site a DNA fragment excised using XbaIlAvrI from pTUMERA2 and encoding a synthetic endoplasmic reticulum (ER) signal sequence from the Adenovirus E1A protein (Persson et al., 1980) (Figure 18a). ER signal sequences have been shown previously to enhance the presentation of both CTL and T helper epitopes in vivo (Ishioka, 1999; Thomson et al., 1998). The plasmids pDVERAC1, pDVERBC1, pDVERCC1 andpDVERAC2, pDVERBC2, pDVERCC2 were then mixed WO 01/90197 PCT/AU01/00622 -122together 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/Xhol (cassette A) or BamHI/SalI (cassettes B and C) and cloned into the marker rescue plasmid, pTK7.5, cleaved with BamHI/Sall. These 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 (Figure 18b).
Two further DNA vaccine plasmids were constructed each encoding a version of the full length HIV Savine (Figure 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 pBSAB1 and pBSAB2 respectively. The joined A/B cassettes in pBSAB1 and pBSAB2 were excised with XbaIIXhoI and cloned into pDVCC1 and pDVCC2, respectively, and cleaved with XbaI/XhoI to generate pDVFLl and pDVFL2. These were then further modified to contain an ER signal sequence using the same cloning strategy as outlined in figure 18a.
Restimulation ofHIV 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. Figure 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 recognised 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 PCT/AUO 1/00622 Received 07 February 2002 -123lysis recorded for any other infected target. This probably reflects the combined CTL responses to gag, pol, and env plus other HIV antigens not analysed 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 segmented, 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 segmentation 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 segmentation 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 AMENDED SHEET !PtIAU WO 01/90197 PCT/AU01/00622 -124mutagenic virus, hence this feature was utilised 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 long oligonucleotides was synthesised, 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 2) Only sequence combinations actually present in the HIV clade 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 synthesised, 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 synthesised 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 rcsolvase treatment. Resolvases are bacteriophage-encoded endonucleases which recognise disruptions to double stranded DNA and are primarily used by bacteriophages to resolve PCT/AU01/00622 Received 07 February 2002 -125- 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 hybridisation step mutated DNA hybridises to correct DNA sequences and results in a mispairing of DNA bases which is able to be recognised by the resolvase. This method resulted in a reduction in the frequency of errors. Further optimisation 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 clade 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 segment 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 (Figure 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.
AMENDED SHEET PEA1AU PCT/AU01/00622 Received 07 February 2002 -126- 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 immunise naive mice. Figure 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 (Figure 22) showed, by INF-y 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 segments 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 AMENDED SHEET PCT/AU01/00622 Received 07 February 2002 -127strains which would normally require two Savines to be constructed. However since this Savine is small, the antigen segments 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 1 aand 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 characterise 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 Saviine Synthetic immunomodulatory molecules have also been designed for treating Hepatitis C. In one example, the algorithm of Figure 25 was applied to a consensus polyprotein sequence of Hepatitis C la to facilitate its segmentation into overlapping segments (30 aa segments overlapping by 15 aa), the rearrangement of these segments into a scrambled order and the output of Savine nucleic acid and amino acid sequences, as shown in Figure 26. Exemplary DNA cassettes B and C) are also shown in Figure 26, which contain suitable restriction enzyme sites at their ends to facilitate their joining into a single expressible open reading frame.
AMENDED
SHEET
.RAAu PCT/AU01/00622 Received 07 February 2002 -128- EXAMPLE 3 Melanoma Savine The algorithm of Figure 25 was also applied to melanocyte differentiation antigens (gpl00, MART, TRP-1, Tyros, Trp-2, MC1R, MUC1F and MUC1R) and to melanoma specific antigens (BAGE, GAGE-1, gpl00In4, MAGE-1, MAGE-3, PRAME, TRP2IN2, NYNSOla, NYNSOlb and LAGE1), as shown in Figure 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 PL 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 1iL of 10xPCR buffer, 2 AL of 20 mM MgC12 and 6 pL of MilliQ T M water (MQW) and Taq DNA polymerase were added to the 10 pL DNA sample. The mixture was subjected to the following thermal profile; 95°C for 5min, 65 0 C for 30min, cooled and held at 37 0 C. Five PL of 10xT7 endonuclease I buffer, 8 jL of 1/50 IL of T7endoI enzyme stock and 17 pL 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 agaros, 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) AMENDE R SHE-T_ WO 01/90197 WO 0190197PCT/AU01/00622 129 Buffers were as follows.
IOx T7endonuclease buffer 2.5m1 IM TRIS pH7.8, 0.5m1 IM MgCl 2 25 ItL 1 M DTT, 50,uL 1lOmg/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 AL 1 M TRIS pH7.8, 0.ljxiL IM EDTA pH8, 5 ptL 100 mM glutathione, 50 JZL l0mg/mL BSA, 2.3 mL MQW, 2.5 mL glycerol made up to a total of 5 mL.
Results The results are summarised in Tables 2 and 3.
TABLE 2 Total Errorsf UntratedResolvase treated AIT to G/G =6 AIT to G/C=1I G/C to AIT 12 GIC to AIT= 7 AlT to deletion =1 A/T to deletion I GIG to deletion 6 GIG to deletion 3 TABLE 3 Clone summary11- Uiitreated Resolvase treated 6/11 contained deletions 3/11 contained deletions 9/11 contained mutations 7/11 contained mutations WO 01/90197 PCT/AU01/00622 -130- Clone summary Untreated Resolvase treated 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 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 optimised e.g, by using proofreading PCR enzymes or optimised 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 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 in ajoining 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 WO 01/90197 PCT/AU01/00622 -131 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.
WO 01/90197 PCT/AU01/00622 -132-
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Claims (64)

1. A synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, wherein the segments in the synthetic polypeptide are linked sequentially in a different order or arrangement relative to their linkage in the parent 00 5 polypeptide to impede or abrogate at least one function associated therewith and wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide. t 2. The synthetic polypeptide of claim 1, further comprising one or more segments from at least one other parent polypeptide. 10 3. The synthetic polypeptide of claim 1, further comprising a plurality of Sdifferent segments from at least one other parent polypeptide, wherein those segments are linked together in a different relationship relative to their linkage in the other parent polypeptide(s) to impede or abrogate at least one function associated therewith and wherein the linkage of the segments does not result in complete or partial reassembly of is the other parent polypeptide(s).
4. The synthetic polypeptide of any one of claims 1 to 3, wherein the segments in the synthetic polypeptide are randomly rearranged relative to their order or arrangement in the or each parent polypeptide. The synthetic polypeptide of any one of claims 1 to 4, wherein the size of an individual segment is at least 4 amino acids.
6. The synthetic polypeptide of claim 5, wherein the size of an individual segment is from about 20 to about 60 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 6, comprising at least 30% of the parent polypeptide sequence.
9. The synthetic polypeptide of any one of claims 1 to 8, wherein at least one of the segments comprises partial sequence identity or homology to one or more other of the segments.
10. The synthetic polypeptide of claim 9, wherein the sequence identity or homology is contained at one or both ends of an individual segment.
11. The synthetic polypeptide of claim 10, wherein one or both ends of the segment comprises at least 4 contiguous amino acids that are identical to, or homologous with, an amino acid sequence contained within one or more other of the segments. IBZ\Vaughan\Prosecutions\752598AU]752598AUclaims.doc:THR N 12. The synthetic polypeptide of any one of claims 9 to 11, wherein the size of San individual segment is about twice the size of the sequence that is identical or homologous to the sequence of the or each other said segment.
13. The synthetic polypeptide of claim 12, wherein the size of an individual 00 5 segment is about 30 amino acids and the size of the sequence that is identical or homologous to the sequence of the or each other said segment is about 15 amino acids.
14. The synthetic polypeptide of any one of claims 1 to 13, wherein an optional spacer is interposed between some or all of the segments. The synthetic polypeptide of claim 14, wherein the spacer alters proteolytic processing and/or presentation of adjacent segment(s).
16. The synthetic polypeptide of claim 14 or claim 15, wherein the spacer Scomprises at least one neutral amino acid.
17. The synthetic polypeptide of any one of claims 14 to 16, wherein the spacer comprises at least one alanine residue.
18. The synthetic polypeptide of any one of claims I to 17, wherein an individual parent polypeptide is associated with a disease or condition.
19. The synthetic polypeptide of any one of claims 1 to 18, wherein an individual parent polypeptide is selected from a polypeptide of a pathogenic organism, a cancer-associated polypeptide, an autoimmune disease-associated polypeptide, an allergy- associated polypeptide or a variant or derivative of these. The synthetic polypeptide of claim 19, wherein an individual parent polypeptide is a polypeptide of a virus.
21. The synthetic polypeptide of claim 20, wherein the virus is selected from a Human Immunodeficiency Virus (HIV) or a Hepatitis virus.
22. The synthetic polypeptide of claim 21, wherein the virus is a Human Immunodeficiency Virus (HIV) and the at least one parent polypeptide is selected from env, gag, pol, vif, vpr, tat, rev, vpu and nef, or a combination thereof.
23. The synthetic polypeptide of claim 19, wherein an individual parent polypeptide is a cancer-associated polypeptide.
24. The synthetic polypeptide of claim 23, wherein the cancer is melanoma. The synthetic polypeptide of claim 24, wherein an individual parent polypeptide is a melanocyte differentiation antigen.
26. The synthetic polypeptide of claim 24, wherein an individual parent polypeptide is a melanocyte differentiation antigen selected from gpl00, MART, TRP-1, Tyros, TRP2, MC1R, MUC1F, MUC1R or a combination thereof. [R:\LI B Z\Vaughan\Prosec u tions\7 52 598AU] 752 598A U_clai ms.doc:TH R N 27. The synthetic polypeptide of claim 24, wherein an individual parent polypeptide is a melanoma-specific antigen.
28. The synthetic polypeptide of claim 24, wherein the at least one parent polypeptide is a melanoma-specific antigen selected from BAGE, GAGE-1, gpl00In4, 00 5 MAGE-1, MAGE-3, PRAME, TRP2IN2, NYNSOla, NYNSOlb, LAGE1 or a O combination thereof.
29. A synthetic polynucleotide encoding the synthetic polypeptide of any one n of claims 1 to 28. A method for producing a synthetic polynucleotide, the method comprising: O- linking together in the same reading frame nucleic acid sequences C encoding a plurality of different segments from a parent polypeptide to form synthetic polynucleotide whose sequence encodes the segments linked sequentially in a different order or arrangement relative to their linkage in the parent polypeptide to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide.
31. The method of claim 30, further comprising linking to the nucleic acid sequences in the same reading frame at least one other nucleic acid sequence that encodes a segment from at least one other parent polypeptide.
32. The method of claim 30, further comprising linking to the nucleic acid sequences in the same reading frame a plurality of other nucleic acid sequence encoding a plurality of different segments from at least one other parent polypeptide, wherein the resulting synthetic polynucleotide encodes those segments linked sequentially in a different order or arrangement relative to their linkage in the other parent polypeptide(s) to impede or abrogate at least one function associated therewith and wherein the linkage of those segments does not result in complete or partial reassembly of the other parent polypeptide(s).
33. The method of any one of claims 30 to 32, further comprising segmenting the sequence of an individual parent polypeptide into segments and linking sequentially the segments together in a different order or arrangement to their linkage in the individual parent polypeptide sequence.
34. The method of claim 33, wherein the segments are randomly linked together. R:\LI BZ\Vaughan\Prosecutions\752598AU]752598A Uclaims.doc:THR O 35. The method of any one of claims 30 to 33, further comprising reverse Stranslating the sequence of an individual parent polypeptide or a segment thereof to provide a nucleic acid sequence encoding the parent polypeptide or the segment.
36. The method of claim 35, wherein an amino acid of an individual parent 00 5 polypeptide sequence is reverse translated to provide a codon, which has higher translational efficiency than other synonymous codons in a cell of interest.
37. The method of claim 35, wherein an amino acid of an individual parent Spolypeptide 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.
38. The method of claim 37, wherein the undesirable sequence is selected Sfrom a palindromic sequence or a duplicated sequence, which is refractory, and the task is selected from cloning or sequencing.
39. The method of any one of claims 30 to 38, further comprising linking a spacer oligonucleotide encoding at least one spacer residue between segment-encoding nucleic acids. The method of claim 39, wherein the spacer oligonucleotide encodes 2 to 3 spacer residues.
41. The method of claim 39 or claim 40, wherein the spacer residue is a neutral amino acid.
42. The method of claim 41, wherein the spacer residue is alanine.
43. The method of any one of claims 30 to 42, further comprising 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.
44. The method of claim 43, wherein the variant segment comprises conserved and/or non-conserved amino acid differences relative to one or more other encoded segments. The method of claim 44, wherein the differences correspond to sequence polymorphisms.
46. The method of any one of claims 43 to 45, wherein degenerate bases are designed or built in to the at least one variant nucleic acid sequence to give rise to all desired homologous sequences. R:\IIBZ\Vaughan\Prosecutions\752598AU] 752598AU-claims.doc:THR I 47. The method of any one of claims 30 to 46, further comprising optimising Sthe codon composition of the synthetic polynucleotide such that it is translated efficiently by a host cell.
48. A synthetic construct comprising the synthetic polynucleotide encoding a 00 5 synthetic polypeptide as defined in any one of claims 1 to 28 operably linked to a regulatory polynucleotide.
49. The synthetic construct of claim 48, further including a nucleic acid sequence encoding an immunostimulatory molecule. The synthetic construct of claim 49, wherein the immunostimulatory C 10 molecule comprises a domain of an invasin protein (Inv).
51. The synthetic construct of claim 49, wherein the immunostimulatory C molecule comprises the sequence set forth in SEQ ID NO: 1467 or an immune stimulatory homologue thereof.
52. The synthetic construct of claim 49, wherein the immunostimulatory molecule is a T cell co-stimulatory molecule.
53. The synthetic construct of claim 49, wherein the immunostimulatory molecule is a T cell co-stimulatory molecule selected from a B7 molecule or an ICAM molecule.
54. The synthetic construct of claim 49, wherein the immunostimulatory molecule is a B7 molecule or a biologically active fragment thereof, or a variant or derivative of these. The synthetic construct of claim 49, wherein the immunostimulatory molecule is a cytokine selected from an interleukin, a lymphokine, tumour necrosis factor or an interferon.
56. The synthetic construct of claim 49, wherein the immunostimulatory molecule is an immunomodulatory oligonucleotide.
57. An immunopotentiating composition, comprising an immunopotentiating agent selected from the synthetic polypeptide of any one of claims 1 to 28, the synthetic polynucleotide of any one of claim 29 or the synthetic construct of any one of claims 48 to 56, together with a pharmaceutically acceptable carrier.
58. The composition of claim 57, further comprising an adjuvant.
59. The composition of claim 57, for use as a medicament. The composition of claim 57, for use in modulating an immune response.
61. Use of an immunopotentiating agent selected from the synthetic polypeptide of any one of claims 1 to 28, the synthetic polynucleotide of claim 29, the R:\LIBZ\Vaughan\Prosecutions\752598AU]752598AU-claims.doc:THR 140 ND synthetic construct of any one of claims 48 to 56, or the composition of claim 57 or claim S58 in the manufacture of a medicament for modulating an immune response.
62. The use of claim 61, wherein the immune response is directed against a pathogen or a cancer. 00 s 63. A computer program product for designing the sequence of a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, the t program product comprising: n code that receives as input the sequence of the parent polypeptide; S- code that segments the sequence of the parent polypeptide into segments; 10 code that links sequentially the segments in a different order or Sarrangement relative to their linkages in the parent polypeptide sequence to impede or C abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide sequence; and a computer readable medium that stores the codes.
64. The computer program product of claim 63, further comprising: code that receives as input the sequence of at least one other parent polypeptide; code that segments the sequence(s) of the other parent polypeptide(s) into segments; code that links sequentially those segments in a different order or arrangement relative to their linkage in the other parent polypeptide sequence(s) to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the other parent polypeptide sequence(s); and a computer readable medium that stores the codes. The computer program product of claim 63 or claim 64, further comprising code that randomly rearranges the segments.
66. The computer program product of claim 63 or claim 64, further comprising code that links the sequence of a spacer to the sequence of an individual segment.
67. A computer program product for designing the sequence of a synthetic polynucleotide that encodes a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, the computer program product comprising: code that receives as input the sequence of the parent polypeptide; code that segments the sequence of the parent polypeptide into segments; [RZ:\LI3Z\Vaughan\Prosecutions\752598AU]752598AU-ciaims.doc:THR ID- code that reverse translates the sequence of an individual segment to Sprovide a nucleic acid sequence encoding the individual segment; code that links sequentially in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence 00 5 in which the segments are linked sequentially in a different order or arrangement relative to their linkage in the parent polypeptide sequence to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide sequence; and a computer readable medium that stores the codes.
68. The computer program product of claim 67, further comprising: code that receives as input the sequence of at least one other parent polypeptide; code that segments the sequence(s) of the other parent polypeptide(s) into segments; code that reverse translates the sequence of an individual segment to provide a nucleic acid sequence encoding the individual segment; code that links sequentially in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which the segments are linked sequentially in a different order or arrangement relative to their linkage in the other parent polypeptide sequence(s) to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the other parent polypeptide sequence(s); and a computer readable medium that stores the codes.
69. The computer program product of claim 67 or claim 68, further comprising code that randomly rearranges the nucleic acid sequences. The computer program product of claim 67 or claim 68, further comprising code that reverse translates an amino acid of an individual parent polypeptide sequence to provide a codon, which has higher translation efficiency than other synonymous codons in a cell of interest.
71. The computer program product of claim 67 or claim 68, further comprising code that reverse translates an amino acid of an individual parent polypeptide sequence 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.
72. The computer program product of claim 67 or claim 68, further comprising code that links a spacer oligonucleotide to one or more of the nucleic acid sequences. I R:\l IBZ \Vaughan\Prosecutions\752598AU]752598AUclaims.doc:TH R
73. A computer for designing the sequence of a synthetic polypeptide comprising a plurality of different segments from a parent polypeptide, the computer comprising: a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein the machine-readable data comprise the sequence of the parent polypeptide; a working memory for storing instructions for processing the machine- readable data; a central-processing unit that processes the machine readable data coupled to to the working memory and to the machine-readable data storage medium for processing the machine readable data to provide the synthetic polypeptide sequence, wherein the processing comprises segmenting the sequence of the parent polypeptide into segments and linking sequentially the segments in a different order or arrangement relative to their linkage in the parent polypeptide to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide; and an output hardware coupled to the central processing unit, for receiving the synthetic polypeptide sequence.
74. The computer of claim 73, wherein the processing further comprises segmenting the sequence of at least one other parent polypeptide into segments and linking sequentially those segments in a different order or arrangement relative to their linkage in the other parent polypeptide(s) to impede or abrogate at least one function associated therewith, wherein the linkage of those segments does not result in complete or partial reassembly of the other parent polypeptide(s).
75. The computer of claim 73 or claim 74, wherein the processing of the machine readable data comprises randomly rearranging said segments.
76. The computer of claim 73 or claim 74, wherein the processing of the machine readable data comprises linking the sequence of a spacer to the sequence of the segments.
77. A computer for designing the sequence of a synthetic polynucleotide that encodes a synthetic polypeptide comprising a plurality of different segment of one or more parent polypeptides, the computer comprising: a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein the machine-readable data comprise the sequence of the parent polypeptide; [R:\LI BZ \Va ughan\Proseutiofs\752598AU I752598AU~caims.RL2do:VN B ND a working memory for storing instructions for processing the machine- 0 O readable data; a central-processing unit that processes the machine readable data coupled to the working memory and to the machine-readable data storage medium for processing 00 s the machine readable data to provide the synthetic polynucleotide sequence, wherein the processing comprises segmenting the sequence of the parent polypeptide into segments, reverse translating the sequence of the segments to provide for each segment a nucleic t acid sequence encoding the segment and linking sequentially in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a to polypeptide sequence in which the segments are linked sequentially in a different order or O arrangement relative to their linkages in the parent polypeptide to impede or abrogate at least one function associated therewith, wherein the linkage of the segments does not result in complete or partial reassembly of the parent polypeptide; and an output hardware coupled to the central processing unit, for receiving the synthetic polynucleotide sequence.
78. The computer of claim 77, wherein the processing further comprises segmenting the sequence of at least one other parent polypeptide into segments, reverse translating the sequence of those segments to provide for each segment a nucleic acid sequence encoding the segment, and linking sequentially in the same reading frame each said nucleic acid sequence to provide a polynucleotide sequence that codes for a polypeptide sequence in which the segment are linked sequentially in a different order or arrangement relative to their linkage in the other parent polypeptide(s) to impede or abrogate at least one function associated therewith, wherein the linkage of those segments does not result in complete or partial reassembly of the other parent polypeptide(s).
79. The computer of claim 77 or claim 78, wherein the processing of the machine readable data comprises randomly rearranging the nucleic acid sequences. The computer of claim 77 or claim 78, wherein the processing of the machine readable data comprises reverse translating an amino acid of an individual parent polypeptide sequence to provide a codon, which has higher translational efficiency than other synonymous codons in a cell of interest.
81. The computer of claim 77 or claim 78, wherein the processing of the machine readable data comprises reverse translating an amino acid of an individual parent polypeptide sequence 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. [RALIIBZ\Vaughan\Prosecutions\752598AU]752598AU-claims.doc:THR I 82. The computer of claim 77 or claim 78, wherein the processing of the Smachine readable data comprises linking a spacer oligonucleotide to one or more of the nucleic acid sequences.
83. A method for modulating an immune response directed against a pathogen 00 5 or a cancer, wherein said method comprises administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from the synthetic polypeptide of any one of claims 1 to 28, the synthetic polynucleotide of claim 29, the t synthetic construct of any one of claims 48 to 56, or the composition of any one of claims 58 to
84. The synthetic polypeptides, synthetic nucleotides, synthetic constructs, Scompositions, computer program products or computers substantially as hereinbefore disclosed with reference to any one of the examples. A method for treatment and/or prophylaxis of a disease or condition, wherein said method comprises administering to a patient in need of such treatment an effective amount of an immunopotentiating agent selected from selected from the synthetic polypeptide of any one of claims 1 to 28 or 84, the synthetic polynucleotide of claim 29, the synthetic construct of any one of claims 48 to 56 or 84, or the composition of any one of claims 58 to 60 or 84.
86. The methods for producing synthetic polynucleotides, substantially as hereinbefore disclosed with reference to any one of the examples. Dated 5 May, 2006 The Australian National University Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON R:\LIBZ\Vaughan\Prosecutions\7 52598AU752 598AU_clai ms.doc:TH R
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