GB2406336A - HIV Pharmaccines - Google Patents

Abstract

A recombinant polypeptide comprising amino acid sequence derived from at least one of an HIV gag gene product; an HIV pol gene product; or an HIV nef gene product, said sequence being mutated with respect to the natural sequence of said gene product, and said sequence maintaining each of the naturally occurring CD8+ T cell epitopes of said gene product as defined in p17 and p24 (gag), amino acids 1-440 of RT (pol) and nef shown in Annex 1. Furthermore the invention relates to nucleic acids encoding same, and viral vectors encoding same, and to their use in medicine and in immunisation and vaccination. In a preferred embodiment the recombinant polypeptide comprises at least two of gag, pol, or nef.

Description

HIV Pharmaccines

FIELD OF THE INVENTION

The invention relates to polypeptides comprising HIV antigens, in particular to polypeptides comprising mutated HIV sequences, said mutated sequences retaining their naturally occurring CD8+ epitopes. Furthermore, the invention relates to mutated HIV sequences into which extra T helper epitopes have been introduced.

BACKGROUND TO THE INVENTION

HIV is a pathogenic virus leading to debilitating and fatal immune deficiencies such as AIDS. Although there are certain therapies for conditions such as AIDS which can prolong life expectancy and increase quality of life for affected individuals, the disease is usually terminal.

Eliminating or controlling the virus in HIV infected patients is a problem.

Prevention of infection with HIV is a problem which has thus far been mostly countered by social solutions such as modification of sexual behaviour and/or greater take up of sterile practices for users of hypodermic needles.

Medical prevention of HIV infection remains a high priority area of research. There is a need for effective HIV vaccination strategies.

Even when immune responses are mounted against certain element(s) of an HIV particle, there are problems of immunodominance, viral escape and presentation of viral proteins which can lead to amelioration of the response. Clearly, it is extremely hazardous to render HIV particles for use in vaccines. Furthermore, use of proteins identical to natural proteins may itself be problematic if these proteins have undesirable effects. Furthermore, the proteins may themselves have immunosuppressive qualities and/or may lack sufficient CD8+ T cell epitopes for a suitably strong or broad immune response to be provoked.

Viral escape by mutation of CD8+ T cell epitopes, especially immunodominant CD8+ T cell epitopes, is a significant problem in viral vaccination. Existing vaccines can exhibit poor recruitment of T helper cells and hence produce a narrow and/or weak immune response. Natural viral sequences, subject to evolutionary pressures in viva, may possess relatively few immunologically significant epitopes. Furthermore, immunodominant effects can skew the clinical importance of certain CD8+ T cell epitopes and focus the response (if any) on these. Clearly, this serves to accentuate immunodominance problems, for example leading to sub selection of viral lines which mutate at this epitope, leading to potentially fatal viral escape (ea. see Barouch et al. 2002 Nature 415 p.335).

W002/32943 (Nabel and Huang) disclose modifications of HIV ENV, GAG and POL to enhance immunogenicity for genetic immunization. In particular, they focus on modification of glycosylation of ENV, and on nucleic acid constructs encoding delta CFI HIV ENV. Furthermore, specific deletions of ENV cleavage site, fusogenic domain, and spacing of heptad repeats 1 and 2 are disclosed. Immunization with DNA plasmids encoding GAG alone or GAGPOL is described, the best results being alleged for the gag-pol plasmid immunization. The bulk of W002/32943 is concerned with the disclosure of specific plasmids listed in table 1 and the claims of W002/32943. Also claimed are 'analogs' of various parts of these plasmids, and segments having at least 95% sequence identity thereto.

The present invention seeks, inter alla, to overcome some of the problem(s) discussed above.

SUMMARY OF THE INVENTION

The present invention is based on the design of particularly effective presentation of HIV derived CD8+ T cell epitopes. In this way, the strongest and broadest immune response can be produced.

This is accomplished by altering the context of the polypeptide on which the epitopes are carried. In this way, the natural biological function(s) of the viral polypeptides can be destroyed, advantageously rendering them safe for vaccination use, whilst carefully preserving naturally occurring epitopes which prior art techniques are known to destroy.

In this and other aspects, supplementary (ie. non-naturally occurring) T helper epitopes are also introduced into the antigenic polypeptides, thereby advantageously strengthening and broadening the immune response.

Thus, in one aspect the invention provides a recombinant polypeptide comprising amino acid sequence derived from at least one of (i) an HIV gag gene product; (ii) an HIV pal gene product; or (iii) an HIV nef gene product, said sequence being mutated with respect to the natural sequence of said gene product, and said sequence maintaining substantially all of the the naturally ocurring CD8+ T cell epitopes of said gene product as defined in ply and p24 (gag), amino acids 1-440 of RT (pot) and nef shown in Annex 1.

In another aspect, the invention relates to a recombinant polypeptide as described above comprising amino acid sequence derived from at least two of (i), (ii) and (iii).

In another aspect, the invention relates to a recombinant polypeptide as described above comprising amino acid sequence derived from (i) and (ii) and (iii).

In another aspect, the invention relates to a recombinant polypeptide as described above wherein the amino acid sequences derived from (i) and/or (ii) and/or (iii) are arranged in the order (i) - (ii) - (iii) from the N terminus to the C terminus of the polypeptide.

In another aspect, the invention relates to a recombinant polypeptide as described above comprising SEQ ID NO:9, or a sequence having at least 95% identity thereto.

In another aspect, the invention relates to a recombinant polypeptide as described above further comprising an antibody recognition tag.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein said tag is an HA tag comprising the sequence as shown in SEQ ID NO:8.

In another aspect, the invention relates to a recombinant polypeptide as described above further comprising a CD8+ T cell epitope tag.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein said tag is a gpl60 derived tag comprising the sequence as shown in SEQ ID NO:7.

In another aspect, the invention relates to a recombinant polypeptide as described above, said polypeptide comprising the sequence as shown in SEQ ID NO: 1.

In another aspect, the invention relates to a recombinant polypeptide as described above, said polypeptide comprising amino acid sequence derived from an HIV nef gene product, said recombinant polypeptide sequence being mutated to disrupt the function of said nef sequence, said nef sequence further comprising one or more T helper epitopes which are not present in the naturally occurring nef gene.

In another aspect, the invention relates to a recombinant polypeptide as described above comprising one or more T helper epitopes which are not present in the naturally occurring nef sequence and are shown in Figure 3A.

In another aspect, the invention relates to a recombinant polypeptide as described above further comprising substantially all of the naturally occurring nef CD8+ T cell epitopes as defined in Annex 1.

In another aspect, the invention relates to a recombinant polypeptide as described above further comprising substantially all of the naturally occurring nef T helper epitopes as defined in Annex 1.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein said polypeptide comprises sequence as shown in SEQ ID NO:6, or a sequence having at least 95% identity thereto.

In another aspect, the invention relates to recombinant polypeptide as described above, said polypeptide comprising amino acid sequence derived from an HIV pot gene product, said recombinant polypeptide sequence being mutated to disrupt the reverse transcriptase activity of the pol sequence, wherein substantially all of the CD8+ T cell epitopes of the naturally occurring pal sequence as defined in amino acids 1-440 of RT (pot) shown in Annex 1 are retained in said recombinant polypeptide.

In another aspect, the invention relates to a recombinant polypeptide as described above, wherein the reverse transcriptase activity of said pot sequence is mutated by duplication of an internal sequence derived from the centre of the naturally occurring pot gene and exchange of the amino and carboxy terminal portions of said pal sequence.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein said duplicated internal sequence comprises TPDKKHQKEPPF (SEQ ID NO:4).

In another aspect, the invention relates to a recombinant polypeptide as described above wherein said polypeptide comprises sequence as shown in SEQ ID NO:12 or a sequence having at least 95% identity thereto.

In another aspect, the invention relates to a recombinant polypeptide as described above, said polypeptide comprising amino acid sequence derived from an HIV gag gene product, said recombinant polypeptide sequence being mutated to disrupt processing of the gag gene product, and said gag sequence further comprising a disrupted myristoylation site, wherein substantially all of the CD8+ T cell epitopes of the naturally occurring gag sequence as defined in pl7 and p24 (gag) shown in Annex I are retained in said recombinant polypeptide.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein the processing of gag is disrupted by exchanging the pl7 and p24 domains and wherein the myristoylation site is disrupted by mutation of the second glycine to alanine.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein said polypeptide comprises sequence as shown in SEQ ID NO:13 or a sequence having at least 95% identity thereto.

In another aspect, the invention relates to a recombinant polypeptide as described above wherein the HIV is a clade B HIV.

In another aspect, the invention relates to a recombinant nucleic acid encoding a polypeptide as described above.

In another aspect, the invention relates to a recombinant nucleic acid sequence comprising SEQ ID NO:11, or a sequence which differs only by silent mutations with respect to the genetic code, or a sequence having at least 95% identity thereto.

In another aspect, the invention relates to a viral vector encoding a polypeptide as described above.

In another aspect, the invention relates to a viral vector as described above wherein said vector is an MVA or MVA derived vector.

In another aspect, the invention relates to a viral vector as described above wherein said vector is a fowlpox or fowlpox derived vector.

In another aspect, the invention relates to a viral vector as described above wherein said vector is a FP9 fowlpox vector. Specific teachings with regard to this vector may be found in WO03/047617 which is incorporated herein by reference.

In another aspect, the invention relates to the use of a polypeptide as described above in medicine.

In another aspect, the invention relates to the use of polypeptide as described above in the preparation of a medicament for the treatment or prevention of HIV infection.

In another aspect, the invention relates to the use of polypeptide as described above in the preparation of a medicament for immunization against HIV infection.

In another aspect, the invention relates to the use of a nucleic acid as described above m medcme.

In another aspect, the invention relates to the use of nucleic acid as described above in the preparation of a medicament for the treatment or prevention of HIV infection.

In another aspect, the invention relates to the use of nucleic acid as described above in the preparation of a medicament for immunization against HIV infection.

In another aspect, the invention relates to a method of immunising a subject against HIV infection comprising administering to said subject a polypeptide or nucleic acid as described above.

In another aspect, the invention relates to the use of a polypeptide or nucleic acid as described above as a priming agent or as a boosting agent in a prime-boost immunisation regime. Prime boost immunization is well known in the art, and specific teachings on this subject may be taken from WO98/056919, which is incorporated herein by reference.

In another aspect, the invention relates to the use of a polypeptide or nucleic acid as described herein in the induction of an immune response. Said immune response may be, for example, a cellular immune response, such as a CD8+ or CD4+ response, or a humoral (antibody) response. The invention moreover provides a method for eliciting an immune response in a subject comprising administering to said subject, which may be in need of such administration, a polypeptide, nucleic acid or vector as herein described.

In another aspect, the invention relates to a nucleic acid vector comprising a nucleic acid sequence as described above or encoding a polypeptide as described above.

In another aspect, the invention relates to an adenovirus vector comprising a nucleic acid sequence as described above or encoding a polypeptide as described above.

In a further aspect, the invention relates to a vector based on VSV (vesicular stomatitis virus), adeno-associated virus (AAV), Sendai virus or Herpes Simplex virus comprising a nucleic acid sequence as described above or encoding a polypeptide as described above.

In another aspect, the invention relates to a poxvirus vector comprising a nucleic acid sequence as described above or encoding a polypeptide as described above.

In another aspect, the invention relates to a plasmid selected from the group consisting of p29D.gpn, pOPK6.gpn and pSG2.gpn.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

As used herein, the term "adenovirus" comprises the members of the Adenoviridae (adenovirus family). This family, in turn, comprises three genera: Mastadenovirus, Aviadenovirus and ATadenovirus. In particular, the invention contemplates the use of ovine adenovirus (an ATadenovirus).

A "CD8+ T cell epitope" is an amino acid sequence which is a peptide recognised by CD8+ T cells usually in conjunction with a class I major histocompatibility complex.

In particular, reference to "all" CD8+ T cell epitopes, and/or "all known" CD8+ T cell epitopes, refers to currently known epitopes, as defined in Annex 1 hereto, HIV Molecular Immunology 2002: Maps of CTL Epitope Locations Plotted by Protein; Theoretical Biology & Biophysics, Los Alamos National Laboratory, August 7, 2003.

CD8+ T cells are synonymous with CTLs (cytotoxic T-lymphocytes).

"Substantially all" means at least 99%; preferably, it means at least 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86% or 85%.

A "T helper epitope", likewise, is a peptide recognised by T helper cells usually in conjunction with a class II major histocompatibility complex; "all" and/or "all known" T helper cell epitopes are as defined in Annex 1, HIV Molecular Immunology 2002: Maps of CTL Epitope Locations Plotted by Protein; Theoretical Biology & Biophysics, Los Alamos National Laboratory, August 7, 2003. In accordance with the present invention, substantially all human T helper cell epitopes are retained in the modified polypeptides; however, T helper epitopes relevant in other mammalian species, such as murine T helper epitopes, may be lost. A T helper cell is synonynous with a helper T cell.

"HIV" is Human Immunodeficiency Virus, a virus that causes immunodeficiency by attacking CD4+ cells in the body. The term "HIV", as used herein, includes any HIV, including all groups and subtypes (clades) of HIV-1 and HIV-2, for example HIV-1 M and HIV-1 O groups; Clade B HIV-1 is preferred. Gag, pot and nef gene produce are well known in the art and are as defined, for instance, in Annex 1 hereto.

A "mutation", as referred to herein, encompasses any addition, deletion or substitution of amino acids in a polypeptide or nucleic acid. Mutations, in general, alter the amino acid sequence of the polypeptide in question such that it differs from a or the naturally occurring polypeptide sequence.

A "recombinant" polypeptide, as referred to herein, is a peptide whose sequence differs from a or the naturally occurring equivalent polypeptide and which may be produced by genetic recombination technologies, including DNA synthesis and manipulation. Recombinant peptides also includes peptides which are not produced by recombinant means, but which have designed using recombinant DNA technology or, preferably, have a sequence identical to a peptide designed by such technology.

An "immune response" as referred to herein, is either a cellular (to include, but not limited to, CD4+ and CD8+) or humoral response to an antigenic sequence or a combination of both.

A 'protective immune response" as referred to herein, is an antigen specific immune response that provides a prophylactic and/or therapeutic benefit.

VIRAL ESCAPE

Mutations can occur in any epitope and lead to viral escape. By providing a greater number of epitopes it is more likely that some of the epitopes will remain unmutated.

Furthermore, by advantageously providing extra T helper epitopes, the immune response can be broadened and/or strengthened according to the present invention.

Thus, in one embodiment, the present invention advantageously counters immunodominant effects which can affect conventional vaccines.

It is demonstrated that all of the identified nef human epitopes (both CD8+ T cell and T helper epitopes) in the HIV molecular immunology database are present in the GPN sequence of the present invention (SEQ ID NO: 1).

As noted above, W002/32943 (Nabel and Huang) makes various disclosures in the field of HIV vaccines. As will be apparent from this specification, the present invention is distinct from disclosures made therein. In preferred embodiments, the present invention relates to materials comprising and/or encoding HIV derived proteins which expressly exclude any of those disclosed in W002/32943, and preferably exclude any having 95% or greater identity to those disclosed in W002/32943.

CD8+ T CELL EPITOPES CD8+ T cell epitopes can be identified experimentally and can be predicted by analysis of the sequence of interest. Preferably these epitopes are predicted/recognised using the ProPred program (epitope prediction program, employing a matrix based prediction algorithm as disclosed in Sturniolo et al. Nat. Biotechnol. 17. 555- 561(1999) and Singh and Raghava (2001) Bioinformatics,17(12), 1236-37, such as may be found at (http://www.imtech.res.in/raghava/propred/)).

It is an advantage of the present invention that naturally occurring epitopes are preserved in the polypeptide(s) of interest and in nucleic acids encoding them.

Addition or introduction of new CD8+ T cell epitopes may occur in the process of mutation and gene construction.

T HELPER EPITOPES

T helper epitopes can be identified experimentally and can be predicted by analysis of the sequence of interest. Preferably these epitopes are predicted/recognised using the ProPred program (epitope prediction program, employing a matrix based prediction algorithm as disclosed in Sturniolo et al. Nat. Biotechnol. 17. 555-561(1999) and Singh and Raghava (2001) Bioinformatics,17(12), 1236-37, such as may be found at (http://www.imtech.res.in/raghava/propred/)).

Preferably, in the present invention naturally occurring T helper epitopes are preserved in the polypeptide(s) of interest and in nucleic acids encoding them.

Furthermore, the present invention advantageously provides novel T helper epitopes which have the beneficial effect of boosting and/or broadening the immune response to an antigen bearing said epitopes. In a preferred embodiment of the present invention, increased numbers of T helper epitopes are provided.

In another preferred embodiment of the invention, new T helper epitopes are created and/or introduced into the polypeptides of the present invention, thereby enhancing the immune response.

In some embodiments, the invention comprises a recombinant strain FP9 fowlpox and/or a recombinant modified vaccinia virus Ankara, (MVA), each expressing a novel fusion protein containing antigenic peptide sequences found in the translation products of the gag, pot and nef genes of human immunodeficiency virus type I, (HIV-1), preferably clade B. The invention also relates to the use of such vectors in vaccination methods such as prime-boost (including single prime-multiple boost versions of primeboost). Indeed, as discussed below, the use of either or both of these two recombinant viruses to boost a DNA plasmid-mediated prime has been shown to induce a strong immune response in mammals such as rodents and finds application in primates such as humans. The invention also relates to therapeutic immunotherapy for people infected with HIV-1, for example using the antigenic gene(s) of the present invention in combination with HAART, and/or a prophylactic immunotherapy for people at risk of infection with HIV- I. s Advantageously, vectors according to the invention employ appropriate codon usage to optimise protein expression from mammalian cells. Advantageously, human codon usage is employed.

In a preferred embodiment, therapeutic antigen(s) of the present invention comprise a fusion protein based on the products of the products of the HIV-I (preferably clade-B) gag, pot and nef genes. Preferably said antigen(s) are delivered by one or both of the two recombinant pox viruses described herein.

In one aspect the invention provides a novel fusion protein containing antigenic peptide sequences derived from the translation products of the gag, pot and nef genes of human immunodeficiency virus type I, (HIV- 1), preferably clade B. A preferred amino acid sequence of the fusion protein is shown in Fig 1. A preferred nucleotide sequence encoding this amino acid sequence is shown in Figure 6.

Naturally a person skilled in the art will appreciate that due to degeneracy of the genetic code, numerous possible nucleotide sequences are possible and this is only one preferred example of same.

The gene products are preferably in the order GAG-POL-NEF in the fusion protein.

This is also the way they are arranged in the HIV genome. Other orders may be equally effective. It is well within the abilities of a person skilled in the art to alter the order to meet the needs of a particular application, or even merely to facilitate an easier construction and/or handling of the reagent(s).

In a preferred embodiment, the constructs of the present invention possess advantageous effects regarding immunodominance. Immunodominance has hindered prior art vaccines, especially with respect to gag epitope(s) . This is discussed above.

Three epitopes in GAG in a preferred GPN construct according to the present invention are studied in the example section and each mounted an immune response. It is further disclosed in a preferred embodiment how the immune response across the entire fusion protein is monitored (see below for more details).

It will be apparent that some of the GPN protein could be deleted, for example to reduce the size of the construct. Indeed, certain individual gene(s) are presented herein for individual application. Preferably the gpn constructs disclosed are not truncated.

Without wishing to be bound by theory, it is suggested that a single protein may be less immunogenic if used alone as a smaller entity rather than used as disclosed within a gene. Thus, in a preferred embodiment, gpn genes disclosed herein are used without truncation/deletion.

It will be apparent to the skilled reader that the immunogenic components of the present invention are generally polypeptides. However, the invention relates both to these polypeptides and to nucleic acids encoding them, whether these take the form of DNA, plasmids, viral vectors or other such entities. Indeed, the actual mode of production of the antigenic polypeptides may be varied by a person skilled in the art according to the particular application to which the invention is being put. The polypeptides may be made in vitro or preferably in vivo, whether intra- or extra cellularly. Specific modes of use are described herein.

GAG

The GAG protein has two functional domains, pl7 matrix protein and p24 core capsid protein, both of which are essential and sufficient for assembly of HIV virus-like particles. In the gag gene(s) of the present invention, the pl7 and p24 domains have advantageously been exchanged to disrupt processing of the GAG protein, in accordance with the International Aids Vaccination Initiative (WO01/47955). Other disruptional rearrangements are possible, such as scrambling as was applied to nef (see below), so long as the naturally occurring CD8+ T cell epitopes are preserved.

The myristoylation site of the ply domain is advantageously mutated in order to prevent binding of the GAG protein to cellular membranes and, consequently, assembly and budding. This mutation is preferably by deletion or replacement of the second glycine residue in the domain with another amino acid, more preferably replacement with an inert non-polar amino acid, most preferably by replacement of the second glycine residue in the domain with an alanine residuue. This prevents GAG from functioning in binding to cellular membranes, in virus assembly and in budding.

Advantageously the immunogenicity of gag is not adversely altered in the recombinant gene(s) of the present invention.

POL

The pol gene encodes a number of proteins, including a protease, (plO), reverse transcriptase, (p51), RNase H. (pl5) and an integrase, (p31).

In the present invention, preferably the reverse transcriptase activity present in the P5 1 subunit is disrupted. This is preferably done by rearrangement of the encoding sequence such that an altered P51 polypeptide is made with disrupted RT activity.

Most preferably the sequence TPDKKHQKEPPF (which is present close to centre of the P5 1 polypeptide), is duplicated and the sequence encoding the amino- and carboxy-terminal parts of the P51 polypeptide exchanged about the point between the duplicated sequences.

This novel technique disrupts the active site in POL to ameliorate reverse transcriptase activity. Furthermore, this is advantageously accomplished according to the present invention while retaining the immunogenicity of the gene product. If the active site is mutated ea. by insertion, deletion or other known replacement type mutation, a T cell epitope would be destroyed. According to the present invention such epitope(s) are retained.

NEF

The nef gene encodes a multifunctional protein which enhances virus growth and mediates immune evasion. The NEF protein encoded by the gene in the current invention has been inactivated (ie. its function disrupted) . In a preferred embodiment this inactivation is by dividing the coding region into eight subregions which were then reordered. Preferably these eight regions comprise 6 regions of 26 amino acid and two regions of 25 amino acids. A highly preferred scrambled NEF sequence is presented below (see Figure 2). Exact junctional boundaries in this sequence are easily identified by comparison to the native nef sequence (also presented below) .

The objective of scrambling according to the present invention is to disrupt the functions of nef whilst preserving its T cell epitopes.

In scrambling nef according to the present invention, attention should be paid to disrupt the function of nef whilst creating a minimal number of surplus CD8+ T cell epitopes.

The preferred choice of 8 sub regions disclosed herein represents an advantageous balance between maintaining CD8+ T cell epitopes and not creating too many new junctional CD8+ T cell epitopes. In a preferred embodiment, creation of new junctional CD8+ T cell epitopes is minimised. In this context 'new' CD8+ T cell epitopes are those not occurring in the natural nef sequence. The average length of a CD8+ T cell epitope is 9 amino acids. The preferred GPN fusion protein according to the present invention is

mapped to show where epitopes have been maintained and where new junctional epitopes have been made. This is discussed further below.

When making a scrambled gene according to the present invention, it is advantageous to introduce junctional linkers in order to maintain any epitopes which span the chosen junctions. This may be easily accomplished by review of the sequence(s) using the ProPred tools and making correlating modifications as necessary.

In a preferred embodiment, twelve residue linkers, spanning these subregion junctions, are inserted between the re-ordered subregions to restore T cell epitopes located at the junctions of the eight subregions in the original NEF protein sequence.

SEQUENCES

to Specific sequences are presented in the sequence listing. In brief, SEQ ID NO:1 comprises a full exemplary gpn gene sequence, including tags. In a preferred embodiment, the tags are removed such as for human use, and an exemplary core GPN sequence is presented in SEQ ID NO:9.

SEQ ID NO:2 is the p24 gag fragment, SEQ ID NO:3 is the pl7 gag fragment. In one embodiment, SEQ ID NO:2 and SEQ ID NO:3 are joined (last residue of SEQ ID NO:2 to first residue of SEQ ID NO:3) to make a preferred gag gene according to the present invention.

SEQ ID NO:4 represents a short pot p51 sequence which is advantageously duplicated in a preferred pot construct according to the present invention as explained herein.

SEQ ID NO:5 is the N and C terminal pot sequence. In one embodiment, a larger sequence is constructed in the order SEQ ID NO:4- SEQ ID NO:5- SEQ ID NO:4, resulting in duplication of SEQ ID NO:4, one repeat each side of SEQ ID NO:5 (ie.

one at the N-terminus and one at the C-terminus of SEQ ID NO:5), thereby making a preferred pot gene according to the present invention.

SEQ ID NO:6 is a scrambled nef according to the present invention.

SEQ ID NO:7 is a tag recognised by murine CD8+ T cells, and SEQ ID NO:8 is an HA tag. Either or both of these tags may be advantageously incorporated into gene sequences of the present invention, for example to monitor expression and/or to monitor immune response(s) and/or to monitor for persistence/processing of the protein via the N- or C- termini (preferably via the C-terminus). However, for embodiments involving human subjects, preferably these tags are not incorporated into the gene sequence(s) of the present invention, or are removed from or not expressed on said gene sequence(s).

SEQ ID NO: 10 is the native HIV nef sequence of the HIV clade B consensus sequence protein.

SEQ ID NO:ll is an exemplary nucleotide sequence coding for GPN. Naturally the person skilled in the art will appreciate that due to the degeneracy of the genetic code, many variant nucleotide sequences could equally code for the GPN of the present invention, especially those related to SEQ ID NO: 11 and varying only by translationally silent differences in nucleotide sequence.

As used herein the term sequence 'derived from' an HIV gene product has its natural meaning in the art. Derived from simply indicates that it is based on the HIV sequence ea. as a starting point, whether this is in silica or actually experimental derivation ea.

by cloning, PCR etc. For example the term would include predicted amino acid sequence from an HIV ORE, and is not limited to experimentally derived sequence. If a skilled person can recognise that the sequence originates from HIV, however much it has been rearranged or mutated, then it will be considered to be 'derived from' HIV.

In a preferred embodiment, the sequence derived from another will possess a number of contiguous residues (amino acid or nucelotide) identical to the sequence from which is is derived. Preferably there will be at least 5 such residues, preferably at least 8 such residues, preferably at least 10 such residues, preferably at least 14 such residues, preferably at least 18 such residues, preferably at least 20 such residues, preferably at least 22 such residues, preferably at least 25 such residues. For example, the gag, pot and nef sequences given herein are each derived from HIV.

The term 'mutated' has its usual meaning and includes deletion, insertion, point mutation, truncation, inversion as well as site directed mutation and scrambling as described herein.

It will be appreciated that the invention also embraces nucleic acid fragment(s) of the disclosed nucleotide sequences. Preferably nucleic acid fragments comprise at least 40 nucleotides, preferably at least 50 nucleotides, preferably at least 100 nucleotides, preferably at least 200 nucleotides, preferably at least 400 nucleotides, preferably at least 600 nucleotides, preferably at least 800 nucleotides, preferably at least 1000 nucleotides, preferably at least 1500 nucleotides, preferably at least 2000 nucleotides, preferably at least 2500 nucleotides, preferably at least 3000 nucleotides, preferably at least 3400 nucleotides, preferably at least 3410 nucleotides.

It will be appreciated that the invention also relates to nucleic acids which differ from those presented only by virtue of the degeneracy of the genetic code. The open reading frame(s) of importance when judging variation connected to degeneracy of the genetic code will be those which encode polypeptide(s) of the present invention, particularly encoding core GPN (SEQ ID NO:9 and nucleic acids encoding it).

Unnatural nucleotide residues or analogues or derivatised moieties may feature in nucleic acids according to the present invention.

It will be appreciated that the invention also embraces polypeptide fragment(s) of the disclosed amino acid sequences. Preferably polypeptide fragments comprise at least 8 amino acids, preferably at least 9 amino acids, preferably at least 10 amino acids, preferably at least 12 amino acids, preferably at least 15 amino acids, preferably at least 20 amino acids, preferably at least 25 amino acids, preferably at least 26 amino acids, preferably at least 30 amino acids, preferably at least 40 amino acids, preferably at least 60 amino acids, preferably at least 80 amino acids, preferably at least 100 amino acids, preferably at least 150 amino acids, preferably at least 200 amino acids, preferably at least 300 amino acids, preferably at least 400 amino acids, preferably at least 600 amino acids, preferably at least 800 amino acids, preferably at least 1000 amino acids, preferably at least 1108 amino acids, preferably at least 1125 amino acids.

Unnatural amino acids or analogues thereof or derivatised moieties may feature in the polypeptides of the present invention.

Relative sequence identity may be determined by computer programs which can calculate the percentage identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters. A typical example of such a computer program is CLUSTAL (see Thompson et al., 1994 (NAR 22:4673-80) or http://www.psc. edu/general/software/packages/clustaVclustal.html). Alternatively, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih. gov/BLAST/blast_help.html, which is incorporated herein by reference.

Other computer programs used to determine identity and/or similarity between sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12:387), FASTA (Atschul et al 1990 J Mol Biol 403- 410) and the GENEWORKS suite of comparison tools.

FASTA uses the method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988)) to search for similarities between one sequence (the query) and any group of sequences. FASTA uses the following search parameters: these can be advantageously set to the defined default parameters: Matrix: as for BLAST (not used by FASTA for nucleotide comparisons). Wordsize - the number of continuous residues or bases which are considered at once in the initial comparison; default is 6 for nucleotide sequences, 2 for amino acid sequences. Gap penalty: This is the number of points deducted from a similarity score when a new gap is created; default is 16 for nucleotide sequences, 12 for amino acid sequences. Gap extension penalty: This is the number of points deducted from a similarity score when an existing gap is enlarged; default is 4 for nucleotide sequences, 2 for amino acid sequences. Expect: this restricts the number of sequences returned according to statistical significance; default is 2.

List: this restricts the number of homologous sequences which are reported; default is 40. Align: this restricts the number of homologous sequences for which alignments are displayed; default is 10.

FASTA is available via Biology WorkBench at the University of Illinois (http://biology.ncsa.uiuc.edul!' or from the Genetics Computer Group (GCG) .

BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih. gov/BLASTlblast_help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994:Nature Genetics 6:119-29).

BLAST uses the following search parameters: these can be advantageously set to the defined default parameters: HISTOGRAM - Displays a histogram of scores for each search; default is yes. DESCRIPTIONS - Restricts the number of descriptions of homologous sequences reported; default is 100. EXPECT - The statistical significance threshold for matches between sequences, according to the stochastic model of Karlin and Altschul (1990: PNAS 87:2264-8); default is 10. ALIGNMENTS - Restricts the number of sequences for which alignments are displayed; default is 50. MATRIX Specifies a scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff 1992:PNAS 89:10915-9).

STRAND - Restrict a search to one or other strands of the sequence, (if a nucleotide sequence); default is both strands. FILTER - Masks off segments of the query sequence which have low complexity, as determined by the SEG program of Wootton & Federhen (1993: Computers in Chemistry 17:149-163), or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993: Computers and Chemistry 17:191- 201) or by the DUST program of Tatusov and Lipman (see http://www.ncbi. nlm.nih.gov); default filtering is DUST for BLASTN, SEG for other programs.

Most preferably, sequence comparisons are conducted using the FASTA alignment tool.

Although in general the sequence comparison techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. The present invention embraces sequences possessing significant identity to the exemplary sequences disclosed herein. Preferably a sequence has at least 40% identity to a sequence disclosed herein, preferably at least 45% identity, preferably at least 50% identity, preferably at least 55% identity, preferably at least 60% identity, preferably at least 65% identity, preferably at least 70% identity, preferably at least 75% identity, preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, more preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, or even more.

OPTIMISATION OF GENE CONSTRUCTS

In a preferred embodiment, a 'Kozak' concensus sequence, GCCGCC, is placed upstream of the initiation codon of the gene construct(s).

In a preferred embodiment, the codon usage of the sequence may be modified to optimize the efficiency of translation of the gag-pol-nef transcript in human cells.

In a preferred embodiment, an Apa I and an Asc I restriction endonuclease recognition site are placed at the 5-prime and 3-prime ends of the gagpol-nef (gpn) gene respectively. This advantageously facilitates subcloning of the gene cassette. In this embodiment, the restriction sites will be present in recombinant virus but are advantageously outside the open reading frame of the GAG-POL-NEF fusion protein.

Consequently they will not have any influence ori the amino acid sequence of the fusion protein nor the immune response generated to that protein. ApaI and AscI were also chosen to advantageously allow direct cloning into the pOPK6 recombinant plasmid. They have the further advantage of exerting no known influence on gene function.

In a preferred embodiment, a sequence encoding a reporter CD8+ T cell epitope, RGPGRAFVTI (SEQ ID NO:7), recognized by murine CD8-positive T cells specific for the gpl60 protein, may be incorporated into a recombinant gene of the present invention. This has the advantage of allowing monitoring of the induction of CD8+ T cells following immunization with GPN-containing vaccines. Advantageously the presence of the epitope is not known to affect the function of the fusion gene. In a highly preferred embodiment, this reporter epitope is absent from construct(s)/recombinant gene(s) for primate vaccination, such as human vaccincation.

In a preferred embodiment, an additional antibody tag, YPYDVPDYA (SEQ ID NO:8), recognized by antibodies specific for this part of the influenza virus haemagglutinin protein, is added to the the gene of the present invention (or to the nucleic acid encoding it). Preferably this is added to the carboxyterminus of the protein to allow the detection of expression in antibody-based immunoassays, such as western blot assays'. In a highly preferred embodiment, this tag is incorporated into the carboxyterminus of a recombinant gag-pol-nef gene, such as in a recombinant vector comprising a recombinant gag-pol-nef gene. In a highly preferred embodiment, this antibody tag is absent from construct(s) /recombinant gene(s) for primate vaccination, such as human vaccincation.

PHARMACEUTICAL COMPOSITIONS

The present invention also provides a pharmaceutical composition comprising administering a therapeutically effective amount of the agent of the present invention (such as a recombinant HIV gene such as the recombinant gpn gene as discussed herein) and a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).

The pharmaceutical composition may comprise two components - wherein a first S component comprises a nucleic acid vector and a second component which comprises a viral vector thereof. The first and second component may be delivered sequentially, simultaneously or together, and even by different administration routes.

The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.

1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.

Alternatively, the formulation may be designed to be delivered by both routes.

Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

PHARMACEUTICAL COMBINATIONS

The agent of the present invention may be administered with one or more other pharmaceutically active substances. By way of example, the present invention covers the simultaneous, or sequential treatments with an agent according to the present invention and one or more steroids, analgesics, antivirals or other pharmaceutically active substance(s).

The invention also finds application in a therapeutic immunotherapy for people infected with HIV such as HIV-1. The invention may be used in combination with HAART, and/or in combination with a prophylactic immunotherapy for people at risk of infection with HIV such as HIV-1.

It will be understood that these regimes include the administration of the substances sequentially, simultaneously or together.

The present invention will now be described by way of example, which is not intended to limit the scope of the appended claims, in which reference will be made to the following figures:

Brief Description of the Figures

Figure 1 shows preferred gpn gene constructs. The sequence is continuous and has only been separated here for clarity. bold = sequence variation between GPN sequence and the HIV Molecular Immunology sequence.

Figure 2 shows scrambled and native nef gene sequences used for MHC Class II epitope comparisions.

Figure 3 shows T helper epitopes for scrambled nef (fig 3A) and native nef (fig 3B).

Figure 4 shows a map of recombination plasmid, p29D.gpn, for the construction of a recombinant fowlpox strain FP9 expressing gag-pol-nef.

Figure 5 shows a map of recombination plasmid, pOPK6.gpn, for the construction of a recombinant MVA expressing gag-pol-nef.

Figure 6 shows the sequence of GPN. The GPN sequence is shown in normal text. The upstream region is shown in italics. 5-prime ApaI and 3-prime AscI sites are underlined. Initiating ATG and terminating TGA are shown in bold.

Figure 7 shows a bar chart of an IFN-y ELISpot as described in Example 3.

Figure 8 shows a bar chart of an IFN-y ELISpot as described in Example 5.

Figure 9 shows overlapping 20mer peptides used in an IFN-y ELISpot assay. Amino acid length shown in brackets.

EXAMPLES

Example 1: Analysis of CD8+ T cell and T helper epitopes in the GPN sequence.

We used ProPred to compare T helper epitopes for native NEF versus scrambled NEF.

This program predicts that there are more T helper epitopes in scrambled NEF according to the present invention. This demonstrates that the scrambled NEF sequence is at least as potent in eliciting T helper and CD8+ T cell responses as native NEF.

CD8+ T cell Epitopes CD8+ T cells can recognise and eliminate virally infected T cells and have been associated with control of viraemia in HIV infection in man and SIV infection in monkeys.

Comparison of the GPN sequence (Figure 1) with the HXB2 reference sequence published in the HIV molecular immunology database ("HIV Molecular Immunology 2002: Maps of CD8+ T cell Epitope Locations Plotted by Protein" Theoretical Biology and Biophysics, Los Alamos National Laboratory. August 7, 203; http://hiv web.lanl.gov/content/immunolocy/maps/ctl/ctl. pdf) is performed.

This comparison reveals that all of the known murine and human CD8+ T cell epitope sequences identified for native gag (ply, p24), truncated- pol (p51) and nef are present in the GPN polypeptide sequence. There are single amino acid differences between the GPN sequence and the HXB2 reference sequence. Epitope sequences are indicated on the protein sequences of HXB2 and provide a relative location of the defined epitopes although they may vary relative to the protein sequence from which they were defined (refer to HIV Molecular Immunology Database 2002: Section II-A-2 - HIV Protein Epitope Maps, p56; http://hiv-web.lanl. cov/content/immunolov/pdf/2002/ immuno2002.pdf).

Therefore, shuffling and rearrangements within the GPN sequence have not altered per se the capacity of this molecule to induce CD8+ T cell responses in mice, monkeys or man against the native gag (ply, p24), truncated-pol (pS1) and nef sequences present in HIV.

Scrambling and rearrangement of the native polypeptides constituting the GPN protein may have created new CD8+ T cell epitopes unique to this molecule. Since these epitopes are unlikely to be present in native gag, pol or nef sequences within the HIV virus, they are therefore unlikely to be of biological relevance for a novel antigen for inclusion in a vaccine against HIV. Consequently, these epitopes have not been investigated further in this example.

T helper epitopes T helper immune responses enhance the CD8+ T cell effecter response in terms of magnitude and breadth and therefore may enhance the efficacy of the CD8+ T cell response against HIV infection.

Comparison of the GPN sequence with the HXB2 reference sequence published in the HIV molecular database ("HIV Molecular Immunology 2002: Maps of T helper Epitope Locations Plotted by Protein" Theoretical Biology and Biophysics, Los Alamos National Laboratory. August 7, 203; http://hivweb.lanl.gov/content/inlmuno]o=Y/maps/helper/lle]per.pdl) reveals that all of the human T helper epitope sequences identified for native gag (ply, p24), truncated-pol (pS1) and nef are present in the GPN polypeptide sequence. Therefore, shuffling and rearrangements within the GPN sequence have not reduced the capacity per se of this molecule to induce T helper responses against the native gag (ply, p24), truncated-pol (p5 1) and nef sequences present in the clade B virus in humans.

Since T helper epitopes are usually longer than 15 amino acids, fusion of the gag, truncated-pol and nef sequences in GPN is likely to have advantageously created novel epitopes bridging the points at which these genes or parts of genes have been fused.

Moreover, shuffling of the nef gene is likely to have created a number of novel epitopes bridging the shuffled sections of resulting polypeptide. Such novel T helper epitopes advantageously may elicit immune responses when GPN is administered as a vaccine, thereby enhancing the breadth and strength of the biologically-relevant CD8+ T cell responses elicited by the polypeptide(s) of the present invention relative to the native GAG, truncated-POL and NEF proteins.

To demonstrate that shuffling had created potential new T helper epitopes, the native NEF and scrambled NEF polypeptide sequences (Figure 2) are compared using the Propred MHC Class II epitope prediction program, employing a matrix based prediction algorithm as disclosed in Sturniolo et al. Nat. Biotechnol. 17. 555 561(1999) and Singh and Raghava (2001) Bioinformatics,17(12), 1236-37, such as may be found at (http://www.imtech.res.in/raghava/propred/).

Comparison of these molecules reveals substantial changes in the predicted human MHC class II restricted T helper epitopes, with a greater number of predicted epitopes in the scrambled NEF sequence than the native NEF sequence (Figures 3A and 3B).

Thus, scrambling of the nef sequence has resulted in the creation of additional T helper epitopes for enhancement of the CD8+ T cell response against this polypeptide according to the present invention. These advantageous novel T helper epitopes comprise those epitopes shown in Figure 3A and which are absent from Figure 3B.

Similar novel T helper epitopes will have been generated across the fusion points between the proteins comprising the GPN polypeptide. Comparison as described above may be used to define/investigate these further.

Example 2: Recombinant GPN gene in DNA, MVA and Fowlpox A recombinant gagpol-nef (gpn) gene is constructed for expression of a GPN fusion protein. This gene construct is used in nucleic acid carrier such as DNA carrier (ea.

plasmid carrier), as well as in MVA and fowlpox vectors and materials for construction of same.

The use of recombinant virus vectors such as MVA and/or fowlpox vectors to boost a DNA plasmid-mediated prime has been shown to induce a strong immune response in rodents.

Essentially the same constructs also find application in primates such as humans.

In this example, the therapeutic antigen delivered by the recombinant pox viruses comprises a fusion protein based on the products of the products of the HIV-I clade-B gag, pot and nef genes.

The recombinant gag-pol-nef gene was synthesized as a series of overlapping oligonucleotides, amplified by use of polymerase chain reaction cloned into the commercially available plasmid, pUCI9, to make pUCl9.gpn, (or 02-213), and the nucleotide sequence determined.

The gag-pol-nef gene is subcloned into three plasmid vectors to make i) a plasmid expression plasmid, pSG2.gpn for use as a priming agent for in vivo use such as preclinical testing of gpn-containing recombinant poxvirus vectors; ii) a recombination plasmid, pOPK6.gpn, for the construction of a recombinant MVA expressing gag-pol-nef; iii) a recombination plasmid, p29D.gpn, for the construction of a recombinant fowlpox strain FP9 expressing gag-pol-nef.

The expression plasmid, pSG2.Mel3, (Palmowski, MJ. et al 2002 J Immunol 168 p4391-4398) contains an expression cassette based on the human cytomegalovirus immediate-early promoter and intron combined with the bovine growth hormone gene polyadenylation signal to express a synthetic epitope string containing melanoma antigens. The expression plasmid pSG2. gpn is made by digesting pUCl9.gpn with ApaI and AscI, blunt-ending the insert, and subcloning into blunted PstI-digested pSG2Mel3.

The MVA recombination plasmid, pOPK6 is made as follows. The plasmid is based on the commercially available cloning plasmid, pSP72, (Promega Inc.) ; the standard multiple cloning site being replaced with a synthetic linker containing unique restriction sites and the vaccinia virus P7.5 late-early promoter and late P11 promoter arranged in a head-to-head orientation. The linker also has the first twenty nucleotides of theEscherichia cold LacZ, (beta-galactosidase), gene as found in the vaccinia virus recombination plasmid pSC11, (Chakrabarti et al 1985 Mol Cell Biol 5 p3403-3409).

The synthetic linker is synthesized as a series of overlapping oligonucleotides, amplified by PCR, and cloned into the commercial cloning plasmid, pcDNA3.1, to make plink, (or 02-363). The sequence of the 300 base pair linker is given below: agatctttaattaatgcctaggcaattgagacggcgcgcccgggcactagtaagggcccgtgcaataaattaga atatattttc tacttttaccagaaattaattgtacaatttattatttatgggtgaaaaacttactataaaaagcgggtgggttt ggaattagtggtac catgcatcttagaatatatgtatgtaaaaatatagtagaatttcattttgtttttttctatgctataaatgaat tcctcaagggatccgt ctcctgcaggcatgctaagctagcggccggccctcgag Part of the LacZ gene from pSC11 is subcloned into pLINK to make pLinkLacZ. The linker and LacZ gene were then subcloned into pSP72 as a XhoI-BglII fragment such that a complete beta-galactosidase-encoding open reading frame is formed 3-prime to the P11 late promoter to make pSP72LinkLacZ.

MVA flanking regions representing 1500 base pairs 5-prime and 3-prime to the EcoRI site in the MVA thymidine kinase (tk) gene were isolated from MVA by PCR using the following primer-pairs: Left-hand, (5-prime) tk flank Forward primer CAATTACAGATTTCTCCGTGATAGGT Reverse primer CGTGCATGCGGCCGCAACAATGTCTGGAAAGAACTG Right-hand, (3-prime) tk flank Forward primer CAGGAATTCGCGGCCGCTGTGAGCGTATGGCAAACG Reverse primer TCATTTGCACTTTCTGGTTCGTA The 1500bp PCR products are cloned into the commercial cloning vector pTOPO-TA, (Invitrogen) and sequenced.

The right-hand flank is subcloned into pSP72LinkLacZ as an EcoRI-MfeI fragment to make p72LinkLacZR. The left-hand flank insert is then subcloned into p72LinkLacZR as an NheI-SphI fragment to make pOPK6.

Subsequent sequence analysis of this plasmid identified an additional 174 base pairs of sequence in the pOPK6 backbone plasmid introduced from the pTOPO-TA vector with the left-hand flank due to non-specific cleavage by the NheI enzyme.

The gag-pol-nef open reading frame was subcloned from pUCl9.gpn into pOPK6 as an ApaI-AscI fragment to make pOPK6.gpn and the gpn insert sequenced to confirm no alterations had occurred during the subcloning steps.

This plasmid functions as a vehicle for introducing the gpn open reading frame, under control of the P7.5 late-early promoter, into the tk locus of MVA.

FP9 vector The FP9 strain fowlpox recombination plasmid, pEFL29, was obtained from Dr Mike Skinner at the Institute for Animal Health, Compton, UK as published by Qingzhong, Y. et al 1994, Vaccine 12(6) p569-573.

The gpn open reading frame was subc]oned from pUCl9.gpn as a blunted KpnISacI fragment into the SmaI site of pEFL29 to make pEFL29.gpn. Sequencing of this plasmid showed that LacY and part of LacA had been introduced with the LacZ marker gene during the construction of pEFL29. Consequently, a linker was synthesized that allowed the deletion of all of the LacY and most of the remaining LacA open reading frames by means of a BsrGI- BlpI digest of pEFL29 to make p29Delta. The sequence of the linker is given below.

TGAGCGCCGGTAGATACCATTATCAGCTGGTGTGGTGTCAGAAGTAATGTA

C

The gpn expression cassette was then subcloned from pEFL29.gpn as an AatII-SphI fragment into AatII-SphI cut p29Delta to make p29D.gpn. The gpn insert was then sequenced to confirm no alterations had occurred during the subcloning steps.

The recombination plasmids, pOPK6.gpn and p29D.gpn are used to introduce the P7.5-gpn expression cassette into MVA and FP9, respectively.

The recombinant poxviruses were made by homologous recombination of the virus genome with the relevant recombination plasmid in chicken embryo fibroblast, (CEF), cultures using standard molecular biology techniques as described in Current Protocols in Molecular Biology, Ed. F.M. Ausubel, John Wiley & Sons; or according to suppliers' or manufacturers' instructions.

MVA.gpn is made by infecting confluent cultures of CEF cells with a nonrecombinant MVA, (stock 575FHE-K), derived from MVA passage number 575, obtained from Anton Mayr, University of Munich, Germany (Mayr et al., (1978) Zentralbl Bakteriol [b]. 167(5-6):375-90) at a multiplicity of infection of 0.1 for one hour. The infected cells are then transfected with between 0.5 and 2.0pg pOPK6.gpn plasmid DNA mixed with lipofectin (Invitrogen), according to the manufacturer's protocol. The cultures are incubated for three days before harvesting. The harvested cells are freeze-thawed three times and titrated as ten-fold dilutions and cultured under CMC-containing medium for three days. The medium is removed and replaced with CMC-medium containing X-Gal and the cultures incubated in this medium for 20 hours.

S Recombinant viruses are identified by a blue plaque phenotype and picked with a pipette into cryotubes containing 100,1 medium. The picked viruses are plaque purified five more times to ensure purity. Punty from non-recombinant parental virus was confirmed by the absence of white plaque phenotype and the absence of a WT PCR product when the virus DNA is screened using appropriate primers.

Purity PCR screening was performed on virus DNA purified from 2ml cultures infected with fifth or sixth round clones of MVA.gpn.

The virus DNA is purified using a Qiagen Blood and Cell Culture DNA minkit (Qiagen GmbH, Max Volmar qtr. 4, 40724 Hilden, Germany) according to the manufacturer's protocol. A PCR reaction was performed using Klentaq proof reading polymerase (Sigma) using appropriate incubation conditions.

Parental virus was screened for using the following primers to give a band of 471 base pairs in the wild-type, but nothing in MVA:

TKU CAATTACAGATTTCTCCGTGATAGGT

TKL TCATTTGCACTTTCTGGTTCGTA

MVA.gpn was screened for using primer TKL and the following primer to give a band of approximately 2000 base pairs from the MVA.gpn construct:

HIV-U ATACCCCCGTGTTCGCCATTAAGA

Expression of the GPN protein from MVA.gpn is confirmed by immunoblotting as described below.

CEF cells are infected with MVA.gpn or MVA.LacZ control virus at a multiplicity of infection of 1. The culture is incubated for 3 days before harvesting. The cells are heated to 100 degrees C in SDS sample buffer and electrophoresed through a 4-20% acrylamide gel and electroblotted onto a nitrocellulose membrane. The transferred protein is then probed with either an antibody specific for the haemagglutinin tag (Abeam), or one specific for HIV GAG P24, (Dako) and visualized using a suitable peroxidase-labelled secondary antibody and a chromogen. The blot assay indicates that the cells infected with MVA.gpn contain a protein of approximately 134kDa containing P24 and HA epitopes that was absent in the cells infected with MVA.LacZ.

The predicted molecular weight of the GPN protein is 128.5 kDa, which is in close agreement with the specific band produced in the MVA.gpninfected cells.

Expression of the GPN protein from FP9.gpn is confirmed by immunoblotting. CEF cells are infected with FP9.gpn or FP9.LacZ, ('FP9.29D'), control virus at a multiplicity of infection of 1. The culture is incubated for 5 days before harvesting.

The cells are heated to 100 degrees C in SDS sample buffer and electrophoresed through a 4-20% acrylamide gel and electroblotted onto a nitrocellulose membrane.

The transferred protein is then probed with either an antibody specific for the haemagglutinin tag (Abeam), or one specific for HIV GAG P24, (Dako) and visualized using a suitable alkaline phosphatase-labelled secondary antibody and a chromogen.

The blot assay indicated that the cells infected with FP9.gpn contained a protein of approximately 134kDa containing P24 and HA epitopes that is absent in the cells infected with FP9.29D. The predicted molecular weight of the GPN protein is 128.5 kDa, which is in close agreement with the specific band produced in the FP9.gpn- infected cells.

The immunogenicity of the MVA.gpn and FP9.gpn viruses was tested in BALB/c mice using a 'prime-boost' protocol. The mice were divided into groups of four animals.

The following test and control groups were established: pSG2.gpn (Soil) prime followed 14 days later by a boost 1 x l of p.f.u. of MVA.gpn.

pSG2.gpn (50) prime followed 14 days later by a boost 1x106 p.f.u. of FP9. gpn.

Ix106p.f.u. of MVA.gpn alone.

l x l o6 p.f.u. of FP9.gpn alone.

7 days after virus infection the peripheral blood lymphocytes were harvested and the number of GPN-specific interferon gamma-secreting lymphocytes determined in an ELISpot assay using the HIV following T cell epitopes (see also example 3): Hz-D CD8 epitopes: AMQMLKETI

TTSTLQEQ

Gpl60 Hz-D CD8 epitope: RGPGRAFVTI Hz-D CD4 epitope: NPPIPVGEIYKRWIILGLNK Both MVA.gpn and FP9.gpn are shown to induce strong CD8+ T cell responses in the mice.

The prime-boost protocol is shown to give greater response than that seen in the groups receiving a single injection with virus.

Example 3: Immunisation with FP9.gpn and MVA.gpn elicits antigen specific CD8+ T cell responses In this example it is demonstrated that FP9.gpn and MVA.gpn elicit enhanced antigen- specific CD8+ T cell responses when administered alone or in a prime- boost immunization regime with pSG2.gpn. In this example, the demonstration is presented in mice.

* Female BALB/c mice (6 - 8 weeks old) are immunised with Song of pSG2.gpn by intramuscular (im.) injection and boosted with l X 106 PFU FP9.gpn or 1 X 1Os MVA.gpn by intravenous injection (iv.) two weeks later. A further group of age matched nave female BALB/c mice are immunised iv. with 1 X 106 PFU FP9.gpn or 1 X 105 MVA.gpn at the same time as the booster immunisation.

Fourteen days after the booster immunisation, all mice are sacrificed by cervical dislocation and the T cell response elicited against three H-24 restricted CD8+ epitopes from the GPN polypeptide (Table A: AMQ, TTS, RGP) is determined by IFN-y ELISpot assay as described below.

Murine IFNy ELISpot protocol: Materials: IFN-y ELISpot ALP Kit Mabtech 3321-2A 600pg anti-IFN-y purified Mab AN18 5ollg anti-IFN-y biotinylated Mab R46A2 5O'll Streptavidin-Alkaline Phosphatase Complete a-MEM medium 500ml MEM a-modification Sigma M-4526 50ml FCS [10%] Sigma F-2442 Sml per/strep [ l OOU penicillin l OOtlg strep] Sigma P-0781 1 Oml L-glutamine [4mM] Sigma G-7513 500)l12-Mercaptoethanol [50pm] Gibco BRL 31350-010 ACK buffer 8.29g NH4CI [O.1 SM] (Sigma A-4514) lg KHCO3[1mM] (Sigma P-9144) 37.2mg Na2EDTA (Sigma ED2SS) 800ml milli-Q water Adjust pH to 7.2-7.4 with HCI (Sigma S-7653) Make up to 1000 ml with water and autoclave Colour Development Buffer: BioRad AP Conjugate Substrate kit (170-6432).

For one plate: 5ml deionised water 200111 of 25x buffer 501 reagent A 50111 reagent B Mix well and use immediately Protocol 1. Preparation of Plates: 1.1. Coating plates: coat MAIP multiscreen plates (Millipore MAIPS4510) with rat anti-mouse IFNy (Mate AN18) antibody. Dilute to lO'lg/ml in Phosphate Buffered Saline (PBS; Sigma P-3813) and add 501 per well to MAIP plates.

Incubate overnight at 4 C in a humidified chamber 1.2.Blocking plates: Flick off coating antibody and wash plates once with 150 ul of sterile PBS (Sigma P-3813) per well using a multi-channel pipette. Flick off the PBS, add lOOul complete a-MEM medium per well, and incubate at room temperature for 1+ hour. It is important to keep the plates sterile at this stage.

2. Splenocyte preparation: 2.1.Crush individual spleens in 2 ml of PBS with the plunger of a 10 ml syringe in a 70 ham cell strainer (Falcon 352350) contained in a petri dish, add 5 ml of PBS, suspend splenocytes by pipetting, and transfer into a 50 ml tube. Rinse cell strainer and dish with a further 10 ml of PBS and add to the 50 ml tube.

Centrifuge at 1500 rpm for 5 min. 2.2.Remove supernatant, re-suspend cells by tapping tube and add 5ml ACK buffer and mix by inversion. Incubate at room temperature for no longer than 5 minutes. Add 25 ml PBS, mix by inversion and centrifuge at 400 X g for 5 min. 2.3.Remove supernatant re-suspend pellet by tapping the tube, add lOml PBS and vortex. Count using an improved Neubauer haemacytometer by diluting 1:10 in 0.4% trypan blue solution (Sigma T-8154). Aliquot amount needed for the Elispot and centrifuge at 1500 rpm for 5 min. resuspend by vortexing in an appropriate volume of complete Alpha MEM medium to give a concentration of 10 million cells/ml.

3. Plate setup: 501 501 501 1 2 3 4 5 6 7 8 9 10 11 12 150 150 _ 150 _ _ = 150 == = 150 = = = 150 == = 150 __ 150 _ 150 = == 150 = = = 150 == = 150 = == 150 _ = _ 150 _ 150 _ 150 _ 150 150 _ 150 _ _ _ 150 __ _ 150 __ _

C C C

Note: Plate layout should be varied according to needs. This layout is convenient for

this example.

3.1.Flick blocking media from plate and add 50111 of complete alpha MEM medium to columns 3, 4, 7, 8, 11&12.

3.2.Add 1501 of splenocytes to columns 2, 6 and 10 in duplicate. (Up to 12 samples per plate) 3.3.Take 501 of splenocytes from columns 2, 6 and 10 and transfer to columns 1, and 9 respectively: these are the negative control wells.

3.4.Serially dilute each sample by taking 501 from columns 2, 6 and 10, to columns 3, 7 and 11, mix well and transfer 501 to 4, 9 and 12. Discard 501 after mixing final columns in dilution.

3.5.Add test peptide and control peptide to twice the desired final concentration to naive splenocytes at 10 million/ml in complete oc-MEM medium. Add 50111 of control peptide and target cells to columns 1, 5 and 9.Add 501 test peptide and target cells to remaining columns.

3.6.Incubate plates at 37 C for 18-20 hours.

4. Developing the Assay 4.1.Wash plates twice with PBS containing 0.05% Tween 20 (Sigma P1379), once with distilled water and twice with PBST.

4.2.Add SOIll/well of biotinylated rat anti-mouse interferon-gamma diluted to 1 1lg/ml in PBS. Incubate for 2 hours at room temperature.

4.3.Wash plates four times with PBST, then add 50 pI Streptavidin Alkaline Phosphatase (Mabtech) diluted to 1 g/ml in PBS. Incubate at room temperature for 1 hour.

4.4.Wash plates four times with PBST, add 501/well of colour development buffer Incubate at room temperature until spots develop (approx. 10 min). Wash plates well with tap water, peel off plastic bottom and leave to dry overnight on paper towels.

Calculation Results are calculated as the number of epitope-specific IFNy spot forming cells/million splenocytes (sfc/million). Differences between groups are determined by one-way ANOVA and a post hoc Tukey-Karamer multiple comparison test on logy transformed data using GraphPad Instat version 3.05.

Results The demonstration that immunization with FP9.gpn and MVA.gpn alone or in prime- boost regimes with pSG2.gpn elicits antigen specific CD8+ T cell responses in mice is illustrated in figure 7. Female BALB/c mice were immunised im. with pSG2.gpn (DNA) or sham immunised with PBS ( - ) and boosted 14 days later with FP9. gpn (FP9) or MVA.gpn (MVA) as described above. The CD8+ T cell response was determined in splenocytes 14 days after the booster immunization using the IFN-y ELISpot assay. Columns represent the mean IFN-y spot forming cells/million splenocytes + standard deviation for 4 mice per group elicited by CD8+ reactive epitopes AMQ, TTS and RGP (see Table A).

Table A: GPN epitopes used in this study ('ND = not determined) Antigen Abbr. Epitope CD4/CD8 MHC reactive restriction HIV- 1 RGP RGPGRAFVTI CD8 Da HIV- 1 AMQ AMQMLKETI CD8 K HIV- 1 TTS TTSTLQEQ CD8 H_2d HIV- 1 NPP NPPIPVGEIYKRWIILG CD4 H_2a gag LNK Single or prime-boost immunisation with either FP9.gpn or MVA.gpn elicited a significantly (P<0. 01) enhanced antigen-specific T cell response against each of the CD8+ T cell epitopes when compared to sham- immunised controls (Figure 7).

In addition, priming with pSG2.gpn and boosting with FP9.gpn or MVA.gpn elicited significantly (P<0.01) enhanced responses against each CD8+ T cell epitope when compared to single immunizations with each virus (Figure 7).

It should be noted that the virus titres were not identical in this experiment. Thus, the relative immune potency of the viruses has not been compared. Naturally it is straightforward to assess this by performing the example with identical viral titres.

Thus it is demonstrated that specific CD8+ T cell responses are elicited against the recombinant HIV genes according to the present invention such as the gpn gene by immunization with either FP9.gpn or MVA.gpn, indicating that these constructs are both potent in eliciting an immune response against the GPN polypeptide.

Moreover, potent immune responses are directed at epitopes lying in the Nand C- terrninal regions of the GPN polypeptide, indicating that the whole polypeptide is expressed, processed and presented after delivery with either FP9.gpn or MVA.gpn.

Both viruses elicit significantly higher immune responses against each epitope when administered to animals that have been primed with pSG2.gpn, indicating that both can act as boosting agents in prime boost immunisation regimes.

Example 4: Immunogenicity of FP9.gpn and MVA.gpn in primates The immunogenicity of the gpn immunogens is demonstrated in viva in primates as follows.

The immunogenicity of the GPN polypeptide consisting of the gag, pot and nef proteins of clade B HIV-I is tested in non-human primates (Macaca mulatta).

The polyprotein (human codon usage) is expressed in recombinant MVA, fowlpoxvirus FP9, adeno virus and a DNA vaccine vector. The polyprotein expressing constructs are administered in a heterologous immunisation regimen in order to induce high levels of antigen-specific CD8+ and CD4+ T cells.

Table B shows the study design of the immunogenicity studies.

Table B

Group No Prime 1 Prime 2 Boost 1 Boost 2 n-5 Day 0 Day 28 Day 56 Day 84 FP9. gpn FP9. gpn MVA. gpn MVA. gpn MVA.gpn MVA.gpn FP9.gpn FP9.gpn 3 FP9.gpn MVA.gpn 4 pDNA+IL2- MVA.gpn FP9.gpn Adeno.gpn Ig/fc The design of the macaque study of this example is shown in Table B. Blood samples are taken pre- immunisation and 7 days after each immunisation and four weeks after the last immunization. PBMCs were cryopreserved and tested in ELISpot and intracellular cytokine assays using pools of overlapping peptides.

Cellular immune responses are tested during and at the end of the study. The frequency of IFN--secreting T cells is tested by IFN-y ELISpot assays (see Figure 9 for overlapping peptides used).

In an additional group, for comparitive purposes, DNA vaccines adjuvanted with IL- 2/Ig fusion proteins (Barouch, D. H., A. Craiu, et al. (2000) Proc Natl Acad Sci U S A 97(8): 4192-7) and recombinant adenovirus are tested. For study design see Table B. There are several parameters that are presented in this study: (a) Order of FP9 and MVA. The most effective combinations of poxvirus vector primes and boosts are demonstrated. This particularly focusses on groups 1 and 2 of the study design (see Table B).

(b) Frequency of primes and boosts. The effectiveness (in terms of immunogenicity) of priming with a single poxvirus immunisation and boosting with a single heterologous poxvirus vector boost is demonstrated. This particularly focuses on animals in group 3 which will be immunised as outlined.

Read-out: The key objective of this vaccination strategy is to induce cellular immune responses. Therefore the following assays are used to monitor cellular immune responses: IFN-y ELISpot using overlapping peptides (20mers overlapping by 10 lO amino acids as shown in Fig 9), intracellular cytokine staining for CD8+ and CD4+ T cells. In ELISpot assays CD4/8 depletion experiments will confirm IFN-y secretion in response to peptides by CD4+ and/or CD8+ T cells.

Thus the effectiveness of the recombinant genes of the present invention is demonstrated in viva in primates.

Example 5: Heterologous prime-boost immunisation regimens with FP9.gpn and MVA.gpn elicit enhanced T cell responses.

In this example it is demonstrated that FP9.gpn and MVA.gpn elicit enhanced antigen- specific CD4+ and CD8+ T cell responses when administered to subjects in heterologous or homologous prime-boost immunisation regimens. In this example, the effect is demonstrated in mice.

Female BALB/c mice (6 - 8 weeks old) are immunised with either 1 X 105 PFU FP9.gpn or the 1 X 106 of MVA.gpn by iv. injection. Animals were boosted two weeks later with either virus in an identical manner to the initial immunization.

Fourteen days after the booster immunization, all mice were sacrificed by cervical dislocation and the T cell responses elicited against three CD8+ epitopes (Table A: AMQ, TTS, RGP) and a CD4+ epitope (Table A: NPP) from GPN were determined by IFN-y ELISpot assay as described in the above examples.

Results were calculated as the sum of the number of epitope-specific IFNy spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by one-way ANOVA and a post hoc Tukey-Karamer multiple comparison test on logy transformed data using GraphPad Instat version 3.05.

Results The demonstration that immunization with FP9.gpn and MVA.gpn in prime-boost ] 0 regimes elicits enhanced T cell responses against GPN in mice is illustrated in figure 8.

Female BALB/c mice were immunised iv. with FP9.gpn (FP9) or MVA.gpn (MVA)sham immunised with PBS ( - ) and boosted 14 days later with either virus as described above. The CD8+ T cell response was determined in splenocytes 14 days after the booster immunization using the IFN-y ELISpot assay. Columns represent the sum of the mean IFN-y spot forming cells/million splenocytes _ standard deviation for 4 mice per group elicited by AMQ, TTS, RGP and NPP (see Table A).

Heterologous prime-boost immunization with FP9.gpn and MVA.gpn in either order elicited a significantly (P<0.001) enhanced overall T cell response against epitopes from GPN when compared to subjects given homologous immunizations with either virus (Figure 8).

Heterologous immunization with FP9.gpn/MVA.gpn or MVA.gpn/FP9.gpn elicits significantly higher T cell responses against GPN than homologous immunization with FP9.gpn/FP9.gpn or MVA.gpn/MVA.gpn.

Moreover, these viruses can advantageously be used interchangeably as priming and boosting agents with each other.

Thus the efficacy of recombinant HIV genes such as the gpn gene is demonstrated, in particular when delivered by viral vector such as fowlpox or MVA based vectors.

Example 6. Ileterologous prime-boost inmunisation with FP9.gpn and MVA. gpn elicits enhanced CDS+ and CD4+ T cell responses against the individual component proteins of 8p,' It is demonstrated that heterologous prime-boost immunisation with FP9.gpn and MVA.gpn or FP9.gpn and MVA.gpn elicits enhanced antigen-specific CD4+ and CD8+ T cell responses against the component parts of the GPN polypeptide. In this example, the demonstration is presented in mice.

Female BALB/c and C57/BL6 mice (6 - 8 weeks old) are immunised with either 1 X 106 PFU FP9.gpn or 1 X 106 PFU MVA.gpn by iv. injection. Animals are boosted two weeks later with either virus in an identical manner to the initial immunization.

Fourteen days after the booster immunization, all mice are sacrificed by cervical dislocation and the T cell responses elicited against a library of peptides (20 amino acids overlapping by to amino acids) derived from the GPN sequence determined using the IFN-y ELISpot assay as described in the above example.

Results were calculated as the sum of the number of epitope-specific IFNy spot forming cells/million splenocytes (sLc/million) for each peptide.

Results Heterologous prime-boost immunization with FP9.gpn and MVA.gpn (administered in either order) elicits enhanced T cell responses across the GPN sequence when compared to homologous immunization regimes with the same vectors.

Moreover, these T cell responses include CD8+ and CD4+ responses, as well as being directed at epitopes derived from several regions of the GPN polypeptide.

Thus it is demonstrated that heterologous prime-boost is a highly effective application of the recombinant gene(s) of the present invention.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Sequence Listing SEQ ID NO:1 Artificial sequence - GPN

M A P I V Q N L Q G Q M V H Q A I S P R T L N A W V K V V E E K A F S

P E V I P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M

L K E T I N E E A A E W D R L H P V H A G P I A P G Q M R E P R G S D

I A G T T S T L Q E Q I G W M T N N P P I P V G E I Y K R W I I L G L

N K I V R M Y S P T S I L D I R Q G P K E P F R D Y V D R F Y K T L R

A E Q A S Q E V K N W M T E T L L V Q N A N P D C K T I L K A L G P A

A T L E E M M T A C Q G V G G P G H K A R V L M A A R A S V L S G G E

L D R W E K I R L R P G G K K K Y K L K H I V W A S R E L E R F A V N

P G L L E T S E G C R Q I L G Q L Q P S L Q T G S E E L R S L Y N T V

A T L Y C V H Q R I E V K D T K E A L E K I E E E Q N K S K K K A Q Q

A A A D T G N S S Q V S Q N Y T P D K K H Q K E P P F L W M G Y E L H

P D K W T V Q P I V L P E K D S W T V N D I Q K L V G K L N W A S Q I

Y A G I K V K Q L C K L L R G T K A L T E V I P L T E E A E L E L A E

N R E I L K E P V H G V Y Y D P S K D L I A E I Q K Q G Q G Q W T Y Q

I Y Q E P F K N L K T G K Y A R M R G A H T N D V K Q L T E A V Q K I

A T E S I V I W G K T P K F K L P I Q K E T W E A W W T E Y W Q A T W

I P E W E F V N T P P L V K L W Y Q L E K E P I V G A E T F P I S P I

E T V P V K L K P G M D G P K V K Q W P L T E E K I K A L V E I C T E

M E K E G K I S K I G P E N P Y N T P V F A I K K K D S T K W R K L V

D F R E L N K R T Q D F W E V Q L G I P H P A G L K K K K S V T V L D

v G D A Y F S V P L D K D F R K Y T A F T I P S I N N E T P G I R Y Q

Y N V L P Q G W K G S P A I F Q S S M T K I L E P F R K Q N P D I V I

Y Q Y M D D L Y V G S D L E I G Q H R T K I E E L R Q H L L R W G F T

T P D K K H Q K E P P F L V W K F D S R L A F H H M A R E L H P E Y Y

K D C D P E K E V L V W K F D A N E G E N N S L L H P M S L H G M D D

P E K E V P E K V E E A N E G E N G P G I R Y P L T F G W C F K L V P

V E P E K V E E W Q N Y T P G P G I R Y Q K R Q D I L D L W V Y H T Q

G Y F P D W Q N Y T P E G L I Y S Q K R Q D I P M T Y K A A L D L S H

F L K E K G G L E G L I Y S P Q V P L R P M T Y K A A D C A W L E A Q

E E E E V G F P V R P Q V P L R N T A A N N A D C A W L A D G V G A V

s R D L E K H G A I T S S N T A A N N R R A E P A A D G V G A M G G K

W S K R S V V G W P T V R E R M R R A E P A R G P G R A F V T I Y P Y

D V P D Y A so

SEQ ID NO:2 Artificial sequence- GAG P24

M A P I V Q N L Q G Q M V H Q A I S P R T L N A W V K V V E E K A F S

P E V I P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M

L K E T I N E E A A E W D R L H P V H A G P I A P G Q M R E P R G S D

I A G T T S T L Q E Q I G W M T N N P P I P V G E I Y K R W I I L G L

N K I V R M Y S P T S I L D I R Q G P K E P F R D Y V D R F Y K T L R

A E Q A S Q E V K N W M T E T L L V Q N A N P D C K T I L K A L G P A

A T L E E M M T A C Q G V G G P G H K A R V L

SEQ ID NO:3 Artificial sequence- GAG P17

M A A R A S V L S G G E L D R W E K I R L R P G G K K K Y K L K H I V W A S R E L E R F A V N P G L L E T S E G C R Q I L G Q L Q P S L Q T

G S E E L R S L Y N T V A T L Y C V H Q R I E V K D T K E A L E K I E

E E Q N K S K K K A Q Q A A A D T G N S S Q V S Q N Y

SEQ ID NO:4 Artificial sequence - POL P51 duplication

T P D K K H Q K E P P F

SEQ ID NO:5 Artificial sequence - POL P51 core

L W M G Y E L H P D K W T V Q P I V L P E K D S W T V N D I Q K L V G

K L N W A S Q I Y A G I K V K Q L C K L L R G T K A L T E V I P L T E

E A E L E L A E N R E I L K E P V H G V Y Y D P S K D L I A E I Q K Q

G Q G Q W T Y Q I Y Q E P F K N L K T G K Y A R M R G A H T N D V K Q

L T E A V Q K I A T E S I V I W G K T P K F K L P I Q K E T W E A W W

T E Y W Q A T W I P E W E F V N T P P L V K L W Y Q L E K E P I V G A

E T F P I S P I E T V P V K L K P G M D G P K V K Q W P L T E E K I K

A L V E I C T E M E K E G K I S K I G P E N P Y N T P V F A I K K K D

S T K W R K L V D F R E L N K R T Q D F W E V Q L G I P H P A G L K K

K K S V T V L D V G D A Y F S V P L D K D F R K Y T A F T I P S I N N

E T P G I R Y Q Y N V L P Q G W K G S P A I F Q S S M T K I L E P F R

K Q N P D I V I Y Q Y M D D L Y V G S D L E I G Q H R T K I E E L R Q

H L L R W G F T

SEQ 1:D NO:6 Artificial sequence - scrambled NEF

L V W K F D S R L A F H H M A R E L H P E Y Y K D C D P E K E V L V W

K F D A N E G E N N S L L H P M S L H G M D D P E K E V P E K V E E A

N E G E N G P G I R Y P L T F G W C F K L V P V E P E K V E E W Q N Y

T P G P G I R Y Q K R Q D I L D L W V Y H T Q G Y F P D W Q N Y T P E

G L I Y S Q K R Q D I P M T Y K A A L D L S H F L K E K G G L E G L I

Y S P Q V P L R P M T Y K A A D C A W L E A Q E E E E V G F P V R P Q

V P L R N T A A N N A D C A W L A D G V G A V S R D L E K H G A I T S

S N T A A N N R R A E P A A D G V G A M G G K W S K R S V V G W P T V

R E R M R R A E P A

SEQ ID NO:7 Artificial sequence - gpl60 murine CD8 tag

R G P G R A F V T I

SEQ ID NO:8 Artificial sequence - HA-tag

Y P Y D V P D Y A

SEQ ID NO: 9 Artificial sequence - core GPN

M A P I V Q N L Q G Q M V H Q A I S P R T L N A W V K V V E E K A F S

P E V I P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M

L K E T I N E E A A E W D R L H P V H A G P I A P G Q M R E P R G S D

I A G T T S T L Q E Q I G W M T N N P P I P V G E I Y K R W I I L G L

N K I V R M Y S P T S I L D I R Q G P K E P F R D Y V D R F Y K T L R

A E Q A S Q E V K N W M T E T L L V Q N A N P D C K T I L K A L G P A

A T L E E M M T A C Q G V G G P G H K A R V L M A A R A S V L S G G E

L D R W E K I R L R P G G K K K Y K L K H I V W A S R E L E R F A V N

P G L L E T S E G C R Q I L G Q L Q P S L Q T G S E E L R S L Y N T V

A T L Y C V H Q R I E V K D T K E A L E K I E E E Q N K S K K K A Q Q

A A A D T G N S S Q V S Q N Y T P D K K H Q K E P P F L W M G Y E L H

P D K W T V Q P I V L P E K D S W T V N D I Q K L V G K L N W A S Q I

Y A G I K V K Q L C K L L R G T K A L T E V I P L T E E A E L E L A E

N R E I L K E P V H G V Y Y D P S K D L I A E I Q K Q G Q G Q W T Y Q

I Y Q E P F K N L K T G K Y A R M R G A H T N D V K Q L T E A V Q K I

A T E S I V I W G K T P K F K L P I Q K E T W E A W W T E Y W Q A T W

I P E W E F V N T P P L V K L W Y Q L E K E P I V G A E T F P I S P I

E T V P V K L K P G M D G P K V K Q W P L T E E K I K A L V E I C T E

M E K E G K I S K I G P E N P Y N T P V F A I K K K D S T K W R K L V

D F R E L N K R T Q D F W E V Q L G I P H P A G L K K K K S V T V L D

V G D A Y F S V P L D K D F R K Y T A F T I P S I N N E T P G I R Y Q

Y N V L P Q G W K G S P A I F Q S S M T K I L E P F R K Q N P D I V I

Y Q Y M D D L Y V G S D L E I G Q H R T K I E E L R Q H L L R W G F T

T P D K K H Q K E P P F L V W K F D S R L A F H H M A R E L H P E Y Y

K D C D P E K E V L V W K F D A N E G E N N S L L H P M S L H G M D D

P E K E V P E K V E E A N E G E N G P G I R Y P L T F G W C F K L V P

V E P E K V E E W Q N Y T P G P G I R Y Q K R Q D I L D L W V Y H T Q

G Y F P D W Q N Y T P E G L I Y S Q K R Q D I P M T Y K A A L D L S H

F L K E K G G L E G L I Y S P Q V P L R P M T Y K A A D C A W L E A Q

E E E E V G F P V R P Q V P L R N T A A N N A D C A W L A D G V G A V

S R D L E K H G A I T S S N T A A N N R R A E P A A D G V G A M G G K

W S K R S V V G W P T V R E R M R R A E P A

SEQ ID NO:10 Human immunodeficiency virus - Native NEF

M G G K W S K R S V V G W P T V R E R M R R A E P A A D G V G A V S R

D L E K H G A I T S S N T A A N N A D C A W L E A Q E E E E V G F P

V R P Q V P L R P M T Y K A A L D L S H F L K E K G G L E G L I Y S Q

K R Q D I L D L W V Y H T Q G Y F P D W Q N Y T P G P G I R Y P L T F

G W C E K L V P V E P E K V E E A N E G E N N S L L H P M S L H G M D

D P E K E V L V W K F D S R L A F H H M A R E L H P E Y Y K

SEQ ID NO: 11 Artificial sequene - gpn nucleotide sequence

C C G G G C C C G C C G C C A C C A T G G C C C C C A T C G T G C A G

A A C C T G C A A G G C C A G A T G G T G C A C C A G G C C A T C A G

C C C C A G A A C C C T G A A T G C C T G G G T G A A G G T G G T G G

A G G A G A A G G C C T T C A G C C C T G A G G T G A T C C C T A T G

T T C A G C G C C C T G A G C G A G G G C G C C A C C C C C C A G G A

C C T G A A C A C C A T G C T G A A T A C C G T G G G C G G C C A T C

A G G C C G C C A T G C A G A T G C T G A A G G A G A C C A T C A A C

G A G G A G G C C G C C G A G T G G G A C A G A C T G C A C C C C G T

G C A C G C C G G A C C T A T C G C C C C T G G C C A G A T G A G A G

A G C C C A G A G G C A G C G A C A T C G C C G G C A C C A C C A G C

A C C C T G C A A G A G C A G A T C G G C T G G A T G A C C A A C A A

C C C C C C T A T C C C T G T G G G C G A G A T C T A C A A G A G A T

G G A T C A T C C T G G G C C T G A A C A A G A T C G T G A G A A T G

T A C A G C C C T A C C A G C A T C C T G G A C A T C A G A C A G G G

A C C C A A G G A G C C C T T C A G A G A C T A C G T G G A C A G G T

T C T A C A A G A C C C T G A G A G C C G A G C A G G C C A G C C A G

G A A G T G A A G A A C T G G A T G A C C G A G A C C C T G C T G G T

G C A G A A C G C C A A C C C C G A C T G C A A G A C C A T C C T G A

A G G C C C T G G G A C C T G C C G C T A C C C T G G A G G A G A T G

A T G A C C G C C T G C C A G G G C G T G G G C G G A C C T G G C C A

C A A G G C C A G A G T G C T C A T G G C C G C C A G A G C C A G C G

T G C T G A G C G G C G G A G A G C T G G A T A G A T G G G A G A A G

A T C A G A C T G A G A C C T G G C G G C A A G A A G A A G T A C A A

G C T G A A G C A C A T C G T G T G G G C C T C T A G G G A G C T G G

A G A G A T T C G C C G T G A A T C C T G G C C T G C T G G A G A C C

A G C G A G G G C T G T A G A C A G A T C C T G G G C C A G C T G C A

A C C C A G C C T G C A A A C C G G C T C T G A G G A G C T G A G A T

C C C T G T A C A A C A C C G T G G C C A C C C T G T A C T G C G T G

C A C C A G A G A A T C G A G G T G A A G G A C A C A A A G G A G G C

C C T G G A G A A G A T C G A G G A G G A G C A G A A C A A G T C C A

A G A A G A A G G C C C A G C A G G C C G C T G C C G A C A C C G G C

A A C A G C A G C C A G G T G A G C C A G A A C T A C A C C C C C G A

C A A G A A G C A C C A G A A G G A G C C C C C T T T C C T G T G G A

T G G G C T A C G A G C T G C A C C C C G A T A A G T G G A C C G T G

C A G C C C A T C G T G C T G C C T G A G A A G G A T A G C T G G A C

C G T G A A C G A C A T C C A G A A G C T G G T G G G C A A G C T G A

A C T G G G C C A G C C A G A T C T A C G C C G G C A T C A A G G T G

A A G C A G C T G T G T A A G C T G C T G A G A G G C A C C A A G G C

C C T C A C A G A A G T G A T C C C C C T C A C A G A G G A G G C C G

A G C T G G A G C T G G C C G A G A A C A G A G A G A T C C T G A A A

G A G C C C G T G C A C G G C G T G T A C T A C G A C C C C A G C A A

G G A T C T C A T C G C C G A G A T T C A G A A G C A G G G C C A G G

G C C A G T G G A C C T A C C A G A T C T A C C A G G A G C C T T T C

A A G A A C C T G A A A A C C G G C A A G T A C G C C A G A A T G A G

A G G C G C T C A C A C C A A C G A C G T G A A G C A G T T G A C C G

A G G C C G T G C A G A A A A T C G C C A C C G A G A G C A T C G T G

A T C T G G G G C A-A G A C C C C C A A G T T C A A G C T G C C T A T

C C A G A A G G A G A C C T G G G A G G C C T G G T G G A C C G A G T

A T T G G C A G G C C A C C T G G A T T C C T G A G T G G G A G T T C

G T G A A C A C C C C C C C T C T G G T G A A G C T G T G G T A T C A

G C T G G A G A A G G A G C C T A T C G T G G G C G C C G A G A C C T

T C C C C A T C A G C C C T A T C G A G A C C G T G C C C G T G A A G

C T G A A G C C C G G C A T G G A C G G C C C C A A G G T G A A A C A

G T G G C C T C T C A C C G A G G A G A A G A T T A A G G C C C T G G

T G G A G A T T T G C A C C G A G A T G G A G A A G G A A G G C A A G

A T C A G C A A G A T C G G C C C C G A G A A T C C C T A C A A T A C

C C C C G T G T T C G C C A T T A A G A A G A A G G A C T C C A C C A

A G T G G A G A A A G C T G G T G G A C T T C A G A G A G C T G A A C

A A G A G A A C C C A G G A C T T C T G G G A G G T G C A G C T G G G

C A T C C C C C A C C C T G C C G G C C T G A A G A A G A A G A A G A

G C G T G A C C G T G C T G G A C G T G G G C G A C G C C T A C T T C

A G C G T G C C C C T G G A C A A G G A C T T C A G A A A G T A C A C

C G C C T T C A C C A T C C C C A G C A T C A A C A A C G A G A C C C

C C G G C A T C A G A T A C C A G T A C A A C G T G C T G C C C C A G

G G C T G G A A G G G C A G C C C C G C C A T C T T C C A G A G C A G

C A T G A C A A A G A T C C T G G A G C C C T T C C G G A A G C A G A

A C C C C G A T A T C G T G A T C T A C C A G T A C A T G G A C G A C

C T G T A C G T G G G C A G C G A T C T G G A G A T C G G C C A G C A

C A G A A C C A A G A T C G A A G A G C T G A G A C A G C A C C T G C

T G A G A T G G G G C T T C A C C A C A C C A G A T A A G A A A C A T

c A G A A A G A A C C A C C A T T C C T G G T G T G G A A G T T T G A

* C A G C A G A C T G G C C T T C C A C C A T A T G G C C A G A G A G C

T G C A T C C T G A G T A C T A C A A G G A C T G C G A T C C C G A G

A A G G A G G T G C T G G T C T G G A A A T T C G A C G C C A A C G A

G G G C G A G A A C A A C A G C C T G C T G C A C C C C A T G A G C C

T G C A C G G C A T G G A T G A T C C T G A G A A A G A A G T G C C C

G A G A A G G T G G A A G A G G C C A A T G A G G G A G A A A A C G G

C C C T G G C A T T A G A T A T C C C C T C A C C T T C G G C T G G T

G C T T C A A G C T G G T G C C T G T G G A G C C T G A G A A A G T G

G A G G A A T G G C A G A A T T A C A C A C C C G G C C C A G G C A T

C C G G T A T C A G A A G A G A C A G G A C A T T C T G G A C C T G T

G G G T G T A C C A C A C C C A G G G C T A C T T C C C C G A C T G G

C A G A A C T A T A C T C C T G A G G G C C T C A T C T A C A G C C A

G A A G C G G C A G G A T A T C C C C A T G A C C T A C A A G G C C G

C C C T G G A T C T G A G C C A C T T T C T G A A A G A G A A G G G C

G G C C T G G A G G G C T T G A T C T A C T C C C C A C A G G T G C C

A C T G A G A C C T A T G A C A T A T A A G G C T G C C G A C T G C G

C C T G G C T G G A G G C C C A G G A A G A G G A G G A A G T G G G C

T T C C C T G T G A G A C C C C A G G T G C C C C T G C G G A A C A C

C G C C G C C A A T A A T G C C G A T T G T G C T T G G C T C G C C G

A C G G C G T G G G A G C C G T G A G C A G a G A C C T G G A A A A G

C A C G G C G C C A T C A C C A G C A G C A A T A C A G C C G C C A A

C A A C A G A A G A G C C G A G C C T G C C G C C G A T G G A G T G G

G C G C C A T G G G C G G C A A G T G G A G C A A G A G A T C C G T G

G T G G G C T G G C C C A C C G T G A G A G A A A G A A T G A G A A G

G G C C G A A C C C G C C A G A G G A C C C G G C A G A G C C T T C G

T G A C C A T C T A C C C C T A C G A C G T G C C C G A C T A C G C T

T G A T G A G G C G C G C C C T

5-prime ApaI and 3-prime AscI sites are underlined. Initiating ATG and terninating TGA are in bold.

SEQ ID NO:12 Artificial sequence - disrupted pol

T P D K K H Q K E P P F L W M G Y E L H P D K W T V Q P I V L P E K D

S W T V N D I Q K L V G K L N W A S Q I Y A G I K V K Q L C K L L R G

T K A L T E V I P L T E E A E L E L A E N R E I L K E P V H G V Y Y D

P S K D L I A E I Q K Q G Q G Q W T Y Q I Y Q E P F K N L K T G K Y A

R M R G A H T N D V K Q L T E A V Q K I A T E S I V I W G K T P K F K

L P I Q K E T W E A W W T E Y W Q A T W I P E W E F V N T P P L V K L

W Y Q L E K E P I V G A E T F P I S P I E T V P V K L K P G M D G P K

V K Q W P L T E E K I K A L V E I C T E M E K E G K I S K I G P E N P

Y N T P V F A I K K K D S T K W R K L V D F R E L N K R T Q D F W E V

Q L G I P H P A G L K K K K S V T V L D V G D A Y F S V P L D K D F R

K Y T A F T I P S I N N E T P G I R Y Q Y N V L P Q G W K G S P A I F

Q S S M T K I L E P F R K Q N P D I V I Y Q Y M D D L Y V G S D L E I

G Q H R T K I E E L R Q H L L R W G F T T P D K K H Q K E P P F

SEQ II) NO:13 Artificial sequence - disrupted gag

M A P I V Q N L Q G Q M V H Q A I S P R T L N A W V K V V E E K A F S

P E V I P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M

L K E T I N E E A A E W D R L H P V H A G P I A P G Q M R E P R G S D

I A G T T S T L Q E Q I G W M T N N P P I P V G E I Y K R W I I L G L

N K I V R M Y S P T S I L D I R Q G P K E P F R D Y V D R F Y K T L R

A E Q A S Q E V K N W M T E T L L V Q N A N P D C K T I L K A L G P A

A T L E E M M T A C Q G V G G P G H K A R V L M A A R A S V L S G G E

L D R W E K I R L R P G G K K K Y K L K H I V W A S R E L E R F A V N

P G L L E T S E G C R Q I L G Q L Q P S L Q T G S E E L R S L Y N T V

A T L Y C V H Q R I E V K D T K E A L E K I E E E Q N K S K K K A Q Q

A A A D T G N S S Q V S Q N Y

Claims (47)

1. A recombinant polypeptide comprising amino acid sequence derived from at least one of (i) an HIV gag gene product; (ii) an HIV pol gene product; or (iii) an HIV nef gene product, said sequence being mutated with respect to the natural sequence of said gene product, l O and said sequence maintaining substantially all of the naturally occurring CD8+ T cell epitopes of said gene product as defined in pl7 and p24 (gag), amino acids 1-440 of RT (pot) and nef shown in Annex 1.

2. A recombinant polypeptide according to claim l comprising amino acid sequence derived from at least two of (i), (ii) and (iii).

3. A recombinant polypeptide according to claim 2 comprising amino acid sequence derived from (i) and (ii) and (iii).

4. A recombinant polypeptide according to claim 2 or claim 3 wherein the amino acid sequences derived from (i) and/or (ii) and/or (iii) are arranged in the order (i) - (ii) - (iii) from the N terminus to the C terminus of the polypeptide.

5. A recombinant polypeptide according to claim 3 or claim 4 comprising SEQ ID NO:9, or a sequence having at least 95% identity thereto.

6. A recombinant polypeptide according to any preceding claim further comprising an antibody recognition tag.

7. A recombinant polypeptide according to claim 6 wherein said tag is an HA tag comprising the sequence as shown in SEQ ID NO:8.

8. A recombinant polypeptide according to any preceding claim further comprising a CD8+ T cell epitope tag.

9. A recombinant polypeptide according to claim 8 wherein said tag is a gpl60 derived tag comprising the sequence as shown in SEQ ID NO:7.

10. A recombinant polypeptide according to any preceding claim, said polypeptide comprising the sequence as shown in SEQ ID NO: l.

1 1. A recombinant polypeptide according to any preceding claim, said polypeptide comprising amino acid sequence derived from an HIV nef gene product, said recombinant polypeptide sequence being mutated to disrupt the function of said nef sequence, said nef sequence further comprising one or more T helper epitopes which are not present in the naturally occurring nef gene.

12. A recombinant polypeptide according to claim 11 comprising one or more T helper epitopes which are not present in the naturally occurring nef sequence and are shown in Figure 3A.

13. A recombinant polypeptide according to claim 11 or claim 12 comprising one or more T helper epitope(s) which are shown in Figure 3A and which are absent from Figure 3B.

14. A recombinant polypeptide according to any of claims 11 to 13 further comprising all naturally ocurring CD8+ T cell epitopes of the nef gene product as defined in Annex 1.

15. A recombinant polypeptide according to any of claims 11 to 14 further comprising all naturally occurring nef human T helper epitopes as defined in Annex 1.

16. A recombinant polypeptide according to any of claims 11 to 15 wherein said polypeptide comprises sequence as shown in SEQ ID NO:6, or a sequence having at least 95% identity thereto.

17. A recombinant polypeptide according to any preceding claim, said polypeptide comprising amino acid sequence derived from an HIV pot gene product, said recombinant polypeptide sequence being mutated to disrupt the reverse transcriptase activity of the pot sequence, wherein substantially all of the CD8+ T cell epitopes of the naturally occurring pol sequence as defined in amino acids 1-440 of RT (pot) l O shown in Annex 1 are retained in said recombinant polypeptide.

18. A recombinant polypeptide according to claim 17, wherein the reverse transcriptase activity of said pol sequence is mutated by duplication of an internal sequence derived from the centre of the naturally occurring pol gene and exchange of the amino and carboxy terminal portions of said pol sequence.

19. A recombinant polypeptide according to claim 18 wherein said duplicated internal sequence comprises TPDKKHQKEPPF (SEQ ID NO:4).

20. A recombinant polypeptide according to claim 18 or claim 19 wherein said polypeptide comprises sequence as shown in SEQ ID NO:12 or a sequence having at least 95% identity thereto.

21. A recombinant polypeptide according to any preceding claim, said polypeptide comprising amino acid sequence derived from an HIV gag gene product, said recombinant polypeptide sequence being mutated to disrupt processing of the gag gene product, and said gag sequence further comprising a disrupted myristoylation site, wherein substantially all of the CD8+ T cell epitopes of the naturally occurring gag sequence as defined in pl7 and p24 (gag) shown in Annex 1 are retained in said recombinant polypeptide.

22. A recombinant polypeptide according to claim 21 wherein the processing of gag is disrupted by exchanging the p 17 and p24 domains and wherein the mynstoylation site is disrupted by mutation of the second glycine to alanine.

23. A recombinant polypeptide according to claim 21 or claim 22 wherein said polypeptide comprises sequence as shown in SEQ ID NO:13 or a sequence having at least 95% identity thereto.

24. A recombinant polypeptide according to any preceding claim wherein the HIV lO is a clade B HIV.

25. A recombinant nucleic acid encoding a polypeptide according to any preceding claim.

26. A recombinant nucleic acid sequence comprising SEQ ID NO:11, or a sequence which differs only by silent mutations with respect to the genetic code, or a sequence having at least 95% identity thereto.

27. A viral vector encoding a polypeptide according to any one of claims 1 to 24, said viral vector optionally being selected from the group consisting of poxviruses, adenoviruses, AAV, VSV, HSV and Sendai virus.

28. A viral vector according to claim 27 wherein said vector is an MVA or MVA derived vector.

29. A viral vector according to claim 27 wherein said vector is a fowlpox or fowlpox derived vector.

30. A viral vector according to claim 29 wherein said vector is an FP9 fowlpox vector.

31. A nucleic acid vector comprising a nucleic acid sequence according to claim 25 or claim 26 or encoding a polypeptide according to any one of claims 1 to 24.

32. An adenovirus vector comprising a nucleic acid sequence according to claim 25 or claim 26 or encoding a polypeptide according to any one of claims I to 24.

33. A poxvirus vector comprising a nucleic acid sequence according to claim 25 or claim 26 or encoding a polypeptide according to any one of claims 1 to 24.

34. A plasmid selected from the group consisting of p29D.gpn, pOPK6.gpn and pSG2.gpn.

35. Use of a polypeptide according to any one of claims 1 to 24 in medicine.

36. Use of polypeptide according to any one of claims 1 to 24 in the preparation of a medicament for the treatment or prevention of HIV infection.

37. Use of polypeptide according to any one of claims 1 to 24 in the preparation of a medicament for immunization against HIV infection.

38. Use of a nucleic acid or a vector according to any one of claims 25 to 34 in mediums.

39. Use of a nucleic acid or a vector according to any one of claims 25 to 34 in the preparation of a medicament for the treatment or prevention of HIV infection.

40. Use of a nucleic acid or a vector according to any one of claims 25 to 34 in the preparation of a medicament for immunization against HIV infection.

41. A method of immunising a subject against HIV infection comprising administering to said subject a polypeptide according to any one of claims 1 to 24 or a nucleic acid or vector according to any one of claims 25 to 34.

42. Use of a polypeptide according to any one of claims I to 24 or a nucleic acid or a vector according to any of claims 25 to 34 as a priming agent or as a boosting agent in a prime-boost immunization regime.

43. Use of a polypeptide according to any one of claims I to 24 or a nucleic acid or a vector according to any of claims 25 to 34 in the induction of an immune response.

44. Use according to claim 43, wherein the immune response is selected from the group consisting of a CD8+ T cell response, a CD4+ T cell response, and a humoral response.

45. A method for inducing an immune response in a subject, comprising administering to said subject a polypeptide according to any one of claims 1 to 24 or a nucleic acid or a vector according to any of claims 25 to 34.

46. A method according to claim 45, wherein the immune response is selected from the group consisting of a CD8+ T cell response, a CD4+ T cell response, and a humoral response.

47. A recombinant polypeptide, recombinant polynucleotide or viral vector substantially as hereinbefore described with reference to the accompanying drawings.