ZA200204260B - Improvements in or relating to immune responses to HIV. - Google Patents

Description

Title: Improvements in or Relating to Immune Responses to HIV

Field of the Invention

This invention relates to an immunogen designed to elicit an anti-HIV immune response in a human subject (especially a cell-mediated response), a nucleic acid molecule encoding the immunogen, compositions comprising the immunogen and/or the nucleic acid molecule, and to a method of inducing an anti-HIV immune response (especially a cell-mediated response) in a human subject.

Background of the Invention

Development of effective human immunodeficiency virus (HIV) vaccines is one of the primary goals of current acquired immunodeficiency syndrome (AIDS) research. Despite progress in prevention and powerful drug combinations to treat HIV infection, an estimated 16,000 people become infected every day. Over 90% of new infections occur in developing countries for which the recent medical advances are not immediately applicable or affordable. The best hope for these countries is the development of an effective, accessible ) HIV vaccine. There is now growing optimism among scientists that an AIDS vaccine may be possible (McMichael & Hanke 1999 Nat. Med. 5, 612-614; Gold 1999 IA VI Report 4, pp 1-2, 8-9, 15-16 & 18).

An ideal prophylactic vaccine should induce sterilizing immunity, so that after exposure, the virus would never be detected in the body. However, this is probably an unrealistic objective. Rather, an attainable goal may be a vaccine-induced immunity that results in a no signs of discsse and no wsmsmission 0 other individuals Alcrmstively, a potentially successial vaccine may induce imsasme responses that at east hold the vires in check at levels 30 low, thet both progression to AIDS aad transmission are entirely or substantially prevented.

To induce sterilizing immunity, a prophylactic vaccine may need to elicit both humoral and cell-mediated immune responses. Since HIV was isolated and sequenced, there has been a considerable effort to develop envelope-based vaccines inducing neutralizing antibodies (nAb). However, this has proved to be exceedingly difficult (Heilman & Baltimore 1998

Nat. Med. 4 (4 Suppl.) 532-534). Although some success was reported in inducing nAb against laboratory HIV strains (Berman et al, 1990 Nature 345, 622-625; Fultz et al, 1992

Science 256, 1687-1690), it has been extremely difficult to neutralize primary isolates (Trkola et al, 1998 J. Virol. 72, 1876-1885; Haynes 1996 Lancet 34, 933-937). An - explanation for the first 15 years of relative failure has been provided by the crystal structure - of the core gp 120, which revealed multiple mechanisms by which HIV prevents efficient induction of nAb (Wyatt et al, 1998 Nature 393, 705-711; Kwong et al, 1998 Nature 393, 638-659). As a result of these difficulties, the emphasis of many vaccine designers has shifted to the induction of cell-mediated immune responses, which are mediated (predominantly) by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) are usually CD8" cells and participate in an organism’s defence in at least two different ways: they kill virus-infected cells; and they secrete a variety of cytokines and chemokines that directly or indirectly contribute to the suppression : of virus replication. CTL-mediated protection afier vaccination may depend on the levels of . CTL present in the circulation and, perhaps, specifically for proteins expressed early . (regulatory proteins) rather than late (structural proteins) in the replication cycle.

The induction and maintenance of CD8" T cell responses require “help” provided by CD4" T lymphocytes (helper T cells). In some HIV-infected individuals, high levels of HIV-specific helper response have been detected.

Identification of methods for induction of strong CD8" T cell responses would provide tools for studying their role(s) in shaping the course of HIV infection and may stimulate progress towards an effective HIV vaccine. Previously, a prototype HIV vaccine was constructed as a string of partially overlapping epitopes recognised by murine, macaque and human CTL, which was delivered by vaccine vehicles that were safe and acceptable for use in humans, a

DNA vector and modified vaccinia virus Ankara (MVA) vector (Hanke et al, 1998 Vaccine

16, 426-435; Hanke er al, 1998 J. Gen. Virol. 79, 83-90). In mice, the most potent protocol for induction of CTL was found to be DNA priming followed by MV A boosting (Hanke es al, 1998 Vaccine 16, 439-445; Schacider ef al, 1998 Nat. Mcd. 4, 397-402) that is, priming mice with nucleic acid encoding the relevant polypeptide, followed by boosting the mice by inoculation with a modified vaccinia virus Ankara (“MVA”) vector expressing the relevant epitopes.

WO 98/56919 discloses a “prime-boost™ vaccination strategy, involving (i) priming with a composition comprising a source of one or more T cell epitopes of a target antigen, together with a pharmaceutically acceptable carrier, and (ii) boosting with a composition comprising a source of one or more T cell epitopes of the target antigen, including at least one T cell epitope that is the same as a T cell epitope of the priming composition.

The present invention aims, infer alia, to provide immunogens which may be useful in eliciting an HIV-specific response in humans. All documents and publications mentioned in this specification are incorporated herein by reference.

Summary of the Invention

In a first aspect the invention provides an imummogen in sterile form suitable for administration to a human subject, the inenunogen comprising: at least a portion of the gag protein of HIV, said gag protein being from an HIV clade or having a consensus sequence for one or more HIV clades, and comprising at least parts of p17 and p24; and a synthetic polypeptide comprising a plurality of amino acid sequences, cach sequence comprising a buman CTL epitope of an HIV protein, and wherein a plurality of HIV proteins are represented in the synthetic polypeptide, said CTL epitopes being selected to stimulate an immune response to one or more HIV clades of imterest.

For present purpeses “stevile™ sefiors to the gencssl shacnce of visuses, bacteria, fangi, yeasts, chlamydia, sycoplesme, and spoees of sxry of the fosegoing (capecially microbes pathogenic in humans). However, the immunogen may comprise onc or mere specific known microbial (c.g. viral or bacterial) vectors which serve to express the gag protein and/or synthetic polypeptide in a lnsnan subject. Such vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses and pox viruses which are inherently non- pathogenic in humans or have been subjected to genetic manipulation or other modification to render them non-pathogenic in humans. Particularly preferred is vaccinia virus, especially modified vaccinia virus Ankara (MVA). Other specific preferred viral vectors include

Semliki Forest Virus (SFV) and Sindbis virus (see Smerdon & Liljestrom 2000, Gene Ther.

Regul. 1, 1-31). Suitable bacterial vectors include BCG, and attenuated strains of

Salmonella Spp. (especially “double aro” mutants of Salmonella which are being developed as vaccines for diarrhoeal diseases), and Shigella (see Shata et al, 2000 Mol. Med. Today 6, © 66-71).

Other expression systems, which may be useful for producing the immunogen include a tobacco mosaic virus (TMV) expression vector (Palmer ef al, 1999 Arch. Virol. 144, 1345- 60) and NS1 tubules of bluetongue virus (Adler et al, 1998 Med. Microbiol. Immunol. (Berl.) 187, 91-96).

The term “synthetic” as used herein, is intended to refer to a polypeptide which is not, in its entirety, present in any naturally-occurring HIV isolate. The term “synthetic” is not intended to indicate that the polypeptide is necessarily synthesised by conventional chemical techniques of solid phase peptide synthesis. Whilst this represents one possibility, it is generally to be preferred that the polypeptide is synthesised by transcription and/or translation from an appropriate nucleic acid molecule encoding the synthetic polypeptide.

Such methods of synthesis are well known to those skilled in the art and form no part of the invention.

It is preferred that the gag protein (or portion thereof) and the synthetic polypeptide are joined in some way on a single entity. For example, a viral vector may express both the gag protein and the synthetic polypeptide as separate components of the immunogen.

Alternatively, both components of the immunogen may be covalently coupled or conjugated to each other or to a common carrier entity (such as a liposome, ISCOM or molecule). In preferred embodiments, both the gag protein and the synthetic polypeptide components of the immunogen are present in a single polypeptide or fusion protein. In such a fusion protein, the gag protein and synthetic polypeptide may be essentially the only components present. Alternatively the fusion protein may comprise other components which may correspond to other HIV antigens (or portions thereof), or may be derived from other . sources. Thus, in some embodiments, the polypeptide will comprise a portion of the gag protein substantially adjacent to the synthetic polypeptide (ic. with fewer than 10 intervening amino acid residues). In other embodiments, the portion of the gag protein and the synthetic polypeptide may be separated by one or more intervening components (i.e. with or more intervening amino acid residues), which will typically comprise one or more further HIV antigens. "Jt is also generally preferred that the immunogen does not contain the entire gag protein amino acid sequence. Typically between 55 and 95% of the gag protein will be present, preferably between 65 and 85%, most preferably about 75%.

The wild type HIV gag protein is known to consist of three portions termed p17, p24 and pls. These are synthesised in infected cells as a single polyprotein, with p17 at the N terminus. Normally, in HIV-infected cells, the N terminus of p17 is myristylated.

It is desirable that the gag portion of the immunogen will comprise at least part of p17 and p24, but it is generally preferred that the p17 component will be modified in some way to prevent myristylation. Conveniently this can be accomplished by reversing the order of the p17 and p24 components in the immunogen, such that p17 is no longer at a free N terminus ’ and cannot therefore be myrisylated. The inventors believe that this may improve the efficiency of presentation of peptides, derived from the immunogen, to a subject’s immune system.

The gag protein component of the immunogen will generally comprise at least onc T-helper cell, HLA Class Il-restricted, peptide epitope and preferably comprise a plurality of such epitopes (preferably such that a number of different HLA Class I-restricted allcles are represented in the gag component). The gag protein component will also typically comprise one or mare CTL HLA Class I-restricted peptide cpitopes.

The synthetic polypeptide componeat of the immunogen will comvenicatly take the form of a string of CTL epitopes, cach repeescated by, or contained within, a respective sequence of about 8-12 amino acids. Desicably st least some (pecficsably most) of the epitopes will be pastially overlapping (such that onc or more amino acids of one epitope will also be contained within the sequence of an adjoining epitope). Some “non-epitopic” amino acid © sequence may be present between neighbouring epitopes, but this is generally to be avoided.

Non-epitopic amino acid sequence between neighbouring epitopes is preferably less than 20 - amino acid residues, more preferably less than 10 residues, and most preferably 1-5 amino acid residues. It will be apparent that such non-epitopic amino acid sequence may comprise linkers, spacers and the like which optimise the expression levels of the synthetic polypeptide or optimise its immunogenicity.

Thus in one extreme embodiment, all of the human CTL epitopes in the synthetic polypeptide may be overlapping and, at the other extreme, every epitope may be separated from its neighbours by at least some non-epitopic amino acid sequence. It is generally to be preferred that at least 50% of the human CTL epitopes are overlapping.

In addition to overlapping epitopes, the synthetic polypeptide may comprise at least some “adjacent epitopes”. The term adjacent epitopes refers to epitopes which are not overlapping but which are not separated by any intervening non-epitopic amino acid sequence.

Thus in preferred embodiments the synthetic polypeptide comprises a mosaic pieced together from small (typically about 10-20 amino acid residue) fragments of different HIV : proteins, which fragments will typically comprise one, two or three known adjacent and/or overlapping human CTL epitopes. Where a plurality of fragments are present in the "synthetic polypeptide from the same HIV protein, the fragments will typically have been selected from discontinuous portions of the protein, so that it is unlikely that the synthetic polypeptide comprises a sequence corresponding to more than 20-25 consecutive amino acid residues of a particular HIV protein. Generally the synthetic polypeptide is designed so as essentially to omit those portions of HIV proteins not known to contain any human CTL epitopes.

A plurality of different HIV proteins will preferably be represented in the synthetic polypeptide. The synthetic polypeptide may contain epitopes present in any HIV antigen, but preferably will comprise at least one epitope present in one or more of the following: p24; pol; gp4l; gpl20; and nef. In one preferred embodiment, the synthetic polypeptide comprises at least one CTL epitope present in each of the aforementioned HIV proteins. The synthetic polypeptide may additionally comprise at least one CTL epitope present in each of the following HIV proteins: vpr, vpu, vif (and especially) tat and rev. . It will be understood that the term “human CTL epitope” as used herein refers not to the origin of the protein from which the epitope derives, but indicates that the epitope is recognized and responded to by the CTL of at least a portion of the human population.

Typically a human CTL epitope will be recognized by at least 0.01%, preferably 0.1%, and more preferably at least 1% of the world’s human population. :

In one particular embodiment, the immunogen specifically excludes any epitope from the env protein generally recognised by the human immune system. Most presently-used diagnostic tests are based on detection of an HIV env-specific immune response, so by excluding env components from the immunogen it is possible to distinguish between immune responses rising from infection with the virus and inoculation with the immunogen.

In one embodiment, the CTL epitopes present in the synthetic polypeptide are selected such that an immune response to HIV clade A will be generated. Preferably however, the synthetic polypeptide is large enough, and the CTL epitopes appropriately selected, such that an immune response which is cross-reactive against different HIV clades will be stimulated.

This can conveniently be achieved by including one or more CTL epitopes which are conserved among different HIV clades. Several such epitopes are known to those skilled in the art (sec Table 1 below).

In a preferred embodiment, the immunogen comprises at least one epitope (conveniently a

CTL epitope) which is recognized by one or more laboratory test mammals, (e.g. mouse . and/or monkey). Such an epitope can readily be incorporated within the synthetic polypeptide. Inclusion of an epitope of this sort allows for the quality, reproducibility and/or stability of difficscnt batches of the immogen to be assayed in a potency assay using the

Inbosatory test mammal (such as a mouse or macaque monkey). Examples of such epitopes inchade the amino acid sequeace ACTPYDINQML (Seq. ID No. 1; containing a dominant epitope derived from simian immwmodeficicacy vias, SIV, gag p27, recogaised by rhesus macaque monkey CTLs) and RGPGRAFVTI (Seq. ID No. 2; an H-2D" restricted CTL mouse epitope derived from HIV env protein).

A list of some 23 human CTL epitopes suitable for inclusion in the synthetic polypeptide is shown in Table 1. The list is not exhaustive. A preferred immunogen will comprise at least of such human CTL epitopes, more preferably at least 15, most preferably at least 20. In a particular embodiment each of the 23 human CTL epitopes listed in Table 1 will be represented in the synthetic polypeptide, optionally together with the macaque and murine epitopes also listed in Table 1.

Desirably the immunogen may additionally comprise a small tag or marker. Such a tag or marker should be as small as possible, in order to minimise the amount of extraneous material present in the immunogen. A convenient tag or marker allows for detection of expression and/or quantification of the amount of immunogen. Suitable tags include epitopes recognized by the monoclonal antibody Pk (Hanke ef al, 1992 J. Gen. Virol. 73, 653-660).

The immunogen of the invention will conveniently be mixed with other substances in order "to provide a vaccine composition. For example, a vaccine composition may additionally : comprise one or more of the following: an adjuvant (e.g. alum), liposome, or immunostimulatory complex (ISCOM). A vaccine will also generally comprise a sterile : diluent, excipient or carrier, such as a physiologically acceptable liquid (e.g. saline or phosphate-buffered saline solutions). The vaccine may be presented as a liquid or, more preferably, as a freeze-dried solid, which is suspended or dissolved in a physiologically acceptable liquid prior to administration.

Methods of administering the immunogen to a human subject will be apparent to those skilled in the art. Such methods include, in particular, intramuscular injection, subcutaneous injection, or delivery through the skin by needleless injection device. Alternatively, vaccines (especially those comprising bacterial vectors e.g. attenuated Salmonella or Shigella spp.) may be given orally, intranasally or by any other suitable route. A suitable dose of immunogen may typically be from 1 to 500 mg, typically from 10 to 100mg, depending on the size of the immunogen, the body mass of the recipient etc. A suitable dose can, if necessary, be ascertained by routine trial-and-error with different groups of subjects being given different doses of immunogen, and the immune response of the subjects to the ] immunogen asayed in order to determine optimum dose size. The immune response of the subjects can be assayed by conventional immunological techniques (e.g. chromium release assay, using peripheral blood lymphocytes obtained from subjects’ blood samples, acting on peptide-pulsed or virus-infected chromium-labelled target cells). . In a second aspect, the invention provides a nucleic acid molecule encoding a fusion protein, the fusion protein comprising an immunogen in accordance with the first aspect of the

The nucleic acid molecule is preferably in isolated, sterile form, suitable for administration to a human subject The mucleic acid molecule is preferably “humanized” ie. employs codons to code particular amino acids, which codons are frequently used in highly expressed human genes (Andre ef al, 1998 J. Virol. 72, 1497-1503), instead of those codons used by

HIV.

Desirably the nucleic acid molecule is contained within a vector, the sequence encoding the immunogen being opcrably linked to a promoter sequence active in human cells.

Conveniently the promoter is a strong viral promoter such as the promoter from human cytomegalovirus (CMV). The vector will preferably also comprise an enhancer and : polyadenylation signals, which are functional in human cells. Ideally, for maximum safety, the vector should not contain any origin of replication functional in human cells, to prevent undesirable replication of the vector.

The vector may be administered to the subject in isolation, as essentially ‘naked’ mucleic acid (preferably DNA), or cise may be packaged within a delivery means, such as a virus, bacterium, liposome, or gold-coated particles and the like. One suitable delivery means is the modified vaccinia virus Ankara (MVA): the inemmogen gene or open reading frame . (ORF) may be inserted into MVA, for cxamplic, at the thymidine kinase locus. kt will be appeecinted by those skilled in fhe art that 2 vector may comprise a mucleic acid " encoding an immunogen (the nucleic acid thesefore being in accosdance with the second aspect of the invention), and that the vector muy also possess, at its surface or internally, the polypeptide immunogen in scoordence with the first sapect of the invention.

Whether administered as naked nucleic acid, or packaged within a delivery means, at least some of the administered nucleic acid enters the cells of the subject and is transcribed (if : necessary) and translated, resulting in the in situ synthesis of the immunogen in the subject, who then develops an immune response to the immunogen. - As with the immunogen preparation per se, a nucleic acid encoding the immunogen may be administered to a human subject by any of a number of known routes e.g. subcutaneous or intramuscular injection, oral delivery, or delivery through the skin by means of needleless injector. Particular examples of administration by needleless injection device are disclosed in WO 97/34652 and WO 97/48485. A suitable dose of nucleic acid may be from 10ug to 10mg, typically from 100ug to 1mg, depending on the size of nucleic acid molecule, route of administration, body mass of the recipient etc. Again, routine trial and error (with the benefit of the present teaching) will enable those skilled in the art to determine an optimum dose of the nucleic acid.

Successful delivery of DNA to animal tissue has been achieved by cationic liposomes [Watanabe et al., Mol. Reprod. Dev. 38:268-274 (1994); Sharkey et al, WO 96/20013], direct injection of naked DNA into animal muscle tissue [Robinson et al., Vacc. 11:957- 960 (1993); Hoffman et al., Vacc. 12:1529-1533; (1994); Xiang et al., Virol. 199:132-140 (1994); Webster et al., Vacc. 12:1495-1498 (1994); Davis et al., Vacc. 12:1503-1509 (1994); and Davis et al., Hum. Molec. Gen. 2:1847-1851 (1993)], and embryos [Naito et al., Mol. Reprod. Dev. 39:153-161 (1994); and Burdon et al., Mol. Reprod. Dev. 33:436- 442 (1992)}, or intradermal injection of DNA using “gene gun” technology [Johnston et al., Meth. Cell Biol. 43:353-365 (1994)]. . For DNA-based vaccination, delivery by injection of naked plasmid DNA has shown potential in mouse models for inducing both humoral and cellular immune responses.

However, in larger animals, using DNA delivery for vaccination has been hampered by requiring large amounts of DNA or inducing persistent expression of an antigen with the potential for developing tolerance to the antigen. Berglund reported a strategy for inducing or enbancing an immune response by injecting mice with plasmid DNA

. containing an alphavirus DNA expression vector having a recombinant Semliki Forest . Virus (SFV) replicon in a eukaryotic expression cassette [Berglund et al.,, Nature

Biotechnol. 16:562-565 (1998)]. The eukaryotic expression cassette controlled expression of the primary mucicar transcription of the SFV replicon. This SFV replicon transcript, encoding the heterologous antigen, was transported to the cytoplasm and amplified by the . scif-encoded SFV replicase complex. The amplified RNA replicon lead to high level production of an mRNA encoding the heterologous antigen. Similar results were described by Polo and his group [Polo ct al, Nature Biotechnol. 16:517-518 (1998); Hariharan et al., J. Virol. 72:950-958 (1998)]. Both groups found strong immume responses could be induced using small amounts of input plasmid DNA.

Alterpatively, a method to deliver DNA to animals that overcomes the disadvantages of conventional delivery methods is by administering attenuated, invasive bacteria containing a bacterial DNA vector having a eukaryotic expression cassette encoding the gene to be expressed. For example, U.S. Patent No. 5,877,159 to Powell et al., describes live "bacteria that can invade animal cells without establishing a productive infection or causing disease to thereby introduce a eukaryotic expression cassette encoding an antigen capable of being expressed by the animal cells.

In a third aspect the invention provides a method of stimulating an anti-HIV immune response in a human subject, the method comprising preparing an immunogen in accordance with the first aspect of the invention, or a nucleic acid molecule in accordance with the second aspect of the invention; and administering said immunogen or nucleic acid molecule to the subject. acid molecule. In pasticuler, the method prcficssbly comprises one or mose administrations of the mucleic acid melecule (“priming”) followed at a switshie interval (c.g 1 week 0 4 months) by ome or more administrations of the imcumnogen (“boosting”). Boosting may be performed, for example, by administering a replication-competent (c.g. attenuated virus or j molecule encoding the immunogen. Preferably boosting is achieved by administering the immunogen as part of an MVA viral particle, which particle may advantageously comprise a nucleic acid encoding the immunogen.

In a preferred embodiments, performance of the method will result in the generation in the . subject of a protective immune response such that, should the subject subsequently be exposed to HIV infection, the subject will not go on to develop the symptoms of AIDS associated with HIV infection. :

In a fourth aspect the invention provides for use of an immunogen in accordance with the first aspect of the invention and/or a nucleic acid in accordance with the second aspect of the invention in the preparation of a medicament to prevent or treat HIV infection in a human subject.

In a fifth aspect, the invention provides a nucleic acid sequence encoding the amino acid sequence shown in Figure 8A. Conveniently the nucleic acid comprises or essentially "consists of the nucleotide sequence shown in Figure 8B.

In a sixth aspect, the invention provides a polypeptide comprising the amino acid sequence shown in Figure 8A.

The nucleic acid of the fifth aspect and/or the polypeptide of the sixth aspect may be used in immunogen/vaccine compositions as described in relation to the other aspects of the invention, and such immunogens and vaccines are accordingly considered within the scope of the invention, and may comprise vectors etc (especially MVA) as aforesaid. The invention further provides, in a seventh aspect, a method of stimulating an anti-HIV immune - response in a human subject, the method comprising administering to the subject a nucleic acid in accordance with the fifth aspect of the invention and/or a polypeptide in accordance with the sixth aspect of the invention. Finally, the invention provides for use of a nucleic acid in accordance with the fifth aspect of the invention and/or a polypeptide in accordance with the sixth aspect of the invention in the preparation of a medicament to prevent or treat

HIV infection in a human subject.

The invention will now be further described by way of illustrative example and with } reference to the accompanying drawings, wherein:

Figure 1A is a schematic representation of immunogens, HIV A, HIVTA and HIVAeT in accordance with the invention; and an immunogen PPA;

Figure 1B shows the amino acid sequence of the HIVA immunogen (Seq. ID No. 26);

Figure 2A shows the nucleotide sequence (Seq. ID No. 27) of a nucleic acid (termed “HIVA gene”) encoding the HIV A immunogen; :

Figure 2B is a schematic representation of the method used to construct the HIVA gence; .

Figure 3 is a schematic representation of a DNA vector molecule (pTHr. HIVA) in accordance with the invention, and the method of its construction;

Figure 4 is a micrograph showing immunofluorescent detection of HIVA expression by ) mouse cells following transfection with pTHr. HIVA;

Figures 5A, B & C are graphs showing the results of chromium release assays using a splenocytes obtained from mice inoculated with a DNA molecule or an immunogen in accordance with the invention;

Figure 6A shows the amino acid sequence of the HIV TA immunogen (Seg. ID No. 28);

Figure 6B shows the nucleotide sequence (Seq. ID No. 29) of a nucleic acid molecule encoding the HIV TA immunogen;

Figure 7A shows the amino acid soquence (Seg. ID No. 30) of the tat polypeptide present in the HIVA:T inunanogesn;

Figure 7B shows the nucleotide sequence (Seq. ID No. 31) of a nucleic acid molecule encoding the HIVAeT immunogen;

Figure 8A shows the amino acid sequence (Seq. ID No. 32) of the PPA immunogen which isin accordance with the sixth aspect of the invention; and

Figure 8B shows the nucleotide sequence (Seq. ID No. 33) of a nucleic acid molecule (in accordance with the fifth aspect of the invention) encoding the PPA immunogen.

EXAMPLES :

Example 1

This example relates to an immunogen for use in a vaccine focusing on the induction of cellular immune responses mediated by a concerted action of CD4" helper and CD8” effector

T lymphocytes. The immunogen, designated HIVA (Hanke & McMichael Nat. Med. 6, 951- 955), was designed for a phase III efficacy trial in Nairobi, Kenya. Figure 1A is a schematic representation of several immunogens, including HIVA. HIVA is derived from the sequences of HIV-1 clade A, the predominant HIV clade in Nairobi and consists of about : 73% of the gag protein fused to a string of 25 partially overlapping CTL epitopes. The gag domain of HIVA contains p24 and pl7 in an order reversed to the viral gag pl17-p24-pl5 : polyprotein. This rearrangement prevents myristylation of the N-terminus of p17, which could direct the recombinant protein to the cell membrane, thus preventing efficient degradation into peptides necessary for the major histocompatibility complex (MHC) class I presentation. . Figure 1B shows the amino acid sequence (Seq. ID No. 26) of the HIVA immunogen.

Amino acids corresponding to the restriction endonuclease sites used to assemble the gene are shown in bold (GS-corresponds to Bam HI, GT corresponds to Kpnl and EF correspnds to EcoRI).

The amino acid sequence of the gag domain was derived from the protein database consensus sequence of HIV-1 clade A (Korber ef al, “Human retroviruses and AIDS; a compilation and analysis of nucleic acid and amino acid sequences” 1997). In the absence of available Kenyan strain sequences, regions without a strong amino acid clade A preference were biased towards Ugandan isolates. The HIV-1 gag protein contained not only important

MHC class I, but also class I-restricted epitopes which stimulate CD4" T helper cells. . The C-terminus of the HIVA protein was designed as a multi-CTL epitope synthetic polypeptide. The CTL epitopes included in HIVA were recognised by CTL in patients infected with HIV-1 clade A strains circulating in Kenya, were 8- to 10-amino acids long, and originate from the gag, pol, nef or env proteins (Rowland-Jones ef al, 1998 J. Clin.

Invest. 102, 1758-1765; Dorrell et al, 1999 J. Virol. 73, 1708-1714). Many of these epitopes are immunodominant and relatively conserved among other HIV-1 clades (Table 1) (and therefore should be able to elicit an immune response which cross-reacts with HIV viruses of clades other than clade A). They arc presented by seventeen different HLA alleles, which include both frequent African alleles as well as alleles common in most ethnic populations.

It has been estimated that optimally selected epitopes presented by the nine commonest HLA alleles could cover the general population irrespective of ethnic descent (Sydney ef al, 1996 "Immunol. Today 17, 261-266). Thus, given that majority of HIV-infected donors make good

CTL responses to gag p17/p24, each vaccines should have the potential to respond to at least two or three CTL epitopes present in the HIVA protein.

The HIVA synthetic polypeptide also comprised SIV gag and HIV env epitopes recognised by macaque and murine CTL, respectively, so that the quality, reproducibility and stability of the clinical batches could easily be assessed in a mouse (or macaque if necessary) potency assay. A monoclonal antibody epitope Pk (Hanke ef al, 1992 J. Gen. Virol. 73, 653-660) was added to the C-terminus of HIVA for easy detection of the full-size protein and estimation of the level of expression. There is no reason to believe that the three non-HLA - epitopes represent a health hazard for the vaccinated individuals.

Table 1. CD8+ cell epitopes included in the HIVA synthetic polypeptide polyepitope region. a Ls — a CO

FOVORNKTL [RLABE |B [BOG-CRORY

Dea SO LA LJ cc

ES Lc Lo CCS a Lc SO CN

ROUT [WAST [=r [wwomRo

LC Lc ic LA Lc am a LS CN Cc

LC LC LS LL

Bie Lc CS CO

DL SO Co Lc

} a- Epitopes are listed (Seq. ID Nos. 3-25, 1, 2) in the order in which they appear in the polyepitope. _ b- A particular epitope sequence is present in about 50% (small letter) or 90% (capital letter) of sequenced HIV clade isolates. c- ‘= indicates that the cpitopes are present in the N-terminal clade A gag domain. d- A dominant epitope derived from STV gag p27 flanked by Ala and Leu at its N- and

C- termini, respectively, recogmised by rhesss macaque CTL, which can be used for potency studies in rhesus macaques. e- A CTL epitope presented by a murine MHC class I used for the mouse potency

Since it was intended to adopt a “prime/boost™ protocol, in which priming was achieved by administering nucleic acid, it was desirable to design the sequence of the mucleic acid encoding the HIVA immunogen in order to increase the expression of HIVA in human cells. Firstly, to ensure an efficient initiation of translation from the first methionine codon, the HIVA open reading frame (ORF) was preceded by a 12-mucleotide-long Kozak consensus sequence (Kozak 1987 Nucl. Acids Res. 15, 8125-8148). Secondly, the : translation of the resulting mRNA was optimised by substituting most of the HIV-1- derived codons with frequently used codons in highly expressed human genes (Andre ez al, 1998, cited above). The HIVA ORF is a part of a 1,608-base pair-long double-stranded

DNA HindIll-Xbal fragment. (Figure 2A shows the mucleotide sequence of the HindIIl-

Xbal insert containing the HIVA ORF; Seq. ID No. 27) In Figure 2A, the endonuclease sites used to assemble the partial PCR products are included (nucleotides 1-6 = HindIII; 319-324 = Bam HI; 712-717 = KpnI; 1135-1140 = EcoRI; and 1603-1608 - Xbal).

Plasmid DNA was prepased and treated wsing standard protocols (Sambrook ef al, "Molecular Closing. A Laboratory Massel” 2* Edition; Cold Spring Harbor). The

HIVA geac was constructed (as indicated in Figure 2B) in vitro in four parts. Each part oligodeoxymucicotides of 70-90 bases in length. The synthetic oligodeaxymucleotides were purified using the EconoPure™ Kit (Perkin Elmer) according to the manufacturer's instructions, annealed and ligated, followed by PCR assembly. The PCR products were gel-purified after each step until fragments with the expected, unique terminal restriction . endonuclease sites were obtained. These four products were cloned and sequenced, and ligated together to generate the complete HIVA gene. The HIVA gene was then inserted into the pTHr construct and MVA vaccine vector, as described below.

The pTHr vector. A vector pTHr for a direct gene transfer was designed with the aim to minimise the number of functional elements and therefore the amount of DNA required to be administered.

The construction of pTH was described previously (Hanke ef al, 1998 Vaccine 16, 426- 435). It contains an expression efficient enhancer/promoter/intron A cassette of the human . cytomegalovirus (hlCMYV) strain AD169 genome (Whittle ef al, 1987 Protein Eng. 1, 499- 505; Bebbington 1991 Methods 2, 136-145). The promoter region is followed by the pRc/CMV (Invitrogen)-derived polylinker and polyadenylation signal of the bovine growth hormone gene. The B-lactamase genc conferring ampicillin resistance to transformed bacteria and prokaryotic origin of double-stranded DNA replication ColEl are both derived from plasmid pUC19. pTH does not contain an origin for replication in mammalian cells. After insertion of the HIVA DNA into the pTH polylinker, the 8- lactamase gene fragment between the Mul and Dral sites was removed and the resulting construct pTHr.HIVA (Fig. 3) was propagated in bacteria using the repressor-titration system developed by Cobra Pharmaceuticals Lid. (Keele, UK), which sclects plasmid- . carrying bacteria without the need for the presence of an antibiotic-resistance gene on the plasmid (Williams ef al, 1998 Nucl. Acids Res. 26, 2120-2124). Therefore, DNA vaccination does not introduce into the human vaccinee large numbers of copies of an antibiotic resistance gene. Construction of pTHr. HIVA is illustrated schematically in

Figure 3 (CMV e/p/i = human CMV enhancer/promoter/intron A cassette; BGHpA = bovine growth hormone polyadenylation signal; ColE1 = origin of dsDNA replication in bacteria; arrow head symbols denote repressor-binding sequences).

293T cells (and chicken embryo fibroblasts [CEF] were maintained in Dulbecco's modified

Eagle’s medium (DMEM; Gibco) supplemented with 10% foetal bovine serum (FBS;

Gibco); 2mM L-giotamine and penicillin/streptomycin. Cells were cultured in a "humidified incubstor in 5% CO, at 37°C. 293T ceils were transiently transfected with pTHr. HIVA using the DEAE-dextran-chloroquine method (Hanke ef al, 1998 Vaccine 16, 426-435). Briefly, 2.5 x 10° 293T cells were grown om coverslips in 6-well tissue culture plates overnigit. The following day, cells were transfected with 5 g per well of DNA.

After 48 hours, the transfected cells were fixed, their membranes were permeabidized and the SVS5-P-k mAb followed by anti-murine FITC-conjugated antibodies were used to detect

A micrograph illustrating specimen results is shown in Figure 4. The Figure shows three transfected cells and one background untransfected cell (top left).

Boosting was to be achieved by administering an MVA vector expressing the HIVA immunogen. MVA is an attermated vaccinia virus safe for clinical application, which has : almost lost its ability to replicate in human cells (Mayr ef al, 1975 Infection 105, 6-14).

The use of MVA as a vaccine vehicle and its features which make it an attractive choice : among the attenuated poxvirus vectors (see, c.g. Sutter ef al, 1994 Vaccine 12, 1032-1040; and Moss ef al, 1996 Adv. Exp. Med. Biol. 397, 7-13) have been described extensively.

The HIVA gene was ligated into plasmid pSC11, which directed the gene into the thymidine kinase locus of the parental MVA (Carroll & Moss 1995 Biotechniques 19, 352- * 356). Bulk stocks of the recombinant MVA were grown on primary CEF obtained from the cggs of a specific pathogen-free flock. MVA was purified by cestrifagation of

Cytoplasmic extracts thesugh 2 36% (w/v) sucrose cushion in a Beckmsn SW28 rotor at 13,500 ron for 80 minmtes. Taking advantage of the co-imserted 8-gaiactosidase geae, the vires stock titres were detcamined from the smber of blue plagues after incobation of the

Vaccine potency assay. The potencies of the DNA and MVA vectors were tested in groups of Balb/c mice taking advantage of the presence of the H-2D"restricted epitope (Takahashi et al, 1993 Int. Immunol. 5, 849-857). For the pTHr.HIVA DNA vaccine, mice were either needle-injected intramuscularly with 100 ug of DNA or immunised twice 3 weeks apart intradermally with total of 2 ug of DNA using the Dermal XR gene delivery device of PowderJect Vaccines Inc. (Madison, WI, USA). The mice were sacrificed 10 or 21 days after the last immunisation. Spleens from the immunized mice were removed and pressed individually through a cell strainer (Falcon) using a 2-ml syringe rubber plunger.

The splenocytes were washed and divided into two halves. One half was frozen for tetramer analysis and the second half was suspended in 5ml of Lymphocyte medium (R10, 20 mM HEPES and 15 mM B-mercaptoethanol) and restimulated in vitro by incubation with 2 pg/ml of the RGPGRAFVTI (Seq. ID No. 2) peptide in an humidified incubator in 5% CO, at 37°C for 5 days.

The effector cells were 2-fold diluted in U-bottom wells (96-well plate; Costar) starting with 100:1 effector to target ratio. Five thousand *'Cr-labeled P815 target cells in a medium without or supplemented with 10° M peptide was then added to the effectors and incubated at 37°C for 5 hours. Spontaneous and total chromium releases were estimated from wells, in which the target cells were kept in a medium alone or with 5% Triton X- ] 100, respectively. The percentage specific lysis was calculated as [(sample release- spontaneous release)/(total release-spontaneous release)] x 100. The spontaneous release was lower than 5% of the total c.p.m. "The results of typical assays are shown in Figures SA-C, which are graphs of % specific lysis against Effector: Target ratio. Figure 5A shows results for splenocytes obtained from mice immunised once with 100 pg of pTHr.HIVA DNA. Figure 5B shows results from mice immunised twice intradermally with pTHr. HIVA DNA. Figure 5C shows results obtained from mice immunised intramuscularly with 10’ pfu of MVA.HIVA. In each of

Figures 5A-5C, each line represents a single mouse. Lysis of peptide-pulsed targets is denoted by filled symbols, unpulsed targets by open symbols.

Both modes of the pTHr.HIVA vector delivery were highly immunogenic and induced high cytolytic activities in all immunised animals (sec Figs. SA & B). Similarly, a single needle intramuscular injection of 107 plaque-forming units of MVA HIVA elicited in all _ vaccinees stromg peptide-specific lytic activitics measured afer a culture restimulation (Fig. 5C). based on HIVA, but comprising farther HIV antigen components. These further constructs and their encoded immunogens were termed HIVTA and HIVAeT, as shown in ‘Figure 1A. Also shown is a construct/immumogen termed PPA. The relevant DNA/amino acid sequences are shown in Figures 6 A/B-8 A/B respectively, although Figure 7A shows only that portion of amino acid sequence in HIVA cT (amtributable to tat) which is additional to that in HIVTA. (Note that the sequence of the HindIll and Xbal sites at the 5’ and 3’ ends of the DNA sequences are not shown in Figures 6B, 7B and 8B). The constructs were prepared in a manner similar to that described above for HIVA. * HIVTA and HIVAeT share the same design rationale with HIVA, but additionally include the HIV-1 clade A tat sequence, expressed cither as part of a fasion protein with gag and the polyepitopic synthetic polypeptide (in the case of HIVTA, the tat sequence being positioned between gag and the synthetic polypeptide), or else being present on the same construct but expressed as a separate polypeptide (in the case of HIVAeT), by virtue of the inclusion of an internal ribosome cutry site (IRES).

Each of the mucieic acid molecules may be used to immunise subjects, in a manner similar to that described above im relation 0 HIVA DNA. Equally, the moiccuics may be imsroduced into agpeopeiste vectors, capecially MVA, again as described above in Example "1, and the resulting vector used to immmmnise subjects.

The significance of the genetic diversity among individual HIV isolates amd its implication for vaccine design have been Jong debated. The predominant HIV-1 clade in Europe and

North America is clade B, which is also the most studied one. In central and Eastern

Africa, the predominant circulating HIV-1 strain is clade A, while clade C is dominating

Southern Africa, India and China. Generally, a clade-specific vaccine design requires a more careful consideration for the induction of nAb than for CTL. Although there are some important inter-clade differences in CTL epitopes, many epitopes are conserved across clades partially due to structure/function constraints. However, to facilitate the interpretation of efficacy studies, vaccines should attempt to match the local strains prevalent in the trial population with the view that any successful approaches can be adapted for other clades if cross-protection is not achieved.

Claims (60)

Claims

1. An immunogen in sterile form suitable for administration to a human subject, the immunogen comprising: at least a portion of the gag protein of HIV, said gag protein being from an HIV clade or having a consensus sequence for one or more HIV clades, and comprising at least parts of p17 and p24; amd a synthetic polypeptide comprising a plurality of amino acid sequences, each sequence comprising a human CTL cpitope of aa HIV prowcin, and wherein a plurality of HIV proteins are represented in the synthetic polypeptide, said CTL epitopes being selected to stimulate an immune response to one or more HIV clades of interest.

2. An immunogen according to claim 1, in which the said at least portion of gag protein and the synthetic polypeptide are present as a fusion protein.

3. An immunogen according to claim 1 or 2, in which at 55-95% of the amino acid sequence of gag protein is present in the portion of gag.

4. An immunogen according to amy onc of claims 1, 2 or 3, in which 65-85% of the : amino acid sequence of gag protein is present in the portion of gag.

5. An immunogen according to any one of the preceding claims, in which said at least part of p17 is modified to prevent N-terminal myristylation.

6. An immunogen according to any onc of the preceding claims, in which at least some of the human CTL epitopes present in the synthetic polypeptide are overlapping.

7. An imsssmogen accosding to claim 6, in which at least S0% of the haman CTL cpitopes presest in the synthetic polypeptide ase overlapping.

8. An immsssegres acconding to any ome of the preceding claims, in which at least some of the human CTL epitopes present in the synthetic polypeptide are adjacent.

9. An immunogen in according to any one of the preceding claims, in which at least some of the human CTL epitopes are separated by non-epitopic amino acid sequence.

10. An immunogen according to any one of the preceding claims, comprising at least one epitope which is recognised by one or more laboratory test mammals.

11. An immunogen according to claim 10, wherein said epitope recognised by one or more laboratory test mammals is a CTL epitope.

12. An immunogen according to any one of the preceding claims, in which the synthetic

. polypeptide comprises at least 10 of the human CTL epitopes identified in Table 1. - 13. An immunogen according to any one of the preceding claims, in which the synthetic polypeptide comprises at least 15 of the human CTL epitopes identified in Table 1.

14. An immunogen according to any one of the preceding claims, in which the synthetic polypeptide comprises at least 20 of the human CTL epitopes identified in Table 1.

15. An immunogen according to any one of the preceding claims, in which the synthetic polypeptide comprises all 23 of the human CTL epitopes identified in Table 1.

16. An HIV immunogen for a human subject comprising at least a portion of a gag protein, said gag protein being from an HIV clade of interest or having a consensus sequence for an HIV clade of interest, and wherein said portion comprises at least parts of p17 and p24 and is modified to prevent N-terminal myristylation; and a synthetic polypeptide comprising a plurality of human CTL epitopes from a plurality of different HIV proteins, each CTL epitope having from 8 to 12 amino acids, and said CTL epitopes being selected to stimulate an immune response to the clade of interest.

17. An HIV immunogen for a human subject comprising at least a portion of a gag i protein, said gag protein being from an HIV clade of interest or having a consensus sequence for an HIV clade of interest, and wherein said portion comprises at least parts of p17 and p24 and is modified to prevent N-terminal myristylation; and a synthetic polypeptide consisting essentially of a plurality of human CTL epitopes from a pierality of different HIV proteiss, each CTL cpitope having from 8 to 12 amino aciis, and said CTL epitopes being selected to stimniste an immune response to said clade of imterest; and optionally, said systhetic polypeptide having at least one immmmogenic epitope recognised by a laboratory animal.

18. An immunogen according to any onc of the preceding claims, comprising the amino acid sequence shown in Figure 1B.

19. An immunogen according to claim 18, consisting essentially of the amino acid sequence shown in Figure 1B.

20. An immunogen according to any one of the preceding claims, wherein said clade of interest is selected from the group consisting of HIV clade A, B, C, D, E, F, G and

: H.

21. An immunogen according to claim 20, wherein said clade of interest is HIV clade A.

22. An immunogen according to claim 20, wherein said clade of interest is HIV clade B.

23. An immmmoges according to claim 20, wherein said clade of interest is HIV clade C.

24. As immssogen according $0 any ome of the preceding claims, wherein said human CTL cpitopes include at icast cue epitope from each of the HIV proteims nef, p24, p17, pol, gpél and gp120.

25. An immunogen according to claim 24, wherein wherein said human CTL epitopes include at least one epitope from each of the HIV proteins nef, p24, p17, pol, gpdl, gp120, tat, and rev.

26. An immunogen according to claim 24 wherein said human CTL epitopes include at least one epitope from each of the HIV proteins nef, p24, p17, pol, gp41, gp120, tat, rev, vpr, vpu and vif.

27. A nucleic acid molecule encoding an immunogen in accordance with any one of the preceding claims.

28. A nucleic acid according to claim 27, wherein the immunogen is encoded as a fusion protein.

29. A nucleic acid molecule according to claim 27 or 28, comprising the nucleotide sequence shown in Figure 2A.

30. A vector comprising a nucleic acid molecule in accordance with any one of claims 27, 28 or 29. : ©

31. A particulate vector according to claim 30, further comprising an immunogen in accordance with any one of claims 1-26.

32. A method of stimulating an anti-HIV immune response in a human subject, the method comprising the steps of: preparing an immunogen in accordance with any one of claims 1-26 and/or a nucleic acid molecule in accordance with any one of claims 27-29; and administering the immunogen and/or the nucleic acid molecule to the subject.

33. A method according to claim 32, wherein the subject is primed by one or more . administrations of the nucleic acid molecule; and subsequently boosted by one or more administrations of the immunogen.

34. Use of an immunogen in accordance with any one of claims 1-26 and/or a nucleic acid molecule in accordance with any ome of claims 27-29, in the preparation of a medicament to prevent or treat HIV infection in a buman subject.

35. A method of stimulating an anti-HIV immune response in a human subject which comprises administering one or more times an amount of a nucleic acid encoding an immunogen in accordance with any one of claims 1-26 to said subject sufficient to prime an immune response to said immunogen, and administering one or more times a modified vaccinia virus Ankara (MVA) particle encoding and/or containing an immunogen in accordance with any one of claims 1-26 to said subject in an amount sufficient to boost the immune response to common portions of said immunogens. :

36. A bacterium comprising an immunogen in accordance with any one of claims 1-26, or comprising a nucleic acid molecule in accordance with any one of claims 27-29.

37. A bacterium according to claim 36 which is an attemmated pathogen suitable for administration to a hnman subject.

38. A mucleic acid molecule encoding a polypeptide comprising the amino acid sequence shown in Figure 8A.

39. A mucieic acid molecuic encoding a polypeptide cossisting csseatially of the amino acid sequence shows in Figure 8A.

40. A mmcieic acid molecule according to claim 38 or 39, comprising the nucleotide soguence shown in Figure 8B.

28 PCT/GB00/04984

41. A polypeptide comprising the amino acid sequence shown in figure 8A.

42. A polypeptide consisting essentially of the amino acid sequence shown in Figure

8A.

43. A particulate vector comprising the nucleic acid sequence of claim 38 or 39 and/or the polypeptide of claim 41 or 42.

44. A method of stimulating an anti-HIV immune response in a human subject, comprising the steps of: preparing a nucleic acid molecule in accordance with claim 38 or 39 and/or a polypeptide in accordance with claim 41 or 42, and administering the nucleic acid molecule and/or the polypeptide to the subject.

45. Use of a nucleic acid molecule in accordance with claim 38 or 39 and/or a polypeptide in accordance with claim 41 or 42, in the preparation of a medicament to prevent or treat HIV infection in a human subject.

46. A substance or composition for use in a method of stimulating an anti-HIV immune response, said substance or composition comprising an immunogen in accordance with any one of claims 1-26 and/or a nucleic acid molecule in accordance with any one of claims 27-29; and said method comprising administering said substance or composition to the subject.

47. A substance or composition for use in a method of treatment according to claim 46, wherein the subject is primed by one or more administrations of the nucleic acid molecule; and subsequently boosted by one or more administrations of the immunogen.

48. A substance or composition for use in a method of stimulating an anti-HIV immune response, said substance or composition comprising a nucleic acid : AMENDED SHEET

29 PCT/GB00/04984 encoding an immunogen in accordance with any one of claims 1-26, and said method comprising administering one or more times an amount of said substance or composition to said subject sufficient to prime an immune response to said immunogen, and administering one or more times a modified vaccinia virus Ankara (MVA) particle encoding and/or containing an immunogen in accordance with any one of claims 1-26 to said subject in an amount sufficient to boost the immune response to common portions of said immunogens.

49. A substance or composition for use in a method of stimulating an anti-HIV immune response, said substance or composition comprising a nucleic acid molecule in accordance with claim 38 or 39 and/or a polypeptide in accordance with claim 41 or 42, and said method comprising administering said substance or composition to the subject.

50. An immunogen substantially as hereinbefore described and with reference to the accompanying drawings.

51. A method of stimulating an anti-HIV immune response in a human subject substantially as hereinbefore described and with reference to the accompanying drawings.

52. An immunogen according to claim 1, or claim 16, or claim 17, substantially as herein described and illustrated.

53. A nucleic acid according to claim 27, or claim 38, or claim 39, substantially as herein described and illustrated.

54. A vector according to claim 30, or claim 43, substantially as herein described and illustrated.

PCT/GB00/04984

55. A method according to claim 32, or claim 35, or claim 44, substantially as herein described and illustrated.

56. Use according to claim 34, or claim 45, substantially as herein described and illustrated.

57. A bacterium according to claim 36, substantially as herein described and illustrated.

58. A polypeptide according to claim 41, or claim 42, substantially as herein described and illustrated.

59. A substance or composition for use in a method of treatment according to any of claims 46 to 49, substantially as herein described and illustrated.

60. A new immunogen; a new nucleic acid; a new vector; a new non-therapeutic method of treatinent; a new use of an immunogen according to any of claims 1 to 26 or a nucleic acid according to any of claims 27 to 29; a new use of a nucleic acid according to claims 38 or 39 or a polypeptide according to claim 41 or 42; a new bacterium; a new polypeptide; or a substance or composition for a new use in a method of treatment, substantially as herein described.