WO1993021332A1

WO1993021332A1 - Hybrid particles

Info

Publication number: WO1993021332A1
Application number: PCT/GB1993/000783
Authority: WO
Inventors: Sally Elizabeth Adams; Nigel Robert Burns; Alan John Kingsman
Original assignee: British Bio-Technology Limited
Priority date: 1992-04-14
Filing date: 1993-04-14
Publication date: 1993-10-28
Also published as: ZA926340B; ZA932624B; AU4076393A; GB9208218D0

Abstract

Fusion proteins comprise first and second amino acid sequences. The first amino acid sequence is derived from a particle-forming retroviral GAG protein, particularly an HIV-1 GAG protein. The second amino acid sequence is derived from a V3 loop of a lentivirus, again such as HIV-1. The fusion proteins can spontaneously assemble into particles, which are useful in diagnosis or immunotherapeutic vaccines. CTL responses have been observed following immunisation with such vaccines.

Description

HYBRID PARTICLES

This invention relates to particle-forming proteins. Particles formed from such proteins are antigenic and can be used as diagnostic agents, or in immunotherapeutic or prophylactic vaccines.

An ideal immunogen is a polymer of multiple antigen determinants assembled into a high molecular weight, particulate complex. A substantial disadvantage of most antigens produced by recombinant DNA techniques for

• vaccines is that they are usually produced as simple monomeric proteins. This is not the ideal configuration of an immunising antigen as it does not readily permit the cross-linking of the components of the immune system that is required for maximum stimulation of humoral and cellular immunity. For these reasons it would be advantageous to develop polyvalent, particulate carrier systems for immunising antigens.

A number of particulate carrier systems have been developed, including one based on the retroelement Ty, which exploits the particle forming ability of the TYA- encoded pi protein to present multiple copies of antigenic determinants as fusion proteins (Adams et al

1987 Mature 329 68 ) .

WO-A-8803562 and WO-A-88-3563 describe the use of certain fusion proteins derived from retrotransposons or RNA retroviruses for pharmaceut i cal , diagnost ic or purif icat ion appli cations . Specifical ly , the above published PCT applications note that polyvalent particles are useful for immunisation purposes because their polyvalent nature provides that more antibodies will be raised against the particulate antigens used . The particles are formed of fusion proteins having a particle- forming sequence and, in some embodiments at least, an antigenic sequence .

US-A-4722840 (Valanzuela) describes antigenic particles based on hepatitis B particle forming proteins . On the other hand, ϋS-A-4925784 (Crow) describes soluble HIV gag and HIV envelope antigens which do not form particles .

Effective prophylaxis , management and treatment of lentiviruses such as Human Immunodeficiency Virus , the causative agent of Acquired Immune Deficiency Syndrome

(AIDS ) , are goals for which the medical research community is currently striving . The fusion protein route, as represented by the disclosures of O-A-8803562 and O-A-8803563 , is one of the most promising . The problem remains, however, of making the right choice of particle-forming sequence and antigenic sequence for further improved results . For example, many of the vaccines so far developed exhibit humoral (antibody) and

T-helper cell responses, but little or no cytotoxic T- lymphocyte (CTL) response . The present invention relates to a particular combination of sequences which gives rise to excellent results, and which lends itself to dealing with the problem posed by the varying antigenicity of different HIV isolates .

According to a first aspect of the invention, there is provided a particle- forming protein comprising first and second amino-acid sequences, wherein the first amino acid sequence comprises a sequence substantially homologous with a particle-forming retroviral GAG protein and wherein the second amino acid sequence comprises a sequence substantially homologous with at least an antigenic portion of a V3 loop of a lentivirus . The expression "substantially homologous", when describing the relationship of an amino acid sequence to a natural protein, means that the amino acid sequence can be identical to the natural protein or can be an effective but truncated form of the natural protein or can share at least 50%, 60%, 70%, 80%, 90%, 95% or 99%, in increasing order of preference, of the residues of the natural protein or its truncated form. Alternatively or in addition, a nucleic acid sequerice encoding the amino acid sequence may hybridise, for example under stringent conditions, to a nucleic acid sequence encoding the natural protein or its truncated form, or would do so but for the degeneracy of the genetic code. Stringent hybridisation conditions are known in the art and are exemplified by approximately 0.9 molar salt concentration at approximately 35° to 65°C.

The hybrid HIV GAG-V3 particles are potentially important HIV vaccines as they contain the major envelope determinants for neutralising antibody and T-helper responses, together with core determinants for T- cytotoxic responses in a single particulate structure. The GAG sequence is itself potentially im unogenic and thus advantageous over other particle-forming proteins in the treatment of retroviral infections. Sequences recognised by cytotoxic T-cells have been recognised within the core proteins of HIV and SIV. It is also thought that antibodies to ρl7 may be protective.

The first amino acid sequence comprises a sequence substantially homologous with a particle-forming retroviral GAG protein. The retrovirus may be HIV-1, HIV- 2, HTLV-I, HTLV-II, HTLV-III, SIV, BIV, ELAV, CIAV, murine Leukaemia virus, Moloney murine leukaemia virus and Feline Leukaemia virus. More preferred are those GAG proteins of lentiviral origin such as HIV-1, HIV-2, SIV,

BIV and FIV. It is most preferred that the GAG protein be derived from HIV-1. Stability of particles formed from the fusion proteins may be enhanced by removing some or all of the basic C-terminus of the GAG precursor protein

(ρ55 in HIV-1) . At least the first 437 amino acids of the GAG protein will preferably be present in the preferred truncated form; alternatively, at least the first 360 amino acids of the GAG protein may be present.

Natural GAG proteins, at least of HIV-1, are post- translationally modified. Such modification usually includes myristylation in natural HIV-1 GAG. Naturally post-translationally modified GAG proteins, non-naturally post-translationally modified GAG proteins and non-post- translationally modified GAG proteins are included within the scope of the invention. The myristylation or other post-transla ional modification site or sites may cause non-optimal expression in certain non-animal host cells (such as yeast) , and for this reason may be absent in certain embodiments of the invention. Its absence is far from mandatory, however, as certain cell lines (such as insect cell lines) readily express myristylated GAG proteins.

Truncated GAG proteins, and fusion proteins containing them, themselves form a second aspect of the invention, according to which there is provided a polypeptide comprising an amino acid sequence substantially homologous with a retroviral GAG protein, but lacking some or all of the C-terminus of the GAG precursor protein . Preferred features of this aspect of the invention are as described above, Λiutatis mutandis . The second amino acid sequence comprises a sequence substantially homologous with an antigenic portion of a

V3 loop of a lentivirus. The third variable domain

(otherwise known as the V3 loop or GPGR loop) is found between amino acids 300 and 330 of the envelope glycoprotein gpl20 of HIV-1 and in analogous positions of other lentiviruses . The V3 loop is defined by two flanking cysteine residues linked by a disulphide bond and, for HIV-1 at least, is the major neutralising epitope of the virus (Putney et al 1986 Science 234, 1392; Rusche et al 1988 Proc. Natl . Acad. Sci 85, 3198; Palker et al 1988 Proc . Natl . Acad . Sci . 85 1932; and Goudsmit et al 1988 AIDS 2 157) . The antigenic portion of choice may constitute the whole of the V3 loop. However, a conserved sequence of the V3 loop may be useful in conferring immunity against more than one isolate of a virus (such as HIV-1) .

The lentivirus may be Feline Immunodeficiency Virus (FIV) , Simian Immunodeficiency Virus (SIV) or Human Immunodeficiency Virus-1 or -2 (HIV-1 or HIV-2) . HIV-1 will be the virus of choice for many clinical applications. A number of isolates of HIV-1, in which the sequence of the V3 loop varies from isolate to isolate, are known. The most common isolates are HXBII, RF and MN; MAL, ELI and BH10 are also important, but the MN isolate may be the most clinically relevant. Laboratory isolate IIIB is a mixture of strains BH10 and HXBII. The invention is not limited to the V3 loop of any particular isolate, some of which are shown below. BH10 SNCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISG HXBIISNCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISG MN SNCTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAHCNISG MAL SNCTRPGNNTRRGIHFGPGQALYTTGIVDIRRAYCTING RF SNCTRPNNNTRKSITKGPGRVIYATGQIIGDIRKAHCNLSGS ELI STCARPYQNTRQRTPIGLGQSLYTTRSRSIIGQAHCNISG. Neither is the invention limited to natural V3 loop sequences. Examples of variant V3 loop sequences which can be used in the invention include:

MAL(var) SNCTRPGNNTRRGIHFGPGQALYTTGIVDEIRRAYCNISG RF (var) SNCTRPNNNTRKSITKQRGPGRVLYATGQIIGDIRKAHCNSIG ELI(var) STCARPYQNTRQRTPIGLGQSLYTTRGRTKIIGQAHCNISG.

A comparison of the sequences of the V3 loop from many different HIV-1 isolates shows great variation between isolates. Antibodies raised against the V3 loop are therefore usually type-specific. However, approximately 60% of isolates to date have the consensus sequence GPGRAF, and more than 80% have a GPGR sequence at the tip of the loop. Recent studies have shown that immunisation with peptides containing the GPGRAF consensus sequence or cross-immunisation with reco binant gpl20 from different isolates can induce antibodies which cross react between isolates.

Other embodiments of the invention involve the use of short sequences of V3 which are not necessarily conserved between various isolates. Whatever V3-derived or V3- related sequence is used, the resulting fusion proteins, or at least particles assembled from them, will be similar antigenically to natural V3 loop sequences in the sense that they cross-react with one or more common antibodies.

More than one V3-derived sequence can be present in a fusion protein of the invention. This embodiment may enable a single fusion protein to be useful in the protection against more than one HIV isolate: therefore, V3-derived sequences from different HIV isolates can be present on the same molecule.

As fusion proteins in accordance with the invention spontaneously assemble into particles, it is possible by means of the invention to prepare multivalent particles: a plurality of different fusion proteins could be prepared in which V3 loops from different isolates are represented as different second amino acid sequences. Particles could then be allowed to form from a cocktail of different fusion particles, all of which may have the same first amino acid sequence to allow multivalent particle formation.

According to a third aspect of the invention, there is provided a particle comprising a plurality of fusion proteins as described above. The fusion proteins may be the same as each other, but as indicated in the last paragraph above this is not essential.

According to a fourth aspect of the invention, there is provided nucleic acid (particularly DNA) coding for a fusion protein as described above. It will generally be the case that the nucleic acid will be capable of being expressed without splicing or anti-termination events. There will generally be no frame shifting, but frame shifting is not necessarily always excluded. For example, a heterologous sequence can be placed in phase, and at the 3' end, of the protease gene of the retrotransposon Ty (Natsoulis and Boeke 1991, Nature 352 632), leading to the assembly of hybrid TyA- and TyB-fusion proteins. The production of hybrid gag-V3 particles is ananlogous to this; the V3 sequence need not necessarily be placed at the 3' end of the protease gene but at any position downstream of the frameshift site. Further according to the present invention is provided a vector including nucleic acid as described above. Expression vectors in accordance with the invention will usually contain a promoter. The nature of the promoter will depend upon the intended host expression cell. For yeast, PGK is a preferred promoter, but any other suitable promoter may be used if necessary or desirable. Examples include GAPD, GAL1-10 , PH05, ADHl , CYCl , Ty delta sequence, P YK and hybrid promoters made from components from more than one promoter (such as those listed) . For insect cells, preferred promoters include the -polyhedrin promoter from Autographa californi ca nuclear polyhedrosis virus (AcNPV) and the plO promoter from AcNPV. Those skilled in the art will be able to determine other appropriate promoters adapted for expression in these or other cells. Vectors not including promoters may be useful as cloning vectors, rather than expression vectors.

The invention also includes host cells, for examples bacterial cells, such as E. coli , which may be used for genetic manipulation, yeast cells such as S. cerevisiae or animal cells. Although mammalian cells including COS or CHO cells can be used, expression in insect cell lines may be preferred in many cases. Suitable cell lines include Spodoptera frυgiperda , such as SF9 and Trichoplasia ni.

Because of the polyvalent nature of antigenic self assembling particles in accordance with the invention, it is likely that it will be easier to produce antibodies than with conventional antigens and that those antibodies will have specific characteristics. The invention thus further provides antibodies raised against particulate antigens of the invention. The antibodies may be polyclonal (obtained for example by injecting antigens into a rabbit) or monoclonal antibodies, produced by hybridoma cells. Because of the polyvalent nature of the particulate antigens it is likely that in vitro immunisation can be achieved more readily than with other forms of antigen; this may facilitate the production of human monoclonal antibodies. Hybridoma cells may be prepared by fusing spleen cells from an immunised animal with a tumour cell. Appropriately secreting hybridoma cells may thereafter be selected.

Particulate antigens in accordance with the invention may be useful in the preparation of vaccines, for example immunotherapeutic vaccines, which form a further aspect of the invention. The vaccine may comprise a particulate antigen and a physiologically acceptable non-toxic carrier, such as sterile physiological saline or sterile PBS. Sterility will generally be essential for parenterally administrable vaccines. One or more appropriate adjuvants may also be present. Examples of suitable adjuvants include muramyl dipeptide, aluminium hydroxide and saponin.

Vaccines in accordance with the invention may present more than one V3 antigen. Either a cocktail of different particulate antigens may be used, or a homogeneous population of particulate antigens having more than one epitope could be used, as described above. It may in practice be simpler for a vaccine to contain a mixture of different particulate antigens.

Fusion protein and particulate antigens of this invention are useful as diagnostic reagents. Particulate antigens for diagnostic purposes are particularly advantageous because they can be physically separated by centrifugation or filtration and can be directly dispersed on solid supports such as glass or plastic slides, dip sticks, macro or micro beads, test tubes, wells of microtiter plates and the like. The particulate antigens of this invention may also be dispersed in fibrous or bibulous materials such as absorbent disk (see US Patent 4,632,901), strips or chromatography columns as the solid support. The particles and fusion proteins readily adhere to solid supports. The particles may after purification be disrupted into fusion proteins and the fusion proteins may be dispersed on surfaces as indicated above. These reagents are useful for a variety of diagnostic tests. For example, a test sample suspected of having antibody to the particulate antigen and a fluorescent, enzyme or radio-labelled antibcdy is competitively reacted with the particulate antigen or fusion protein on a solid support and the amount of labelled antibody which binds to the particulate antigen on the solid support. Particulate antigens of this invention are also useful for agglutination reactions with antibodies . Those skilled in the diagnostic arts will recognise a wide variety of application of particulate antigens and fusion proteins of this invention for diagnostic purposes.

The invention will now be illustrated by the following examples, which are not limiting. The examples refer to the accompanying drawings, in which:

FIGURE 1 is a schematic diagram showing the construction of pOGS15;

FIGURE 2 shows the sequence details of the construction of pOGS554; FIGURE 3 is a schematic diagram of the construction of pOGS554, pOGS555, pOGS562 and pOGS563;

FIGURE 4 shows the sequence details of the construction of pOGS562;

FIGURE 5 is a schematic diagram of the general purpose baculovirus expression vector pAcRP23;

FIGURE 6 shows a Western blot of extracts from insect cells infected with wild-type baculovirus AcNPV, ACOGS553 (full-length GAG) or AcOGS556 (full- length GAG/V3 loop) . a) shows the result using anti- GAG monoclonal antibody; b) shows the result using anti-GPGR monoclonal antibody;

FIGURE 7 shows a Western blot of extracts from insect cells infected with wild-type baculovirus AcNPV, AcOGS572 (truncated GAG) or AcOGS574

(truncated GAG/V3 loop) . a) shows the result using anti-GAG monoclonal antibody; b) shows the result using anti-GPGR monoclonal antibody;

FIGURE 8 is a schematic diagram of pOGS700;

FIGURE 9 shows the sequence details of the construction of pOGS564;

FIGURE 10 is a schematic diagram of the construction of pOGS565;

FIGURE 11 is a schematic diagram of the construction of pOGS571; FIGURE 12 is a schematic diagram of the construction of pOGS570; '

FIGURE 13 is a protein sequence of the GAG:V3 loop protein derived from pOGS555;

FIGURE 14 is a protein sequence of the GAG.-V3 loop protein derived from pOGS563;

FIGURE 15 is a protein sequence of the GAG:V3 loop protein derived from pOGS568; and

FIGURE 16 is a protein sequence of the GAG:V3 loop protein derived from pOGS570.

13

EXAMPLE 1: HIV GAG Fusion Proteins Formed Usinσ Insect Calls

This example describes the construction and expression of HIV GAG fusion proteins in insect cells. The E . coli strain used was 11W87 (araD139, ( ara -leυ) del 7696, (laclPOZY)dell74, gaiυ, gaK, hsdr, rpsL, srl, rccA56) . The use of this particular strain is not critical; many other commercially available strains could be used. E. coli media were prepared according to Miller (Miller 1972 Experiments in Molecular Genetics, CSH p433) . E . coli were transformed using standard methods (Maniatis et al 1982 "Molecular Cloning- A Laboratory Manual", CSH pl99) . Standard procedures were used for restriction digestion and plasmid constructions (Maniatis et al 1982 op cit ) . Restriction enzymes, T4 polynucleotide kinase and T4 DNA ligase were used according to the suppliers' instructions.

Plasmid DNA was isolated from E. coli preparatively as described by Chinault and Carbon (1979 Gene 5, 111) and for rapid analysis by the method of Holmes and Quigley (1981 Anal . Biochem 114, 193) .

The oligonucleotides were synthesized by automated phosphoramidite chemistry using cyanoethyl phosphora idites (Beaucage and Caruthers 1981 Tetrahedron Letters 24, 245) . Following de-blocking and removal from the controlled pore glass support the oligomers were purified on denaturing polyacrylamide gels, further purified by ethanol precipitation and finally dissolved in water prior to estimation of their concentration. The oligomers were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed prior to ligation into the relevant plasmid vector. The sequence of the synthetic oligomers was confirmed by dideoxy sequencing. The protocol used was essentially as has been described (Biggin et al 1983 Proc. Natl . Acad. Sci . 80, 3963) and modified to allow sequencing on plasmid DNA as described (Guo and Wu 1983 Nucl . Acids Res . 11, 5521) .

HIV-1 gag constructs were derived from pOGS2. Plasmid pOGS2 contains nucleotides 678-2470 of HIV-1 BH10 (Ratner et al 1985 Nature 313, 277) inserted into plasmid pSP64 (Melton et al 1984 Nucl . Acids Res . 12, 7035) as an Sstl-Bcll restriction fragment. The gag gene is encoded by nucleotides 790-2325. In order to provide the gag gene on a convenient BamRI fragment, pOGS2 was modified using Bal31 exonuclease and BamRI linkers (Figure 1) . To modify the 3' end of the gag fragment, plasmid pOGS2 was cleaved with Sail, digested with Bal31 exonuclease for various times and re-ligated in the presence of excess Ba_mHI linkers (CCGGATCCGG) . The deletion end points of the resulting plasmids were determined by DNA sequencing. Plasmid pOGSll is a deletion derivative in which the BamRI site is at position +49 with respect to the gag termination codon. To modify the 5' end of the gag fragment, pOGS2 was cleaved with BcoRl, digested with Ba231 exonuclease for various times and re-ligated in the presence of excess BajnHI linkers (CCGGATCCGG) . The deletion endpoints were determined by DNA sequencing. Plasmid pOGS12 is a deletion derivative in which the BayπHI site is at position -22 with respect to the gag initiating ATG codon. In order to provide a gag gene flanked by BawHI sites, the 3* end of the gag gene in pOGS12 was replaced with^' a Bglll-Bg-lI fragment from pOGSll containing the modified sequence. The resulting plasmid was designated pOGS15 (Figure 1) . 15

A fusion gene containing the whole of HIV-1 gag and a V3 (GPGR) loop sequence was made by inserting synthetic oligomers encoding the V3 loop sequence from HIV-1 isolate HXB2 (Fisher et al 1986 Science 233, 655) at the Bgl l l site at position 2096 in the gag gene. This required modification of the sequence at the Bglll site so that the gag reading frame was maintained following insertion of the V3 loop sequence. pOGS15 was digested with Bglll and ligated with synthetic oligomers SEAGAG/03 and SEAGAG/04 (Figure 2) . This insertion re-creates a Bglll site -1 with respect to the original Bglll site within the gag gene. The resulting plasmid was designated pOGS554. Synthetic oligomers encoding the ^'HXB2 V3 loop sequence were inserted into pOGS554 that had been cleaved at the new Bglll site. This generated plasmid pOGS555 which contains the gag(full-length) :V3 loop fusion gene as a BamRI cassette (Figure 3: SEQ ID 1) .

In order to produce a gag gene in which nucleotides encoding the C-terminal 75 residues have been removed, synthetic oligomers SEAGAG/08 and SEAGAG/09 were inserted into ρOGS554 which had been cleaved with Bglll. This insertion results in the generation of a stop codon and a BamHI site downstream of the Bglll site. The resulting plasmid was designated pOGS562 and contains a truncated gag gene as a BamRI cassette (Figures 3 and 4) .

A fusion gene containing the truncated HIV-1 gag gene and a V3 loop sequence was made by inserting synthetic oligomers encoding the V3 loop sequence from HIV-1 isolate HXB2 (Fisher et al 1986 op cit) at the Bglll site in plasmid pOGS562. The resulting plasmid was designated pOGS563 and contains the gag(truncated) :V3 loop fusion gene as a BamRI cassette (Figure 3: SEQ ID 3) . 16

Each of the four gag cassettes (full-length gag; full- length gag/V3 loop; truncated gag; truncated gag/V3 loop) were excised from pOGS15, pOGS555, pOGS562 and pOGS563 as a BamHI fragment and cloned into the baculovirus expression vector pAcRP23 (Figure 5) to generate pOGS553, pOGS556, pOGS572 and pOGS574 respectively. Plasmid pAcRP23 contains the powerful polyhedrin promoter from Autographa calif ornica nuclear polyhedrosis virus (AcNPV) and a unique BamRI site for insertion of foreign genes flanked by AcNPV sequences. Plasmid pAcRP23 also contains sequences for replication and selection of the plasmid in E . coli (Matsuura et al 1987 J. Gen . Virol 68 1233) . When cloned into the BamRI site of this vector, the gag gene derivatives come under the control of the polyhedrin promoter (Figure 5) .

The cell line designated Spodoptera frugiperda 9 (SF9) (Summers and Smith 1987 Texas Agricultural Experiment Station Bulletin No. 1555), which is a clonal derivative of the lepidopteran line IPLB-Sf21-AE (Vaughn et al 1977 In vitro 13 213), was used for all experiments. Cells were grown in suspension culture in medium comprising TC100 (GIBCO-BRL, UK) containing 10% heat inactivated foetal calf serum (ICN-FLOW Laboratories, UK) plus 50IU/ ml penicillin and 50mg/ml streptomycin (GIBCO-BRL, UK) .

Recombinant baculoviruses were prepared using the phenomenon of in vivo recombination between homologous DNA sequences . The viral (AcNPV) DNA and plasmid DNA (pOGS553, pOGS556, pOGS572 or pOGS574) were introduced into SF9 insect cells by the method of calcium chloride precipitation and progeny were recovered after 48 hours

(Summers and Smith, op ci t ) . Recombinant baculoviruses were selected on the basis of a polyhedrin-negative phenotype in a Brown & Faulkner plaque assay (Brown and 17

Faulkner 1 977 J. Gen . Virol 36 361 ) . After plaque purification , high tit re viral stocks of the recombinant viruses were prepared and thei r t itres determined in plaque assays .

Titres were in excess of 5x10⁷ plaque forming units (pfu) per ml . Recombinant viruses were designated AcOGS553

( ful l-length ) , AcOGS 556 ( ful l- length ga g/V3 loop) ,

AcOGS57 4 ( t runcated ) and AcOGS574 ( truncated gag/V3 loop) .

Expression of the full-length GAG and full-length GAG/V3 loop proteins .was examined by Western blot analysis. Confluent mσnolayers of SF9 cells were infected with the recombinant virus AcOGS553, recombinant virus AcOGS556 or wild-type AcNPV at a multiplicity of infection of 5pfu per cell. The cells were harvested at 48 hours post- infection and pelleted by centrifugation at lOOOg. After washing with phosphate buffered saline (PBS) , the cells were lysed and the proteins resolved by SDS-PAGE as described in Laemmli (Laemmli 1970 Na ture 227 68) . The separated proteins were transferred to nitrocellulose as described by Towbin (Towbin et al 1979 Proc. Natl . Acad. Sci 76 4350) and probed with either anti-GAG or anti-V3 monoclonal antibodies (MAb; Du Pont-UK) . Cells infected with AcOGS553 contained a protein of approximately 55kD which reacted with the anti-GAG MAb but not with the anti-V3 MAb. In contrast, cells infected with AcOGS556 contained a protein of approximately 59kD which reacted with both the anti-GAG and anti-GPGR MAbs, confirming the expression of the full-length GAG/V3 loop fusion gene as a fusion protein (Figure 6) .

When thin sections of cells infected with AcOGS553 (full- length GAG) were examined using electron microscopy, virus-like particles could be seen budding from the plasma membrane. The budded particles showed some irregularity, although they had a similar morphology to immature HIV virions as has been observed previously by Gheyson (Gheyson et al 1989 Cell 59 103) . In contrast, electron microscopic analysis of cells infected with AcOGS556 (full-length GAG/V3 loop) showed that the hybrid protein accumulated as largely aggregated material in the nucleus. In some instances partially spherical structures were evident, which, although predominantly irregular, occasionally had the appearance bf virus-like particles. Thes^'e results suggested that it may be possible to generate hybrid- GAG particles, but that modification of the particle-forming sequence would be desirable for the formation of regular, stable structures.

Expression of the truncated GAG and truncated GAG/V3 loop proteins was also examined by Western blot analysis. Confluent monolayers of SF9 cells were infected with the recombinant virus AcOGS572, recombinant virus AcOGS574 or wild-type AcNPV at a multiplicity of infection of 5pfu per cell. The cells were harvested, lysed and the proteins separated as described above. As expected, cells infected with AcOGS572 contained a protein of approximately 47kD which reacted with the anti-GAG MAb but not with the anti-V3 MAb. Cells infected with AcOGS574 contained a protein of approximately 54kD which reacted with both the anti-GAG and anti-V3 MAbs, confirming the expression of the truncated GAG/V3 loop fusion gene as a fusion protein (Figure 7) .

Electron microscopic analysis of cells infected with AcOGS572 demonstrated the presence of virus-like particles budding from the plasma membrane. In contrast to the virus-like particles budding from cells infected with AcOGS553 (full-length GAG) , the truncated GAG 19

particles appeared to be more regular. Even more striking was the appearance of regular particles, mainly in the nucleus, of cells infected with AcOGS574 (truncated GAG/V3 loop) . In addition, some of the hybrid truncated GAG:V3 loop particles could be seen budding from the cell surface. These demonstrate that the first 437 amino acids of the HIV-1 GAG protein contain sufficient information to form virus-like particles.

The nuclear location of these GAG/V3 loop particles should facilitate purification using standard procedures o isolate nuclei followed by purification methods based on physicochemical properties such as the size and density of the particles, such as centrifugation and size-exclusion chromatography. It is not necessarily the case that all particles produced from GAG fusion genes will be located primarily in the nucleus. They may assemble in the cytoplasm or, as observed with some of the truncated GAG:V3 loop particles, bud from the cell.

20

EXAMPLE 2 : H TV GAG Fusion Proteins Formed Usinσ Yeast Cells

The general procedures described in Example 1 were followed, except that the gag cassettes were inserted into yeast expression vectors and then introduced into yeast cells by transformation .

The S. cerevisiae strain used was BJ2168 ( ura3-52, trp 1, leu 2-3, leu 2-112, prb 1-1122, pep 4-3, pre 1-407) .

Yeast media were prepared according to Hawthorne and

^' Mortimer (1960 Genetics 45 1085) . Yeast was transformed as described by Hinnen et al ( 1978 Proc. Natl . Acad. Sci

75 1929) .

The construction containing the full-length HIV gag gene, pOGS15, is described in Example 1. The full-length gag cassette was excised from pOGS15 as a BamRI fragment and cloned into he yeast expression vector pOGS700" (Figure 9) to generate pOGS577. ρOGS700 is an E. coli/yeast shuttle vector and contains sequences which allow replication and selection in E. coli , the yeast 2 micron plasmid origin of replication and the yeast LEU2 gene for selection in yeast. Expression is driven from a hybrid promoter comprising sequences from the yeast phosphoglycerate kinase (PGK) and GAL-10 promoters. This hybrid PGK:GAL promoter allows high level expression which is induced by growth on galactose (Kingsman et al, Methods in Enzymology 185 329) . The gag cassette was cloned into the Bglll site of pOGS700.

Yeast cells were transformed with pOGS577 and expression analysed by electron microscopy. Thin sections of yeast cells transformed with ρOGS577 showed that the GAG protein was associated with the cell membrane, as demonstrated by the darkly-staining layer below the cell surface. This electron-dense layer is not present in control non-transformed BJ2J68 cells. Large protrusions were also seen from the cell surface, suggesting that budding and formation of GAG particles is hindered by the presence of the yeast cell wall.

The HIV-1 gag gene includes, at its 5' end, a signal for post-translational myristylation of the GAG protein. Myristylation is dependent on the presence of a glycine residue immediately following the initiating methionine residue. The fatty acid modification is involved in membrane localization. The absence of particle formation in yeast cells transformed with pOGS577 may therefore be due to localization of GAG protein at the cell membrane combined with prevention of budding because of the yeast cell wall. By removing the myristylation signal, it may be possible to generate intracellular GAG particles in yeast.

The myristylation signal was removed from the full-length gag gene by insertion of synthetic oligomers SEAGAG/10 and SEAGAG/11 at the BavnHI site of pOGS14 (Figure 9) . pOGS14 is identical to pOGS12 (see Example 1) except that the BainHI site is at position +3 with respect to the initiating ATG codon of full-length gag. The synthetic oligomers inserted at the Ba/nHI site in pOGS14 and the resulting sequences at the 5¹ end of the Myr- gag gene are shown in Figure 9. The resulting plasmid was designated pOGS564.

In order to provide a Myr-gag gene flanked by BamR I sites, the 5' end of the gag gene in pOGS15 was replaced with a Pstl-Bgll fragment from pOGS564 containing the modified sequence (Figure 10) . The resulting plasmid was designated pOGS565. For expression in yeast, the full- length Myr-gag cassette was excised from pOGS565 as a BaΛiHI fragment and inserted at the Bglll site in pOGS700 to generate pOGS566.

Yeast cells were transformed with pOGS566 and analysed by electron microscopy. The Myr-gag protein accumulates in the cell cytoplasm as an amorphous mass rather than as discrete particles. However, at higher magnifications, some partially spherical, irregular structures were evident, which occasionally had the appearance of virus¬ like particles. The presence of these semi-particulate structures suggested that it may be possible to generate Myr-gag particles, but that modification of the particle sequence would be necessary.

To produce a Myr-gag gene in which the C-terminal 75 residues have been removed, the 3* end of the Myr-gag gene in pOGS564 was replaced with a Pstl-Bgll fragment from pOGS562 (see Example 1) in which the 3* end of the full-length, myristylated gag gene had been truncated

(Figure 11) . The resulting plasmid was designated pOGS571. For expression in yeast, the truncated Myr-gag cassette was excised from pOGS571 as a BainHI fragment and inserted at the Bglll site in pOGS700 to generate POGS575.

In order to produce a fusion protein containing non- myristylated, truncated GAG and a V3 loop sequence, the 3' end of the Myr-gag gene in pOGS564 was replaced with a Pstl-Bgll fragment from pOGS563 (see Example 1) . pOGS563 contains a myristylated, truncated GAG:V3 cassette. The resulting plasmid .was designated pOGS570 (Figure 12: SEQ ID 7) . For expression in yeast, the truncated, Myr- gag:GPGR cassette was excised from pOGS570 as a BamRI fragment and inserted at the Bgl l l site of pOGS700 to generate pOGS579 (Figure 13) .

Yeast cells were transformed with pOGS575 (truncated, Myr-gag) and pOGS579 (truncated, Myr-GAG:V3) and analysed by electron microscopy. The removal of the 75 C-terminal amino acids from the Myr-GAG protein (pOGS575) restores the ability of the protein to assemble into regular particulate structures. Therefore, the first 400 amino acids of the HIV-1, non-myristylated GAG protein contain sufficient information to form GAG virus-like particles in yeast cells.

Electron microscopic analysis of yeast cells transformed with pOGS579 (truncated, Myr-GAG:V3) demonstrated that the non-myristylated fusion protein retained the ability to assemble into particles. Furthermore, the majority of the Myr-GAG:V3 particles were located in the nucleus.

This observation is consistent with the presence of truncated GAG:V3 particles in the nucleus of insect cells

(see Example 1) .

It is not necessarily the case that all particles produced in yeast cells from Myr-GAG fusion proteins will be located in the nucleus. Purification strategies will depend on their location, although exploitation of their physical properties is likely to result in simple purification procedures.

A gene encoding a non-myristilated GAG:V3 fusion protein was also made by a Pstl-Bgll replacement of the 5' end of the gag gene in pOGS555 with that in pOGS564 and the resulting plasmid designated pOGS568 . The BamRI fragment from pOGS568 (SEQ ID 5) was inserted into pOGS700 to generate pOGS569. pOGS569 is the expression plasmid for full-length, non-myristilated GAG:V3. 24

EXAMPLE 3: Cytotoxic T-cell Responses

Cytotoxic T-lymphocyte (CTL) responses have been observed following immunisation with OGS574 (truncated GAG:V3) particles. Mice received a single intramuscular dose of lOOμg in the absence of adjuvant. Splenocytes were removed after 20 days and expanded in vitro for 7 or 12 days with a 40mer peptide corresponding to the sequence of the cognate (ie IIIB) V3 loop. The effector cells induced in this way were tested for their cytotoxic activity against control p815 cells or ρ815 cells labelled with either the IIIB peptide, or an MN V3 loop peptide (p815 cells express no MHC class II and will therefore only present the peptide in conjunction with MHC class I. CTLs will recognise certain peptide sequences, but only in conjunction with MHC class I) .

Specific lysis of IIIB peptide labelled target cells was observed after both 7 days and 12 days in vitro expansion, whereas there was no significant lysis of MN peptide labelled cells, or of the control p815 cells. The effect titrated out to low effector:target (E:T) ratios.

Further groups of mice were immunised with lOOμg of OGS574 or OGS553 (full-length GAG particles, without the V3 sequence) . Immunisations were again intramuscular with no adjuvant. Splenocytes were prepared from each group on day 14 following immunisation and expanded in vitro with 40mer IIIB V3 peptide. Splenocytes from non- immunised mice were treated in a similar way. Effector cells were tested for their ability to lyse unlabelled cells and target cells labelled with IIIB V3 peptide over a range of E:T ratios. Specific V3 associated lysis was observed in the immunised mice with only background lysis in the control group.

Further splenocytes were prepared from these groups at day 39 following immunisation and these were again expanded in vi tro with IIIB 40mer peptide for 7 days. The effector cells were tested against target cells labelled with a number of peptides derived from the V3 loop sequence. Immunised mice showed specific lysis of all targets as long as they were labelled with peptides containing the sequence RIQRGPGRAFVTIG. This sequence has been previously described in publications as a .human and murine CTL epitope.

EXAMPLE 4: Trunr.at.fid GA :V3 fusions The truncated HIV GAG protein described in Example 1 has a C-terminal deletion of 75 amino acids. Further deletions can be made from the C-terminus of GAG, and V3 loop fusions generated, without disrupting particle assembly. These are constructed by introducing an appropriate restriction enzyme site (eg Bglll) towards the 3' end of the ga g gene using site-directed mutagenesis, and inserting a V3 loop sequence by the methods described in Example 1. As an alternative to site-directed mutagenesis, truncated gag genes can also be generated by cleaving the gag gene with Bglll and digesting with Bal31 exonuclease for various times before religating in the presence of excess linkers and insertion of a V3 loop sequence. Such truncated GAG/V3 loop fusion proteins are then expressed in insect or yeast cells as described in examples 1 and 2. GAG/V3 fusion proteins in which the majority, if not all, of the C-terminal pl5 protein (p6 and p7) has been deleted still retain the ability to self-assemble into particles.

Claims

26CLAIMS

1. A particle-forming protein comprising first and second amino-acid sequences, wherein the first amino acid sequence comprises a sequence substantially homologous with a particle-forming retroviral GAG protein and wherein the second amino acid sequence comprises a sequence substantially homologous with at least an antigenic portion of a V3 loop of a lentivirus.

2. A protein as claimed in claim 1, wherein the retrovirus is a lentivirus.

3. A protein as claimed in claim 1 or 2, wherein the lentivirus is HIV-1.

4. A protein as claimed in claim 1, 2 or 3, wherein the GAG protein is truncated by removal of some or all of the C-terminus of the GAG precursor protein.

5. A protein as claimed in claim 4, wherein at least the first 437 amino acids of the GAG protein are present.

6. A protein as claimed in claim 4, wherein at least the first 360 amino acids of the GAG protein are present.

7. A protein as claimed in any one of claims 1 to 6, wherein the myristylation signal is removed.

8. A particle comprising a plurality of fusion proteins as claimed in any one of claims 1 to 7.

9. Nucleic acid.coding for a fusion protein as claimed in any one of claim 1 to 8. 27

10. A vector comprising nucleic acid as claimed in claim 9.

11. A vector as claimed in claim 10, which is an expression vector comprising an operatively linked promoter.

12. A vector as claimed in claim 11, which is a yeast expression vector, wherein the promoter is PGK.

13. A vector as claimed in claim 11, which is a yeast expression vector, wherein the promoter is the polyhedrin promoter from Autographa californica nuclear polyhedrosis virus (AcNPV) or the plO promoter from AcNPV.

14. A host cell comprising a vector as claimed in any one of claims 10 to 13.

15. A host cell as claimed in claim 14 which is of the species Saccharomyces cerevisiae .

16. A host cell as claimed in claim 14, which is of the species Spodoptera frugiperda or Trichoplasia ni .

17. Antibodies raised against particles as claimed in claim 8.

18. A vaccine comprising particles as claimed in claim 8.

19 . A particle as claimed in claim 8 for use in medicine, particularly immunotherapy .

20 . The use of a particle as claimed in claim 8 in the manufacture of a vaccine .

21. A polypeptide comprising an amino acid sequence substantially homologous with a retroviral GAG protein, but lacking some or all of a basic C-terminus of the GAG protein or its natural precursor .