Title of Invention	CHIMAERIC HIV-1 SUBTYPE C GAG-VIRUS-LIKE PARTICLES
Abstract	A method of producing immunogenic chimaeric proteins which self-assemble into virus-like particles is described. A first DNA sequence encoding a HIV-1 subtype C protein or portion thereof of greater than 200 amino acids is fused to a second DNA sequence encoding at least p17 and p24 of a Gag protein by a direct in-frame C-terminal fusion. A host cell is infected with a vector containing the fused sequence, and the chimaeric protein encoded by the fused sequence is expressed. The second DNA sequence may alternatively be a full-length gag sequence or a truncated gag sequence. Examples of the first DNA sequence are provided for tat, nef and reverse transcriptase, and combinations thereof.

Title of Invention

CHIMAERIC HIV-1 SUBTYPE C GAG-VIRUS-LIKE PARTICLES

Abstract

A method of producing immunogenic chimaeric proteins which self-assemble into virus-like particles is described. A first DNA sequence encoding a HIV-1 subtype C protein or portion thereof of greater than 200 amino acids is fused to a second DNA sequence encoding at least p17 and p24 of a Gag protein by a direct in-frame C-terminal fusion. A host cell is infected with a vector containing the fused sequence, and the chimaeric protein encoded by the fused sequence is expressed. The second DNA sequence may alternatively be a full-length gag sequence or a truncated gag sequence. Examples of the first DNA sequence are provided for tat, nef and reverse transcriptase, and combinations thereof.

Full Text	CHIMAERIC HIV-1 SUBTYPE C GAG-VIRUS-LIKE PARTICLES BACKGROUND OF THE INVENTION The invention relates to a method of constructing chimaeric virus-like particles (VLPs) based on the human immunodeficiency virus (HIV) Gag protein, in particular for vaccine purposes. HIV is one of the greatest problems facing mankind in the 21st century, with about 15 000 new infections daily, particularly in Sub-Saharan Africa where subtype C predominates 31. Although highly active anti-retroviral therapy (HAART) can prolong the survival of those infected with HIV, the progression to acquired immunodeficiency syndrome (AIDS) cannot be prevented at present. In addition, HAART (and other similar treatment regimes) is only economically feasible for treating high proportions of infected populations in the areas that are least affected by the pandemic, such as North America and Europe. These, together with numerous other socio-economic and medical reasons, highlight the need for effective and inexpensive HIV vaccines. Vaccine strategies Over the last 20 years a number of strategies have been developed toward producing HIV vaccines, and representatives from most of these are at various stages of clinical trial; however, there is still no vaccine on the market. The list includes live attenuated virus, whole killed virus, DNA vaccines, various recombinant viral and bacterial vectors, subunit protein vaccines and pseudovirions (including virus-like particles (VLPs)). Clearly there are merits and problems associated with each approach, and a number of recent reviews have investigated these concerns 2,15,18. VLPs as vaccines Vaccine approaches for more than 30 different viruses, including hepatitis B virus and human papilloma virus 7, have included the production of VLPs. There are a number of advantages to using VLPs 8 • They can be produced to relatively high yield in heterologous systems, where protocols are well established and can be purified from expression system culture supernatants by a variety of methods, including centrifugation and column fractionation.They are non-repiicative and non-infectious, which reduces some safety concerns. • Expression systems established to date for production of HIV-1 VLPs include: baculovirus, vaccinia virus, adenovirus and yeast systems. • The vertebrate immune system responds well to particulate antigens of viral size. • They have been shown to stimulate both humoral and cellular immune responses in rodents and non-human primates without addition of adjuvants, and may indeed act as adjuvants themselves. • Efficient epitope presentation on MHC class I and -II molecules and activation of antigen presenting cells (APCs). • They can be incorporated as part of a prime boost vaccination strategy with DNA vaccines. Disadvantages: • Manufacture can be expensive and technically demanding. • immunogenicity is comparatively weaker than related replicating vectors. • Since VLP preparations are essentially homogeneous with regard to specific antigen composition, cross clade reactivity is likely to be difficult to achieve. This is, however, common to most HIV vaccination strategies. HIV Gag VLPs Recent studies have indicated that VLPs can activate the innate immune system through 'danger signals' and lead to dendritic cell (DC) maturation and increased cytokine production 30. This is thought to be a result of the particulate nature of VLPs and residual components of the host/cell-system used in production. An example of this would be co-purified baculovirus in the insect culture system. More classically, humoral responses have been generated in mice 4, rabbits 6,34 and rhesus macaques. However the antibodies produced were generally either weak or non-neutralizing. Chimaeric HIV Gag VLPs Gag VLPs have the potential to act as an antigen carrier or delivery system, where the Gag essentially acts as a scaffold for the insertion or attachment of foreign antigens. The inclusion of foreign antigens (epitopes, polypeptides or folded proteins) into Gag VLPs can be roughly grouped into three classes, designated here as a-, B- and y-VLPs. a-VLPs involve integrating the antigen into the Pr55gag itself, either by direct insertion into the Gag sequence, replacing non-essential portions of Gag or as a C-termina! fusion; yielding.a chimaeric protein that forms the functional unit of the VLP. When proteins are incorporated on the outside of the particle it is termed a B-VLP. Although not much has been reported on it in the literature, a y-VLP would be the synergy of the □- and B- types. B-VLPs have received the most attention, since trimeric envelope glycoprotein (env) is a major target of neutralizing antibodies 28. Initial difficulties with insufficient Env incorporation have been mostly solved by the deletion of part of the cytoplasmic domain gp41, or replacement of it with heterologous membrane anchors 6. Studies in rodents and non-human primates have shown encouraging humoral and cellular responses but without neutralizing antibody production See Deml et.al 2004 for an extensive analysis. To date, chimaeric Gag a-VLPs based on the integration and C-terminal fusion approach have been limited to short peptides and epitopes of less than 200 amino acids; such as the V3 loop from external glycoprotein gp120 11,16,17,33, a CD4 binding domain 15,29 and a Nef-protein derived epitope 35,36 These studies generally reported high titre antibody responses (but with negligible neutralizing activity) in mice and rabbits to the Pr55gag carrier but substantially lower responses to the inserted antigen Up until now it has been believed that the maximum size of inserted or C-terminally fused epitopes is at most around 200 amino acids 7. It is also recognized that both the location of the insert and the nature of the insert itself affect the formation of VLPs from these recombinant proteins 7. Abbreviations used in the specification: aa amino acid AIDS acquired immunodeficiency syndrome APCs antigen presenting cells bp base pair CAT chloramphenicol acetyl transferase CTL cytotoxic T-lymphocyte DC dendritic cell DNA deoxyribonucleic acid EM electron microscopy g gram GFP green fluorescent protein HIV human immunodeficiency virus kDa kilo Dalton ml miililitre myr+ N-terminal myristoylation positive myr N-terminal myristoylation negative NAb neutralizing antibody ng nanogram PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction SIV simian immunodeficiency virus SDS sodium dodecyl sulphate ug microgram ul microlitre VLP virus-like particle WHO World Health Organization wt wild type SUMMARY OF THE INVENTION According to a first embodiment of the invention, there is provided a method of producing immunogenic chimaeric proteins which self-assemble into virus-like particles, the method including the steps of fusing a first DNA sequence encoding a H1V-1 subtype C protein sequence or portion thereof of greater than 200 amino acids to a second DMA sequence encoding at least p17 and p24 of a Gag protein by a direct in-frame C-terminal fusion, infecting a host cell with a vector containing the fused sequence, and causing expression of the chimaeric protein encoded by the fused sequence. The chimaeric protein assembles spontaneously at the cell membrane and buds externally to form a virus-like particle (VLP) that resembles an immature HIV virion. These virus-like particles may be recovered for use in a vaccine. The DNA sequences for both the Gag protein and the HIV protein may be human codon optimised Apart from including only p17 and p24 (p41), the gag DNA sequence may be the full-length gag sequence, or may be a truncated gag sequence, for example, a p6-truncated gag sequence, such as the nucleotide sequences of any one of SEQ I.D. Nos. 1 to 5 (or the corresponding amino acid sequences of SEQ I.D. Nos. 6 to 9), or a sequence which is at least 80%, 90% or 95% identical thereto. The first DNA sequence may be a reverse transcriptase sequence, a tat sequence or a nef sequence, or a combination thereof. These sequences may be modified to be non-functional for stability and safety purposes, such as by mutation, shuffling and/or truncation. For example, the first DNA sequence may be the nucleotide sequence of any one of SEQ I.D. Nos. 10, 12, 14, 16, 18, 40 or 42 (or the corresponding amino acid sequences of SEQ I.D. Nos. 11, 13, 15, 17, 41 or 43), or a sequence which is at least 80%, 90% or 95% identical thereto. The fused sequence may have a nucleotide or amino acid sequence of SEQ I.D. Nos. 20 to 39, or a sequence which is at least 80% identical thereto, or more preferably at least 90% identical thereto, or even more preferably, at least 95% identical thereto. The host cell may be a human, animal or insect cell, wherein the insect cell may be infected by the baculovirus expression system The chimaeric protein typically forms budded particles or virus-like particles including up to about 1 000 amino acids, and especially up to about 800 amino acids, of the first protein. These budded or virus-like particles may have a particle size of between about 100nm and about 450nm in diameter. According to a second embodiment of the invention, there is provided a chimaeric virus-like particle or budded particle produced by the method described above. According to a third embodiment of the invention, there is provided a vaccine or pharmaceutical composition including a chimaeric virus-like particle or budded particle or mixtures thereof as described above. The vaccine or pharmaceutical composition may be for the treatment or prophylaxis of HIV or AIDS. According to a fourth embodiment of the invention, there is provided a method of treating or preventing HIV infection in a mammal, the method including the steps of administering to the mammal a pharmaceutical composition, and in particular a vaccine, including chimaeric budded or virus-like particles or mixtures thereof as described above. According to a fifth embodiment of the invention, there is provided the use of budded or virus-like particles or mixtures thereof as described above in a method of making a pharmaceutical composition, and in particular a vaccine, for the treatment or prophylaxis of HIV or AIDS. BRIEF DESCRIPTION OF THE DRAWINGS FIG 1: Schematic representation depicting shuffling of tat in pTHgrttnC FIG. 2: Schematic representation of chimaeric Gag constructs. Fourteen proteins are shown with the N-terminus on the left - aa, amino acid; RT, Reverse transcriptase; TN, shuffled Tat-Nef, M, methione; G, glycine; A, alanine. (1. Rounded off to nearest kDa.) FIG 3: Nucleotide and amino acid sequences inserted into constructs (a) humanised myristoylated Gag HMGagC (Sequence I.D. Nos. 6 and 8); (b) humanised myristoylated truncated Gag THMGagC (Sequence I.D. Nos. 9 and 7); (c) pTHgagC (Sequence I.D. No. 1); (d) pTHgrttnC. FIG 4: Amino acid sequences of chimaeric full-length Gag constructs The 5 chimaeric Gag sequences shown are the constructs containing full-length Gag. The sequences of the corresponding constructs containing p6-truncated Gag are identical but without the sequence shown in bold (Sequence I.D. Nos. 8 and 9). Amino acid position numbers are shown on the left and sequence identifiers on the right, (a) HMgag3 RT -humanized myristoylated Gag fused to 3' end of RT gene (Sequence I.D. Nos. 11, 21 and 23); (b) HMgag3TN - Gag fused to 3' end of Tat-Nef polygene fragment (Sequence I.D. Nos. 25, 27 and 13); (c) HMgagTN - Gag fused to Tat-Nef polygene fragment (Sequence I.D. Nos. 29, 31, 41 and 43); (d) HMgagRT - Gag fused to RT gene (Sequence I.D. Nos. 33, 35 and 17); (e) HMgagRTTN - Gag fused to RT-Tat-Nef polygene fragment (Sequence I.D. Nos. 37, 39 and 19). FIG. 5: DNA sequences of chimaeric full-length Gag constructs The 5 chimaeric gag sequences shown are the constructs encoding full-length Gag. The sequences of the corresponding constructs encoding p6-truncated Gag are identical but without the sequence shown in bold (Sequence I.D. Nos. 2 to 5). Nucleotide position numbers are shown on the left, (a) HMgag3'RT - encoding humanized myristoylated Gag fused to 3' end of RT gene (Sequence I.D. Nos. 20, 22 and 10); (b) HMgag3TN - encoding Gag fused to 3' end of Tat-Nef polygene fragment (Sequence I.D. Nos. 24, 26 and 12); (c) HMgagTN - encoding Gag fused to Tat-Nef polygene fragment (Sequence I.D. Nos. 28, 30, 14, 40 and 42); (d) HMgagRT - encoding Gag fused to RT gene (Sequence I.D. Nos. 32, 34 and 16); (e) HMgagRTTN - encoding Gag fused to RT-Tat-Nef polygene fragment (Sequence I.D. Nos. 36, 38 and 18), sequence identifiers are indicated above the relevant section of the DNA sequence. FIG. 6: Western blots of control Gag proteins in High Five ™ cell pellets and supernatants High 5 cells were infected with recombinant baculovirus expressing 4 control Gag proteins. Three days post infection cell pellets were collected and equal amounts of cell lysate (CL) or equal volumes of culture supernatant (SUP) were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432) Lanes: M, Molecular weight marker; 1, HMgagC CL; 2, HMgagC SUP; 3, HAMgagC CL; 4, HAMgagC SUP; 5, THMgagC CL; 6, THMgagC SUP; 7, THAMgagC CL; 8, THAMgagC SUP; 9, Baculovirus only CL. Arrowheads indicate the full and truncated Gag protein bands. FIG 7: Western blots of chimaeric Gag proteins in High 5 cell pellets High 5 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell pellets were collected and equal amounts of cell lysate were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432). (A) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgag3'RT; 3, THMgag3 RT; 4, HMgag3TN; 5, THMgag3TN; 6, Baculovirus; 7 Uninfected. (B) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgagTN; 3, THMgagTN; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, Baculovirus; 9 Uninfected. FIG. 8: Western blots of chimaeric Gag proteins in High Five ™ cell pellets High 5 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell pellets were collected and equal amounts of cell lysate were loaded in each lane. Blots were probed with anti-RT polyclonal antiserum (A, ARP428) and anti-nef monoclonal antibody (B, 01-003}. (A) Lanes: M, Molecular weight marker; 1, RT control protein; 2, HMgag3'RT; 3, THMgag3'RT; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, HMgagC; 9 THMgagC. Arrowheads indicate recombinant Gag proteins. (B) Lanes: M, Molecular weight marker; 1, Recombinant Nef control protein; 2, HMgag3TN; 3, THMgag3TN; 4, HMgagTN; 5, THMgagTN; 6, HMgagRTTN; 7, THMgagRTTN; 8, HMgagC; 9 THMgagC FIG. 9: Western blots of Gag proteins in High 5 cell culture supernatant High 5 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell supematants were collected and equal volumes were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432). (A) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgag3'RT; 3, THMgag3'RT; 4, HMgag3TN; 5, THMgag3TN; 6, Baculovirus; 7 Uninfected. (B) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgagTN; 3, THMgagTN; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, Baculovirus; 9 Uninfected. Arrowheads indicate recombinant Gag proteins in (A) and (B). FIG 10: Western blots of Gag proteins in Sf 21 cell culture supernatant Sf21 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell supematants were collected and equal volumes were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432). (A) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgag3'RT; 3, THMgag3'RT; 4, HMgag3TN; 5, THMgag3TN; 6, Baculovirus; 7 Uninfected. (B) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgagTN; 3, THMgagTN; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, Baculovirus; 9 Uninfected. Arrowheads indicate recombinant Gag proteins in (A) and (B). FIG. 11: VLPs and budded structures produced by chimaeric Gag proteins in Sf21 cells. (A) HAMgagC (B) THAMgagC (C) HMgagC (D) THMgagC (E) HMgag3'RT (F) THMgag3'RT (G) HMgag3'TN (H) THMgag3'TN (I) HMgagTN (J) THMgagTN (K) HMgagRT (L) THMgagRT (M) HMgagRTTN (N) THMgagRTTN. Bar = 10Onm, all photos at same scale. FIG. 12 Example of VLP band visible by trans-illumination after sucrose gradient sedimentation. The arrow indicates the VLP band in the 30-70% sucrose gradient visible by trans-illumination, in this example THMgagC VLPs. FIG. 13: Western blots of chimaeric Gag proteins in Sf9 particle purifications VLP purifications were run on the same blot and probed with different antibodies to show that the predominant protein contained desired protein components. FIG 14: Western blots of chimaeric Gag proteins in Sf9 particle purifications VLP purifications were run on the same blot and probed with different antibodies to show that the predominant protein contained desired protein components. FIG. 15: Western blots of chimaeric Gag proteins in Sf9 particle purifications VLP purifications were run on the same blot and probed with different antibodies to show that the predominant protein contained desired protein components. FIG. 16: Schematic representation of HIV-1 Gag precursor protein with functional domains N-Terminus to the left. FIG. 17: Response of mice to a prime with pTHgrttnC and a boost with GagRT VLPs Results show ELISPOT counts of -interferon producing peripheral blood mononuclear cells from mice immunized with pTHr.GrttnC DNA and boosted with HMGagRT VLPs. Med = medium only; irrel pep = unrelated peptide; other peptides derived as shown. DETAILED DESCRIPTION OF THE INVENTION The invention describes the construction of immunogenic chimaeric virus-like particles (VLPs), budded particles or mixtures thereof, based on the human immunodeficiency virus (HIV) Gag protein for vaccine purposes. VLPs are essentially protein-based antigens that structurally resemble the pathogenic virus but are non-infectious and cannot replicate. In this case, the structural protein Gag of HIV (or at least the portion encoding p17 and p24 thereof) forms the basis of the VLP, which produces particles of 100-140nm in diameter. These bud from cells infected with suitable vectors expressing Gag. To produce chimaeric Gag based VLPs, HIV-derived DNA sequences were fused to the 3' end of the gag gene sequence in order to produce chimaeric proteins and thus VLPs of the recombinant protein. Since chimaeric VLPs contain a greater range of H!V protein sequence, they generally elicit a broader immune response compared with Gag-only VLPs. The chimaeric VLPs produced herein contain significantly larger protein fusions than previously reported, or were thought possible, i.e. VLPs containing fusion proteins having up to 778 C-terminal addition amino acids are described herein, whereas it was previously thought that it was not possible to obtain chimaeric VLPs with fusion proteins of greater than 200 amino acids. The plasmids pTHgagC 32 and pTHgrrtnC 40 listed in Table 1 contain HIV-1 subtype C DNA sequences obtained from 2 individuals within three months of infection. These isolates (Du151 and Du422) were selected on having closest amino acid similarity to a South African subtype C consensus sequence 32,38. Full length Gag (non-myristylated) is encoded by pTHgagC, while pTHgrttnC encodes a polyprotein of p6-truncated Gag (non-myristoylated), Reverse transcriptase (RT), shuffled Tat and Nef. As components of DNA vaccines for expression of these proteins in vivo, all genes were human codon optimized {gag by Operon Technologies Inc., USA and RT, tat and nef by GeneArt, Germany) and inhibitory sequence sites were removed. In addition, the polyprotein components were modified, at the genetic level, to be non-functional for stability and safety considerations. Briefly, Gag myristylation site was removed by mutating MGA to MAA (aa number 2) by site-directed mutagenesis; the active site of RT was inactivated by mutation (YMDDL —> YMAAL); Tat was inactivated through shuffling of the three gene regions known to be important to function (while maintaining all potential epitopes) (Fig. 1); and Nef was inactivated by removal of 30 bp at the 5'-end coding for 10 N-terminal amino acids (known to be essential for functionality). The proteins were also shown to be non-functional experimentally: infected HEK 293 cell lysates contained full length GrttnC polyprotein (approximately 150 kDa) but no RT activity. Expression of a shuffled Tat-GFP was visualized in HLCD4-CAT indicator cells but no CAT activity was detectable - which was demonstrated with wild-type (functional) Tat-GFP. Immunofluorescent labeling of infected cells with anti-Nef antibody showed cytoplasmic localization, whereas active Nef is localized to the cell membrane. Preliminary immunogenicity studies have shown that vaccination of BALB/c mice with pTHgrttnC lead to high induction levels of cytotoxic T lymphocytes (CTLs) against multiple epitopes, and strong IFN-y responses to RT were stimulated 40. Therefore, these components were chosen as the fusion sequences for the chimaeric VLPs, with the added advantage of providing a protein analogue to the DNA vaccine for possible use in a heterologous prime-boost. The invention is further described by the following examples. Such examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention. Examples: To test whether it is possible to generate chimaeric VLPs incorporating large inserts, the applicants made C-terminal fusions to full-length Gag and a p6-truncated Gag. It was reasoned that a truncated Gag still capable of forming VLPs might offer more potential for larger fusions. Rather than disrupt the Gag protein itself by inserting protein sequences internally, all fusions were made on the C-terminus of both Gag versions. The 10 Gag fusion constructs, in addition to the 4 Gag positive controls, are shown in Fig. 2. Corresponding amino acid sequences are shown in Figs. 3 and 4. Corresponding DNA sequences are shown in Fig. 5. For each control and chimaeric protein, the encoding DNA sequence was assembled and transposed into baculovirus under the polyhedron promoter using the Bac-to-Bac® Baculovirus Expression System (Invitrogen™). The resulting recombinant baculovirus was used to infect Sf9, Sf21 and High Five™ cells for expression of the protein in either monolayer or suspension culture. Electron microscopy together with Western blotting of cell lysates, culture supernatants and particle preparations was used to ascertain that the 10 Gag fusion constructs formed VLPs. Firstly, it was shown that the correct chimaeric proteins and controls were being expressed. Both myr+ (HMgagC and THMgagC) and myr (HMgagC and THMgagC) controls expressed protein of the expected size in cell lysates that reacted strongly with anti-p24 antibody (Fig. 6). Only protein from the myr1' controls should bud into the media as VLPs, and indeed the expected protein was only present in significant quantities in the corresponding supernatants. (Fig. 6 lanes 2 & 4). It was also shown by electron microscopy (EM) that neither myr Gag construct forms budding structures (Fig. 11A & B). The 10 chimaeric Gag constructs all produced protein of the expected size in the cell lysates (Fig. 7A & B). Fig 8A confirms that the RT fusions do indeed produce RT containing protein, while Fig. 8B shows likewise for Nef. in the -TN and -RTTN fusions, Nef is C-terminal to the shuffled Tat, and so the presence of shuffled Tat can be inferred from the presence of the Nef. Three days post-infection, 8 of the 10 chimaeric Gag proteins were visible in High Five cell supernatant by western blot (Fig. 9A & B). Although the multiplicity of infection (m.o.l.) has not yet been standardized, all other conditions were kept constant between infections with different constructs. The pattern in overall band intensity (and relative to p41 intensity for each lane on the blot) for each chimaeric protein seems to indicate that the larger Gag fusions result in lower protein levels in the supernatant. A similar trend was observed with infection of Sf21 cells (Fig. 10), but three days post-infection the -RTTN constructs were visible in the supernatant. Irrespective of the dynamics of cellular expression level and extra-cellular movement of each construct, all 10 Gag fusions are present in the supernatant. Furthermore, the -3'RT, -3TN and -TN fusions result in distinct VLPs as visible by EM (Fig. 11E-J) and the -RT and -RTTN fusions produce budded structures which appear to be not as defined in structure as classic VLPs. Taken together, the data from the western blots and EM shows that expression of each construct leads to that protein being present in culture supernatant and that VLPs or budded particles are produced. Since there are some specific breakdown products visible in the supernatant for some of the constructs (Fig. 9A & B), particularly those containing shuffled Tat and Nef regions, gradient preparations were analysed to ascertain if the predominant protein is indeed the full-length one. Particulate matter is visible as a band in the sucrose gradient after ultracentrifugation (Fig. 12). Although these particle purifications are fairly crude, they clearly exclude free soluble protein such as p41 and BSA from the supernatant. Three examples of these blots, for HMgagC fusions, are shown in Figs. 13-15. Similar results were obtained for THMgagC fusions. Certainly for the smaller fusions (-3'RT, -3TN and TN) the full length protein is the only distinct band present after VLP purification (Fig. 13, 14). The RT fusions gave full length protein as the major band, while -RTTN fusions had several prominent bands with the full length protein having the greatest band intensity (Fig. 15). Certainly for the -RTTN fusions it seems some of the specific cleavage products form VLPs - which could account for the substantial heterogeneity in particle size observed in EM (Fig. 11 panel M & N). However, the fact that the full-length -RTTN fusion is present after VLP purification implies that this construct can form budded particles. Of the 10 fusion constructs, it was consistently found that the HMgagC fusion gave a greater particle yield than the THMgagC fusions, although the difference has not been accurately quantified as yet. Fig. 17 shows good preliminary evidence that chimaeric Gag-based VLPs containing RT sequences can be used to boost appropriate cellular immune responses - by both CD4+ and CD8+ T cells - in mice primed with a Gag-RT-Tat-Nef -encoding DNA vaccine. The evidence presented here demonstrates that the applicants have constructed Gag-based particles with much larger inserts than previously reported or thought possible. Furthermore, the antigenic nature of the fusions suggests that these chimaeric VLPs may be superior vaccine candidates to the Gag-based VLPs that have previously been reported. Materials and methods Plasmid construction All DNA manipulations were carried out according to standard procedures 26. Cloning into pGEM® - T easy was done according to manufacturers instructions. Tabie 1: Raw materials: plasmids and cloning vectors (Table Removed) Construction of sub-cloning plasmids Note: Introduced restriction enzyme sites and stop codons are bold and underlined respectively. All new plasmids were confirmed by appropriate restriction enzyme digests. pGEM-5 HMgag (3611bp) The 5' end of the gag gene in pTHgagC (van Harmelen et a/., 2003) was PCR amplified with primers HMgagFI (5'-CTT GCC ACC ATG GGT GCT CGC GCA TC-3) and HMgagR1 (5 -GGT GTC CTC CCA CTG TTC AGC ATA GTG TTC-3'); where HMgagFI restores the myristoyiation signal (ala2 —> gly2) at the N-terminus of the protein. This initial PCR product was used as template in a second PCR reaction with HMgagF2 (5 -ATT AGG ATC CAA GCT TGC CAC CAT GGG TGC-3') and HMgagRI where a BamH\ site is introduced upstream of the start codon. This second PCR product was cloned into pGEM® - T easy. pGEM-3 HgagNS (3984bp) The 3' end of the gag gene in pTHgagC was PCR amplified with primers HMgagF3 (5 -GCG AAG GCG CCA CTC CTC-3') and HMgagR2 (5-CTT GAA TTC TTG GCT GAG GGG GTC GCT AC-3), where HMgagR2 eliminates the stop codon and introduces an EcoRI site. This PCR product was cloned into pGEM® - T easy. pGEM-3THgag (3837bp) The 3' end of the gag gene in pTHgagC was PCR amplified with primers HMgagF3 (5 -GCG AAG GCG CCA CTC CTC-3') and HMgagR3 (5'- AGC GAA TTC TTA GCC AGG GCG GCC CTT ATG-3'), where HMgagR3 inserts a stop codon (TAA) and introduces an EcoRI site upstream of the p6 region. This PCR product, which is effectively a p6 truncated gag gene, was cloned into pGEM® - T easy. pFBD-5'HMgag (5817bp) pGEM-5 HMgag was digested with BamHl and EcoRI and the resulting 601 bp fragment was cloned into pFastBac™Dual digested with the same enzymes. pFBD- THMgagNS (6560bp) pTHRepgrttnC (Burgers et ai, 2006) was digested with Narl and EcoRI and the resulting 803bp fragment was cloned into pFBD-5'HMgag digested with the same enzymes. pFBD- HMgagNS (671 Obp) pGEM-3'HgagNS was digested with A/art and EcoRI and the resulting 953bp fragment was cloned into pFBD-5'HMgag digested with the same enzymes. Construction of plasmids for generation of recombinant baculovirus Note: All pFastBac™Dual constructs contain HIV1-C protein sequences cloned into the multiple cloning site (MCS) of the polyhedron promoter present in this vector. All protein sequences have an in-frame stop codon at the 3'-end of the sequence. pFBD-HMgagC (6713bp) pTHgagC was digested with Narl and EcoRI and the resulting 956bp fragment was cloned into pFBD-5'HMgag digested with the same enzymes. pFBD-THMgagC (6563bp) pGEM-3THgag was digested with A/ari and EcoRI and the resulting 806bp fragment was cloned into pFBD-5'HMgag digested with the same enzymes. pFBD-HAMgagC (6707bp) pFBD-HMgagC and pTHgagC were digested with Narl and H/ndlll, yielding 1027bp and 535bp fragments respectively. Both these fragments were sequentially cloned into pFBD-HMgagC digested with Hindlll only. pFBD-THAMgagC (6557bp) pFBD-THMgagC and pTHgagC were digested with Nar\ and H/ndlll, yielding 877bp and 535bp fragments respectively. Both these fragments were sequentially cloned into pFBD-THMgagC digested with H/ndlll only. pFBD-HMgag3 RT (5997bp) pTHRepgrttnC was digested with Alw44\ and blunted with mung bean nuclease. Further digestion with Notl gave a 314bp fragment that was cloned into pFBD-HMgagNS digested with Stul and Notl . pFBD-THMgag3'RT (6847bp) pTHRepgrttnC was digested with Alw44\ and blunted with mung bean nuclease. Further digestion with A/ofl gave a 314bp fragment that was cloned into pFBD-THMgagNS digested with Stul and Notl . pFBD-HMgag3TN (7176bp) pTHRepgrttnC was digested with A/ari and blunted with Klenow enzyme. Further digestion with Xbal gave a 506bp fragment that was cloned into pFBD-HMgagNS digested with Stul and Xbal . pFBD-THMgag3TN (7026bp) pTHRepgrttnC was digested with Narl and blunted with Klenow enzyme. Further digestion with Xbal gave a 506bp fragment that was cloned into pFBD-THMgagNS digested with Stul and Xbal . pFBD-HWIgagTN (7635bp) pTHRepgrttnC was digested with Ncol and blunted with Klenow enzyme. Further digestion with Xbal gave a 965bp fragment that was cloned into pFBD-HMgagNS digested with Stul and Xbal . pFBD-THMgagTN (7485bp) pTHRepgrttnC was digested with A/col and blunted with Klenow enzyme. Further digestion with Xbal gave a 965bp fragment that was cloned into pFBD-THMgagNS digested with Stul and Xbal . pFBD-HMgagRT (8035bp) pTHRepgrttnC was digested with EcoRI and Noti and the resulting 1361bp fragment was cloned into pFBD-HMgagNS digested with the same enzymes. In this case the stop codon (TAG) is 18bp downstream of RT gene. pFBD-THMgagRT (7885bp) pTHRepgrttnC was digested with EcoRI and Wofl and the resulting 1361bp fragment was cloned into pFBD-THMgagNS digested with the same enzymes. In this case the stop codon (TAG) is 18bp downstream of RT gene. pFBD-HNIgagRTTN (9047bp) pTHRepgrttnC was digested with EcoRI and the resulting 2337bp fragment was cloned into pFBD-HMgagNS digested with the same enzyme. pFBD-THMgagRTTN (8897bp) pTHRepgrttnC was digested with EcoRI and the resulting 2337bp fragment was cloned into pFBD-THMgagNS digested with the same enzyme. Sequencing • pFBD-HAMgagC and pFBD-THAMgagC were sequenced with the primer FBDS1 (5-TAA AGG TCC GTA TAC TCC GG-3') to confirm the gag sequence does not contain the myristoylation signal. • All pFastBac™Dual constructs where blunting reactions were included in construction were sequenced with the primer HMgagS1 (5'-GTG CTT CAA TTG TGG CAA GGA GGG-3') to confirm the sequence near the blunting site. • All pGEM® - T easy constructs were sequenced in both directions using an M13 primer set. Insect cell culture Sf21 and Sf9 insect cells (derived from the fall army worm, Spodoptera frugiperda) and High Five™ cells (derived from the cabbage looper, Trichoplusia ni) were all obtained from Invitrogen™ and cell lines were maintained as recommended. For infection with recombinant baculovirus in monolayer, Sf21 cells were grown at 27°C in TC-100 Insect medium (Sigma) supplemented with: 10% (v/v) foetal bovine serum (FBS), 50 µg/ml neomycin, 69.2 ug/ml Penicillin G and 100 µg/ml Streptomycin. Sf9 cells were grown under the same conditions with the addition of 0.1% Pluronic F-68 (Sigma): a surfactant to decrease membrane shearing in suspension culture. Glassware was prepared by coating with Repelcote (VWR International Ltd, Poole, UK), washing with detergent, autoclaving and baking at 200°C for 2 hr. Cultures were incubated in a rotary shaker at 120 RPM. For expression of proteins in serum-free media, High Five™ cells were grown in Express Five® media (Gibco) with 10 µg/ml Gentamycin and 18mM L-glutamine. Generation of recombinant baculovirus. Recombinant FastBac™Dual constructs were individually transformed into competent Escherichia coli DH10Bac cells (invitrogen™) by standard methods 26 to generate corresponding recombinant bacmid DNA using the Bac-to-Bac® Baculovirus Expression System (Invitrogen™) This bacmid DNA was extracted using a modified alkaline lysis method. Sf21 cells (1 x 105) were seeded in sterile 35mm2 tissue culture wells and transfected with recombinant bacmid DNA and Cellfectin™ (Invitrogen™) and recombinant baculovirus (Autographa californica nuclear polyhedrosis virus) was amplified by repeated passages. Immunoblotting Protein samples were diluted in 5x loading buffer14, heated at 90°C for 5 min and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis 26 (SDS-PAGE). For immunoblotting, proteins were transferred to nitrocellulose membranes (NitroBind, Osmonics Inc.) using a Trans Blot® semi-dry transfer cell (Bio-Rad). Membranes were then soaked in blocking buffer [3% (w/v) bovine serum albumen (BSA) and 0.1% (v/v) Tween 20 in PBS] overnight and probed with appropriate primary antibodies (Table 1) for 4 hr. Following 3 sets of washes for 10 min each blots were reacted with appropriate secondary antibodies (Table 1.) for 2 hr. After a second set of washes blots were developed with Nitro blue tetrazolium chloride/5-Bromo-4-chloro-3-indolyi phosphate (NBT/BCIP, Roche, Germany). The pre-stained molecular weight marker (PageRuler™) was obtained from Fermentas (Maryland, USA). Table 2: Positive control proteins used in Western blotting (Table Removed) Table 3: Primary antibodies used in Western blotting (Table Removed) Table 4: Secondary antibodies used in Western blotting (Table Removed) Electron microscopy Sf21 cells (1 x 106) were seeded into 35mm2 tissue culture wells and infected with recombinant baculovirus. Cells were fixed 72hrs post-infection with 2.5% giutaraldehyde in PBS overnight and resuspended in low melting point agarose. Samples were post-fixed with 1% osmium tetroxide in PBS, dehydrated through an ethanol series and embedded in Spurrs resin. Ultrathin sections, cut with a Leica Reichert Ultracuts microtome, were mounted on copper grids and stained with 2% uranyl acetate for 10 min. After 5 washes in ultrapure water, samples were stained with Reynolds lead citrate for 10 min. Grids were then rinsed in a stream of ultrapure water, dried, and samples were visualized with a LEO 912 transmission electron microscope. VLP purification Supernatant from 60ml Sf9 suspension culture, infected with 600µl of amplified viral stock, was centrifuged at 30 000 RPM for 3 hr in 70ml polycarbonate tubes in a Beckman L7-55 ultracentrifuge. The pellet containing VLPs was resuspended in PBS pH 7.4 and overlayed on a 30-70% sucrose gradient and centrifuged at 26 000 RPM for 90 min. The VLP band visible by trans-illumination was extracted by syringe through a side puncture and resuspended in 40mls PBS. The VLPs were pelleted by further centrifugation at 26 000 RPM for 1 hr and resuspended in a final volume of 50Dul PBS with Complete protease inhibitor cocktail (Roche, Germany). Celiuiar immune responses Two groups of mice were primed with pTHGrttnC (100ug, im). Twenty eight days later one group was given an intraperitoneal inoculation of 20ng GagRT VLPs in a volume of 150 ul PBS. Mice were sacrificed on day 40 and splenocytes prepared. Immune responses to CD8 and CD4 epitopes in Gag and RT were investigated in a mouse IFN-y ELISPOT assay. A group of mice given just 20ng of GagRT VLPs did not show an immune response to Gag and RT (Fig. 17). While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention. It is therefore intended that the claims cover or encompass all such modifications, alterations and/or changes. Reference list: 1- Accola, M. A., B. Strack, and H. G. Gottiinger 2000. Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J. Virol. 74:5395-5402. 2. Bojak, A., L. Demi, and R. Wagner. 2002. The past, present and future of HIV-vaccine development: a critical view. Drug Discov. Today 7:36-46. 3. Bryant M. and L. Ratner. 1990. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc. Natl. Acad. Sci. U. S. A 87:523-527. 4. Buonaguro, L., M. L. Visciano, M. L. Tornesello, M. Tagliamonte, B. Biryahwabo, and F. M. Buonaguro. 2005. Induction of systemic and mucosal cross-clade neutralizing antibodies in BALB/c mice immunized with human immunodeficiency virus type 1 clade A virus-iike particles administered by different routes of inoculation. J. Virol. 79:7059-7067. 5 Delchambre, NL, D. Gbeysen, D. Thines, C. Thiriart, E. Jacobs, E. Verdin, M. Horth, A. Burny, and F. Bex. 1989. The GAG precursor of simian immunodeficiency virus assembles into virus-like particles. EMBO J. 8:2653-2660. 6. Demi, L, R. Schirmbeck, J. Reimann, H. Wolf, and R. Wagner. 1997. 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Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques. J Virol 75:5879-5890. 21. Morikawa, Y., D. J. Hockley, M. V. Nermut, and I. M. Jones 2000. Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly. J. Virol. 74:16-23. 22. Paliard, X., Y. Liu, R. Wagner, H. Wolf, J. Baenziger, and C. M. Walker. 2000. Priming of strong, broad, and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T cells after administration of a virus-like particle vaccine in rhesus macaques. AIDS Res. Hum. Retroviruses 16:273-282. 23. Perez-Caballero, D., T. Hatziioannou, J. Martin-Serrano, and P. D. Bieniasz 2004. Human immunodeficiency virus type 1 matrix inhibits and confers cooperativity on gag precursor-membrane interactions. J. Virol. 78:9560-9563. 24. Royer, M., M. Cerutti, B. Gay, S. S. Hong, G. Devauchelle, and P. Boulanger. 1991. 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Demi, R. Schirmbeck, M. Niedrig, J. Reimann, and H. Wolf. 1996. Construction, expression, and immunogenicity of chimaeric HIV-1 virus-like particles. Virology 220:128-140. 35. Wagner, R., L. Demi, V. Teeuwsen, J. Heeney, S. Yiming, and H. Wolf. 1996. A recombinant HIV-1 viruslike particle vaccine: from concepts to a field study. Antibiot. Chemother. 48:68-83. 36. Wagner, R., L. Demi, and H. Wolf. 1994. Polyvalent, recombinant HIV-1 virus-like particles: novel HIV-1 vaccine strategies. Antibiot. Chemother. 46:48-61. 37. Wang, C. T., H. Y. Lai, and J, J. Li. 1998. Analysis of minimal human immunodeficiency virus type 1 gag coding sequences capable of virus-like particle assembly and release. J. Virol. 72:7950-7959. 38. Williamson, C, L. Morris, M. F. Maughan, L. H. Ping, S. A. Dryga, R. Thomas, E. A. Reap, T. Cilliers, H. J. van, A. Pascual, G. Ramjee, G. Gray, R. Johnston, S. A. Karim, and R. Swanstrorn. 2003. Characterization and selection of HIV-1 subtype C isolates for use in vaccine development. AIDS Res. Hum. Retroviruses 19:133-144. 39. Wu, Z., J. Alexandratos, B. Ericksen, J. Lubkowski, R. C. Gallo, and W. Lu. 2004. Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: structural and mechanistic implications of p17 myristoylatior, Proc. Natl. Acad. Sci. U. S. A 101:11587-11592. 40. Burgers, A, Van Harmelen, J. H., Shephard, E., Adams, C, Mgwebi, T., Bourn, W., Hanke, T., Williamson, A.L., and Williamson, C. 2006. Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial. Journal of General Virology 87 399-410. CLAIMS: 1. A method of producing immunogenic chimaeric proteins which self-assemble into virus-like particles, the method including the steps of fusing a first DNA sequence encoding a HIV-1 subtype C protein or portion thereof of greater than 200 amino acids to a second DNA sequence encoding at least p17 and p24 of a Gag protein by a direct in-frame C-terminal fusion, infecting a host cell with a vector containing the fused sequence, and causing expression of the chimaeric protein encoded by the fused sequence. 2. A method according to claim 1, wherein the first and/or second DNA sequence are/is human codon optimized. 3. A method according to either of claims 1 or 2, wherein the second DNA sequence is a full-length or truncated gag sequence selected from the group consisting of SEQ I.D. Nos. 1 to 5, or a sequence which is at least 80% identical thereto. 4. A method according to any one of the preceding claims, wherein the first DNA sequence is modified to be non-functional for stability and safety purposes. 5. A method according to any one of the preceding claims, wherein the first DNA sequence is a reverse transcriptase sequence of SEQ I.D. No. 10, or a sequence which is at least 80% identical thereto. 6. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a tat sequence of SEQ I.D. No. 40, or a sequence which is at least 80% identical thereto. 7. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a nef sequence of SEQ I.D. No. 42, or a sequence which is at least 80% identical thereto. 8. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a combination of tat and nef sequences, or portions thereof, having a nucleotide sequence of SEQ ID No. 14, or a sequence which is least 80% identical thereto. 9. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a combination of reverse transcriptase, tat and nef sequences, or portions thereof, having a nucleotide sequence of SEQ ID No. 18, or a sequence which is at least 80% identical thereto. 10. A method according to any one of the preceding claims, wherein the host cell is a human, animal, plant or insect cell. 11. A method according to any one of the preceding claims, wherein from greater than 200 to about 1 000 amino acids of each virus-like particle are from the first DNA sequence. 12. An immunogenic chimaeric virus-like particle including a first HIV protein or portion thereof of greater than 200 amino acids fused by an in-frame C-terminal fusion to a second HIV protein containing at least p17 and p24 of a Gag protein. 13. A chimaeric virus-like particle according to claim 12, wherein the second protein is a full-length or truncated Gag protein having an amino acid sequence selected from any one of SEQ ID Nos. 6 to 9, or a sequence which is at least 80% identical thereto. 14. A chimaeric virus-like particle according to any one of claims 12 to 13, wherein the first protein has been modified so that it is non-functional for stability and safety purposes. 15. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a Reverse Transcriptase protein having an amino acid sequence of SEQ l.D. No. 11, or a sequence which is at least 80% identical thereto. 16. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a Tat protein having an amino acid sequence of SEQ l.D. No. 41, or a sequence which is at least 80% identical thereto. 17. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a Nef protein having an amino acid sequence of SEQ l.D. No. 43, or a sequence which is at least 80% identical thereto. 18. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a combination of Tat and Nef protein sequences having an amino acid sequence of SEQ ID Nos. 13 or 15, or a sequence which is at least 80% identical thereto. 19. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a combination of Reverse Transcriptase, Tat and Nef protein sequences having an amino acid sequence of SEQ ID No. 19, or a sequence which is at least 80% identical thereto. 20. A chimaeric virus-like particle according to any one of claims 12 to 19 for the treatment or prophylaxis of HIV or AIDS.

Full Text

CHIMAERIC HIV-1 SUBTYPE C GAG-VIRUS-LIKE PARTICLES
BACKGROUND OF THE INVENTION
The invention relates to a method of constructing chimaeric virus-like particles (VLPs) based on the human immunodeficiency virus (HIV) Gag protein, in particular for vaccine purposes.
HIV is one of the greatest problems facing mankind in the 21st century, with about 15 000 new infections daily, particularly in Sub-Saharan Africa where subtype C predominates 31. Although highly active anti-retroviral therapy (HAART) can prolong the survival of those infected with HIV, the progression to acquired immunodeficiency syndrome (AIDS) cannot be prevented at present. In addition, HAART (and other similar treatment regimes) is only economically feasible for treating high proportions of infected populations in the areas that are least affected by the pandemic, such as North America and Europe. These, together with numerous other socio-economic and medical reasons, highlight the need for effective and inexpensive HIV vaccines.
Vaccine strategies
Over the last 20 years a number of strategies have been developed toward producing HIV vaccines, and representatives from most of these are at various stages of clinical trial; however, there is still no vaccine on the market. The list includes live attenuated virus, whole killed virus, DNA vaccines, various recombinant viral and bacterial vectors, subunit protein vaccines and pseudovirions (including virus-like particles (VLPs)). Clearly there are merits and problems associated with each approach, and a number of recent reviews have investigated these concerns 2,15,18.
VLPs as vaccines
Vaccine approaches for more than 30 different viruses, including hepatitis B virus and human papilloma virus 7, have included the production of VLPs. There are a number of advantages to using VLPs 8
• They can be produced to relatively high yield in heterologous systems, where protocols are well established and can be purified from expression system culture supernatants by a variety of methods, including centrifugation and column fractionation.They are non-repiicative and non-infectious, which reduces some safety concerns.
• Expression systems established to date for production of HIV-1 VLPs include:
baculovirus, vaccinia virus, adenovirus and yeast systems.
• The vertebrate immune system responds well to particulate antigens of viral size.
• They have been shown to stimulate both humoral and cellular immune responses in rodents and non-human primates without addition of adjuvants, and may indeed act as adjuvants themselves.
• Efficient epitope presentation on MHC class I and -II molecules and activation of antigen presenting cells (APCs).
• They can be incorporated as part of a prime boost vaccination strategy with DNA vaccines.
Disadvantages:
• Manufacture can be expensive and technically demanding.
• immunogenicity is comparatively weaker than related replicating vectors.
• Since VLP preparations are essentially homogeneous with regard to specific antigen composition, cross clade reactivity is likely to be difficult to achieve. This is, however, common to most HIV vaccination strategies.
HIV Gag VLPs
Recent studies have indicated that VLPs can activate the innate immune system through 'danger signals' and lead to dendritic cell (DC) maturation and increased cytokine production 30. This is thought to be a result of the particulate nature of VLPs and residual components of the host/cell-system used in production. An example of this would be co-purified baculovirus in the insect culture system. More classically, humoral responses have been generated in mice 4, rabbits 6,34 and rhesus macaques. However the antibodies produced were generally either weak or non-neutralizing.
Chimaeric HIV Gag VLPs
Gag VLPs have the potential to act as an antigen carrier or delivery system, where the Gag essentially acts as a scaffold for the insertion or attachment of foreign antigens.
The inclusion of foreign antigens (epitopes, polypeptides or folded proteins) into Gag VLPs can be roughly grouped into three classes, designated here as a-, B- and y-VLPs. a-VLPs involve integrating the antigen into the Pr55gag itself, either by direct insertion into the Gag sequence, replacing non-essential portions of Gag or as a C-termina! fusion; yielding.a chimaeric protein that forms the functional unit of the VLP. When proteins are incorporated on the outside of the particle it is termed a B-VLP. Although not much has been reported on it in the literature, a y-VLP would be the synergy of the □- and B- types.
B-VLPs have received the most attention, since trimeric envelope glycoprotein (env) is a major target of neutralizing antibodies 28. Initial difficulties with insufficient Env incorporation have
been mostly solved by the deletion of part of the cytoplasmic domain gp41, or replacement of it with heterologous membrane anchors 6. Studies in rodents and non-human primates have shown encouraging humoral and cellular responses but without neutralizing antibody production See Deml et.al 2004 for an extensive analysis.
To date, chimaeric Gag a-VLPs based on the integration and C-terminal fusion approach have been limited to short peptides and epitopes of less than 200 amino acids; such as the V3 loop from external glycoprotein gp120 11,16,17,33, a CD4 binding domain 15,29 and a Nef-protein derived epitope 35,36 These studies generally reported high titre antibody responses (but with negligible neutralizing activity) in mice and rabbits to the Pr55gag carrier but substantially lower responses to the inserted antigen Up until now it has been believed that the maximum size of inserted or C-terminally fused epitopes is at most around 200 amino acids 7. It is also recognized that both the location of the insert and the nature of the insert itself affect the formation of VLPs from these recombinant proteins 7.
Abbreviations used in the specification:
aa amino acid
AIDS acquired immunodeficiency syndrome
APCs antigen presenting cells
bp base pair
CAT chloramphenicol acetyl transferase
CTL cytotoxic T-lymphocyte
DC dendritic cell
DNA deoxyribonucleic acid
EM electron microscopy
g gram
GFP green fluorescent protein
HIV human immunodeficiency virus
kDa kilo Dalton
ml miililitre
myr+ N-terminal myristoylation positive
myr N-terminal myristoylation negative
NAb neutralizing antibody
ng nanogram
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
SIV simian immunodeficiency virus
SDS sodium dodecyl sulphate
ug microgram
ul microlitre
VLP virus-like particle
WHO World Health Organization
wt wild type
SUMMARY OF THE INVENTION
According to a first embodiment of the invention, there is provided a method of producing immunogenic chimaeric proteins which self-assemble into virus-like particles, the method including the steps of fusing a first DNA sequence encoding a H1V-1 subtype C protein sequence or portion thereof of greater than 200 amino acids to a second DMA sequence encoding at least p17 and p24 of a Gag protein by a direct in-frame C-terminal fusion, infecting a host cell with a vector containing the fused sequence, and causing expression of the chimaeric protein encoded by the fused sequence.
The chimaeric protein assembles spontaneously at the cell membrane and buds externally to form a virus-like particle (VLP) that resembles an immature HIV virion. These virus-like particles may be recovered for use in a vaccine.
The DNA sequences for both the Gag protein and the HIV protein may be human codon optimised
Apart from including only p17 and p24 (p41), the gag DNA sequence may be the full-length gag sequence, or may be a truncated gag sequence, for example, a p6-truncated gag sequence, such as the nucleotide sequences of any one of SEQ I.D. Nos. 1 to 5 (or the corresponding amino acid sequences of SEQ I.D. Nos. 6 to 9), or a sequence which is at least 80%, 90% or 95% identical thereto.
The first DNA sequence may be a reverse transcriptase sequence, a tat sequence or a nef sequence, or a combination thereof. These sequences may be modified to be non-functional for stability and safety purposes, such as by mutation, shuffling and/or truncation. For example, the first DNA sequence may be the nucleotide sequence of any one of SEQ I.D. Nos. 10, 12, 14, 16, 18, 40 or 42 (or the corresponding amino acid sequences of SEQ I.D. Nos. 11, 13, 15, 17, 41 or 43), or a sequence which is at least 80%, 90% or 95% identical thereto.
The fused sequence may have a nucleotide or amino acid sequence of SEQ I.D. Nos. 20 to 39, or a sequence which is at least 80% identical thereto, or more preferably at least 90% identical
thereto, or even more preferably, at least 95% identical thereto.
The host cell may be a human, animal or insect cell, wherein the insect cell may be infected by the baculovirus expression system
The chimaeric protein typically forms budded particles or virus-like particles including up to about 1 000 amino acids, and especially up to about 800 amino acids, of the first protein. These budded or virus-like particles may have a particle size of between about 100nm and about 450nm in diameter.
According to a second embodiment of the invention, there is provided a chimaeric virus-like particle or budded particle produced by the method described above.
According to a third embodiment of the invention, there is provided a vaccine or pharmaceutical composition including a chimaeric virus-like particle or budded particle or mixtures thereof as described above.
The vaccine or pharmaceutical composition may be for the treatment or prophylaxis of HIV or AIDS.
According to a fourth embodiment of the invention, there is provided a method of treating or preventing HIV infection in a mammal, the method including the steps of administering to the mammal a pharmaceutical composition, and in particular a vaccine, including chimaeric budded or virus-like particles or mixtures thereof as described above.
According to a fifth embodiment of the invention, there is provided the use of budded or virus-like particles or mixtures thereof as described above in a method of making a pharmaceutical composition, and in particular a vaccine, for the treatment or prophylaxis of HIV or AIDS.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1: Schematic representation depicting shuffling of tat in pTHgrttnC
FIG. 2: Schematic representation of chimaeric Gag constructs.
Fourteen proteins are shown with the N-terminus on the left - aa, amino acid; RT, Reverse transcriptase; TN, shuffled Tat-Nef, M, methione; G, glycine; A, alanine. (1. Rounded off to nearest kDa.)
FIG 3: Nucleotide and amino acid sequences inserted into constructs
(a) humanised myristoylated Gag HMGagC (Sequence I.D. Nos. 6 and 8); (b) humanised myristoylated truncated Gag THMGagC (Sequence I.D. Nos. 9 and 7); (c) pTHgagC (Sequence I.D. No. 1); (d) pTHgrttnC.
FIG 4: Amino acid sequences of chimaeric full-length Gag constructs
The 5 chimaeric Gag sequences shown are the constructs containing full-length Gag. The sequences of the corresponding constructs containing p6-truncated Gag are identical but without the sequence shown in bold (Sequence I.D. Nos. 8 and 9). Amino acid position numbers are shown on the left and sequence identifiers on the right, (a) HMgag3 RT -humanized myristoylated Gag fused to 3' end of RT gene (Sequence I.D. Nos. 11, 21 and 23); (b) HMgag3TN - Gag fused to 3' end of Tat-Nef polygene fragment (Sequence I.D. Nos. 25, 27 and 13); (c) HMgagTN - Gag fused to Tat-Nef polygene fragment (Sequence I.D. Nos. 29, 31, 41 and 43); (d) HMgagRT - Gag fused to RT gene (Sequence I.D. Nos. 33, 35 and 17); (e) HMgagRTTN - Gag fused to RT-Tat-Nef polygene fragment (Sequence I.D. Nos. 37, 39 and 19).
FIG. 5: DNA sequences of chimaeric full-length Gag constructs
The 5 chimaeric gag sequences shown are the constructs encoding full-length Gag. The sequences of the corresponding constructs encoding p6-truncated Gag are identical but without the sequence shown in bold (Sequence I.D. Nos. 2 to 5). Nucleotide position numbers are shown on the left, (a) HMgag3'RT - encoding humanized myristoylated Gag fused to 3' end of RT gene (Sequence I.D. Nos. 20, 22 and 10); (b) HMgag3TN - encoding Gag fused to 3' end of Tat-Nef polygene fragment (Sequence I.D. Nos. 24, 26 and 12); (c) HMgagTN - encoding Gag fused to Tat-Nef polygene fragment (Sequence I.D. Nos. 28, 30, 14, 40 and 42); (d) HMgagRT - encoding Gag fused to RT gene (Sequence I.D. Nos. 32, 34 and 16); (e) HMgagRTTN - encoding Gag fused to RT-Tat-Nef polygene fragment (Sequence I.D. Nos. 36, 38 and 18), sequence identifiers are indicated above the relevant section of the DNA sequence.
FIG. 6: Western blots of control Gag proteins in High Five ™ cell pellets and
supernatants
High 5 cells were infected with recombinant baculovirus expressing 4 control Gag proteins. Three days post infection cell pellets were collected and equal amounts of cell lysate (CL) or equal volumes of culture supernatant (SUP) were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432) Lanes: M, Molecular weight marker; 1, HMgagC CL; 2, HMgagC SUP; 3, HAMgagC CL; 4, HAMgagC SUP; 5, THMgagC CL; 6, THMgagC SUP; 7, THAMgagC CL; 8, THAMgagC SUP; 9, Baculovirus only CL. Arrowheads indicate the full and truncated Gag protein bands.
FIG 7: Western blots of chimaeric Gag proteins in High 5 cell pellets
High 5 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell pellets were collected and equal amounts of cell lysate were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432). (A) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgag3'RT; 3, THMgag3 RT; 4, HMgag3TN; 5, THMgag3TN; 6, Baculovirus; 7 Uninfected. (B) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgagTN; 3, THMgagTN; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, Baculovirus; 9 Uninfected.
FIG. 8: Western blots of chimaeric Gag proteins in High Five ™ cell pellets
High 5 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell pellets were collected and equal amounts of cell lysate were loaded in each lane. Blots were probed with anti-RT polyclonal antiserum (A, ARP428) and anti-nef monoclonal antibody (B, 01-003}. (A) Lanes: M, Molecular weight marker; 1, RT control protein; 2, HMgag3'RT; 3, THMgag3'RT; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, HMgagC; 9 THMgagC. Arrowheads indicate recombinant Gag proteins. (B) Lanes: M, Molecular weight marker; 1, Recombinant Nef control protein; 2, HMgag3TN; 3, THMgag3TN; 4, HMgagTN; 5, THMgagTN; 6, HMgagRTTN; 7, THMgagRTTN; 8, HMgagC; 9 THMgagC
FIG. 9: Western blots of Gag proteins in High 5 cell culture supernatant
High 5 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell supematants were collected and equal volumes were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432). (A) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgag3'RT; 3, THMgag3'RT; 4, HMgag3TN; 5, THMgag3TN; 6, Baculovirus; 7 Uninfected. (B) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgagTN; 3, THMgagTN; 4, HMgagRT; 5, THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, Baculovirus; 9 Uninfected. Arrowheads indicate recombinant Gag proteins in (A) and (B).
FIG 10: Western blots of Gag proteins in Sf 21 cell culture supernatant
Sf21 cells were infected with recombinant baculovirus expressing different Gag chimaeric proteins. Three days post infection cell supematants were collected and equal volumes were loaded in each lane. Blots were probed with anti-p24 polyclonal antiserum (ARP432). (A) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgag3'RT; 3, THMgag3'RT; 4, HMgag3TN; 5, THMgag3TN; 6, Baculovirus; 7 Uninfected. (B) Lanes: M, Molecular weight marker; 1, HIV-1B Gag protein; 2, HMgagTN; 3, THMgagTN; 4, HMgagRT; 5,
THMgagRT; 6, HMgagRTTN; 7, THMgagRTTN; 8, Baculovirus; 9 Uninfected. Arrowheads indicate recombinant Gag proteins in (A) and (B).
FIG. 11: VLPs and budded structures produced by chimaeric Gag proteins in Sf21
cells.
(A) HAMgagC (B) THAMgagC (C) HMgagC (D) THMgagC (E) HMgag3'RT (F) THMgag3'RT (G) HMgag3'TN (H) THMgag3'TN (I) HMgagTN (J) THMgagTN (K) HMgagRT (L) THMgagRT (M) HMgagRTTN (N) THMgagRTTN. Bar = 10Onm, all photos at same scale.
FIG. 12 Example of VLP band visible by trans-illumination after sucrose gradient
sedimentation.
The arrow indicates the VLP band in the 30-70% sucrose gradient visible by trans-illumination, in this example THMgagC VLPs.
FIG. 13: Western blots of chimaeric Gag proteins in Sf9 particle purifications
VLP purifications were run on the same blot and probed with different antibodies to show that the predominant protein contained desired protein components.
FIG 14: Western blots of chimaeric Gag proteins in Sf9 particle purifications
VLP purifications were run on the same blot and probed with different antibodies to show that the predominant protein contained desired protein components.
FIG. 15: Western blots of chimaeric Gag proteins in Sf9 particle purifications
VLP purifications were run on the same blot and probed with different antibodies to show that the predominant protein contained desired protein components.
FIG. 16: Schematic representation of HIV-1 Gag precursor protein with functional
domains
N-Terminus to the left.
FIG. 17: Response of mice to a prime with pTHgrttnC and a boost with GagRT
VLPs
Results show ELISPOT counts of -interferon producing peripheral blood mononuclear cells from mice immunized with pTHr.GrttnC DNA and boosted with HMGagRT VLPs. Med = medium only; irrel pep = unrelated peptide; other peptides derived as shown.
DETAILED DESCRIPTION OF THE INVENTION
The invention describes the construction of immunogenic chimaeric virus-like particles (VLPs), budded particles or mixtures thereof, based on the human immunodeficiency virus (HIV) Gag protein for vaccine purposes. VLPs are essentially protein-based antigens that structurally resemble the pathogenic virus but are non-infectious and cannot replicate. In this case, the structural protein Gag of HIV (or at least the portion encoding p17 and p24 thereof) forms the basis of the VLP, which produces particles of 100-140nm in diameter. These bud from cells infected with suitable vectors expressing Gag.
To produce chimaeric Gag based VLPs, HIV-derived DNA sequences were fused to the 3' end of the gag gene sequence in order to produce chimaeric proteins and thus VLPs of the recombinant protein. Since chimaeric VLPs contain a greater range of H!V protein sequence, they generally elicit a broader immune response compared with Gag-only VLPs. The chimaeric VLPs produced herein contain significantly larger protein fusions than previously reported, or were thought possible, i.e. VLPs containing fusion proteins having up to 778 C-terminal addition amino acids are described herein, whereas it was previously thought that it was not possible to obtain chimaeric VLPs with fusion proteins of greater than 200 amino acids.
The plasmids pTHgagC 32 and pTHgrrtnC 40 listed in Table 1 contain HIV-1 subtype C DNA sequences obtained from 2 individuals within three months of infection. These isolates (Du151 and Du422) were selected on having closest amino acid similarity to a South African subtype C consensus sequence 32,38. Full length Gag (non-myristylated) is encoded by pTHgagC, while pTHgrttnC encodes a polyprotein of p6-truncated Gag (non-myristoylated), Reverse transcriptase (RT), shuffled Tat and Nef. As components of DNA vaccines for expression of these proteins in vivo, all genes were human codon optimized {gag by Operon Technologies Inc., USA and RT, tat and nef by GeneArt, Germany) and inhibitory sequence sites were removed. In addition, the polyprotein components were modified, at the genetic level, to be non-functional for stability and safety considerations. Briefly, Gag myristylation site was removed by mutating MGA to MAA (aa number 2) by site-directed mutagenesis; the active site of RT was inactivated by mutation (YMDDL —> YMAAL); Tat was inactivated through shuffling of the three gene regions known to be important to function (while maintaining all potential epitopes) (Fig. 1); and Nef was inactivated by removal of 30 bp at the 5'-end coding for 10 N-terminal amino acids (known to be essential for functionality). The proteins were also shown to be non-functional experimentally: infected HEK 293 cell lysates contained full length GrttnC polyprotein (approximately 150 kDa) but no RT activity. Expression of a shuffled Tat-GFP was visualized in HLCD4-CAT indicator cells but no CAT activity was detectable - which was demonstrated with wild-type (functional) Tat-GFP. Immunofluorescent labeling of infected cells with anti-Nef antibody showed cytoplasmic localization, whereas active Nef is localized to the cell membrane. Preliminary immunogenicity studies have shown that vaccination of BALB/c mice with pTHgrttnC lead to high induction levels of cytotoxic T lymphocytes (CTLs) against multiple epitopes, and strong IFN-y responses to RT were stimulated 40. Therefore, these components were chosen as the fusion sequences for the chimaeric VLPs, with the added advantage of providing a protein analogue to the DNA vaccine for possible use in a heterologous prime-boost.
The invention is further described by the following examples. Such examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention.
Examples:
To test whether it is possible to generate chimaeric VLPs incorporating large inserts, the applicants made C-terminal fusions to full-length Gag and a p6-truncated Gag. It was reasoned that a truncated Gag still capable of forming VLPs might offer more potential for larger fusions. Rather than disrupt the Gag protein itself by inserting protein sequences internally, all fusions were made on the C-terminus of both Gag versions. The 10 Gag fusion constructs, in addition to the 4 Gag positive controls, are shown in Fig. 2. Corresponding amino acid sequences are shown in Figs. 3 and 4. Corresponding DNA sequences are shown in Fig. 5. For each control and chimaeric protein, the encoding DNA sequence was assembled and transposed into baculovirus under the polyhedron promoter using the Bac-to-Bac® Baculovirus Expression System (Invitrogen™). The resulting recombinant baculovirus was used to infect Sf9, Sf21 and High Five™ cells for expression of the protein in either monolayer or suspension culture. Electron microscopy together with Western blotting of cell lysates, culture supernatants and particle preparations was used to ascertain that the 10 Gag fusion constructs formed VLPs.
Firstly, it was shown that the correct chimaeric proteins and controls were being expressed. Both myr+ (HMgagC and THMgagC) and myr (HMgagC and THMgagC) controls expressed protein of the expected size in cell lysates that reacted strongly with anti-p24 antibody (Fig. 6). Only protein from the myr1' controls should bud into the media as VLPs, and indeed the expected protein was only present in significant quantities in the corresponding supernatants. (Fig. 6 lanes 2 & 4). It was also shown by electron microscopy (EM) that neither myr Gag construct forms budding structures (Fig. 11A & B).
The 10 chimaeric Gag constructs all produced protein of the expected size in the cell lysates (Fig. 7A & B). Fig 8A confirms that the RT fusions do indeed produce RT containing protein, while Fig. 8B shows likewise for Nef. in the -TN and -RTTN fusions, Nef is C-terminal to the shuffled Tat, and so the presence of shuffled Tat can be inferred from the presence of the Nef.
Three days post-infection, 8 of the 10 chimaeric Gag proteins were visible in High Five cell supernatant by western blot (Fig. 9A & B). Although the multiplicity of infection (m.o.l.) has not yet been standardized, all other conditions were kept constant between infections with different constructs. The pattern in overall band intensity (and relative to p41 intensity for each lane on the blot) for each chimaeric protein seems to indicate that the larger Gag fusions result in lower protein levels in the supernatant. A similar trend was observed with infection of Sf21 cells (Fig. 10), but three days post-infection the -RTTN constructs were visible in the supernatant. Irrespective of the dynamics of cellular expression level and extra-cellular movement of each construct, all 10 Gag fusions are present in the supernatant. Furthermore, the -3'RT, -3TN and -TN fusions result in distinct VLPs as visible by EM (Fig. 11E-J) and the -RT and -RTTN fusions produce budded structures which appear to be not as defined in structure as classic VLPs. Taken together, the data from the western blots and EM shows that expression of each construct leads to that protein being present in culture supernatant and that VLPs or budded particles are produced. Since there are some specific breakdown products visible in the supernatant for some of the constructs (Fig. 9A & B), particularly those containing shuffled Tat and Nef regions, gradient preparations were analysed to ascertain if the predominant protein is indeed the full-length one. Particulate matter is visible as a band in the sucrose gradient after ultracentrifugation (Fig. 12). Although these particle purifications are fairly crude, they clearly exclude free soluble protein such as p41 and BSA from the supernatant. Three examples of these blots, for HMgagC fusions, are shown in Figs. 13-15. Similar results were obtained for THMgagC fusions. Certainly for the smaller fusions (-3'RT, -3TN and TN) the full length protein is the only distinct band present after VLP purification (Fig. 13, 14). The RT fusions gave full length protein as the major band, while -RTTN fusions had several prominent bands with the full length protein having the greatest band intensity (Fig. 15). Certainly for the -RTTN fusions it seems some of the specific cleavage products form VLPs - which could account for the substantial heterogeneity in particle size observed in EM (Fig. 11 panel M & N). However, the fact that the full-length -RTTN fusion is present after VLP purification implies that this construct can form budded particles. Of the 10 fusion constructs, it was consistently found that the HMgagC fusion gave a greater particle yield than the THMgagC fusions, although the difference has not been accurately quantified as yet.
Fig. 17 shows good preliminary evidence that chimaeric Gag-based VLPs containing RT sequences can be used to boost appropriate cellular immune responses - by both CD4+ and CD8+ T cells - in mice primed with a Gag-RT-Tat-Nef -encoding DNA vaccine.
The evidence presented here demonstrates that the applicants have constructed Gag-based particles with much larger inserts than previously reported or thought possible. Furthermore, the antigenic nature of the fusions suggests that these chimaeric VLPs may be superior vaccine candidates to the Gag-based VLPs that have previously been reported.
Materials and methods
Plasmid construction
All DNA manipulations were carried out according to standard procedures 26. Cloning into pGEM® - T easy was done according to manufacturers instructions.
Tabie 1: Raw materials: plasmids and cloning vectors
(Table Removed)
Construction of sub-cloning plasmids
Note:
Introduced restriction enzyme sites and stop codons are bold and underlined respectively.
All new plasmids were confirmed by appropriate restriction enzyme digests.
pGEM-5 HMgag (3611bp)
The 5' end of the gag gene in pTHgagC (van Harmelen et a/., 2003) was PCR amplified with primers HMgagFI (5'-CTT GCC ACC ATG GGT GCT CGC GCA TC-3) and HMgagR1 (5 -GGT GTC CTC CCA CTG TTC AGC ATA GTG TTC-3'); where HMgagFI restores the myristoyiation signal (ala2 —> gly2) at the N-terminus of the protein. This initial PCR product was used as template in a second PCR reaction with HMgagF2 (5 -ATT AGG ATC CAA GCT TGC CAC CAT GGG TGC-3') and HMgagRI where a BamH\ site is introduced upstream of the start
codon. This second PCR product was cloned into pGEM® - T easy.
pGEM-3 HgagNS (3984bp)
The 3' end of the gag gene in pTHgagC was PCR amplified with primers HMgagF3 (5 -GCG AAG GCG CCA CTC CTC-3') and HMgagR2 (5-CTT GAA TTC TTG GCT GAG GGG GTC GCT AC-3), where HMgagR2 eliminates the stop codon and introduces an EcoRI site. This PCR product was cloned into pGEM® - T easy.
pGEM-3THgag (3837bp)
The 3' end of the gag gene in pTHgagC was PCR amplified with primers HMgagF3 (5 -GCG AAG GCG CCA CTC CTC-3') and HMgagR3 (5'- AGC GAA TTC TTA GCC AGG GCG GCC CTT ATG-3'), where HMgagR3 inserts a stop codon (TAA) and introduces an EcoRI site upstream of the p6 region. This PCR product, which is effectively a p6 truncated gag gene, was cloned into pGEM® - T easy.
pFBD-5'HMgag (5817bp)
pGEM-5 HMgag was digested with BamHl and EcoRI and the resulting 601 bp fragment was
cloned into pFastBac™Dual digested with the same enzymes.
pFBD- THMgagNS (6560bp)
pTHRepgrttnC (Burgers et ai, 2006) was digested with Narl and EcoRI and the resulting 803bp
fragment was cloned into pFBD-5'HMgag digested with the same enzymes.
pFBD- HMgagNS (671 Obp)
pGEM-3'HgagNS was digested with A/art and EcoRI and the resulting 953bp fragment was
cloned into pFBD-5'HMgag digested with the same enzymes.
Construction of plasmids for generation of recombinant baculovirus
Note:
All pFastBac™Dual constructs contain HIV1-C protein sequences cloned into the multiple cloning site (MCS) of the polyhedron promoter present in this vector. All protein sequences have an in-frame stop codon at the 3'-end of the sequence.
pFBD-HMgagC (6713bp)
pTHgagC was digested with Narl and EcoRI and the resulting 956bp fragment was cloned into
pFBD-5'HMgag digested with the same enzymes.
pFBD-THMgagC (6563bp)
pGEM-3THgag was digested with A/ari and EcoRI and the resulting 806bp fragment was cloned into pFBD-5'HMgag digested with the same enzymes.
pFBD-HAMgagC (6707bp)
pFBD-HMgagC and pTHgagC were digested with Narl and H/ndlll, yielding 1027bp and 535bp fragments respectively. Both these fragments were sequentially cloned into pFBD-HMgagC digested with Hindlll only.
pFBD-THAMgagC (6557bp)
pFBD-THMgagC and pTHgagC were digested with Nar\ and H/ndlll, yielding 877bp and 535bp fragments respectively. Both these fragments were sequentially cloned into pFBD-THMgagC digested with H/ndlll only.
pFBD-HMgag3 RT (5997bp)
pTHRepgrttnC was digested with Alw44\ and blunted with mung bean nuclease. Further digestion with Notl gave a 314bp fragment that was cloned into pFBD-HMgagNS digested with Stul and Notl .
pFBD-THMgag3'RT (6847bp)
pTHRepgrttnC was digested with Alw44\ and blunted with mung bean nuclease. Further digestion with A/ofl gave a 314bp fragment that was cloned into pFBD-THMgagNS digested with Stul and Notl .
pFBD-HMgag3TN (7176bp)
pTHRepgrttnC was digested with A/ari and blunted with Klenow enzyme. Further digestion with
Xbal gave a 506bp fragment that was cloned into pFBD-HMgagNS digested with Stul and Xbal .
pFBD-THMgag3TN (7026bp)
pTHRepgrttnC was digested with Narl and blunted with Klenow enzyme. Further digestion with Xbal gave a 506bp fragment that was cloned into pFBD-THMgagNS digested with Stul and Xbal .
pFBD-HWIgagTN (7635bp)
pTHRepgrttnC was digested with Ncol and blunted with Klenow enzyme. Further digestion with
Xbal gave a 965bp fragment that was cloned into pFBD-HMgagNS digested with Stul and Xbal .
pFBD-THMgagTN (7485bp)
pTHRepgrttnC was digested with A/col and blunted with Klenow enzyme. Further digestion with
Xbal gave a 965bp fragment that was cloned into pFBD-THMgagNS digested with Stul and
Xbal .
pFBD-HMgagRT (8035bp)
pTHRepgrttnC was digested with EcoRI and Noti and the resulting 1361bp fragment was cloned into pFBD-HMgagNS digested with the same enzymes. In this case the stop codon (TAG) is 18bp downstream of RT gene.
pFBD-THMgagRT (7885bp)
pTHRepgrttnC was digested with EcoRI and Wofl and the resulting 1361bp fragment was cloned into pFBD-THMgagNS digested with the same enzymes. In this case the stop codon (TAG) is 18bp downstream of RT gene.
pFBD-HNIgagRTTN (9047bp)
pTHRepgrttnC was digested with EcoRI and the resulting 2337bp fragment was cloned into pFBD-HMgagNS digested with the same enzyme.
pFBD-THMgagRTTN (8897bp)
pTHRepgrttnC was digested with EcoRI and the resulting 2337bp fragment was cloned into
pFBD-THMgagNS digested with the same enzyme.
Sequencing
• pFBD-HAMgagC and pFBD-THAMgagC were sequenced with the primer FBDS1 (5-TAA AGG TCC GTA TAC TCC GG-3') to confirm the gag sequence does not contain the myristoylation signal.
• All pFastBac™Dual constructs where blunting reactions were included in construction were sequenced with the primer HMgagS1 (5'-GTG CTT CAA TTG TGG CAA GGA GGG-3') to confirm the sequence near the blunting site.
• All pGEM® - T easy constructs were sequenced in both directions using an M13 primer set.
Insect cell culture
Sf21 and Sf9 insect cells (derived from the fall army worm, Spodoptera frugiperda) and High Five™ cells (derived from the cabbage looper, Trichoplusia ni) were all obtained from Invitrogen™ and cell lines were maintained as recommended. For infection with recombinant baculovirus in monolayer, Sf21 cells were grown at 27°C in TC-100 Insect medium (Sigma) supplemented with: 10% (v/v) foetal bovine serum (FBS), 50 µg/ml neomycin, 69.2 ug/ml Penicillin G and 100 µg/ml Streptomycin. Sf9 cells were grown under the same conditions with
the addition of 0.1% Pluronic F-68 (Sigma): a surfactant to decrease membrane shearing in suspension culture. Glassware was prepared by coating with Repelcote (VWR International Ltd, Poole, UK), washing with detergent, autoclaving and baking at 200°C for 2 hr. Cultures were incubated in a rotary shaker at 120 RPM. For expression of proteins in serum-free media, High Five™ cells were grown in Express Five® media (Gibco) with 10 µg/ml Gentamycin and 18mM L-glutamine.
Generation of recombinant baculovirus.
Recombinant FastBac™Dual constructs were individually transformed into competent Escherichia coli DH10Bac cells (invitrogen™) by standard methods 26 to generate corresponding recombinant bacmid DNA using the Bac-to-Bac® Baculovirus Expression System (Invitrogen™) This bacmid DNA was extracted using a modified alkaline lysis method. Sf21 cells (1 x 105) were seeded in sterile 35mm2 tissue culture wells and transfected with recombinant bacmid DNA and Cellfectin™ (Invitrogen™) and recombinant baculovirus (Autographa californica nuclear polyhedrosis virus) was amplified by repeated passages.
Immunoblotting
Protein samples were diluted in 5x loading buffer14, heated at 90°C for 5 min and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis 26 (SDS-PAGE). For immunoblotting, proteins were transferred to nitrocellulose membranes (NitroBind, Osmonics Inc.) using a Trans Blot® semi-dry transfer cell (Bio-Rad). Membranes were then soaked in blocking buffer [3% (w/v) bovine serum albumen (BSA) and 0.1% (v/v) Tween 20 in PBS] overnight and probed with appropriate primary antibodies (Table 1) for 4 hr. Following 3 sets of washes for 10 min each blots were reacted with appropriate secondary antibodies (Table 1.) for 2 hr. After a second set of washes blots were developed with Nitro blue tetrazolium chloride/5-Bromo-4-chloro-3-indolyi phosphate (NBT/BCIP, Roche, Germany). The pre-stained molecular weight marker (PageRuler™) was obtained from Fermentas (Maryland, USA).
Table 2: Positive control proteins used in Western blotting
(Table Removed)
Table 3: Primary antibodies used in Western blotting
(Table Removed)
Table 4: Secondary antibodies used in Western blotting
(Table Removed)
Electron microscopy
Sf21 cells (1 x 106) were seeded into 35mm2 tissue culture wells and infected with recombinant baculovirus. Cells were fixed 72hrs post-infection with 2.5% giutaraldehyde in PBS overnight and resuspended in low melting point agarose. Samples were post-fixed with 1% osmium tetroxide in PBS, dehydrated through an ethanol series and embedded in Spurrs resin.
Ultrathin sections, cut with a Leica Reichert Ultracuts microtome, were mounted on copper grids and stained with 2% uranyl acetate for 10 min. After 5 washes in ultrapure water, samples were stained with Reynolds lead citrate for 10 min. Grids were then rinsed in a stream of ultrapure water, dried, and samples were visualized with a LEO 912 transmission electron microscope.
VLP purification
Supernatant from 60ml Sf9 suspension culture, infected with 600µl of amplified viral stock, was centrifuged at 30 000 RPM for 3 hr in 70ml polycarbonate tubes in a Beckman L7-55 ultracentrifuge. The pellet containing VLPs was resuspended in PBS pH 7.4 and overlayed on a 30-70% sucrose gradient and centrifuged at 26 000 RPM for 90 min. The VLP band visible by trans-illumination was extracted by syringe through a side puncture and resuspended in 40mls PBS. The VLPs were pelleted by further centrifugation at 26 000 RPM for 1 hr and resuspended in a final volume of 50Dul PBS with Complete protease inhibitor cocktail (Roche, Germany).
Celiuiar immune responses
Two groups of mice were primed with pTHGrttnC (100ug, im). Twenty eight days later one group was given an intraperitoneal inoculation of 20ng GagRT VLPs in a volume of 150 ul PBS. Mice were sacrificed on day 40 and splenocytes prepared. Immune responses to CD8 and CD4 epitopes in Gag and RT were investigated in a mouse IFN-y ELISPOT assay. A group of mice given just 20ng of GagRT VLPs did not show an immune response to Gag and RT (Fig. 17).
While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention. It is therefore intended that the claims cover or encompass all such modifications, alterations and/or changes.
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CLAIMS:
1. A method of producing immunogenic chimaeric proteins which self-assemble into virus-like particles, the method including the steps of fusing a first DNA sequence encoding a HIV-1 subtype C protein or portion thereof of greater than 200 amino acids to a second DNA sequence encoding at least p17 and p24 of a Gag protein by a direct in-frame C-terminal fusion, infecting a host cell with a vector containing the fused sequence, and causing expression of the chimaeric protein encoded by the fused sequence.
2. A method according to claim 1, wherein the first and/or second DNA sequence are/is human codon optimized.
3. A method according to either of claims 1 or 2, wherein the second DNA sequence is a full-length or truncated gag sequence selected from the group consisting of SEQ I.D. Nos. 1 to 5, or a sequence which is at least 80% identical thereto.
4. A method according to any one of the preceding claims, wherein the first DNA sequence is modified to be non-functional for stability and safety purposes.
5. A method according to any one of the preceding claims, wherein the first DNA sequence is a reverse transcriptase sequence of SEQ I.D. No. 10, or a sequence which is at least 80% identical thereto.
6. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a tat sequence of SEQ I.D. No. 40, or a sequence which is at least 80% identical thereto.
7. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a nef sequence of SEQ I.D. No. 42, or a sequence which is at least 80% identical thereto.
8. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a combination of tat and nef sequences, or portions thereof, having a nucleotide sequence of SEQ ID No. 14, or a sequence which is least 80% identical thereto.
9. A method according to any one of claims 1 to 4, wherein the first DNA sequence is a combination of reverse transcriptase, tat and nef sequences, or portions thereof, having a nucleotide sequence of SEQ ID No. 18, or a sequence which is at least 80% identical thereto.
10. A method according to any one of the preceding claims, wherein the host cell is a human, animal, plant or insect cell.

11. A method according to any one of the preceding claims, wherein from greater than 200 to about 1 000 amino acids of each virus-like particle are from the first DNA sequence.
12. An immunogenic chimaeric virus-like particle including a first HIV protein or portion thereof of greater than 200 amino acids fused by an in-frame C-terminal fusion to a second HIV protein containing at least p17 and p24 of a Gag protein.
13. A chimaeric virus-like particle according to claim 12, wherein the second protein is a full-length or truncated Gag protein having an amino acid sequence selected from any one of SEQ ID Nos. 6 to 9, or a sequence which is at least 80% identical thereto.
14. A chimaeric virus-like particle according to any one of claims 12 to 13, wherein the first protein has been modified so that it is non-functional for stability and safety purposes.
15. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a Reverse Transcriptase protein having an amino acid sequence of SEQ l.D. No. 11, or a sequence which is at least 80% identical thereto.
16. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a Tat protein having an amino acid sequence of SEQ l.D. No. 41, or a sequence which is at least 80% identical thereto.
17. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a Nef protein having an amino acid sequence of SEQ l.D. No. 43, or a sequence which is at least 80% identical thereto.
18. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a combination of Tat and Nef protein sequences having an amino acid sequence of SEQ ID Nos. 13 or 15, or a sequence which is at least 80% identical thereto.
19. A chimaeric virus-like particle according to any one of claims 12 to 14, wherein the first protein is a combination of Reverse Transcriptase, Tat and Nef protein sequences having an amino acid sequence of SEQ ID No. 19, or a sequence which is at least 80% identical thereto.
20. A chimaeric virus-like particle according to any one of claims 12 to 19 for the treatment or prophylaxis of HIV or AIDS.

Documents:

4859-delnp-2008-Abstract-(19-08-2014).pdf

4859-delnp-2008-abstract.pdf

4859-delnp-2008-Claims-(19-08-2014).pdf

4859-delnp-2008-claims.pdf

4859-delnp-2008-Correspondence Others-(06-02-2014).pdf

4859-DELNP-2008-Correspondence Others-(19-08-2014).pdf

4859-DELNP-2008-Correspondence-Others (20-10-2009).pdf

4859-DELNP-2008-Correspondence-Others (21-10-2009).pdf

4859-delnp-2008-correspondence-others.pdf

4859-delnp-2008-description (complete).pdf

4859-delnp-2008-drawings.pdf

4859-delnp-2008-form-1.pdf

4859-DELNP-2008-Form-13-(05-06-2008).pdf

4859-delnp-2008-form-13.pdf

4859-DELNP-2008-Form-18 (20-10-2009).pdf

4859-DELNP-2008-Form-18.pdf

4859-delnp-2008-form-2.pdf

4859-delnp-2008-Form-3-(06-02-2014).pdf

4859-delnp-2008-form-3.pdf

4859-delnp-2008-form-5.pdf

4859-delnp-2008-pct-210.pdf

4859-delnp-2008-pct-304.pdf

4859-delnp-2008-pct-306.pdf

4859-delnp-2008-pct-409.pdf

4859-delnp-2008-pct-416.pdf

4859-delnp-2008-Petition-137-(19-08-2014)-1.pdf

4859-delnp-2008-Petition-137-(19-08-2014).pdf

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Patent Number

263129

Indian Patent Application Number

4859/DELNP/2008

PG Journal Number

41/2014

Publication Date

10-Oct-2014

Grant Date

08-Oct-2014

Date of Filing

05-Jun-2008

Name of Patentee

SOUTH AFRICAN MEDICAL RESEARCH COUNCIL

Applicant Address

FRANCIE VAN ZIJI DRIVE, PAROW, 7925, CAPE TOWN, SOUTH AFRICA.

Inventors:

#	Inventor's Name	Inventor's Address
1	HALSEY, RICHARD JAMES	39 GREENWOOD ROAD, CLAREMONT, 7708 CAPETOWN, SOUTH AFRICA.
2	TANZER, FLONA LESLEY	23 HAWTHORN WAY, PINELANDS, 7405, CAPE TOWN, SOUTH AFRICA.
3	WILLIAMSON, ANNA-LISE	34 UITVLUGH ROAD, PINELANDS, 7405 CAPE TOWN, SOUTH AFRICA.
4	RYBICKI, EDWARD PETER	34 UITVLUGH ROAD, PINELANDS, 7405, CAPE TOWN, SOUTH AFRICA.

PCT International Classification Number

C07K 14/16

PCT International Application Number

PCT/IB2006/003155

PCT International Filing date

2006-11-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2005/09035	2005-11-08	South Africa