A vector for eliciting an immune response to a host comprising a gene encoding the gp140 protein of the primary isolate of HIV-1, BX08, under the control of a promotor for expression of the protein in the host, specifically plasmids pCMV.gp140.BX08, pMP83, pMP84 and pMP88. Murine and human MHC class 1-restricted binding motifs contained in BX08 are identified.

SUMMARY OF THE INVENTION

The present effort has turned to design and construction of HIV DNA-based immunogens capable of eliciting cell-mediated immunity (CMI). In this context, the inventors have focused interest on the extracellular envelope fragment, gp140, expressed in a primary HIV-1 isolate, HIV-1 (BX08), for the reason that this protein is rich in motifs restricted to both the murine and human Major Histocompatibility Complex (MHC) class 1 alleles.

Immunization with an appropriately constructed immunogen expressing the gp140 protein leads to the generation of peptides with class 1 binding capability to allow the induction of HIV-1-specific CTLs capable of killing virus infected cells to limit infection.

The invention described by the inventors is that they have found a plasmid designated, pCMV.gp140.BX08, expressing the gp140 gene under the control of a CMV promotor was immunogenic in BALB/c mice in the elicitation of CTL response directed against multiple epitopes of the gp140 protein that are restricted to different H-2.sup.d class 1 gene products. It was also found that plasmids based on Semliki Forest Virus (SFV) vectors, namely pMP83, pMP84 and pMP88, also requiring the gp140 gene under the control of a CMV promoter were similarly immunogenic.

Accordingly, in one aspect of the present invention, there is provided a vector, comprising a gene encoding the extracellular fragments of gp140 of a primary HIV-1 isolate, preferably BX08, under the control of a promotor for expression of the gene product in a host organism, thereby eliciting a cytotoxic T-cell response.

The promotor preferably is the cytomegalovirus promotor. The vector may preferably be a plasmid vector having the identifying characteristics of plasmid pCMV.gp140.BX08, as shown in FIG. 1. The vector also may preferably be a plasmid vector having the identifying characteristics of plasmids pMP88, pMP84 or pMP83.

The invention further includes an immunogenic composition containing the vector as well as a method of generating a cytotoxic T-cell response to HIV-1 in a host by administering to the host the immunogenic composition provided herein. Such immunogenic composition may be formulated for intramuscular immunization with a suitable carrier or may be formulated for gene gun delivery with gold particles.

The invention extends to the vector when used as an immunogen for generating a cytotoxic T-cell response to HIV-1 in a host and to the use of the vector in the manufacture of an immunogen for the generation of a cytotoxic T-cell response to the HIV-1 in a host.

DETAILED DESCRIPTION OF THE INVENTION

A DNA immunogen is constructed using recombinant DNA technology to molecularly clone a gene of interest into a plasmid expression vector. A unique feature involving vaccination with a DNA-based immunogen is that, once delivered into a cell, the intracellular production of the immunogen favours the induction of MHC class 1-restricted cytotoxic T-cells as compared to other forms of vaccination involving the use of killed whole cell and formulated sub-unit immunogens, which tend to favour the elicitation of MHC class 2-restricted immuno-regulatory responses in the majority of cases studied (ref. 2). In this context, it is, therefore, favourable to use DNA technology to construct naked DNA immunogens for vaccination purposes in order to optimize the induction of cellular effector response against intracellular organisms, such as viruses as well as certain tumours. The other advantages DNA vaccines offer include: (i) the ease to produce them; and (ii) their stability over a wide temperature range.

A common model which has been used recently to predict murine and human CTL antigenic determinants has involved the identification of binding motifs for the respective MHC class 1 molecules from the primary sequences of the native protein molecules (see refs. 3 to 5). Thus, it has been proposed that motifs which are most favoured to bind and lodge into the peptide-binding groove of the H-2D.sup.d gene product is usually 8 to 10 amino acids long. In the majority of cases, these peptides are found to contain anchor residues, such as glycine and proline (GP), at positions 2 and 3 near the amino- (N-) terminus, and either a leucine or phenylalanine at the carboxy- (C-) terminus, which serve to interact with the respective `pockets` of the peptide-binding groove of a membrane-bound H-2D.sup.d molecule. The motifs restricted to the other class 1 allele, K.sup.d, of the H-2.sup.d haplotype were reported to contain a tyrosine at position 2, and could be an isoleucine, valine or leucine at the C-terminus. Studies of the peptides isolated from the human MHC class 1 molecules, HLA-A0201, had similarly revealed that the anchor residues were leucine or methionine at position 2 and valine or leucine at the C-terminus in the majority of cases.

The suitability of the HIV-1(BX08) gp140 gene product as a CTL-inducing imnunogen was assessed by prediction algorithms to determine the number of both the murine and human MHC class 1-restricted binding motifs it contained. The amino acid sequences of the binding motifs and the designation of the peptides representing them are shown in Table 1 below. Such peptides are novel and are claimed herein. The presence of binding motifs towards the different H-2.sup.d restricted class 1 alleles, i.e. D.sup.d and K.sup.d, allows the immunogenicity of plasmids, pCMV.gp140.BX08, pMP83, pMP84 and pMP88, expressing gp140 of HIV-1 of the primary isolate, BX08, and constructed as described in the Examples below, to be studied in the inbred mouse strain BALB/c of the H-2.sup.d haplotype. The elements and restriction sites of plasmid pCMV.gp140.BX08 are shown in FIG. 1. The construction of the plasmids pMP83, pMP84 and pMP88 is described in Example 5 below. The nucleotide sequence (SEQ ID No: 1) and the deduced amino acid sequence (SEQ ID No: 2) of the gp140 open reading frame of the plasmid pCMV.gp140.BX08, pMP83, pMP84 and pMP88, is shown in FIG. 2, which appear to be unique sequences and are claimed herein along with their complements.

The location of several binding motifs against the human MHC class 1 allele, HLA-A0201, as seen in Table 1, implied that, under an appropriate immunization regimen, the plasmid has the potential to elicit CTL response directed to these epitopes in the context of this class 1 molecule in human subjects.

The immunogenicity of the plasmid pCMV.gp140.BX08 was studied in BALB/c mice. The results of the study involving three injections of the plasmid at 100.0 .mu.g per dose using the intramuscular route are shown in FIG. 3. Upon in vitro re-stimulation of the spleenocytes of the plasmid-immunized animals with irradiated autologous LPS blasts pulsed individually with the D.sup.d- and K.sup.d-restricted motif containing peptides, namely, CLP-501 and CLP-504 (SEQ ID Nos.: 3, 5), respectively, it was found that CTLs were generated that killed P815 targets presented with the respective peptides (FIGS. 3A and 3B). The amino acid sequences of the peptides are shown in Table 1. A comparison of the magnitude of the responses at the same effector to target (E:T) ratio revealed that the D.sup.d-restricted response to the CLP-501 peptide is immuno-dominant and that the K.sup.d-restricted response to the CLP-504 peptide is sub-dominant. The in vitro re-stimulation leading to the expansion of the effectors was specific because the addition of the same number of irradiated LPS blasts alone (not treated with peptide) did not lead to any generation of effectors in the bulk culture able to kill either of the specific targets tested. The findings that the control group of mice injected with the pCMV vector without the gp140 insert alone failed to generate any of the two sub-populations of CTLs (FIG. 3C) confirmed that the plasmid, pCMV.gp 140.BX08, was indeed immunogenic.

The pCMV.gp140.BX08 plasmid, when delivered with the gene gun, was similarly found to be immunogenic. The results shown in FIG. 4 show that following two injections at a dose of 0.7 .mu.g of the plasmid, and using the same in vitro re-stimulation condition described above that CTLs recognizing the CLP-501 and CLP-504 peptides were detected (FIGS. 4A and 4B), while no effector response was elicited by the group of animals given the vector, pCMV, alone (FIG. 4C).

The immunogenicity of the plasmids pMP83, pMP84 and pMP88 was separately studied, delivered intramuscularly, in BALB/c mice, in comparison to pCMV.gp140.BX08, at three different dosage levels, namely 1.0, 10.0 and 100.0 .mu.g of DNA following the above described procedures. The results obtained are contained in Tables II and III below. CTL activation is achieved at significantly lower doses of the alphavirus vectors than with pCMV.gp140.BX08.

It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis and treatment of HIV infections. A further non-limiting discussion of such uses is further presented below.

Immunogenic compositions, including vaccines, containing the DNA vector may be prepared as injectables, in physiologically-acceptable liquid solutions or emulsions for polynucleotide administration. The DNA vector may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 93/24640) or the DNA vector may be associated with an adjuvant, as described in more detail below.

Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions, such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to 100% of the polynucleotide. In addition, the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses the degradative enzymes of the lysosomal compartment. Published PCT application WO 94/27435 describes compositions for genetic immunization comprising cationic lipids and polynucleotides.

Agents which assist in the cellular uptake of nucleic acid, such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used with the vector.

Polynucleotide immunogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles. Thus, U.S. Pat. No. 5,151,264 describes a particular carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vecteurs Supra Moleculaires (BVSM). The particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof.

U.S. Pat. No. 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly (DL-lactideco-glycolide). Other polymers for encapsulation are suggested, such as poly(glycolide), poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone, poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters and poly(8-hydroxybutyric acid), and polyanhydrides.

Published PCT application WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens. The microspheres being of starch, gelatin, dextran, collagen or albumin. This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosa. The delivery vehicle may additionally contain and absorption enhancer.

In particular embodiments of the present invention, the vector may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immune system.

The vectors may be delivered to the host by a variety of procedures, for example, Tang et al (ref. 6) discloses that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice, while Furth et al. (ref. 7) showed that a jet injector could be used to transfect skin, muscle, fat and mammary tissues of living animals.

Biological Deposits

Certain vectors that contain nucleic acid coding for an extracellular fragment of gp140 of a primary isolate that are described and referred to herein as well as precursor alphavirus vectors have been deposited with the America Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA, pursuant the Budapest Treaty and prior to the filing of this application. Samples of the deposited vectors will become available to the public and all restrictions imposed or access to the deposits will be received upon grant of a patent based on this United States patent application. In addition, the deposit will be replaced if viable samples cannot be dispensed by the Depository. The invention described and claimed herein is not limited in scope by the biological materials deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar vectors that contain nucleic acid which encodes equivalent or similar antigens as described in this application are within the scope of the invention.
 

Claim 1 of 2 Claims

1. A peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID Nos.: 4, 5, 7, 8, 9, 11, 13 and 14, as shown In Table 1.

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