Expressing gp140 fragment of primary HIV-1 isolate

Abstract

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 plasmid pCMV.gp140.BX08. Murine and human MHC class 1-restricted binding motifs contained in BX08 are identified.

Description

FIELD OF INVENTION

The present invention relates to the field of immunology, specifically HIV Vaccine Technology, and, in particular, is concerned with expressing the extracellular fragment of the envelope gene, gp140, of a primary human immunodeficiency virus type 1 (HIV-1) isolate.

BACKGROUND OF THE INVENTION

Acquired immunodeficiency syndrome (AIDS) is a disease which is the ultimate result of infection with human immunodeficiency virus (HIV). Currently, there is no effective vaccine which can protect the human population from HIV infection and hence the development of an efficacious HIV-vaccine and protocol for administering the same is urgently required. Previously, HIV-1 particles exhaustively inactivated by chemical treatments, a vaccinia vector encoding the whole envelope gene (gp140) of HIV-1, and purified recombinant gp120 have been evaluated as candidate HIV vaccines. Although inactivated HIV-1 virus preparations elicited a T-cell-mediated Delayed-Type Hypersensitivity (DTH) reaction in humans, and vaccinia/gp160 and gp120 recombinant vaccine candidates induced virus neutralizing antibodies, non of these immunogens have been shown to be efficacious human HIV vaccines (ref. 1, throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). The inventors' interest in HIV vaccinology is to develop immunogenic and cost-effective HIV-1 DNA vaccines and consider that their use alone or in conjunction with other forms of HIV-1 vaccine candidates will lead to the elicitation of more effective immune responses against HIV-1.

There has previously been described in granted European Patent No. 470,980 and U.S. Pat. No. 5,639,854, assigned to the assignee hereof, the disclosures of which are incorporated herein by reference, inter alia, the identification and characterization of a T-cell epitope of the core protein, p24E, of HIV-1. There has further been described in granted U.S. Pat. Nos. 5,759,769 and 5,795,955, assigned to the assignee hereof, and disclosures of which are incorporated by reference, the use of the T-cell epitope in the construction of immunogenic synthetic HIV-1 chimeric peptides comprising p24E linked to amino acid sequences of different B-cell epitopes of an envelope or core protein of HIV-1.

SUMMARY OF THE INVENTION

The present effort has turned to design and construct 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. Upon 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 d class 1 gene products.

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 thereby is the cytomegalovirus promotor. The vector is preferably one having the identifying characteristics of plasmid pCMV.gp140.BX08, as shown in FIG. 1 .

The invention further includes an immunogenic composition containing the vector as well as a method of generating a cytotoxic T-cell response 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.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows details of the elements of plasmid pCMV.gp140.BX08;

FIGS. 2A to 2 F show the nucleotide (SEQ ID No: 1) and deduced amino acid sequence (SEQ ID No: 2) of the gp140 open reading frame of the plasmid pCMV.gp140.BX08;

FIGS. 3A , 3 B and 3 C show the effector responses elicited by intramuscular injection of the plasmid, pCMV.gp140.BX08, in BALB/c mice; and

FIGS. 4A , 4 B and 4 C show the effector responses elicited by gene gun delivery of the plasmid, pCMV.gp140.BX08, in BALB/c mice.

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; 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 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 d molecule. The motifs restricted to the other class 1 allele, K d , of the H-2 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 immunogen 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. The presence of binding motifs towards the different H-2 d restricted class 1 alleles, i.e. D d and K d , allows the immunogenicity of a plasmid, pCMV.gp140.BX08, expressing gp140 of HIV-1 of the primary isolate, BX08, and constructed as described in the Example below, to be studied in the inbred mouse strain BALB/c of the H-2 d hapotype. The elements and restriction sites of plasmid pCMV.gp140.BX08 are shown in FIG. 1 . The nucleotide (SEQ ID No: 1) and the deduced amino acid sequences (SEQ ID No: 2) of the gp140 open reading frame of the plasmid pCMV.gp140.BX08 is shown in FIGS. 2A to 2 F, which appear to be unique sequences.

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 μg 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 d - and K 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. 3 A and 3 B). 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 d -restricted response to the CLP-501 peptide is immuno-dominant and that the K d -restricted response to the CLP-504 peptide is sub-dominant. The in vitro re-stimulating 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.gp140.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 μg 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. 4 A and 4 B), while no effector response was elicited by the group of animals given the vector, pCMV, alone (FIG. 4 C).

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 preparation 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 Moléculaires (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 a high molecular weight protein of a non-typeable strain of Haemophilus that are described and referred to herein 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.

Deposit Summary
	Plasmid	ATCC	Deposited Date
	pCMV.gp140.BX08	203839	March 9, 1999

EXAMPLES

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Methods of plasmid construction, peptide synthesis, cell culture, CTL assay and other testing procedures that are not explicitly described in this disclosure are amply reported in the scientific literature and are will within the scope of those skilled in the art.

Example 1

This Example illustrates the construction of the plasmid, pCMV.gp140.BX08.

The plasmid, pCMV.gp140.BX08, contains sequence segments from various sources, and the elements of construction are depicted in FIG. 1 . The nucleotide (SEQ ID No: 1) and derived amino acid sequences (SEQ ID No: 2) of the gp140 open reading frame of the plasmid are shown in FIGS. 2A to 2 F.

The prokaryotic vector pBluescript SK (Stratagene) is the backbone of the plasmid pCMV.gp140.BX08 and was modified by the replacement of the Amp R with Kan R gene and the deletion of the fl and the LacZ region. To achieve the desired modifications, the sequence between Ahdl (nucleotide 2,041) and Sacl (nucleotide 759) of pBluescript SK, which contains the Amp R , fl origin and the LacZ, was deleted. A 1.2 kb Pstl fragment from the plasmid pUC-4K (Pharmacia) containing the Kan R gene, was blunt end ligated to the Ahdl site of pBluescript SK in a counter-clockwise orientation relative to it's transcription. A 1.6 kb Sspl/Pstl DNA fragment containing the human cytomegalovirus immediate-early gene promotor, enhancer and intron A sequences (CMV) was ligated to the other end of the Kan R gene so that the transcription from the CMV promotor proceeds in the clockwise orientation. A synthetic oligonucleotide segment containing translation initiation sequence and sequences encoding the human tissue plasminogen activator signal peptide (TPA) was used to link the CMV promotor and the sequences encoding the gp140 of the primary isolate HIV-1 BX08 . The gp140 sequence encodes a portion of the envelope protein between amino acid 33 and 666 which ends before the transmembrane domain of gp41 (see FIG. 2 ). A translation termination codon was placed at the end of the gp140 coding sequence. Next to the gp140 coding region is a 0.2 kb fragment containing the bovine growth hormone (BGH) polyadenylation signal sequence that is PCR amplified from pRC/CMV (Invitrogen). A remnant 80 bp DNA segment from the SV40 polyadenylation signal remained between the BGH poly A sequence and the Sacl site of pBluescript SK due to DNA manipulation and it serves no purpose in this plasmid.

The pCMV.gp140.BX08 construct was introduced into HB101 competent cells according to manufacturer's recommendations (GibcoBRL). Correct molecular clones were identified by restriction and sequencing analysis and their expression of gp140 was examined in transient transfections followed by Western blot analysis.

All DNAs used for immunizations were prepared using EndoFree Plasmid Kit (Qiagen). For intramuscular immunizations in mice, 100 μg of pCMV.gp140.BX08, in 100 μl PBS was injected into the tibialis anterior muscles at 4 weeks intervals. Gene gun immunizations were accomplished with the Helios Gene Gun System (Biorad). Cartridges were prepared according to manufacturer's recommendations. Specifically, each cartridge was made to contain 0.7 μg of the DNA and 0.5 mg gold. Immunizations were carried out by applying two cartridges to each animal onto the shaved abdominal area at 4 week intervals.

Example 2

This Example illustrates the synthesis of peptides.

Solid phase peptide synthesis of peptide CLP-501 and CLP-504 were conducted on an ABI 430A automated peptide synthesizer according to the manufacturer's standard protocols. The peptides were cleaved from solid support by treatment with liquid hydrogen fluoride in the presence of thiocresole, anisole, and methyl sulfide. The crude products were extracted with trifluoroacetic acid (TFA) and precipitated with diethyl ether.

The amino acid sequences of these peptides are shown in Table 1.

Example 3

This Example illustrates in vitro cell culture protocols to re-stimulate and expand CTLs and assay for their effector functions.

Spleens of BALB/c mice injected with the plasmid, pCMV.gp140.BX08, prepared and formulated as described in Example 1, using the intramuscular route or gene gun delivery method, were removed 10 to 11 days post final booster injection. Spleenocytes at 3.0×10 7 were co-cultured with 1.3×10 7 autogolous LPS blasts which had been pulsed with the test peptide for 5 hr at 37° C. and irradiated at 3000 rads in 10.0 ml of complete medium (RPMI 1640 supplemented with 10.0% 56° C. heat-inactivated bovine serum, 120.0 units per ml of penicillin G sodium, 120.0 μg per ml of streptomycin sulphate and 0.35 mg per ml of L-glutamine) in a 25 cm 2 tissue culture flask. The cultures were kept at 37° C. in a humidified CO 2 incubator for days, and the responders were then tested against peptide-pulsed P815 target cells in a standard in vitro 4 hr CTL assay as follows:

The responders were harvested from the 7-day cultures and washed once with RPMI 1640 medium without added bovine serum. The positive target was created by incubating 3 to 5×10 6 P815 cells with 100.0 μg of the specified peptide overnight in a 26° C. water bath. The target cells were then labeled with 51 Cr at 250.0 uCi per 1×10 6 cells in the presence of 25.0 μg of the same test peptides for 60 to 75 minutes at 26° C. After washing twice with complete medium to remove excess 51 Cr, the targets were incubated at 2.5×10 3 with different numbers of the responders per well in a V-bottomed 96 will tissue culture plates for 4 hr in a 37° C. CO 2 incubator. Half amount of the supernatant from each micro-assay culture was then removed and counted for radioactivity. Results were expressed as % which was calculated using the equation:

% lysis=(spontaneous lysis in cpm of experimental sample−spontaneous lysis in cpm of labeled target cells alone) devided by (totally lysis in com of target cells alone−spontaneous lysis in cpm of target cells alone)×100.

The results obtained employing intramuscular injection are shown in FIGS. 3A , 3 B and 3 C while those obtained employing the gene gun delivery are shown in FIGS. 4A , 4 B and 4 C and are disable alone.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novel plasmid expressing, in vitro and in vivo, the gp140 protein of the primary HIV-1 isolate BX08 and the generation of MHC class 1-restricted cytotoxic T-cells in animals. Modifications are possible within the scope of this invention.

TABLE 1
MHC class 1-restricted motifs of the extracellular envelope fragment, gp140, of HIV-1(BX08)
H-2 d -restricted *
Peptide ***	D d -	Peptide ***	K d -	HLA-A0201-restricted **
CLP-501	IGP GRAFY TT (274-283)	CLP-503	A Y DTEVHN V (29-37) (SEQ ID No:4)	K L TPLCVT L (91-98) (SEQ ID No:10)
	(SEQ ID No:3)
		CLP-504	F Y SLKIVP I (141-149) (SEQ ID No:5)	T L FRVAIK L (305-313) (SEQ ID No:11)
		CLP-505	L Y KYKVVK I (443-451) (SEQ ID No:6)	T L TVQARQ L (403-411) (SEQ ID No:12)
		CLP-506	K Y KVVKIEP L (445-454) (SEQ ID No:7)	T L TVQARA L (496-504) (SEQ ID No:13)
		CLP-507	R Y LQDQRF L (545-553) (SEQ ID No:8)	Q L QARVLA L (535-543) (SEQ ID No:14)
		CLP-508	N Y TEIIYS L (597-605) (SEQ ID No:9)
* or ** Anchors residues were typed in bolded letters
*** Peptides chosen for the study reported herein are bolded.

REFERENCES

1. B. J. Spalding. Biotechnology, vol. 10, pp 24-28, 1992

2. H. L. Robinson and C. A. T. Torres. Seminars in Immunology, vol. 9, pp 271-283, 1997.

3. Ian A. Wilson and Daved H. Fremont. Seminars in Immunology, vol. 5, pp 75-80, 1993.

4. Kristen Falk and Olaf Rotzschke. Seminars in Immunology. Vol. 5, pp 81-94, 1993.

5. Victor H. Englefard. Current Opinion in Immunology, Vol. 6, pp 13-23, 1994.

6. Tang et al, Nature, 1992, 356:152-154.

7. Furth et al, Analytical Biochemistry, 1992, 205:365-368.

Claims

1. A method of generating a cytotoxic T-cell response in a host, which comprises administering to the host an immunogenic composition comprising a plasmid vector comprising a gene encoding the extracellular fragment of gp140 of the primary HIV-1 isolate BX08 under the control of a promoter which expresses the gene product in the host.

2. The method of claim 1, wherein the promoter is the cytomegalovirus promotor.

3. The method of claim 1 wherein the plasmid vector is pCMV.gp140.BX08 (ATCC No. 203839).

4. A vector which is pCMV.gp140.BX08 (ATCC No. 203839), as shown in FIG. 1.

5. An immunogenic composition comprising a vector which is pCMV.gp140.BX08 (ATCC No. 203839).

6. The immunogenic composition of claim 5 formulated for intramuscular immunization with a pharmaceutically-acceptable liquid carrier.

7. The immunogenic composition of claim 5 formulated for gene gun delivery with gold particles.