Title of Invention	REPLICATION DEFECTIVE HIV VACCINE
Abstract	A replication-defective HIV particle pseudotyped with vesicular stomatitis virus G protein (VSV-G). The pol gene of the HIV genome in the particle is modified to inactivate the pol reverse transcriptase and protease activity. This pseudotyped HIV particle can infect many cell types, including human and simian cells, and only undergoes one round of replication. Furthermore, a virus-specific immune response can be detected in mice immunized with the VSV-G pseudotyped replication-defective HIV.

Title of Invention

REPLICATION DEFECTIVE HIV VACCINE

Abstract

A replication-defective HIV particle pseudotyped with vesicular stomatitis virus G protein (VSV-G). The pol gene of the HIV genome in the particle is modified to inactivate the pol reverse transcriptase and protease activity. This pseudotyped HIV particle can infect many cell types, including human and simian cells, and only undergoes one round of replication. Furthermore, a virus-specific immune response can be detected in mice immunized with the VSV-G pseudotyped replication-defective HIV.

Full Text	REPLICATION DEFECTIVE HIV VACCINE BACKGROUND OF THE INVENTION 1. Field of the Invention A method is described for making a replication- defective HIV virus particle. The present invention is also directed to a HIV virus particle produced according to the method and to a vaccine including the particle. 2. Description of the Related Art A variety of strategies have evolved in the pursuit of an effective HIV vaccine. One strategy is the use of attenuated viruses. Recent advances in SIV vaccine studies indicate that live attenuated vaccines provide the best protection against challenge with the pathogenic strain of SIV in vaccinated animals (Clements J.E. et al., Journal of Virology 69 (1995): 2737-2744; Daniel, M.D. et al., Science 258 (1992): 1938-1941; Stahl-Hennig C. et al., Journal of General Virology 77 (1996) : 2969-2981; Norley, S. et al., Journal of Virology 219 (1996): 195-205; Almond, N. et al., The Lancet 345 (1995): 1342-1344; Wyand, M.S. et al., Journal of Virology 70 (1996): 3724-3733). This animal model suggests that the protective immunity can be elicited by vaccination with attenuated viruses. However, there is a potential risk in using an attenuated virus as a vaccine in humans. This concern is strengthened by studies in which attenuated viruses are shown to cause disease in neonatal rhesus monkeys (Wyand, M.S. et al., Nature Medicine 3 (1997): 32-36; Baba, T.W. et al., Science 267 (1995): 1820-1825) and, further, by the high rate of reversion of some attenuated SIV strains inoculated in monkeys (Cohen, J., Science 278 (1997): 24-25). Humoral and cellular immune responses can be elicited in rhesus monkeys inoculated with a naked DNA vaccine. However, there is little or no protective immunity observed in vaccinated animals after challenge with SIV (Lu, S. et al., Journal of Virology 70 (1996): 3978-3991) . The mechanisms of protective immunity elicited by attenuated live vaccines in vaccinated animals have not been identified. Protective immunity may be due to the continuous expression of viral proteins from the persisting viral genome in the vaccinated monkeys, to the expression of all (or part of) the SIV proteins from endogenous pathways, or to other aspects (e.g., viral replication site) of the immune response to the live, attenuated virus. United States Patent No. 5,571,712, to Haynes et al. discloses replication-defective HIV virus particles which do not include retroviral RNA. These retroviral particles are acceptable for safety reasons due to their inability to replicate. However, these particles differ from vaccines capable of replication in their lesser ability to elicit protective immunity. Thus, it is expected that larger numbers of particles must be administered to a subject in a course of vaccination. Further, lack of expression of viral proteins in a vaccinated subject will affect the quality of the protective immunity. Other vaccines are currently under development, which include peptide vaccines, DNA vaccines, multi-valent virus (i.e., recombinant canarypox vaccines) and combination vaccines (i.e., a DNA vaccine combined with a fowlpox booster, vaccine) with varying results. (Gold, D., IAVI Report 3 (1998): 1+.) The efficacy of HIV vaccines is, generally, difficult to evaluate due to the lack of readily accessible animal models (Girard, M. et al., Virology 232 (1997): 98- 104). Therefore, it is most desirable to have a replication-defective virus particle with broad tropism, both with regard to range of species it will infect and with regard to the cell types it will infect in a given subject. It is also highly desirable to have a replication-defective virus particle which can produce antigenic HIV proteins in levels and for a duration suitable to elicit a protective immune response, with both cellular and humoral components. These goals have been achieved in the present invention, a replication-defective HIV vaccine pseudotyped with vesicular stomatitis virus G (VSV-G) protein. SUMMARY OF THE INVENTION A method is provided for producing a replication- defective retrovirus particle, including the steps of providing a DNA molecule which includes a complete retroviral genome; modifying a portion of the pol gene of the DNA molecule including the protease and reverse transcriptase activity coding regions to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome, thereby forming a pol- construct; transferring the pol- construct into a suitable host cell; prior to, during or after the step of transferring the pol- construct into the host cell, transferring into the host cell a pseudotyping construct and a packaging construct; growing the host cell under conditions suitable for expression of the constructs and for production of replication-defective retrovirus particles; and collecting the virus particles. The retroviral genome is preferably an HIV virus genome and most preferably an HIV-1 genome. In one embodiment, the pol- construct is plasmid pHXB2 with nucleotides 2621-4552 deleted. The method may further include the step of modifying the retroviral genome to represent protein sequence variations in immunologically variant retroviruses. The pseudotyping construct is preferably a construct for expressing the vesicular stomatitis virus G protein. The pol- construct, the packaging construct and the pseudotyping construct may be co-transfected or, alternately, at least one of the pseudotyping construct and the packaging construct are carried stably in the genome of the host cell. The present invention further includes a replication-defective retrovirus particle, including a retroviral genome in which a portion of the pol gene including the protease and reverse transcriptase activity coding regions is modified to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome. As described above in connection with the method embodiment, the retroviral genome can be an HIV genome and, preferably, an HIV-1 genome. In one embodiment, the retroviral genome is a pol- construct that is derived from plasmid pHXB2 with nucleotides 2621-4552 thereof deleted. The pseudotyping protein may be the vesicular stomatitis virus G protein. The present invention further includes a vaccine including the above-described virus particle in its variously described embodiment in combination with a pharmaceutically and/or veterinarilly suitable excipient. The excipient can include mono-, di-, oligo- and polysaccharides, viscosity enhancing and/or tackifying agents, polymers, block polymers and cross-linked polymers. The vaccine can be lyophilized for use in reconstituted form or in immunization by scarification. Further, the vaccine can be formulated into a form selected from the group consisting of an oral liquid, an oral capsule, a liquid for parenteral injection, a transdermal or transmucosal device and a suppository and includes a suitable excipient for preparation of the selected form. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic representation of the three plasmid expression system used for generating a VSV-G pseudotyped HIV-1 vector. The Ball restriction site was used to truncate the pol gene. The arrows indicated the primer sites for PCR analysis; Figs. 2A-C are micrographs showing transduction of sMAGI cells with HIVpol-/VSV-G. Three days after transduction, cells were stained with X-gal. Fig 2A shows cells infected with wild-type SIVmac 239, as a positive control; Fig 2B shows HIVpol"/VSV-G infected cells; and Fig 2C shows mock-infected cells; Fig. 3 is an autoradiograph of the immunoprecipitation of pseudotyped viruses. 35S labeled cell-free viruses were immunoprecipitated with anti-HIV-1 serum. Lane 1: mock-transfected cells, lane 2: cells transfected with pHIVpol- and pCMVAR8.2 constructs, lane 3: cells transfected with three plasmids. Molecular weight markers are indicated on the left. Fig. 4 is autoradiograph of the immunoprecipitation of 293T cells transduced with HIVpol" /VSV-G. Transduced (lane 1) and untransduced (lane 2) 293 T cells were immunoprecipitated with anti-HIV-1 serum; Figs. 5A and 5B are photographs of agarose gels showing PCR analysis of HIVpol"/VSV-G transduced CEM174 cells. CEM174 cells were transduced with HIVpol"/VSV-G (Fig. 5A) or were cultured with second round cell-free medium (Fig. 5B); Fig. 5A, lane 1: negative control for PCR (no template DNA), lane 2: mock-transduced CEM174 cells, lane 3: HIVpol"/VSV-G transduced CEM174 cells, lane 4: positive control of PCR (100 pg pHIVpol"), lane 5: positive control of PCR (1 ng of pHIVHxB2). A 0.7 kb band indicated the deletion in the pol gene as compared to the wild-type 3 kb band; Fig. 5B, a nested PCR analysis of DNA extracted from CEM174. Lane 1: negative control of PCR (no template DNA), lane 2: mock transduced cells, lane 3: second round cell-free medium cultured cells, lane 4: HIVIIIB-infected cells, and lanes on the right are HIV plasmid DNA with indicated copies number. The one kb fragment indicates the pol gene product. M: 1 kb size marker (BRL/Life Tech.); Fig. 6 is a graph showing the results of a standard chromium release assay. Autologous B cells from HIV infected individuals were infected with HIVpol"/VSV-G (MOI=0.1) or vaccinia vectors expressing gag (rVV-gag) or env (rVV-env) genes (M0I=5) as target cells one day before assay; and Fig. 7 is a photograph of a Western blot analysis showing antibody responses in mice immunized with VSV-G pseudotyped HIVpol" viruses. Lane 1: positive control, lane 2: prebleed sera, and lane 3: representative of immunized mice sera. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is a method for producing a replication-defective HIV virus particle, a virus particle prepared according to the method and to a vaccine including the virus particles. The virus particles exhibit increased ability to produce antigen, expanded cell tropism and the ability to elicit strong humoral and cellular immunity. As a result of the expanded tropism, the virus particles can infect rhesus monkeys in a single cycle of replication. Thus, the protective immunity can be further evaluated in rhesus monkeys by challenge with an SIV/HIV chimera virus (SHIV). The method of the present invention is a method for preparing a replication-defective retroviral particle including the steps of 1) preparing a DNA molecule which includes a complete retroviral genome; 2) modifying a portion of the pol gene including the protease and reverse transcriptase coding regions, to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome (the pol- construct); 3) transferring the pol- construct into a suitable host cell prior to, along with or after the step of transferring a pseudotyping construct and a packaging construct into the host cell or a predecessor cell or a progeny cell thereof; 4) growing the host cell under conditions suitable for expression of the constructs and for production of the replication-defective retrovirus particle; and 5) collecting the virus particles. The DNA molecule can be prepared from any DNA containing a complete retroviral genome. In theory, a genome suitable for preparing the pol- construct is any substantially complete retroviral genome which is intact, and encodes and is capable of expressing all viral proteins other than those inactivated by the described modification of the pol gene, whether or not one or more of the viral proteins are modified or unmodified as compared to the wild-type retrovirus. It later may be found that expression of certain viral proteins (non-pol proteins) are unnecessary to elicit protective immunity. A genome having these viral proteins modified or deleted could be a suitable genome for modification according to the methods of the present invention. According to the present invention, the pol gene of the retroviral genome is modified to cause the protease and the reverse transcriptase functions of the pol gene to lose their viral replication-associated functionality. This loss of function can be induced by a variety of methods, including: by insertions of amino acids, by replacement of amino acids and by deletions of amino acids. Preferably, a significant portion of the pol gene is deleted, as in the example, below. The packaging construct can be any construct which provides in trans all functions missing from the pol- construct to allow packaging of the pol- genome into a replication-defective viral particle which, when transduced, can achieve one round of viral replication. The packaging construct will at least encode a complete and functional reverse transcriptase for incorporation into the replication-defective virus particles and to copy the pol- genome into DNA once the replication-defective virus particle infects a target cell. The pseudotyping construct is, preferably, a recombinant construct for expressing the VSV-G protein. Expression of the protein is under transcriptional control of a suitable promoter. In the example below, the common constitutive CMV promoter is used. Other promoters, whether inducible or constitutive, are suitable so long as the construct encodes the VSV-G protein. Alternative pseudotyping proteins may be encoded by and expressed by the pseudotyping construct. An example of an alternative tropism-modifying protein is the Murine Leukemia Virus (MLV) envelope protein. The appropriate pseudotyping protein can be selected according to the application of the vaccine. The VSV-G protein is an appropriate pseudotyping protein for human or simian applications. Different pseudotyping proteins are appropriate for specific veterinary applications, such as if the virus particle is to serve as a Feline Leukemia Virus vaccine. The host cell for production of replication- defective retrovirus particles can be virtually any mammalian cell so long as the cell is capable of producing infectious virus particles according to the methods described herein. In the examples described below, the preferred, and widely available, 293 cells (human transformed embryonal kidney cells; ATCC CRL-1573, American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, U.S.A. 20852) are used as host cells to produce the HIV pol- particles. This cell line is particularly useful due to its ease of growth, its rapid growth characteristics and its ease of transformation by standard protocols. Cells with similar characteristics are particularly suited as host cells for preparation of the replication-defective retrovirus particles. The example provided below utilizes a three component transfection to produce virus particles. In the example, a first plasmid contains a DNA fragment corresponding to the pol" HIV genome ("the HIVpol- construct"). A second plasmid provides suitable functions in trans which are necessary for production of complete retroviral virus particles (the packaging construct). A third plasmid provides the genetic material for expression of the VSV-G protein (a VSV G-encoding or pseudotyping construct). Transfection of the host cell can be accomplished by a variety of methods, including, without limitation: calcium phosphate, liposome and electroporation methods. Virtually any transfection method is suitable, so long as the functionality of each construct is maintained. Further, transfer of the constructs into the host cell can be accomplished simultaneously, or in any desired order. It should be noted that, although the examples herein utilize plasmid DNA to provide the three constructs, other equivalent vector systems may be utilized to transfer the constructs to the host cell, including, for example and without limitation: linear DNA fragments, plasmids and other recombinant DNA vectors and recombinant virus genomes or particles. In the case of recombinant virus particles, the method for transferring the constructs to the host cell is by transduction with the virus particles. For instance, virus particles including the transfer vectors described in Naldini et al., Science 272 (1996): 263-267 would be suitable for delivery of one or both of the packaging construct and the VSV-G-encoding construct. Further, the replication-defective HIV virus particles of the present invention can be used as a suitable source of the particles to transduce the HIV pol- construct. Further, a host cell may be established having one or both of the packaging construct and the pseudotyping construct integrated permanently into the host cell DNA. Packaging cell lines for retroviruses are well known in the art. (Delwart, E.L. et al., Aids Research and Human Retroviruses 8 (1992): 1669-1677). The integrated packaging construct and pseudotyping construct may be under the control of a constitutive promoter such as the cytomegalovirus (CMV) promoter, as described in the examples, or under the control of an inducible promoter, allowing exact control of virus production and preventing interference with host cell growth during passage of the packaging cell line. Transfection inefficiencies are lessened by the integration of one or more of the constructs into the host cell genome. Virus particles prepared according to the above- described method include all components of a wild-type virus particle with the exception that the particles include, at least, the above-described modified (pol- retroviral) genome, as opposed to the wild-type RNA genome. Further, the virus particles preferably include the VSV-G (pseudotyping) protein in order to alter the tropism of the virus particles. Virus particles are prepared according to the above-described methods and are, then, collected by standard methodology. In the example provided herein, the virus particles are purified by sucrose gradient ultracentrifugation. Other methods for purification can be employed, such as affinity chromatography, so long as the method enables concentrate of the titer of the infectious replication-defective virus particles. In use, the replication-defective HIV virus particles are formulated as a vaccine. The only limitation is that the vaccine includes, in addition to the virus particles a pharmaceutically or veterinarilly acceptable excipient, which can include, without limitation: virus stabilizing compounds (i.e., sugars such as sucrose or other saccharides or polysaccharides); buffer systems (i.e., TNE or its equivalents); antibiotic compounds (i.e., bacteriocides, bacteriostatic agents and fungicides); viscosity enhancing agents (i.e., polyethylene glycol); polymeric materials (i.e., polymers, copolymers, block polymers and cross-linked polymers) for use as viscosity enhancing agents, as tackifying agents or as a matrix or reservoir for harboring the particles; tackifying agents which promote adhesion of the vaccine mixture to the skin or mucosa; viral transduction-enhancing agents; penetration enhancing agents; flavoring agents; coloring agents; adsorption enhancing agents and adjuvants. For example, as shown below, mice were immunized with virus particles in tissue culture media, which served as a suitable excipient. The vaccine can be prepared in many forms (and with an excipient suitable for each form), such as, without limitation: an oral liquid for either swallowing or for application to a surface of the mouth, including sublingual or buccal application; an oral capsule or tablet; a liquid for parenteral injection; a cream or ointment for topical application; a lyophilized product for reconstitution or for vaccination by scarification; a transdermal preparation; and a liquid or a suppository for rectal or intravaginal application. A "lyophilized product" is understood to include virus particles freeze-dried, lyophilized or vitrified in the presence of a suitable buffer system, such as TNE, and a suitable carrier, such as a sugar (i.e., trehalose or sucrose), a polysaccharide, or other compounds suitable for stabilizing labile biological compounds. The vaccine can also contain two or more pol~ variants of the same retrovirus. Therefore, a subject can be immunized once or multiple times with two or more variants of the virus which reflect common antigenic variations of HIV. Production of a variant virus would be straight-forward once the nucleotide sequence of the variant HIV is known. Appropriate modification of DNA encoding the replication-defective genome would be accomplished through well known cloning and mutagenesis methods. Example A. Materials and Methods 1. Plasmid constructs and virus production. An HIV-1 genome with a deletion in the Pol gene was constructed by deleting a 1.9 kb Ball fragment (2621- 4552 nt) of molecular clone pHXB2 (Ratner L. et. al, Nature 313 (1985): 277-284, GeneBank No. K03455). The resulting plasmid is called pHIVpol". A helper expression vector (pCMVAR8.2) encoding HIV-1 gag and pol genes was obtained. The packaging sequence (?) and long terminal repeat (LTR) of HIV-1 were deleted from this expression vector. Therefore, the viral RNA transcribed from this expression vector cannot be packaged into viral particles. Plasmid pCMVAR8.2 was constructed from an infectious molecular clone of proviral HIV-1, pR9 (D. Trono, M.B. Feinberg, D. Baltimore, Cell 59, 113, 1989). A 1.3 K base pair BgIII fragment (6308-7611) was deleted from the envelope gene. A 39 base pair internal deletion in the packaging signal sequence was introduced (A. Lever et al., Journal of Virology 63 (1989): 4085), and the 3'HIV long terminal repeat (LTR) was replaced with poly-A site of insulin genomic DNA. The 5'LTR and leader sequence of HIV were substituted with a 0.8 K base pair fragment containing the CMV promoter (Naldini, Science 272 (1996): 263-268) A VSV-G protein expression construct (pCMV.VSV.G) was obtained. Plasmid pCMV.VSV.G was constructed by inserting a 1.7k base pair fragment encoding the VSV.G protein (GeneBank No. X03633) into the BamHI site of pCMVneo. The pseudotyped virus was produced by co- transfecting these three plasmids into a highly transfectable cell line 293T, as shown in Fig. 1. About 106 293T cells were transfected with 5 µq of each plasmid by the calcium phosphate method. Seventy-two hours after transfection, cell-free virus supernatant ("HIVpol"/VSV-G") was harvested and stored at -80°C. Some viruses were concentrated by sucrose density gradient ultracentrifugation at 100,000 x g for 2 hours and were resuspended into TNE buffer (50 mM Tris.HCl pH 7.8, 130 mM NaCl, 1 mM EDTA). The viral titer was determined on MAGI and sMAGI cells as described. In brief, cells were transduced with serial dilutions of viruses and stained with 5-bromo-4-chloro-3-indolyl-li-D-galactopyranoside (X- Gal) after 3 days of incubation. 2. Transduction of sMAGI cell line. An indicator cell line, sMAGI (Chackerian, B. et al., Journal of Virology 71 (1997): 3932-3939), was used for the transduction assays. sMAGI cells are derived from macaque mammary tumor cells. They express human CD4 and encode an HIV LTR fused to the (3-galactosidase ((3-Gal) reporter gene. This cell line allows detection of productive infection by a single virus particle by exploiting the ability of the SIV or HIV Tat protein to transactivate the 3-Gal gene through the HIV LTR promoter. sMAGI cells were transduced with one ml of 1:1 medium- diluted virions in the presence of 8 µg/ml of polybrene. Transduced cells were incubated for 3 days at 37 °C before X-Gal staining. 3. Immunoprecipitation. Approximately 106 293T cells were transduced with 105 transducing units/ml HIVpol7VSV-G. Forty-eight hours later, transduced cells were labeled with 35S methionine for 16 hours. A cellular lysate of transduced cells was immunoprecipitated with anti-HIV serum from AIDS patients. For analysis of pseudotyped virus particles, 106 293T cells were transfected with three plasmids (pHIVpol", pCMVAR8.2, and pVSV-G) or two plasmids (without pVSV-G). Twenty-four hours later, transfected cells were labeled with 35S methionine for 16-24 hours. The cell-free medium was then immunoprecipitated with anti-HIV serum. 4. PCR analysis. Total DNA was extracted from about 105 CEM174 cells transduced with 1 ml of cell-free virus and subjected to PCR analysis of the pol gene with primers P1 and P3 (see Fig. 1). To monitor the replication-competent virus in the virus preparation, the cell-free medium harvested from one week culture of the transduced CEM17 4 cells was used to culture CEM174 cells again for another week. The total DNA was isolated from the second CEM174 cell culture and was subjected to a sensitive nested PCR analysis of pol gene with primers PI and P2 (Tung, F. T., Serodiagnosis and Immunotherapy 6 (1994): 218-220). 5. Cytotoxic T lymphocyte assay. Immortalized B cells from HIV infected individuals were transduced with HIVpol"/VSV-G at MOI = 0.1 and used as target cells in a standard chromium release assay (Rinaldo, C. et al., Journal of Virology 69 (1995): 5838-5842). B cells infected with vaccinia virus vectors (MOI= 5) expressing HIV-1 proteins were used as a control. Different ratios of effector cells were added to 104 B cells for each assay. 6. Immunization of mice. BALB/c mice were immunized intramuscularly with 0.1 ml (approximately 20ng of p24) of cell-free pseudotyped viral supernatant (titer of 105 transducing units TU/ml) at 7-10 day intervals for a total of 5 times. Sera were collected (retro-orbital venous plexus during Metophane- induced anesthesia) and tested (1:100 dilution) using an HIV-1 Western blot kit (BioRad, CA). Prebleed mouse sera and HIV seropositive sera were used as controls. Alkaline phosphatase conjugated goat anti-mouse antibody (Sigma, St. Louis, MO) was used as the second antibody for mouse serum samples. Alkaline phosphatase conjugated goat anti-human antibody (included in the kit) was used as the second antibody for positive control. B. Results 1. Construction of plasmid pHIVpol". pHIVpol" was constructed by deleting a 1.9 kb Ball fragment of the pol gene of an HIV-1 genome and was self- ligated by T4 ligase. pHIVpol" encodes all HIV-1 genes including a truncated pol gene. This construct cannot generate infectious viruses when transfected into CEM174 cells (data not shown). 2. Production of replication-defective HIV. Cell-free virus stocks were produced by co- transfecting three plasmids (or without pVSV-G) into a highly transfectable cell line 293T (Fig. 1). The cell- free viruses were then used to transduce sMAGI cells to assess virus infectivity. The sMAGI cells can only be infected with SIV; none of the HIV-1 strains (macrophage or T-cell tropic) tested to date can establish productive infection (data not shown) (Chackerian, B. : 3932-3939). The HIVpol"/VSV-G produced from co-transfection of the three plasmids can infect sMAGI, as shown in Figs. 2A-C. When any one of the three plasmids was omitted in co- transfection to produce pseudotyped virus, no productive infection was established in sMAGI cells (data not shown). 3. The titer of HIVpol"/VSV-G can be concentrated by ultracentrifugation. The titer of HIVpol"/VSV-G was determined on sMAGI and MAGI cells (Table 1). Like sMAGI cells, MAGI cells were derived from human HeLa CD4 cells encoding an LTR-driven fi-gal gene (Kimpton, J. et al., Journal of Virology 66 (1992): 2232-2239). A viral titer of 2-4xl05 transduction units/ml, which is higher than that of HIV-1 cultured in CEM cells, can be prepared routinely from transient transfected cells without concentration. Virus particles can be concentrated by sucrose density gradient ultracentrifugation without losing infectivity (>95% recovery). The virus was quite stable even after two freeze-thaw cycles. 4. The formation of pseudotyped viral particles. To confirm the formation of pseudotyped virus, the HIVpol-/VSV-G pseudotyped viral particles were analyzed by immunoprecipitation. About 106 293T cells were co- transfected with the three plasmids (or without pVSV-G). The transfected cells were labeled with 35S methionine for 24 hours. Cell-free medium was immunoprecipitated with anti-serum from an AIDS patient. The results indicated that both the HIV-1 gag and env were precipitated with anti-HIV-1 antiserum in pseudotyped and non-pseudotyped viruses (Fig. 3). However, in the presence of VSV-G protein, fewer env proteins were precipitated, suggesting that VSV-G protein is competing with env for incorporation into the pseudotyped viruses. 5. Transduction of HIVpol"/VSV-G into CD4 and non-CD4 cells. To confirm that HIVpol"/VSV-G can express HIV-1 genes in transduced cell lines, HeLa-CD4 clone 1022 (CD4+) and 293T(CD4_) cells were transduced with HIVpolWSV-G. Significant numbers of multinuclear syncytia were observed in HeLa-CD4 clone 1022 cells 48 hours after transduction with HIVpol"/VSV-G (data not shown), suggesting a high level of env expression. The expression of HIVpol"/VSV-G transduced HIV genes was further analyzed by immunoprecipitation in 293T cells. The results indicated that gag (p50, precursor Gag) and env (gpl60/120) proteins were detected in the pseudotyped virus-transduced 293T cells (Fig. 4). The gag cannot be processed into p24 and pl7 because no viral protease activity is present in the HIVpolWSV-G transduced 293T cells. 6. PCR analysis of transduced cells and replication- competent virus. To further verify the presence of HIV-1 genes in the pseudotyped virus-transduced cells, PCR analysis of total DNA isolated from HIVpol'/VSV-G transduced CEM174 cells was positive for the truncated pol gene (Fig. 5A). A 0.7 kb PCR fragment indicated the cells were transduced with the pol deletion virus. To demonstrate the absence of replication-competent virus in the pseudotyped virus preparation, cell-free culture medium from a one-week culture of the transduced CEM174 cells was used to culture CEM174 cells for another week. The two-week culture medium was checked for p24 and for reverse transcriptase activity. The results indicated no viral activity in the two-week culture medium. The CEM174 cells from the two-week culture were also checked for provirus sequence by a sensitive nested PCR. This nested PCR can detect less than 10 copies of provirus. The results indicated that no provirus was detected in the second round of infection, suggesting no replication-competent virus in the viral preparation (Fig. 5B). 7. HIV pseudotyped virus transduced B cells can function as target cells for cytotoxic T lymphocytes. To check the HIV-1 protein expression in pseudotyped virus transduced primary cells, immortalized B cells from AIDS patients were transduced with HIVpol"/VSV-G and were used as target cells in a standard chromium release assay. Autologous T cells recognized and lysed the HIVpolVVSV-G transduced B cells with an efficiency equal to (or better than) B cells infected with vaccinia vectors expressing HIV-1 genes (Fig. 6). Thirty percent specific lysis was observed at the effector-to-target cell ratio of 20:1. These data suggest that the HIV-1 protein can be expressed and processed normally in transduced primary B cells. 8. Antibody responses in immunized mice. To demonstrate the immunogenicity of pseudotyped viruses, BALB/c mice were immunized with cell-free viruses and antibody responses were checked by Western blot. The results indicated that weak but clearly visible bands appeared in Western blot assay (Fig. 7). Three major viral proteins (p24, p50 and gpl60) were detected two months after immunization. Prebleed sera were negative. These data indicated that cell-free viral supernatant is immunogenic in the vaccinated mice. Lyophilization Cell-free virus supernatant (HIVpol-/VSV-G) was prepared as described above, in 293T cells in DMEM with 10% fetal bovine serum. One (1) ml of cell-free culture supernatant was dispensed into a 1.5 ml plastic tube and the sample was stored for about two months at -80°C. The frozen sample containing the pseudotyped virus particles was placed into a standard SpeedVac connected to a vacuum pump. The sample was dried in the SpeedVac for one hour under a vacuum. The lyophilized sample was assayed for infectivity as described above. Substantial virus infectivity was retained in the lyophilized sample. This data indicates that the pseudotyped virus particles are exceptionally stable as compared to native retrovirus particles. Further, typical retrovirus particles do not retain substantial infectivity after sucrose density gradient ultracentrifugation. That the pseudotyped virus particles survive both ultracentrifugation and lyophilization is unexpected and indicates that the HIVpol-/VSV-G is a superior vaccine product. The described replication-defective HIV-1 vector can establish only a single cycle of viral replication. Viral antigens were expressed in transduced cells and functioned as target cells for cytotoxic T lymphocytes from HIV infected individuals. Furthermore, an antibody response to HIV-1 has been demonstrated in mice immunized with the described replication-defective HIV. The replication-defective HIV-1 described herein provides a safe vaccine. The replication defective virus presented herein encodes all HIV-1 proteins and a truncated pol gene. Because the important protease and reverse transcriptase activities are nonfunctional in the described construct, provirus cannot initiate a second round of replication. The VSV-G pseudotyped virus expands cell tropism to all primate species as well as expanding the types of cells which may be infected in a single animal or individual. Therefore, the safety and efficacy of the same vaccine preparations can be evaluated in monkeys, chimpanzees and humans. A recent report indicates that VSV-G pseudotyped HIV-1 not only can expand cell tropism but also can enhance infectivity by using a different receptor pathway (Aiken, C, Journal of Virology 71 (1997): 5871-5877). A unique feature of the replication-defective retroviral vector described herein is the absence of co- expression of foreign protein derived from the vector systems (e.g., adenovirus, poliovirus and vaccinia virus vectors) during the single round of replication. Therefore, an advantage over the attenuated retroviral and pox vaccines is that it is feasible to repeatedly immunize a subject in order to boost the immune response. It is believed that cell-mediated immunity plays a very important role in protective immunity of viral infection and is associated with long-term survival of HIV- 1 infected patients (Miller, M.D. et al., Journal of Immunology 144 (1989): 122-128; Kent, S.J. et al., Journal of Virology 70 (1996): 4941-4947). The class-I restricted antigen presenting pathway is responsible for cell-mediated immunity (Salk, Jonas et al., Science 260 (1993): 1270- 1272). It has been demonstrated that the live attenuated vaccines provided the best protective immunity against SIV infection in vaccinated animals. In that system, the protective immunity could be elicited due to cell-mediated immune response as the result of endogenously expressed viral antigens (Johnson, R.P. et al., Journal of Virology 71 (1997): 7711-7718). The replication-defective HIV-1 vector described herein provides a safe vaccine candidate to express viral antigens endogenously. Despite the low MOI (0.1) of defective HIV that was used in the cytotoxic T lymphocyte assay, strong (30%) specific lysis was observed in the chromium release assay. These data suggest either high level of viral protein expression or high efficiency of transduction in primary B lymphocytes. Furthermore, the efficacy of protective immunity can be further tested in the rhesus monkeys and in other nonhuman primates. It is not clear which viral antigen elicits the protective immunity in attenuated viruses vaccinated animals. Using replication-defective HIV-1 vectors with different deletions (e.g., gag, gag-pol, env), the viral factors contributing to the protective immunity (cellular or humoral immune responses) can be assessed in this system. Because viral antigens (about 200 ng/ml p24) are present in the viral supernatants used for the immunization, it is not absolutely certain whether the antibody response elicited in mice is due to soluble viral antigens or endogenously expressed viral proteins. Due to the fact that no adjuvant was used for immunization and little (total of 100 ng p24 per mouse) viral supernatant (antigens) was inoculated into the mice, the antibody responses were likely elicited by the transduced cells. It is intriguing and unexpected that defective HIV particles cannot establish productive infection in MAGI cells when the VSV-G protein is omitted from the viral particles. Since all the viral structure proteins are present in the co-transfected cells, this phenomenon suggests that VSV-G protein is much more efficient than envelope protein for viral infectivity in this assay system. The above invention has been described with reference to the preferred embodiment. Obvious modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the invention be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. REFERENCES 1. Clements J.E. et al., "Cross-protective Immune Responses Induced in Rhesus Macaques by Immunization with Attenuated Macrophage-Tropic Simian Immunodeficiency Virus." Journal of Virol 69 (1995): 2737-2744. 2. Daniel M.D. et al., "Protective Effects of a Live Attenuated SIV Vaccine with a Deletion in the nef Gene." Science 258 (1992): 1938-1941. 3. Stahl-Hennig C. et al., "Rapid Development of Vaccine Protection in Macaques by Live-Attenuated Simian Immunodeficiency Virus." Journal of General Virology 77 (1996): 2969-81. 4. Norley S. et al., "Protection from Pathogenic SIVmac Challenge Following Short-term Infection with a nef- Deficient Attenuated Virus." Virology 219 (1996): 195-205. 5. Almond N. et al., "Protection by Attenuated Simian Immunodeficiency Virus in Macaques Against Challenge with Virus-Infected Cells." Lancet 34 5 (1995): 1342-1344. 6. Wyand M. et al., "Vaccine Protection by a Triple Deletion Mutant of Simian Immunodeficiency Virus." Journal of Virology 70 (1996): 3724-3733. 7. Wyand M.S. et al., "Resistance of Neonatal Monkeys to Live Attenuated Vaccine Strains of Simian Immunodeficiency Virus." Nature Medicine 3 (1997): 32-36. 8. Baba T.W. et al., "Pathogenicity of Live, Attenuated SIV After Mucosal Infection of Neonatal Macaques." Science 267 (1995): 1820-1825. 9. Cohen J., "Weakened SIV Vaccines Still Kills." Science 278 (1997): 24-25. 10. Lu S. et al., "Simian Immunodeficiency Virus DNA Vaccine Trial in Macaques." Journal of Virology 70 (1996): 3978-3991. 11. Gold, D., "Geneva Overview-Vaccines at the 12th World AIDS Conference." IAVI Report 3 (1998): 1+. 12. Girard M. et al., "Challenge of Chimpanzees Immunized with a Recombinant Canarypox-HIV-1 Virus." Virology 232 (1997): 98-104. 13. Naldini L. et al., "In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector." Science 272 (1996): 263-267. 14. Delwart, E.L. et al., "Analysis of HIV-1 Envelope Mutants and Pseudotyping of Replication-Defective HIV-1 Vectors by Genetic Complementation." Aids Research and Human Retroviruses 8 (1992): 1669-1677. 15. Ratner, L. et al., "Complete Nucleotide Sequence of the AIDS Virus, HTLV-III." Nature 313 (1985): 277-284. 16. Chackerian B., "Human Immunodeficiency Virus Type 1 Coreceptors Participate in Postentry Stages in the Virus Replication Cycle and Function in Simian Immunodeficiency Virus Infection." Journal of Virology 71 (1997): 3932- 3939. 17. Tung F.T., "Detection of Human Immunodeficiency Virus Type 1 by a Highly Sensitive and Specific Polymerase Chain Reaction Method." Serodiaanosis and Immunotherapy 6 (1994): 218-220. 18. Rinaldo C et al., "High Levels of Anti-Human Immunodeficiency Virus Type l(HIV-l) Memory Cytotoxic T- Lymphocyte Activity and Low Viral Load Are Associated with Lack of Disease in HIV-i Infected Long-term Nonprogressor." Journal of Virology 69 (1995): 5838-5842. 19. Kimpton J. et al., "Detection of Replication- Competent and Pseudotyped Human Immunodeficiency Virus with a Sensitive Cell Line on the Basis of Activation of an Integrated Ji-Galactosidase Gene." Journal of Virology 66 (1992): 2232-2239. 20. Aiken C, "Pseudotyping Human Immunodeficiency Virus Type 1 (HIV-1) by the Glycoprotein of Vesicular Stomatitis Virus Targets HIV-1 Entry to an Endocytic Pathway and Suppresses Both the Requirement for nef and the Sensitivity to Cyclosporin A." Journal of Virology 71 (1997): 5871- 5877. 21. Miller M.D. et al., "The Gag Specific Cytotoxic T Lymphocyte in Rhesus Monkeys Infected with the Simian Immunodeficiency Virus of Macaques." Journal of Immunology 144 (1990): 122-128. 22. Kent S.J. et al., "Detection of Simian Immunodeficiency Virus. (SIV)-specific Cd8+ T Cells in Macaques Protected from SIV Challenge by Prior SIV Subunit Vaccination." Journal of Virology 70 (1996): 4941-4947. 23. Salk J. et al., "A Strategy for Prophylactic Vaccination Against HIV." Science 260 (1993): 1270-1272. 24. Johnson P.R. et al., "Induction of Vigorous Cytotoxic T-Lymphocyte Responses by Live Attenuated Simian Immunodeficiency Virus." Journal of Virology 71 (1997): 7711-7718. I claim: 1. A method for producing replication-defective retrovirus particles, comprising the steps of: a. providing a DNA molecule which includes a complete retroviral genome; b. modifying a portion of the pol gene of the DNA molecule including the protease and reverse transcriptase activity coding regions to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome, thereby forming a pol- construct; c. transferring the pol" construct into a suitable host cell; d. prior to, during or after the step of transferring the pol- construct into the host cell, transferring into the host cell or a predecessor cell or a progeny cell thereof, a pseudotyping construct and a packaging construct; e. growing the host cell under conditions suitable for expression of the constructs and for production of the replication-defective retrovirus particles; and f. collecting the replication-defective virus particles. 2. The method for producing replication-defective retrovirus particles as claimed in claim 1, wherein the retroviral genome is an HIV virus genome. 3. The method for producing replication-defective retrovirus particles as claimed in claim 1, wherein the retroviral genome is an HIV-1 virus genome. 4. The method for producing replication—defective retrovirus particles as claimed in claim 3, wherein the pol gene is modified by deleting a portion thereof. 5. The method for producing replication-defective retrovirus particles as claimed in claim 1, wherein the pol- construct is plasmid pHXB2 with nucleotides 2621-4552 thereof deleted. 6. The method for producing replication-defective retrovirus particles as claimed in claim 1, wherein the retroviral genome is a Feline Leukemia Virus genome. 7. The method for producing replication-defective retrovirus particles as claimed in claim 1, further comprising the step of modifying the retroviral genome to represent protein sequence variations in immunologically variant retroviruses. 8. The method for producing replication-defective retrovirus particles as claimed in claim 3, wherein the pseudotyping construct encodes the vesicular stomatitis virus G protein. 9. The method for producing replication-defective retrovirus particles as claimed in claim 1, wherein at least one of the pol- construct, the pseudotyping construct and the packaging construct are co- transfected. 10. The method for producing replication-defective retrovirus particles as claimed in claim 1, wherein the collecting step includes a step of concentrating the titer of the replication-defective virus particles. 11. A replication-defective retrovirus particle, comprising: a. a retroviral genome in which a portion of the pol gene including the protease and reverse transcriptase activity coding regions is modified to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome; and b. a pseudotyping protein. 12. The replication-defective retrovirus particle as claimed in claim 11, wherein the retroviral genome is an HIV genome. 13. The replication-defective retrovirus particle as claimed in claim 11, wherein the retroviral genome is an HIV-1 genome. 14. The replication-defective retrovirus particle as claimed in claim 10, wherein the pol gene is modified by deletion of nucleotide sequences thereof. 15. The replication-defective retrovirus particle as claimed in claim 11, wherein the retroviral genome is a pol- construct derived from plasmid pHXB2 with nucleotides 2621-4552 thereof deleted. 16. The replication-defective retrovirus particle as claimed in claim 13, wherein the pseudotyping protein is vesicular stomatitis virus G protein. 17. The replication-defective retrovirus particle as claimed in claim 11, wherein the particle includes a retroviral protein which is an immunological variant of a wild-type retroviral protein. 18. A vaccine comprising a virus particle as claimed in claim 9 in combination with a pharmaceutically and/or veterinarilly suitable excipient. 19. The vaccine as claimed in claim 18, wherein the excipient is selected from the group consisting of a buffer system, a mono-, di-, oligo- or polysaccharide, a viscosity enhancing or tackifying agent, a polymer, a copolymer, a block polymer and a cross-linked polymer. 20. The vaccine as claimed in claim 18, wherein the vaccine is lyophilized. 21. The vaccine as claimed in claim 18, wherein the vaccine is prepared in a form selected from the group consisting of an oral liquid, an oral capsule, a liquid for parenteral injection, a transdermal or transmucosal device and a suppository, and the excipient is a suitable excipient for preparation of the selected form. 22. The vaccine as claimed in claim 18, wherein the retroviral genome is an HIV-1 genome and the pseudotyping protein is VSV-G. 23. A vaccine as claimed in claim 18, further comprising a second replication-defective pol- virus particle representing an immunological variant of the virus particle. A replication-defective HIV particle pseudotyped with vesicular stomatitis virus G protein (VSV-G). The pol gene of the HIV genome in the particle is modified to inactivate the pol reverse transcriptase and protease activity. This pseudotyped HIV particle can infect many cell types, including human and simian cells, and only undergoes one round of replication. Furthermore, a virus-specific immune response can be detected in mice immunized with the VSV-G pseudotyped replication-defective HIV.

Full Text

REPLICATION DEFECTIVE HIV VACCINE
BACKGROUND OF THE INVENTION
1. Field of the Invention
A method is described for making a replication-
defective HIV virus particle. The present invention is
also directed to a HIV virus particle produced according to
the method and to a vaccine including the particle.
2. Description of the Related Art
A variety of strategies have evolved in the
pursuit of an effective HIV vaccine. One strategy is the
use of attenuated viruses. Recent advances in SIV vaccine
studies indicate that live attenuated vaccines provide the
best protection against challenge with the pathogenic
strain of SIV in vaccinated animals (Clements J.E. et al.,
Journal of Virology 69 (1995): 2737-2744; Daniel, M.D. et
al., Science 258 (1992): 1938-1941; Stahl-Hennig C. et al.,
Journal of General Virology 77 (1996) : 2969-2981; Norley,
S. et al., Journal of Virology 219 (1996): 195-205; Almond,
N. et al., The Lancet 345 (1995): 1342-1344; Wyand, M.S. et
al., Journal of Virology 70 (1996): 3724-3733). This
animal model suggests that the protective immunity can be
elicited by vaccination with attenuated viruses. However,
there is a potential risk in using an attenuated virus as
a vaccine in humans. This concern is strengthened by
studies in which attenuated viruses are shown to cause
disease in neonatal rhesus monkeys (Wyand, M.S. et al.,
Nature Medicine 3 (1997): 32-36; Baba, T.W. et al., Science
267 (1995): 1820-1825) and, further, by the high rate of
reversion of some attenuated SIV strains inoculated in
monkeys (Cohen, J., Science 278 (1997): 24-25).
Humoral and cellular immune responses can be
elicited in rhesus monkeys inoculated with a naked DNA
vaccine. However, there is little or no protective
immunity observed in vaccinated animals after challenge

with SIV (Lu, S. et al., Journal of Virology 70 (1996):
3978-3991) . The mechanisms of protective immunity elicited
by attenuated live vaccines in vaccinated animals have not
been identified. Protective immunity may be due to the
continuous expression of viral proteins from the persisting
viral genome in the vaccinated monkeys, to the expression
of all (or part of) the SIV proteins from endogenous
pathways, or to other aspects (e.g., viral replication
site) of the immune response to the live, attenuated virus.
United States Patent No. 5,571,712, to Haynes et
al. discloses replication-defective HIV virus particles
which do not include retroviral RNA. These retroviral
particles are acceptable for safety reasons due to their
inability to replicate. However, these particles differ
from vaccines capable of replication in their lesser
ability to elicit protective immunity. Thus, it is
expected that larger numbers of particles must be
administered to a subject in a course of vaccination.
Further, lack of expression of viral proteins in a
vaccinated subject will affect the quality of the
protective immunity.
Other vaccines are currently under development,
which include peptide vaccines, DNA vaccines, multi-valent
virus (i.e., recombinant canarypox vaccines) and
combination vaccines (i.e., a DNA vaccine combined with a
fowlpox booster, vaccine) with varying results. (Gold, D.,
IAVI Report 3 (1998): 1+.)
The efficacy of HIV vaccines is, generally,
difficult to evaluate due to the lack of readily accessible
animal models (Girard, M. et al., Virology 232 (1997): 98-
104). Therefore, it is most desirable to have a
replication-defective virus particle with broad tropism,
both with regard to range of species it will infect and
with regard to the cell types it will infect in a given
subject. It is also highly desirable to have a
replication-defective virus particle which can produce
antigenic HIV proteins in levels and for a duration
suitable to elicit a protective immune response, with both
cellular and humoral components. These goals have been
achieved in the present invention, a replication-defective
HIV vaccine pseudotyped with vesicular stomatitis virus G
(VSV-G) protein.
SUMMARY OF THE INVENTION
A method is provided for producing a replication-
defective retrovirus particle, including the steps of
providing a DNA molecule which includes a complete
retroviral genome; modifying a portion of the pol gene of
the DNA molecule including the protease and reverse
transcriptase activity coding regions to the extent that
the remaining pol gene cannot produce a protease and
reverse transcriptase capable of functioning in replication
of the genome, thereby forming a pol- construct;
transferring the pol- construct into a suitable host cell;
prior to, during or after the step of transferring the pol-
construct into the host cell, transferring into the host
cell a pseudotyping construct and a packaging construct;
growing the host cell under conditions suitable for
expression of the constructs and for production of
replication-defective retrovirus particles; and collecting
the virus particles.
The retroviral genome is preferably an HIV virus
genome and most preferably an HIV-1 genome. In one
embodiment, the pol- construct is plasmid pHXB2 with
nucleotides 2621-4552 deleted. The method may further
include the step of modifying the retroviral genome to
represent protein sequence variations in immunologically
variant retroviruses.
The pseudotyping construct is preferably a
construct for expressing the vesicular stomatitis virus G
protein. The pol- construct, the packaging construct and
the pseudotyping construct may be co-transfected or,
alternately, at least one of the pseudotyping construct and
the packaging construct are carried stably in the genome of
the host cell.
The present invention further includes a
replication-defective retrovirus particle, including a
retroviral genome in which a portion of the pol gene
including the protease and reverse transcriptase activity
coding regions is modified to the extent that the remaining
pol gene cannot produce a protease and reverse
transcriptase capable of functioning in replication of the
genome. As described above in connection with the method
embodiment, the retroviral genome can be an HIV genome and,
preferably, an HIV-1 genome. In one embodiment, the
retroviral genome is a pol- construct that is derived from
plasmid pHXB2 with nucleotides 2621-4552 thereof deleted.
The pseudotyping protein may be the vesicular stomatitis
virus G protein.
The present invention further includes a vaccine
including the above-described virus particle in its
variously described embodiment in combination with a
pharmaceutically and/or veterinarilly suitable excipient.
The excipient can include mono-, di-, oligo- and
polysaccharides, viscosity enhancing and/or tackifying
agents, polymers, block polymers and cross-linked polymers.
The vaccine can be lyophilized for use in reconstituted
form or in immunization by scarification. Further, the
vaccine can be formulated into a form selected from the
group consisting of an oral liquid, an oral capsule, a
liquid for parenteral injection, a transdermal or
transmucosal device and a suppository and includes a
suitable excipient for preparation of the selected form.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic representation of the three
plasmid expression system used for generating a VSV-G
pseudotyped HIV-1 vector. The Ball restriction site was
used to truncate the pol gene. The arrows indicated the
primer sites for PCR analysis;
Figs. 2A-C are micrographs showing transduction
of sMAGI cells with HIVpol-/VSV-G. Three days after
transduction, cells were stained with X-gal. Fig 2A shows
cells infected with wild-type SIVmac 239, as a positive
control; Fig 2B shows HIVpol"/VSV-G infected cells; and Fig
2C shows mock-infected cells;
Fig. 3 is an autoradiograph of the
immunoprecipitation of pseudotyped viruses. 35S labeled
cell-free viruses were immunoprecipitated with anti-HIV-1
serum. Lane 1: mock-transfected cells, lane 2: cells
transfected with pHIVpol- and pCMVAR8.2 constructs, lane 3:
cells transfected with three plasmids. Molecular weight
markers are indicated on the left.
Fig. 4 is autoradiograph of the
immunoprecipitation of 293T cells transduced with HIVpol"
/VSV-G. Transduced (lane 1) and untransduced (lane 2) 293
T cells were immunoprecipitated with anti-HIV-1 serum;
Figs. 5A and 5B are photographs of agarose gels
showing PCR analysis of HIVpol"/VSV-G transduced CEM174
cells. CEM174 cells were transduced with HIVpol"/VSV-G
(Fig. 5A) or were cultured with second round cell-free
medium (Fig. 5B);
Fig. 5A, lane 1: negative control for PCR (no
template DNA), lane 2: mock-transduced CEM174 cells, lane
3: HIVpol"/VSV-G transduced CEM174 cells, lane 4: positive
control of PCR (100 pg pHIVpol"), lane 5: positive control
of PCR (1 ng of pHIVHxB2). A 0.7 kb band indicated the
deletion in the pol gene as compared to the wild-type 3 kb
band;
Fig. 5B, a nested PCR analysis of DNA extracted
from CEM174. Lane 1: negative control of PCR (no template
DNA), lane 2: mock transduced cells, lane 3: second round
cell-free medium cultured cells, lane 4: HIVIIIB-infected
cells, and lanes on the right are HIV plasmid DNA with
indicated copies number. The one kb fragment indicates the
pol gene product. M: 1 kb size marker (BRL/Life Tech.);
Fig. 6 is a graph showing the results of a
standard chromium release assay. Autologous B cells from
HIV infected individuals were infected with HIVpol"/VSV-G
(MOI=0.1) or vaccinia vectors expressing gag (rVV-gag) or
env (rVV-env) genes (M0I=5) as target cells one day before
assay; and
Fig. 7 is a photograph of a Western blot analysis
showing antibody responses in mice immunized with VSV-G
pseudotyped HIVpol" viruses. Lane 1: positive control, lane
2: prebleed sera, and lane 3: representative of immunized
mice sera.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is a method for producing
a replication-defective HIV virus particle, a virus
particle prepared according to the method and to a vaccine
including the virus particles. The virus particles exhibit
increased ability to produce antigen, expanded cell tropism
and the ability to elicit strong humoral and cellular
immunity. As a result of the expanded tropism, the virus
particles can infect rhesus monkeys in a single cycle of
replication. Thus, the protective immunity can be further
evaluated in rhesus monkeys by challenge with an SIV/HIV
chimera virus (SHIV).
The method of the present invention is a method
for preparing a replication-defective retroviral particle
including the steps of 1) preparing a DNA molecule which
includes a complete retroviral genome; 2) modifying a
portion of the pol gene including the protease and reverse
transcriptase coding regions, to the extent that the
remaining pol gene cannot produce a protease and reverse
transcriptase capable of functioning in replication of the
genome (the pol- construct); 3) transferring the pol-
construct into a suitable host cell prior to, along with or
after the step of transferring a pseudotyping construct and
a packaging construct into the host cell or a predecessor
cell or a progeny cell thereof; 4) growing the host cell
under conditions suitable for expression of the constructs
and for production of the replication-defective retrovirus
particle; and 5) collecting the virus particles.
The DNA molecule can be prepared from any DNA
containing a complete retroviral genome. In theory, a
genome suitable for preparing the pol- construct is any
substantially complete retroviral genome which is intact,
and encodes and is capable of expressing all viral proteins
other than those inactivated by the described modification
of the pol gene, whether or not one or more of the viral
proteins are modified or unmodified as compared to the
wild-type retrovirus. It later may be found that
expression of certain viral proteins (non-pol proteins) are
unnecessary to elicit protective immunity. A genome having
these viral proteins modified or deleted could be a
suitable genome for modification according to the methods
of the present invention.
According to the present invention, the pol gene
of the retroviral genome is modified to cause the protease
and the reverse transcriptase functions of the pol gene to
lose their viral replication-associated functionality.
This loss of function can be induced by a variety of
methods, including: by insertions of amino acids, by
replacement of amino acids and by deletions of amino acids.
Preferably, a significant portion of the pol gene is
deleted, as in the example, below.
The packaging construct can be any construct
which provides in trans all functions missing from the pol-
construct to allow packaging of the pol- genome into a
replication-defective viral particle which, when
transduced, can achieve one round of viral replication.
The packaging construct will at least encode a complete and
functional reverse transcriptase for incorporation into the
replication-defective virus particles and to copy the pol-
genome into DNA once the replication-defective virus
particle infects a target cell.
The pseudotyping construct is, preferably, a
recombinant construct for expressing the VSV-G protein.
Expression of the protein is under transcriptional control
of a suitable promoter. In the example below, the common
constitutive CMV promoter is used. Other promoters,
whether inducible or constitutive, are suitable so long as
the construct encodes the VSV-G protein. Alternative
pseudotyping proteins may be encoded by and expressed by
the pseudotyping construct. An example of an alternative
tropism-modifying protein is the Murine Leukemia Virus
(MLV) envelope protein. The appropriate pseudotyping
protein can be selected according to the application of the
vaccine. The VSV-G protein is an appropriate pseudotyping
protein for human or simian applications. Different
pseudotyping proteins are appropriate for specific
veterinary applications, such as if the virus particle is
to serve as a Feline Leukemia Virus vaccine.
The host cell for production of replication-
defective retrovirus particles can be virtually any
mammalian cell so long as the cell is capable of producing
infectious virus particles according to the methods
described herein. In the examples described below, the
preferred, and widely available, 293 cells (human
transformed embryonal kidney cells; ATCC CRL-1573, American
Type Culture Collection, 12301 Parklawn Drive, Rockville,
MD, U.S.A. 20852) are used as host cells to produce the
HIV pol- particles. This cell line is particularly useful
due to its ease of growth, its rapid growth characteristics
and its ease of transformation by standard protocols.
Cells with similar characteristics are particularly suited
as host cells for preparation of the replication-defective
retrovirus particles.
The example provided below utilizes a three
component transfection to produce virus particles. In the
example, a first plasmid contains a DNA fragment
corresponding to the pol" HIV genome ("the HIVpol-
construct"). A second plasmid provides suitable functions
in trans which are necessary for production of complete
retroviral virus particles (the packaging construct). A
third plasmid provides the genetic material for expression
of the VSV-G protein (a VSV G-encoding or pseudotyping
construct). Transfection of the host cell can be
accomplished by a variety of methods, including, without
limitation: calcium phosphate, liposome and electroporation
methods. Virtually any transfection method is suitable, so
long as the functionality of each construct is maintained.
Further, transfer of the constructs into the host cell can
be accomplished simultaneously, or in any desired order.
It should be noted that, although the examples
herein utilize plasmid DNA to provide the three constructs,
other equivalent vector systems may be utilized to transfer
the constructs to the host cell, including, for example and
without limitation: linear DNA fragments, plasmids and
other recombinant DNA vectors and recombinant virus genomes
or particles. In the case of recombinant virus particles,
the method for transferring the constructs to the host cell
is by transduction with the virus particles. For instance,
virus particles including the transfer vectors described in
Naldini et al., Science 272 (1996): 263-267 would be
suitable for delivery of one or both of the packaging
construct and the VSV-G-encoding construct. Further, the
replication-defective HIV virus particles of the present
invention can be used as a suitable source of the particles
to transduce the HIV pol- construct.
Further, a host cell may be established having
one or both of the packaging construct and the pseudotyping
construct integrated permanently into the host cell DNA.
Packaging cell lines for retroviruses are well known in the
art. (Delwart, E.L. et al., Aids Research and Human
Retroviruses 8 (1992): 1669-1677). The integrated
packaging construct and pseudotyping construct may be under
the control of a constitutive promoter such as the
cytomegalovirus (CMV) promoter, as described in the
examples, or under the control of an inducible promoter,
allowing exact control of virus production and preventing
interference with host cell growth during passage of the
packaging cell line. Transfection inefficiencies are
lessened by the integration of one or more of the
constructs into the host cell genome.
Virus particles prepared according to the above-
described method include all components of a wild-type
virus particle with the exception that the particles
include, at least, the above-described modified (pol-
retroviral) genome, as opposed to the wild-type RNA genome.
Further, the virus particles preferably include the VSV-G
(pseudotyping) protein in order to alter the tropism of the
virus particles. Virus particles are prepared according to
the above-described methods and are, then, collected by
standard methodology. In the example provided herein, the
virus particles are purified by sucrose gradient
ultracentrifugation. Other methods for purification can be
employed, such as affinity chromatography, so long as the
method enables concentrate of the titer of the infectious
replication-defective virus particles.
In use, the replication-defective HIV virus
particles are formulated as a vaccine. The only limitation
is that the vaccine includes, in addition to the virus
particles a pharmaceutically or veterinarilly acceptable
excipient, which can include, without limitation: virus
stabilizing compounds (i.e., sugars such as sucrose or
other saccharides or polysaccharides); buffer systems
(i.e., TNE or its equivalents); antibiotic compounds (i.e.,
bacteriocides, bacteriostatic agents and fungicides);
viscosity enhancing agents (i.e., polyethylene glycol);
polymeric materials (i.e., polymers, copolymers, block
polymers and cross-linked polymers) for use as viscosity
enhancing agents, as tackifying agents or as a matrix or
reservoir for harboring the particles; tackifying agents
which promote adhesion of the vaccine mixture to the skin
or mucosa; viral transduction-enhancing agents; penetration
enhancing agents; flavoring agents; coloring agents;
adsorption enhancing agents and adjuvants. For example, as
shown below, mice were immunized with virus particles in
tissue culture media, which served as a suitable excipient.
The vaccine can be prepared in many forms (and
with an excipient suitable for each form), such as, without
limitation: an oral liquid for either swallowing or for
application to a surface of the mouth, including sublingual
or buccal application; an oral capsule or tablet; a liquid
for parenteral injection; a cream or ointment for topical
application; a lyophilized product for reconstitution or
for vaccination by scarification; a transdermal
preparation; and a liquid or a suppository for rectal or
intravaginal application. A "lyophilized product" is
understood to include virus particles freeze-dried,
lyophilized or vitrified in the presence of a suitable
buffer system, such as TNE, and a suitable carrier, such as
a sugar (i.e., trehalose or sucrose), a polysaccharide, or
other compounds suitable for stabilizing labile biological
compounds.
The vaccine can also contain two or more pol~
variants of the same retrovirus. Therefore, a subject can
be immunized once or multiple times with two or more
variants of the virus which reflect common antigenic
variations of HIV. Production of a variant virus would be
straight-forward once the nucleotide sequence of the
variant HIV is known. Appropriate modification of DNA
encoding the replication-defective genome would be
accomplished through well known cloning and mutagenesis
methods.
Example
A. Materials and Methods
1. Plasmid constructs and virus production.
An HIV-1 genome with a deletion in the Pol gene
was constructed by deleting a 1.9 kb Ball fragment (2621-
4552 nt) of molecular clone pHXB2 (Ratner L. et. al, Nature
313 (1985): 277-284, GeneBank No. K03455). The resulting
plasmid is called pHIVpol". A helper expression vector
(pCMVAR8.2) encoding HIV-1 gag and pol genes was obtained.
The packaging sequence (?) and long terminal repeat (LTR)
of HIV-1 were deleted from this expression vector.
Therefore, the viral RNA transcribed from this expression
vector cannot be packaged into viral particles.
Plasmid pCMVAR8.2 was constructed from an
infectious molecular clone of proviral HIV-1, pR9 (D.
Trono, M.B. Feinberg, D. Baltimore, Cell 59, 113, 1989).
A 1.3 K base pair BgIII fragment (6308-7611) was deleted
from the envelope gene. A 39 base pair internal deletion
in the packaging signal sequence was introduced (A. Lever
et al., Journal of Virology 63 (1989): 4085), and the 3'HIV
long terminal repeat (LTR) was replaced with poly-A site of
insulin genomic DNA. The 5'LTR and leader sequence of HIV
were substituted with a 0.8 K base pair fragment containing
the CMV promoter (Naldini, Science 272 (1996): 263-268)
A VSV-G protein expression construct (pCMV.VSV.G)
was obtained. Plasmid pCMV.VSV.G was constructed by
inserting a 1.7k base pair fragment encoding the VSV.G
protein (GeneBank No. X03633) into the BamHI site of
pCMVneo.
The pseudotyped virus was produced by co-
transfecting these three plasmids into a highly
transfectable cell line 293T, as shown in Fig. 1. About 106
293T cells were transfected with 5 µq of each plasmid by
the calcium phosphate method. Seventy-two hours after
transfection, cell-free virus supernatant ("HIVpol"/VSV-G")
was harvested and stored at -80°C. Some viruses were
concentrated by sucrose density gradient
ultracentrifugation at 100,000 x g for 2 hours and were
resuspended into TNE buffer (50 mM Tris.HCl pH 7.8, 130 mM
NaCl, 1 mM EDTA). The viral titer was determined on MAGI
and sMAGI cells as described. In brief, cells were
transduced with serial dilutions of viruses and stained
with 5-bromo-4-chloro-3-indolyl-li-D-galactopyranoside (X-
Gal) after 3 days of incubation.
2. Transduction of sMAGI cell line.
An indicator cell line, sMAGI (Chackerian, B. et
al., Journal of Virology 71 (1997): 3932-3939), was used
for the transduction assays. sMAGI cells are derived from
macaque mammary tumor cells. They express human CD4 and
encode an HIV LTR fused to the (3-galactosidase ((3-Gal)
reporter gene. This cell line allows detection of
productive infection by a single virus particle by
exploiting the ability of the SIV or HIV Tat protein to
transactivate the 3-Gal gene through the HIV LTR promoter.
sMAGI cells were transduced with one ml of 1:1 medium-
diluted virions in the presence of 8 µg/ml of polybrene.
Transduced cells were incubated for 3 days at 37 °C before
X-Gal staining.
3. Immunoprecipitation.
Approximately 106 293T cells were transduced with
105 transducing units/ml HIVpol7VSV-G. Forty-eight hours
later, transduced cells were labeled with 35S methionine for
16 hours. A cellular lysate of transduced cells was
immunoprecipitated with anti-HIV serum from AIDS patients.
For analysis of pseudotyped virus particles, 106 293T cells
were transfected with three plasmids (pHIVpol", pCMVAR8.2,
and pVSV-G) or two plasmids (without pVSV-G). Twenty-four
hours later, transfected cells were labeled with 35S
methionine for 16-24 hours. The cell-free medium was then
immunoprecipitated with anti-HIV serum.
4. PCR analysis.
Total DNA was extracted from about 105 CEM174
cells transduced with 1 ml of cell-free virus and subjected
to PCR analysis of the pol gene with primers P1 and P3
(see Fig. 1). To monitor the replication-competent virus
in the virus preparation, the cell-free medium harvested
from one week culture of the transduced CEM17 4 cells was
used to culture CEM174 cells again for another week. The
total DNA was isolated from the second CEM174 cell culture
and was subjected to a sensitive nested PCR analysis of pol
gene with primers PI and P2 (Tung, F. T., Serodiagnosis and
Immunotherapy 6 (1994): 218-220).
5. Cytotoxic T lymphocyte assay.
Immortalized B cells from HIV infected
individuals were transduced with HIVpol"/VSV-G at MOI = 0.1
and used as target cells in a standard chromium release
assay (Rinaldo, C. et al., Journal of Virology 69 (1995):
5838-5842). B cells infected with vaccinia virus vectors
(MOI= 5) expressing HIV-1 proteins were used as a control.
Different ratios of effector cells were added to 104 B cells
for each assay.
6. Immunization of mice.
BALB/c mice were immunized intramuscularly with
0.1 ml (approximately 20ng of p24) of cell-free pseudotyped
viral supernatant (titer of 105 transducing units TU/ml) at
7-10 day intervals for a total of 5 times. Sera were
collected (retro-orbital venous plexus during Metophane-
induced anesthesia) and tested (1:100 dilution) using an
HIV-1 Western blot kit (BioRad, CA). Prebleed mouse sera
and HIV seropositive sera were used as controls. Alkaline
phosphatase conjugated goat anti-mouse antibody (Sigma, St.
Louis, MO) was used as the second antibody for mouse serum
samples. Alkaline phosphatase conjugated goat anti-human
antibody (included in the kit) was used as the second
antibody for positive control.
B. Results
1. Construction of plasmid pHIVpol".
pHIVpol" was constructed by deleting a 1.9 kb Ball
fragment of the pol gene of an HIV-1 genome and was self-
ligated by T4 ligase. pHIVpol" encodes all HIV-1 genes
including a truncated pol gene. This construct cannot
generate infectious viruses when transfected into CEM174
cells (data not shown).
2. Production of replication-defective HIV.
Cell-free virus stocks were produced by co-
transfecting three plasmids (or without pVSV-G) into a
highly transfectable cell line 293T (Fig. 1). The cell-
free viruses were then used to transduce sMAGI cells to
assess virus infectivity. The sMAGI cells can only be
infected with SIV; none of the HIV-1 strains (macrophage
or T-cell tropic) tested to date can establish productive
infection (data not shown) (Chackerian, B. : 3932-3939).
The HIVpol"/VSV-G produced from co-transfection of the three
plasmids can infect sMAGI, as shown in Figs. 2A-C. When
any one of the three plasmids was omitted in co-
transfection to produce pseudotyped virus, no productive
infection was established in sMAGI cells (data not shown).
3. The titer of HIVpol"/VSV-G can be concentrated by
ultracentrifugation.
The titer of HIVpol"/VSV-G was determined on
sMAGI and MAGI cells (Table 1). Like sMAGI cells, MAGI
cells were derived from human HeLa CD4 cells encoding an
LTR-driven fi-gal gene (Kimpton, J. et al., Journal of
Virology 66 (1992): 2232-2239). A viral titer of 2-4xl05
transduction units/ml, which is higher than that of HIV-1
cultured in CEM cells, can be prepared routinely from
transient transfected cells without concentration. Virus
particles can be concentrated by sucrose density gradient
ultracentrifugation without losing infectivity (>95%
recovery). The virus was quite stable even after two
freeze-thaw cycles.
4. The formation of pseudotyped viral particles.
To confirm the formation of pseudotyped virus,
the HIVpol-/VSV-G pseudotyped viral particles were analyzed
by immunoprecipitation. About 106 293T cells were co-
transfected with the three plasmids (or without pVSV-G).
The transfected cells were labeled with 35S methionine for
24 hours. Cell-free medium was immunoprecipitated with
anti-serum from an AIDS patient. The results indicated
that both the HIV-1 gag and env were precipitated with
anti-HIV-1 antiserum in pseudotyped and non-pseudotyped
viruses (Fig. 3). However, in the presence of VSV-G
protein, fewer env proteins were precipitated, suggesting
that VSV-G protein is competing with env for incorporation
into the pseudotyped viruses.
5. Transduction of HIVpol"/VSV-G into CD4 and non-CD4
cells.
To confirm that HIVpol"/VSV-G can express HIV-1
genes in transduced cell lines, HeLa-CD4 clone 1022 (CD4+)
and 293T(CD4_) cells were transduced with HIVpolWSV-G.
Significant numbers of multinuclear syncytia were observed
in HeLa-CD4 clone 1022 cells 48 hours after transduction
with HIVpol"/VSV-G (data not shown), suggesting a high level
of env expression. The expression of HIVpol"/VSV-G
transduced HIV genes was further analyzed by
immunoprecipitation in 293T cells. The results indicated
that gag (p50, precursor Gag) and env (gpl60/120) proteins
were detected in the pseudotyped virus-transduced 293T
cells (Fig. 4). The gag cannot be processed into p24 and
pl7 because no viral protease activity is present in the
HIVpolWSV-G transduced 293T cells.
6. PCR analysis of transduced cells and replication-
competent virus.
To further verify the presence of HIV-1 genes in
the pseudotyped virus-transduced cells, PCR analysis of
total DNA isolated from HIVpol'/VSV-G transduced CEM174
cells was positive for the truncated pol gene (Fig. 5A).
A 0.7 kb PCR fragment indicated the cells were transduced
with the pol deletion virus. To demonstrate the absence of
replication-competent virus in the pseudotyped virus
preparation, cell-free culture medium from a one-week
culture of the transduced CEM174 cells was used to culture
CEM174 cells for another week. The two-week culture medium
was checked for p24 and for reverse transcriptase activity.
The results indicated no viral activity in the two-week
culture medium. The CEM174 cells from the two-week culture
were also checked for provirus sequence by a sensitive
nested PCR. This nested PCR can detect less than 10 copies
of provirus. The results indicated that no provirus was
detected in the second round of infection, suggesting no
replication-competent virus in the viral preparation (Fig.
5B).
7. HIV pseudotyped virus transduced B cells can
function as target cells for cytotoxic T lymphocytes.
To check the HIV-1 protein expression in
pseudotyped virus transduced primary cells, immortalized B
cells from AIDS patients were transduced with HIVpol"/VSV-G
and were used as target cells in a standard chromium
release assay. Autologous T cells recognized and lysed the
HIVpolVVSV-G transduced B cells with an efficiency equal to
(or better than) B cells infected with vaccinia vectors
expressing HIV-1 genes (Fig. 6). Thirty percent specific
lysis was observed at the effector-to-target cell ratio of
20:1. These data suggest that the HIV-1 protein can be
expressed and processed normally in transduced primary B
cells.
8. Antibody responses in immunized mice.
To demonstrate the immunogenicity of pseudotyped
viruses, BALB/c mice were immunized with cell-free viruses
and antibody responses were checked by Western blot. The
results indicated that weak but clearly visible bands
appeared in Western blot assay (Fig. 7). Three major viral
proteins (p24, p50 and gpl60) were detected two months
after immunization. Prebleed sera were negative. These
data indicated that cell-free viral supernatant is
immunogenic in the vaccinated mice.
Lyophilization
Cell-free virus supernatant (HIVpol-/VSV-G) was
prepared as described above, in 293T cells in DMEM with 10%
fetal bovine serum. One (1) ml of cell-free culture
supernatant was dispensed into a 1.5 ml plastic tube and
the sample was stored for about two months at -80°C. The
frozen sample containing the pseudotyped virus particles
was placed into a standard SpeedVac connected to a vacuum
pump. The sample was dried in the SpeedVac for one hour
under a vacuum. The lyophilized sample was assayed for
infectivity as described above. Substantial virus
infectivity was retained in the lyophilized sample.
This data indicates that the pseudotyped virus
particles are exceptionally stable as compared to native
retrovirus particles. Further, typical retrovirus
particles do not retain substantial infectivity after
sucrose density gradient ultracentrifugation. That the
pseudotyped virus particles survive both
ultracentrifugation and lyophilization is unexpected and
indicates that the HIVpol-/VSV-G is a superior vaccine
product.
The described replication-defective HIV-1 vector
can establish only a single cycle of viral replication.
Viral antigens were expressed in transduced cells and
functioned as target cells for cytotoxic T lymphocytes from
HIV infected individuals. Furthermore, an antibody
response to HIV-1 has been demonstrated in mice immunized
with the described replication-defective HIV.
The replication-defective HIV-1 described herein
provides a safe vaccine. The replication defective virus
presented herein encodes all HIV-1 proteins and a truncated
pol gene. Because the important protease and reverse
transcriptase activities are nonfunctional in the described
construct, provirus cannot initiate a second round of
replication. The VSV-G pseudotyped virus expands cell
tropism to all primate species as well as expanding the
types of cells which may be infected in a single animal or
individual. Therefore, the safety and efficacy of the same
vaccine preparations can be evaluated in monkeys,
chimpanzees and humans. A recent report indicates that
VSV-G pseudotyped HIV-1 not only can expand cell tropism
but also can enhance infectivity by using a different
receptor pathway (Aiken, C, Journal of Virology 71 (1997):
5871-5877). A unique feature of the replication-defective
retroviral vector described herein is the absence of co-
expression of foreign protein derived from the vector
systems (e.g., adenovirus, poliovirus and vaccinia virus
vectors) during the single round of replication.
Therefore, an advantage over the attenuated retroviral and
pox vaccines is that it is feasible to repeatedly immunize
a subject in order to boost the immune response.
It is believed that cell-mediated immunity plays
a very important role in protective immunity of viral
infection and is associated with long-term survival of HIV-
1 infected patients (Miller, M.D. et al., Journal of
Immunology 144 (1989): 122-128; Kent, S.J. et al., Journal
of Virology 70 (1996): 4941-4947). The class-I restricted
antigen presenting pathway is responsible for cell-mediated
immunity (Salk, Jonas et al., Science 260 (1993): 1270-
1272). It has been demonstrated that the live attenuated
vaccines provided the best protective immunity against SIV
infection in vaccinated animals. In that system, the
protective immunity could be elicited due to cell-mediated
immune response as the result of endogenously expressed
viral antigens (Johnson, R.P. et al., Journal of Virology
71 (1997): 7711-7718). The replication-defective HIV-1
vector described herein provides a safe vaccine candidate
to express viral antigens endogenously. Despite the low MOI
(0.1) of defective HIV that was used in the cytotoxic T
lymphocyte assay, strong (30%) specific lysis was observed
in the chromium release assay. These data suggest either
high level of viral protein expression or high efficiency
of transduction in primary B lymphocytes. Furthermore, the
efficacy of protective immunity can be further tested in
the rhesus monkeys and in other nonhuman primates.
It is not clear which viral antigen elicits the
protective immunity in attenuated viruses vaccinated
animals. Using replication-defective HIV-1 vectors with
different deletions (e.g., gag, gag-pol, env), the viral
factors contributing to the protective immunity (cellular
or humoral immune responses) can be assessed in this
system. Because viral antigens (about 200 ng/ml p24) are
present in the viral supernatants used for the
immunization, it is not absolutely certain whether the
antibody response elicited in mice is due to soluble viral
antigens or endogenously expressed viral proteins. Due to
the fact that no adjuvant was used for immunization and
little (total of 100 ng p24 per mouse) viral supernatant
(antigens) was inoculated into the mice, the antibody
responses were likely elicited by the transduced cells.
It is intriguing and unexpected that defective
HIV particles cannot establish productive infection in MAGI
cells when the VSV-G protein is omitted from the viral
particles. Since all the viral structure proteins are
present in the co-transfected cells, this phenomenon
suggests that VSV-G protein is much more efficient than
envelope protein for viral infectivity in this assay
system.
The above invention has been described with
reference to the preferred embodiment. Obvious
modifications and alterations will occur to others upon
reading and understanding the preceding detailed
description. It is intended that the invention be
construed as including all such modifications and
alterations insofar as they come within the scope of the
appended claims or the equivalents thereof.
REFERENCES
1. Clements J.E. et al., "Cross-protective Immune
Responses Induced in Rhesus Macaques by Immunization with
Attenuated Macrophage-Tropic Simian Immunodeficiency
Virus." Journal of Virol 69 (1995): 2737-2744.
2. Daniel M.D. et al., "Protective Effects of a Live
Attenuated SIV Vaccine with a Deletion in the nef Gene."
Science 258 (1992): 1938-1941.
3. Stahl-Hennig C. et al., "Rapid Development of Vaccine
Protection in Macaques by Live-Attenuated Simian
Immunodeficiency Virus." Journal of General Virology 77
(1996): 2969-81.
4. Norley S. et al., "Protection from Pathogenic SIVmac
Challenge Following Short-term Infection with a nef-
Deficient Attenuated Virus." Virology 219 (1996): 195-205.
5. Almond N. et al., "Protection by Attenuated Simian
Immunodeficiency Virus in Macaques Against Challenge with
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6. Wyand M. et al., "Vaccine Protection by a Triple
Deletion Mutant of Simian Immunodeficiency Virus." Journal
of Virology 70 (1996): 3724-3733.
7. Wyand M.S. et al., "Resistance of Neonatal Monkeys to
Live Attenuated Vaccine Strains of Simian Immunodeficiency
Virus." Nature Medicine 3 (1997): 32-36.
8. Baba T.W. et al., "Pathogenicity of Live, Attenuated
SIV After Mucosal Infection of Neonatal Macaques." Science
267 (1995): 1820-1825.
9. Cohen J., "Weakened SIV Vaccines Still Kills."
Science 278 (1997): 24-25.
10. Lu S. et al., "Simian Immunodeficiency Virus DNA
Vaccine Trial in Macaques." Journal of Virology 70 (1996):
3978-3991.
11. Gold, D., "Geneva Overview-Vaccines at the 12th World
AIDS Conference." IAVI Report 3 (1998): 1+.
12. Girard M. et al., "Challenge of Chimpanzees Immunized
with a Recombinant Canarypox-HIV-1 Virus." Virology 232
(1997): 98-104.
13. Naldini L. et al., "In Vivo Gene Delivery and Stable
Transduction of Nondividing Cells by a Lentiviral Vector."
Science 272 (1996): 263-267.
14. Delwart, E.L. et al., "Analysis of HIV-1 Envelope
Mutants and Pseudotyping of Replication-Defective HIV-1
Vectors by Genetic Complementation." Aids Research and
Human Retroviruses 8 (1992): 1669-1677.
15. Ratner, L. et al., "Complete Nucleotide Sequence of
the AIDS Virus, HTLV-III." Nature 313 (1985): 277-284.
16. Chackerian B., "Human Immunodeficiency Virus Type 1
Coreceptors Participate in Postentry Stages in the Virus
Replication Cycle and Function in Simian Immunodeficiency
Virus Infection." Journal of Virology 71 (1997): 3932-
3939.
17. Tung F.T., "Detection of Human Immunodeficiency Virus
Type 1 by a Highly Sensitive and Specific Polymerase Chain
Reaction Method." Serodiaanosis and Immunotherapy 6 (1994):
218-220.
18. Rinaldo C et al., "High Levels of Anti-Human
Immunodeficiency Virus Type l(HIV-l) Memory Cytotoxic T-
Lymphocyte Activity and Low Viral Load Are Associated with
Lack of Disease in HIV-i Infected Long-term Nonprogressor."
Journal of Virology 69 (1995): 5838-5842.
19. Kimpton J. et al., "Detection of Replication-
Competent and Pseudotyped Human Immunodeficiency Virus with
a Sensitive Cell Line on the Basis of Activation of an
Integrated Ji-Galactosidase Gene." Journal of Virology 66
(1992): 2232-2239.
20. Aiken C, "Pseudotyping Human Immunodeficiency Virus
Type 1 (HIV-1) by the Glycoprotein of Vesicular Stomatitis
Virus Targets HIV-1 Entry to an Endocytic Pathway and
Suppresses Both the Requirement for nef and the Sensitivity
to Cyclosporin A." Journal of Virology 71 (1997): 5871-
5877.
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Lymphocyte in Rhesus Monkeys Infected with the Simian
Immunodeficiency Virus of Macaques." Journal of Immunology
144 (1990): 122-128.
22. Kent S.J. et al., "Detection of Simian
Immunodeficiency Virus. (SIV)-specific Cd8+ T Cells in
Macaques Protected from SIV Challenge by Prior SIV Subunit
Vaccination." Journal of Virology 70 (1996): 4941-4947.
23. Salk J. et al., "A Strategy for Prophylactic
Vaccination Against HIV." Science 260 (1993): 1270-1272.
24. Johnson P.R. et al., "Induction of Vigorous Cytotoxic
T-Lymphocyte Responses by Live Attenuated Simian
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7711-7718.
I claim:
1. A method for producing replication-defective
retrovirus particles, comprising the steps of:
a. providing a DNA molecule which includes a
complete retroviral genome;
b. modifying a portion of the pol gene of the DNA
molecule including the protease and reverse
transcriptase activity coding regions to the
extent that the remaining pol gene cannot produce
a protease and reverse transcriptase capable of
functioning in replication of the genome, thereby
forming a pol- construct;
c. transferring the pol" construct into a suitable
host cell;
d. prior to, during or after the step of
transferring the pol- construct into the host
cell, transferring into the host cell or a
predecessor cell or a progeny cell thereof, a
pseudotyping construct and a packaging construct;
e. growing the host cell under conditions suitable
for expression of the constructs and for
production of the replication-defective
retrovirus particles; and
f. collecting the replication-defective virus
particles.
2. The method for producing replication-defective
retrovirus particles as claimed in claim 1, wherein
the retroviral genome is an HIV virus genome.
3. The method for producing replication-defective
retrovirus particles as claimed in claim 1, wherein
the retroviral genome is an HIV-1 virus genome.
4. The method for producing replication—defective
retrovirus particles as claimed in claim 3, wherein
the pol gene is modified by deleting a portion
thereof.
5. The method for producing replication-defective
retrovirus particles as claimed in claim 1, wherein
the pol- construct is plasmid pHXB2 with nucleotides
2621-4552 thereof deleted.
6. The method for producing replication-defective
retrovirus particles as claimed in claim 1, wherein
the retroviral genome is a Feline Leukemia Virus
genome.
7. The method for producing replication-defective
retrovirus particles as claimed in claim 1, further
comprising the step of modifying the retroviral genome
to represent protein sequence variations in
immunologically variant retroviruses.
8. The method for producing replication-defective
retrovirus particles as claimed in claim 3, wherein
the pseudotyping construct encodes the vesicular
stomatitis virus G protein.
9. The method for producing replication-defective
retrovirus particles as claimed in claim 1, wherein at
least one of the pol- construct, the pseudotyping
construct and the packaging construct are co-
transfected.
10. The method for producing replication-defective
retrovirus particles as claimed in claim 1, wherein
the collecting step includes a step of concentrating
the titer of the replication-defective virus
particles.
11. A replication-defective retrovirus particle,
comprising:
a. a retroviral genome in which a portion of the pol
gene including the protease and reverse
transcriptase activity coding regions is modified
to the extent that the remaining pol gene cannot
produce a protease and reverse transcriptase
capable of functioning in replication of the
genome; and
b. a pseudotyping protein.
12. The replication-defective retrovirus particle as
claimed in claim 11, wherein the retroviral genome is
an HIV genome.
13. The replication-defective retrovirus particle as
claimed in claim 11, wherein the retroviral genome is
an HIV-1 genome.
14. The replication-defective retrovirus particle as
claimed in claim 10, wherein the pol gene is modified
by deletion of nucleotide sequences thereof.
15. The replication-defective retrovirus particle as
claimed in claim 11, wherein the retroviral genome is
a pol- construct derived from plasmid pHXB2 with
nucleotides 2621-4552 thereof deleted.
16. The replication-defective retrovirus particle as
claimed in claim 13, wherein the pseudotyping protein
is vesicular stomatitis virus G protein.
17. The replication-defective retrovirus particle as
claimed in claim 11, wherein the particle includes a
retroviral protein which is an immunological variant
of a wild-type retroviral protein.
18. A vaccine comprising a virus particle as claimed in
claim 9 in combination with a pharmaceutically and/or
veterinarilly suitable excipient.
19. The vaccine as claimed in claim 18, wherein the
excipient is selected from the group consisting of a
buffer system, a mono-, di-, oligo- or polysaccharide,
a viscosity enhancing or tackifying agent, a polymer,
a copolymer, a block polymer and a cross-linked
polymer.
20. The vaccine as claimed in claim 18, wherein the
vaccine is lyophilized.
21. The vaccine as claimed in claim 18, wherein the
vaccine is prepared in a form selected from the group
consisting of an oral liquid, an oral capsule, a
liquid for parenteral injection, a transdermal or
transmucosal device and a suppository, and the
excipient is a suitable excipient for preparation of
the selected form.
22. The vaccine as claimed in claim 18, wherein the
retroviral genome is an HIV-1 genome and the
pseudotyping protein is VSV-G.
23. A vaccine as claimed in claim 18, further comprising
a second replication-defective pol- virus particle
representing an immunological variant of the virus
particle.

A replication-defective HIV particle pseudotyped with vesicular stomatitis virus G protein (VSV-G). The pol gene of the HIV genome in the particle is modified to inactivate the pol reverse transcriptase and protease activity. This pseudotyped HIV particle can infect many cell types, including human and simian cells, and only undergoes one round of replication. Furthermore, a virus-specific immune response can be detected in mice immunized with the VSV-G pseudotyped replication-defective HIV.

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Patent Number

233888

Indian Patent Application Number

443/CAL/1999

PG Journal Number

16/2009

Publication Date

17-Apr-2009

Grant Date

16-Apr-2009

Date of Filing

12-May-1999

Name of Patentee

GENECURE LLC

Applicant Address

24 PEACHTREE CENTER AVENUE, SUITE 525, ATLNTA, GEORGIA

Inventors:

#	Inventor's Name	Inventor's Address
1	YAO TSUNG TUNG	1390 ROYAL OAK DRIVE, WEXFORD, PENNSYLVANIA 15090

PCT International Classification Number

G01N 33/53

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/085,115	1998-05-12	U.S.A.