PRIMATE LENTIVIRUS VACCINES

Abstract: Molecular clones of primate lentiviruses that harbor an engineered non-revertible null mutation in the nef gene and which may further include a non-revertible null mutation in one or more of the NRE, vpr, vpx, and/or vpu sequences are disclosed. Intact viruses containing such mutant genomes are also disclosed. These infectious, nonpathogenic viruses are capable of eliciting a protective host immune response and, thus, are useful as a vaccine that protects against AIDS in human subjects.

Description: PRIMATE LENTIVIRUS VACCINES Background of the Invention This invention is in the general field of primate lentiviruses and their utilization as protective vaccines. Human immunodeficiency virus type 1 (HIV-1) , a member of the lentivirus subfamily of retroviruses , is the etiologic agent of the disease, acquired immune deficiency syndrome (AIDS) (F. Barre-Sinoussi et. al., Science 220:868, 1983, R. C. Gallo et. al. , -Science 224:500. 1984). This virus suppresses the immune system rendering the host highly susceptible to a variety of opportunistic infections and neoplasms. The AIDS epidemic is worldwide, with thousands of Europeans and possibly millions of Africans infected with the virus (T. C. Quinn et al., Science 234:955. 1986). As of December 1987, there were 47,000 cases of AIDS in the United States, and it was estimated that an additional 1 to 2 million individuals were infected but, at that time, asymptomatic (U.S. Public Health Service, Public Health Rep. 101:341, 1986). HIV infection is spread by sexual contact, by infected blood or blood products, and perinatally from mother to infant (A. S. Fauci et al., Ann . Intern . Med. , 100:92. 1984; J. . Curran et al.. Science 229:1352. 1985). A closely related but distinct virus, human immunodeficiency virus type 2 (HIV-2) has been isolated from patients with a clinical syndrome comparable to HIV- 1-induced AIDS. HIV-2 shares a significant degree of sequence homology and serologic reactivity with HIV-1 (F. Clavel, N. Eng. J. of Med. 116:1180, 1987). B. Hahn et. al. (Nature 312:166. 1984) and G. M. Shaw et. al. (Science 226:1165. 1984) report the isolation of HIV-1 DNA clones capable of transmitting infection to cultured cells. L. Ratner et. al. (Nature 313:277. 1985,) and S. ain-Hobson et. al. (Cell 40:9. 1985) report the complete nucleotide sequence of HIV-1. The HIV-1 genome contains at least nine open reading frames. As catalogued by Cullen and W. C. Green (Cell 58:423, 1989), these encode structural proteins (Gag, Pol, Env) , proteins required for virion morphogenesis and maturation (Vif, Vpu), nonstructural, regulatory proteins (Tat, Rev, Νef) , and a protein of unknown function (Vpr) . Simian immunodeficiency viruses (SIVs) are nonhu an primate lentiviruses that are the closest known relatives of HXV-l and HIV-2. They closely parallel their human counterparts in genetic organization and biological properties. Similarities between HIV and SIV include lentiviral morphology; tropism for CD4 lymphocytes and macrophages; extra genes called tat, rev, vif. vpf. and nef generally not present in other retroviruses; interaction with the CD4 receptor on host cells; cytopathicity; and the ability, of at least some, to cause chronic disease and death after long-term persistent infection (R.C. Desrosiers, Annu . Rev. Microbiol . 42.:607, 1988). SIV molecular clones have been isolated from macaque monkeys, although other species such as mangabeys may be their natural host, and are generally designated SIVmac (L. Chakrabarti et. al., Nature 328:543, 1987, V. Hirsch et. al., Cell 4£:307, 1987, G. Franchini et. al., Nature 328:539. 1987). One clone, chosen for its ability to grow well in macaque peripheral blood lymphocytes, has been designated SIVmac239. (Y.M. Νaidu et. al., J. Virol . 62.:4691, 1988). Rhesus monkeys inoculated with stock virus derived from cloned SIVmac239 DΝA develop an AIDS-like disease ari eventually die (H. Kestler et. al., Science 248:1107. 1 )) . To date, efforts at provoking a protective immune response against HIV have generally focused on specific antigens, such as HIV-encoded proteins. The sera of patients infected with HIV-l contain antibodies that recognize the precursor and processed forms of the viral envelope (env) gene product, gpl20 (J. S. Allan et. al., Science 228:1091, 1985, F. Barin et. al., Science 228:1094, 1985). These antibodies reportedly are not sufficient to impart immunity against AIDS, at least in the vast majority of infected individuals (J. Schupbach et. al., -Science 224:503. 1984, M. Essex et. al., Science 220:859. 1983, Barre-Sinoussi et. al., Science 220:868 P 1983) . However, various Env-based vaccines are currently being studied (see, for example, D. Zagury et. al., Nature 3.32:728, 1988, . C. Koff and A. S. Fauci, AT-D≤r 19892 (supEl) : S125, 1989). A killed, non-infectious, envelope-depleted whole HIV-l virus is also being tested in patients already infected with HIV-l (A. Levine et. al., -Fifth International Conference on AIDS. The Scientific and Social Challenge . , p. 219, 1989) D. Baltimore (Nature 335:395. 1988) reports a proposed procedure for introducing, into a human subject, bone-marrow stem cells that are "infected or transfected with a virus or DNA construct that encodes an RNA or protein able dominantly to interfere with the intracellular growth of HIV, and propose that it be called intracellular immunization". M. H. Malim et. al. (Cell 58:205, 1989) report the isolation of a Rev mutant that trans-dominantly inhibits wild-type Rev function and inhibits HIV-l replication and say that trans-dominant Rev protein may be useful as a means of providing "intracellular immunization against HIV-l". D. Trono et. al. (Cell 59:113, 1989) report that replication of wild-type HIV-l virus is inhibited by co- expression of dominant negative Gag protein and say that these mutant proteins "appear to constitute suitable substrates for developing an antiviral scheme based on intracellular immunization". M. Green et. al. (Cell 58:215, 1989) report that defective Tat peptides block trans-activation of the HIV- 1 LTR and say that their "results suggest an attractive approach for the development of an AIDS therapy". Summary of the Invention In one aspect, the invention features a DNA clone of an infectious nonpathogenic primate lentivirus (PLV) genome harboring a non-revertible null mutation of the nef gene. By "primate lentivirus genome" is meant the genetic material derived from a retrovirus, that, in wild-type form, (a) includes tat, rev, and nef genes, (b) is capable of infecting T4 cells of a primate host, and (c) possesses a viral morphogenesis and morphology characteristic of the lentivirus subfamily. The term includes, without limitation, all variants of HIV and SIV, including HTLV-III, ARV, LAV, HIV-l, HIV-2, SIVmac, SIVagm, SIVmnd, SlVsmm, SIVman, SIVmand, and SIVcpz. By "null mutation" is meant an alteration in the nucleotide sequence that renders the gene incapable of expressing a functional protein product. By "non-revertible" is meant unable to regain the ability to produce a functional protein product in the absence of wild-type virus or intentional genetic manipulation. Preferred clones according to this invention are derived from isolated primate lentiviruses, for example, they are produced by modifying a naturally occurring isolate to create the required nef mutation or by synthesizing nucleic acid to generally correspond to the sequence of a naturally occurring isolate, but with the required nef mutation. Thus, this invention is not limited to any specific viral clone. Those of ordinary skill in the art can readily isolate or engineer equivalent DNA clones which may differ in nucleic acid sequence by conservative substitutions such that the altered codon encodes a very similar amino acid, for example, a substitution of an alanine codon for a glycine codon. In addition, the nucleic acid sequence may have one or more non-conservative codon substitutions or one or more deletions which do not destroy the ability of the virus to produce progeny virus. Such equivalent DNA clones may be engineered by standard techniques of recombinant DNA technology, e.g., random or site-specific in vitro mutagenesis or deletion of sequences between restriction sites, and isolated by standard techniques of recombinant DNA technology including, but not limited to, the use of PCR and lambda, plasmid, cosmid, and/or other cloning vectors. Further, the invention is not limited to the specific non-revertible null mutation of the nef gene provided in the drawing; those skilled in the art can readily isolate other mutations which interfere with the ability of the nef gene to produce a functional protein product and which are non-revertible, e. g. , larger or smaller deletions of the nef gene sequence. Such equivalent mutations may be engineered or isolated by standard techniques of recombinant DNA technology, e.g., by in vitro mutagenesis. In a related aspect, the invention features a primate lentivirus (i.e., an RNA clone or virally packaged RNA) whose genome contains an engineered (i.e., intentionally created or intentionally selected and isolated) non-revertible, null mutation in the nef gene. Such a virus is infectious but not pathogenic, and it may be readily isolated by transfecting cultured primate cells with one of the DNA clones described above and harvesting progeny virus. In preferred embodiments of both aspects, the nucleic acid sequence of the DNA clone of the primate lentivirus is derived from a primate lentivirus, specifically HIV or SIV, particularly SIVmac, and the mutation leaves intact the env gene sequence. In other preferred embodiments, the nucleic acid sequence of the DNA clone of the primate lentivirus includes, in addition to a nef mutation (as described above) , a non-revertible null mutation in one or more of the following sequences: NRE, vpr. or vpx (in the case of a simian lentivirus) or vpu (in the case of a human lentivirus) . Particularly preferred DNA clones according to the invention include SIVmac239ΔnefΔNRE, SIVmac239Δ3, SIVmac239Δ4, HIV-lΔnefΔNRE, HIV-1Δ3, and HIV-1Δ4. In other aspects, the invention features a vaccine which evokes protection against AIDS and associated diseases including purified primate lentivirus, or a DNA clone thereof, in a pharmaceutically acceptable carrier. Such a DNA clone harbors a non-revertible null mutation of the nef gene and may also include a non-revertible null mutation in one or more of the NRE, vpr, vpx, and/or vpu sequences. Further, the invention features a method for administering an immunizing amount of the vaccine to a patient (i.e., a host for the live pathogenic virus). Finally, the invention features a method of producing such a vaccine by transfecting cultured primate cells with a primate lentiviral nucleic acid according to the invention (i.e., one which contains an engineered non-revertible null mutation of the nef gene and which may also contain a non-revertible null mutation in the NRE, vpr, vpx, and/or vpu sequences) , isolating lentivirus whose genome contains the mutation of the nef gene, and compounding the virus into a pharmaceutically acceptable vaccine. The vaccine of the present invention provides immunological protection against primate lentiviruses using a live and infectious, but nonpathogenic, derivative of the parent virus. The introduction of non- revertible mutations provides an advantage over methods previously employed to produce attenuated viral vaccines, for example, isolation of viral stocks harboring genomic point mutation(s) , capable of reverting to the wild-type sequence. In addition, construction of the engineered viral DNA clone is rapid, and the method of producing the vaccine by propagation of attenuated virus in mammalian cell culture is both rapid and inexpensive. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims. Description of the Preferred Embodiments The drawings will first be described. Brief Description of Drawings Fig. 1 is a representation of the complete nucleic acid and amino acid sequence of the SIVmac239 genome, one example of a primate lentivirus useful in this invention. The boundaries of the nef open reading frame and the extent of the nef deletion in the mutant clone, pSIVmac239 nef-deletion, are shown. Fig. 2 is a representation of the complete nucleic acid and amino acid sequence of an isolate of HIV-l. The nef open reading frame is denoted E 1 , and its boundaries are shown. Fig. 3 (SEQ ID NO:5) is a representation of the complete nucleic acid sequence of HIV NL43. Primate lentivirus clones There now follow examples of DNA clones according to the invention which include a deletion in the nef gene of SIVmac239 or HIV-l. All nef gene sequences isolated to date exhibit homology at the nucleotide and amino acid levels (Human Retroviruses and AIDS 1990. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, ed. G. Myers et. al., Los Alamos National Laboratory, Los Alamos, NM) . In addition, the position of the nef gene in the lentiviral genome (i.e., between the env gene and the 3' LTR) is conserved (Human Retroviruses and AIDS 1990. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, ed. G. Myers et. al., Los Alamos National Laboratory, Los Alamos, NM) . It is therefore a matter within the skill in the art to identify the nef gene sequence in any known or newly isolated primate lentivirus. Using standard techniques, such as those described below, said nef gene can be appropriately mutated, tested for pathogenicity, and compounded into a suitable vaccine that evokes protection against the AIDS virus. The following clones are described to illustrate, not to limit, the invention. As described below, those of ordinary skill in the art can readily produce DNA clones from other SIV or HIV isolates having similar utility in the production of a nonpathogenic viral vaccine against AIDS. SIVmac239 nef-deletion Parental virus SIVmac239 was isolated from a macaque monkey (New England Regional Primate Research Center) that exhibited impaired T-cell function, lymphoproliferative disorders, and opportunistic infections, symptoms characteristic of SIV infection. Cell free serum samples from this animal were cocultivated with HuT-78 cells (a human tumor T-cell line available from American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 under accession No. TIB 161) and infectious SIVmac239 virus isolated from the HuT-78 cell supernatant as described in M. D. Daniel et al. (-Science 228:1201. 1985). The cell supernatant was shown to possess detectable reverse transcriptase activity (by the method of M. D. Daniel et. al.. Science 228:1201 r 1985) beginning 12 to 18 days after initiation of cocultivation. The presence of viral particles in this cell line was confirmed by electron microscopy; SIVmac239 infected cells displayed budding viral particles with a morphology characteristic of primate lentiviruses. To facilitate cloning of the full length viral genome, endonuclease EcoRI was identified as a noncutter of SIVmac239 DNA. Total cell DNA was prepared from SIVmac239-infected HuT-78 cells and digested with EcoRI. Sucrose density gradient size-fractionated 10- to 20- kilobase pair EcoRI fragments were inserted into the EcoRI site of lambda cloning vector, EMBL4. This library was screened using pK2 BamA (described in V. Hirsch et. al.. Cell 49.:307, 1987) as a probe, and full length molecular clone, SIVmac239, was isolated. The nucleotide sequence of both strands was determined by the primer- directed dideoxy-chain termination method of Sanger et. al. (Proc. Natl . Acad. Sci . USA 74.:5463, 1977) using Sequenase (i.e., T7 DNA polymerase) versions 1.0 or 2.0 (United States Biochemical, Cleveland, OH) . 35 S-labelled sequencing reactions were electrophoresed on a buffer gradient (0.75 to 2.5% Tris-borate buffer) 6% polyacrylamide/8M urea gel. Sequences were entered into an IBM-PC computer with an IBI gel reader and digitizer and were analyzed with IBI-Pustell DNA analysis software (International Biotechnologies, Inc., New Haven, CT) . The SIVmac239 clone in EMBL4 was then used to generate two plasmid subclones as follows. EMBL- SIVmac239 was digested with SphI, and a 6706 base pair fragment, containing the sequence from the SphI site in the left flanking cellular DNA sequence to the SphI site at viral nucleotide number 6451, was inserted into the SphI site of vector pBS(+) (Stratagene, La Jolla, CA) to produce subclone, p239SpSp5'. In a separate reaction, EMBL-SIVmac239 was digested with SphI and EcoRI, and a 6361 base pair fragment, containing the sequence from the SphI site at viral nucleotide 6452 to the EcoRI site in the right flanking cellular sequence, was inserted into Sphl/EcoRI cleaved vector pBS(-) (Stratagene, La Jolla, CA) to produce subclone, p239SpE3'. These subclones were used to generate a full length genomic sequence as follows. Both p239SpSp5'and p239SpE3' were digested with SphI, ligated together, and used to directly transfect cultured cells (e.g., macaque peripheral blood lymphocytes) . A non-revertible null mutation (a deletion) was engineered into the nef gene of the SIVmac239 clone as follows. The C-terminus of the nef gene, contained within an SstI fragment of p239SpE3' and encompassing nucleotides 9230 to 11,436, was cloned into M13 to produce M13-SSTB. This M13 clone was subjected to oligonucleotide site specific deletion mutagenesis using procedures described in M. J. Zoller and M. Smith (DNA 2:479, 1984) or M. Strauss et. al. (Gene 19:331, 1986). A 73 base pair oligonucleotide complementary to bases (9215 through 9250) and (9433 through 9469) was synthesized. The two segments were contiguous, i.e., bases 9251 through 9432 were not present on the 73-mer. This oligonucleotide was annealed to single stranded M13- SSTB in the presence of Klenow polymerase and the four deoxyribonucleotides to promote synthesis of the complementary strand. Since bases 9251 through 9432 were not included in the oligonucleotide primer, their complement was similarly not included in the newly synthesized negative strand. Double stranded replicative form (RF) phage DNA was transformed into E. coli host, JM109. DNA from the resulting M13 plaques was immobilized on nitrocellulose filters by standard techniques ( . D. Benton and R. . Davis, Science 196:180. 1977) and screened for the sequence in the deleted region, using an oligonucleotide probe complementary to the deleted sequence. A negative signal was taken as an indication that a clone contained a nef deletion. One M13 clone, confirmed to have the expected deletion by DNA sequencing, was subsequently used for reconstruction of the full length viral sequence as follows. The fragment, containing the deletion, was liberated by digestion of deleted M13-SSTB, with Sstl. Said fragment was then cloned into p239SpE3' to yield p239SpE3• (nef-deletion) . Plasmid subclones, p239SpSp5' and p239SpE3* (nef-deletion) , were digested with SphI, ligated together, and used to directly transfect cultured cells (e.g., macaque peripheral blood lymphocytes). As shown in Figure 1, the SIVmac239 nef gene overlaps the env gene sequence. Disruption of the env gene sequence of the SIVmac239 genome, or, generally, any PLV genome, can render the lentivirus non-infectious and, hence, not useful for the production of an effective live vaccine. For this reason, the nef deletion, contained in p239SpE3' (nef-deletion) , leaves the env gene intact. Plasmid clones p239SpSp5' and p239SpE3 » (nef- deletion) have been deposited with the American Type Culture Collection and they, respectively, bear the accession numbers: ATCC No. 68365 and No. 68364. Applicant's assignee, the President and Fellows of Harvard University, acknowledge their responsibility to replace these cultures should they die before the end of the term of a patent issued hereon, and their responsibility to notify the ATCC of the issuance of such a patent, at which time the deposits will be made available to the public. Until that time the deposits will be made available to the Commissioner of Patents under the terms of 37 CFR §1-14 and 35 USC §112. Using the SIVmac239 sequence provided in Figure l, one skilled in the art can engineer other non-revertible null mutations in the nef gene sequence. Deletions that remove a portion (> 10%) of the SIV nef open reading frame can be easily verified by restriction mapping. Such deletions of the Nef encoding nucleic acid, or deletions that disrupt Nef control or response sequences, in most cases, render the gene incapable of producing a Nef gene product. The absence of Nef gene product can be verified by immunoassay (e.g.. Western blot analysis), using purified antibodies directed to the Nef protein or serum from primates infected with a primate lentivirus. When other types of null mutations are accomplished (e.g., smaller deletions), their efficacy can be tested using an in vivo assay. Briefly, for a simian immunodeficiency virus, an aliquot of purified virus derived from a mutant nef clone can be injected intravenously into a live, permissive host, preferably a rhesus monkey, and tested for pathogenicity (i.e., ability to induce AIDS-like disease symptoms) . Virus harboring a genome with a null mutation in the nef gene (e.g. , SIVmac239 nef-deletion) are nonpathogenic. SIVmac239ΔnefΔNRE Another example of an SIV DNA clone according to the invention, i.e., one which includes a deletion in the nef gene and a deletion in the NRE sequence of the lonq terminal repeat (LTR) , was constructed as follows. Plasmid p239SpE3 • (nef-deletion) was digested with Nsil and StuI, enzymes which cut at unique sites within the NRE region of the SIVmac LTR. The DNA fragment comprising most of the plasmid sequence (but lacking the internal NRE fragment) was isolated, the protruding ends blunt-ended using T4 DNA polymerase, and the blunt ends ligated together to recreate a circular plasmid. The plasmid was re-isolated, and was confirmed, by nucleotide sequence analysis, to be lacking nucleotides 9660 to 9831 (in the NRE region) ; this plasmid was termed p239SpE3'ΔnefΔNRE. Plasmids p239SpSp5' and p239SpE3'ΔnefΔNRE were each digested with SphI, ligated together, and used to directly transfect permissive cultured cells (e.g., macaque peripheral blood lymphocytes) as described above. Such cells produced an SIVmac239 virus whose genome included a deletion in the nef gene and a deletion in the NRE sequence (i.e., SIV mac239ΔnefΔNRE) . SIVmac239Δ3 Another example of an SIV DNA clone according to the invention, i.e., one which includes deletions in the nef and vpr genes and in the NRE sequence was constructed as follows. Plasmid p239SpSp5' was subjected to polymerase chain reaction (PCR) site specific mutagenesis by the method of Ho et al. (Gene 7_7:51, 1989). 35 bp oligonucleotides, complementary to bases (6135 through 6152) and (6254 through 6270) of SIVmac239, respectively, were synthesized and used as the PCR primers. Specifically such oligonucleotides were of sequence: 5' TCCAGGACTAGCATAAATTTGATCCTCGCTTGCTA 3' (SEQ ID NO:l) and 5' TAGCAAGCGAGGATCAAATTTATGCTAGTCCTGGA 3' (SEQ ID NO:2) . The resultant plasmid, termed p239SpSp5'Δvpr, included a deletion of nucleotides 6153 through 6253 of the vpr gene. Plasmids p239SpSp5'Δvpr and p239SpE3'ΔnefΔNRE were each digested with SphI, ligated together, and used to directly transfect permissive cultured cells (e.g., macaque peripheral blood lymphocytes) as described above. Such cells produced an SIVmac239 virus whose genome included deletions in the nef and vpr genes and in the NRE sequence (i.e., SIVmac239Δ3) . SIVmac239Δ4 Another example of an SIV DNA clone according to the invention, i.e., one which includes a deletion in the nef. vpr, and vpx genes and in the NRE sequence was constructed as follows. Plasmid p239SpSp5' was subjected to PCR-site specific mutagenesis as described above. 30 bp oligonucleotides, complementary to bases (5973 through 5987) and (6254 through 6268) of SIVmac239, respectively, were synthesized and used as PCR primers. Specifically, such oligonucleotides were of sequence: 5' ATACTGGCATGATGATTGATCCTCGCTTGC 3' (SEQ ID NO:3) and 5' GCAAGCGAGGATCAATCATCATGCCAGTAT 3' (SEQ ID NO: 4) . The resultant plasmid, termed p239SpSp5'ΔvpxΔvpr, included a deletion of nucleotides 5988 through 6253 of the vpx and vpr genes. Plasmids p239SpSp5'ΔvpxΔvpr and p239SpE3•ΔnefΔNRE were each digested with SphI, ligated together, and used to directly transfect permissive cultured cells (e.g., macaque peripheral blood lymphocytes) as described above. Such cells produced an SIVmac239 virus whose genome included deletions in the nef. vpr, and vox genes and in the NRE sequence (i.e., SIVmac239Δ4) . Non-revertible null mutations can also be engineered into the genomic nef sequence of other primate lentivirus genomes, for example, HIV-l. Figure 2 shows the complete nucleotide and amino acid sequence of an isolate of HIV-l; as indicated in this figure, the nef gene (referred to as E') includes the sequence between nucleotides 8,347 and 8,992. Said sequence is located between the env gene and the 3' LTR and exhibits nucleotide and amino acid sequence homology to the nef gene of the simian lentivirus isolate, SIVmac239. Non- revertible null mutations can be introduced into the HIV- 1 nef sequence by standard recombinant DNA techniques, for example, site directed in vitro mutagenesis or deletion of sequence between restriction sites. Again, deletions that remove all or most of the HIV-l nef open reading frame can be verified by restriction mapping or by immunoassay. Deletions which disrupt Nef control or response elements, in most cases, also render the gene incapable of producing a Nef gene product, and this event also could be tested by immunoassay as described above for SIV Nef. When other types of null mutations (e.g., smaller deletions) are accomplished, their efficacy should be tested using an in vivo assay. No permissive host (except humans) exists for directly testing the pathogenicity of a mutant HIV clone in vivo. However, as described below, due to the similarity in the human and simian nef sequences, an equivalent SIV nef-mutant clone (i.e., with a deletion in the corresponding SIV Nef domain) can be engineered and can be tested for pathogenicity in rhesus monkeys as described above. There now follow specific examples of HIV-l clones according to the invention. These examples are for the purpose of illustrating, not limiting, the invention. HIV-lΔnefΔNRE - 16 - An example of an HIV DNA clone according to the invention, i.e., one which includes a deletion in the nef gene and in the NRE sequence is constructed as follows. Beginning with the proviral clone, termed pNL4-3 (Adachi et al., J. Virol . £59:284, 1986; available from the AIDS Research and Reference Reagent Program, NIAID, National Institute of Health, Bethesda, MD) , the HIV-l sequence is sub-cloned into two separate vectors. Specifically, pNL4-3 is digested with ApaLI and EcoRI and a 5897 bp fragment including bp -154 (within the flanking cellular sequence) to bp 5743 (within the flanking cellular sequence) (i.e., the viral genes, gag, pol. vif, and vpr) is inserted into a plasmid vector, termed pDR8, to produce pLEFT. Plasmid pDR8 was constructed by digesting pUC19 with Ndel and Earl (i.e., deleting the lacZ and lad genes and the polylinker) and rendering the protruding ends blunt with T4 DNA polymerase. Into this backbone was inserted, by blunt-end ligation, an oligonucleotide of sequence: 5' TGGTGACCTTCGAAGGATCCCATATGTCTAGAGAATTCGGTCACCA 3' 3' ACCACTGGAAGCTTCCTAGGGTATACAGATCTCTTAAGCCAGTGGT 5 1 (SEQ ID NO:14) , to produce pDR8. In a separate reaction pNL4-3 is digested with EcoRI and Thai and a 4002 bp fragment including bp 5743 of Fig. 3 (SEQ ID NO:5) to bp 9745 (in the flanking cellular sequence) (i.e., the viral genes, tat, rev. vpu. env. nef. and LTR) is inserted into plasmid vector, pDR8, to produce pRIGHT. To mutate the nef and NRE genes, pRIGHT is digested with Ndel or ApaLI and Thai and at 3247 bp or 3136 bp viral fragment, respectively, is isolated and inserted into plasmid vector pDR8, to create pRIGHT-SUBl. The nef gene and NRE sequence are mutated by PCR-site specific mutagenesis by the method of Ho et al. (supra) . PCR primers are chosen such that most or all of the nef and NRE sequences are deleted. For example, deletion of the nef gene may be accomplished using PCR primers of sequence: 5' AAGGATTTTGCTATAATAGCCACTTTTTTAAAA 3' (SEQ ID NO:6) and 5 i TTTTAAAAAAGTGGCTTATTATAGCAAAATCCTT 3' (SEQ ID NO: 7) , complementary to bases (8770 through 8785) and (9047 through 9062) of the HIV-l sequence of Fig. 3 (SEQ ID NO:5). Deletion of the NRE sequence may be accomplished using PCR primers of sequence: 5' ACTGACCTTTGGATGGCATCCGGAGTACTTCA (SEQ ID NO:8) and 5' TGAAGTACTCCGGATGCCATCCAAAGGTCAGT 3' (SEQ ID NO:9) , complementary to bases (9194 through 9209) and (9381 through 9396) of the HIV-l sequence of Fig. 3 (SEQ ID NO:5). The mutated version of pRIGHT-SUBl is digested with Ndel or ApaLI (corresponding to the enzyme chosen above) and Thai and the 3075 bp or 2964 bp fragment, respectively isolated and re-inserted into Ndel- or ApaLI- and Thai-digested pRIGHT to create pRIGHT-MUTl. pRIGHT-MUTl and pLEFT are digested with EcoRI, ligated together, and used to directly transfect permissive cultured cells (e.g., human peripheral blood lymphocytes) as described in Sompayrac and Danna (Proc. Natl . Acad. Sci . USA 78.:7575, 1981) or Milman and Herzberg (Somat . Cell Genet . 7:161, 1981) or Naidu et al. (J " . Virol . £2:4691, 1988). Such cells produce an HIV-l virus whose genome includes a deletion in the nef gene and in the NRE sequence (i . e. , HIV-lΔnef ΔNRE) . HIV-1Δ3 Another example of an HIV DNA clone according to the invention, i.e., one which includes deletions in the nef and vpr genes and in the NRE sequence is constructed as follows. Plasmids pRIGHT-MUTl and pLEFT are constructed as described above. A fragment containing the vpr gene is further subcloned. Specifically, pLEFT is digested with BspMI or Ndel and EcoRI and a 697 bp or 621 bp viral fragment, respectively, is isolated and inserted into plasmid vector, pDR8, to create pLEFT-SUBl. The vpr gene is mutated by PCR-site specific mutagenesis (by the method of Ho et al., supra) . PCR primers are designed such that most or all of the vpr gene is deleted. For example, primers may be of sequence: 5' AATGAATGGACACTAGTAATAAGAATTC 3' (SEQ ID NO:10) and 5 1 GAATTCTTATTACTAGTGTCCATTCATT 3 » (SEQ ID NO:11), complementary to bases (5604 through 5619) and (5737 through 5748) of the HIV-l sequence of Fig. 3 (SEQ ID NO:5). The mutated version of pLEFT-SUBl is digested with BspMI or Ndel (corresponding to the enzyme chosen above) and EcoRI, and the 579 bp or 503 bp viral fragment, respectively, isolated and re-inserted into BspMI- or Ndel- and EcoRI-digested pLEFT to create pLEFT- MUT. pLEFT-MUT and pRIGHT-MUTl are digested with EcoRI, ligated together, and used to directly transfect permissive cultured cells (e.g., human peripheral blood lymphocytes) as described in Sompayrac and Danna (Proc. Natl . Acad. Sci . USA 78.:7575, 1981) or Milman and Herzberg (Somat. Cell Genet . 2:161, 1981) or Naidu et al. (J. Virol . 62:4691, 1988). Such cells produce an HIV-l virus whose genome includes deletions in the nef and vpr genes and in the NRE sequence (i.e., HIV-1Δ3) . HIV-1Δ4 Another example of an HIV DNA clone according to the invention, i.e., one which includes deletions in the nef, vpr. and vpu genes and in the NRE sequence is constructed as follows. Plasmids pLEFT-MUT, pRIGHT, and pRIGHT-MUTl are constructed as described above. A fragment containing the vpu gene is subcloned. Specifically, pRIGHT is digested with EcoRI and Ndel or ApaLI and a 864 bp or 656 bp fragment, respectively, is isolated and inserted into plasmid vector, pDR8, to create pRIGHT-SUB2. The vpu gene is mutated by PCR-site specific mutagenesis by the method of Ho et al. (supra) . PCR primers are chosen such that most or all of the vpu gene is deleted. For example, primers may be of sequence: 5' GTAAGTAGTACATGTAATGAGAGTGAAGGAGA 3' (SEQ ID NO:12) and 5 i TCTCCTTCACTCTCATTACATGTACTACTTAC 3' (SEQ ID NO:13) , complementary to bases (6045 through 6060) and (6221 through 6236) of the HIV-l sequence of Fig. 3 (SEQ ID NO:5). The mutated version of pRIGHT-SUB2 is digested with EcoRI and Ndel or ApaLI (corresponding to the enzyme chosen above) and the 475 bp or 686 bp (respectively) viral fragment isolated and inserted into an EcoRI- and Ndel- or ApaLI-digested pRIGHT-MUTl backbone, to create pRIGHT-MUT2. pRIGHT-MUT2 and pLEFT-MUT are digested with EcoRI, ligated together, and used to directly transfect permissive cultured cells (e.g., human peripheral blood lymphocytes) as described in Sompayrac and Danna (Proc. Natl . Acad. Sci . USA 22:7575, 1981) or Milman and Herzberg (Somat . Cell Genet . 2:161, 1981) or Naidu et al. (J. Virol . £2:4691, 1988). Such cells produce an HIV-l virus whose genome includes deletions in the nef, vpr. and vpu genes and in the NRE sequence (i.e., HIV1Δ4) . Isolation of Primate Lentivirus Having produced an infectious DNA clone containing a nef mutation according to the invention, that clone is used to transfect appropriate cells, for example, immortalized lymphocytes, such as H9 cells or HuT-78 cells, or peripheral blood lymphocytes from donor mammals. After culturing the infected cells, primate lentiviruses, suitable for making a vaccine, can be isolated from the cell supernatant following centrifugation and filtration, using procedures identical to those utilized to isolate wild-type virus. In order to more fully illustrate the manner of isolating primate lentivirus, the following example is presented. Isolation of SIVmac239 nef-deletion Virus SIVmac239 nef-deletion virus can be isolated by digesting p239SpSp5* and p239SpE3 » (nef-deletion) DNA with SphI, ligating together, and transfecting into HuT-78 cells by the DEAE-dextran technique of Sompayrac and Danna (Proc. Natl . Acad. Sci . USA 22:7575, 1981) and Milman and Herzberg (Somatic Cell Genet . 2:161, 1981) followed by isolation of progeny virus as follows. Five aliquots of viral DNA (3 ug of ligated DNA/aliquot) are sequentially added, with intermittent mixing, to 1.4 ml of serum-free Dulbecco's modified Eagle medium (GIBCO, Grand Island, N.Y.) containing 125 ug/ml DEAE-dextran and 50 mM Tris (pH 7.3). HuT-78 cells are split 1:2 or 1:3, grown to 3 x 10 6 cells/plate (i.e., 24 hours or less), washed twice with Dulbecco's modified Eagle medium, mixed with the 1.4 ml DNA solution, and incubated at 37°C for 1 hr. The cells are then washed with serum-free Dulbecco modified Eagle medium, washed with serum-free RPMI 1640 medium (GIBCO, Grand Island, NY) , and incubated at 37°C in RPMI 1640 medium containing 10% fetal calf serum. Transfected cells are split 1:2 or 1:3, twice per week. Alternatively, SIVmac239 nef-deletion may be propagated by transfecting H9 cells by the above procedure or by transfecting macaque peripheral blood lymphocytes essentially by the above procedure, except that the cells are stimulated with 1 ug/ml phytohemagglutinin (GIBCO, Grand Island, NY) for 48 hrs prior to transfection and grown in RPMI 1640 medium containing 10% interleukin-2 (lectin-free, T-cell growth factor; Electro-Nucleonics, Inc., Fairfield, N.J.) following transfection. Generally, seven to fourteen days after transfection, cells are removed by centrifugation (1500g, 10 minutes, 4°C) , and the supernatant is filtered through a disposable 0.45 micron filter (Corning, Corning, NY). Cell-free supernatant is monitored for reverse transcriptase activity, using the methods of M. D. Daniel et. al. (Science 228:1201. 1985), or for the amount of Gag antigen, by antigen capture (Coulter Immunology, Hialeah, FL) . Cell free supernatant, possessing detectable reverse transcriptase activity and viral antigen, is saved as virus stock. Stocks may be stored in liquid nitrogen for long periods of time, e.g., one to two years, without significant loss of virus viability. A cycle of freezing and thawing, however, may reduce viral infectivity approximately ten-fold. Vaccine Production A PLV molecular clone containing a non-revertible null mutation in the nef gene or a virus which harbors such a mutant genome is used to transfect or infect appropriate primate cells, e.g., immortalized HuT-78 or H f cells, or peripheral blood lymphocytes, using p -o-edures described above. After a standard incubation t .: i, for example, seven to fourteen days, the culture mt-άium is removed by aspiration, the cells are washed twice with phosphate-buffered saline (137mM NaCl, 2.7mM - 22 - KC1, 4.3mM Na 2 HP0 4 * 7H 2 0, 1.4mM KH 2 P0 4 , pH 7.3) and the medium is replaced with serum-free RPMI 1640. Following an appropriate incubation time in serum-free medium (approximately 12 to 24 hours) , virus is harvested by centrifugation and further purified by passage of the cell free supernatant through a 0.45 micron filter as described above. Viral stock is titered by standard techniques (Techniques in HIV Research , ed. A. Aldovini and B. D. Walker, Stockton Press, New York, NY) , diluted, if necessary, in a pharmaceutically acceptable carrier, such as physiological saline, and stored frozen in aliquots appropriate for a single immunizing dose. Stabilizers (e.g., MgCl 2 , hydrolyzed gelatin, or sorbitol) , antibiotics (e.g., streptomycin or neomycin) , and pH indicators (e.g., phenol red), may also be added to the vaccine just prior to storage. Method of Vaccination An ύnmunizing amount (i.e., an infecting dose) of this vaccine is administered by any standard procedure, for example, by intramuscular injection. This dose is effective over the course of at least twelve months. Other embodiments are within the following claims. For example, the genome may be substantially identical to a PLV genome, but for the nef mutation discussed above and a non-revertible null mutation in one or more of the other PLV genes that are not essential to infectivity, for example, vpr, vpu, or vif (e.g., as described above), or the deletion of LTR sequences that are not essential for virus replication.

Claims: Claims 1. A DNA clone of a primate lentivirus containing an engineered non-revertible null mutation in the nef gene. 2. The DNA clone of claim 1, wherein said mutation leaves intact the genomic env gene sequence. 3. The DNA clone of claim 1, wherein said clone is derived from the genome of a human immunodeficiency virus. 4. The DNA clone of claim 3, wherein said clone is derived from the genome of HIV-l. 5. The DNA clone of claim 1, wherein said clone is derived from the genome of a simian immunodeficiency virus. 6. The DNA clone of claim 5, wherein said clone is derived from the genome of SIVmac. 7. The DNA clone of claim 6, comprising the DNA clones deposited with the ATCC and designated No. 68364 and No. 68365. 8. The DNA clone of claim 1, further comprising an engineered non-revertible null mutation in the NRE sequence. 9. The DNA clone of claim 1, further comprising an engineered non-revertible null mutation in the vpr gene sequence. 10. The DNA clone of claim 1, further comprising an engineered non-revertible null mutation in the vpx gene sequence. 11. The DNA clone of claim 1, further comprising an engineered non-revertible null mutation in the vpu gene sequence. 12. The DNA clone of claim 6, wherein said clone is SIVmac239ΔnefΔNRE. 13. The DNA clone of claim 6, wherein said clone is SIVmac239Δ3. 14. The DNA clone of claim 6, wherein said clone is SIVmac239Δ4. 15. The DNA clone of claim 4, wherein said clone is HIV-lΔnefΔNRE. 16. The DNA clone of claim 4, wherein said clone is HIV-1Δ3. 17. The DNA clone of claim 4, wherein said clone is HIV-1Δ4. 18. A primate lentivirus whose genome contains an engineered non-revertible null mutation in the nef gene. 19. The primate lentivirus of claim 18, wherein said virus is derived from human immunodeficiency virus. 20. The primate lentivirus of claim 19, wherein said virus is derived from HIV-l. 21. The primate lentivirus of claim 18, wherein said virus is derived from simian immunodeficiency virus. 22. The primate lentivirus of claim 21, wherein said virus is derived from SIVmac. 23. The primate lentivirus of claim 22, wherein said virus is derived from the DNA clones deposited with the ATCC and designated No. 68364 and No. 68365. 24. The primate lentivirus of claim 18, wherein said genome further comprised an engineered non- revertible null mutation in the NRE sequence. 25. The primate lentivirus of claim 18, wherein said genome further comprises an engineered non- revertible null mutation in the vpr gene sequence. 26. The primate lentivirus of claim 18, wherein said genome further comprises an engineered non- revertible null mutation in the vpx gene sequence. 27. The primate lentivirus of claim 18, wherein said genome further comprises an engineered non- revertible null mutation in the vpu gene sequence. 28. The primate lentivirus of claim 22, wherein said lentivirus is derived from SIVmac239ΔnefΔNRE. 29. The primate lentivirus of claim 22, wherein said lentivirus is derived from SIVmac239Δ3. 30. The primate lentivirus of claim 22, wherein said lentivirus is derived from SIVmac239Δ4. 31. The primate lentivirus of claim 20, wherein said lentivirus is derived from HIV-lΔnefΔNRE. 32. The primate lentivirus of claim 20, wherein said lentivirus is derived from HIV-1Δ3. 33. The primate lentivirus of claim 20, wherein said lentivirus is derived from HIV-1Δ4. 34. A method of producing a vaccine that provides protection against a primate lentivirus comprising: transfecting cultured primate cells with primate lentiviral nucleic acid containing an engineered non- revertible null mutation of the nef gene, isolating lentivirus whose genome contains said mutation of the nef gene, and compounding said virus into a pharmaceutically acceptable vaccine. 35. The method of claim 34, wherein said lentiviral nucleic acid further comprises an engineered non-revertible null mutation in the NRE sequence. 36. The method of claim 34, wherein said lentiviral nucleic acid further comprises an engineered non-revertible null mutation in the vpr gene sequence. 37. The method of claim 34, wherein said lentiviral nucleic acid further comprises an engineered non-revertible null mutation in the vpx gene sequence. 38. The method of claim 34, wherein said primate lentiviral nucleic acid further comprises an engineered non-revertible null mutation in the vpu gene sequence. 39. A vaccine comprising isolated primate lentivirus whose genome contains an engineered non- revertible null mutation in the nef gene, or an infectious DNA clone thereof, in a pharmaceutically acceptable carrier. 40. The vaccine of claim 39, wherein said primate lentivirus is derived from human immunodeficiency virus. 41. The vaccine of claim 40, wherein said primate lentivirus is derived from HIV-l. 42. The vaccine of claim 39, wherein said primate lentivirus is derived from simian immunodeficiency virus. 43. The vaccine of claim 42, wherein said primate lentivirus is derived from SIVmac. 44. The vaccine of claim 43, wherein said primate lentivirus is derived from the DNA clones deposited with the ATCC and designated No. 68364 and No. 68365. 45. The vaccine of claim 39, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the NRE sequence. 46. The vaccine of claim 39, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the vpr gene sequence. 47. The vaccine of claim 39, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the vpx gene sequence. 48. The vaccine of claim 39, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the vpu gene sequence. 49. The vaccine of claim 43, wherein said primate lentivirus is derived from SIVmac239ΔnefΔNRE. 50. The vaccine of claim 43, wherein said primate lentivirus is derived from SIVmac239Δ3. 51. The vaccine of claim 43, wherein said primate lentivirus is derived from SIVmac239Δ4. 52. The vaccine of claim 41, wherein said primate lentivirus is derived from HIV-lΔnefΔNRE. 53. The vaccine of claim 41, wherein said primate lentivirus is derived from HIV-1Δ3. 54. The vaccine of claim 41, wherein said primate lentivirus is derived from HIV-1Δ4. 55. A method of vaccination comprising administering, to a patient, a vaccine comprising an isolated primate lentivirus whose genome contains an engineered non-revertible null mutation in the nef gene, or an infectious DNA clone thereof, in a pharmaceutically acceptable carrier. 56. The method of claim 55, wherein said primate lentivirus is derived from human immunodeficiency virus. 57. The method of claim 56, wherein said primate lentivirus is derived form HIV-l. 58. The method of claim 55, wherein said primate lentivirus is derived from simian immunodeficiency virus. 59. The method of claim 58, wherein said primate lentivirus is derived from SIVmac. 60. The method of claim 59, wherein said primate lentivirus is derived from the DNA clones deposited with the ATCC and designated No. 68364 and No. 68365. 61. The method of claim 55, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the NRE sequence. 62. The method of claim 55, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the vpr gene sequence. 63. The method of claim 55, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the vpx gene sequence. 64. The method of claim 55, wherein said primate lentivirus genome further comprises an engineered non- revertible null mutation in the vpu gene sequence. 65. The method of claim 59, wherein said lentivirus is derived from SIVmac239ΔnefΔNRE. 66. The method of claim 59, wherein said lentivirus is derived from SIVmac239Δ3. 67. The method of claim 59, wherein said lentivirus is derived from SIVmac239Δ4. 68. The method of claim 57, wherein said lentivirus is derived from HIV-lΔnefΔNRE. 69. The method of claim 57, wherein said lentivirus is derived from HIV-1Δ3. 70. The method of claim 57, wherein said lentivirus is derived from HIV-1Δ4.

Inventors: DESROSIERS RONALD C, DESROSIERS, RONALD, C.
Applicants: HARVARD COLLEGE, PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Application year: 1991
Main classification: C07H15
IPC codes: A61K39, C07K14, C12N7, C12N15