AU678152B2 - Non-infectious HIV particles and uses therefor - Google Patents

Description

OPT DATE 08/11/93 AOJP DATE 13/01/94 APPLN. In' 37994/93 111 I I III PCT NUMBER PCT/US93/02142 111 N1111 11 I~JII u AU9337994 (51) International Patent Classification 5 (11) International Publication Number: WO 93/20220 C1 2N 15/86, 15/39, 7/04 Al C1 2N 5/10, G01IN 33/50 (43) International Publication Date: 14 October 1993 (14.10.93) (21) International Application Number: PCT/US93/02142 (74) Agents: GRANAHAN, Patricia et al.; Hamilton, Brook, I Smith Reynolds, Two Militia Drive, Lexington, MA (22) Int rnational Filing Date: 10 Marrch 1993(f%10.03.93) 02173 (US).

Priority data: (81) Designated States: AU, CA, JP, European patent (AT, BE, 859,346 27 March 1992 (27,03.92) us CH-, DE, DK. ES, FR, GB, OR, IE, IT, LU, MC, NL, PT, S E), (71) Applicant: WHITEHEAD INSTITUTE FOR BIOMEDI- CAL RESEARCH [US/US]; Nine Cambridge Center, Published Cambridge, MA 02142 Withi international search report.

(72) Inventors: ALDOVINI, Anna YOUNG, Richard, A. Sawmill Brook Road, Winchester, MA 01890 (US).

FEINBERG, Mark, B. 43A Worth Street, San Francisco, CA 94114 TRONO, Didier 457 Dell Court, Solana Beach, CA 92075 BALTIMORE, David7 U 1230 York Avenue, New York, NY 10021 (US).

(54)Title: NON-INFECTIOUS HIV PARTICLES AND USES THIEREFOR G i 1920 2 GC4GGCCCTA 2 013 GCAG CATCGCGAG CCTA

A

288 WT sequence bCA2O sequence: Cys-His box! Cys Phe Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys 3'Cys -His box: Cys Trp Lys Cys Gly Trp Gtu Gly His Gln Met Lys Asp Cys Intervening sequence:.

wild type -~Arg Ala Pro Arg Lys Lys Gly bCA2O -Ser le AlaoMel ProArgLysLys Gly (57) Abstract This invention related to constructs comprising mutant HIV genomes having an alteration in a nucleotide sequence which is critical for genomic RNA packaging and non-infectious, immunogenic H]V particles produced by expression of these constructs in mammalian cells. Cell lines which stably produce non-infectious, immunogenic HIV particles are also included. Prophylactic and therapeutic vaccines, diagnostic rtagents, and related methods are further described.

WO093/20220 PCr/US93/02142 NON-INFECTIOUS liIV PARTICLES AND USES THEREFOR packeround Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS), which is characterized by immune suppression resulting from selective infection and death of T lymphocytes (Sarin, Ann, _Revo 2rmacoj. 2&:411-428 (1988)).

Clinical manifestations of the disease include severe immune deficiency, which is generally accompanied by malignancies and opportunistic infections. According to current estimates from the World Health Organization, 1 in 250 people are infected with HIV worldwide.

Due to the devastating effects of the virus and the high mortality rate among HIV-infected individuals, much effort, time and money have been expended in the attempt to develop methods for preventing HIV infection (prophylactic methods) and for treating already infected individuals (therapeutic methods). However, only limited progress has been made to date.

The potential efficacy of a HIV vaccine is suggested by studies in the simian AIDS model system.

Vaccines composed of whole, inactivated virions of simian immunodeficiency virus (SIV) were found to confer at least partial protection against challenge with either live virus or cell-associated virus (Langlois, A.

I jCn 2=5:292-293 (1992); Le Grand, R., et Al., Nu :684 (1992); Osterhaus, and P. De Vries, ib., p 684-685; Cranage, M. etAl., pp. 685-6bi). It has been observed that "whole, inactivated SIV preparations induce the strongest and most consistent protection thus far experienced in experimental animal studies" (Langlois, 1992 supr).

WO 93/20220 PCT/US93/02142 -2- A major problem in obtaining whole, inactivated HIV vaccines, however, has been presented by the tradeoff between safety and immunogenicity. Killed HIV currently used in human immunotherapy trials is required to be prepared through two independent inactivation protocols, each of which must be adequate to completely inactivate the virus on its own. The physical and chemical inactivation treatments currently used have resulted in some loss of immunogenicity of the vaccine due to partial destruction of the virions. A method which leaves the virion structure intact, yet which renders the virions completely noninfectious, would be a significant improvement in vaccine development.

Besides vaccines, drugs which inhibit various stages of HIV infection of T cells and the HIV life cycle in infected cells have been suggested as another approach in the development of therapies against HIV infection. A considerable amount of information is available on viral entry, reverse transcription of the RNA genome, and expression of viral gene products. In contrast, little is known about packaging of the viral genome, assembly cf the virion, and budding of the mature virion from the infected cell. One hindrance to HIV research and drug development is the risk of infection to researchers working with reagents which are contaminated with or derived from live HIV. Thus, a means to produce HIV reagents which are totally noninfectious would relieve some of the cost, in terms of risks to workers, and necessary equipment and facilities, of drug development and HIV research.

WO 93/20220 WO 9320220PCr/US93/02142 -3- Summary of the Invention The present invention relates to HIV mutant constructs comprising a mutant HIV genome which has an alteration of a nucleotide sequence critical for packaging the HIV RNA genome, and which, when expressed in mammalian cells, produce non-inf ectious, immunogenic viral particles. HIV mutant constructs based on HIV-l and HIV-2 genomes are included. This invention further relates to cell lines, which are stably transfected with the above-mentioned HIV mutant constructs, and which stably produce non-infectious, immunogenic HIV virions.

Methods are further included for producing HIV particles which are similar in protein content and morphology to infectious HIV particles, and which are immunogenic, but which are completely non-infectious; these noninfectious mutant HIV particles have been shown to be deficient for the viral genome. The production of these mutant HIV particles, described herein, provides a means to obtain vaccines and diagnostic reagents which are based on immunogenic, but non-infectious virus particles. The production of non-infectious HIV particles further provides an alternative and advantageous method of virus inactivation, referred to as genetic inactivation, for preparation of whole virus vaccines. Such vaccines can be used~ to induce an anti- HIV response in an 4 individual, either prior to or after infection with HIV, resulting in enhanced resistance by the individual to the virus. Vaccines and reagents which contain non-infectious HIV mutant virions, and methods of prophylactic and therapeutic treatment against HIV are included in the present invention.

In particular, the present invention relates to HIV mutants defective for RNA packaging as a result of WO 93/20220 PCT/US93/02142 -4nucleotide alterations of the cij-acting RNA packaging site, referred to as the site, and amino acid alterations of the cysteine-rich motifs, alternatively referred to as the CysHis boxes or zinc-knuckle, in the carboxyl-terminal region of the Gag precursor.

HIV mutant constructs for preparing non-infectious HIV particles wit, additional improvements are described. Multiply defective HIV mutants are expected to produce non-infectious HIV virions with a very low probability of reversion to infectivity. These include HIV mutants with multiple defects in both RNA packaging functions, the cleavage site of the gpl60 envelope precursor protein, and the primer-binding site. In addition, non-infectious HIV particles with advantageous antigenic properties can be produced. HIV mutants with defective cleavage of the qpl60 precursor are expected to have increased retention of the gpl20 antigen on the surface of HIV virions. Mutant constructs containing variant envelope genes derived from different HIV strains or isolates can be used to obtain vaccines and diagnostic reagents which are tailored for particular purposes. Variant envelope genes can also be engineered by mutagenesis to increase antigenicity of the vaccines and diagnostic reagents.

Brief Description of the Drawings Figure 1 is a diagram of HIV-1 mutations. A: location and size of deletions affecting the HIV-1 site (SEQ ID No:l). B: amino acid changes in the CysHis boxes of HIV-1 Gag produced by various point mutations (SEQ ID No:2-4).

Figures 2A and 2B are a partial HIV-1 nucleotide sequence (nucleotides 1351-1980; SEQ ID No:5) and the WO 93/20220 PCT/US93/02142 deduced amino acid sequence (SEQ ID No:6) for that partial sequence. Downward arrows and box indicate the location of the mutations in HIV-1 g described herein.

Nucleotide locations are as indicated in Ratner, &t al., alre 31.:277-284 Figure 3 is a diagram of the gag-coding region of HIV-1 with nucleotide numbers indicating the initiation codon, the cleavage sites between pl7, p24 and p15, and the gag termination codon. The nucleotide differences between wild type and bCA20 (SEQ ID No:7) are indicated.

Below is shown the amino acid sequence of the two HIV-1 CysHis boxes, and of the intervening sequence, where the mutation (SEQ ID'No:8) was introduced.

Figure 4 shows results of Western blot analysis of HIV proteins in HIV-1 mutant particles.

Figure 5 shows results of Northern blot analysis of HIV RNA in HIV-1 mutant particles.

Figure 6 is a diagram of the HIV-1 mutant constructs: pAPAC1 and pAPAC-Hygro.

Figure 7 is a diagram of HIV-1 mutant construct pAPAC2.

Figure 8 is a diagram of HIV-1 mutant construct pAPAC3.

Figure 9 is a diagram of pR7neo.

Figure 10 shows the result of immunoblot analysis of cytoplasmic extracts from HT4(A-dhfr) cells.

Figure 11 shows the results of Northern slot blot analysis of viral RNA in the supernatant from HT4(AEdhfr) cells.

Figure 12 shows results of examination of HT4(R7dhfr), HT4(WT-AE-dhfr) and HT4(bCA20-Adhfr) by electron microscopy. Panel A: HT4(R7-dhfr); Panel B: HT4(WT- AE-dhfr); Panel C: HT4(bCA20-AE-dhfr). Pictures were WO 93/20220 PCT/US93/02142 -6taken at a magnification of x38,500 for panels A, B, and C, and x4,500 for the negative control in panel D.

Biological DeDosit Three deposits have been made (October 13, 1989 and October 16, 1989) at the American Type Culture Collection, Rockville, MD, in support of the subject application: a HIV-1 Gag CysHis box mutant, designated pA14-15HXB (ATCC Accession #68123); an HIV-1 Gag insertion mutant designated plasmid bCA20-dhfr (ATCC Accession #40682); and a HIV-1 site mutant, designated pA3HXB (ATCC Accession #68122). These deposits have been made under the terms of the Budapest Treaty and, upon grant of a U.S. patent, all restrictions on their availability will be irrevocably removed.

Detailed Description of the Invention This invention is based on work described herein, which demonstrates for the first time, that RNA packaging defects in HIV can result in the production of mutant virions which are similar in morphology and protein content to wild type HIV virions and are immunogenic, but which are completely non-infectious.

As described herein, mutant virions are produced by constructing plasmids containing mutated HIV genomes using recombinant DNA techniques and expressing the HIV mutant constructs in mammalian cells. Mutant virions are produced and bud off the cells into the culture medium, where they can be collected. HIV mutant constructs are also used to produce cell lines which stably produce non-infectious HIV particles. Methods for producing the non-infectious HIV particles are described herein which provide improved inactivated WO 93/20220 PCT/US93/02142 -7vaccines for prophylaxis against and therapeutic treatment of HIV infection, as well as improved methods for obtaining HIV diagnostic reagents. Methods for producing non-infectious HIV particles, HIV mutant constructs d cell lines for producing the particles, and related aterials and methods for commercial and medical use are further described below.

HTV-1 RNA Packaging: Site Mutants The central event of the packaging step is the interaction of the nucleocapsid proteins with the genomic viral RNA to form the core of the virus. This step occurs after transcription and translation of the viral proteins, and before the entire array of interacting viral proteins buds through the cell membranes as mature virions. The packaging ste[ s a very specific and efficient process, during which 'iral proteins discriminate the genomic RNA from the many spliced viral RNAs and cellular RNAs that exist in the infected cell. For instance, particles containing a spliced mRNA would be defective. Since the retrovirus preferentially packages full length genomic RNA, sequences present exclusively in this RNA but not in spliced viral or cellular RNAs must be involved in the specific RNA-protein interaction that leads to the production of infectious particles.

Viral genomic sequences required for specific packaging have been mapped in several avian and murine retroviruses. These gis-acting sequences, referred to as sites, have been located to a region near the end of the viral genome. The exact boundaries of the sites in the various retroviruses are not known, but sequences between the first splice donor site and the WO 93/20220 WO 9320220PCT/US93/02142 -8- Gag translational start site have been shown to be critical for wild type RNA packaging (Shank, P.R. and M. Linial, 2, .i.rgl. U:450-456 (1980) Mann, R. and D.

Baltimore, J, Mio V 4401-407 (1985); Linial, M. et al., gal J&1371-1381 (1978) Koyamat, Harada F. and S. Kawai, 2,Vra. 5.1:154-162 (1984); Watanabe, S. and H. M. Temin, Proc. Nati. Acad. Sci. USA 21:5980-5990 (1982); Mann, R. and D. Baltimore, J. Vig.. :401-407 (1985)).

U~sing the defined sites of murine leukemia, spleen necrosis and avian sarcoma viruses as a guide, deletion mutations were constructed in homologous sequences in the HIV-1 genome to investigate whether this region between the first donor splice site and the Gag initiation codon acts as a packaging signal for HIV.

Two site mutant constructs were constructed, whooe expression resulted in production of non-infectious HIV particles: pA3HXB, which contains a 39 bp deletion of nucleotides 293-331 (inclusive) and pA4HXB, which contains a 21 bp deletion of nucleotides 293-313 (Figure 2A). The construction of pA3HXB and pA4HXB are described in Example 2.

HJIV-1 ENA Packgaing; Gzag Mutants in studies of avian and murine retroviruses, there is also evidence that the carboxyl-terminus of the Gag precursor can interact with viral genomic RNA. In particular, a cysteine-rich motif, referred to herein as the cysHis box has been shown to be critical for RNA packaging in Rous sarcoma and Moloney leukemia viruses (Karpel, et al., o1 Chem, M:4961-4967 (1987); lleric, C. and Spahr, a. Viro.L §_0:450-45 9 WO 93/20220 WO 9320220PCT/US931021 42 -9- (1986); Meric, C. It Al. asVr 3328-3333 (1988); Meric, C. and S.P. Goff, jVr. §:1558-1568 (1989); Prats, A.C. t,_j o LDuj 2: 1777-1783 (1988); Gorelick, R. Z70J Proc. Natj. AcAd. Sci.

M Uj:8420-8424 (1988)). The cysHis box is present in the carboxyl-terminus of all retroviral Gag precursors and has the consensus sequence: CysX 2

CYSX

3 GlyHiSX 4 Cys (Berg, Sence =:485-487 (1986)). This motif occurs once in the murine retroviruses, and twice in most other retroviruses studied thus far. Cysteine-rich motifs have been implicated in nucleic acid binding through analogy with the "zinc-finger" motifs present in a wide variety of eukaryotic transcription factors (Evans, R. M4. and S. M4. Hollenberg, go," U:1-3 (1988); Berg, 1986 fiU= In retrrvi'jral nucleocapsid proteins, these sequences may also play a role in protein-protein interactions.

in the HIV infectious cycle, the Mg gene is expressed as a protein precursor, Pr 5 5 9ag, which is processed and cleaved into the inatur- viral proteins, p17, p24, and p15. p15 is believed to be cleaved f urther into p9 dnd p7 (Mervis, R. Z~ Mj, 3993-4002 (1988) Veronese, F. D. M4., I sl J. iro.j: 1:95-801 (1988)). The exceedingly basic character of p15 suggests that it might be associated with the viral R~NA (Gelderblom, H. etJ..

JjU:l71-176 (1987)).

The HIV p15 is 123 amino acids long and encoded by the 3' end of the _Aaq gene (Figure It carries striking similarities with other retroviral nucleocapsid proteins. These similarities include, in the p9 region, two tandem copies, separated by seven amino acids, of a CysHis box (see Ficrures 1B, and 3) (Covey, S. N., WO 93/ 2220 PC/US93/02142 Nucleic Acids Resg. i:623-633 (1986)). To investigate the role of the CysHis box region in packaging of the HIV genome, five HIV-1 mutants were constructed, as shown in Figures 1B and Mutant constructs pA14HXB and pA15HXB each encode alteration of a single CysHis box: pA14HXB encodes tyrosine substitutions for Cys, and Cys 3 in the 5' CysHis box (SEQ ID No:2) and encodes corresponding substitutions in the 3' CysHis box (SEQ ID No:3). Mutant construct pA14-15HXB encodes tyrosine substitutions for Cys, and Cys 3 in both CysHis boxes (SEQ ID No:4). pACH1-2HXB encodes a mutant Gag protein with a 35 amino acid deletion of both CysHis boxes and their intervening sequence. Mutant construct (Figure 3) encodes an addition of Ser-Ile-Ala-Met to the intervening peptide sequence immediately after Cys 14 of the 5' CysHis box (SEQ ID No:8), thus, changing the distance between the two CysHis boxes. Mutant constructs pA14HXB, pA15HXB, pA14-15HXB, pACH1-2HXB and were each found to produce non-infectious HIV particles. The construction of the gAg mutants are described in the Examples.

Production of HIV-1 Mutant Particles To observe the effect of the RNA packaging mutations, COS-1 (African Green Monkey kidney) cells were transfected with the above-described HIV mutant constructs for transient expression. The constructs contain the mutated HIV genomes in vectors with an origin of replication. In COS cells, which express the SV40 large T-antigen, these vectors are replicated in high copy numbers. HIV cannot normally infect COS cells because they lack the CD4 receptor, but once the HIV genome is transfected into these cells, virus is WUa 9 3 /2 4221 PCT/US93/02142 -11efficiently produced. The expression of viral gene products in these cells can be monitored, and the viral particles released into the-supernatant can be collected and characterized.

The virions produced by expression of an HIV construct are referred to herein by the name of the construct but without the preceding for example, A3HXB mutant virions are produced from the pA3HXB mutant construct.

Viral gene expression in the transfected cells was monitored by Northern Blot analysis and by metabolic labelling and immunoprecipitation of viral proteins.

Northern blot analysis showed that the patterns of viral RNA from COS cells transfected with the mutant HIV constructs, pA3HXB, pA4HXB, pA14HXB, pA15HXB, and pA14were identical to RNA from cells transfected with a wild type HIV construct, pHXB2gpt. In each case, all three classes of HIV-1 RNA were present: the 9.2 kb genomic RNA, a 4.3 kb spliced mRNA encoding the Mnl and yij genes, and the heterogeneous RNAs at about 2 kb, which includes tat-IlI, rev, and neg mRNAs. Thus, these HIV-1 mutations do not appear to affect the expression of viral RNAs.

Immunoprecipitation of viral proteins expressed in the transfected COS cells revealed that all of the major structural proteins of HIV-1 are present in cells transfected with either wild type (pHXB2gpt) or mutant (pA3HXB, pA4HXB, pA14HXB, pA15HXB, and pA14-15HXB) HIV-1 constructs. The presence of gpl60, gpl20, gp41, p24, p17, and p15 in all of the transfected cells indicates that the HIV-1 mutants do not produce major alterations in the synthesis and processing of Gag and envelope precursors.

WO 93/20220 PCT/US93/02142 -12- The amount of HIV virions produced was quantitated by two assays. One was an ELISA that permits assessment of the level of virus-associated p24 capsid protein. The other was an enzymatic assay that measures the amount of reverse transcriptase activity associated with viral particles. After transfection of COS cells with the HIV mutant constructs, viral particles were pelleted, and analyzed as described above. The results of the p24 ELISA and reverse transcriptase assays are shown in Table 1 (at the end of the Detailed Description). The plasmid pHXB2BAMp3 was included as a negative control; it does not produce virus, due to a post-transcriptional defect. These results show that no major differences were observed in the amounts of the two proteins between wild type and mutant particles, indicating that similar levels of wild-type and mutant particles were produced by the transiently transfected celis.

Infectivity of the HIV-1 Mutant Virions The infectivity of the wild type and packaging mutant virions were assayed on H9 T lymphocytes, which are susceptible to HIV infection. Three different assays were performed during a time course experiment after exposing H9 cells to the supernatants from the transfected cells. The three assays were the following: 1) immunofluorescent staining (IF) with a mouse monoclonal antibody for p24 to measure of infected cells; 2) core protein p24; and 3) reverse transcriptase (RT) in the supernatant of H9 cells to measure virus released from infected H9 cells. Samples were taken at 3, 6, 9, 12, 16, and 30 days after infection. As shown in Table 2, all the packaging WO 93/20220 PCT/US93/02142 -13mutants were negative in all three assays up to 30 days after infection. Only wild type virions produced from the pHXB2gpt construct scored positive in these assays.

These data indicate that the HIV packaging mutant particles are completely non-infectious.

Biochemical Composition and Morphology of the Mutant Virions The Northern blot and immunoprecipitation analyses described above indicated no difference in the accumulation of viral RNA and proteins in cells transfected with the mutant and wild type constructs.

Western blot analysis was further performed to compare the viral protein composition of the mutant and wild type viral particles; the blot was probed with HIV positive human serum. As shown in Figure 4, the protein composition of the site mutant virions, A3HXB and A4HXB, and the gag mutant virions, A15HXB and A14-15HXB, was similar to wild type HXB2gpt virions. A14HXB virions, not shown in this figure, gave results similar to A15HXB virions. In each case, viral proteins p66, p51, gp41, p31 and p24 were observed in about the same relative amounts.

site mutant virions A3HXB and Mgg mutant virions A14-15HXB were then examined for RNA content by Northern dot blot analysis. As shown in Figure 5, no genomic RNA was detected in the mutant particles in contrast to wild type HXB2gpt virions. These data indicate that there is at least a 100-fold reduction in the RNA content in both site and CysHis box mutants relative to wild type.

The morphology of mutant HIV parti~ss was examined by electron microscopy. This analysis owed that viral capsids could assemble in the absence of RNA packaging, WO 93/20220 PCT/US93/02142 -14indicating that mutations in the CysHis box of p 7 9'9 can abolish infectivity without affecting virion assembly.

Careful scoring of the sections indicated that the majority of the mutant particles were less electron dense than wild type viral particles. This morphology is typical of an immature particle in which viral protein precursors have not been processed. It is possible that the lack of RNA affects the rate of particle maturation or the structural condensation of processed precursors.

Further Improvements of HIV Mutant Constructs Additional HIV-1 mutant constructs were made for producing non-infectious virions with further improvements. These improvements are expected to possess advantages with regard to safety and antigenicity of mutant virion preparations.

RNA packaging mutants with multiple defects are expected to result in non-infectious HIV virions with extremely low probability of reversion to infectivity.

Mutant constructs, pAPACl and pAPAC-Hygro, were made which contain efficacious alterations in both the site and the Gag CysHis region (Figure The alterations are referred to as efficacious because each alteration alone results in non-infectivity.

Defects in other HIV functions can be added to the packaging defects to decrease even further the risk of reversion. Mutation of the cleavage site of the envelope precursor gpl60 has been shown to result in non-infectivity (McCune, J. &t Al., Cell 5.1:55-67 (1988)). A triple defective HIV mutant construct, pAPAC2, has been made, which contains efficacious alterations of the site, the Gag CysHis box region, WO 93/20220 PCT/US93/02142 and the gpl60 cleavage site (SEQ ID No:9) (Figure 7).

In addition, deletion of the primer binding site, the site near the 5' end of the HIV genome at which a tRNA primer initiates reverse transcription of the viral genome, is expected to abolish infectivity (Prats, A.

et Al., J. EMBO 7:1777-1783 (1988)). A primer binding site deletion is shown in Figure 8.

Multiply defective HIV mutant constructs can be made containing any combination of the efficacious mutations in RNA packaging and gpl60 cleavage and primer binding functions described herein. The site mutation can be the 39 bp deletion of pA3HXB or the 21 bp deletion of pA4HXB (Figure 1A). The Gag CysHis mutation can result in substitutions in the 5' CysHis box, as in pA14HXB, the 3' CysHis box, as in pA15HXB, or both CysHis boxes, as in pA14-15HXB; or deletion of one or both CysHis boxes, as in pACHl-2HXB (Figure 1B).

Additional mutations of the Env gpl60 cleavage site, as in pAPAC2, and of the primer binding site can be included. An example of a multiply defective HIV construct which contains mutations in all four functions is pAPAC3 shown in Figure 8.

Improvements with regard to immunogenic properties of the non-infectious HIV particles can also be made.

Mutations of the gpl60 cleavage site which block the processing of the gpl60 envelope precursor to gpl20 and gp41 may reduce the "shedding" of gpl20 antigen usually observed during preparation of concentrated HIV virions; the gp41 portion of gpl60 is a transmembrane peptide and may serve to anchor more firmly the gpl20 antigen to the surface of the virion. This increased retention of antigen is expected to improve the immunogenic properties of HIV virions produced from gpl60 cleavage W( 3/20220 W( 3/0220PC'/US93/02142 -16site mutants. The gp16O cleavage site mutation in pAPAC3 is defective for both gpl6O processing and infectivity, and thus, can be used to produce noninfectious virions with the potentially improved immunogenic properties.

Another means for improving the immunogenic performance of the non-infectious HIV particles in vaccines is to use a strategy in constructing the HIV mutant constructs which will be referred to herein as My replacement technique. The envelope protein is the the surface antigen of HIV and also the most variable protein among HIV isolates. For example, HIV isolates from different geographical locations will contain variable gjzv gene sequences. Many variant genes have been cloned and sequenced, and are available from the AIDS Research and Reference Reagent Program of the U.S. Department of Health and Human Services, National Institutes of Health (National institute of Allergy and Infectious Diseases, 6003 Executive Boulevard, Bethesda, MD 20892). The Wfly replacement technique involves replacing the native Vjv gene in a HIV mutant construct with a variant gene from aiiother HIV strain or isolate in order to tailor the resulting non-infectious HIV particles to a specific use. For example, the mutant particles can be used to vaccinate against HIV strains which are prevalent in a population or against particularly virulent strains. In another ex~iple, noninfectious particles used in therapeutic treatment of an infected patient, for instance, to prevent the onset of AIDS symptoms, can be tailored to the particular strain of HIV infecting the patient by isolating virus from the patient, cloning the &nv gene and replacing the env gene in the mutant construct with the cloned sequence.

WO 93/20220 PCT/US93/02142 -17- Alternatively, vaccines can be prepared by combining HIV particles containing a spectrum of various Env proteins for wider protection. The env replacement technique is also useful for obtaining HIV diagnostic reagents which are more specific or more general in detecting various strains of HIV. Variant en genes can also be engineered by mutagenesis of naturally occurring genes in order to produce HIV mutant particles with improved antigenic or immunogenic properties. For example, the Env glycosylation sites could be removed. Env replacement in HIV mutant constructs can be carried out using polymerase chain reaction and other recombinant DNA techniques, as described in the Examples.

Stable Producer Cell Lines Mammalian cell lines have been generated which stably produce non-infectious HIV virions. The cell lines are produced by stable transfection of cultured cells with HIV mutant constructs containing the packaging and other functional defects described above.

Stable transfection procedure includes a selection step for cells which have incorporated the constructs; thus, the transfecting DNA usually contains a selectable marker, such as the ngf gene for G418 resistance or hy=r for hygromycin resistance. A method of selection for stable transfectants is described herein which is improved over other methods. Previously described methods involved cotransfection of DNA containing the selectable marker with DNA containing the construct of interest, or transfecting with constructs in which the selectable marker is placed under a separate promoter from the coding sequence of interest, usually in the vector. In the selection method described herein, the WO 93/20220 PCT/US93/02142 -18selectable marker is transcribed from the same promoter as the coding sequence(s) of interest, in this case, the HIV LTR. Figure 9 shows an HIV construct, pR7neo, in which the nef gene is replaced by a selectable marker (Dng). The ne gene does not appear to be essential for the replicative cycle of HIV, since cell lines stably transfected with pR7neo produce wild type, infectious HIV virions. The advantage of this selection method is that expression of the selectable marker is indicative of expression of the coding sequence of interest, and that integration of the construct in the cell genome is such that the transcriptional unit containing the coding sequence and the selectable marker is functional in the cell. This selection method is, thus, more stringent than the former methods, in which expression of the selectable marker is not always indicative of the desired clone.

The construct, pR7neo, is the parent vector from which the HIV mutant constructs used to produce stable producer cell lines were derived. Other HIV mutant constructs for producing stable producer cell lines can be made by engineering combinations of the RNA packaging, Env gpl60 cleavage site, and primer binding site defects described above into pR7neo. In addition, selectable markers other than neo can be used to replace the n gene.

Stable producer cell lines have been generated from COS, HeLa, and H9 cells, as described in the Examples.

All three types of cell lines have been found to produce large amounts of HIV particles. In the case of H9derived cell lines, the amount of particles produced per cell has been observed to exceed production from live WO 93/20220 PCT/US93/02142 -19virus infection by 40-100 fold. This is a significant additional advantage in terms of production cost.

Other cell lines may also be used to produce noninfectious HIV particles. For example, FDA-approved cell lines may be more convenient for clinical trials.

ImmJunenic erties of Non-infectious HIV Particles Non-inf& tious HIV particles produced as described herein were shown to induce antibody responses in mice upon injecting the animals with the HIV mutant particles mixed with an appropriate adjuvant. These results indicate that the non-infectious HIV particles provided by this invention are immunogenic.

Further Commercial and TheraDeutic Uses The present invention provides means for producing HIV mutant particles, which have a similar protein composition and morphology to wild type virons and are immunogenic, but which are completely non-infectious.

Furthermore, means are provided for producing HIV mutant particles which are expected to have a very low probability of reversion to infectivity and improved immunogenic properties. The non-infectious HIV particles provided by this invention can be used to obtain improved anti-HIV vaccines and diagnostic reagents. Vaccine compositions and diagnostic reagents related to HIV can now be obtained by the expression in mammalian cells of HIV mutant constructs containing RNA packaging and other mutations, as described herein.

These materials can be produced by transient transfection using the constructs or by generating stable producer cell lines by stable transfection with the constructs. Production of non-infectious HIV WO 93/20220 PCT/US93/02142 particles by growing stable producer cell lines is a safer, faster, and more cost-effective method of preparing vaccines and diagnostic reagents than infection with live virus.

The HIV mutant constructs described above are based on HIV-1 genomes. However, constructs based on HIV-2 genomes can also be made using similar methods.

Analogous site mutations can be made in the region between the first splice site and the Gag initiation codon. The CysHis box sequences of HIV-2 and HIV-1 are identical; thus, similar tyrosine substitutions of the first two cysteines of either or both CysHis boxes and deletion of the entire CysHis region are expected to result in non-infectious HIV-2 particles. The Env cleavage site of HIV-2 is defined, and can be altered in an analogous manner to the HIV-1 mutation described herein. The HIV-2 primer binding site is also defined, and can be entirely deleted.

Production of non-infectious HIV particles by efficacious mutation of the HIV genome, particularly in sequences critical for RNA packaging, provides a method of inactivating HIV virus for whole virus vaccines.

This method of genetic inactivation is less destructive to the virion structure and antigenic surface than heat, chemical cross-linking, and irradiation inactivation methods. Vaccines prepared by a combination of genetic inactivation and one of the other methods are expected to have improved immunogenic properties than virus preparations twice undergoing physical or chemical inactivation. Immunogenicity can be further improved by increased retention of the gpl20 surface antigen and by replacement of the env gene with heterologous env genes, as described above.

WO 93/20220 WO 9320220PC/US93/02142 -21- Vaccines can be prepared which contain inactivated, whole virus particles or antigenic portions of the noninfectious virions. The vaccines can be delivered in an appropriate physiological carrier, such as saline. The carrier can contain an adjuvant, such as BCG (Mycobacterium k2A bacillus Calmette-Guerin) Diagnostic reagents can also be prepared which contain whole or protein derivatives of the noninfectious HIV particles. Protein derivatives can be obtained by disrupting the viral particles, for example, in a buffer containing a detergent. Reagents made from non-infectious HIV particles and their protein derivatives can be used in place of the live virus and live virus derivatives currently used in diagnostic methods. For-examzple, ELISAs or Western blots may be performed to detect anti-HIV antibodies in blood saauples. Reagents made from non-infectious HIV particles and protein derivatives would be safer, easier, and less costly to prepare. As described above, stable producer cell lines can produce up to 40-IOOX the amount of HIV particles per cell than that obtained by infection with live virus.

In addition, stable producer cell lines can be used to produce non-infectious STV particles for immunotherapy and prophylaxis trials in simian animal models for AIDS.* SIV mutant constructs with RNA packaging, Env gp16O cleavage and primer binding site mutations corresponding to the HIV mutations described herein have been made in parallel for this purpose.

in addition, stable producer cell lines provide a convenient and safe in~ vir model system to study postinfection events in the HIV life cycle. Increased knowledge of mechanisms by which HIIV reproduce in WO 93/20220 PCT/US93/02142 -22mammalian cells may lead to novel therapies for preventing or controlling HIV infection.

Furthermore, stable producer cell lines provide a safe and convenient method for identifying drugs which inhibit production of HIV particles from infected cells.

For instance, the drugs may disrupt virion assembly or budding. HIV inhibitory drugs could be used to reduce the severity of disease in an infected individual.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

Cell Lines and Viruses The simian virus 40-transformed African green monkey cell line, COS-1, has been described (Gluzman, 2a:175-182 (1981)). The cell line was obtained from G. Khoury (National Institutes of Health) and is available from the American Type Culture Collection (Rockville, MD). COS-1 was maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. The H9 and H9 IIIB T-lymphoid cell lines were maintained as described previously (Popovic, et Al., 22A4497-500 (1984)). Viral infections and reverse transcriptass assays were carried out as described previously after filtration of culture supernatants through a 0.45-gm-pore-size filter (Millipore Corp., Bedford, MA) (Popovic, I= 1984 Daniel, M. pt Al., M61201-1204 (1985)).

WO 93/20220 WO 9320220PCr/US93/02 142 -23- Mutacienesis of the HIV-1 G~enome and Plasmid- Congjtrugtiqfl The parent DNA used for these studies is the biologically active clone, pHXB2gtp (Fisher, Al., HA= (London) 2U~:262-265 (1985)). The =I51 5.3 kilobase pair (kb) fragment (nucleotides 39 to 5366) was cloned in Ml3mpl8, and oligo-mediatei site-directed mutagenesis was performed on the single stranded DNA according to the protocol of Kunkel, T. ItAl (Methods ill Enzymo~ocitv ;U:367-382 (1987)). Nucleotide positions in the HIV-1 genome are numbered as previously described (Ratner, H AlAt3ar =:277-284 (1985)).

Deletions and substitutions of the H-IV-1 genome were made by site-directed mutagenesis using synthetic primers having appropriate segments of the genomic sequence. For example, the following oligomers were used to obtain the site mutations: A3: TGACGC~rcTCGCACCCATCTCTCACCAGTCGCCGCCCTC (SEQ ID for dgleting nucleotides 293 to 331; and M4: CTCTCTCrTTCTAGCCTCCGCTCACCAGTCGCCGCCCCT(', (SEQ ID Nor11) for deleting nucleotides 293 to 313; and the gpq mutations: A14: TGCCCTTCTTTGCCATAATTGAAATACTT. ACAA'TCTTTC (SEQ ID No:12), for changing guanines at positions 1508 and 1517 to adenines and TGTCCTTCCT?1TCGATATTTCCAATAGCCCTTTTTCCTAG (SEQ ID No:13), for changing G at positions 1571 and 1580 to A.

The RNA packaging mutations in pA3HXB, pA4I{XBO pA14HXB, pA14-15HXB were made using these primers.

Deletion of the entire CysHis box' coding region for constructing pACfl1-2HXB and of the 18 bp primer binding site, as illustrated in Figure 8, was performed similarly using appropriate primers.

WO 93/20220 PCT/US93/02142 -24- The introduction of the mutations was verified by sequencing different M13mpl8 recombinant DNAs using two specific oligomers: AS: CCATCGATCTAATTCTC (SEQ ID No:14) and A16: GGCCAGATCTTCCCTAA (SEQ ID (Ausubel, t Al., Current Protocol in Molecular Biolo y, Green Publishing Associates and Wiley Interscience (1987)).

A &UhII/BalI 1.9 kb DNA fragment from the mutated Ml3mpl8 recombinant clones was used to transfer all the different mutations to the wild-type plasmid pHXB2gpt.

The entire region that had been transferred from the M13mpl8 plasmids was sequenced using double stranded DNA sequencing methods (Ausubel, F.M. at A 1987 supra) to confirm the presence of the desired mutations and the absence of any other alterations. All DNA manipulations were according to standard procedures.

Mutation of the Env gp160 cleavage site, as illustrated in Figure 8, has been described in McCune, J. Lt aL., which is hereby incorporated by reference .1:55-67 (1986)).

Replacement of the nLe gene with a selectable marker has been described in Trono, et Al., which is hereby incorporated by referencel :113-120 (1989)).

Replacement of the native env gene with a variant gene is carried out by polymerase chain reaction using DNA containing the variant HIV genomic sequence as templates and primers containing restriction sites appropriate for cloning the amplified DNA into the HIV construct. Restriction sites can be engineered into the HIV genome by site-directed mutagenesis, if necessary.

DNA containing HIV genomic sequences of various HIV strains and isolates can be obtained from the National WO 93/20220 PCT/US93/02142 Institutes of Health repository, or can be obtained from HIV-inrected patients by isolating the virus and cloning the viral genome. In addition, mutated SnX genes, including genes encoding defective glycosylation sites, can be engineered by site-directed mutagenesis.

Transfection of Mammalian Cells with HIV Constructs COS-1 cells were seeded at a density of 106 per 100 mm plate, 24 hours prior to transfection, in DME medium supplemented with 10% fetal calf serum and incubated at 37°C in 5% CO 2 Transfection was carried out using Ag of plasmid DNA/plate as described (Chen, C. and H.

Okoyama, Mol. Cell. Biol. 72:745-2752 (1987)).

When viral particles were destined for RNA analysis, transfections in COS cells were performed according to the method of Seldon t Aal. (Seldon, R. F., Si MHol. Ce Blol, :3173-3179 (1986)) to avoid the heavy DNA contamination of samples that was observed when calcium phosphate transformation was performed.

Stable producer cell lines were generated by selecting for stable transfectants possessing antibiotic resistance conferred by the selectable marker in the construct. Transfection was carried out by either the CaPO 4 or electroporation methods. Stable transfectants were selected using G418 or hygromycin (Gibco) at appropriate concentrations for the cell line, as determined by killing assays. In general, a concentration was used which gave 80% cell death in a week. The resistant clones were collected and pooled, and grown in the presence of the antibiotic. Over time, the pooled stable producer cells increased their HIV particle production as the cell population purified under continued selective pressure. Alternatively, WO 93/2022C PCT/US93/02142 -26resistant clones could be purified through several rounds of replating rather than pooled. Individual clones could be tested for maximum HIV production.

Analysis of Viral RNA For analysis of RNA in transfected cells: RNA was extracted from COS-1 cells 48 hours after transfection, using the hot phenol method described by Queen and Baltimore (Queen, C. and D. Baltimore, gell 21:741-748 (1983)). For Northern blot analysis, 10 jig of DNase Itreated total cellular RNA was used per lane. RNA from mock transfected COS-1 cells was used as a negative control and 5 Ag of RNA from H9 cells, chronically infected with HXB2 virus, was used as a positive control The HIV-1 specific probe was a 3 2 P-labelled full length viral DNA.

For analysis of RNA in viral particles: RNA was extracted from supernatants containing equal amounts of p24 in the presence of equal amounts of added tRNA; the tRNA was used to monitor the final recovery of RNA. RNA samples were resuspended in water at identical concentrations of tRNA (1 Ag/ml) and 1, 0.3 and 0.1 equivalents of RNA were loaded on nitrocellulose, where one equivalent represents the amount of RNA obtained from-COS-1 supernatants containing 18 ng of p24. RNA Slot Blot analysis was performed as previously described (Ausubel Al., 1987 su ra). A 3<8 kb =AI/=ERI gAgpol fragment from pHXB2gpt was labelled with 32 P by random priming and used as probe for the Slot Blot.

WO 93/20220 PCT/US93/02142 -27- Analysis of Viral Proteins For analysis of proteins in transfected cells: COS-1 cells (4x10 6 transfected 48 hours earlier with gg of each plasmid were labelled for 4 hours with 500 gCi of 35 S methionine. As a negative control, cei.ls were transfected with the plasmid pHXB2Bamp3, which does not produce virus due to a post-transcriptional defect (Feinberg, M. et Al., el 46:807 (1986)). Cell lysates were prepared and immunoprecipitations were performed as described (Veronese, F. et Al., Cll 46:807 (1986)), using HIV-1 positive human serum which had demonstrated reactivity with all known viral structural proteins (Feinberg et al., 1986 sup2r)).

Immunoprecipitated proteins were resolved using a SDS-polyacrylamide gel (Laemmli, U. Nature 2f:680 (1970)).

For analysis of proteins in HIV particles: virus was pelleted by centrifugation for three hours at 27,000 rpm in a SW27 rotor. The pellet was resuspended in dissociation buffer (0.01 M Tris-HCL pH 7.3, 0.2% Triton X-100, 0.001M EDTA, 0.005M dithiothreitol (DTT), 0.006M KCL), if reverse transcriptase activity was to be measured, or in 0.2% Triton and Laemli buffer if protein analysis was to be performed. Western blot analysis and radio-immunoprecipitations (RIP) followed the procedure of Veronese et Al. (Veronese, F. et Al., Proc, Natl. Acad. Sci. USA 9:5199-5202 (1985)). p24 analysis on tissue culture supernatants or on pelleted virus was performed.

Amounts of p24 gag protein (ng/ml) in the supernatant of each mutant were determined 48 hours after transfection. Each transfection was overlayed with 10 ml of medium. A DuPont p24 ELISA kit was used WO 93/20220 PCT/US93/02142 -28and three different dilutions of each supernatant were analyzed. RT activity was measured after concentrating 3 ml of COS-1 supernatant from transfections of each mutant by centrifugation for three hours at 27,000 rpm.

:umbers refer to 1 ml of supernatant and are the mean of three experiments. Analysis of protein content of wildtype and mutant particles by radio-immunoprecipitation was also done.

Infectivity Assays H9 cells were infected by filtered (0.45 Am, Millipore) supernatants from COS-1 cells that had been transfected 48 hours. Immunofluorescence assays were performed with murine monoclonal antibody specific for the p 24 9ag protein (Veronese, F. st al., Pro atl.

Acad. Sci. USA 1.:5199-5202 (1985)) and the h9-HIV IIIB cell line as a positive control. p24 assays were performed with a p24 ELISA (DuPont Reverse transcriptase assays were carried out after filtration of culture supernatants (Daniel, M. t, Al., Science 22:1201-1204 (1985)).

Construction and Analysis of To address the role of HIV-1 p15gag, a mutation was introduced into plasmid W13 (Kim, e tal J. Virol.

63:3708-3713 (1989)), which contains an infectious copy of the HIV-HXB2-D proviral DNA. (Shaw, Al., Science 26:1165-1171 (1984)). W13 was modified by inserting an 8-nucleotide-long =AI linker in a unique AbaI site present at position 1549, and then blunting this lAI site with Klenow to rectify the Gag reading frame. The mutated construct thereby obtained is called bCA20-W13.

The mutation results in the replacement of the two PCT/US93/02142 WO 93/20220 -29residues which immediately follow the first CysHis box, arginine-alanine, by a stretch of four amino acids, serine-isoleucine-alanine-methionine (Figure 3).

Therefore, both the amino acid sequence and length of the intervening sequence between the two CysHis boxes is altered.

To analyze the phenotypic consequences of the mutation, COS cells were transfected with bCA20 and generation of viral particles was scored by measuring the amount of p24 antigen as well as the reverse trans-riptase activity released in the supernatant (Table 3).

p24 and reverse transcriptase activities were approximately 40% and 30% of wild-type, respectively. This indicated that the mutation present in bCA20 only mildly interfered with the release of viral particles. The COS cell supernatant was then used to infect H9 cells, which were followed by an indirect immunofluorescence as6iy (Ho, et A3, Scince Zj6:451-453 (1984)), using serum from an HIV-infected individual as detector antibody. After three weeks, no positive cells were seen. This showed that the particles generated following transfection were noninfectious. Therefore, it could be concluded that the bCA20 mutation was lethal for viral replication.

To further study the consequences of the p 15 9 ga mutation contained in bCA20, a cell line which constitutively expresses an Env- version of this mutant was generated. Expression of the HIV gag gene products is sufficient to generate viral particles in the absence of Env. Such ce, 1 lines are made as follows: Briefly, HT4-6C cells (a HeLa cell line expressing the CD4 molecule at its surface were transfected with construct WO 93/20220 PCT/US93/02142 a modified version of bCA20-W13 which contains a translational frameshift in the env gene and the mutant dihydrofolate-reductase gene in place of the ni£ reading frame. The HT4-6C cells were a gift from B.

Chasebro (Chasebro, B. and Wehrly, J. Virol.

2a:3779-3788 (1988)). Cells were selected for resistance to methotrexate, cloned, and analyzed by polymerase chain reaction to check for the presence of the viral integrant. Indirect immuno-fluorescence, using serum of an HIV-infected individual as detector antibody, was also performed. The immunofluorescence seen in HT4(bCA20-AE-dhfr) was similar to that observed in HT4(WT-AE-dhfr), which expresses a wild-type ga sequence. p24 antigen and reverse transcriptase activity were also measured in the supernatants of these cell lines; the ratio of activity of bCA20 to wild-type was grossly similar to those observed with the transient transfection of the corresponding W13 viral constructs (Table In addition, Western blot analysis of cytoplasmic proteins was performed as described previously by Trono and co-workers (Trono, F A~l., Cel, October 6, 1989), using an anti-p24 monoclonal antibody (a gift from F. Veronese) as detector antibody.

HT4(bCA20-AE-dhfr) and HT4(WT-AE-dhfr) g've similar patterns (Figure 10). Therefore, it could be concluded that the mutation present in bCA20 did not affect the synthesis, the cleavage or the stability of the Gag precursor.

To ask whether the mutation contained in bCA20 had deleterious consequences on the packaging of the viral RNA, a slot-blot analysis of the supernatant from HT4(WT-AE-dhfr) and HT4(bCA20-AE-dhfr) cells was performed. For this, 700 Al of culture medium was mixed WO 93/20220 PCT/US93/02142 -31with 35 i1 of 10 mg/ml proteinase K (Boehringer, Mannheim), 7 Al of culture medium was mixed with 35 p1 of 10 mg/ml tRNA, 3.5 jul 0.5 M EDTA, 17.5 Al 20% SDS, incubated at 37° C for 45 minutes, phenol-extracted and ethanol precipitated in 0.4 M NaCl. The RNA was resuspended in 20 Al mM EDTA, denatured in formamide-17% formaldehyde, heated to 50° C for minutes, mixed with 120 Al 15XSSC, and bound to nitrocellulose by aspiration through a slot blot apparatus. Hybridization was then performed as previously described (Trono, et Ai., J. Virol.

§2:2291-2299 (1988)), using a [32p] probe generated with T7 polymerase, which is complementary to nucleotides 8475 to 8900 of the HIV-1 genome. After hybridization, the filter was washed in 0.2xSSC three times at 680 C and exposed to X-ray film. Results showed that the amount of viral RNA present in the supernatant from HT4(bCA20-AE-dhfr) was dramatically reduced, compared to the control cell line, HT4(WT-AE-dhfr) (Figure 11).

Therefore, it was concluded that the bCA20 mutation specifically inhibited the packaging of the viral genomic RNA into particles.

Both cell lines were also examined by electron microscopy, to see if the defect in viral RNA packaging correlated with morphological differences (Figure 12).

The morphology of the virus particles observed in HT4(WT-AE-dhfr) was very similar to that observed in HT4(R7-dhfr), a cell line infected with an Env replication competent version of the same virus: cellreleased, "mature" particles contained a condensed core surrounded by the viral lipid bilayer (Figure 12, Panel A and Panel By contrast, in particles released from HT4(bCA20-AE-dhfr), two dramatic differences were noted.

WO 93/20220 PCT/US93/02142 -32- First, the diameter of the particles was approximately larger than observed in the controls; second, the electron-dense region was tightly apposed to the membrane, but the center of the particles was strikingly electron-luscent (Figure 12, Panel Still, approximately 1% of the bCA20 particles had a morphology close to that of wild-type, probably because of some leakage in the bCA20 phenotype, as already suggested by the RNA hybridization study on the cell supernatant.

These electron microscopy findings indicate that the inability to package the viral RNA in bCA20 particles is accompanied by an increased diameter and an absence of "collapse" of the inner componenrts of the virion, which normally reflects the final steps of maturation. It remains to be determined whether this block of maturation is primarily due to the absence of viral RNA in the particle, or is a direct consequence of the aberrant p 15 g ag protein. Interestingly, the HIV-l virions produced by cells transfected with the p 15 9 gag 9 variant provirus bear a notable resemblance to the virus-like particles released from Spodontera ruierda insect cells infected with a recombinant baculovirus expression vector encoding the p 5 7 gag precursor of the simian immunodeficiency virus, SIVmac (Delchambre, et Al., EBO J. 8:2653-2660 (1989)).

Comparison of the morphologic features of the RNA-minus particles produced in these diverse setting suggests that the viral RNA itself may play an important role in the structural organization and maturation of the mature retroviral virion.

In conclusion, as a result of this assessment of the phenotype of an in vitro-engineered HIV-l variant, which contains a mutation between the two CysHis WO 93/20220 PCT/US93/02142 -33boxes of the p 15 9ag protein, it has been demonstrated that this domain is critical to the packaging of the genomic RNA into the virus particle. In addition, it has been shown that the RNA-deficient phenotype generated by the p15ga9 lesion is associated with striking morphological anomalies, as shown by electron microscopy. Importantly, results indicate that it is possible to generate HIV particles that do not contain the viral genome. This is of primary relevance for the development of a vaccine strategy based on intact, fully immunogenic, but non-infectious virus particles.

TABLE 1 p24 Content and Reverse Transcriptase Activity Virus Assc-liated p24 (ng/ ml) Reverse Transcriptase Activity 32 P dGTP cpm/ml/min) Plasmid Construct pHXB2gpt 1.90 5060 pA311XB 1.70 3133 pA4HXB 1.60 2718 1.75 3436 pAl4-ISHXB 1.65 2949 pflXB2Banp3 0.00 740 WO 93/20220 WO 930220P/US93/02142 TABLE 2 Plasmid Construct pI{XB2 gpt pA3HXB pA4HXB pAl 4HXB pHXB2gpt pA3HXB pA4HXB pAl 4HXB pHXB2gpt pA3HXB pA4HXB pAl 5HXB pAl4HXB IF of positive control) 0.5 5 30 65 90 negative negative negative negative pz4 (na/mll >20 >20 >20 >20 >20 negative negative negative negative Reverse Transcriptase 3 2 p dGTp C]m/ml/min X 103) nd negative negative negjative negative 32.0 91.4 156.6 56.7 56.6

TABLE

p24 Antigena and Reverse Transcriptase (RT) Activityb In the Supernatant of Transfected COS Cells and 11T4 (AE-dhfr) Cell Lines p24 antigen (ng/ml) liT activity (cpxn/ml) COS Transfectants W13 (WT 120 23,000 bCA2O-W13 50 7,300 HT4 Cell Lines HT4 (WT-AE-dhfr) 400 75,000 11T4 (bCA20-AE-dhfr) 180 20,000 a p24 antigen was measured using an Elisa assay system (DuPont-NEN, Inc., Billerica, MA).

b RT activity was determined as described (Kim et al., J. Virol. 63:3708-3713 (1989)).

WO 93/20220 PCT/US93/02142 -37- Ecu jjvlalts Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the ucope of the following claims.

WO 9 3/2022O PCT/US93/02142 -38- SEQUENCE LISTIN0 GENERAL INFORMATION: APPLICANT: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH (ii) TITLE OF INVENTION: NON-INFECTIOUS HIV PARTICLES AND USES THERE FOR (iii) NUMBER OF SEQUENCi3S: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: HAMILTON, BROOK, SHITH &REYNOLDS, P.C.

STREET: TWO Militia Drive CITY: Lexington STATE: Massachusetts COUNTRY: U.S.A.

ZIP: 02173 COMPUTER READAB3LE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatiLle OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWAREt Patentln Release Version 11.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 10-MAR-1993

CLASSIFICATION:

(vii) PRIORITY APPLICATION DATA: APPLICATION NUMBER: US 07/859,346 FILING DATE: 27-MAR-1992

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: GRANAHAN, PATRICIA REGISTR.ATION NM~ERt 32,227 REFERENCE/DOCKET NUMBER: WHI89-IOA PCT (ix) TELECOMMUNICATION INFORMTION: TELEPHONEt (617) 861-624J TELEFAX: (617) 861-9D4O TELEX: 951794 INFORMATION FOR SEQ ID NOil: SEQUENCE CHARACTERISZICS1 LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: double T0O"LOGY: linear (Ui) MOLECULE TYPE: DNA (gonomic) WO 93/20220 WO 9320220PCT/tVS93/02142 (xi) SEQUENCE DESCRIP". ION: SEQ ID 110: 1: GCGACTGGTG ROTACGCCMA AAATITGAC TAGCGGPLGGC TAGAAGGAGA GAGA71G 57 INORj4 7I0N FOR SEQ 10 1*:2: ji) SEQUENCE CHARACTERISTICS: LENGTH: 39 amino acids TYPE: amino acid TOPOLOGY: tUnoar (LI) MOLECULE TYPE: protein (xk) SEQUENC1.1 DESC-RIPTION: SEQ ID NOv2: Lye Tyr Ph. Ann Tyr gly L~ye GlU Gly His Thr Ala Arg Ann Cya Arg 1 5 10 Ala Pro Arg Lye Lye Oly Cys Trp Lys Cys Gly Lye Glu G).y His Gln 2530 Hot Lys Asp Cys TAVs Glu ArqT INFORMATION FOR SEQ ID 1*0:3: (i SEQUXNCZ CHARACTERISTICS: LENGTH: 39 amino acids TYPE: amino acid TOPOLOGY: linear (iL) 11OLECULE TY1.E: protein 1,t SEQUENCE DESCRIPTION; SIEQ ID10 3: L~ys Cys Ph. Ann Cys Cys Lys Glu Gly His Thr Ala Arg Ann Cyu krg 1 5 1.O Ala Pro Arg Lys Lys Gly Tyr TrI7 Lys Tyr Gly Lys Glu Gly liai Gin 25 3 Hot Lys Asp Ol~e Thr Glu Alg INFORMATION FOR SEQ ID 1*0:4: Ci) SEQUENCE MWAACTERISTICS: LENGTH: 3! amino acids TYPE: amino aoid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 93/20220 WO 93/02.'AIPC1/U593/02142 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Lys Tyr Gly Ann Tyr Gly Lys G7hu Gly His Thr Ala Arg hen Cys Arg 1 5 10 Ala Pro Arg Lys Lys Gly Tyr Trp Lye Tyr CyB Lys Glu Gly Hie Gin 25 Hot Asp Cys Thr G. Arg ,70RXKTION FOR SEQ ID SEQUENCE CHARACTERISTICS:a LENGTH: 630 base pairs TYPEz nuclaic &aid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATIONt 3..518 OTHER INYORMATTON: /product- "Gag" (xi) SZQUh...E DESCRIPTION: SEQ XD NO:S: CG GCU ACA CUA GAA GAA AUG AUG ACA OCA UGO CAG GGA QUA GGA (WA 47 Ala Tkhr Lou Glz Glu Hot Het Th~r Ala Cys Gin Gly Val Gly Ofly 1 5 10 is CCC GGC CAU MfC4 GCA AGA GUU UUG GCU GMA OCA AUG AGC CMA QUA ACA P~re Gly His Lym Ala Arq Val Lou Ala Glu Ala Hot Sor Gin Val Thr 25 MAU ACA GCU ACC AUA AUG AUG CAG AGA GGC MAU UVU AGO MAC CMA AGA 143 Aen Thr Ala Thr Ile Met Met Gin Arg Gly Ann Phe Arg hen Gin hrg 40 MAG AUG GUU MAG UGU UUC MAU UGU GGC AM GMA G00 CAC ACA GCC AGA 191 Lys Met Val Lys Cys Phis Ann Cym Gly Lyu Glu Gly His Thr Ala Arg so 55 MAU UGC AGO GCC CCU AGG AAA MAG GGC UGU UGO AAA UGU GGA MAG GMh 239 Ann Cys Arg AiA Pro hrg Lye Lyn Gly Cys Trp Lys Cys Gly Lys Giu 70 GGA CAC CMA AUG AMA GAb bGU ACU GAG AGA CAG GCU MAU U UT3A 00 287 Gly Hie Oln Met Lys hsp Cys Thr Glu Arg Gin Ala Ann Phe Lou Gly so as 90 MAG AUC UGO CCU DCC UAC MAG GGA AGO CCA 0=G MAU UUU CDU CAG AOC 335 Lys I. Trp Pro 5cr Tyr Lys Gly Arg Pro Gly hnn Ph. Lou Gin 100 105 3-10 AGA CCA GAG CCA ACA GCC CCA CC.A Ubti CUU CAG AGC AGA CCA GAG CCA 383 Arg Pro Glu Pro Thr Pro Pro Phe Lou Gin Ser Arg Pro Glu Pro 115 120 125 WO 93/20220 PCr/US93/02 142 -41- ACA GCC CCA CCA GAA GAG AGC UUC AGG UCU GGG QUA GAG ACA ACA ACU 431 Thr Ala Pro Pro Glu Glu Ser Ph* Ar; Ser Gly Val Glu Thr Thr Thr 130 135 140 CCC CCU CAG AAG CAG GAG CCG AUh GAC hAG GhA CUG UAU CCU UUA ACU 479 Pro Pro Gin Lys GIn Glu Pro Ile Asp Lys Glu Lou Tyr Pro Laiu Thr 145 150 155 UCC CUC AGA UCA CUC UUU 0CC AhC GAC CCC UCO UCA CAA UAAAGAUAGO 528 Ser Lou Ar; Ser Lou Phe Gly Aen Asp Pro Ser Ser Gin 160 165 170 GCGGCAACUA AAGGAAGCUC UAUUAGAUAC AGGAGCAGAU GAUACAGUAU UAGAAGAAAU 588 GAGUUUGCCA GGAAGAUGGA AACCAAAAAU GAUAGOGGGA AU 630 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 172 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NOt6t Ala Thr Lou Glu Glu. Met Met Thr Ala Cyu Gin Gly Val Gly Gly Pro 1 5 10 Gly His Lys Ala Arg Val Lou Ala Glu Ala Met Ser Gin Val Thr Ann 25 Thr Ala Thr Ile Met Met Gin Ar; Gly Ann Phe Ar; Ann Gin Arg Lyn 40 Met Val Lys Cym Phe Ann Cys Gly Lys Glu Gly Hie Thr Ala et,' Ann 55 Cys Ar; Ala Pro hrg Lye Lyn Gly Cys Trp Lys Cys Gly Lys Glu Gly 70 75 His Gin Met Lye Asp Cys Thr Glu Ar; Gin Ala Ann Ph. Lou Gly Lye 90 Ile Trp Pro Sar Tyr Lye Gly Ar; Pro Gly Ann Phe Lou Gin Ser Arg 100 105 110 Pro Giu Pro Thr Ala Pro Pro Ph. Lou Gin Ser Ar; Pro Glu Pro Thr 115 120 125 Ala Pro Pro Glu Glu Ser Ph* Ar; Ser Gly Val Glu Thr Thr Thr Pro 130 135 140 Pro Gln Lye Gin GIL, Pro 110 Asp Lys Glu Lou Tyr Pro Leu Thr Ser 145 150 155 160 WO 93/20220 WO 9320220.PCT/U593/02142 -42- Lou Arg Ser Lou Ph@ Gly Ann Asp Pro Ser Ser Gln 165 170 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 18 bass pair.

TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ganeamic) (ix) FEATURE: NAME/KEY:. CUS LOCATION: 3..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GC AGC ATC GCG ATG CCT A 18 Ser Ile Ala Met Pro 1 INFORMATION FOR SEQ ID NOaS:- SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acid.

TYPE: amino acid T~OPOOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NOzS: Ser Ile Ala Het Pro 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CH ARA CTERISTICS: LENGTH: 10 amino acid.

TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:0% Val Val Gin Gly Glu Glu Ph. Ala Val Oly 1 5 WO 93/20220 WO 9320220PCT/US93/02142 -43- INFORMATION FOR SEQ ID NO:lO: SEQUENCE CHARACTERISTICS: LENGTH: 39 basns pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCS DESCRXiTIONt SEQ ID NO:l0: TGACGCTCTC GCACCCATCT CTCACCAGTC GCCGCCCTC 39 INFORMATION FOR SEQ ID NOzil: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: CTCTCTCCTT CTAGCCTCCG CTCACCAGTC GCCGCCCCTC INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 40 barse pair.

TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TGCCCTTCTT TGCCATAATT GAAATACTTA ACAATCTTTC INFORMATION FOR SEQ ID NOt,13: SEQUENCE CHARACTERISTICS: LENGTH: 40 basns pair.

TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID 14:13: TGTCCTTCCT TTCGATATTT CChATAGCCC T~TCCTAG INFORMATION FOR SEQ ID 140:14: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pair.

TYPE: nucleic acid.

STRANDEDNESS: single TOPOLOGY: linear WO 93/20220 PCr/US93/02 142 -44- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CCATCGATCT AATTCTC 17 INFORMATION FOR SEQ ID NO:1S: SEQUENCE CHARACTERISTICS: LENGTH: 17 bass pairs TYPE: nucleic acid STP.ANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION, SEQ ID NOziS: GGCCAGATCT1 TCCCTAA 17

Claims (24)

1. A construct, comprising an HIV mutant genome in which there is an alteration in the amino acid sequence of the gag gene of wild-type HIV, wherein the alteration is a deletion of the 5' CysHis box, the 3' CysHis box, and their intervening sequence, and results in production of non-infectious immunogenic HIV particles.

2. A construct which produces non-infectious immunogenic HIV particles and which comprises an alteration of wild-type HIV genome, wherein the alteration consists of a deletion in the site of nucleotides 293 to 331, inclusive, a substitution in the gag gene of tyrosine for the first two cysteines of the 3' CysHis box, and a substitution of the nef gene with the neomycin resistance gene.

3. The construct of claim 2, further comprising an alteration of a sequence selected from the group consisting of: a) a sequence which encodes the envelope precursor cleavage site; b) the primer binding site; and c) combinations of these.

4. The construct of claim 3, wherein said primer binding site alteration is an 18 base pair deletion of the entire primer binding site, as shown in Figure 8.

5. A mammalian cell line, which stably produces non-infectious immunogenic HIV particles, wherein the non-infectious immunogenic HIV particles are encoded by an 20 HIV mutant genome selected from the group consisting of: a) an HIV mutant genome in which there is an alteration in the amino acid sequence of the gag -3 e as llq( I JJlfiI 4fill J 6 KC IN M NC 10 M 4-0 0 1 4H1 i 1O rm 7 (iH 1 +1 IU U .du 'I'i -46- gene of wild-type HIV, wherein the alteration is a deletion of the 5' CysHis box, the 3' CysHis bo)g, and their intervening sequence; and b) an HIV mutant genome in which there is an alteration of the wild-type HIV genomq, wherein the alteration consists of a deletion in the iU site of nucleotides 293 to 331, inclusive, a substitution in the g£aq 7ene of tyrosine for the first two cysteinos of the 3' Cys)iis box, and a substitution of the net gene with the neomycin resistance gene.

6. The mammalian cell line of Claim'5, wherein in the HIV mutant genome further comprises 4n alteration o£ a sequence selected from the group consisting of: a) a sequence which encodes the envelope precursor cleavage site; b) the primer binding site; and c) combinations of these.

7. The mammaalian cell line of Claim 5, wherein the primer binding site alteration is an 18 base pair deletion of the entire primer binding site, as shown in Figure 8.

8. A method of producing a cell line, which stably produces HIV non-infectious particles, comprising the steps of: a) obtaining a construct selected from the group consisting of: 1) the construct of Claim 1; 2) the construct of Claim 2; and 3) the construct of Claim 3; b) transfecting a mammalian cell line with said construct; c) selecting said transfected cells for stable R AJ kll0 ff L 0 ,li~ti( j transfectants; and d) collecting said stable transfectants.

9. A method for producing non-infectious immunogenic HIV particles, or antigenic portions thereof, comprising the steps of: a) obtaining a construct selected from the group consisting of: 1) the construct of claim 1; 2) the construct of claim 2; and 3) the construct of claim 3; b) transfecting a mammalian cell line with said construct; and c) expressing said construct in said cell line, thereby, producing non-infectious immunogenic HIV particles, or antigenic portions thereof.

A method for producing non-infectious immunogenic HIV particles, or antigenic portions thereof, comprising growing a cell line selected from the group consisting of: a) the cell line of claim b) the cell line of claim 6; and c) the cell line of claim 7.

11. Non-infectious immunogenic HIV particles, or antigenic portions thereof, which are produced by the method of claim 9.

12. Non-infectious immunogenic HIV particles, or antigenic portions thereof, which are produced by the method of claim

13. A vaccine, comprising non-infectious immunogenic HIV particles, or antigenic portions thereof, which are produced by the method of claim 9, in a physiologically acceptable vehicle.

14. A vaccine, comprising non-infectious immunogenic HIV particles, or antigenic portions thereof, which are produced by the method of claim 10, in a physiologically acceptable vehicle.

A diagnostic reagent, comprising non-infectious immunogenic HIV particles, or non-infectious protein derivatives thereof, produced by a construct selected from the group consisting of: a) the construct of claim 1; b) the construct of claim 2; and c) the construct of claim 3.

16. A method for identifying a purified and isolated substance that caecA production of non-infectious immunogenic HIV virions from mammnalian cells, said method comprising the steps of: a) obtaining a mammalian cell line, transfected with a construct selected from the group consisting of: 1) the construct of claim 1; 40 2) the construct of claim 2; and S. S S 3) the construct of claim 3, wherein the construct is stably integrated in the genome of the cell line, and the cell line stably produces non-infectious immunogenic HIV particles; b) exposing said cell line to said substance; and c) determining HIV virion production from said exposed cells, wherein a difference in production from said exposed cells compared to production from unexposed cells indicates that said substance affects production of HIV virions from mammalian cells.

17. A purified and isolated substance, which is identified by the method of claim 16.

18. The substance of claim 17, which inhibits production of non-infectious HIV virions from mammalian cells.

19. A method for identifying a drug which inhibits the production of HIV particles by HIV-infected cells, comprising the steps of: a) obtaining a mammalian cell line, transfected with a construct selected from the group consisting of: 1) the construct of claim 1; 2) the construct of claim 2; and 3) the construct of claim 3; 20 wherein the construct is stably integrated in the genome of the cell line, and the cell line stably produces non-infectious HIV particles; b) exposing said cell line to said drug; and c) determining HIV particle production from said exposed cells, wherein an absence in production of HIV particles from said exposed cells compared with a presence 25 in production of HIV particles from unexposed cells indicates that said drug inhibits the production of HIV particles from the HIV-infected cells.

20. A drug, which is identified by the method of claim 19.

21. A construct, comprising an HIV mutant genome in which there is an alteration in the amino acid sequence of the gag of wild-type HIV wherein the alteration is a deletion of the 5' CysHis box, the 3'CysHis box, and their intervening sequence, and results in the production of non-infectious immunogenic HIV particles, substantially as hereinbefore described with reference to any one of the Examples.

22. A method for producing non-infectious immunogenic HIV particles, or antigenic portions thereof, comprising the steps of: a) obtaining the construct of claim 21; b) transfecting a mammalian cell with said construct; and c) expressing said construct in cell line; thereby, producing non-infectious immunogenic HIV particles, or antigenic portions thereof.

23. Non-infectious immunogenic HIV particles, or antigenic portions thereof, 40 which are produced by the method of claim 22. R.~c tNW'LIFIrlO0465:MCN 49

24. A vacci prising non-infectious immunogenic HIV particles or antigenic portions thereof, produced by the method of claim 22, in a physiologically acceptable carrier diluent and/or adjuvant. A diagnostic reagent, comprising non-infectious immunogenic HIV particles, or non-infectious protein derivatives thereof produced by the construct of claim 21. Dated 10 March, 1997 Whitehead Institute for Biomedical Research Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON r r o s i34( 'j (N:\tLUIB(IOX)6MCN