EP0633942A1 - Nicht-infektiöse HIV-Partikeln und ihre Verwendungen - Google Patents
Nicht-infektiöse HIV-Partikeln und ihre VerwendungenInfo
- Publication number
- EP0633942A1 EP0633942A1 EP93907366A EP93907366A EP0633942A1 EP 0633942 A1 EP0633942 A1 EP 0633942A1 EP 93907366 A EP93907366 A EP 93907366A EP 93907366 A EP93907366 A EP 93907366A EP 0633942 A1 EP0633942 A1 EP 0633942A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- hiv
- construct
- particles
- infectious
- alteration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N2740/00—Reverse transcribing RNA viruses
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- C12N2740/16011—Human Immunodeficiency Virus, HIV
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16051—Methods of production or purification of viral material
- C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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- C12N2740/10011—Retroviridae
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- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- HIV Human immunodeficiency virus
- AIDS acquired immune deficiency syndrome
- 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, l in 250 people are infected with HIV worldwide.
- SIV simian immunodeficiency virus
- 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-infectious, immunogenic viral particles.
- HIV mutant constructs based on HIV-1 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 non- infectious mutant HIV particles have been shown to be deficient for the viral genome.
- the production of these mutant HIV particles 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 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.
- the present invention relates to HIV mutants defective for RNA packaging as a result of nucleotide alterations of the cis-actin ⁇ RNA packaging site, referred to as the f 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 with 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.
- non-infectious HIV particles with advantageous antigenic properties can be produced.
- HIV mutants with defective cleavage of the gpl60 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.
- Figure 1 is a diagram of HIV-1 mutations.
- A location and size of deletions affecting the HIV-1 f 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-l nucleotide sequence (nucleotides 1351-1980; SEQ ID No:5) and the 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 gag described herein. Nucleotide locations are as indicated in Ratner, L.. et al., fiaSaJEa 212:277-284 (1985).
- Figure 3 is a diagram of the gag-coding region of HIV-l with nucleotide numbers indicating the initiation codon, the cleavage sites between pl7, p24 and pl5, and the gag termination codon.
- the nucleotide differences between wild type and bCA20 are indicated.
- Figure 4 shows results of Western blot analysis of HIV proteins in HIV-l mutant particles.
- Figure 5 shows results of Northern blot analysis of HIV RNA in HIV-l mutant particles.
- Figure 6 is a diagram of the HIV-l mutant constructs: (A) p ⁇ PACl and (B) p ⁇ PAC-Hygro.
- Figure 7 is a diagram of HIV-l mutant construct p ⁇ PAC2.
- Figure 8 is a diagram of HIV-l mutant construct pAPAC3.
- Figure 9 is a diagram of pR7neo.
- Figure 10 shows the result of immunoblot analysis of cytoplasmic extracts from HT4( ⁇ -dhfr) cells.
- Figure 11 shows the results of Northern slot blot analysis of viral RNA in the supernatant from HT4( ⁇ E- dhfr) cells.
- Figure 12 shows results of examination of HT4(R7- dhfr), HT4(WT- ⁇ E-dhfr) and HT4(bCA20- ⁇ dhfr) by electron microscopy.
- Panel A HT4(R7-dh r)
- Panel B HT4(WT- ⁇ E-dhfr)
- Panel C HT (bCA20- ⁇ E-dhfr) .
- Pictures were taken at a magnification of x38,500 for panels A, B, and C, and x4,500 for the negative control in panel D.
- 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 of 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.
- Non-infectious HIV particles are described herein which provide improved inactivated vaccines 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 and cell lines for producing the particles, and related materials and methods for commercial and medical use are further described below.
- 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 step is a very specific and efficient process, during which viral 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 retroyirus 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 sis-acting sequences, referred to as f sites, have been located to a region near the 5' end of the viral genome. The exact boundaries of the if sites in the various retroviruses are not known, but sequences between the first splice donor site and the Gag translational start site have been shown to be critical for wild type RNA packaging (Shank, P.R. and M. Linial, J. Virol. 36:450-456 (1980); Mann, R. and D. Baltimore, J. Virol. 54:401-407 (1985); Linial, M. et al.. ££ll 15.:1371-1381 (1978); Koyamat, Harada F.
- pA3HXB which contains a 39 bp deletion of nucleotides 293-331 (inclusive)
- pA4HXB which contains a 21 bp deletion of nucleotides 293-313 ( Figure 2A) .
- the construction of pA3HXB and pA4HXB are described in Example 2.
- HIV-l RNA Packaging Gag Mutants
- CysHis box 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, R.L., et al.. J. Biol. Chem. 262:4961-4967 (1987); Meric, C. and P.-F. Spahr, J. Virol. 60:450-459 (1986) Meric, C, eial- / J. Virol. 62:3328-3333 (1988) Meric, C. and S.P.
- 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, J. , Science 232:485-487 (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. M. and S. M. Hollenberg, Cell 52:1-3 (1988); Berg, 1986 supra ⁇ . In retroviral nucleocapsid proteins, these sequences may also play a role in protein-protein interactions.
- the gag gene is expressed as a protein precursor, Pr55 9ag , which is processed and cleaved into the mature viral proteins, pl7, p24, and pl5.
- pl5 is believed to be cleaved further into p9 and p7 (Mervis, R. J. , e£ al. , J. V ojj. £2:3993-4002 (1988); Veronese, F. D. M. , et al. , J. Virol. 62:795-801 (1988)).
- the exceedingly basic character of pl5 suggests that it might be associated with the viral RNA (Gelderblom, H. R. , et al. , Virology 15.6:171-176 (1987)).
- the HIV pl5 is 123 amino acids long and encoded by the 3' end of the gag gene ( Figure 2). 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 Figures IB and 3) (Covey, S. N. , Nucleic Acids Res. 14:623-633 (1986)). To investigate the role of the CysHis box region in packaging of the HIV genome, five HIV-l mutants were constructed, as shown in Figures IB and 3) .
- Mutant constructs pA14HXB and pA15HXB each encode alteration of a single CysHis box: pA14HXB encodes tyrosine substitutions for Cy ⁇ 1 and Cy ⁇ 3 in the 5' CysHis box (SEQ ID No:2) and pA15HXB encodes corresponding substitutions in the 3' CysHis box (SEQ ID No:3).
- Mutant construct pA14-15HXB encodes tyrosine substitutions for Cys x and Cys 3 in both CysHis boxes (SEQ ID No:4) .
- p ⁇ CHl-2HXB encodes a mutant Gag protein with a 35 amino acid deletion of both CysHis boxes and their intervening sequence.
- Mutant construct bCA20 ( 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, p ⁇ CHl-2HXB and bCA20 were each found to produce non-infectious HIV particles. The construction of the gag mutants are described in the Examples.
- COS-1 African Green Monkey kidney
- the constructs contain the mutated HIV genomes in vectors with an SV40 origin of replication.
- 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 -li ⁇ efficiently 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 "p"; 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 pA14- 15HXB, were identical to RNA from cells transfected with a wild type HIV construct, pHXB2gpt.
- HIV-l RNA all three classes of HIV-l RNA were present: the 9.2 kb genomic RNA, a 4.3 kb spliced mRNA encoding the env and vif genes, and the heterogeneous RNAs at about 2 kb, which includes tat-III. rev, and nef mRNAs.
- these HIV-l mutations do not appear to affect the expression of viral RNAs.
- HIV-l constructs The presence of gpl60, gpl20, gp41, p24, pl7, and pl5 in all of the transfected cells indicates that the HIV-l mutants do not produce major alterations in the synthesis and processing of Gag and envelope precursors. The amount of HIV virions produced was quantitated by two assays.
- the plasmid pHXB2BAMp3 was included as a negative control; it does not produce virus, due to a post-transcriptional defect.
- 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 mutants 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.
- site mutant virions A3HXB and gag 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.
- mutant HIV particles The morphology of mutant HIV particles was examined by electron microscopy. This analysis showed that viral capsids could assemble in the absence of RNA packaging, indicating that mutations in the CysHis box of p7 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.
- HIV-l 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, p ⁇ PACl and p ⁇ PAC-Hygro were made which contain efficacious alterations in both the f site and the Gag CysHis region ( Figure 6). The alterations are referred to as ef icacious because each alteration alone results in non-infectivity.
- 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 if site mutation can be the 39 bp deletion of pA3HXB or the 21 bp deletion of pA4HXB ( Figure IA) .
- 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 p ⁇ CHl-2HXB ( Figure IB) . Additional mutations of the Env gpl ⁇ o cleavage site, as in p ⁇ PAC2, 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 p ⁇ PAC3 shown in Figure 8.
- 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 gpl20 antigen is expected to improve the immunogenic properties of HIV virions produced from gpl60 cleavage site mutants.
- the gpl60 cleavage site mutation in p ⁇ PAC3 is defective for both gpl60 processing and infectivity, and thus, can be used to produce non- infectious virions with the potentially improved immunogenic properties.
- the envelope protein is the the surface antigen of HIV and also the most variable protein among HIV isolates.
- HIV isolates from different geographical locations will contain variable env gene sequences.
- Many variant env genes have been cloned and sequenced, and are available from the AIDS Research and Re erence 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 env replacement technique involves replacing the native env gene in a HIV mutant construct with a variant gene from another HIV strain or isolate in order to tailor the resulting non-infectious HIV particles to a specific use.
- the mutant particles can be used to vaccinate against HIV strains which are prevalent in a population or against particularly virulent strains.
- non- infectious 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 env gene and replacing the env gene in the mutant construct with the cloned sequence.
- 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 env 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.
- 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 neo gene for G418 resistance or hv ⁇ xo 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.
- the selectable marker is transcribed from the same promoter as the coding sequence( ⁇ ) 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 (neo) .
- the nef 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.
- selectable markers other than neo can be used to replace the nef gene.
- Stable producer cell lines have been generated from COS, He a, 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 H9- derived cell lines, the amount of particles produced per cell has been observed to exceed production from live virus infection by 40-100 fold. This is a significant additional advantage in terms of production cost.
- cell lines may also be used to produce non- infectious HIV particles.
- FDA-approved cell lines may be more convenient for clinical trials.
- Non-infectious 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.
- 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.
- The.HIV mutant constructs described above are based on HIV-l genomes. However, constructs based on HIV-2 genomes can also be made using similar methods. Analogous f 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-l 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-l 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.
- Vaccine ⁇ can be prepared which contain inactivated, whole virus particles or antigenic portions of the non- ⁇ infectious virions.
- the vaccines can be delivered in an appropriate physiological carrier, such as saline.
- the carrier can contain an adjuvant, such as BCG (Mvcobacterium bovis bacillus Calmette-Guerin) .
- Diagnostic reagents can also be prepared which contain whole or protein derivatives of the non- infectious 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 example, ELISAs or Western blots may be performed to detect anti-HIV antibodies in blood samples. 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 40-100X the amount of HIV particles per cell than that obtained by infection with live virus.
- stable producer cell lines can be used to produce non-infectious SIV particles for immunotherapy and prophylaxis trials in simian animal models for AIDS.
- SIV mutant constructs with RNA packaging, Env gpl60 cleavage and primer binding site mutations corresponding to the HIV mutations described herein have been made in parallel for this purpose.
- stable producer cell lines provide a convenient and safe j.n vitro model system to study post- infection events in the HIV life cycle. Increased knowledge of mechanisms by which HIV reproduce in mammalian cells may lead to novel therapies for preventing or controlling HIV infection.
- stable producer cell lines provide a safe and convenient method for identifying drugs which inhibit production of HIV particles from infected cells.
- 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 simian virus 40-transformed African green monkey cell line, COS-1 has been described (Gluzman, Y., Cell 23:175-182 (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, M. , et al. , Science 224:497-500 (1984)).
- the parent DNA used for these studies is the biologically active clone, pHXB2gtp (Fisher, A.G., et al. , Nature (London) 116:262-265 (1985)).
- the Sacl-Sall 5.3 kilobase pair (kb) fragment (nucleotides 39 to 5366) was cloned in M13mpl8, and oligo-mediated site-directed mutagenesis was performed on the single stranded DNA according to the protocol of Kunkel, T. A., ⁇ t al. (Methods in Enzymologv 154:367-382 (1987)). Nucleotide positions in the HIV-l genome are numbered as previously described (Ratner, L. , et al « . Nature 313:277-284 (1985)).
- A3 TGACGCTCTCGCACCCATCTCTCACCAGTCGCCGCCCTC (SEQ ID No:10), for deleting nucleotides 293 to 331; and A4: CTCrCTCCTTCTAGCCTCCGCTCACCAGTCGCCGCCCCTC (SEQ ID No:11), for deleting nucleotides 293 to 313; and the gag mutations:
- A14 TGCCCTTCTTTGCCATAATTGAAATACTTAACAATCTTTC (SEQ ID No:12), for changing guanines (G) at positions 1508 and 1517 to adenines (A) ;
- A15 TGTCCTTCCTTTCGATATTTCCAATAGCCL'T'l l'TCCTAG (SEQ ID No:13), for changing G at positions 1571 and 1580 to A.
- the RNA packaging mutations in pA3HXB, pA4HXB, pA14HXB, pA15HXB, pA14-15HXB were made using these primers. Deletion of the entire CysHis box coding region for constructing p ⁇ CHl-2HXB and of the 18 bp primer binding site, as illustrated in Figure 8, was performed similarly using appropriate primers.
- a Bsshll/Ball 1.9 kb DNA fragment from the mutated M13mpl8 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. e£ al. , 1987 suprai to confirm the presence of the desired mutations and the absence of any other alterations. All DNA manipulations were according to standard procedures.
- Replacement of the nef gene with a selectable marker has been described in Trono, D., et al. , which is hereby incorporated by reference (Cell 59:113-120 (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 Institutes of Health repository, or can be obtained from HIV-infected patients by isolating the virus and cloning the viral genome.
- mutated env genes including genes encoding defective glycosylation sites, can be engineered by site-directed mutagenesi ⁇ .
- COS-1 cells were seeded at a density of 10 6 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% C0 2 . Transfection was carried out using 10 ⁇ g of plasmid DNA/plate as described (Chen, C. and H. Okoyama, Mol. Cell. Biol. 7:2745-2752 (1987)). When viral particles were destined for RNA analysis, transfections in COS cells were performed according to the method of Seldon et a . (Seldon, R. F. , fiial., Mol. Cell. Biol.
- Stable producer cell lines were generated by selecting for stable transfectant ⁇ possessing antibiotic resistance conferred by the selectable marker in the construct. Transfection was carried out by either the CaP0 4 or electroporation methods. Stable transfectants were selected using G418 or hygromycin (Gibco) at appropriate concentrations for the cell line, as determined by killing a ⁇ says. 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, resistant clones could be purified through several rounds of replating rather than pooled. Individual clones could be tested for maximum HIV production.
- 10 ⁇ g of DNase I- treated total cellular RNA was used per lane.
- RNA from mock transfected COS-l cells was used as a negative control and 5 ⁇ g of RNA from H9 cells, chronically infected with HXB2 virus, was used as a positive control
- the HTV-l specific probe was a 32 P-labelled full length viral DNA.
- 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 (l ⁇ g/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-l supernatants containing 18 ng of p24.
- RNA Slot Blot analysis was performed as previously described (Ausubel et al., 1987 suprai .
- a 3.8 kb Clal/EcoRI oag- pol fragment from pHXB2gpt was labelled with 3 P by random priming and used as probe for the Slot Blot.
- COS-l cells (4xl0 6 ) transfected 48 hours earlier with 10 ⁇ g of each plasmid were labelled for 4 hours with 500 ⁇ Ci of 35 S methionine.
- As a negative control cells were transfected with the plasmid pHXB2Bamp3, which does not produce virus due to a post-transcriptional defect (Feinberg, M. B. , e£ al. , ££1146:807 (1986)).
- Cell lysates were prepared and immunoprecipitations were performed as described (Veronese, F. D.
- virus was pelleted by centrifugation for three hours at 27,000 rpm in a SW27 rotor. The pellet was resu ⁇ pended in di ⁇ sociation 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 analysi ⁇ wa ⁇ to be performed.
- Western blot analysis and radio-immunoprecipitations (RIP) followed the procedure of Veronese et al. (Veronese, F. D.
- 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 and three dif erent dilutions of each supernatant were analyzed. RT activity was measured after concentrating 3 ml of COS-l supernatant from transfections of each mutant by centrifugation for three hours at 27,000 rpm. Numbers refer to l ml of supernatant and are the mean of three experiments. Analysis of protein content of wild- type and mutant particles by radio-immunoprecipitation was also done.
- H9 cells were infected by filtered (0.45 ⁇ m, Millipore) supernatants from COS-l cells that had been transfected 48 hours. Immunofluorescence assays were performed with murine monoclonal antibody specific for the p24 9 - 9 protein (Veronese, F. D., efe al., Proc. Natl. Acad. Sci. USA £2_:5199-5202 (1985)) and the h9-HIV IIIB cell line as a positive control. p24 assays were performed with a p24 ELISA (DuPont Co.). Reverse transcriptase assays were carried out after filtration of culture supernatants (Daniel, M. D., gt al. , Science 228:1201-1204 (1985)) .
- 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 transcriptase activity released in the supernatant (Table 3).
- bCA20-induced 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 assay (Ho, D.D., et al, Science 226:451-453 (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 non- infectious. Therefore, it could be concluded that the bCA20 mutation was lethal for viral replication.
- 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 cell lines are made as follows: Briefly, HT4-6C cells (a HeLa cell line expressing the CD4 molecule at its surface were transfected with construct bCA20- ⁇ E-dhfr, a modified version of bCA20-W13 which contains a translational fra e ⁇ hift in the env gene and the mutant dihydrofolate-reducta ⁇ e gene in place of the nef reading frame. The HT4-6C cell ⁇ were a gift from B.
- a slot-blot analysis of the supernatant from HT4(WT- ⁇ E-dhfr) and HT4(bCA20- ⁇ E-dhfr) cells was performed.
- 700 ⁇ l of culture medium was mixed with 35 ⁇ l of 10 mg/ml proteinase K (Boehringer, Mannheim)
- 7 ⁇ l of culture medium was mixed with 35 ⁇ l of 10 mg/ml tRNA, 3.5 ⁇ l 0.5 M EDTA, 17.5 ⁇ l 20% SDS, incubated at 37° C for 45 minute ⁇ , phenol-extracted and ethanol precipitated in 0.4 M NaCl.
- Hybridization was then performed as previously described (Trono, D. , et al. , J. Virol. £2:2291-2299 (1988)), using a [ 32 P] probe generated with T7 polymerase, which is complementary to nucleotides 8475 to 8900 of the HIV-l genome.
- the filter was washed in 0.2xSSC three times at 68° C and exposed to X-ray film.
- Results showed that the amount of viral RNA present in the supernatant from HT4(bCA20- ⁇ E-dhfr) was dramatically reduced, compared to the control cell line, HT4(WT- ⁇ E-dhfr) ( Figure 11). Therefore, it was concluded that the bCA20 mutation specifically inhibited the packaging of the viral genomic RNA into particles.
- the diameter of the particles was approximately 50% 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 C) . Still, approximately 1% of the bCA20 particles had a morphology clo ⁇ e 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 components of the virion, which normally reflects the final steps of maturation.
- Plasmid Construct (ng/ml) ( 32 P dGTP cpm/ml/min)
- pHXB2gpt >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20
- p24 antigen was measured using an Elisa assay system (DuPont-NEN, Inc. ,
- ADDRESSEE HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE protein
- SEQUENCE DESCRIPTION SEQ ID NO: :
- AGA CCA GAG CCA ACA GCC CCA CCA UUU CUU CAG AGC AGA CCA GAG CCA 383 Arg Pro Glu Pro Thr Ala Pro Pro Phe Leu Gin Ser Arg Pro Glu Pro 115 120 125 ACA GCC CCA CCA GAA GAG AGC UUC AGG UCU GGG GUA GAG ACA ACA ACU 431 Thr Ala Pro Pro Glu Glu Ser Phe Arg Ser Gly Val Glu Thr Thr Thr 130 135 140
- MOLECULE TYPE DNA (genomic)
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US6589734B1 (en) | 1989-07-11 | 2003-07-08 | Gen-Probe Incorporated | Detection of HIV |
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WO1995025806A2 (en) * | 1994-03-24 | 1995-09-28 | Syngenix Limited | Packaging-deficient lentiviruses |
US5858838A (en) | 1998-02-23 | 1999-01-12 | Taiwan Semiconductor Manufacturing Company, Ltd. | Method for increasing DRAM capacitance via use of a roughened surface bottom capacitor plate |
US5955342A (en) * | 1994-08-15 | 1999-09-21 | Connaught Laboratories Limited | Non-infectious, replication-defective, self-assembling HIV-1 viral particles containing antigenic markers in the gag coding region |
US6291157B1 (en) | 1998-02-23 | 2001-09-18 | Connaught Laboratories Limited | Antigenically-marked non-infectious retrovirus-like particles |
US6080408A (en) * | 1994-08-22 | 2000-06-27 | Connaught Laboratories Limited | Human immunodeficiency virus type 1 nucleic acids devoid of long terminal repeats capable of encoding for non-infectious, immunogenic, retrovirus-like particles |
US6013516A (en) * | 1995-10-06 | 2000-01-11 | The Salk Institute For Biological Studies | Vector and method of use for nucleic acid delivery to non-dividing cells |
FR2747046B1 (fr) * | 1996-04-05 | 1998-06-19 | Univ Paris Curie | Nouveaux vaccins issus de plasmovirus |
US6121021A (en) * | 1997-12-16 | 2000-09-19 | Connaught Laboratories Limited | Constitutive expression of non-infectious HIV-like particles |
US6218181B1 (en) | 1998-03-18 | 2001-04-17 | The Salk Institute For Biological Studies | Retroviral packaging cell line |
AU4936500A (en) * | 1999-05-21 | 2000-12-12 | Oxford Biomedica (Uk) Limited | Method for selecting improved vectors |
MY144532A (en) | 2001-08-20 | 2011-09-30 | Lundbeck & Co As H | Novel method for down-regulation of amyloid |
MA50213A (fr) * | 2016-12-16 | 2020-07-22 | Avixgen Inc | Peptide de transduction cytoplasmique et messager intracellulaire le comprenant |
KR101933217B1 (ko) | 2017-12-28 | 2018-12-27 | (주) 에빅스젠 | 피부 염증 억제용 펩티드 및 이를 포함하는 피부 염증 예방 또는 치료용 조성물 |
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CA2084659A1 (en) * | 1990-06-20 | 1991-12-21 | Joseph G. Sodroski | Vectors containing hiv packaging sequences, packaging defective hiv vectors, and uses thereof |
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