Expression of HIV-1 envelope gene by recombinant avipox viruses.
Recombinant canarypox (CP) and fowlpox (FP) viruses that contained two forms of the HIV-1 (SF2 strain) env gene were engineered and their expression analysed in chick, simian and human cells. These vectors can efficiently replicate in avian but not in mammalian cells, in which infection is abortive. The two forms, consisting of the entire env open reading frame (IS+) or of the same gene lacking the putative immunosuppressive (IS-) region (amino acids 583-599), were individually inserted into the two virus vector backgrounds. In order to avoid premature transcription termination of the foreign gene and to improve protein expression, a mutagenesis was also performed within the T5NT motif without altering the amino acid sequence. By immunoprecipitation analyses, cells infected with CP and FP recombinants expressed HIV-1 env polypeptides of the appropriate molecular weight. We observed that the gp160 precursor was proteolytically cleaved except in MRC-5 cells infected with the IS- recombinants and that these polypeptides were glycosylated. Further analysis of these recombinant viruses by indirect immunofluorescence and syncytia inhibition assays indicated that the gp120 gp41 complex was present on the surface of infected cells, the number of syncytia being significantly lower when cells were infected by the CPIS- or FPIS- recombinants. Moreover, sera of immunized rabbits revealed the presence of specific antibodies in animals inoculated either with CP or with FP recombinants. These new constructs, which are unable to support a productive infection in human cells, might therefore also be a good anti-HIV-1 candidate vaccine in seropositive hosts.
Radaelli
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A
Institute of Pharmacological Sciences, University of Milano, Italy.
De Giuli Morghen
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C
D000595:Amino Acid Sequence; D000818:Animals; D018150:Avipoxvirus / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D015752:Genes, env / Q000235:genetics*; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000096:biosynthesis; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000235:genetics; D011817:Rabbits; D015531:Radioimmunoprecipitation Assay; D013047:Specific Pathogen-Free Organisms; D014158:Transcription, Genetic / Q000235:genetics; D014709:Vero Cells
D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; C076451:HIV envelope protein gp41 (519-541)