RESUMO
Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIVmne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIVmne. Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp 130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core (gag-pol) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10-100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorporate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).
Assuntos
Proteína gp160 do Envelope de HIV/imunologia , Infecções por Lentivirus/imunologia , Infecções por Lentivirus/prevenção & controle , Fragmentos de Peptídeos/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Macaca fascicularisRESUMO
Anti-human immunodeficiency virus type 1 (Anti-HIV-1) antibody response was compared in four groups of mice following inoculation with HIV-1 gp160, with live recombinant vaccinia virus expressing HIV-1 envelope glycoproteins, or with both immunogens in alternate orders for primary or secondary immunizations. Both subunit and recombinant virus immunogens induced similar levels of antibody response following primary immunization. However, after secondary immunization, mice primed with live recombinant virus and then boosted with subunit gp160 immunogen showed significantly higher antibody response than those in the other three groups. Neutralizing antibodies were generated only in this group of mice and were shown to neutralize both the homologous virus (BRU) and a divergent isolate (SF2) of HIV-1. On the other hand, their reactivities to peptide sequences from the principal neutralizing determinant (PND) of gp120 were limited to the BRU isolate, not SF2 or MN, indicating that the cross-neutralizing activities were directed against determinants other than the linear epitope(s) within the PND. These results also indicate that combined immunization by priming with liver recombinant virus and boosting with subunit immunogen may be more effective than immunization by either immunogen alone.
Assuntos
Vacinas contra a AIDS/genética , Produtos do Gene env/imunologia , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Precursores de Proteínas/imunologia , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Animais , Reações Cruzadas , Anticorpos Anti-HIV/imunologia , Proteína gp160 do Envelope de HIV , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Testes de Neutralização , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vaccinia virus/genéticaAssuntos
Produtos do Gene env/imunologia , Glicoproteínas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos/imunologia , Anticorpos Antivirais/biossíntese , Sequência de Bases , Humanos , Macaca fascicularis/imunologia , Dados de Sequência Molecular , Vírus da Imunodeficiência Símia/isolamento & purificação , Especificidade da Espécie , Linfócitos T/imunologia , Vaccinia virusRESUMO
Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultra violet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, our results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.
Assuntos
Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Imunidade Celular/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Ficusina/farmacologia , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Antígenos HIV/genética , Antígenos HIV/imunologia , Proteína do Núcleo p24 do HIV , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/efeitos da radiação , Immunoblotting , Testes de Neutralização , Coelhos , Vacinas Sintéticas/genética , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Vacinas Virais/genéticaRESUMO
Recombinant vaccinia viruses that contained regions of the gag-pol open reading frames of human immunodeficiency virus type 1 (HIV-1) were constructed. Cells infected with recombinants containing both gag and protease genes expressed and processed HIV gag antigens efficiently. Processing was much reduced in cells infected with recombinants containing only gag, but not the protease gene. However, significant amounts of p41 were produced by protease-defective recombinants. This protein was immunoreactive with p24-specific monoclonal antibodies and was produced in a truncated form by a recombinant containing a 3' deletion in the p15 coding region of gag ORF. These results indicate that p41 could represent an alternative gag precursor with N-terminal sequences derived from p24 and C-terminal from p15. Ultrastructural analysis of recombinant-infected cells revealed that the gag antigens expressed were assembled into retrovirus-like particles and were secreted into culture medium. This assembly process was not dependent on HIV protease function, because immature core particles were produced by recombinants lacking HIV-1 protease functions. Immunization of mice and chimpanzees with vaccinia-HIVgag recombinant viruses generated both antibody and cell-mediated immune responses to HIV gag antigens. These recombinants are therefore useful not only for studying HIV virion processing and assembly, but also for designing immunogens for the prophylaxis and immunotherapy against AIDS.
Assuntos
Proteínas de Fusão gag-pol/genética , HIV-1/genética , Fases de Leitura Aberta , Vaccinia virus/genética , Proteínas do Core Viral/genética , Animais , Linhagem Celular , Anticorpos Anti-HIV , HIV-1/ultraestrutura , Immunoblotting , Ativação Linfocitária , Microscopia Eletrônica , Pan troglodytes , Recombinação Genética , Proteínas do Core Viral/ultraestruturaRESUMO
To study the roles of the V3 hypervariable region (amino acid residues 301-336) of the HIV-1 envelope glycoprotein (gp160) during infection, we constructed recombinant vaccinia viruses that expressed either wild-type gp160 (v-env10) or mutant gp160 in which the V3 region was deleted (v-dl29.1 and v-dl29.2). In v-dl29.1 the V3 loop, formed by disulfide bonding between cysteine residues 301 and 336, was deleted from cys301 to cys336 (inclusive) and replaced by one serine residue. In v-dl29.2 the V3 loop was deleted from arg303 to ala334 and replaced by three residues: gly-ala-gly. Cells infected with all three recombinant vaccinia viruses expressed gp160 on the cell surface, but v-dl29.1-derived gp160 was not cleaved into gp120 and gp41 and did not bind the CD4 glycoprotein. In contrast, gp160 produced by recombinant v-dl29.2 was cleaved normally, and the mutant gp120 produced was secreted and retained binding activity to CD4+ cells. However, both mutants failed to induce syncytia in HeLa CD4+ cells. Thus a disulfide loop at the V3 portion of gp160 is required for cleavage into gp120 and gp41, presumably because the loop is required for proper tertiary structure. The sequence within the V3 loop, however, is not required for cleavage and secretion of gp160, or for binding to CD4+, but this region is essential for gp120-mediated syncytia formation.
Assuntos
Produtos do Gene env/metabolismo , HIV-1/metabolismo , Precursores de Proteínas/metabolismo , Antígenos CD4/metabolismo , Fusão Celular , Células Cultivadas , Análise Mutacional de DNA , Produtos do Gene env/genética , Proteína gp160 do Envelope de HIV , Células HeLa , Humanos , Técnicas In Vitro , Ligação Proteica , Precursores de Proteínas/genética , Processamento de Proteína Pós-TraducionalRESUMO
Simian AIDS (SAIDS) is an endemic disease of macaques that shares many characteristics with AIDS in humans. SAIDS is etiologically linked to infection by a type D retrovirus, SAIDS retrovirus (SRV). Immunization with an inactivated whole-virus vaccine was shown to protect macaques against infection by SRV serotype 1. To identify the antigen(s) responsible for eliciting protective immunity, we have constructed a recombinant vaccinia virus (v-senv5) that expresses the envelope glycoproteins of SRV serotype 2 (SRV-2/W). Pig-tailed macaques (Macaca nemestrina) immunized with v-senv5 showed lymphoproliferative responses to purified SRV-2/W. They also generated antibodies that neutralized SRV-2/W infectivity in vitro and mediated antibody-dependent cellular cytotoxicity against SRV-2-infected cells. Four v-senv5-immunized animals, together with four control animals, were challenged intravenously with 5 x 10(3) tissue culture infectious doses of SRV-2/W. As early as 2 weeks after challenge, three of four control animals became viremic, and two of these three animals also seroconverted. The animal that was viremic but remained antibody negative died of symptoms of SRV infection 6 1/2 weeks after challenge. In contrast, all four v-senv5-immunized animals remained healthy, virus-free, and seropositive against only the immunizing envelope antigens. These results indicate that immunization with a recombinant vaccinia virus expressing the envelope antigens of SRV-2/W protects primates from infection by a retrovirus that causes immunodeficiency diseases.