RESUMO
BACKGROUND: The development of a prophylactic vaccine against HIV-1 has so far not been successful. Therefore, attention has shifted more and more toward the development of novel therapeutic vaccines. Here, we evaluated a new mRNA-based therapeutic vaccine against HIV-1-encoding activation signals (TriMix: CD40Lâ+âCD70â+âcaTLR4) combined with rationally selected antigenic sequences [HIVACAT T-cell immunogen (HTI)] sequence: comprises 16 joined fragments from Gag, Pol, Vif, and Nef). METHODS: For this purpose, peripheral blood mononuclear cells from HIV-1-infected individuals on cART, lymph node explants from noninfected humans, and splenocytes from immunized mice were collected and several immune functions were measured. RESULTS: Electroporation of immature monocyte-derived dendritic cells from HIV-infected patients with mRNA encoding HTIâ+âTriMix potently activated dendritic cells which resulted in upregulation of maturation markers and cytokine production and T-cell stimulation, as evidenced by enhanced proliferation and cytokine secretion (IFN-γ). Responses were HIV specific and were predominantly targeted against the sequences included in HTI. These findings were confirmed in human lymph node explants exposed to HTIâ+âTriMix mRNA. Intranodal immunizations with HTI mRNA in a mouse model increased antigen-specific cytotoxic T-lymphocyte responses. The addition of TriMix further enhanced cytotoxic responses. CONCLUSION: Our results suggest that uptake of mRNA, encoding strong activation signals and a potent HIV antigen, confers a T-cell stimulatory capacity to dendritic cells and enhances their ability to stimulate antigen-specific immunity. These findings may pave the way for therapeutic HIV vaccine strategies based on antigen-encoding RNA to specifically target antigen-presenting cells.
Assuntos
Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos/administração & dosagem , Antígenos HIV/imunologia , Infecções por HIV/prevenção & controle , RNA Mensageiro/genética , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Adjuvantes Imunológicos/genética , Animais , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Antígenos HIV/genética , Humanos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The generation of a vaccine against HIV/AIDS is extremely challenging, as evidenced by more than 20 years of attempts. Here are highlighted the strategies adopted within the AIDS Vaccine Integrated project (AVIP) to speed up the clinical evaluation of novel vaccine candidates and to increase the chances to get an effective preventive and/or therapeutic vaccine.
Assuntos
Vacinas contra a AIDS , HIV-1/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Genes Virais , Antígenos HIV/genética , Antígenos HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/genética , Humanos , Modelos Animais , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/análise , Infecções por HIV/imunologia , Soropositividade para HIV/imunologia , HIV-1/imunologia , Antígenos HLA-A/genética , Antígenos HLA-C/genética , Alelos , Sequência de Aminoácidos , Células Cultivadas , Epitopos de Linfócito T/genética , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Antígenos HIV/genética , Antígenos HIV/imunologia , Infecções por HIV/sangue , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Leucócitos Mononucleares , Contagem de Linfócitos , Dados de Sequência Molecular , Poliproteínas/genética , Poliproteínas/imunologia , Especificidade da Espécie , Linfócitos T Citotóxicos/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Produtos do Gene rev/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Anticorpos Anti-HIV/biossíntese , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/administração & dosagem , Proteína gp160 do Envelope de HIV/administração & dosagem , HIV-1/imunologia , Interleucina-12/genética , Fragmentos de Peptídeos/administração & dosagem , Vacinas contra a AIDS/imunologia , Administração Cutânea , Animais , Biomarcadores , Biópsia , Dermabrasão , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Produtos do Gene rev/genética , Produtos do Gene rev/imunologia , Antígenos HIV/genética , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/genética , Hipersensibilidade Tardia/imunologia , Imunidade Celular , Células de Langerhans/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas S100/análise , Pele/imunologia , Pele/patologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The adjuvant properties of Montanide CSA 720 were assessed in a comparison with alum. BALB/c mice were immunized with recombinant HIV-1 gag protein p17 administered in either of the two adjuvants. The serum antibody response to p17 with Montanide CSA 720 appeared faster and reached a higher titre than with alum. The serum antibody response to p17 in Montanide CSA 720 was further characterized by a higher titre antibody directed against a 30 amino acid segment from the entire protein. The Montanide CSA 720 adjuvant was sufficiently strong to induce an antibody response against a weak synthetic peptide immunogen after two immunizations, while immunization with the peptide in alum generated no detectable serum antibody. The p17-specific proliferative response of splenocytes from animals immunized with recombinant protein in either adjuvant was similar.