RESUMEN
Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4+ Th cell-proliferative response and by inducing an antigen-specific IFN-gamma ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4+ Th responses and IFN-gamma ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4+ T cell responses.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD4-Positivos/inmunología , Quimiocina CCL5/inmunología , VIH-1/inmunología , Virus Reordenados/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunación , Síndrome de Inmunodeficiencia Adquirida/virología , Adyuvantes Inmunológicos , Animales , Recuento de Linfocito CD4 , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Antígenos VIH/inmunología , VIH-1/genética , Interferón gamma/biosíntesis , Macaca mulatta , Masculino , Virus Reordenados/genética , Virus Reordenados/metabolismo , Virus de la Inmunodeficiencia de los Simios/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Carga ViralRESUMEN
Chimeric simian-human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES gene (SHIV-RANTES) and characterized its properties in vitro. SHIV-RANTES replicated both in human and monkey T cell lines. Along with SHIV-RANTES replication, RANTES was detected in the supernatant of human and monkey cell cultures, at maximal levels of 98.5 and 4.1 ng/ml, respectively. A flow cytometric analysis showed that the expressed RANTES down-modulated CC-chemokine receptor 5 (CCR5) on PM1 cells, which was restored by adding anti-RANTES antibody. UV-irradiated culture supernatants from the SHIV-RANTES-infected cells suppressed replication of CCR5-tropic HIV-1 BaL in PM-1 cells. Differentiating real-time RT-PCR showed that pre-infection of SHIV-RANTES in C8166 cells expressing CCR5 suppressed the replication of HIV-1 BaL. Biological activity of the expressed RANTES and the inserted RANTES gene in SHIV-RANTES remained stable after 10 passages. These results suggest that SHIV-RANTES is worth testing in macaque models.
Asunto(s)
Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Ingeniería Genética , VIH/genética , VIH/fisiología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Línea Celular , Regulación hacia Abajo , Humanos , Macaca fascicularis , Receptores CCR5/metabolismo , Recombinación Genética , Factores de Tiempo , Replicación ViralRESUMEN
Chimeric simian and human immunodeficiency viruses (SHIVs) are useful tool for investigating AIDS pathogenesis and for development of vaccine. We constructed a SHIV-vpr vector (designated as SHIV-3sj) by replacing vpr region with restriction enzyme sites. SHIV-3sj was designed to express inserted gene along with its viral replication. Five cytokine genes were inserted into SHIV-3sj, and ability of viral replication and expression of the inserted genes were examined. The short insert including RANTES and IL-5 resulted in the successful expression from SHIV-3sj, while the construct having longer genes including IL-2, IL-6 and IL-12p35 failed to become replication competent. These results suggest that the length of the insert is an important factor for the replication ability of SHIV-3sj vector.
Asunto(s)
ADN Recombinante/genética , Genes prv/genética , Vectores Genéticos/genética , Vectores Genéticos/fisiología , VIH-1/genética , Virus de la Inmunodeficiencia de los Simios/genética , Replicación Viral/genética , Animales , Secuencia de Bases , Línea Celular , Quimiocina CCL5/genética , Expresión Génica , VIH-1/fisiología , Humanos , Interleucina-12/genética , Interleucina-2/genética , Interleucina-5/genética , Interleucina-6/genética , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/virologíaRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) has been reported to be involved in the development and progression of acquired immunodeficiency syndrome (AIDS). To study the role of this cytokine in AIDS pathogenesis, we constructed a chimeric simian and human immunodeficiency virus (SHIV) having the human TNF-alpha gene (SHIV-TNF) and characterized its properties in vitro. SHIV-TNF replicated both in M8166, a human T cell line, and in monkey peripheral blood mononuclear cells (PBMCs). Along with SHIV-TNF replication, TNF-alpha was detected in the culture supernatant by ELISA. The maximum expression level of TNF-alpha reached 120 ng/ml in M8166 cells, and 2.5 ng/ml in monkey PBMCs. The expressed TNF was biologically active, as shown by a cytotoxic assay using TNF-sensitive L929 mouse fibroblasts. This activity was detected at least until 10 passages of SHIV-TNF (74 days after the initial infection). In monkey PBMCs, SHIV-TNF replicated much better than the parental SHIV-NI. Flow cytometric analysis showed that the death of monkey PBMCs infected with SHIV-TNF was severer than that caused by the parental SHIV-NI. These results suggest that SHIV-TNF would be useful for inducing the disease in a monkey model, which may contribute to a better understanding of the role of TNF-alpha in AIDS etiology.