RESUMEN
The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/ß receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNß. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.
Asunto(s)
Interferón Tipo I/química , Interferón Tipo I/metabolismo , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Proliferación Celular/efectos de los fármacos , Chlamydia/efectos de los fármacos , Femenino , Humanos , Inmunidad Mucosa , Interferón Tipo I/farmacología , Ratones , Fosforilación , Conformación Proteica en Hélice alfa , Células RAW 264.7 , Receptores de Interferón/metabolismo , Reproducción , Factor de Transcripción STAT1/metabolismo , Transducción de SeñalRESUMEN
Viremic nonprogressors (VNPs) constitute a very scarce group of untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain stable CD4+ T cell counts despite high levels of HIV-1 replication. The specific factors associated with this atypical control of the HIV infection have been poorly described. Since specific T cell responses seem to be one of the main causes of HIV-1 control in elite controllers, we studied whether HIV-1 Gag-specific cytotoxic T lymphocyte (CTL) responses could also modulate disease control in VNPs. We characterized the immune responses from four VNPs compared to those of five standard progressors (SPs) during the first years of HIV-1 infection. We observed no differences in the breadth and frequency of Gag-specific cellular responses. Furthermore, we obtained 217 HIV-1Gag clonal sequences in which the viral variability of Gag increased over 3 years of infection for synonymous and nonsynonymous mutations in both VNPs and SPs. VNPs evolution rates in gag were comparable to SPs. This observation is in line with a similar accumulation of CTL putative escape mutations in Gag epitopes targeted by CTL responses. Altogether, the absence of viral pathogenesis in VNP individuals seems to be independent of HIV-Gag-specific CTL responses. This novel information guides to the study of alternative mechanism of HIV-1 pathogenesis control.IMPORTANCE Control of HIV infection has been widely studied in elite controllers or long-term nonprogressor models. However, there is a less-known group of individuals, termed viremic nonprogressors (VNPs), who maintain stable CD4+ T cell counts despite high plasma viremia. The mechanisms involved in this remarkable control of HIV-1 pathogenesis clearly have implications for the development of new drugs and vaccines. We show here for the first time that VNPs have immune responses and HIV-gag evolution similar to those of standard progressors. Remarkably, we demonstrate that the mechanism of pathogenesis control in these individuals differs from some elite controllers that are reported to have improved immune control. This is noteworthy since it opens the door to new, as-yet-unknown mechanisms for HIV control. Our novel results advance the understanding of mechanisms involved in viremic nonprogression and suggest that there are alternative mechanisms to the adaptive immune responses for an effective control of viral pathogenesis.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/patología , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Viremia/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Carga Viral , Viremia/virología , Replicación Viral/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Interferon epsilon (IFNÉ) is a type I IFN that is expressed constitutively in the female reproductive tract (FRT), and contributes to protection in models of sexually transmitted infections. Using multiple cell systems, including reporter cell lines and activated peripheral blood lymphocytes (PBLs), we show that recombinant IFNÉ impairs HIV infection at stage(s) post HIV entry and up to the translation of viral proteins. Consistent with this, IFNÉ upregulated a number of host cell restriction factors that block HIV at these stages of the replication cycle. The potency of IFNÉ induction of these HIV restriction factors was comparable to conventional type I IFNs, namely IFNα and IFNß. IFNÉ also significantly reduced the infectivity of progeny virion particles likely by inducing expression of HIV restriction factors, such as IFITM3, which act at that stage of infection. Thus, our data demonstrate that human IFNÉ suppresses HIV replication at multiple stages of infection.
Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Interferones/metabolismo , Replicación Viral , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/virología , Infecciones por VIH/patología , Células HeLa , Humanos , Interferón-alfa/metabolismo , Fitohemaglutininas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Linfocitos T/virología , Virión/efectos de los fármacos , Virión/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.
Asunto(s)
Reactores Biológicos , Vectores Genéticos , Lentivirus , Lentivirus/genética , Humanos , Vectores Genéticos/genética , Medio de Cultivo Libre de Suero , Línea Celular , Técnicas de Cultivo de Célula/métodos , Cultivo de Virus/métodos , Células HEK293 , Transfección/métodosRESUMEN
Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary T cells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement.