RESUMEN
Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8(+) T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC(42-50), GLVGLVTFL; GPC(60-68), SLYKGVYEL; and GPC(441-449), YLISIFLHL) that displayed high-affinity binding (< or =98 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC(42-50) or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.
Asunto(s)
Mapeo Epitopo , Epítopos de Linfocito T/química , Antígeno HLA-A2/metabolismo , Virus Lassa/inmunología , Proteínas de la Nucleocápside/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Humanos , Inmunización , Fiebre de Lassa/inmunología , Fiebre de Lassa/prevención & control , Virus Lassa/genética , Virus Lassa/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Péptidos/administración & dosificación , Péptidos/química , Péptidos/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.
Asunto(s)
Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/genética , Coriomeningitis Linfocítica/inmunología , Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/genética , Vacunas de ADN/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/virología , Cisteína Endopeptidasas/fisiología , Relación Dosis-Respuesta Inmunológica , Antígenos de Histocompatibilidad Clase I/biosíntesis , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/biosíntesis , Epítopos Inmunodominantes/inmunología , Recuento de Linfocitos , Coriomeningitis Linfocítica/enzimología , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Complejos Multienzimáticos/fisiología , Nucleoproteínas/administración & dosificación , Nucleoproteínas/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/virología , Vacunas de ADN/administración & dosificación , Carga ViralRESUMEN
Several viruses in the Arenavirus genus of the family Arenaviridae cause severe, often fatal, hemorrhagic fever. One such virus, Lassa virus (LV), is a frequent cause of disease in Africa, and survivors often are left with substantial neurological impairment. The feasibility of protective immunization against LV infection, and the associated disease, has been demonstrated in animal models, using recombinant vaccinia viruses to deliver Lassa proteins. Circumstantial evidence implicates cellular immunity in this Lassa-induced protection, but this has not been confirmed. Here, we describe DNA vaccines that encode LV proteins. A single inoculation of a plasmid encoding full-length Lassa nucleoprotein (LNP) can induce CD8(+) T cell responses in mice and can protect against challenge with two arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). A DNA minigene vaccine encoding a 9 amino acid sequence from LNP also induces CD8(+) T cells and protects against arenavirus challenge, thus confirming prior speculation that protective cellular immunity is induced by LV proteins.
Asunto(s)
Infecciones por Arenaviridae/prevención & control , Linfocitos T CD8-positivos/inmunología , Virus Lassa/inmunología , Coriomeningitis Linfocítica/prevención & control , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Infecciones por Arenaviridae/virología , Células HeLa , Humanos , Inmunización , Virus Lassa/genética , Virus Lassa/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Nucleoproteínas/metabolismo , Virus Pichinde/patogenicidad , Vacunas de ADN/administración & dosificación , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Vacunas Virales/administración & dosificaciónRESUMEN
The antigen presentation pathways constitute a fulcrum on which adaptive immunity is balanced, and their manipulation should allow us to induce designer immune responses. The ease and rapidity with which DNA vaccines can be constructed and altered make them ideal candidates with which to test the various targeting strategies that have been conceived to date. These approaches and the mechanisms that may (or may not) underlie their success are reviewed in this article.