RESUMEN
Glycoprotein K (gK) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays an important role in virion morphogenesis and egress. We previously demonstrated that immunization of mice with gK, but not with any of the 10 other HSV-1 glycoproteins, resulted in exacerbation of corneal scarring and herpetic dermatitis following ocular HSV-1 infection. However, little is known about the gK epitope(s) that is (are) involved in T cell activities in vitro or in vivo. Thus, epitope mapping of gK was performed using a panel of 15-mer peptides with five-amino acid overlaps spanning the full-length gK, and four expressed gK recombinant proteins representing different regions of gK. Epitope mapping within the gK polypeptide defined the amino acid sequence STVVLITAYGLVLVW as the predominant CD4(+) and CD8(+) T cell stimulatory region both in vitro and in vivo. IFN-gamma expression by CD4(+) T cells was CD8(+) T cells-dependent. This immunodominant epitope is located within the signal sequence of the gK polypeptide and is highly conserved in HSV-1 and HSV-2 strains. Using prediction algorithms, the peptide is predicted to bind to numerous MHC class I and class II molecules.
Asunto(s)
Mapeo Epitopo , Epítopos de Linfocito T/inmunología , Herpesvirus Humano 1/inmunología , Señales de Clasificación de Proteína , Linfocitos T/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Femenino , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Interferón gamma/biosíntesis , Activación de Linfocitos , Ratones , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Señales de Clasificación de Proteína/genética , Conejos , Proteínas Recombinantes , Transducción de Señal , Spodoptera , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
In this study, we have taken advantage of the unique property of a potent dendritic cell (DC) growth factor, Flt-3 ligand (FL), which could act as a vaccine adjuvant. Accordingly, a single injection of plasmid DNA coding for soluble FL (FLex) was shown to induce large numbers of DCs in various tissue compartments and was critical for generating high frequencies of antigen-specific (HIV gp120 and LCMV NP) immune responses in mice. Interestingly, this enhanced level of immune response is strictly dependent on the co-delivery (i.m.) of the DNA vaccines and hFLex DNA to mice harboring large numbers of DCs. The high frequencies of antigen-specific CD8(+) T cells were largely associated with the expansion phase of DCs in vivo. However, DC expansion and immune enhancement have not reciprocally maintained a linear correlation, suggesting that other factors, cytokines/chemokines, which have the potential to modulate the microenvironment of DCs, could influence immunological outcome in this vaccination modality.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/genética , Animales , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Proliferación Celular , ADN Viral/genética , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Memoria Inmunológica , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos BALB C , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Plásmidos/genética , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
DNA vaccines exploit the inherent abilities of professional antigen-presenting cells to prime the immune system and to elicit immunity against diverse pathogens. In this study, we explored the possibility of augmenting human immunodeficiency virus type 1 gp120-specific immune responses by a DNA vaccine coding for a fusion protein, CTLA4:gp120, in mice. In vitro binding studies revealed that secreted CTLA4:gp120 protein induced a mean florescence intensity shift, when incubated with Raji B cells, indicating its binding to B7 proteins on Raji B cells. Importantly, we instituted three different vaccination regimens to test the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of both cellular (CD8(+)) and antibody responses. Each of the vaccination regimens incorporated a single intramuscular (i.m.) injection of the DNA vaccines to prime the immune system, followed by two booster injections. The i.m.-i.m.-i.m. regimen induced only modest levels of gp120-specific CD8(+) T cells, but the antibody response by CTLA4:gp120 DNA was nearly 16-fold higher than that induced by gp120 DNA. In contrast, using the i.m.-subcutaneous (s.c.)-i.m. regimen, it was found that gp120 and CTLA4:gp120 DNAs were capable of inducing significant levels of gp120-specific CD8(+) T cells (3.5 and 11%), with antibody titers showing a modest twofold increase for CTLA4:gp120 DNA. In the i.m.-gene gun (g.g.)-g.g. regimen, the mice immunized with gp120 and CTLA4:gp120 harbored gp120-specific CD8(+) T cells at frequencies of 0.9 and 2.9%, with the latter showing an eightfold increase in antibody titers. Thus, covalent antigen modification and the routes of genetic vaccination have the potential to modulate antigen-specific immune responses in mice.
Asunto(s)
Vacunas contra el SIDA/inmunología , Antígenos de Diferenciación/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Animales , Antígenos CD/análisis , Antígenos de Diferenciación/genética , Antígeno B7-1/análisis , Antígeno B7-2 , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4 , Femenino , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/genética , Inmunización Secundaria , Glicoproteínas de Membrana/análisis , Ratones , Ratones Endogámicos BALB C , Vacunación , Vacunas de ADN/administración & dosificaciónRESUMEN
DNA vaccines target dendritic cells (DC) to induce Ag-specific immune responses in animals. Potent HIV-specific immunity could be achieved by efficient priming of the immune system by DNA vaccines. We investigated a novel DNA vaccine approach based on the role of growth factors in DC expansion and differentiation. To this end, we constructed chimeric genes encoding the HIV envelope glycoproteins physically linked to the extracellular domain of Fms-like tyrosine kinase receptor-3 ligand (FLex; a DC growth factor; both mouse (m)FLex and human (h)FLex). These chimeric gene constructs synthesized biologically active, oligomeric FLex:gp120 fusion proteins and induced DC expansion (CD11c(+)CD11b(+)) when injected i.v. into mice. This DC expansion is comparable to that achieved by FLex DNA encoding native FLex protein. When delivered intramuscularly as DNA vaccines, hFLex:gp120 induced high frequencies of gp120-specific CD8(+) T cells in the presence or absence of FLex DNA-induced DC expansion, but gp120 and mFLex:gp120 elicited only low to moderate levels of Ag-specific CD8(+) T cells. In contrast, mFLex:gp120 induced high levels of anti-gp120 Abs under identical conditions of DNA vaccination. However, the Ab levels in mice immunized with DNA vaccines encoding hFLex:gp120 and gp120 proteins were low without DC expansion, but reached high levels comparable to that elicited by mFLex:gp120 only after the second boost in the presence of DC expansion. Importantly, the gp120-specific CD8(+) T cells persisted at high frequency for 114 days (16 wk) after a booster injection. These experiments provide insight into the importance of modulating DC function in vivo for effective genetic vaccination in animals.