RESUMEN
In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.
Asunto(s)
Inmunidad Adaptativa , Antígenos Virales/inmunología , Baculoviridae/genética , Proteínas de la Cápside/inmunología , Virión/inmunología , Animales , Presentación de Antígeno , Antígenos Virales/genética , Baculoviridae/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Técnicas de Visualización de Superficie Celular , Inmunidad Humoral , Ovalbúmina/inmunología , Vesiculovirus/genética , Vesiculovirus/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/inmunologíaRESUMEN
Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.
Asunto(s)
Epítopos/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Glicoproteínas/inmunología , Vesiculovirus/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Epítopos/genética , Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Glicoproteínas/genética , Inmunoglobulina G/sangre , Memoria Inmunológica , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Secuencias Repetidas en Tándem , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vesiculovirus/genética , Proteínas Estructurales Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Vesicular stomatitis (VS) viruses have been classified into two serotypes: New Jersey (VSNJV) and Indiana (VSIV). Here, we have characterized field isolates causing vesicular stomatitis in Brazil and Argentina over a 35-year span. Cluster analysis based on either serological relatedness, as inferred from virus neutralization and complement fixation assays, or nucleotide sequences of two separate genes (phosphoprotein or glycoprotein) grouped the field isolates into two distinct monophyletic groups within the Indiana serogroup. One group included seven viruses from Brazil and Argentina that were serologically classified as Indiana-2 and Cocal virus (COCV). The other group contained three viruses from Brazil that were serologically classified as Indiana-3 and the prototype of this group, Alagoas virus (VSAV). Interestingly, two vesiculoviruses that were isolated from insects but do not cause disease in animals, one from Brazil (Maraba virus; MARAV) and the other from Colombia (CoAr 171638), grouped into two separate genetic lineages within the Indiana serotype. Our data provide support for the classification of viruses causing clinical VS in livestock in Brazil and Argentina into two distinct groups: Indiana-2 (VSIV-2) and Indiana-3 (VSIV-3). We suggest using nomenclature for these viruses that includes the serotype, year and place of occurrence, and affected host. This nomenclature is consistent with that currently utilized to describe field isolates of VSNJV or VSIV in scientific literature.
Asunto(s)
Antígenos Virales/inmunología , Enfermedades de los Bovinos/virología , Enfermedades de los Caballos/virología , Insectos/virología , Infecciones por Rhabdoviridae/veterinaria , Vesiculovirus/genética , Vesiculovirus/inmunología , Animales , Antígenos Virales/genética , Bovinos , Caballos , Datos de Secuencia Molecular , Pruebas de Neutralización , Filogenia , Infecciones por Rhabdoviridae/virología , América del Sur , Vesiculovirus/clasificación , Vesiculovirus/aislamiento & purificaciónRESUMEN
An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p(delta)GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and deltaGF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 microg each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of deltaGF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with deltaGF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-microg doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-deltaGF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 microg/ml, was quite limited in its ability to inhibit binding of anti-deltaGF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.
Asunto(s)
Antígenos Virales/inmunología , Aphthovirus/inmunología , Cápside/inmunología , Glicoproteínas de Membrana , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Aphthovirus/genética , Baculoviridae/genética , Cápside/genética , Proteínas de la Cápside , Línea Celular , Chlorocebus aethiops , Cricetinae , Fiebre Aftosa/inmunología , Vectores Genéticos , Inmunogenética , Pruebas de Neutralización , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Spodoptera/citología , Vesiculovirus/genética , Proteínas del Envoltorio Viral/genéticaAsunto(s)
Humanos , Masculino , Femenino , Niño , Adulto , Persona de Mediana Edad , Anticuerpos Antivirales/análisis , Vesiculovirus/inmunología , BrasilRESUMEN
A serological survey of Piry was undertaken of the sera of inhabitants of Catolância-Bahia State, Brazil. Serum results obtained by a vesiculovirus neutralization test of C6/36 cells read by ELISA are compared with those obtained by the classic technique, carried out on newborn mice. The agreement between the results was as high as 98.7% of the 204 sera tested and the neutralization test of C6/36 cells was chosen as the most suitable technique for the sero-survey testing.
Asunto(s)
Anticuerpos Antivirales/sangre , Pruebas de Neutralización/métodos , Vesiculovirus/inmunología , Animales , Brasil , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Ratones , Vesiculovirus/aislamiento & purificaciónAsunto(s)
Anticuerpos Antivirales/análisis , Vesiculovirus/inmunología , Adulto , Anciano , Brasil , Niño , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Vesicular stomatitis New Jersey (VS NJ) virus is capable of undergoing rapid evolution in nature and therefore has the potential for antigenic variation. We selected an area of Costa Rica where VS NJ virus is enzootic to study whether this virus used the mechanism of antigenic variation to persist in nature. Three sentinel herds and three nonsentinel herds were observed from 1986 to 1988. Eleven VS NJ virus isolates were collected from naturally infected cattle. Remarkably, nine animals that were bled prior to reinfection with VS NJ virus had neutralizing antibody titers up to 1: 102,400 yet virus was isolated from, and disease was observed in, these animals. Sequence analysis of the portion of the glycoprotein gene coding for the neutralizing epitopes demonstrated that all virus isolates were 98-100% similar with no indication of specific genetic variation. The 3' end of the glycoprotein gene also remained stable in that all isolates were again 98-100% similar in nucleotide sequence. Each isolate was neutralized to equivalent titers with monoclonal antibodies directed against four neutralizing epitopes on the glycoprotein. Additionally, preisolation sera from each animal were able to neutralize the virus that caused the subsequent disease. These results provide evidence that antigenic variation is not a mechanism used by VS NJ virus to persist in an enzootic focus of Costa Rica.
Asunto(s)
Epítopos/análisis , Genes Virales , Glicoproteínas/genética , Vesiculovirus/genética , Proteínas Virales/genética , Proteínas Estructurales Virales/genética , Animales , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/inmunología , Costa Rica , Datos de Secuencia Molecular , Pruebas de Neutralización , New Jersey , ARN Viral/genética , Infecciones por Togaviridae/inmunología , Infecciones por Togaviridae/veterinaria , Vesiculovirus/inmunología , Vesiculovirus/aislamiento & purificaciónRESUMEN
Strains of vesicular stomatitis virus, New Jersey serotype (VSV-NJ), isolated from diseased cattle or swine were examined by genomic RNA sequencing for genetic diversity potentially leading to antigenic variations in their type-specific glycoproteins as determined by reactivity with epitope-specific monoclonal antibodies (MAbs). Seven field isolates recovered in Colorado, New Mexico, Georgia, and Mexico during the widespread 1982-1985 epizootic in the western United States resembled the prototypic 1952 Hazelhurst subtype by partial sequence homology, but amino acid reversions to the 1949 Ogden subtype occurred frequently. When studies were performed with MAbs directed to the Ogden subtype glycoprotein, relatively limited antigenic variation, and only in neutralization epitope VIII, was noted among two of five epizootic isolates from Colorado and New Mexico. However, amino acid differences in the glycoprotein of a 1983 isolate from an enzootic region of Georgia resulted in major antigenic deficiencies in epitopes V, VI, and VII as determined by Western blotting and neutralization of infectivity with epitope-specific MAbs. Quite a few genetic but no antigenic differences were noted in an enzootic 1984 isolate from Mexico, a potential origin of the United States epizootic. Marked or complete loss of epitopes VII, VI, VIII, and V can be traced to spontaneous mutations leading to amino acid substitutions at glycoprotein positions 199, 263, 275, and 317, respectively, in the enzootic Georgia isolate 07/83-GA-P and the epizootic New Mexico isolate 06/85-NM-B. By comparison, closely adjacent amino acid substitutions at glycoprotein positions 210, 268, 277, and 364 occurred in epitope-deficient mutants selected for resistance to neutralization by MAbs specific for epitopes VII, VI, VIII, and V, respectively. Two neutralization epitopes designated X and XI were found to be unique for the G protein of the 1952 Hazelhurst isolate..../52-GA-P. The epitope X-specific MAb H21, in particular, failed to neutralize the infectivity not only of the Ogden subtype..../49-UT-B but also was ineffective against all the 1982-1985 field isolates. The classical 1952 Hazelhurst strain of VSV-NJ is genetically and antigenically quite different from those viruses isolated during the 1982-1985 epizootic.