RESUMO
In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.
Assuntos
Anticorpos Monoclonais , Síndrome Respiratória Aguda Grave/diagnóstico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Animais , Chlorocebus aethiops , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Sensibilidade e Especificidade , Células VeroRESUMO
To establish a mucosal vaccine system, we evaluated the immunogenicity of a recombinant influenza virus, designated NS2Acat, in which the chloramphenicol acetyltransferase (CAT) reporter gene is fused in frame to the NS1 gene in the NS gene segment. The NS2Acat replicated slightly within the lungs in BALB/c mice after intranasal administration, accompanying expression of the CAT and the viral HA mRNA. NS2Acat induced delayed-type hypersensitivity (DTH) and sensitized the CAT-specific T lymphocytes in the regional lymph nodes, which proliferated and synthesized several cytokines in vitro upon antigenic challenge. These results suggest that NS2Acat is capable of eliciting a respiratory immune response against the fused gene product.
Assuntos
Vacinas contra Influenza/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antivirais/biossíntese , Bovinos , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Feminino , Genes Reporter , Hipersensibilidade Tardia , Imunidade nas Mucosas , Técnicas In Vitro , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/genética , Interferon gama/biossíntese , Interleucina-4/biossíntese , Pulmão/imunologia , Pulmão/virologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Replicação ViralRESUMO
The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.