RESUMO
RNA interference (RNAi) is a post-transcriptional, gene silencing mechanism which uses small interfering RNA molecules (siRNA) for gene silencing. Respiratory Syncytial Virus (RSV) is an important respiratory pathogen of medical significance that causes high mortality in infants. The fusion (F) protein of RSV is a good target for therapeutic purposes as it is primarily responsible for penetration of the virus into host cells and subsequent syncytium formation during infection. In the present study, four siRNAs were designed and used individually as well as a mixture, to silence the RSV F gene. The relationship between siRNA design, target RNA structure, and their thermodynamics was also investigated. Silencing of F gene was observed using indirect immunofluorescence, western blot, reverse transcription PCR, and progeny viral titers. Our results show F gene silencing by all the four siRNAs individually and collectively. RT-PCR analysis revealed a decrease in mRNA level which corresponded to decreased F protein expression. siRNAs also inhibited RSV progeny as shown by viral titer estimation on infected HEp-2 cells. The present study demonstrates the silencing of the F gene using siRNA. Thermodynamic characteristics of the target RSV mRNA and siRNA seem to play an important role in siRNA gene silencing efficiency.
Assuntos
Conformação de Ácido Nucleico , Interferência de RNA , RNA Mensageiro/química , RNA Interferente Pequeno/química , Vírus Sinciciais Respiratórios/genética , Proteínas Virais de Fusão/genética , Linhagem Celular Tumoral , Técnica Indireta de Fluorescência para Anticorpo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Relação Estrutura-Atividade , Termodinâmica , Proteínas Virais de Fusão/metabolismo , Ensaio de Placa ViralRESUMO
RSV vaccine development has constraints due to safety issues encountered by formalin-inactivated FI-RSV vaccines. A desirable vaccine should induce Th(1) responses and a strong mucosal immunity to provide complete protection from RSV infection. In the present paper, we developed and evaluated a mucosal vaccine against RSV in a mouse model. The antigenic regions corresponding to residues 412-524 of RSV-F protein were amplified by RT-PCR and cloned into a vector containing the ctxA(2)B gene of the cholera toxin. The recombinant protein was expressed in E. coli and properties of the recombinant protein were analyzed by SDS-PAGE, Western blot and G(M1)-ELISA. The purified recombinant protein (rRF-412) was used to immunize BALB/c mice intranasally. The results from our studies show that the rRF-412 immunogen induced mucosal (IgA) and systemic antibody (IgG, IgG1, IgG2a, and IgG2b) responses which neutralized RSV. The IgG1/IgG2a ratios indicated a Th(1)-biased antibody response. The Th(1) (TNF-alpha, IL-12p70, IFN-gamma, IL-2) and Th(2) (IL-10, IL-4 and IL-5) cytokine profiles were analyzed after stimulation of spleen cells from mice immunized with purified RF-412 protein. Similar to the antibody response, we observed that the rRF-412 immunogen induced a mixed Th(1)/Th(2) cytokine immune response with a Th(1)-bias response. Serum antibodies were capable of neutralizing RSV and mice immunized with rRF-412 were significantly protected from live RSV challenge. Our data provides evidence that the rRF-412 immunogen may be a potential mucosal vaccine candidate against RSV.
Assuntos
Modelos Animais de Doenças , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Antivirais/sangue , Feminino , Regulação Viral da Expressão Gênica , Imunidade nas Mucosas , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus Sinciciais Respiratórios/metabolismo , Proteínas Virais de Fusão/genéticaRESUMO
We have developed and evaluated an immunodominant respiratory syncytial virus (RSV) F antigen in a mouse model. The antigenic region corresponding to amino acids 255-278 of the RSV F protein was cloned into a vector containing the ctxA(2)B gene of cholera toxin (CT). The recombinant protein was expressed in Escherichia coli and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The purified protein was evaluated by immunoblot and ganglioside GM(1) enzyme-linked immunosorbent assay to confirm the expression of the RSV F protein and to correct association of the recombinant protein to form a holotoxin-like chimera, respectively. We hypothesized that genetic fusion of modified CT-based adjuvant with RSV F immunodominant epitopes (rRF-255) would induce protective humoral and cellular immune responses in mice. Intranasal immunization of mice with rRF-255 overall induced higher concentrations of anti-RSV F-specific antibodies in both serum and saliva as compared with mice immunized intranasally with RSV or phosphate-buffered saline (PBS). Antibody isotype analysis (IgA, IgG1, IgG2a, and IgG2b) was also performed. The predominant IgG2a antibody isotype response in combination with cytokine analysis of helper T cell type 1 (interferon-gamma, interleukin [IL]-2, IL-12 p70, and tumor necrosis factor-alpha) and helper T cell type 2 (IL-4 and IL-10) responses revealed that rRF-255 antigen induces a prominent helper T cell type 1 immune response in mice. The rRF-255 antigen also induced serum neutralizing antibodies in immunized mice. Analysis of RSV load in lungs showed that rRF-255 immunization provided significant protection compared with PBS control animals.
