[Research on the methods for titrating respiratory syncytial virus].

OBJECTIVE: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection in infants. It is very important to quantitative assay of RSV titer in the study on RSV pathogenesis, candidate vaccine and antiviral treatment. Therefore, we develop Real-time Quantitative PCR (Q-PCR) assay and enzyme immunospots (EIS) for titrating RSV and compare them with traditional 50% tissue culture infectious doses (TCID50). METHODS: Q-PCR, based upon the RSV-L genes, and EIS were utilized to titrate samples from RSV culture supernatants and RSV infected mouse lungs. Then, the results were compared with TCID50. RESULTS: For the samples from RSV culture supernatants, the ratio of Q-PCR and EIS (plaque forming unit, pfu) was 10:1 and the ratio of EIS and TCID50 was 10:1 when TCID50 was converted as pfu. For the samples from RSV infected mouse lungs, the ratio of Q-PCR and EIS was 1000:1 and the ratio of EIS and TCID50 was 5:1. CONCLUSION: We have successfully established Q-PCR and EIS to titrate RSV and compared them with TCID50. We concluded EIS is a cost-effective method to titrate RSV.

A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV), for which no clinically approved vaccine is available yet, is globally a serious pediatric pathogen of the lower respiratory tract. Several approaches have been used to develop vaccines against RSV, but none of these have been approved for use in humans. An efficient vaccine-enhancing strategy for RSV is still urgently needed. We found previously that oral SL7207/pcDNA3.1/F and intranasal FGAd/F were able to induce an effective protective immune response against RSV. The heterologous prime-boost immunization regime has been reported recently to be an efficient vaccine-enhancing strategy. Therefore, we investigated the ability of an oral SL7207/pcDNA3.1/F prime and intranasal (i.n.) FGAd/F boost regimen to generate immune responses to RSV. The SL7207/pcDNA3.1/F prime-FGAd/F boost regimen generated stronger RSV-specific humoral and mucosal immune responses in BALB/c mice than the oral SL7207/pcDNA3.1/F regimen alone, and stronger specific cellular immune responses than the i.n. FGAd/F regimen alone. Histopathological analysis showed an increased efficacy against RSV challenge by the heterologous prime-boost regimen. These results suggest that such a heterologous prime-boost strategy can enhance the efficacy of either the SL7207 or the FGAd vector regimen in generating immune responses in BALB/c mice.

Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus.

[Expression and purification of a secreted form of fusion glycoprotein of human respiratory syncytial virus encoded by recombinant baculovirus].

OBJECTIVE: To study the expression and purification of a secreted form of fusion glycoprotein (sF) of human respiratory syncytial virus (RSV) encoded by recombinant baculovirus. METHODS: According the ORF of F protein, a pair of specific primers was designed and PCR technique was exploited to amplify the gene of sF in which the gene sequence of the transmembrane and cytoplasmic tail domains were replaced by a C-terminal six-histidine tag. Then, a recombinant baculovirus encoding sF-His was constructed, and transfected into sf9 insect cells by Lipofectamine cellfectine reagent. Finally, the expressed sF was purified by Ni2+ -affinity chromatograph. RESULTS: The gene encoding sF-His was obtained. The resulting construct of recombinant baculovirus is capable of expressing sF protein. The concentration of Ni2+ -affinity chromatograph purified sF is 1.084 mg/ml with the purity of no less than 90%. CONCLUSION: Baculovirus expression system is a good method for large scale of preparation of sF. The purified F paves the way for the development of potential RSV vaccine and diagnostic kit, etc.

Oral respiratory syncytial virus (RSV) DNA vaccine expressing RSV F protein delivered by attenuated Salmonella typhimurium.

Human respiratory syncytial virus (RSV) is a major viral pathogen of the lower respiratory tract of infants and young children worldwide. No effective prevention measure is available. Attenuated Salmonella strains expressing heterologous antigens can be delivered by the oral route, triggering efficient antigen-specific humoral, cellular, and mucosal immunity. In this study, we orally administered attenuated Salmonella strain SL7207, carrying the plasmid pcDNA3.1/F expressing the RSV F gene, to BALB/c mice and showed significant elevations of serum anti-RSV IgG and bronchoalveolar lavage secretory IgA as compared with the control group carrying empty plasmid (p<0.001). The ratio of IgG1 and IgG2a was 0.96. The experimental group also showed a stronger cytotoxic T cell response (p<0.01 at effector:target ratios of 100:1 and 50:1) and a higher stimulation index value of T cell proliferation (p<0.05) than the respective control group. RSV titers in the lung homogenates of the experimental group on day 3 and day 5 postchallenge were lower than in the control group (p<0.05). Histopathological analysis showed obvious differences in infiltration of inflammatory cells and pulmonary alveolar wall thickness (p<0.01) between the two groups. In summary, our results demonstrate the potential of orally administered SL7207-based DNA vaccines against RSV infection.