Immunogenicity of chimeric peptide gene of Xinjiang hemorrhagic fever virus glycoprotein / 国际生物医学工程杂志

Objective:To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein.Methods:The 5' end was added or not added with interleukin-2 (IL-2) signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies. GcⅠ and GcⅡ and the epitopes previously identified on GcⅠ (Gc 233-248, Gc 241-256 and Gc 281-296) were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a. The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified, and the recombinant eukaryotes were extracted by indirect immunofluorescent assay. BALB/c mice were immunized by protein immunity, gene immunity, and DNA prime-protein boost immunity. The IgG antibody level was measured by ELISA. The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen. Results:The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed, and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy. After three times of immunization, the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group ( P<0.01). The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma (IFN-γ) revealed that the response of the DNA prime-protein boost immunity was biased to Th1. Conclusions:The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed, and the target antigen could be expressed effectively in vivo. The immune groups stimulated stronger humoral and cellular immune responses compared with the control group. Among them, the immune effect of pVAX1-ST(GcⅠe+GcⅡ) combined with recombinant protein r(GcⅠe+GcⅡ) was the best, and it is expected to be a new candidate vaccine for XHFV.

Effects of various prime-boost regimes on immunities in mice by using DNA and reocmbinant vaccin-ia-based H5N1 vaccines / 中华微生物学和免疫学杂志

Objective To develop an effective and broad immune protective vaccination strategy by using DNA and recombinant vaccinia-based H5N1 vaccines.Methods BALB/c mice were immunized with various prime-boost regimens by using different DNA ( pIRES-based or pVRC-based) and recombinant vaccinia (Tiantan strain, rTTV)-based H5N1 vaccines expressing multivalent antigens (HA, NA, M1 and M2).The differences of immunity induced by two DNA vaccines were compared between intradermal electro -poration (IDE) and intramuscular electroporation (IME) deliveries.Immune responses were analyzed by hemagglutination inhibition( HAI) assay, neuraminidase ( NA)-specific antibody measured by ELISA , mi-croneutralization assay and IFN-γELISPOT assay .Results High levels of humoral immunity and T cell re -sponses were induced in mice primed with DNA-based vaccine than those primed with rTTVb-ased vaccine . DNA priming by IDE resulted in higher levels of neutralizing antibody in mice than those by IME delivery . Higher levels of HAI and anti-NA antibodies as well as NA-specific T cell responses were induced by pVRC-based DNA prime than those by pIRES-based DNA prime .HA-specific T cell responses were also enhanced in mice primed with pVRC-based DNA than those primed with pIRES-based DNA by IME .Conclusion The prime-boost strategies by using DNA-based vaccine in combination with rTTV-based H5N1 vaccine could induce humoral and T cell responses in mice against multi-antigens .Immunities induced by vaccines in com-bination might be modulated by various prime regimes .The study provided references for the further develop-ment of novel H5N1 vaccine and the optimization of immunization programs of combined multi-antigen vac-cine candidates .

Evaluation of systemic and mucosal immune responses in mice administered with recombinant Salmonella Typhimurium expressing IutA protein / 대한수의학회지

Avian pathogenic Escherichia coli (APEC) are known to cause extraintestinal disease in poultry, leading to substantial losses in the industry. IutA, iron-regulated aerobactin receptor is firmly associated with APEC. To assess the potential of IutA to induce protective immune responses, attenuated Salmonella Typhimurium strain expressing IutA was constructed and administered orally to BALB/c mice. The IutA-specific immune responses were measured with sera, vaginal and fecal samples by an enzyme-linked immunosorbent assay. We found that the Salmonella-IutA vaccine induced significantly higher immune responses as compared to the control inoculated with the attenuated S. Typhimurium containing the plasmid only. The IutA-specific immune responses were increased by second immunization at third week after initial immunization, whereas triple immunization induced lower immune responses than those induced by the double immunization. The Salmonella-IutA vaccine induced a nature of immunity biased to the Th1-type, as judged by the ratio of IutA-specific IgG isotypes (IgG2a/IgG1). Overall, these results suggest that the Salmonella-IutA vaccine appear to be suitable candidate for a vaccine against APEC.

Immune responses induced by DNA vaccine combined with recombinant adenovirus containing MOMP gene of Chlamydia trachomatis serovar E / 中华微生物学和免疫学杂志

Objective To investigate the specific immune responses induced by DNA vaccine com-bined with recombinant adenovirus carrying major outer membrane protein ( MOMP) gene of Chlamydia trachom-atis (Ct) serovar E.Methods Recombinant eukaryotic expression plasmid pVAX 1-MOMP and recombinant adenovirus Ad-MOMP were constructed and purified .Four immunization strategies were designed including DNA immunization (DNA group), recombinant adenovirus immunization (Ad group), DNA prime-recombinant ade-novirus boost regimen ( DNA/Ad group ) and recombinant adenovirus prime-DNA boost regimen ( Ad/DNA group) .Two weeks after the final immunization , vaginal wash specimens , blood samples and spleens were col-lected from all mice for the evaluation of humoral and cellular immune responses .Results Th1 responses and mucosal responses were not found in DNA group .Ad group induced both Th 1 responses and local mucosal re-sponses , and its IgG and IgA levels were significantly higher than those in DNA group .DNA/Ad group and Ad/DNA group induced stronger Th 1 responses and humoral responses than DNA and Ad group .Serum antibodies and mucosal antibodies in DNA/Ad group were higher than those in Ad/DNA group, but Th1 responses in DNA/Ad group were impaired than those in Ad/DNA group.Conclusion The recombinant adenovirus could induce Th1 responses and genital mucosal responses .Combined immunization of DNA/Ad or Ad/DNA showed enhanced specific immune responses in comparison with the single immunization with DNA or Ad .The differ-ences between the two combination methods were that DNA /Ad group stimulated higher levels of specific anti-body responses , while Ad/DNA group produced stronger Th 1 responses .The study provided a reference for the enhancement and development of vaccines against Chlamydia trachomatis in further studies .

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

The immunological effect of Ad/MDC-VP1 combined with DNA vaccine against Coxsackievirus infection / 中华微生物学和免疫学杂志

Objective To construct recombinant adenovirus Ad/MDC-VP1 and investigate its im-muno-boosting effect of the mice primed with the experimental DNA vaccine against Coxsackievirus infection. Methods The recombinant adenovirus Ad/MDC-VP1 was constructed and packaged. The Western blot analysis was used to verify the target protein. BALB/c mice were divided into four groups: Ad/MDC-VP1 group, pcDNA3/MDC-VP1 group, pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group and PBS group. The mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assay, respectively. The lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with le-thal dose of Coxsackievirus, and the assay of the serum virus titers and the observation of protection efficacy against Coxsackievirus infection were carried out. Results The recombinant adenovirus Ad/MDC-VP1 was successfully constructed and the target protein was expressed. It was observed that the titers of CVB3 VP1 specific antibody, lymphocyte stimulation index, CTL cytotoxicity activities and protection rate of the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group were much higher than those of the rest groups( P < 0.05), and the titer of serum virus was lower after CVB3 challenged ( P < 0.05 ). Conclusion Both the cellular and humoral immune responses in mice could been significantly enhanced by the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost strategy.