Serological Study of An Imported Case of Middle East Respiratory Syndrome and His Close Contacts in China, 2015.

The first imported Middle East respiratory syndrome (MERS) case in China was identified in May 2015. We determined the kinetics of antibody (IgG and IgM) and neutralizing antibodies against MERS-coronavirus (MERS-CoV) in this case before discharge. Moreover, no seroconversion was found among 53 close contacts by anti-MERS IgG antibody enzyme-linked immunosorbent assay (ELISA) of paired serum samples. These findings suggest that neither community nor nosocomial transmission of MERS-CoV occurred in China.

[Study on combined immunization of rAd/MDC-VP1 and pcDNA3/MDC-VP1 against Coxsackievirus B3 challenge in mice].

AIM: To immunize the mice using the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost strategy and observe its immunological effect against Coxsackievirus B3(CVB3). METHODS: BALB/c mice were randomly divided into four groups: PBS group, rAd/MDC-VP1 group, pcDNA3/MDC-VP1 group and rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost group. Mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assays respectively. The Lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with lethal dose of CVB3, and the serum virus titer was assayed and the protection efficacy against Coxsackievirus infection was observed. RESULTS: It was observed that the titers of CVB3 VP1 specific IgG and neutralizing antibody, non-specific lymphocytic proliferation activity and specific lymphocytic CTL cytotoxic activity of the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost group were much higher than those of the rest groups(P<0.05), what's more, after CVB3 challenged, the serum virus titer of this group was lower and the protection rate(41.67%) was higher (P<0.05). CONCLUSION: Both the cellular and humoral immune responses in mice could be significantly enhanced by the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost strategy and the protection rate after challenged by lethal dose of virus could be increased.

[The effects of inoculation route and adjuvant type on the immunizing potency of CVB3 VP1 protein].

AIM: To explore the effects of inoculation route and adjuvant type on the immunizing potency of coxsackievrus B type 3 (CVB3) VP1 protein. METHODS: The recombinant plasmid pET-His/VP1 expressed CVB3 VP1 was transformed into E.coli BL21(DE3) pLysS and induced to express VP1 protein by IPTG, and verified by Western blot analysis. The fusion VP1 protein was purified with Ni affinity chromatography. Firstly, BALB/c mice were administered via different inoculation route(subcutaneous, intraperitoneal, intramuscular), with twelves mice in each group. Secondly, combined with various adjuvants (Alum, Freund's adjuvant, Montanide ISA720), with eighteen mice in each group. The mice were immunized three times at a three week interval with 50 µg of VP1 protein. The titers of sera IgG and neutralizing antibody were detected by ELISA and neutralization assay. Cell mediated immune response was tested by the lymphocytes proliferation activity and specific CTL cytotoxic activity. The mice were challenged with lethal dose of CVB3, the titers of the sera virus were titrated. Furthermore, the survival rates of mice were observed. RESULTS: The VP1 protein was expressed in E.coli successfully and the fusion protein was purified. In different inoculation route, the titers of neutralizing antibody and specific IgG in intramuscular injection group was much higer than other groups (P<0.01). VP1 protein in combination with Montanide ISA720 and Freund's adjuvant elicit higher titer antibodies and cell mediated immune response, and the virus titers in blood were lower in comparison to Alum adjuvant group (P<0.05).The survival rate of Freund' adjuvant group was better than adjuvant AL(OH)(3); group (P<0.05). CONCLUSION: The VP1 protein combined with ISA720 and Freund's adjuvant given by intramuscular injection may induce an improved immune responses and the better survival rates of the mice after virus challenge.

[Construction of the recombinant adenovirus expressing the CVB3 sVP1-C3d3 protein and study on its immunological effects in mice].

AIM: To construct recombinant adenovirus Ad/C3d3-sVP1 and investigate the immune effects against coxsackievirus infection in mouse. METHODS: The recombinant adenovirus Ad/sVP1-C3d3 was constructed and packaged. BALB/c mouse were divided into four groups: Ad/sVP1-C3d3 group, Ad/VP1 group, Ad group and PBS group. The mice in each group were immunized by intramuscular injection. The titers of sera IgG and neutralizing antibody were detected by ELISA method and trace neutralization assay, respectively.The specific CTL cytotoxic activity was detected by CCK-8 assay. The mice in each group were challenged with lethal dose of coxsackievirus, the titers of the sera virus were titrated. RESULTS: The recombinant adenovirus Ad/sVP1-C3d3 was successfully constructed. It's observed that the titers of CVB3 VP1 specific antibody and neutralizing antibody were much higher than those of the other three groups(P<0.01). CTL cytotoxicity activities was much higher than PBS and Ad group(P<0.01), but little higher than Ad/VP1 group(P<0.05).The titer of sera virus was lower than Ad and PBS groups after CVB3 challenged(P<0.05). CONCLUSION: Both the celluar and humoral immune responses in mice could been significantly enhanced by Ad/sVP1-C3d3.

[Immune effects of four coxsackievirus B3 VP1 DNA fusion vaccines in mice].

AIM: To compare the immunogenicity and protective effects on CVB3 infected mice of four DNA fusion vaccines coupling coxsackievirus B3 (CVB3) VP1 with macrophage-derived chemokine (MDC), C3d3, shiga toxin B subuit (STxB) and mouse beta-defensin-2 (mBD2), respectively. METHODS: BALB/c mice were divided into 6 groups randomly and inoculated in quadriceps at 3-week interval for 3 times with pcDNA3, pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1, respectively. Fourteen days after every inoculation, serum samples were collected and CVB3 specific neutralizing antibodies were determined. Three weeks after the last immunization, the mice were treated in three ways. First, the spleen cells were isolated from 3 mice of each group and specific CTL activities were tested. Second, 3 mice of each group were further challenged with 3LDLD(50); CVB3 and sacrificed 7 days later, and their blood viral titers were evaluated. Third, the rest mice of each group were subjected to intraperitoneal (i.p.) challenge with 5LDLD(50); CVB3 and their survival was observed. RESULTS: The neutralizing antibodies against CVB3 were induced in pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1 groups, and antibody titers correlated with the number of injections (P<0.01). After three immunizations, the antibody titers in pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2 -VP1 groups were higher than the ones in pcDNA3/VP1and pcDNA3/STxB-VP1 groups (P<0.01). The specific CTL activities in both pcDNA3/STxB-VP1 and pcDNA3/mBD2-VP1 groups were significantly stronger than those in the other groups (P<0.01). After CVB3 challenge, the blood viral titers in the pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2-VP1 groups were lower than those in the other groups (P<0.01), and the pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 mice survived longer than the others (P<0.05). CONCLUSION: Both pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 vaccines could induce stronger immune responses, resulting in higher survival rates and better protective effects on CVB3 infection than pcDNA3/STxB-VP1, pcDNA3/mBD2-VP1 and pcDNA3/VP1 vaccines.