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Objective To develop a novel anti-Enterovirus 71 (EV71) vaccine as recombinant virus-like particles.Methods By utilizing the foreign antigen presentation and virus-like particles forming features of Norovirus casipid VP1 P domain (NoVP), two pET-28a (+)-based recombinant expression plasmids containing either NoVP alone or NoVP with three specific epitopes SP55, SP70 and VP2-28 of EV71 capsid proteins tandemly inserted at the surface loop site were constructed and transferred to Escherichia coli.The recombinant fusion proteins of NoVP + EV71-SP55-SP70-VP2-28 and NoVP were induced expression and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and transmission electron microscopy (TEM) observation.BALB/C mice were randomly divided into three groups:group A immunized with the recombinant fusion protein, group B immunized with NoVP and group C injected with 10 mmol/L Tris plus 20 mmol/L NaCl (pH 9.0).Enzymelinked immunosorbent assay (ELISA) was used to test specific antibodies in the serum of the mice, besides, the serums were mixed with the EV71 H3-TY strain and Vero cells, then specific antibody titer was examined by microneutralization test.One way ANOVA and Bonferroni test were used to analyze data.Results Both recombinant fusion protein and NoVP were expressed in Escherichia coli in inclusion bodies form.SDS-PAGE demonstrated that the relative molecular weights of recombinant fusion protein and NoVP protein were approximately 43 × 103 and 36 × 103, respectively;positive protein band of about 43 × 103 (relative molecular mass) was detected in recombinant fusion protein by Western Blot.Virus-like particles derived from the recombinant fusion proteins were observed under TEM.ELISA showed that absorbance 490 (A490) of mice serum added in SP70 peptide was significantly higher than those of group B and C (F =13.860,P ＜0.05).And microneutralization test demonstrated that the serum from group A was able to neutralize EV71 at a geometric mean titer above 1:38.Conclusion A novel virus-like particles vaccine against EV71 with good antigenicity and specificity has been prepared, which is able to induce high titer of neutralizing antibody against EV71 in mice.
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Objective To promote the progress in varicella-zoster virus (VZV) immunoglobulin preparation,a rapid microneutralization test ( RMNt) was set up for screening plasmapheresis donations with high titers of special neutralizing antibodies to VZV.Methods With reference to the VZV immunoglobulin (VZIG) preparation standard of FDA and VZIG international unit ( IU) , a screening standard was formulated; the amount of virus was analyzed to determine the optimal conditions for RMNt;screening technology was established and the IU was introduced as quality control ;twenty samples of apheresis plasma and fifteen samples of pooled plasma were diluted at 1∶2 to 1∶256 and tested by RMNt respectively;and the sensitivity of RMNt was also analyzed by the commercial ELISA kit .Results Plasma samples that were diluted at 1∶16 and had a titer more than 0.4 U/ml could be used in the production of VZIG .1500 PFU/ml titers of virus in RMNt pro-duced readable results in plasma screening .Eight apheresis plasma samples tested met the screening standard , but none of the pooled samples tested positive .RMNt had a good linear relationship with ELISA (r=0.895 24,P<0.0001).Conclu-sion The sensitivity, throughput and operability of the established RMNt can be used in the screening of plasma donations as key techniques for the production of VZIG .
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OBJECTIVE: To prepare the first batch of national standard for enterovirus 71 immunoglobulin for the efficacy test of EV71 human immunology products. METHODS: The domestic intravenous immunoglobulin products with batch release certification and high efficacy EV71 immunoglobulin products were mixed, filled, and lyophilized under aseptic conditions to get the first batch of national standard for enterovirus 71 immunoglobulin. The standards were distributed to five laboratories for cooperative calibration according to the unified SOP for microneutralization test. Neutralizing titer which corresponded to the reciprocal of the highest serum dilution that neutralized enterovirus 71 was defined as the efficacy (reported as unit) of the national standard. Sterility test, moisture determination, precision for filling test, and stability of potency were verified. RESULTS: A total of 63 calibration tests were earned out by the five collaboration laboratories, and the results were statistically analyzed after logistic convertion. The inter-laboratories variations varied from 1.5%-4.1% and the intra-laboratories variation was 3.1%. The geometric mean of the prepared national EV71 immunoglobulin standard was 327 U and defined as 330 U for convenience of use. The potency of the prepared standard was stable after 22 m and the contents of monomer plus dimer determined by HPLC-SEC were more than 98.0% during storage at a wide range of temperatures. The prepared national EV71 immunoglobulin standard was qualified in the sterility test, and the moisture content and precision for filling were 0.6% and 0.56%, respectively. CONCLUSION: The prepared national EV71 immunoglobulin standard met all the relevant requirements and may be served as the first generation of national standard for the potency test of EV71 immunoglobulin products.
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BACKGROUND: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus. METHODS: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed. RESULTS: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results. CONCLUSIONS: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.