| Section I Prepration and effectiveness evaluation of RV subunit vaccineBackground:Rubella virus (RV) belongs to the Togaviridae family of positive-strand RNA viruses, which causes a mild, self-limiting disease in humans. However, it occasionally causes death and congenital rubella syndrome (CRS) of the fetus in pregnant women. The rubella vaccine presently used consists of an attenuated strain of the rubella virus that should not be used during pregnancy. Additionally, AIDS children and children with other severe immunodeficiencies can not be vaccinated with the attenuated rubella virus vaccine, and can severely suffer from rubella infection. The RV subunit vaccine which is a non-replicaion rubella vaccine is considered to be safer than the attenuated vaccine, and could be thus particularly useful for individualitys who can not immunized with the attenuated vaccine.The E1protein is transmembrane glycoprotein inserted in the membrane, and contains T-cell epitopes, B-cell epitopes and neutralizing epitopes which can induce humoral immunity and cellular immunity. Four neutralizing epitopes are contained in E1protein, which are E1(221-239), E1(234-252), E1(254-285) and E1(272-285).Of these, one region, E1(221-239) has been shown preciously to be the main neutralizing epitope and other region E1(272-285) contain overlapping T-cell and B-cell epitopes. Soluble fragments or full-length of El could be produced in baby hamster kidney (BHK-21), Vero cells, CHO cells and Pichia pastoris cell. And they have been shown to induce RV neutralizing antiboies in BALB/c mice and protect mice from RV infection.Objectives:To obtain the high expression level of purified E72-296protein of rubella virus (RV), to measure the immunogenicity of E72-296protein in BALB/c mice and to evaluate the safety and protection efficacy of the RV E72-296subunit vaccine.Methods:Four cloning and expression vectors-pGAPZaA-E25-395、pGAPZaA-E72-395、 pGAPZαA-E72-304and pGAPZaA-E72-296were transfered into the host Pichia pastoris, and expressed proteins induced by Zeocin. By the identification, E72-296protein expressed as a soluble, secreted form in Pichia pastoris cells. After purified on the anon exchange column SP-Sepharose and ZebaTM Desalt Spin Column, sample of E72-296was of high purity. SDS-PAGE, Mass spectrometry analysis, the BCA Protein Assay Kit and Western blot (WB) were also carried out to identify the physico-chemical property like molecular weight, purity, concentration and biologic activity of the protein, respectively.E72-296protein was combined with alum adjuvants to prepare RV E72-296vaccine. Then, BALB/c mice at6weeks of age were immunized with the RV E72-296vaccine, RV JR23strain or attenuated RV vaccine. Anti-RV antibodies were tested by indirect ELISA, and cell immune responses were examined by lymphocyte proliferation assay. NK cell activity and challenge tests were assayed to evaluate the safety of the E72-296protein. Secreted cytokines (IL-2, IL-5, IL-10and IL-12) were measured by ELISA and immunohistochemistry was conducted to evaluate protective efficacy of the E72-296protein.E72-296protein was used as coating antigen in ELISA for a better definition of specificity antibodies generated in response to RV. Different dilutions (1:20,1:40,1:80,1:160,1:320,1:640,1:1280and1:2560) of purified E72-296peptide (0.46mg/ml) and different dilutions of serum (1:25,1:50,1:100,1:200and1:400) were tested for proper concentration in ELISA.Results:1. Preparation of the RV subunit vaccineThe results of SDS-PAGE, Mass spectrometry analysis, the BCA Protein Assay Kit and WB showed the molecular weight and yield of purified E72-296protein were21kDa and106.88mg/L, respectively.2. Immunogenicity evaluation of the RV subunit vaccine Results showed that the level of specific IgG (P=1.000, P=0.002), neutralizing antibody (P=1.000, P=0.000), BHK21cell survival rate (P=1.000, P=0.000) and lymphocytes proliferation (P=1.000, P=0.002) in the RV E72-296Vaccine group were all significantly higher than that of control group but not of Attenuted RV Vaccine group. No significant difference was found between the level of the cytokines in mice immunized with or with out RV E72-296Vaccine. These results indicated E72-296protein could induce effect humoral immunity (A450nm=1.1448±0.1405), limited cellular immunity (SI=2.0656±0.2944), and was less toxic to NK cells than Attenuated RV vaccine [36.01%±7.04%vs21.36%±2.61%(P<0.05)].3Protection efficacy evaluation of the RV subunit vaccineThe results of HE and immunohistochemical stainings showed no statistically significant difference between RV E72-296Vaccine group and negative control group, and all tissues of the former group had RV viremia negative. The results of immunohistochemical analysis were further verified by the RT-PCR analyses. These results indicated RV E72-296protein can induced complete protection against RV challenge in mice.4Detection of anti-RV IgG for use with E72-296protein antigenThe purified E72-296protein was used in ELISA for detecting RV-specific antibodies. The optimum concentration of the purified E72-296protein and the optimum serial dilution which showed maximum difference between the positive and negative sera were1.44μg/well and1:25, respectively. The results of patient sera which were tested by the anti-E72-296IgG ELISA and commercial kit showed that the positive rate tested by the former kit was higher than the other, and it indicated that E72-296protein was a candidate antigen for ELISA because of its superior characteristic.Conclusions:In this study, we obtained purified E72-296protein by using the anon exchange column SP-Sepharose and ZebaTM Desalt Spin Colum. High specific RV IgG titer, limited proliferative activity of splenolymphocytes and less toxic to NK cells than Attenuated RV vaccine was induced by E72-296protein in BALB/c mice. Meanwhile, the results of challenge test showed effective protection of the subunit vaccine also was a superior vaccine instead of attenuated RV vaccine. Section Ⅱ Mutational analysis of hydrophobic amino acids and charged amino acids in neutralizing epitope of rubella virus E1protein Background:Residues221-239of rubella virus El glycoprotein contain antibody neutralization domains. On one hand, it was found that antibodies binding to amino acids221-239of RV El were abundant in RV-positive individuals but there were few antibodies in congenital rubella syndrome patients who were persistently infected with RV. On the other hand, the RV BCH-178peptide has been used as a coated antigen in ELISA for the screening of RV neutralizing antibodies. However, which amino acid residue on the BCH-178peptide affects the affinity of peptide-antibody binding is unknown.Antigen and antibody are attracted by Van der Waals forces and ionic and hydrogen bonds. These bonds require hydrophobic amino acids and charged amino acids in the antibody binding sites of antigen. Previous studies showed that polar/aromatic residues dominate the interaction in the antibody-antigen complexes averaging81%of the energy and50%of the antigen energy is attributable to four residue types, Arg, Lys, Asn and Asp. And other studies reported that the solvent exposed charged amino acids at the binding interface may be crucial for binding ability. But the roles of hydrophobic amino acids and charged amino acids on RV E1epitope were not determined.Objectives:The study was aimed to study the effect of hydrophobic amino acid mutatuions and charged amino acids mutations in the immunoreactivity and the immunogenicity of BCH-178peptide and identify the key amino acids in the important neutralizing epitope of E1protein. Further, the study also revealed the function mechanism of hydrophobic amino acids and charged amino acids in the BCH-178peptide-antibody binding.Methods:Site-directed mutational analyses were performed to explore the role of the hydrophobic amino acids (W218and W230), charged amino acids (D229, R237and H238) and uncharged (Q236) on the BCH-178peptide. The relationships between the binding of the anti-El antibody to E221-239peptide, BCH-178peptide and BCH-178 peptide mutations were detected by ELISA, and the binding kinetics of the peptide-antibody interaction was determined by biolayer interferometry. BALB/c mice at6weeks of age were immunized with the peptides and Freund’s Adjuvants. RV-specific antibody levels were detected by peptide-ELISA and Virion-based ELISA, and cell immune responses were examined by lymphocyte proliferation assay.Results:The R237H mutation, H238R mutation or R237A/H238A mutation in E213-239peptide was sufficient to abolish the binding activity. Furthermore, mutants W230F, W230A, W218A, Q236D and W218A/W230A mutation were found to significantly reduce the binding activity. However, mutants W230Y and D229A were found to increase the binding activity.The mutants W230F, W230A, W218A/W230A, D229A, Q236D, R237H, H238R and R237A/H238A were associated with a loss in imunogencity of BCH-178peptide and mutants W230Y and W218A did not affect the imunogencity. In addition, mutants W230F, W230A, W218A, W218A/W230A, D229A, Q236D, R237H, H238R and R237A/H238A were found to reduce the cellular immune response.Conclusions:R237and H238are key amino acids in the rubella virus El neutralization epitope. Replacement of R237or H238by other amino acids in BCH-178peptide results in losing the antigenicity and the peptide-antibody binding affinity. The mutant (W230A, W230F, W218A, D229A and Q236D) in BCH-178peptide abolish the antigenicity and reduce the peptide-antibody binding affinity. |
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