| Porcine reproductive and respiratory syndrome (PRRS) caused by Porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease characterized by severe reproduction failure in sow and serious respiratory disturbance in piglets. PRRSV has restricted tropism for porcine alveolar macrophages and antigenicity variation, resulting in the variant strain in prevalence currently. The traditional commercial vaccines to prevent PRRS are mainly attenuated and inactivated vaccines. Although attenuated vaccines can provide solid protection against PRRSV, there exist dangers of reversion to more virulent strain. Compared with attenuated vaccine, inactived vaccine is safe, but its effect is not stable. Therefore, it is of great importance to develop novel vaccine that is safer, more effective and cheaper for PRRS prevention.Modified vaccinia virus ankara (MVA) is considered to be one of the most promising vectors for expressing foreign antigens. Recombinant MVA was designed to mimic viral infections hereby eliciting the approriate innate 'danger' signals to the adaptive immune system. Additionally, the MVA life cycle allows for the synthesis of vaccine antigens in the cytosol and appears particularly suitable for efficient presentation via MHC-I molecules, the prerequisite for CD8+ T cell responses. Unlike reproducible vaccinia virus, MVA is not able to efficiently grow in human body and the other mammalians, so it is safe even for immunosuppressive animals. By now, preclinical vaccine research with MVA vectors has shown promise in a variety of animal models demonstrating protective humoral and cell-mediated immunity. Consequently, all these made MVA be the hot spot for recombinant living vector vaccine study. In this report, recombinant MVA coexpressing the two major protective antigens, modified GP5 and M protein of PRRSV were constructed and the immunogenic effect was evaluated. This paper includes:1. Immunogenicity for the recombinant Modified Vaccinia Virus Ankara coexpressing GP5 and M protein of Porcine Respiratory and Reproductive Syndrome VirusIn this study, four recombinant viruses that expressed GP5 and M protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in the Modified Vaccinia Virus Ankara (MVA) with different expression pattern were made. In these recombinant viruses (rMVAs), GP5 and M were expressed in MVA in a same virus but under the control of two promoters (rMVA-GP5/M), or as a fusion protein under one promoter (rMVA-GP5-M), or separately (rMVA-GP5 and rMVA-M). The humoral and cellular immune responses of the four recombinant viruses were evaluated with mouse model. Neutralizing antibody titer for each group was detected with virus neutralization test assay weekly after the primary vaccination for 13 weeks to evaluate the humoral immune response. The production of gamma interferon (γ-IFN), interleukin-2 (IL-2) and interleukin-4 (IL-4) was detected in splenocytes of rMVAs-inoculated mice at 30, 60 and 90 days post inoculation to evaluate the cellular immune response. Results showed, for both humoral and cellular immune response, the rMVA-GP5/M inoculation group got the earliest and highest immune response. The rMVA-GP5-M inoculation group, which inoculated with the rMVA of fusion protein of GP5 and M, got the immune response less than that of rMVA-GP5/M inoculation group but higher than that of groups inoculated with separately expressed GP5 or M protein. The strategy of coexpressing PRRSV GP5 and M protein in MVA under the control of two promoters might be an attractive method for future PRRSV vaccine design.2. Influence of epitope "A" modification and N-Linked glycosylated site mutation for GPS of PRRSV on the ability to induce neutralizing antibodies and T cell responseTo enhance the immunogenicity of ORF5 gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), CpG and the universal helper T cell antigen epitope (PADRE) were inserted between the decoy epitope (A) and the neutralizing epitope (B). At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 (MORF5) and ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot, the expression in vitro for the two genes was confirmed, then six-week-old Balb/C mouse were inoculated with the recombinant plasmid pcDNA-M5A-6A at 100μg/mouse. The recombinant plasmid pcDNA-5A-6A containning wild type ORF5 gene, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. PRRSV specific neutralizing antibody titer was evaluated with virus neutralization test assay, furthermore, the production ofγ-IFN, IL-2 and IL-4 in splenocytes was detected with commercial ELISA Kit. Results showed that recombinant plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous Th1 type response compared with the recombinant plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising vaccine candidate for PRRS prevention.3. Reconstruction for the transfer vector of the Modified Vaccinia Virus Ankara expression systemThe screening marker for the Modified Vaccinia Virus Ankara (MVA) expression system used in our laboratory is lacZ. Considering the shortage of this selecting marker, and combining with the BHK-21cell line that can express Cre recombinant enzyme in our lab (BHK-Cre), a novel transfer vactor for MVA was constructed with Eco gpt marker. In this new transfer vector, Eco gpt gene followed with P7.5 promoter of vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning sites that can coexpress two foreign genes under the control of other two vaccinia virus promoters were constructed outside the LoxP sites. With this new transfer vector, the Eco gpt marker in recombinant MVA can be deleted on BHK-Cre with the interaction between the Cre recombinant enzyme and LoxP sequence.4. The construction and biological characteristics for the recombinant Modified vaccinia virus ankara that coexpressing modified GPS and M protein of Porcine Reproductive and Respiratory Syndrome VirusThe modified ORF5 (MORF5) and ORF6 gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) were cloned into the two multiple cloning sites of the Modified vaccinia virus ankara (MVA) transfer vector pLR-gpt which was obtained in the former chapter to construct the recombinant transfer vector pLR-MORF5/ORF6. Homologous recombination between the recombinant plasmid pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell was mediated with liposome by infecting the cell with wtMVA at 0.01 MOI two hours before transfecting. When the cytopathic effect (CPE) was obvious, cell cultures was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2%MXHAT). After twelve cycles of selection, the recombinant MVA with a selecting marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this recombinant MVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 gene into the MVA genome was confirmed with PCR analysis, the expression of MGP5 and M protein was identified with Western-blot analysis and IFA and the deletion of Eco gpt gene was identified with PCR. Biolgical study for rMVAs indicated that there is no obvious difference among rMVAgpt-MGP5/M, rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve.5. The immunogenic efficacy on piglets for the rMVA-MGP5/M coexpressing modified GPS and M protein of Porcine Reproductive and Respiratory Syndrome VirusIn order to test the immunogenic efficacy of rMVA-MGP5/M coexpressing modified GP5 and M protein of PRRSV constructed in the sixth chapter, sixty of PRRSV negative piglets (fourth-week-old) were randomly divided into six groups with ten of them in each group. Piglets in group I were injected with purified rMVA-MGP5/M; piglets in group II and III were injected with purified pcDNA-M5A-6A, constructed in the chapter four; piglets in groupIV were injected with commercial inactived vaccine. At the same time, the V and VI groups served as negative and blank control, were inoculated with the same amount of wtMVA and 2 mL steriled PBS, respectively. Injections were done with the quantity of rMVA-MGP5/M and wtMVA at 5×105 TCID50 or 200μg purified plasmid via intramuscular (i.m.) route. Four weeks post the primary vaccination, the piglets were boosted with same dose of corresponding pcDNA-M5A-6A, rMVA-MGP5/M, wtMVA and PBS, excepted that the groupIII was boosted with rMVA-MGP5/M instead of pcDNA-M5A-6A. Neutralizing antibody titer for each group was detected with virus neutralization test assay at two weeks interval after the primary vaccination for five times to evaluate the humoral immune response. The production ofγ-IFN, IL-2 and IL-4 was detected from blood lympholeukocyte of the immunized piglets at 30, 50 and 70days post primary immunization to evaluate the cellular immune response. Results showed, both wtMVA and rMVA-MGP5/M can not replicate in the immunized piglets; for both humoral and cellular immune responses, the rMVA-MGP5/M inoculation group got the earliest immune responses, however, the pcDNA-M5A-6A prime and rMVA-MGP5/M boosted group obtained the highest neutralizing antibody titer and the most vigorous Th1 type response.
|
PDF Full Text Request