Studies On Molecular Epidemiology Of Haemophilus Parasuis And The Immuno-Enhancing Effect Of HSP70 From Haemophilus Parasuis On Structural Protein GP3/GP5 Of PRRSV

Haemophilus parasuis (H.parasuis), the causative agent of Glasser's disease, is a member of the family Pasteurellaceae and commensal organism of the upper respiratory tract of conventional pigs.Under appropriate conditions it can invade and cause severe systemic disease, characterized by fibrinous polyserositis, arthritis and meningitis. Currently, H.parasuis infection, which causing significant mortality and morbidity in piglets and great economic losses in pig industry, has been one of the most important swine diseases in China. However, Studies based on serotyping demonstrated that a high antigenic heterogeneity exists among H. parasuis strains; Moreover, virulence factors and protective antigens of H.parasuis are not known, so there is no universal vaccine to prevent and control the outbreaks of this disease.Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases in the swine industry. It is characterized by reproductive problems in sows such as poor farrowing rates, premature farrowings, and increased stillbirths, as well as respiratory problems in piglets such as pneumonia and atrophic rhinitis. Current commercial PRRS vaccines, including both live attenuated and killed vaccines, have been wildly used, but they cannot provide effective protection against PRRS. Recently, the emergence and prevalence of highly pathogenic PRRSV strains across China, which led to great economic losses, bring new challenge to the prevention and control of the disease.In this study, H.parasuis field strains isolated from several provinces or municipality of China were genotyped and heat shock protein 70 (HSP70) gene was cloned from one of the strains; The recombinant adenoviruses expressing HSP70 or truncated HSP70 (tHSP70) and structural protein of PRRSV GP3/GP5 were constructed and the immonoenhancing property of HSP70 or tHSP70 was studied in mouse model by inoculation of mice with these recombinant adenoviruses; Protective efficacy of these recombinant adenoviruses against highly pathogenic PRRSV infection was examined in pigs, which provides new strategy for construction of PRRSV genetic engineering vaccine. The contents of the paper contain five parts as following:1. Characterization of Haemophilus parasuis field isolates from China by PCR-RFLP analysis of the tbpA geneFifty-seven of Heamophilus parasuis isolates along with 15 reference strains of all known serovars were subjected to PCR-FRLP (restriction fragment length polymorphism) based on tbpA gene. The analysis of the 1.9-kb tbpA amplicon using Taq I, Ava I and Afa I endonucleases produced 9 RFLP patterns for the 15 reference strains and 15 patterns for the 57 field isolates. And the first 3 prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). It is confirmed that Haemophilus parasuis existed widely in China with numerous genotype. It provides significant basis for prevention and control of the disease in China.2. Sequencing and expression of the HSP70 gene of Haemophilus parasuis and antigenicity of heat shock protein 70The HSP70 (dnaK) gene of Haemophilus parasuis was cloned and sequenced by PCR with designed primers, which having GenBank accession NO. EU693116。Nucleotide sequencing showed that the gene consists of an open reading frame of 1,908 bp and encodes a protein of 635 amino acids with a high degree of homology to that of Mannheimia haemolytica, Haemophilus ducreyi and Actinobacillus pleuropneumoniae, which were calculated to be 91%,91% and 87%, respectively. After cloning of the C terminal fragment of the HSP70 coding region into pET-32a(+), it was expressed in E. coli and confirmed by SDS-PAGE and Western-blotting using experimentally infected pig serum and mice serum immunized with purified recombinant HSP70. It could be used to study on the foundation of this protein in future.3. Construction and Identification of recombinant adenovirus expressing HSP70 of Haemophilus parasuis fused with GP3 and GP5 of Porcine reproductive and respiratory syndrome virusGenes of GP3 and GP5 were amplified from a highly pathogenic PRRSV isolate SY0608, and cloned into p-shuttle-CMV vetor tandem inframe which resulted in the recombinant pshuttle-CMV-GP3-GP5. Then, HSP70 gene of Haemophilus parasuis was colned into pshuttle-CMV-GP3-GP5 using two different linkers (5×Glycine or 2A gene of FMDV virus) beteen HSP70 and GP3gene, which leads to the production of recombinant pshuttle-CMV-HSP-GP3-GP5 and pshuttle-CMV-HSP-2A-GP3-GP5. Positive clones were identified by restriction enzyme ananlysis and further confirmed by sequencing. Recombinant plasmids were linearized with Pme I and homologously recombined with pAdEasy-1 backbone vector in BJ5183 host bacteria. The recombinant adenovirus DNA produced by recombination were linearized with Pac I and transfected into HEK-293 A cells for packaging of intact adenovirus. The expression of HSP70/GP3/GP5 by recombinant adenovirus in 293A cells were confirmed by RT-PCR, indirect immuno-fluorescence assay (IFA), and western blot. It shows that HSP70/GP3/GP5 could be expressed by recombinant adenovirus constructed in this study as expected, which provides the basis for the further immunogenicity experiment.4. Analysis of immunogenicity of recombinant adenovirus co-expressing HSP70 of H.parasuis and GP3-GP5 of PRRSV in miceIn this study, three replication-defective adenovirus recombinants were developed as potential vaccine against PRRSV in a mouse model. Three groups of BALB/c mice were inoculated subcutaneously twice at 3-week intervals with the recombinants expressing GP3-GP5 (rAd-GP35), HSP-GP3-GP5 (rAd-HS35) and HSP-2A-GP3-GP5 fusion protein (rAd-HSA35). Two additional groups were injected with wild type adenovirus (wtAd) or PBS as control. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response at 2 weeks post second inoculation. However, only mice immunized with recombinant adenovirus rAd-HSA35 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP35. It was also found that lymphocytes of mice immunized with rAd-HS35 or rAd-HSA35 were primed for significant higher levels of PRRSV specific IFN-y responses upon stimulation with purified PRRSV antigen in vitro than that of mice immunized with rAd-GP35. It suggested that HSP70 of haemophilus parasuis has the immuno-enhancing effect on the PRRSV antigens chaperoned.5. Analysis of protective efficacy of recombinant adenovirus co-expressing HSP70 of H.parasuis and GP3-GP5 of PRRSV against highly pathogenic PRRSV challenge in pigletsIn this study, twenty piglets were divided into 4 groups of five, and immunized twice with wtAd, rAd-GP35, rAd-HS35 and rAd-HSA35 with three weeks intervals, then challenged with SY0608 strain of highly pathogenic PRRSV by 3 weeks post boost. Immune responses of pigs immunized with these recombinant adenoviruses were detected and protective efficacy against homologous challenge were examined. The results showed that all animals vaccinated with rAd-GP35, rAd-HS35 and rAd-HSA35 developed specific anti-PRRSV ELISA antibody, neutralizing antibody. The humoral immune responses of rAd-HS35, especially rAd-HSA35 containing 2A of FMDV between HSP70 and GP3 gene, were significantly higher than that from rAd-GP35. Both IFN-y and IL-4 in serum of pigs were markedly induced by fusing with HSP70. Following challenge with PRRSV, piglets inoculated with recombinant rAd-HS35 and rAd-HSA35 showed lighter clinical signs, lower viremia and less gross lesion of lungs, comparing with those in rAd-GP35 group. Moreover, the protective efficiency induced by rAd-HSA35 was higher than that rAd-HS35. It indicated that HSP70 fused with GP3 and GP5 of PRRSV could markedly increase the immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant adenovirus rAd-HSA35 might be an attractive candidate vaccine for preventing and control of highly pathogenic PRRSV infections.6. Construction and immunogenicity of recombinant adenovirus expressing truncated HSP70 of Haemophilus parasuis fused with GP3 and GP5 of PRRSV in miceGene of tHSP70 (384-635 amino acid)was amplified from a H.parasuis isolate, and cloned into pshuttle-CMV-GP3-GP5 using two different linkers (5×Glycine or 2A gene of FMDV virus) beteen HSP70 and GP3gene, which leads to the production of recombinant pshuttle-CMV-tHSP-GP3-GP5 and pshuttle-CMV-tHSP-2A-GP3-GP5. Positive clones were identified by restriction enzyme ananlysis and further confirmed by sequencing. Recombinant plasmids were linearized with Pme I and homologously recombined with pAdEasy-1 backbone vector in BJ5183 host bacteria. The recombinant adenovirus DNA produced by recombination were linearized with Pac I and transfected into HEK-293 A cells for packaging of intact adenovirus. The expression of HSP70/GP3/GP5 by recombinant adenovirus in 293A cells were confirmed by RT-PCR, indirect immuno-fluorescence assay (IFA), and western blot. It shows that tHSP70/GP3/GP5 could be expressed by recombinant adenovirus constructed in this study as expected, which provides the basis for the further immunogenicity experiment. In order to detect the immunogenicity of the recombinant adenoviruses, five groups of BALB/c mice (15 mice per group) were inoculated subcutaneously twice at 3-week intervals with the recombinants expressing GP3-GP5 (rAd-GP35), HSP-GP3-GP5 (rAd-HS35), HSP-2A-GP3-GP5 (rAd-HSA35), tHSP-GP3-GP5 (rAd-tHS35), tHSP-2A-GP3-GP5 (rAd-tHSA35) fusion protein. Two additional groups were injected with wild type adenovirus (wtAd) or PBS as control. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response at 2 weeks post second inoculation. However, mice immunized with recombinant adenovirus rAd-HS35, rAd-HSA35, rAd-tHS35 and rAd-tHSA35 developed higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP35. It was also found that lymphocytes of mice immunized with rAd-HS35, rAd-HSA35, rAd-tHS35 and rAd-tHSA35 were primed for significant higher levels of PRRSV specific IFN-γresponses upon stimulation with purified PRRSV antigen in vitro than that of mice immunized with rAd-GP35. It suggested that truncated HSP70 of haemophilus parasuis has the immuno-enhancing effect on the PRRSV antigens chaperoned.In summary, H.parasuis exist widely in diseased pig herds with numerous genotypes; HSP70 of H.parasuis shows strong antigenicity; Fusion proteins HSP-GP3-GP5 and HSP-2A-GP3-GP5 expressed by recombinant adenoviruses could significantly improve both PRRSV-specific neutralizing antibodies and cell-mediated immune responses. Moreover, it is indicated that HSP70 fused with GP3 and GP5 of PRRSV could markedly increase the immune responses and provide protection against virulent PRRSV challenge in pigs. This study provides the foundation for developing the genetic engineering vaccine against PRRSV.