Screening Of Important Immunogenic Protein And Studing Of The Immune Regulation Mechanism Of GAPDH Of Haemophilus Parasuis

Haemophilus parasuis (H. parasuis), the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. In naive pigs disease onset occurs few days after H. parasuis exposure and is usually lethal. These animals recovering from the acute phase of the disease can develop chronic arthritis.To date,15serovars of H. parasuis, which typically have a range of different virulence potentials, have been described. In addition, a high percentage of strains are non-serotypeable. To date, only a few virulence related factors have been reported in relation to the pathogenicity of Glasser’s disease. Limited cross-protection among strains has complicated the control of Glasser’s disease. Maternally derived antibodies also have an important role in protection, allowing the colonization of piglets and the development of a protective immune response without the induction of disease. Animals protected against H. parasuis infection after immunization with a bacterin may develop antibodies against outer membrane proteins (OMPs). The currently commercially available inactivated vaccine confers protection against homologous challenge, but further research is needed to develop a new generation of vaccine that can stimulate long-term immunity and provide cross-protection against infections by several H. parasuis serovars.The sequencing of H. parasuis SH0165has been completed by our group. The current knowledge of the genomic sequence of pathogenic bacteria has facilitated recent vaccine development. Because OMPs are the primary bacterial components that interact with host cells, targeting OMPs for the development of recombinant vaccines is of value. Based on this, we focused on putative OMPs of H. parasuis which were predicted by SignalP2.0software. They were cloned, expressed, and purified as HIS fusion proteins and screened for protective efficacy in a piglet model of infection. Furthermore, we have studied the immune biological activity of the OMPs and constructed a DNA vaccine to prevent and control the infection of H. parasuis. Below is detailed information on this study:1. Immunogenicity and protective efficacy of recombinant Haemophilus parasuis SH0165putative outer membrane proteins as vaccine candidatesH. parasuis putative outer membrane proteins (OMPs) are potentially essential components of more effective vaccines. Recently, the genomic sequence of H. parasuis serovar5strain SH0165was completed in our laboratory, which allow us to target OMPs for the development of recombinant vaccines. In this study, we focused on10putative OMPs and all the putative OMPs were cloned, expressed and purified as HIS fusion proteins. Primary screening for immunoprotective potential was performed in mice challenged with an LD50challenge. Out of these10OMPs three fusion proteins rGAPDH, rOapA, and rHPS-0675were found to be protective in a mouse model of H. parasuis infection. We further evaluated the immune responses and protective efficacy of rGAPDH, rOapA, and rHPS-0675in pig models. All three proteins elicited humoral antibody responses and conferred different levels of protection against challenge with a lethal dose of H. parasuis SH0165in pig models. In addition, the antisera against the three individual proteins and the the synergistic protein efficiently inhibited bacterial growth in a whole blood assay. The data demonstrated that the three proteins showed high value individually and the combination of rGAPDH, rOapA, and rHPS-0675offered the best protection. Our results indicate that rGAPDH, rOapA, and rHPS-0675induced protection against H. parasuis SH0165infection, which may facilitate the development of a multi-component vaccine.2. Construction and immune effect of Haemophilus parasuis DNA vaccine encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in miceIn this study the gap A gene that encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be present and highly conserved in various serotypes of H. parasuis and we constructed a novel DNA vaccine encoding GAPDH (pCgap) to evaluate the immune response and protective efficacy against infection with H. parasuis MD0322serovar4or SH0165serovar5in mice. A significant antibody response against GAPDH was generated following pCgap intramuscular immunization; moreover, antibodies to the pCgap DNA vaccine were bactericidal, suggesting that it was expressed in vivo. The gapA transcript was detected in muscle, liver, spleen, and kidney of the mice seven days post-vaccination. The IgG subclass (IgGl and IgG2a) analysis indicated that the DNA vaccine induced both Thl and Th2immune responses, but the IgG1response was greater than the IgG2a response. Moreover, the groups vaccinated with the pCgap vaccine exhibited83.3%and50%protective efficacy against the H. parasuis MD0322serovar4or SH0165serovar5challenge, respectively. The pCgap DNA vaccine provided significantly greater protective efficacy compared to the negative control groups or blank control groups (P<0.05for both). Taken together, these findings indicate that the pCgap DNA vaccine provides a novel strategy against infection of H. parasuis and offer insight concerning the underlying immune mechanisms of a bacterial DNA vaccine.3. Multifunctional Glyceraldehyde-3-phosphate Dehydrogenase of Haemophilus parasuis enhances host immune responseSome research showed that some OMPs of the diverse pathogenic microbes impact bacteria colonization by modulation of the host immune system. And these OMPs also elicit the production of IL-2and or IL-10in the host cells. So we studied whether the outer membrane proteins play an important role in regulating the host’s immune response. In this study, the GAPDH protein was screened from12OMPs which had a significant B cell stimulatoty effect regulated CD69expression observed on lymphocytes. In the MTT assay the GAPDH protein induces lymphocyte proliferation and B cell differentiation. A H. parasuis strain GAO overexpressing GAPDH protein demonstrated that it can promote the clearance of body against H. parasuis and induce the host IL-2production. Compared with the WT strain, the GAO strain was less able to resist killing by PMN cells and the RAW cells. Furthermore, infection with the GAO strain significantly upregulated the expression of all the examined immune-related genes except that encoding MHC II a. Taken together, GAPDH protein involved in regulating the host immune defense system which promote the immune response and bacterial clearance in vivo.