Study Of The Immuneogenicity Of The Major Antigens Of Rickettesia Ricketsii

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), is a small, obligate, intracellular bacterium. The outer envelope or cell wall resembles that of other gram-negative bacteria. Peoples are susceptible to R. rickettsii which is highly pathogenic and infectious. Vaccination is an effective mean for the prevention of RMSF. However, no effective RMSF vaccine has been made yet since several whole-cell vaccines tried in humans had significant side effects. The current RMSF molecular vaccines focused on few outer membrane proteins of R. rickettsii, such as outer membrane protein A (OmpA) and outer membrane protein B (OmpB), and however they are not applied for vaccination due to their weak immune protectivity against R. rickettsii.In order to discover new protective antigens of R. rickettsii, the immune-associated genes of R. rickettsii were chosen based on its genome sequence (CP000848) published. The 46 major protein antigen genes were applified with primer pairs designed based on the whole genomic sequence of R. rickettsii with Primer 5.0 software. The restriction enzymes sequences homologous to the cloning sites of prokaryotic expression plasmid pET-32a(+) were added to the primer pairs which allow the PCR products to recombine with the expression plasmid. The restriction enzyme-digested PCR products were ligated with pET-32a(+) and the E. coli competent cells were transfomed with the recombinant plasmids. According to analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 36 genes were successfully expressed in E. coli cells.Using nickel-ion affinity chromatography (Ni-NTA), 36 recombinant proteins were purified from the cellular debris of IPTG-induced transformed E. coli cells. In immunoblot assay, 9 of the 36 recombinant proteins were recognized by sera from guinea pigs experimentally infected with R. rickettsii and 5 of the 9 recombinant proteins were also recognized by sera from the patients suffering from spotted fever. The 5 recombinant proteins recognized by patient's sera are encoded by genes, A1G₀0130, A1G₀1995, A1G₀2015, A1G₀2300, A1G₀2430 in GenBank, respectively. The ²00kDa protein encoded by A1G₀0130 gene is a cell surface antigen whose function is unknown. The protein encoded by A1G₀1995 gene is a GTP-binding protein playing roles in signal transduction, transport, secretion, phagocytosis, and exocytosis. The protein encoded by A1G₀2015 is one of out membrane proteins (OmpH, OmpH-like) associated with bacterial adaptation to external environmental changes, which is more immunogenic and immuno-protective than OmpA in other bacteria. The protein encoded by A1G₀2300 gene is a lipoprotein B precursor protein of outer membrane, which is associated with the metalloendoprotease and its function needs further study. The protein encoded by A1G₀2430 gene is called alkaline protease secretion protein (AprE), which is a membrane fusion protein of type I secretion system and may play an important role in bacterial surviving under stringent status.BALB/c mice were immunized with the 5 recombinant proteins and the sera specific to single protein were prepared. R. rickettsii organisms in growth medium were incubated with the immunosera at room temperature for 30 min before infecting Vero cells, respectively. The Vero cells infected with the serum-incubated organisms were determined for R. rickettsii-burden at 1 to 11 days after infection. It was found that R. rickettsii's entry into Vero cells was inhibited.by the immunosera. The ranking of inhibiting effects of the immunosera was antiserum to A1G₀1995 > antiserum to A1G₀0130 > antiserum to A1G₀2015 > antiserum to A1G₀2300 > antiserum to A1G₀2430 > antiserum to whole cell antigens. The inhibiting effects of antisera to these proteins may be postulated that the antibodies in sera block the surface protein(s) whose function is necessary for attachment to and/or penetration of host cells.We also observed that the phagocytosis of R. rickettsii organisms inactivated with heat is not affected by the antisera, suggesting that the surface structures of the heat-inactivated organisms were destroyed under heat so that the antibodies could not specifically bind to them and the heat-inactivated organisms were phagocytized by host cells.Our results in this study indicate that the five recombinant proteins recognized by sera from patients suffering from spotted fever were major surface antigens of R. rickettsii and they have a capability to induce specific immune responses to produce netrilization antibodies which can bind to it to inhibite it's entry into host cells. These protein antigens have not be reported in published literatures and they may be considered as new candidates for developing diagnosis reagents and/or subunit vaccines of spotted fever.