Identification And Immunoprotectivity Of Major Surface-exposed Protein Of Rickettsia Rickettsii

Rickettsia rickettsii is an obligate intracellular Gram-negative bacterium and the pathogen responsible for Rocky Mountain spotted fever（RMSF）. RMSF is mainly transmitted by tick bites in nature, but it also can be efficiently transmitted by aerosols in man-made. Patients suffering from RMSF usually have fever, headache, myalgias, and rash, and will develop symptoms of acute lung edema, renal failure, and/or encephalitis in serious infection. R. rickettsii is a potential bio-warfare/bio-terrorism agent due to its highpathogenicity. Therefore, the development of effective vaccines against RMSF is required since vaccination is still the best way to prevent the endemic and man-made infectious diseases.The surface-exposed proteins（SEPs） of rickettsiae play key roles in rickettsial adherence and invasion of host cells, migration among host cells, and activation of host’s immune system. There are no other SEPs, except Omp A and Omp B, have been found to effectively protect the host from rickettsial infection. Therefore, it is very necessary to identify novel protective antigens for the development of safe and effective molecular vaccine against RMSF.In the present study, the major SEPs of R. rickettsii were separated and identified with proteomics and ESI-MS/MS, the subcellular location and functions of the identified SEPs were predicted by bioinformatics method and determined by indirect immune fluorescent assay（IFA） and transmission electron microscopy（TEM）. Subsequently, the potential protective SEPs were screened by immunological methods and animal protection tests. Finally, the protection mechanism of those SEPs in the humoral and cellular immunity was also explored.Firstly, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five（Omp A, Omp B, Gro EL, Gro ES, and a DNA-binding protein） of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins（Adr1, Adr2, Omp W, Porin₄, and Tol C） were first recognized as SEPs of R. rickettsii herein. Bioinformatics analysis found that all of the 5 novel SEPs contain β-pleated sheets and signal peptides, which indicates that they are membrane-spanning proteins. To further define their location, R. rickettsii organisms were stained with specific immunosera to each SEP using IFA and TEM, the results showed that all of them presented in the inner and/or outer membrane. The recombinant SEPs（r Adr1, r Adr2, r Omp W, r Porin₄, and r Tol C） were prepared and used to immunize mice. After challenge with viable R. rickettsii cells,the rickettsial load in lung, spleen, or liver of mice immunized with r Adr2 and that in lung of mice immunized with r Adr1, r Omp W, or r Porin₄, but not r Tol C, were significantly lower than that in cognate organs of PBS mock-immunized mice. This result proved that r Adr1, r Adr2, r Omp W, and r Porin₄, particularly r Adr2, were protective antigens of R. rickettsii.In a previous study, Ybg F was recognized as a protective antigen of Rickettsia heilongjiangensis. A comparison of the amino acid sequences of Ybg F from R. rickettsii and R. heilongjiangensis showed that only six of 245 amino acids were different, suggesting that Ybg F is a conservative antigen at least among the SFG rickettsiae. Sera from mice immunized with r Ybg F were used to neutralize R. rickettsii in vitro, the result showed that anti-r Ybg F sera could significantly reduce R. rickettsii invasion of vascular endothelial cells, suggesting that Ybg F can mediate the interaction of R. rickettsii with vascular endothelial cells. Additionally, for the protective efficacy assay, the results showed that the rickettsial load and pathological alters in the lungs, spleens, or livers of mice immunized with r Ybg F were significantly lower or sligher than those in PBS mock-immunized mice. These results demonstrated that Ybg F was a protective antigen of R. rickettsii.To explore the potential protective mechanisms of Adr2 and Ybg F, mice were immunized with them, respectively. The levels of Ig G, Ig G1, and Ig G2 a to Adr2 or Ybg F were determined by indirect enzyme-linked immunosorbent assays（ELISAs）, and the results showed that mice immunized with either r Adr2 or r Ybg F produced high levels of specific antibodies, particularly Ig G2 a which has been proved to be involved in T-helper cell type 1（Th1）-immune responses and enhanced efficacy of protein vaccination. Additionally, after in vitro stimulation with either r Adr2 or r Ybg F, the antigen-specific CD4+ and CD8+ T cells rapidly proliferated and differentiated into IFN-γ-producing CD4+ and CD8+ T lymphocytes cells. These results strongly indicated that the protection conferred by r Adr2 or r Ybg F was mainly dependent upon Th1-type cell-mediated immune responses.In previous studies, Omp B of rickettsiae was demonstrated to be an important protective antigen. In this study, the whole gene（4965 bp） encoding Omp B of R. rickettsii were divided into 5 fragments to express in prokaryotic cells, resulting in 5 recombinant proteins（r Omp B-1 to 5）. Following the analysis of immunoprotective efficacy, r Omp B-4 was proved to be the best one to confer protection against R. rickettsii infection in mice. Herein, immunization with r Omp B-4 and/or r Adr2 was performed in mice so as to explore whether their combination could induce an enhanced immunoprotection against R. rickettsii infection. The results showed that the rickettsial loads and pathological alters in mice immunized by the combination of r Adr2 and r Omp B-4 were significantly lower or slighter than those in mice immunized with either r Adr2 and r Omp B-4, suggesting that their combination could confer an enhanced protection against R. rickettsii infection. The further study revealed that the enhanced protection was depends on a stronger Th1-oriented immunoresponse with greater INF-γ and TNF-α secretion by antigen-specific T cells and specific Ig G2 a production by antigen-specific B cells.Coxiella burnetii, a rickettsia-like bacterium belonging to order Legionellales, is the etiological agent of Q fever in humans. C. burnetii is also recognized as a potential agent of bio-warfare/bio-terrorism. Previous studies have revealed that animals treated with inactivated phase I C. burnetii organisms had a significant increase in resistance to tumors, virus, bacteria or protozoans by the specific and nonspecific immunity modulated by the organisms, indicating that phase I C. burnetii is a potent immunopotentiator. Herein, r Omp B-4 mixed with the chloroform-methanol residue of phase I（CMR） of C. burnetii was applied to immunize mice. After challenge with R. rickettsii, mice immunized with r Omp B-4 combined with CMR-C had significant lower rickettsial loads and slighter pathological lesions in organs compared with mice immunized with only r Omp B-4, indicating CMR of C. burnetii could potentiate the r Omp B-4-specific immunoprotection to effectively resist R. rickettsii infection in mice. While, after challenge with C. burnetii, mice immunized with r Omp B-4 combined with CMR of C. burnetii had significant lower coxiella loads, indicating that CMR of C. burnetii could offer a specific protection to counter C. burnetii infection in mice. The further studies uncoved that the r Omp B-4 combined with CMR of C. burnetii could elicite hosts to produce greater amounts of IFN-γ and TNF-α, which play key roles in clearance of intracellular bacteria.In summary, a total of five novel SEPs of R. rickettsii were indentified in the present study, and all of them were located in inner and/or outer membrane. Adr2 and Ybg F were main protective antigens of R. rickettsii due to their ability to confer a stronger protection against R. rickettsii infection. Additionally, immunization with r Omp B-4 combined with Adr2 or combined with CMR of C. burnetii could provide a more effective protection than immunization with only r Omp B-4 in mice. The efficient or enhanced protection is mainly dependent on antigen-specific cell-mediated immunoresponses, including the effective activation of CD4+ and CD8+ T cells to secrete massive IFN-γ and TNF-α as well as B cells to produce greater amount of specific Ig G2 a, which synergistically function to effectively kill and eradicate rickettsiae in hosts. The present studies have provided new candidate molecules and ideals for development of RMSF subunit vaccine, and have layed a new molecular foundation for clarifying pathogenic mechanism of R. rickettsii. In addition, the protective antigens of R. rickettsii combined with CMR of C. burnetii may be a candidate design for development of vaccines against both R. rickettsii and C. burnetii infection.