| Rickettsia heilongjiangensis is an obligate, intracellular, gram-negative bacterium that causes Far Eastern tick-borne spotted fever. It is a new species of spotted fever group rickettsia firstly isolated from Dermacentor silvarum ticks collected in Heilongjiang Province of China. Vascular endothelial cells are the main target cells of R. heilongjiangensis in infection and outer membrane protein B (OmpB) is it's abundant surface-exposed protein.Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in vitro. R. heilongjiangensis organisms cultured in Vero cells and purified by renografin gradient centrifugation were applied to infect HUVEC and rickettsiae in HUVEC were examined by both indirect immune fluorescence assay (IFA) and scanning electron micrograph (SEM) after infection. During the first 24 hours of infection, two infection peaks were found at 6 and 24 hours post-infection (pi), respectively, which is similar to R. rickettaii infection of HUVEC. Multiplication of rickettsiae and pathological changes in the host cells were observed at 5 days pi, while the amount of intracellular rickettsiae was found to markedly increase in the host cells and rickettsial dissemination between neighbor cells was observed within 5 to 9 days pi. A few of rickettsiae were observed in the nucleus of host cells that appeared severe pathological changes within 8 to 9 days pi. Due to the overwhelming rickettsial infection, most of the host cells shed 12 days pi. Our results demonstrated that R. heilongjiangensis has a capability to infect vascular endothelial cells and cause vascular injury.Outer membrane protein B gene (ompB, 4 875bp) of R. heilongjiangensis was divided into 4 fragments based on immunogenic epitope prediction of OmpB by computer analysis. Four fragments of ompB were amplified from the genomic DNA of R. heilongjiangensis, and subsequently 4 recombinant OmpB proteins (OmpB-P1, OmpB-P2, OmpB-P3, and OmpB-P4) were highly expressed in Escherichia coli cells transformed by ompB-p1-, ompB-p2-, ompB-p3-, ompB-p4-insered plasmids (pET32a), respectively. In western blotting analysis, all of the 4 recombinant OmpB proteins reacted with sera from mice infected with R. heilongjiangensis. The 4 OmpB proteins were used to immune C3H/HeN mice, respectively, and high levels of specific antibody titers (≧1 :5 120) were determined in mouse sera by IFA. The results suggest the OmpB proteins have good immunogenicity.Dendritic cells (DC) are antigen presenting cells that are specialized to capture, process, and present antigens to T lymphocytes to cause immunity or tolerance to the antigens. To investigate their role in Far Eastern tick-borne spotted fever, we analyzed the responses of murine bone marrow–derived dendritic cells (BMDCs) stimulated by whole cell antigen of R. heilongjiangensis and the 4 OmpB proteins in vitro and their protective roles of the antigen-pulsed BMDCs were evaluated. BMDCs pulsed by whole cell antigen, OmpB-P1, OmpB-P2, OmpB-P3, and OmpB-P4 were intraperitoneally transferred to C3H/HeN mice, respectively. On day 14 post-transfer of BMDCs, mice were intraperitoneally challenged with R. heilongjiangensis, and the challenged mice were sacrificed and their spleen, lung, liver, and brain were harvested on day 7 pi for detection of R. heilongjiangensis load by a quantitative PCR analysis. Compared with mice receiving unpulsed BMDCs (negative control), mice receiving BMDCs pulsed with whole cell antigen, OmpB-P2, OmpB-P3, or OmpB-P4 exhibited significantly lower R. heilongjiangensis load, while mice receiving BMDCs pulsed with OmpB-P1 or TrxA encoded by pET32a displayed high levels of R. heilongjiangensis load similar to that of negative control. This result suggests that OmpB-P2, OmpB-P3, and OmpB-P4 are protective antigens, but OmpB-P1 is not.CD4~+ and CD8~+ T cells from spleens of C3H/HeN mice immunized with antigen-pulsed BMDCs were isolated with T Cell Isolation Kit, respectively. The antigens-pulsed BMDCs were cocultured with the CD4~+ and CD8~+ T cells. Phenotypic molecules and intra-cellular cytokines of CD4~+ and CD8~+ T cells were analyzed on a FACScalibur flow cytometer. CD4~+/CD8~+ T cells from mice immunized with whole cell antigen-, OmpB-P2-, OmpB-P3-, or OmpB-P4-pulsed BMDCs were efficiently activated with high expression of IFN-γthat were significantly higher than that from mice immunized with OmpB-P1-pulsed BMDCs. This result suggests that protection against R. heilongjiangensis offered by BMDCs activated with the protective antigens were associated with proliferation and activation of Th1-type CD4~+ T cells and of Cytotoxic T lymphocyte (CTL) that are more potent to produce IFN-γagainst this intracellular bacterium.
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