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Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs) of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-A(b) based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4(+) T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4(+) T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4(+) T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.

From the point of view of material multiscale analysis, the deterioration of the fatigue properties of the cable is due to the micro-damage inside the strand. To describe the damage failure process of the cable as accurately as possible, many micro damage details need to be implanted in the strand model. However, with the increase of the number of wires in the cable body, this modeling method will produce a large number of elements in the finite element model of the stay cable, which makes the calculation cannot be completed iteratively. Based on this, a time-step adaptive simulation method for corrosion fatigue damage evolution of stay cables was proposed in this paper. In this method, the corrosion fatigue damage evolution model was implanted in the local part of the strand model, and a damage variable was used to comprehensively consider the evolution behavior of distributed micro-damage in the strand, which satisfies the calculation accuracy and realizes the simulation of the whole process of fatigue deterioration of the stay cable, greatly improving the calculation accuracy and convenience. Then taking an in-service cable-stayed bridge as an example, the stress of the main beam and the local spatial stress of the main beam under the special condition of the cable damage failure were studied by combining the rod model with the local model, which provides data reference for the replacement of the cable of the cable-stayed bridge with a long construction life.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, 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 (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

•Adr2 is a novel protective antigen of R. rickttsii.•Protection offered by Adr2 is mainly dependent on antigen-specific cell-mediated immune responses.•Adr2 could activate CD4+ and CD8+ T cells to produce great amount of TNF-α and/or IFN-γ as well as elicit rapid increase of specific IgG2a.•Anti-serum against Adr2 could prevent R. rickttsii from invasion of host cells. Rickettsia rickettsii is the pathogen of Rocky Mountain spotted fever (RMSF), a life-threatening tick-transmitted infection. Adr2 was a surface-exposed adhesion protein of R. rickettsii and its immunoprotection against RMSF was investigated in mice. Recombinant Adr2 (rAdr2) was used to immunize C3H/HeN mice, and the rickettsial loads in organs of the mice were detected after challenge with R. rickettsii. The levels of specific antibodies of sera from the immunized mice were determined and the sera from immunized mice were applied to neutralize R. rickettsii. Proliferation and cytokine secretion of CD4+ and CD8+ T cells isolated from R. rickettsii-infected mice were also assayed after rAdr2 stimulation. After R. rickettsii challenge, the rickettsial loads in spleens, livers, and lungs were significantly lower and the impairment degrees of these organs in rAdr2-immunized mice were markedly slighter, compared with those in negative control mice. The ratio of specific IgG2a/IgG1 of rAdr2-immunized mice kept increasing during the immunization. After treatment with rAdr2-immunized sera, the total number of R. rickettsii organisms adhering and invading host cells was significantly lower than that treated with PBS-immunized sera. Interferon-γ secretion by CD4+ or CD8+ T cells and tumor necrosis factor-α secretion by CD4+ T cells from R. rickettsii-infected mice were respectively significantly greater than those from uninfected mice after rAdr2 stimulation. Adr2 is a protective antigen of R. rickettsii. Protection offered by Adr2 is mainly dependent on antigen-specific cell-mediated immune responses, including efficient activity of CD4+ and CD8+ T cells to produce great amount of TNF-α and/or IFN-γ as well as rapid increase of specific IgG2a, which synergistically activate and opsonize host cells to killing intracellular rickettsiae.

•rYbgF is a new discovered protective antigen against Far-Eastern spotted fever.•Protective mechanism of rYbgF relies on Th1-type immune response.•Anti-serum against rYbgF inhibits invasion of R. heilongjiangensis into host cells. Surface proteins YbgF and PrsA are major seroreactive antigens of Rickettsia heilongjiangensis, the etiological agent of Far-Eastern spotted fever. This study investigated their potential immunogenicity for protective immunity. Recombinant YbgF and PrsA were used to immunize C3H/HeN mice and rickettsial loads in immunized mouse organs were assessed after R. heilongjiangensis challenge. Anti-sera from immunized mice were applied to neutralize rickettsiae. CD4+ and CD8+ T cells isolated from R. heilongjiangensis-infected mice were stimulated with YbgF or PrsA, and proliferation and cytokine production assessed. The IgG2a/IgG1 ratio of sera was markedly increased in YbgF-immunized mice but was unaltered in PrsA-immunized mice after immunization. The rickettsial load in YbgF-immunized mice was significantly lower than in PrsA-immunized mice after R. heilongjiangensis challenge. Incubation with anti-serum to YbgF, but not PrsA, significantly reduced the number of rickettsiae adhering to and invading endothelial cells. The proliferation level and tumor necrosis factor-α secretion level of CD4+ T cells from R. heilongjiangensis-infected mice were significantly higher than in uninfected mice after stimulation with YbgF but not PrsA. YbgF is a novel protective antigen that induces a Th1-type of protective immune response against R. heilongjiangensis infection.

Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF), is an obligate intracellular bacterium. The surface-exposed proteins (SEPs) of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis. R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA). Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever. Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease.

Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B（Omp B） is an important surface protein antigen of rickettsiae. In the present study, the omp B gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins（OmpB-p1, Omp B-p2, Omp B-p3, and Omp B-p4）. Each Omp B was used in vitro to stimulate murine bone marrow-derived dendritic cells（BMDCs） of C3H/He N mice, and the Omp B-pulsed BMDCs were transferred to naive C3H/He N mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with Omp B-p2, Omp B-p3, or Omp B-p4 exhibited significantly lower bacterial load compared with mice receiving Omp B-p1-pulsed BMDCs. CD4＋ and CD8＋ T cells isolated from the spleen of C3H/He N mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4＋ or CD8＋ T cells from mice receiving BMDCs pulsed with Omp B-p2, OmpB-p3, or Omp B-p4 was higher than that on cells from mice receiving Omp B-p1-pulsed BMDCs, while the expression level of tumor necrosis factor（TNF）-α on CD8＋ T cells and interferon（IFN）-γ on the CD4＋ and CD8＋ T cells from mice receiving Omp B-p2,-p3, or-p4 was significantly higher than on cells from mice receiving Omp B-p1-pulsed BMDCs. Our results suggest that the protective Omp Bs could activate CD4＋ and CD8＋ T cells and drive their differentiation toward CD4＋ Th1 and CD8＋ Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.

► Recombinant proteins Com1, Mip and GroEL were cloned and used to stimulate BMDCs. ► Mice receiving Com1 or Mip-pulsed DCs showed lower bacterial loads than control. ► Mice receiving Com1 or Mip-pulsed DCs exhibited greater IFN-γ-positive cells. The recombinant membrane-associated proteins of Coxiella burnetii, Com1, Mip and GroEL, were used in vitro to stimulate BALB/c mouse bone marrow-derived dendritic cells (BMDCs). The antigen-activated BMDCs were transferred into naïve BALB/c mice. Seven days after challenge of C. burnetii, the bacterial loads of mice receiving BMDCs activated with Com1 or Mip, but not GroEL, were significantly lower than that of mice receiving BMDCs pulsed with TrxA (Esherichia coli thioredoxin) in a quantitative polymerase chain reaction assay. After in vitro interaction with cognate antigen-pulsed BMDCs, the percentages of CD69-positive cells and TNF-α-positive cells in CD4+ and CD8+ T cells isolated from the spleens of mice receiving Com1-, Mip-, or GroEL-pulsed BMDCs were significantly higher than that of mice receiving mock-pulsed BMDCs in flow cytometric analysis. The percentages of IFN-γ-positive cells in CD4+ and CD8+ T cells from mice receiving Com1- or Mip-pulsed BMDCs were significantly greater than that of mice receiving GroEL-pulsed BMDCs. However, the percentage of IL-4-positive cells in CD4+ T cells of mice receiving GroEL-pulsed BMDCs was obviously higher than that of mice receiving Com1- or Mip-pulsed BMDCs. Our results demonstrate that Com1 and Mip are protective antigens and strongly indicate that they favor to induce IFN-γ-producing Th1 and Tc1 cells, whereas the non-protective antigen GroEL is biased to induce a Th2 response. Therefore, Com1 and Mip are key antigens to induce a protective immune response against C. burnetii infection.

Background Rickettsia rickettsiiis the pathogen of Rocky Mountain spotted fever (RMSF), a life-threatening tick-transmitted infection. Adr2 was a surface-exposed adhesion protein ofR. rickettsiiand its immunoprotection against RMSF was investigated in mice. Methods Recombinant Adr2 (rAdr2) was used to immunize C3H/HeN mice, and the rickettsial loads in organs of the mice were detected after challenge withR. rickettsii. The levels of specific antibodies of sera from the immunized mice were determined and the sera from immunized mice were applied to neutralizeR. rickettsii. Proliferation and cytokine secretion of CD4+and CD8+T cells isolated fromR. rickettsii-infected mice were also assayed after rAdr2 stimulation. Results AfterR. rickettsiichallenge, the rickettsial loads in spleens, livers, and lungs were significantly lower and the impairment degrees of these organs in rAdr2-immunized mice were markedly slighter, compared with those in negative control mice. The ratio of specific IgG2a/IgG1 of rAdr2-immunized mice kept increasing during the immunization. After treatment with rAdr2-immunized sera, the total number ofR. rickettsiiorganisms adhering and invading host cells was significantly lower than that treated with PBS-immunized sera. Interferon-γ secretion by CD4+or CD8+T cells and tumor necrosis factor-[alpha] secretion by CD4+T cells fromR. rickettsii-infected mice were respectively significantly greater than those from uninfected mice after rAdr2 stimulation. Conclusion Adr2 is a protective antigen ofR. rickettsii. Protection offered by Adr2 is mainly dependent on antigen-specific cell-mediated immune responses, including efficient activity of CD4+and CD8+T cells to produce great amount of TNF-[alpha] and/or IFN-γ as well as rapid increase of specific IgG2a, which synergistically activate and opsonize host cells to killing intracellular rickettsiae.

Far-eastern spotted fever (FESF) is an important emerging infectious disease in Northeast Asia. The laboratory diagnosis of FESF in hospitals is mainly based on serological methods. However, these methods need to cultivate rickettsial cells as diagnostic antigens, which is both burdensome and dangerous. Eleven surface-exposed proteins (SEPs) were identified in our previous study and their recombinant proteins (rSEPs) fabricated on a microarray were serologically analyzed with seventeen paired sera from patients suffered from FESF in this study. All the rSEPs showed sensitivities of between 53% and 82% to acute-phase sera and of between 65% and 82% to convalescent-phase sera, and all the rSEPs except rRplA showed specificities of between 80% and 95%. The combination assay of two, three, or four of the four rSEPs (rOmpA-2, rOmpB-3, rRpsB, and rSdhB) showed better sensitivities of between 76% and 94% to the acute-phase sera or between 82% and 100% to the convalescent-phase sera and acceptable specificities of between 75% and 90%. Our results suggest that the four rSEPs are more likely candidate antigens for serological diagnosis of FESF.