GroEL is an immunodominant surface-exposed antigen of Rickettsia typhi.

Rickettsioses are neglected and emerging potentially fatal febrile diseases that are caused by obligate intracellular bacteria, rickettsiae. Rickettsia (R.) typhi and R. prowazekii constitute the typhus group (TG) of rickettsiae and are the causative agents of endemic and epidemic typhus, respectively. We recently generated a monoclonal antibody (BNI52) against R. typhi. Characterization of BNI52 revealed that it specifically recognizes TG rickettsiae but not the members of the spotted fever group (SFG) rickettsiae. We further show that BNI52 binds to protein fragments of ±30 kDa that are exposed on the bacterial surface and also present in the periplasmic space. These protein fragments apparently derive from the cytosolic GroEL protein of R. typhi and are also recognized by antibodies in the sera from patients and infected mice. Furthermore, BNI52 opsonizes the bacteria for the uptake by antigen presenting cells (APC), indicating a contribution of GroEL-specific antibodies to protective immunity. Finally, it is interesting that the GroEL protein belongs to 32 proteins that are differentially downregulated by R. typhi after passage through immunodeficient BALB/c CB17 SCID mice. This could be a hint that the rickettsia GroEL protein may have immunomodulatory properties as shown for the homologous protein from several other bacteria, too. Overall, the results of this study provide evidence that GroEL represents an immunodominant antigen of TG rickettsiae that is recognized by the humoral immune response against these pathogens and that may be interesting as a vaccine candidate. Apart from that, the BNI52 antibody represents a new tool for specific detection of TG rickettsiae in various diagnostic and experimental setups.

Evaluation of Sporicidal Disinfectants for the Disinfection of Personal Protective Equipment During Biological Hazards.

A fast, effective, and safe disinfection of personal protective equipment (PPE) is vitally important for emergency forces involved in biological hazards. This study aimed to investigate a broad range of disinfectants to improve the established disinfection procedure. We analyzed the efficacy of chlorine-, peracetic acid-, and oxygen-based disinfectants against Bacillus spores on PPE. Therefore, spores of different Bacillus species were exposed to disinfectants on PPE material by using a standardized procedure covering the dried spores with disinfectants and applying mechanical distribution. Efficacy of disinfectants was quantified by determining the reduction factor (log10 levels) and number of viable spores left afterward. The chlorine-based granulate Hypochlorit CA G (2% chlorine) sufficiently inactivated Bacillus spores of risk groups 1 and 2, even with temperatures ranging from -20 to 35°C. Wofasteril® SC super (1.75% peracetic acid) achieved a reliable reduction of risk groups 1 and 2 and even fully virulent Bacillus spores by ≥5 log10 levels on PPE. With this, Hypochlorit-CA G and Wofasteril® SC super proved to be promising alternatives to the previously proven and widely used peracetic acid compound Wofasteril® (2% peracetic acid) for the disinfection of PPE when bacterial spores are known to be the contaminating agent. These results will help to improve the disinfection of PPE during biological hazards by providing new data on promising alternative compounds.

Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology.

Endemic typhus caused by Rickettsia (R.) typhi is an emerging febrile disease that can be fatal due to multiple organ pathology. Here we analyzed the requirements for protection against R. typhi by T cells in the CB17 SCID model of infection. BALB/c wild-type mice generate CD4+ TH1 and cytotoxic CD8+ T cells both of which are sporadically reactivated in persistent infection. Either adoptively transferred CD8+ or CD4+ T cells protected R. typhi-infected CB17 SCID mice from death and provided long-term control. CD8+ T cells lacking either IFNγ or Perforin were still protective, demonstrating that the cytotoxic function of CD8+ T cells is not essential for protection. Immune wild-type CD4+ T cells produced high amounts of IFNγ, induced the release of nitric oxide in R. typhi-infected macrophages and inhibited bacterial growth in vitro via IFNγ and TNFα. However, adoptive transfer of CD4+IFNγ-/- T cells still protected 30-90% of R. typhi-infected CB17 SCID mice. These cells acquired a TH17 phenotype, producing high amounts of IL-17A and IL-22 in addition to TNFα, and inhibited bacterial growth in vitro. Surprisingly, the neutralization of either TNFα or IL-17A in CD4+IFNγ-/- T cell recipient mice did not alter bacterial elimination by these cells in vivo, led to faster recovery and enhanced survival compared to isotype-treated animals. Thus, collectively these data show that although CD4+ TH1 cells are clearly efficient in protection against R. typhi, CD4+ TH17 cells are similarly protective if the harmful effects of combined production of TNFα and IL-17A can be inhibited.

Comparative evaluation of two Rickettsia typhi-specific quantitative real-time PCRs for research and diagnostic purposes.

Rickettsioses are caused by intracellular bacteria of the family of Rickettsiaceae. Rickettsia (R.) typhi is the causative agent of endemic typhus. The disease occurs worldwide and is one of the most prevalent rickettsioses. Rickettsial diseases, however, are generally underdiagnosed which is mainly due to the lack of sensitive and specific methods. In addition, methods for quantitative detection of the bacteria for research purposes are rare. We established two qPCRs for the detection of R. typhi by amplification of the outer membrane protein B (ompB) and parvulin-type PPIase (prsA) genes. Both qPCRs are specific and exclusively recognize R. typhi but no other rickettsiae including the closest relative, R. prowazekii. The prsA-based qPCR revealed to be much more sensitive than the amplification of ompB and provided highly reproducible results in the detection of R. typhi in organs of infected mice. Furthermore, as a nested PCR the prsA qPCR was applicable for the detection of R. typhi in human blood samples. Collectively, the prsA-based qPCR represents a reliable method for the quantitative detection of R. typhi for research purposes and is a promising candidate for differential diagnosis.

CD4+ T Cells Are as Protective as CD8+ T Cells against Rickettsia typhi Infection by Activating Macrophage Bactericidal Activity.

Rickettsia typhi is an intracellular bacterium that causes endemic typhus, a febrile disease that can be fatal due to complications including pneumonia, hepatitis and meningoencephalitis, the latter being a regular outcome in T and B cell-deficient C57BL/6 RAG1-/- mice upon Rickettsia typhi infection. Here, we show that CD4+ TH1 cells that are generated in C57BL/6 mice upon R. typhi infection are as protective as cytotoxic CD8+ T cells. CD4+- as well as CD8+-deficient C57BL/6 survived the infection without showing symptoms of disease at any point in time. Moreover, adoptively transferred CD8+ and CD4+ immune T cells entered the CNS of C57BL/6 RAG1-/- mice with advanced infection and both eradicated the bacteria. However, immune CD4+ T cells protected only approximately 60% of the animals from death. They induced the expression of iNOS in infiltrating macrophages as well as in resident microglia in the CNS which can contribute to bacterial killing but also accelerate pathology. In vitro immune CD4+ T cells inhibited bacterial growth in infected macrophages which was in part mediated by the release of IFNγ. Collectively, our data demonstrate that CD4+ T cells are as protective as CD8+ T cells against R. typhi, provided that CD4+ TH1 effector cells are present in time to support bactericidal activity of phagocytes via the release of IFNγ and other factors. With regard to vaccination against TG Rickettsiae, our findings suggest that the induction of CD4+ TH1 effector cells is sufficient for protection.

Liver Necrosis and Lethal Systemic Inflammation in a Murine Model of Rickettsia typhi Infection: Role of Neutrophils, Macrophages and NK Cells.

Rickettsia (R.) typhi is the causative agent of endemic typhus, an emerging febrile disease that is associated with complications such as pneumonia, encephalitis and liver dysfunction. To elucidate how innate immune mechanisms contribute to defense and pathology we here analyzed R. typhi infection of CB17 SCID mice that are congenic to BALB/c mice but lack adaptive immunity. CB17 SCID mice succumbed to R. typhi infection within 21 days and showed high bacterial load in spleen, brain, lung, and liver. Most evident pathological changes in R. typhi-infected CB17 SCID mice were massive liver necrosis and splenomegaly due to the disproportionate accumulation of neutrophils and macrophages (MΦ). Both neutrophils and MΦ infiltrated the liver and harbored R. typhi. Both cell populations expressed iNOS and produced reactive oxygen species (ROS) and, thus, exhibited an inflammatory and bactericidal phenotype. Surprisingly, depletion of neutrophils completely prevented liver necrosis but neither altered bacterial load nor protected CB17 SCID mice from death. Furthermore, the absence of neutrophils had no impact on the overwhelming systemic inflammatory response in these mice. This response was predominantly driven by activated MΦ and NK cells both of which expressed IFNγ and is considered as the reason of death. Finally, we observed that iNOS expression by MΦ and neutrophils did not correlate with R. typhi uptake in vivo. Moreover, we demonstrate that MΦ hardly respond to R. typhi in vitro. These findings indicate that R. typhi enters MΦ and also neutrophils unrecognized and that activation of these cells is mediated by other mechanisms in the context of tissue damage in vivo.

Persisting Rickettsia typhi Causes Fatal Central Nervous System Inflammation.

Rickettsioses are emerging febrile diseases caused by obligate intracellular bacteria belonging to the family Rickettsiaceae. Rickettsia typhi belongs to the typhus group (TG) of this family and is the causative agent of endemic typhus, a disease that can be fatal. In the present study, we analyzed the course of R. typhi infection in C57BL/6 RAG1(-/-) mice. Although these mice lack adaptive immunity, they developed only mild and temporary symptoms of disease and survived R. typhi infection for a long period of time. To our surprise, 3 to 4 months after infection, C57BL/6 RAG1(-/-) mice suddenly developed lethal neurological disorders. Analysis of these mice at the time of death revealed high bacterial loads, predominantly in the brain. This was accompanied by a massive expansion of microglia and by neuronal cell death. Furthermore, high numbers of infiltrating CD11b(+) macrophages were detectable in the brain. In contrast to the microglia, these cells harbored R. typhi and showed an inflammatory phenotype, as indicated by inducible nitric oxide synthase (iNOS) expression, which was not observed in the periphery. Having shown that R. typhi persists in immunocompromised mice, we finally asked whether the bacteria are also able to persist in resistant C57BL/6 and BALB/c wild-type mice. Indeed, R. typhi could be recultivated from lung, spleen, and brain tissues from both strains even up to 1 year after infection. This is the first report demonstrating persistence and reappearance of R. typhi, mainly restricted to the central nervous system in immunocompromised mice.

A new monoclonal anti-human CD83 antibody for flow cytometry, western blot analysis, and ELISA.

CD83 is expressed on mature dendritic cells (DCs) and many immune cells, including B and T lymphocytes upon activation. CD83 is essential for the development of CD4 single positive T cells and involved in the regulation of peripheral T and B cell responses. Here we generated a new monoclonal antibody against human CD83, which we named ELBE-1. ELBE-1 recognizes a linear epitope and cannot only be applied for Western blot analysis and flow cytometry, but also detects soluble CD83 (sCD83) by ELISA. As with other anti-CD83 antibodies described thus far, ELBE-1 does not influence T cell activation. Nevertheless, it represents a useful new tool for the detection and functional characterization of CD83.