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FEATURE
Pursuing Immunopathophysiology Research to Support Typhus Vaccine Development
Daniel H. Paris
Mahidol-Oxford Tropical Medicine Research Unit (MORU), Mahidol University, Thailand

"Scrub typhus is probably one of the most underdiagnosed and underreported febrile illnesses requiring hospitalization in the region". (WHO 1999) Current situation and problems

What is typhus? – Typhus is a majorly under-recognized infectious disease in Asia, accounting for up to 28% of malaria-negative fevers in the central regions of southeast Asia [1]- [3]. It is caused by bacteria transmitted by chigger mites that need to infect specific cells of the human body to survive; these cells are within or surrounding the blood vessels of the human body resulting in widespread systemic disease including the brain, the lungs, the liver and other sites leading to severe clinical complications if left untreated.

Scrub typhus, caused by Orientia tsutsugamushi, and murine typhus caused by Rickettsia typhi are the two main typhus disease groups in Thailand and the Lao PDR [2]- [5], but serological data from Malaysia, Indonesia, Korea, Japan and Vietnam suggests scrub typhus being the dominant form of rickettsial disease in these countries as well. Together they probably represent two of the most frequent, under-recognized and neglected treatable infections in the world. In Southeast Asia alone an estimated 1 million cases of scrub typhus occur annually [6], which translates into approximately over 80,000 deaths per year, based on the limited available mortality rates. This is almost certainly an underestimate, which, together with treatment delays due to lack of appropriate diagnostics and awareness, must lead to a substantial disease burden and economic impact in the world’s most densely populated regions.

The discovery of Chloramphenicol and later the Tetracycline antibiotics as highly effective chemotherapeutics led to the belief that typhus is easily controllable [7], [8]. Unfortunately, this resulted in a decline of scientific interest and an almost complete lack of awareness for this disease. The absence of any structured and precise epidemiological studies that delineate the extent of morbidity and mortality associated with scrub typhus underlines this dramatically. Further, the understanding of how the bacteria are maintained in nature and the exact natural disease course in patients remains enigmatic, as do the roles of the immune response in protection from re-infection.

Unfortunately, therapeutic interventions have no impact on the distribution of typhus, in contrast to malaria for example, as humans are not involved in the cycle between pathogen and vectors. To date no effective and reliable vaccine has been developed for scrub typhus, due to difficulties of the enormous heterogenicity of strains, which also obstructs development of precise diagnostics. Recently, reports of antibiotic-resistant strains emerging in northern Thailand and southern India are additional causes for concern [9]- [11].

The current research

The rickettsial research program at the Mahidol-Oxford Research Unit at the Faculty of Tropical Medicine of Mahidol University in Bangkok involves multiple aspects: a) development and evaluation of diagnostic tools to improve the diagnosis of these diseases, which are notoriously difficult to diagnose, but easy to treat, b) performing prospective clinical studies in typhus, c) molecular characterization of new rickettsial species in the region
(Figure 1), d) investigating the natural immune response to infection in patients with typhus from different regions infected with different strains, and d) the development of immunopathophysiological studies in animal models allowing for detailed time course studies leading to baseline data for future vaccine evaluation.

Diagnostics: The Rickettsiology program has an ongoing effort in evaluating and developing diagnostic tests for typhus [12]. Prospective clinical studies require serological gold standard diagnostics, which enable cross-comparisons of the diagnostic accuracies for novel diagnostic tests [13]. Our group has designed and improved multiple PCR-based diagnostic assays for the diagnosis of scrub typhus [14]- [16], including a novel Loop-Mediated Isothermal PCR Assay (LAMP) for detection of O. tsutsugamushi. The diagnostic accuracy of this LAMP assay was similar to real-time and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course [15], [17]. The combination of DNA- and antibody- based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub
typhus [17].

Host-pathogen interactions and im-munopathophysiology: We are expanding our research activities in these areas with emphasis on immuno-pathophysiological investigations, meaning the interactions between host and causative pathogen with a view to what factors are causing disease. As the hallmark of rickettsial illness is a disease of the blood vessels (termed vasculitis), we have focused on endothelial cell function and the blood clotting system to study differences between scrub and murine typhus.

Previous postmortem and in vitro reports demonstrated infection of endothelial cells, and data from our group indicated a more prevalent host leucocyte than endothelial cell response in vivo in pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation [18]. We examined eschar skin biopsies from patients with scrub typhus and characterized the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy [19]. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells (Figure 2), which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an inflammatory monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/ S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells (Figure 3). Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.

Further, correlations of surrogate markers of endothelial activation, inflammation and coagulation with the type and severity of clinical features of scrub and murine typhus in a prospective cohort of sympatric scrub and murine typhus clearly revealed that the pathological interactions of the coagulation system and the blood vessel walls differed substantially between the two diseases [20]. In scrub typhus, the in vivo coagulation activation is prominent and related to a strong pro-inflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways follow an endothelial cell perturbation pattern. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation.

These findings are now being further corroborated in studies addressing the detailed characterization of the natural immune response. It is crucial to understand the aspects of immune protection in both the humoral, antibody-based and cell-mediated immune systems in disease, to be able to translate this knowledge into a suitable vaccine. A successful vaccine requires not only good target(s), but also an antigen delivery system, that induces the right immune response with long-term memory. This is where the development of animal models in parallel to ongoing specific investigations in humans with typhus is very valuable. The feedback of clinico-pathological data obtained from humans suffering from the disease synergistically interacts with investigations on the disease mechanisms from animal models.

This clinically-focused research strategy of performing animal model investigations closely guided by clinically relevant questions and data obtained from patients with acute typhus is bearing fruit, as time course studies, disease challenge models, bacterial immune escape mechanisms and the dissection of the human immune response are much more clinically driven and focused on the next relevant steps respectively.

This approach will benefit the patient and contribute to the development of improved diagnostics and the evaluation and design of vaccine candidates, which again will feed into a pre-existing and gradually growing clinical network within the region of endemicity of tropical typhus.

About the Author

Daniel H. Paris, graduated in Human Medicine at the University of Zurich, Switzerland and pursued a career combining Internal Medicine and Infectious / Tropical Diseases.

In 2005 he began work in Thailand with the Welcome Trust Mahidol University Oxford Tropical Medicine Research Programme, a collaborative research programme between Mahidol University and Oxford University where he completed his PhD on the pathophysiology of rickettsial illness in 2010.

He currently co-ordinates rickettsial research across various clinical centres in Southeast Asia with emphasis on clinically-driven investigations directed towards understanding the natural history and immune-pathophysiology of tropical typhus and thus supporting the development of diagnostics
and vaccines.

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