A NONHUMAN PRIMATE MODEL OF RICKETTSIA PROWAZEKII INFECTION (EPIDEMIC TYPHUS)

Tulane University Of Louisiana, New Orleans LA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rickettsia prowazekii is the causative agent of epidemic typhus, a vector (louse)-borne disease characterized by a sudden onset of febrile symptoms with significant pathologic sequelae. In addition to causing epidemic disease in poor and unhygienic environments, R. prowazekii has also been identified as possessing the characteristics of a biological weapon. There presently is no vaccine available for protection against epidemic typhus, although development of new products is currently underway. A fully characterized primate model of disease is needed to determine immune response, safety, and protective efficacy of vaccine products developed to protect against R. prowazekii infection. The goal of this project is to develop a robust nonhuman model of disease for epidemic typhus in order to accelerate testing of newly developed vaccine products. The specific hypothesis is that use of the Rhesus macaque (Macaca mulatta) of Chinese origin will provide an optimal test system for an infection model of epidemic typhus. We base this hypothesis upon the observations from limited past studies that detailed 1) a clear dose-response relationship in prior model development efforts with the Breinl strain using Rhesus macaques when directly compared to experimental infection with avirulent strains, and 2) typical pathology in the form of disseminated vasculitis including typhus nodules in the central nervous system that emulated the human clinical syndrome. Based on these observations, the experimental focus of this proposal is on the model development of epidemic typhus in the Rhesus macaque. The specific aims are to: 1. Reestablish a nonhuman primate model for intravenous R. prowazekii infection. Studies over 25 years ago reported on model development of R. prowazekii infection in the nonhuman primate without the benefit of advances in immunologic and physiologic measurement. We will utilize the state of the art methodology during experimental infection to achieve precise account of biological response in infected animals. This will reestablish the model in the context of immune and pathologic response for future nonhuman primate studies involving R. prowazekii infection. 2. Develop an aerosol challenge model R. prowazekii challenge. Rickettsia prowazekii, in addition to causing epidemic disease, is considered a biological threat agent because of its low dose, aerosol infectivity and history of weaponization. As such, new products must provide protective efficacy against a realistic aerosol challenge. Clinical and immunologic parameters obtained from the IV Rickettsia prowazekii infections will serve as a guide to development of the aerosol model of disease. The aerosol disease model will provide an optimal system for testing of products developed expressly for protective efficacy against potential mucosal challenge with R. prowazekii. Six rhesus macaques were exposed intravenously to Breinl strain of Rickettsia prowazekii. Three groups (n=2) were administered graduated doses from 2.5E+04 to 1.0E+06 PFU. All animals became clinically ill two to three days postexposure;noted signs included depression, anorexia, weakness, and general malaise. Changes in stool were also noted. Local edema and thick discharge was observed in the mucus membranes (eyes) of three of the four animals 96 hours postexposure. Clinical signs improved over the next 5-7 days postexposure with a return to clinically normal appearance thereafter. Mild anorexia was the only lasting clinical sign that endured for up to 14 days postexposure. Subsequently, all animals experienced significant weight loss postexposure of between 10-15% of preexposure weight. Weights of three of the four animals gradually returned to preexposure weights. Telemetric monitoring of animals showed acute core temperature spikes of up to +2.5 degrees centigrade in all animals for up to 96 hours postexposure, gradually returning to homeostasis, although without a return to normal diurnal variation. Gradual increase in core temperature was observed thereafter (from day 10-30) postexposure;low grade (+1.0-1.5 degrees centigrade) fever was observed in all animals (4/4) to day 30. Similarly, disturbances in cardiovascular rhythm was recorded. Clinical chemistries of peripheral blood showed initial decreases of globulin, blood urea nitrogen, and blood glucose in all animals exposed. Increases in albumin, abumin-globulin ratio, and calreticulin were also observed. All animals initially showed a dramatic drop in circulating neutrophils and monocytes and concurrent increase in lymphocytes +5 days postexposure. Neutrophils gradually increased +20 days postexposure, with lymphocytes and monocytes declining to below normal levels by the time of euthanasia. Exposed animals also showed a dose-dependent increase in CD3+ and CD8+ lymphocytes +10 days postexposure. Both animals administered 1.0E+06 PFU showed dramatic increases in CD20+ B cells relative to the lower dosage group;CD14+ monocytes also increased in these animals +10 days postexposure. Total NK cell counts increased in all animals exposed;CD11+ dendritic cells also increased in all animals while the CD123+ count of dendritic cells decreased. Histopathological analysis of tissues from infected animals indicated mild lymphoid hyperplasia and mild chronic inflammation throughout the major organ systems. Animals exposed to the higher inoculating dose (1.0E+06 PFU) showed both lymphoid hyperplasia and chronic inflammation in the brain, lung, and liver that was consistent with previously described pathology associated with typhus infection. Chronic focal inflammation and vascular lesions in the cerebral cortex were noted in both of these animals. Further analysis of the brain showed the presence of R. prowazekii in areas with vascular lesions and upregulating of CD163 on microglia. These stains were colocalized with Ab-CD163 which also indicated upregulation of CD163 in many of the microglia affected. The increased presence of the double-stained positive cells was observed as a dose dependent phenomenon. Thus far, the results from the model development studies are positive in that we have observed dose-dependent changes associated with experimental infection of R. prowazekii in rhesus macaques. The preliminary results from the IV exposures showed clinical illness characterized by many of the signs associated with the human disease, including core temperature and cardiovascular system alternation during active infection. Similarly, clinical chemistry and hematology values changed dramatically when infected, showing many of the same trends observed in previous studies involving R. prowazekii and nonhuman primates. Advanced analysis of peripheral blood also showed increases in particular lymphocyte subpopulations in response to infection. Pathologically, animals at the highest inoculating dose (1.0E+06 PFU) showed disease-specific chronic focal inflammation in the form of vascular lesions in the brain;a noted hallmark of R. prowazekii infection. Taken together, these preliminary results have shown that the rhesus macaque is susceptible to IV infection with R. prowazekii. These data will provide baseline infection data in future studies where IV exposure is appropriate and serve as a basis for model development studies employing an alternative route of exposure to study infection.