We are conducting studies to examine the role of human herpesviruses in CNS disease. In particular, we have focused on the ubiquitous virus HHV-6 (and HHV6B) that is associated with a variety of neurologic diseases including multiple sclerosis, encephalitis, epilepsy, and brain cancer. The generation of an animal model of HHV-6 infection would allow studies on the potential of this virus to cause neurologic disease. Human exposure to HHV-6 occurs most likely through mucous membranes, and we have recently shown that nasal mucous is a reservoir for the virus. We therefore inoculated marmosets intranasally with HHV-6A, to examine a more physiologic route of exposure with the virus that was comparatively more immunogenic and neuropathogenic. In contrast to what we observed with the intravenous inoculations, marmosets inoculated intranasally with HHV-6A did not exhibit clinical symptoms, did not mount robust anti-HHV-6 antibody responses, and had a marked increase of detectable virus in the periphery (plasma, PBMC, saliva). These data are suggestive of an inverse correlation between antibody production and circulation of viral DNA in the periphery, and moreover, that the clinical symptoms observed in the intravenously inoculated marmosets may have been tied to the robust antibody responses. With this model, we have generated a system in which to independently study the biology of the two HHV-6 species. This is an ongoing problem in the field, due to the high homology between HHV-6A and HHV-6B, the early exposure time to HHV-6B and the unknown time of exposure to HHV-6A. This also enables us to compare the development of neurologic disease with experimental autoimmune encephalomyelitis (EAE), a well-known established model for MS. EAE in the common marmoset develops cortical and white matter lesions with remarkable immunological and pathological similarity to those seen in MS. We have successfully established EAE in the marmoset, using human white matter homogenate as the auto-antigen to drive inflammatory CNS demyelination, with progressive or relapsing phenotypes. Using these methods of induction, we are able to implement clinical and MRI parameters that will enable us to test new disease modifying therapies. Using the 7T MRI, we now have a MRI protocol optimized to follow lesion development and quantitate lesion loads in affected marmosets. We are investigating the radiological correlates of the tissue changes that accompany the formation of inflammatory, demyelinating brain lesions, the histopathological hallmarks of MS. We recently completed experiments to investigate the outcomes of EAE in marmosets previously inoculated with HHV-6, as viruses are considered a trigger of disease onset and progression in MS. Marmosets were intranasally inoculated with HHV-6A, HHV-6B or control material, and then several months later induced with white matter homogenate EAE. Viral inoculation was asymptomatic, and did not result in the induction of inflammatory cytokines in plasma. Moreover, during ex vivo PBMC stimulations with viral lysates, cytokine production was observed in only a subset of virus inoculated marmosets. Moreover, an HHV-6 specific antibody response was observed in a subset of the HHV-6B inoculated marmosets, but rarely in HHV-6A inoculated marmosets, consistent with our previous observations. Similar to what is observed in healthy adults, HHV-6B was preferentially detected in the saliva. All HHV-6B inoculated marmosets had detectable virus in saliva at multiple time points, but rarely in HHV-6A inoculated animals. Viral DNA was not observed in the PBMC of any HHV-6B inoculated marmosets. Interestingly, viral DNA was detected in the PBMC of the HHV-6A marmosets following EAE induction, reminiscent of findings of increased HHV-6A in MS patient PBMC. In one marmoset who was sacrificed following the last HHV-6B intranasal inoculation, viral antigen was detected sparsely in the brain parenchyma in the absence of any other detectable neuropathology suggesting that intranasal inoculation is sufficient for viral entry into the CNS. Following the induction of EAE, we noted significantly accelerated clinical disease in virus inoculated marmosets. Examination of brain tissue from a marmoset inoculated with HHV-6B demonstrated the localization and upregulation of HHV-6 viral antigen within inflammatory nodules and perivascular cuffs of EAE lesions. This finding is significant in light of the fact that localization of HHV-6 viral nucleic acid and/or viral antigen around MS lesions has provided some of the most compelling data for its hypothesized role in MS pathogenesis. We are performing cytokine analyses and intracellular cytokine staining to examine the mechanism of accelerated disease in the virus inoculated marmosets. Post-EAE, the virus inoculated marmosets exhibited increased effector/memory CD8 cells, which significantly correlated with time post-EAE induction, while this was not observed in the control marmosets. As this CD8 subset is the highest producer of interferon gamma, this observation suggests a proinflammatory cytokine-mediated enhancement of EAE, wherein EAE induction served as a boost of CD8 effector function to a pool of virus-specific? cytotoxic T cells primed by the HHV-6 inoculations. Collectively, these observations support the hypothesis that asymptomatic intranasal viral acquisition accelerated and exacerbated subsequent CNS inflammatory disease through CNS infection and peripheral immune activation, both of which may promote blood brain barrier permeability and enhance inflammatory processes. These pre-clinical data further refine the association of HHV-6 with neuroinflammation and lend further rationale for prophylactic or therapeutic anti-viral interventions in patients affected by chronic or acute neuroinflammatory conditions. Our marmoset studies on the interactions between an experimental viral inoculation and an experimental neuroinflammatory disease (HHV-6 and EAE, respectively) provide further rationale for the development of anti-herpesvirus interventions in acute and chronic neuroinflammatory conditions. While the HHV-6 inoculation studies were highly informative, marmosets are not the natural hosts for this virus, and there is at present no known marmoset HHV-6 homolog. There is however, a marmoset EBV homolog, termed Callitrichine herpesvirus 3 (CalHV3). EBV has also been considered one of the environmental triggers in MS based on epidemiological, serological and histological studies suggesting that EBV may be associated with the pathogenesis of MS. CalHV3 is a marmoset gamma herpesvirus with significant homology to EBV. CalHV3 can be detected in the blood and saliva of about 55% NINDS colony marmosets, consistent with reports of other captive colonies. Like EBV, CalHV3 is oncogenic, and was originally isolated from a marmoset B cell lymphoma 50. CalHV3 levels also show longitudinal fluctuation in blood and saliva, as we have demonstrated for EBV. Moreover, analogous to the hypothesis of herpesviruses in MS, some groups hypothesize that CalHV3 transformed B cells may play a role in marmoset EAE, specifically in the activation of a core pathogenic subset of cytotoxic T cells. Based on these observation, we have begun to investigate the use of a therapeutic vaccine against the marmoset gamma herpesvirus in the context of CNS neuroinflammation as a valid preclinical model for proposed clinical trials of an EBV vaccine in MS patients. The goal of these preclinical studies is to inform the clinical testing of a similarly designed EBV vaccine in humans.
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