Immunologic Mechanisms In Experimental Autoimmune Diseases Of The Nervous System

National Institute Of Neurological Disorders And Stroke

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Abstract

We have examined 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 shown that nasal mucous is a reservoir for the virus. Rodents lack the appropriate receptor for virus binding; however, marmosets are susceptible to HHV6 infection. We have successfully infected 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 suggested 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 is one model of autoimmune inflammatory-mediated CNS demyelination, created by immunizing animals with CNS (usually white matter) peptides or proteins. Although EAE is typically studied in rodents, marmoset EAE presents with greater radiologic and pathologic similarities to MS, reflecting the genetic, immunological, and CNS anatomical proximity of marmosets to humans. 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 antigen(s) 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. We have demonstrated that intranasal inoculations with HHV-6A and HHV-6B accelerate the MS-like neuroinflammatory disease, EAE. Although those animals inoculated intranasally with HHV-6 were asymptomatic, they exhibited significantly accelerated clinical EAE compared with control animals. In addition, expansion of a proinflammatory CD8+ T cell subsets correlated with post-EAE survival in virus/EAE marmosets, suggesting that a peripheral (viral?) antigen-driven expansion may have occurred post-EAE induction. HHV-6 viral antigen in virus/EAE marmosets was markedly elevated and concentrated in brain lesions, similar to previously reported localizations of HHV-6 in MS brain lesions. Collectively, we demonstrate that asymptomatic intranasal viral acquisition accelerates subsequent neuroinflammation in a nonhuman primate model of MS. Recently, interest in the infectious hypothesis of AD, which suggests that microbes such as herpesviruses may contribute to AD onset or progression, has grown. Novel therapeutics for AD that are successful in rodent preclinical trails often fail in human clinical trials, suggesting that animal models that more closely mimic human AD are needed. Hallmarks of AD pathology, including plaques and tangles, have previously been described as spontaneous pathology in aged marmosets. We are exploring the role of viral infections on AD-related biomarkers in the marmoset model, including pathologic hallmarks, fluid neurodegenerative protein levels, and MRI and PET/CT imaging features. In addition to being competent to experimental HHV-6 infection, marmosets can be naturally infected with CalHV3, an EBV-related gammaherpesvirus and lymphocryptovirus. Similar to human EBV infection, CalHV3 has been associated with chronic persistent infection and, in rare cases, has been associated with lymphoma and lymphoproliferative disease in the marmoset. We have developed a droplet digital PCR (ddPCR) assay that allows for the quantification of CalHV3 viral load in infected animals. We have characterized prevalence and demographics associated with CalHV3 infection in the NINDS colony in order to further explore the utility of CalHV3 infection as a model of chronic herpesvirus infection and its potential role in neurodegenerative disease. Using the marmoset model of experimental HHV-6 infection and natural CalHV3 infection, we are investigating the effects of herpesvirus infection on biomarkers of AD in a translationally relevant comparative animal model. We are identifying and quantifying levels of AD-related hallmarks, such as beta-amyloid and tau, in our extensive marmoset tissue brain and blood bank specimens, including both herpesvirus infected and uninfected marmosets to determine if viral infection enhances biomarker levels, which would suggest a role for herpesviruses in AD pathogenesis and help dictate future preventative measures and disease therapeutics. In addition to experimental HHV-6 infection, we have characterized the prevalence and demographics related to Callitrichine herpesvirus 3 (CalHV-3), a naturally occurring Epstein-Barr (EBV) related gammaherpesvirus and lymphocryptovirus, in the NINDS marmoset colony as a model of chronic persistent herpesvirus infection. We have determined the prevalence of this virus in a number of NIH colonies and is as high as 20% in both the NINDS and NIMH facilities. In addition, we have found similar levels of CalHV3 infected marmosets in the University of Pittsburgh colony. It is therefore believed that this virus is much more widespread that previously recognized. Moreover, we have confirmed that CalHV3 is the causative agent of B cell lymphomas since all animals to date, that have developed these specific B cell malignancies have levels of CalHV3 in the millions of copies of virus. Marmosets without B cell lymphoma had no detectable CalHV3. The mechanism(s) associated with this oncogenic virus is currently being explored in collaboration with NCI. Lastly, we have submitted a proposal to test the antiviral properties of a broad-spectrum viral DNA polymerase inhibitor, Brincidofovir, in CalHv3 infected marmosets. These preclinical studies will inform the possible use of this drug in clinical trials in patients with MS since EBV has been suggested to play a role in this disorder.