Human Parechovirus and Enterovirus Initiate Divergent Innate Immune Responses in the CNS: Pathogenic and Diagnostic Implications

The picornaviruses human parechovirus (HPeV) and enterovirus (EV) cause a wide range of diseases, including CNS infections, which can be severe and potentially fatal. EV causes most cases of pediatric meningoencephalitis worldwide, and HPeV type 3 (HPeV3) is the most common cause of viral meningitis in young infants. Each year in the United States, there are over 75,000 cases of aseptic meningitis. Despite reassuring short-term outcomes, negative neurodevelopmental sequalae are increasingly associated with HPeV and EV. The pathogenesis and severity of HPeV and EV infections are undoubtedly linked to the innate and adaptive immune responses elicited by these viruses. Until this work, the innate immune response mounted against HPeV was largely unknown. Pattern recognition receptors in the CNS, including a number of Toll-like receptors located in different cells and subcellular compartments, detect invading pathogens and cause the release of cytokines and chemokines almost immediately into the CSF compartment at measurable levels. Essentially, this allows for determination of an amplified, infectious agent-specific pattern. These virus specific patterns of innate immune activation may provide insight into the pathogenesis of the corresponding disease states. Also, since these infections have similar clinical presentations, the immune profiles may be useful for rapid pathogen diagnosis in the clinical setting

Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research

Elite Suppressors Harbor Low Levels of Integrated HIV DNA and High Levels of 2-LTR Circular HIV DNA Compared to HIV+ Patients On and Off HAART

Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population

Clinical measures of HIV provide insight into determinants of reservoir size

Although current therapy regimens can suppress virus to levels that are undetectable by most clinical assays, there is still no cure for HIV. This is due to a stable pool of latently infected cells that cannot be targeted by antiretroviral therapy. Consequently, understanding the properties of this reservoir is vital to curing HIV. Existing in vitro latency models do not always reflect latency in vivo. Therefore this thesis presents both in vitro and in vivo experiments to examine reservoir levels as well as immune visibility of and potential therapeutic strategies to remove the reservoir. Specifically we examined the reservoir in a unique patient population called elite controllers, who control viremia without therapy, and compared it to that of treated and untreated non-controllers. This experimental paradigm enables us to probe the natural mechanism of reservoir control in controllers, which may be translated to new therapeutic avenues for all HIV infected individuals. By measuring HIV DNA intermediates in controllers, we found they contain very low levels of integrated HIV but relatively high levels of 2-LTR circles compared to non-controllers. This was not due to differences in cell susceptibility to virus. As integrated HIV DNA is far more efficient at expressing HIV proteins than unintegrated HIV DNA, we hypothesized the immune response may be preferentially clearing the former. Certain elite controller cohorts are known to have robust CD8+T cell responses to HIV, so we probed whether controller CD8+T cells could be responsible for low reservoir levels by targeting and clearing latently infected cells. We found CD8+T cells from controllers more efficiency cleared HIV protein-expressing resting cells using an in vitro coculture system, suggesting this is a mechanism of reservoir control in controllers. Furthermore, we found a correlation between reservoir size, as measured by our integration assay, and CTL clearance in our in vitro latent model. To test the in vivo relevance of these findings, we sorted HIV protein-expressing resting cells from HAART treated patients. The sorted cells were highly enriched for integrated HIV, suggesting that low level HIV protein expression is a feature of reservoir cells in HIV patients. In summary, these studies combined in vitro latency techniques with a unique patient population to identify a potential role for the immune system in controlling the HIV reservoir. These findings provide an important rationale for pursuing therapeutic vaccination to boost the immune response. As a portion of the reservoir may be visible without additional stimulation, vaccinations that boost CD8+T cell responses may be effective at clearing a portion of the HIV reservoir

Clinical measures of HIV provide insight into determinants of reservoir size

Although current therapy regimens can suppress virus to levels that are undetectable by most clinical assays, there is still no cure for HIV. This is due to a stable pool of latently infected cells that cannot be targeted by antiretroviral therapy. Consequently, understanding the properties of this reservoir is vital to curing HIV. Existing in vitro latency models do not always reflect latency in vivo. Therefore this thesis presents both in vitro and in vivo experiments to examine reservoir levels as well as immune visibility of and potential therapeutic strategies to remove the reservoir. Specifically we examined the reservoir in a unique patient population called elite controllers, who control viremia without therapy, and compared it to that of treated and untreated non-controllers. This experimental paradigm enables us to probe the natural mechanism of reservoir control in controllers, which may be translated to new therapeutic avenues for all HIV infected individuals. By measuring HIV DNA intermediates in controllers, we found they contain very low levels of integrated HIV but relatively high levels of 2-LTR circles compared to non-controllers. This was not due to differences in cell susceptibility to virus. As integrated HIV DNA is far more efficient at expressing HIV proteins than unintegrated HIV DNA, we hypothesized the immune response may be preferentially clearing the former. Certain elite controller cohorts are known to have robust CD8+T cell responses to HIV, so we probed whether controller CD8+T cells could be responsible for low reservoir levels by targeting and clearing latently infected cells. We found CD8+T cells from controllers more efficiency cleared HIV protein-expressing resting cells using an in vitro coculture system, suggesting this is a mechanism of reservoir control in controllers. Furthermore, we found a correlation between reservoir size, as measured by our integration assay, and CTL clearance in our in vitro latent model. To test the in vivo relevance of these findings, we sorted HIV protein-expressing resting cells from HAART treated patients. The sorted cells were highly enriched for integrated HIV, suggesting that low level HIV protein expression is a feature of reservoir cells in HIV patients. In summary, these studies combined in vitro latency techniques with a unique patient population to identify a potential role for the immune system in controlling the HIV reservoir. These findings provide an important rationale for pursuing therapeutic vaccination to boost the immune response. As a portion of the reservoir may be visible without additional stimulation, vaccinations that boost CD8+T cell responses may be effective at clearing a portion of the HIV reservoir

Directly Infected Resting CD4+T Cells Can Produce HIV Gag without Spreading Infection in a Model of HIV Latency

<div><p>Despite the effectiveness of highly active antiretroviral therapy (HAART) in treating individuals infected with HIV, HAART is not a cure. A latent reservoir, composed mainly of resting CD4+T cells, drives viral rebound once therapy is stopped. Understanding the formation and maintenance of latently infected cells could provide clues to eradicating this reservoir. However, there have been discrepancies regarding the susceptibility of resting cells to HIV infection <em>in vitro</em> and <em>in vivo</em>. As we have previously shown that resting CD4+T cells are susceptible to HIV integration, we asked whether these cells were capable of producing viral proteins and if so, why resting cells were incapable of supporting productive infection. To answer this question, we spinoculated resting CD4+T cells with or without prior stimulation, and measured integration, transcription, and translation of viral proteins. We found that resting cells were capable of producing HIV Gag without supporting spreading infection. This block corresponded with low HIV envelope levels both at the level of protein and RNA and was not an artifact of spinoculation. The defect was reversed upon stimulation with IL-7 or CD3/28 beads. Thus, a population of latent cells can produce viral proteins without resulting in spreading infection. These results have implications for therapies targeting the latent reservoir and suggest that some latent cells could be cleared by a robust immune response.</p> </div