Profiling for KSHV and Other Herpesviruses In Vivo In Clinical Specimens

The 1994 discovery of Kaposi Sarcoma-associated herpesvirus (KSHV) was an important finding, especially as the sharp rise in HIV infections in the early 1990's led to an explosive Kaposi Sarcoma (KS) epidemic in men who have sex with men. This cancer-causing herpesvirus is the causative agent of three diseases in addition to KS: Multicentric Castlemen Disease (MCD), Primary Effusion Lymphoma (PEL), and the newly described condition KSHV Inflammatory Cytokine Syndrome (KICS). By understanding viral gene expression in human patients, we hope to better understand KSHV infection and its diseases, with the hope to shed light on novel therapeutic techniques. In order to quantify KSHV viral gene expression, I devised methods to profile human clinical specimens using high-throughput qPCR. This technique was used to profile microRNA expression and was published as a methods video journal in the Journal of Visualized Experiments (Chugh, Tamburro, and Dittmer, 2010). The accompanying manuscript is found in the Appendix chapter. This same high-throughput technique was employed in Chapter II to determine KSHV viral load and to determine expression of viral kinases, and in Chapter III to determine KSHV gene expression profiles. In Chapter II, we hypothesized that HIV patients with newly diagnosed KS in Malawi could potentially benefit from treatment with the antiherpesviral drug Ganciclovir (GCV). This was addressed through a pilot study with the AIDS Malignancy Consortium (AMC). By using qPCR to assess gene expression of two viral kinases critical to drug responsiveness, we found evidence that 78% of patients express a viral kinase and would potentially benefit from this therapy. This finding is important, as it indicates that patients can be stratified for treatment based on kinase expression in a tumor biopsy, which will maximize potential effectiveness and limit waste of drugs in a resource limited setting. This work will lead to upcoming treatment studies in this population. Next, we used whole-genome KSHV transcriptome profiling to understand whether all KS patients from the Malawian cohort express similar gene expression profiles or if subgroups can be distinguished. Our results indicate the gene expression profiles vary significantly between patients. Specifically, two subpopulations were discovered- those with and those without high levels of genome-wide transcription. Patients with high transcription levels demonstrated significantly higher CD4 counts and KSHV viremia in plasma, although HIV viremia, number of tumors, or size of biopsied lesion were not significantly associated with KSHV transcription. This work is described in Chapter III. In Chapter IV, we employed high-throughput DNA sequencing as a method to diagnose co-infections in an end-stage AIDS patient. This was published in the Journal Virology (Tamburro et al. 2012). The patient had minimal KS skin lesions despite having one of the highest viral loads on record- leading to the diagnosis of the newly described condition KSHV Inflammatory Cytokine Syndrome (KICS). Additionally, sequencing uncovered a co-infection with HHV6a, which may have accounted for Systemic Inflammatory Response Syndrome (SIRS) that ultimately led to the patient's death. Notable to the research community, this work resulted in the first complete sequence of KSHV using direct methods, rather than relying on pre-sequencing cloning or cell passage methods.Doctor of Philosoph

Recent cancer incidence trends in an observational clinical cohort of HIV-infected patients in the US, 2000 to 2011

Abstract Background In HIV-infected populations in developed countries, the most recent published cancer incidence trend analyses are only updated through 2008. We assessed changes in the distribution of cancer types and incidence trends among HIV-infected patients in North Carolina up until 2011. Methods We linked the University of North Carolina Center for AIDS Research HIV Clinical Cohort, an observational clinical cohort of 3141 HIV-infected patients, with the North Carolina Cancer registry. Cancer incidence rates were estimated across calendar years from 2000 to 2011. The distribution of cancer types was described. Incidence trends were assessed with linear regression. Results Across 15,022 person-years of follow-up, 202 cancers were identified (incidence rate per 100,000 person-years [IR]: 1345; 95% confidence interval [CI]: 1166, 1544). The majority of cancers were virus-related (61%), including Kaposi sarcoma (N = 32) (IR: 213; 95%CI: 146, 301), non-Hodgkin lymphoma (N = 34) (IR: 226; 95%CI: 157, 316), and anal cancer (N = 16) (IR: 107; 95%CI: 61, 173). Non-Hodgkin lymphoma was observed to decrease from 2000 to 2011 (decline of 15 cases per 100,000 person-years per calendar year, 95%CI: -27, -3). No other changes in incidence or changes in incidence trends were observed for other cancers (all P > 0.20). Conclusions We observed a substantial burden of a variety of cancers in this population in the last decade. Kaposi sarcoma and non-Hodgkin lymphoma were consistently two of the greatest contributors to cancer burden across calendar time. Cancer rates appeared stable across calendar years, except for non-Hodgkin lymphoma, which appeared to decrease throughout the study period

Tousled-like Kinases Modulate Reactivation of Gammaherpesviruses from Latency

Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread but the cellular players involved in the switch between the two viral lifecycle phases are not clearly understood. We conducted a siRNA screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus (EBV). Our results establish the TLKs as cellular repressors of gammaherpesviral reactivation

mTOR Inhibitors Block Kaposi Sarcoma Growth by Inhibiting Essential Autocrine Growth Factors and Tumor Angiogenesis

Kaposi’s Sarcoma (KS) originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi Sarcoma-associated Herpes Virus (KSHV) are endemic, KS is the most common cancer overall, but model systems for disease study are insufficient. Here we report the development of a novel mouse model of KS where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results demonstrated that mTOR inhibitors exert a direct anti-KS effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for KS and other cancers of endothelial origin

Vironome of Kaposi sarcoma associated herpesvirus-inflammatory cytokine syndrome in an AIDS patient reveals co-infection of human herpesvirus 8 and human herpesvirus 6A

KSHV inflammatory cytokine syndrome (KICS) is a newly described condition characterized by systemic illness as a result of systemic, lytic KSHV infection. We used Illumina sequencing to establish the DNA vironome of blood from such a patient. It identified concurrent high-level viremia of human herpesvirus (HHV) 8 and 6a. The HHV8 plasma viral load was 5,300,000 copies/ml, which is the highest reported to date; this despite <5 skin lesions and no HHV8 associated lymphoma. This is the first report of systemic HHV6a/KSHV co-infection in a patient. It is the first whole genome KSHV sequence to be determined directly from patient plasma rather than cultured or biopsied tumor material. This case supports KICS as a new clinical entity associated with KSHV

3-Hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin)-induced 28-kDa interleukin-1beta interferes with mature IL-1beta signaling

Multiple clinical trials have shown that the 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors known as statins have anti-inflammatory effects. However, the underlying molecular mechanism remains unclear. The proinflammatory cytokine interleukin-1beta (IL-1beta) is synthesized as a non-active precursor. The 31-kDa pro-IL-1beta is processed into the 17-kDa active form by caspase-1-activating inflammasomes. Here, we report a novel signaling pathway induced by statins, which leads to processing of pro-IL-1beta into an intermediate 28-kDa form. This statin-induced IL-1beta processing is independent of caspase-1- activating inflammasomes. The 28-kDa form of IL-1beta cannot activate interleukin-1 receptor-1 (IL1R1) to signal inflammatory responses. Instead, it interferes with mature IL-1beta signaling through IL-1R1 and therefore may dampen inflammatory responses initiated by mature IL-1beta. These results may provide new clues to explain the anti-inflammatory effects of statins

Oral shedding of herpesviruses in HIV-infected patients with varying degrees of immune status

ObjectiveHerpesvirus shedding in the oral cavity was analyzed to determine if presence in the oral compartment correlates with systemic changes in HIV-associated immune deficiency as measured by CD4 cell counts, plasma HIV viral load and presence of AIDS-defining events.DesignA5254 is a multicenter, cross-sectional, single-visit study to evaluate oral complications of HIV/AIDS and determine the association between clinical appearance, herpesvirus shedding, and immune status as ascertained by CD4 cell count and HIV viral load. In total, 307 HIV-infected individuals were evaluated and throat wash collected.MethodsFisher's exact test and Kruskal-Wallis test were used to assess the association between presence of herpesviruses and the state of immunodeficiency as stratified by a combination of CD4 cell count and HIV viral load. Relationship between pathogens and HIV viral load in plasma was modeled by logistic regression.ResultsThe presence of cytomegalovirus (CMV) and herpes simplex virus-1 in throat wash was associated with decreased CD4 cell counts. By contrast, Kaposi sarcoma-associated herpesvirus and Epstein-Barr virus were similarly detectable across all levels of CD4 cell counts. One unit increase in log10 (HIV viral load) was associated with 1.31 times higher odds of detecting CMV in throat wash when controlling for oral candidiasis, CD4 cell count, and sites (95% confidence interval 1.04-1.65, P = 0.02).ConclusionOral CMV shedding was significantly higher in highly immunocompromised HIV participants. Our finding supports the recommendations to start antiretroviral therapy independent of CD4 cell count as this may have the added benefit to lower the risk of herpesvirus transmission among persons infected with HIV and their partners

mTOR Inhibitors Block Kaposi Sarcoma Growth by Inhibiting Essential Autocrine Growth Factors and Tumor Angiogenesis

Kaposi’s Sarcoma (KS) originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi Sarcoma-associated Herpes Virus (KSHV) are endemic, KS is the most common cancer overall, but model systems for disease study are insufficient. Here we report the development of a novel mouse model of KS where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results demonstrated that mTOR inhibitors exert a direct anti-KS effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for KS and other cancers of endothelial origin