Comparative transcriptome analysis of endemic and epidemic Kaposi's sarcoma (KS) lesions and the secondary role of HIV-1 in KS pathogenesis.

In sub-Saharan Africa, endemic Kaposi's sarcoma (EnKS) is still prevalent despite high incidence of epidemic Kaposi's sarcoma (EpKS) resulting from the on-going HIV-1 epidemic. While KSHV is clearly the etiologic agent of KS, the mechanisms underlying KS development are not fully understood. For example, HIV-1 co-infection and concomitant immune dysfunction have been associated with EpKS development. However, the direct or indirect role(s) of HIV-1, and therefore of immune suppression, in EpKS remains unclear. How, or whether, EpKS is mechanistically distinct from EnKS is unknown. Thus, the absence of HIV-1 co-infection in EnKS provides a unique control for investigating and deciphering whether HIV-1 plays a direct or indirect role in the EpKS tumor microenvironment. We hypothesized that HIV-1 co-infection would induce transcriptome changes that differentiate EpKS from EnKS, thereby defining the direct intra-tumor role of HIV-1 in KS. Comparison of ART-treated and -naïve patients would further define the impact of ART on the KS transcriptome. We utilized RNA-seq followed by multiparameter bioinformatics analysis to compare transcriptomes from KS lesions to uninvolved control skin. We provide the first transcriptomic comparison of EpKS versus EnKS, ART-treated vs-naïve EpKS and male vs female EpKS to define the roles of HIV-1 co-infection, the impact of ART, and gender on KS gene expression profiles. Our findings suggest that ART-use and gender have minimal impact on transcriptome profiles of KS lesions. Gene expression profiles strongly correlated between EpKS and EnKS patients (Spearman r = 0.83, p<10-10). A subset of genes involved in tumorigenesis and inflammation/immune responses showed higher magnitude, but not unique dysregulation in EnKS compared to EpKS. While gender and ART had no detectable contribution, the trend toward higher magnitude of gene dysregulation in EnKS coupled with the absence of HIV-1 transcripts in EpKS may suggest an indirect or systemic effect of HIV-1 to promote KS tumorigenesis.

RNA-Seq of Kaposi's sarcoma reveals alterations in glucose and lipid metabolism.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS). It is endemic in a number of sub-Saharan African countries with infection rate of >50%. The high prevalence of HIV-1 coupled with late presentation of advanced cancer staging make KS the leading cancer in the region with poor prognosis and high mortality. Disease markers and cellular functions associated with KS tumorigenesis remain ill-defined. Several studies have attempted to investigate changes of the gene profile with in vitro infection of monoculture models, which are not likely to reflect the cellular complexity of the in vivo lesion environment. Our approach is to characterize and compare the gene expression profile in KS lesions versus non-cancer tissues from the same individual. Such comparisons could identify pathways critical for KS formation and maintenance. This is the first study that utilized high throughput RNA-seq to characterize the viral and cellular transcriptome in tumor and non-cancer biopsies of African epidemic KS patients. These patients were treated anti-retroviral therapy with undetectable HIV-1 plasma viral load. We found remarkable variability in the viral transcriptome among these patients, with viral latency and immune modulation genes most abundantly expressed. The presence of KSHV also significantly affected the cellular transcriptome profile. Specifically, genes involved in lipid and glucose metabolism disorder pathways were substantially affected. Moreover, infiltration of immune cells into the tumor did not prevent KS formation, suggesting some functional deficits of these cells. Lastly, we found only minimal overlaps between our in vivo cellular transcriptome dataset with those from in vitro studies, reflecting the limitation of in vitro models in representing tumor lesions. These findings could lead to the identification of diagnostic and therapeutic markers for KS, and will provide bases for further mechanistic studies on the functions of both viral and cellular genes that are involved.

Similar Immunological Profiles Between African Endemic and Human Immunodeficiency Virus Type 1-Associated Epidemic Kaposi Sarcoma (KS) Patients Reveal the Primary Role of KS-Associated Herpesvirus in KS Pathogenesis.

BACKGROUND: Kaposi sarcoma (KS)-associated herpesvirus (KSHV) is etiologically linked to all KS forms, but mechanisms underlying KS development are unclear. The incidence of KS in human immunodeficiency virus type 1-infected (HIV-1+) individuals implicates immune dysregulation; however, the lack of characterization of KSHV immune responses in endemic KS makes the role of HIV-1 unclear. The study objective was to investigate the HIV-1 and KSHV roles in viral nucleic acid detection, antibody responses, and cytokine responses in polymerase chain reaction-confirmed epidemic KS and endemic KS patients and non-cancer controls from sub-Saharan Africa. METHODS: KSHV viral DNA (vDNA), total anti-KSHV antibody, KSHV neutralizing antibody (nAb), and cytokines were quantified. RESULTS: KSHV vDNA was detectable in tumors but variably in plasma and peripheral blood mononuclear cells. Consistent with elevated antibody-associated cytokines (interleukin [IL] 6, IL-5, and IL-10), nAb titers were higher in epidemic KS and endemic KS patients than in controls (P < .05). Despite HIV-1 coinfection in epidemic KS, nAb titers were similar between epidemic KS and endemic KS patients (P = 0.3). CONCLUSIONS: Similarities in antibody and cytokine responses between epidemic and endemic KS patients suggest that KSHV drives KS pathogenesis, whereas HIV-1 exacerbates it.

SARS-CoV-2-specific T cell and humoral immunity in individuals with and without HIV in an African population: a prospective cohort study.

OBJECTIVES: To longitudinally compare SARS-CoV-2-specific T cell and humoral immune responses between convalescent individuals who are HIV-positive (HIV+) and HIV-negative (HIV-). METHODS: We conducted enzyme-linked immunospots to determine the SARS-CoV-2-specific T cell responses to spike and nucleocapsid, membrane protein, and other open reading frame proteins (NMO), whereas an immunofluorescence assay was used to determine the humoral responses. Participants were sampled at baseline and after 8 weeks of follow-up. RESULTS: Individuals who are HIV- had significantly more T cell responses to NMO and spike than individuals who are HIV+ at baseline, P-value = 0.026 and P-value = 0.029, respectively. At follow-up, T cell responses to NMO and spike in individuals who are HIV+ increased to levels comparable with individuals who are HIV-. T cell responses in the HIV- group significantly decreased from baseline levels at the time of follow-up (spike [P-value = 0.011] and NMO [P-value = 0.014]). A significantly higher number of individuals in the HIV+ group had an increase in T cell responses to spike (P-value = 0.01) and NMO (P-value = 0.026) during the follow-up period than the HIV- group. Antispike and antinucleocapsid antibody titers were high (1: 1280) and not significantly different between individuals who were HIV- and HIV+ at baseline. A significant decrease in antinucleocapsid titer was observed in the HIV- (P-value = 0.0001) and the HIV+ (P-value = 0.001) groups at follow-up. SARS-CoV-2 vaccination was more effective in boosting the T cell than antibody responses shortly after infection. CONCLUSION: There is an impairment of SARS-CoV-2-specific T cell immunity in individuals who are HIV+ with advanced immunosuppression. SARS-CoV-2-specific T cell immune responses may be delayed in individuals who are HIV+, even in those on antiretroviral therapy. There is no difference in SARS-CoV-2-specific humoral immunity between individuals who are HIV- and HIV+.

Comparative polar and lipid plasma metabolomics differentiate KSHV infection and disease states.

BACKGROUND: Kaposi sarcoma (KS) is a neoplastic disease etiologically associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV). KS manifests primarily as cutaneous lesions in individuals due to either age (classical KS), HIV infection (epidemic KS), or tissue rejection preventatives in transplantation (iatrogenic KS) but can also occur in individuals, predominantly in sub-Saharan Africa (SSA), lacking any obvious immune suppression (endemic KS). The high endemicity of KSHV and human immunodeficiency virus-1 (HIV) co-infection in Africa results in KS being one of the top 5 cancers there. As with most viral cancers, infection with KSHV alone is insufficient to induce tumorigenesis. Indeed, KSHV infection of primary human endothelial cell cultures, even at high levels, is rarely associated with long-term culture, transformation, or growth deregulation, yet infection in vivo is sustained for life. Investigations of immune mediators that distinguish KSHV infection, KSHV/HIV co-infection, and symptomatic KS disease have yet to reveal consistent correlates of protection against or progression to KS. In addition to viral infection, it is plausible that pathogenesis also requires an immunological and metabolic environment permissive to the abnormal endothelial cell growth evident in KS tumors. In this study, we explored whether plasma metabolomes could differentiate asymptomatic KSHV-infected individuals with or without HIV co-infection and symptomatic KS from each other. METHODS: To investigate how metabolic changes may correlate with co-infections and tumorigenesis, plasma samples derived from KSHV seropositive sub-Saharan African subjects in three groups, (A) asymptomatic (lacking neoplastic disease) with KSHV infection only, (B) asymptomatic co-infected with KSHV and HIV, and (C) symptomatic with clinically diagnosed KS, were subjected to analysis of lipid and polar metabolite profiles RESULTS: Polar and nonpolar plasma metabolic differentials were evident in both comparisons. Integration of the metabolic findings with our previously reported KS transcriptomics data suggests dysregulation of amino acid/urea cycle and purine metabolic pathways, in concert with viral infection in KS disease progression. CONCLUSIONS: This study is, to our knowledge, the first to report human plasma metabolic differentials between in vivo KSHV infection and co-infection with HIV, as well as differentials between co-infection and epidemic KS.

Lower SARS-CoV-2 Seroprevalence among Cancer Patients in Sub-Saharan Africa.

BACKGROUND: Despite the high COVID-19 morbidity and mortality rates across the world, the reported rates in sub-Saharan Africa (SSA), which has a higher burden of other infectious diseases and overwhelmed healthcare systems, remain relatively low. This study aims to better understand the potential factors that contribute to this phenomenon, especially among cancer patients who are considered as a high-risk group for developing severe COVID-19. METHODS: Plasma samples collected during the COVID-19 pandemic from SARS-CoV-2 unvaccinated cancer and potential blood donor populations were analyzed for SARS-CoV-2 (spike and nucleocapsid proteins) antibodies by an immunofluorescence assay. The relationships between SARS-CoV-2 seroprevalences and study variables were determined using a logistic regression analysis. RESULTS: High seroprevalence against the SARS-CoV-2 spike and nucleocapsid proteins were found among the SARS-CoV-2 unvaccinated COVID-19 pandemic populations in SSA. However, the cancer patients demonstrated a lower seroprevalence compared to potential blood donors. There was also an association between mild COVID-19 symptoms with prior tuberculosis vaccination among cancer patients. CONCLUSION: Cancer patients in SSA tend to have a relatively lower SARS-CoV-2 seroprevalence compared to potential blood donors recruited from the same geographic locations during the COVID-19 pandemic. More study is required to determine its cause and potential impact on SARS-CoV-2 vaccination among cancer patients.

High prevalence of pre-existing serological cross-reactivity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in sub-Saharan Africa.

OBJECTIVE: Significant morbidity and mortality have occurred in the USA, Europe, and Asia due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), whereas the numbers of infections and deaths in sub-Saharan Africa (SSA) have remained comparatively low. It has been hypothesized that exposure of the population in SSA to other coronaviruses prior to the COVID-19 pandemic resulted in some degree of cross-protection against SARS-CoV-2 infection and pathogenesis. We evaluated this hypothesis by comparing SARS-CoV-2 cross-reactive antibodies in pre-pandemic plasma samples collected from SSA and the USA. METHOD: Pre-COVID-19 pandemic plasma samples from SSA and the USA were collected and tested by immunofluorescence assay against the spike and nucleocapsid proteins of all known human coronaviruses (HCoVs). RESULTS: The prevalence of SARS-CoV-2 serological cross-reactivity was significantly higher in samples from SSA compared with the USA. Most of these cross-reactive samples cross-recognized the SARS-CoV-2 nucleocapsid protein and the spike proteins of other HCoVs. Nucleocapsid proteins from HCoV-NL63 and HCoV-229E were detected in most samples, thereby implicating prior exposure to these two HCoVs as the likely source of cross-reactive antibodies against SARS-CoV-2. CONCLUSION: The low incidences of SARS-CoV-2 infection and disease in SSA appear to be correlated with the pre-pandemic serological cross-recognition of HCoVs, which are substantially more prevalent in SSA than the USA.

Presence of antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 in COVID-19 plasma.

BACKGROUND: Neutralizing-antibody (nAb) is the major focus of most ongoing COVID-19 vaccine trials. However, nAb response against SARS-CoV-2, when present, decays rapidly. Given the myriad roles of antibodies in immune responses, it is possible that antibodies could also mediate protection against SARS-CoV-2 via effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), which we sought to explore here. METHODS: Plasma of 3 uninfected controls and 20 subjects exposed to, or recovering from, SARS-CoV-2 infection were collected from U.S. and sub-Saharan Africa. Immunofluorescence assay was used to detect the presence of SARS-CoV-2 specific IgG antibodies in the plasma samples. SARS-CoV-2 specific neutralizing capability of these plasmas was assessed with SARS-CoV-2 spike pseudotyped virus. ADCC activity was assessed with a calcein release assay. RESULTS: SARS-CoV-2 specific IgG antibodies were detected in all COVID-19 subjects studied. All but three COVID-19 subjects contained nAb at high potency (>80% neutralization). Plasma from 19/20 of COVID-19 subjects also demonstrated strong ADCC activity against SARS-CoV-2 spike glycoprotein, including two individuals without nAb against SARS-CoV-2. CONCLUSION: Both neutralizing and non-neutralizing COVID-19 plasmas can mediate ADCC. Our findings argue that evaluation of potential vaccines against SARS-CoV-2 should include investigation of the magnitude and durability of ADCC, in addition to nAb.

Lack of CD8+ T-cell co-localization with Kaposi's sarcoma-associated herpesvirus infected cells in Kaposi's sarcoma tumors.

Despite the close association between Kaposi's sarcoma (KS) and immune dysfunction, it remains unclear whether tumor infiltrating immune cells (TIIC), by their absence, presence, or dysfunction, are mechanistically correlated with KS pathogenesis. Therefore, their potential capacity to serve as prognostic biomarkers of KS disease progression or control is unclear. Because epidemic-KS (EpKS) occurs with HIV-1 co-infection, it is particularly important to compare TIIC between EpKS and HIV-negative African endemic-KS (EnKS) to dissect the roles of HIV-1 and Kaposi Sarcoma-associated herpesvirus (KSHV) in KS pathogenesis. This cross-sectional study of 13 advanced KS (4 EnKS, 9 EpKS) patients and 3 healthy controls utilized single-color immunohistochemistry and dual-color immunofluorescence assays to characterize and quantify KSHV infected cells in relation to various TIIC in KS biopsies. Analysis of variance (ANOVA) and Mann-Whitney tests were used to assess differences between groups where P-values < 0.05 were considered significant. The abundance of KSHV infected cells was heterogeneous in KS biopsies. Despite the presence of T-cell chemoattractant chemokine CxCL-9 in biopsies, CD8+ T-cells were sparsely distributed in regions with evident KSHV infected cells but were readily detectable in regions devoid of KSHV infected cells (P < 0.0001). CD68+ (M1) macrophages were evenly and diffusely distributed in KS biopsies, whereas, the majority of CD163+ (M2) macrophages were localized in regions devoid of KSHV infected cells (P < 0.0001). Overall, the poor immune cell infiltration or co-localization in KS biopsies independent of HIV-1 co-infection suggests a fundamental tumor immune evasion mechanism that warrants further investigation.

Prevalence of Kaposi's sarcoma-associated herpesvirus and transfusion-transmissible infections in Tanzanian blood donors.

OBJECTIVE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS), one of the most common cancers in Tanzania. We have investigated KSHV prevalence and factors associated with KSHV infection in Tanzania. METHODS: This is a cross-sectional study of voluntary blood-donors from Dar es Salaam, Tanzania. Plasma was screened for KSHV, HIV-1, HBV, HCV and Treponema pallidum (syphilis). Associations between KSHV sero-status and risk factors were analyzed. Odds ratios (OR) and 95% confidence intervals (CI) are reported to evaluate risk factors of KSHV infection. All tests were 2-tailed, and P-values <0.05 were considered statistically significant. RESULTS: The overall KSHV seroprevalence was 56.9%. Significantly increased risk of KSHV infection was detected in persons from the Lake and Central Zones (OR=6.4, 95% CI=1.6-25.3, P=0.008 and OR=5.7, 95% CI=1.0-32.5, P=0.048 respectively). A trend toward increased risk of KSHV infection with HIV-1 co-infection was not significant (OR=2.8, 95% CI=1.0-8.0, P=0.06). Seroreactivity to T. pallidum was surprisingly high (14.9%). CONCLUSION: The prevalence of KSHV infection and syphilis was high among Tanzanian blood-donors. The most common transfusion-transmissible infections did not associate with KSHV infection. Regions of focal KSHV infection need further investigation for underappreciated risk factors.

Viral and Immunological Analytes are Poor Predictors of the Clinical Treatment Response in Kaposi's Sarcoma Patients.

Kaposi's sarcoma-associated herpes virus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS). The prognostic utility of KSHV and HIV-1 (human immunodeficiency virus) viremia as well as immunological parameters in clinical management of participants with KS is unclear. The objective of this study was to investigate viral and immunological parameters as predictors of KS treatment responses in participants with KS from sub-Saharan Africa (SSA). Plasma KSHV-DNA, HIV-1 viral load, total anti-KSHV antibody, KSHV-neutralizing antibody (nAb), cytokine/chemokine levels, and T-cell differentiation subsets were quantified before and after KS treatment in 13 participants with KS and in 13 KSHV-infected asymptomatic control individuals. One-way analysis of variance and the Mann-Whitney t-test were used to assess differences between groups where p-values <0.05 were considered significant. Subjects with patch and plaque KS lesions responded more favorably to treatment than those with nodular lesions. Pre-treatment and post-treatment levels of plasma KSHV-DNA, HIV-1 viral load, KSHV-Ab responses, cytokines, and T-cell populations did not predict the KS treatment response. Elevated KSHV-humoral and cytokine responses persisted in participants with KS despite a clinical KS response. While patch and plaque KS lesions were more common among treatment responders, none of the analyzed viral and immunological parameters distinguished responders from non-responders at baseline or after treatment.