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 \u3c 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 \u3c 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 \u3c 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

The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) gH/gL Complex Is the Predominant Neutralizing Antigenic Determinant in KSHV-Infected Individuals

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), one of the most prevalent cancers of people living with HIV/AIDS in sub-Saharan Africa. The seroprevalence for KSHV is high in the region, and no prophylactic vaccine against the virus is available. In this study, we characterized the antigenic targets of KSHV-specific neutralizing antibodies (nAbs) in asymptomatic KSHV-infected individuals and KS patients with high nAbs titers. We quantified the extent to which various KSHV envelope glycoproteins (gB, ORF28, ORF68, gH, gL, gM, gN and gpK8.1) adsorbed/removed KSHV-specific nAbs from the plasma of infected individuals. Our study revealed that plasma from a majority of KSHV neutralizers recognizes multiple viral glycoproteins. Moreover, the breadth of nAbs responses against these viral glycoproteins varies among endemic KS, epidemic KS and asymptomatic KSHV-infected individuals. Importantly, among the KSHV glycoproteins, the gH/gL complex, but neither gH nor gL alone, showed the highest adsorption of KSHV-specific nAbs. This activity was detected in 80% of the KSHV-infected individuals regardless of their KS status. The findings suggest that the gH/gL complex is the predominant antigenic determinant of KSHV-specific nAbs. Therefore, gH/gL is a potential target for development of KSHV prophylactic vaccines

Antibody profiling and predictive modeling discriminate between Kaposi sarcoma and asymptomatic KSHV infection

Protein-level immunodominance patterns against Kaposi sarcoma-associated herpesvirus (KSHV), the aetiologic agent of Kaposi sarcoma (KS), have been revealed from serological probing of whole protein arrays, however, the epitopes that underlie these patterns have not been defined. We recently demonstrated the utility of phage display in high-resolution linear epitope mapping of the KSHV latency-associated nuclear antigen (LANA/ORF73). Here, a VirScan phage immunoprecipitation and sequencing approach, employing a library of 1,988 KSHV proteome-derived peptides, was used to quantify the breadth and magnitude of responses of 59 sub-Saharan African KS patients and 22 KSHV-infected asymptomatic individuals (ASY), and ultimately to support an application of machine-learning-based predictive modeling using the peptide-level responses. Comparing anti-KSHV antibody repertoire revealed that magnitude, not breadth, increased in KS. The most targeted epitopes in both KS and ASY were in the immunodominant proteins, notably, K8.129−56 and ORF65140-168, in addition to LANA. Finally, using unbiased machine-learning-based predictive models, reactivity to a subset of 25 discriminative peptides was demonstrated to successfully classify KS patients from asymptomatic individuals. Our study provides the highest resolution mapping of antigenicity across the entire KSHV proteome to date, which is vital to discern mechanisms of viral pathogenesis, to define prognostic biomarkers, and to design effective vaccine and therapeutic strategies. Future studies will investigate the diagnostic, prognostic, and therapeutic potential of the 25 discriminative peptides

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 \u3c 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 \u3c 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 \u3c 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

RNA-Seq of Kaposi\u27s sarcoma reveals alterations in glucose and lipid metabolism

Kaposi\u27s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi\u27s sarcoma (KS). It is endemic in a number of sub-Saharan African countries with infection rate of \u3e50%. 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

Immunological and Molecular Virological Studies of Kaposi’s Sarcoma Patients with and Without HIV-1 Infection

The African endemic Kaposi’s sarcoma (EnKS) is still prevalent in sub-Saharan Africa (SSA) despite high incidence of HIV-1 associated/epidemic Kaposi’s sarcoma (EpKS). While KSHV is clearly the etiologic agent of KS, 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 KS remains unclear. The objective of this research was to investigate the role of HIV-1 in KS by comparative analysis of immune responses and transcriptome changes in EpKS and EnKS patients. Cytokines and antibody responses in circulation of EpKS and EnKS patients from Tanzania and Zambia were characterized. For the first time, we reported that EnKS patients mount high KSHV neutralizing antibody responses similar to EpKS patients and were higher than asymptomatic controls. Regulatory and anti-inflammatory cytokines (IL-10, IL-5, and TGF-β) were also elevated in EpKS and EnKS patients irrespective of HIV-1 co-infection. This suggested that in the circulation, KSHV induces B-cell instead of T-cell responses required to control KSHV infected and cancer cells. The lack of clear effects of HIV-1 on humoral responses of KS patients suggested that possibly HIV-1 exerts its effect locally at the site of the tumor. Therefore, we moved to investigate the impact of HIV-1 in KS tumors. Similarly, transcriptomes analysis revealed high concordance in gene expression profiles between EpKS and EnKS with undetectable or minimal HIV-1 transcripts in EpKS lesions. However, interferon related chemokines (CxCL-9, -10 and -11) were significantly upregulated in KS lesions compared to uninvolved control skins. Although, a T-cell chemoattractant CxCL-9 was demonstrated to be overexpressed at protein level in KS lesions compared to normal skin, there was poor immune cell infiltration and co-localization with KSHV infected cells in KS tumors independent of HIV-1 co-infection, suggesting a fundamental tumor immune evasion mechanism. Overall, the findings of this study suggest that KSHV drives KS pathogenesis while perhaps HIV-1 indirectly or systemically accelerates and exacerbates KSHV pathogenesis and KS development

Immunological and Molecular Virological Studies of Kaposi’s Sarcoma Patients with and Without HIV-1 Infection

The African endemic Kaposi’s sarcoma (EnKS) is still prevalent in sub-Saharan Africa (SSA) despite high incidence of HIV-1 associated/epidemic Kaposi’s sarcoma (EpKS). While KSHV is clearly the etiologic agent of KS, 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 KS remains unclear. The objective of this research was to investigate the role of HIV-1 in KS by comparative analysis of immune responses and transcriptome changes in EpKS and EnKS patients. Cytokines and antibody responses in circulation of EpKS and EnKS patients from Tanzania and Zambia were characterized. For the first time, we reported that EnKS patients mount high KSHV neutralizing antibody responses similar to EpKS patients and were higher than asymptomatic controls. Regulatory and anti-inflammatory cytokines (IL-10, IL-5, and TGF-β) were also elevated in EpKS and EnKS patients irrespective of HIV-1 co-infection. This suggested that in the circulation, KSHV induces B-cell instead of T-cell responses required to control KSHV infected and cancer cells. The lack of clear effects of HIV-1 on humoral responses of KS patients suggested that possibly HIV-1 exerts its effect locally at the site of the tumor. Therefore, we moved to investigate the impact of HIV-1 in KS tumors. Similarly, transcriptomes analysis revealed high concordance in gene expression profiles between EpKS and EnKS with undetectable or minimal HIV-1 transcripts in EpKS lesions. However, interferon related chemokines (CxCL-9, -10 and -11) were significantly upregulated in KS lesions compared to uninvolved control skins. Although, a T-cell chemoattractant CxCL-9 was demonstrated to be overexpressed at protein level in KS lesions compared to normal skin, there was poor immune cell infiltration and co-localization with KSHV infected cells in KS tumors independent of HIV-1 co-infection, suggesting a fundamental tumor immune evasion mechanism. Overall, the findings of this study suggest that KSHV drives KS pathogenesis while perhaps HIV-1 indirectly or systemically accelerates and exacerbates KSHV pathogenesis and KS development

Antiretroviral Therapy for HIV-Associated Cutaneous Kaposi’s Sarcoma: Clinical, HIV-Related, and Sociodemographic Predictors of Outcome

Kaposi’s sarcoma (KS) is an AIDS-defining malignancy that can improve or worsen with antiretroviral therapy (ART). We aimed at identifying clinical, HIV-related, and sociodemographic factors associated with either progression or nonprogression (regression or stable disease) of ART-treated HIV-associated KS in patients with limited cutaneous disease. We conducted a prospective cohort study of ART-treated HIV-associated KS cases. Clinical, HIV-related, and sociodemographic variables were collected at baseline, and patients were followed up to determine treatment outcomes. Cox regression, linear mixed effects model, and Spearman’s rank correlation were used for analysis. Half (50%) of the study participants had KS regression or stable disease, whereas the other half (50%) had disease progression during the treatment and follow-up period. Among the data analyzed, presence of KS nodules at baseline (hazard ratio = 5.47; 95% confidence interval = 1.32–22.65; p = .02) was an independent predictor of poor treatment outcome. Progressors and nonprogressors were in distinguishable in the changes they experienced in the HIV plasma viral load and CD4 counts as a result of ART. Even when cutaneous presentation is limited, the presence of nodular morphotype KS lesions should be considered an indicator for combined ART plus chemotherapy. Temporal trends in CD4 counts and HIV viral loads did not correlate with treatment outcome in ART-treated HIV-associated KS

Prevalence of Kaposi\u27s sarcoma-associated herpesvirus and transfusion-transmissible infections in Tanzanian blood donors

Objective: Kaposi\u27s sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi\u27s 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 \u3c0.05 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

Clinicopathological Characteristics and Outcomes of Anal Squamous Cell Carcinoma Patients With and Without HIV Infection in Sub-Saharan Africa

PURPOSEIn the past 20 years, the burden of anal cancer (AC) increased by 60% in the United States and over three-fold in Africa. Rates of AC have increased by 20× in people living with HIV and the highest (50×) in men with HIV who have sex with men. However, in sub-Saharan Africa (SSA) where HIV is endemic, data on clinicopathological characteristics and outcomes of patients with AC are lacking. To address this, we have investigated AC disease presentation, treatment outcomes, and its predictors in a cohort of patients who were either HIV-infected or HIV-uninfected in SSA.METHODSWe conducted a retrospective cohort study of patients with anal squamous cell carcinoma (SCC) treated at Ocean Road Cancer Institute in Dar es Salaam, Tanzania from January 2014 to December 2019. Associations between the study outcomes and their predictors were analyzed using univariate and multivariate analysis models.RESULTSA total of 59 patients with anal SCC were retrieved and had at least 2-year follow-up. The mean age was 53.9 (standard deviation ±10.5) years. While none of the patients presented with stage I disease, 64.4% had locally advanced disease. HIV infection was the major comorbidity (64.4%). The rate of complete remission at the end of treatment was at 49% while the 2-year overall survival (OS) and local recurrence-free survival were 86.4% and 91.3%, respectively. Despite high HIV coinfection in the cohort, AC treatment outcomes were not significantly associated with HIV status. Disease stage (P = .012) and grade (P = .030) were significantly associated with 2-year OS.CONCLUSIONPatients with anal SCC in Tanzania present mainly with locally advanced disease associated with high HIV prevalence. In this cohort, the SCC grade was independently associated with treatment outcomes unlike other factors such as HIV coinfection