Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) by SARS-CoV-2 in non-hospitalised HIV-infected patients.

BACKGROUND: While acute SARS-CoV-2 infection and associated inflammation resulted in substantial morbidity and mortality during the COVID-19 pandemic, particularly in unvaccinated patients, long-term effects of SARS-CoV-2 exposure for reactivation of latent oncogenic herpesviruses, such as KSHV, is unknown. METHODS: We performed a longitudinal observational cross-sectional study on 407 non-hospitalised adult HIV-infected (CD4 count <350 cells/µL) patients attending antiretroviral therapy services in Gugulethu, South Africa, from October 2020 to April 2023. FINDINGS: KSHV seroprevalence was 53.5%; the quarterly SARS-CoV-2 seroprevalence increased from 76.2% (before roll-out of COVID-19 vaccinations) to 94.9%, with 32.2% being self-reportedly vaccinated against COVID-19. Over the course of recruitment, the quarterly percentage of patients with detectable KSHV viral load (VL) in the peripheral blood increased from 3.3% to 69.2%. The presence of KSHV VL was significantly associated with SARS-CoV-2 RBD antibody titers in unvaccinated (median RBD IgG OD 1.24 [IQR 0.82-2.42] in non-reactivated versus 2.83 [IQR 1.08-4.72] in reactivated patients, p = 0.0030) but not in vaccinated patients (median RBD IgG OD 5.13 [IQR 4.11-6.36] in non-reactivated versus 4.53 [IQR 2.90-5.92] in reactivated patients, p = 0.086). Further logistic regression revealed significantly higher odds of KSHV reactivation in unvaccinated, previously SARS-CoV-2 exposed patients (p = 0.015, adjusted OR 1.28 [95% CI: 1.05-1.55]), but not vaccinated patients (p = 0.080, adjusted OR 0.83 [95% CI: 0.67-1.02]). Interestingly, detectable KSHV VL was not associated with increased inflammatory markers such as C-reactive protein and interleukin-6. INTERPRETATION: High, and most likely repeated, exposure to SARS-CoV-2 in unvaccinated individuals may have long-term consequences for reactivation of KSHV infection as shown here in the context of HIV-infected patients with impaired immune functions. Post-pandemic prevention and/or monitoring strategies of potential KSHV-associated pathologies in high-risk patients with immunodeficiencies are therefore highly recommended. FUNDING: This research was funded by the EDCTP2 programme (Training and Mobility Action TMA2018SF-2446).

The Impact of Co-Infections for Human Gammaherpesvirus Infection and Associated Pathologies.

The two oncogenic human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause significant disease burden, particularly in immunosuppressed individuals. Both viruses display latent and lytic phases of their life cycle with different outcomes for their associated pathologies. The high prevalence of infectious diseases in Sub-Saharan Africa (SSA), particularly HIV/AIDS, tuberculosis, malaria, and more recently, COVID-19, as well as their associated inflammatory responses, could potentially impact either virus' infectious course. However, acute or lytically active EBV and/or KSHV infections often present with symptoms mimicking these predominant diseases leading to misdiagnosis or underdiagnosis of oncogenic herpesvirus-associated pathologies. EBV and/or KSHV infections are generally acquired early in life and remain latent until lytic reactivation is triggered by various stimuli. This review summarizes known associations between infectious agents prevalent in SSA and underlying EBV and/or KSHV infection. While presenting an overview of both viruses' biphasic life cycles, this review aims to highlight the importance of co-infections in the correct identification of risk factors for and diagnoses of EBV- and/or KSHV-associated pathologies, particularly in SSA, where both oncogenic herpesviruses as well as other infectious agents are highly pervasive and can lead to substantial morbidity and mortality.

LSDV-Vectored SARS-CoV-2 S and N Vaccine Protects against Severe Clinical Disease in Hamsters.

The SARS-CoV-2 pandemic demonstrated the need for potent and broad-spectrum vaccines. This study reports the development and testing of a lumpy skin disease virus (LSDV)-vectored vaccine against SARS-CoV-2, utilizing stabilized spike and conserved nucleocapsid proteins as antigens to develop robust immunogenicity. Construction of the vaccine (LSDV-SARS2-S,N) was confirmed by polymerase chain reaction (PCR) amplification and sequencing. In vitro characterization confirmed that cells infected with LSDV-SARS2-S,N expressed SARS-CoV-2 spike and nucleocapsid protein. In BALB/c mice, the vaccine elicited high magnitude IFN-γ ELISpot responses (spike: 2808 SFU/106 splenocytes) and neutralizing antibodies (ID50 = 6552). Testing in hamsters, which emulate human COVID-19 disease progression, showed the development of high titers of neutralizing antibodies against the Wuhan and Delta SARS-CoV-2 variants (Wuhan ID50 = 2905; Delta ID50 = 4648). Additionally, hamsters vaccinated with LSDV-SARS2-S,N displayed significantly less weight loss, lung damage, and reduced viral RNA copies following SARS-CoV-2 infection with the Delta variant as compared to controls, demonstrating protection against disease. These data demonstrate that LSDV-vectored vaccines display promise as an effective SARS-CoV-2 vaccine and as a potential vaccine platform for communicable diseases in humans and animals. Further efficacy testing and immune response analysis, particularly in non-human primates, are warranted.

SARS-CoV-2 Infection Is Associated with Uncontrolled HIV Viral Load in Non-Hospitalized HIV-Infected Patients from Gugulethu, South Africa.

In South Africa, high exposure to SARS-CoV-2 occurs primarily in densely populated, low-income communities, which are additionally burdened by highly prevalent Human Immunodeficiency Virus (HIV). With the aim to assess SARS-CoV-2 seroprevalence and its association with HIV-related clinical parameters in non-hospitalized patients likely to be highly exposed to SARS-CoV-2, this observational cross-sectional study was conducted at the Gugulethu Community Health Centre Antiretroviral clinic between October 2020 and June 2021, after the first COVID-19 wave in South Africa and during the second and beginning of the third wave. A total of 150 adult (median age 39 years [range 20−65 years]) HIV-infected patients (69% female; 31% male) were recruited. 95.3% of the cohort was on antiretroviral therapy (ART), had a median CD4 count of 220 cells/µL (range 17−604 cells/µL) and a median HIV viral load (VL) of 49 copies/mL (range 1−1,050,867 copies/mL). Furthermore, 106 patients (70.7%) were SARS-CoV-2 seropositive, and 0% were vaccinated. When stratified for HIV VL, patients with uncontrolled HIV viremia (HIV VL > 1000 copies/mL) had significantly higher odds of SARS-CoV-2 seropositivity than patients with HIV VL < 1000 copies/mL, after adjusting for age, sex and ART status (p = 0.035, adjusted OR 2.961 [95% CI: 1.078−8.133]). Although the cause−effect relationship could not be determined due to the cross-sectional study design, these results point towards a higher risk of SARS-CoV-2 susceptibility among viremic HIV patients, or impaired HIV viral control due to previous co-infection with SARS-CoV-2.

Cellular Receptors Involved in KSHV Infection.

The process of Kaposi's Sarcoma Herpes Virus' (KSHV) entry into target cells is complex and engages several viral glycoproteins which bind to a large range of host cell surface molecules. Receptors for KSHV include heparan sulphate proteoglycans (HSPGs), several integrins and Eph receptors, cystine/glutamate antiporter (xCT) and Dendritic Cell-Specific Intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This diverse range of potential binding and entry sites allows KSHV to have a broad cell tropism, and entry into specific cells is dependent on the available receptor repertoire. Several molecules involved in KSHV entry have been well characterized, particularly those postulated to be associated with KSHV-associated pathologies such as Kaposi's Sarcoma (KS). In this review, KSHV infection of specific cell types pertinent to its pathogenesis will be comprehensively summarized with a focus on the specific cell surface binding and entry receptors KSHV exploits to gain access to a variety of cell types. Gaps in the current literature regarding understanding interactions between KSHV glycoproteins and cellular receptors in virus infection are identified which will lead to the development of virus infection intervention strategies.

The Role of the Eph Receptor Family in Tumorigenesis.

The Eph receptor tyrosine kinase family, activated by binding to their cognate ephrin ligands, are important components of signalling pathways involved in animal development. More recently, they have received significant interest due to their involvement in oncogenesis. In most cases, their expression is altered, affecting the likes of cell proliferation and migration. Depending on the context, Eph receptors have the potential to act as both tumour promoters and suppressors in a number of cancers, such as breast cancer, colorectal cancer, lung cancer, prostate cancer, brain cancer and Kaposi's sarcoma (KS), the latter being intrinsically linked to EphA2 as this is the receptor used for endothelial cell entry by the Kaposi's sarcoma-associated herpesvirus (KSHV). In addition, EphA2 deregulation is associated with KS, indicating that it has a dual role in this case. Associations between EphA2 sequence variation and KSHV infection/KS progression have been detected, but further work is required to formally establish the links between EphA2 signalling and KS oncogenesis. This review consolidates the available literature of the role of the Eph receptor family, and particularly EphA2, in tumorigenesis, with the aim to develop a better understanding of Eph signalling pathways for potential targeting in novel cancer therapies.

Kaposi's Sarcoma-Associated Herpesvirus, but Not Epstein-Barr Virus, Co-infection Associates With Coronavirus Disease 2019 Severity and Outcome in South African Patients.

In South Africa, the Coronavirus Disease 2019 (COVID-19) pandemic is occurring against the backdrop of high Human Immunodeficiency Virus (HIV), tuberculosis and non-communicable disease burdens as well as prevalent herpesviruses infections such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). As part of an observational study of adults admitted to Groote Schuur Hospital, Cape Town, South Africa during the period June-August 2020 and assessed for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, we measured KSHV serology and KSHV and EBV viral load (VL) in peripheral blood in relation to COVID-19 severity and outcome. A total of 104 patients with PCR-confirmed SARS-CoV-2 infection were included in this study. 61% were men and 39% women with a median age of 53 years (range 21-86). 29.8% (95% CI: 21.7-39.1%) of the cohort was HIV positive and 41.1% (95% CI: 31.6-51.1%) were KSHV seropositive. EBV VL was detectable in 84.4% (95% CI: 76.1-84.4%) of the cohort while KSHV DNA was detected in 20.6% (95% CI: 13.6-29.2%), with dual EBV/KSHV infection in 17.7% (95% CI: 11.1-26.2%). On enrollment, 48 [46.2% (95% CI: 36.8-55.7%)] COVID-19 patients were classified as severe on the WHO ordinal scale reflecting oxygen therapy and supportive care requirements and 30 of these patients [28.8% (95% CI: 20.8-38.0%)] later died. In COVID-19 patients, detectable KSHV VL was associated with death after adjusting for age, sex, HIV status and detectable EBV VL [p = 0.036, adjusted OR = 3.17 (95% CI: 1.08-9.32)]. Furthermore, in HIV negative COVID-19 patients, there was a trend indicating that KSHV VL may be related to COVID-19 disease severity [p = 0.054, unstandardized co-efficient 0.86 (95% CI: -0.015-1.74)] in addition to death [p = 0.008, adjusted OR = 7.34 (95% CI: 1.69-31.49)]. While the design of our study cannot distinguish if disease synergy exists between COVID-19 and KSHV nor if either viral infection is indeed fueling the other, these data point to a potential contribution of KSHV infection to COVID-19 outcome, or SARS-CoV-2 infection to KSHV reactivation, particularly in the South African context of high disease burden, that warrants further investigation.

Evidence for altered host genetic factors in KSHV infection and KSHV-related disease development.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), the most common AIDS-related malignancy. It also causes other rare, but certainly underreported, KSHV-associated pathologies, namely primary effusion lymphoma, multicentric Castleman disease and KSHV inflammatory cytokine syndrome. Epidemiology and pathogenicity studies point to the potential for host genetic predisposition to KSHV infection and/or the subsequent development of KSHV-associated pathologies partly explaining the peculiar geographic and population-specific incidence of KSHV and associated pathologies and discrepancies in KSHV exposure and infection and KSHV infection and disease development. This review consolidates the current knowledge of host genetic factors involved in the KSHV-driven pathogenesis. Studies reviewed here indicate a plausible connection between KSHV susceptibility and host genetic factors that affect either viral access to host cells via entry mechanisms or host innate immunity to viral infection. Subsequent to infection, KSHV-associated pathogenesis, reviewed here primarily in the context of KS, is likely influenced by an orchestrated concert of innate immune system interactions, downstream inflammatory pathways and oncogenic mechanisms. The association studies reviewed here point to interesting candidate genes that may prove important in achieving a more nuanced understanding of the pathogenesis and therapeutic targeting of KSHV and associated diseases. Recent studies on host genetic factors suggest numerous candidate genes strongly associated with KSHV infection or subsequent disease development, particularly innate immune system mediators. Taken together, these contribute toward our understanding of the geographic prevalence and population susceptibility to KSHV and KSHV-associated diseases.

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus to Mortality in Hospitalized Human Immunodeficiency Virus-Infected Patients Being Investigated for Tuberculosis in South Africa.

BACKGROUND: Despite increasing numbers of human immunodeficiency virus (HIV)-infected South Africans receiving antiretroviral therapy (ART), tuberculosis (TB) remains the leading cause of mortality. Approximately 25% of patients treated for TB have microbiologically unconfirmed diagnoses. We assessed whether elevated Kaposi's sarcoma-associated herpesvirus (KSHV) viral load (VL) contributes to mortality in hospitalized HIV-infected patients investigated for TB. METHODS: Six hundred eighty-two HIV-infected patients admitted to Khayelitsha Hospital, South Africa, were recruited, investigated for TB, and followed for 12 weeks. KSHV serostatus, peripheral blood KSHV-VL, and KSHV-associated clinical correlates were evaluated. RESULTS: Median CD4 count was 62 (range, 0-526) cells/µL; KSHV seropositivity was 30.7% (95% confidence interval [CI], 27%-34%); 5.8% had detectable KSHV-VL (median, 199.1 [range, 13.4-2.2 × 106] copies/106 cells); 22% died. Elevated KSHV-VL was associated with mortality (adjusted odds ratio, 6.5 [95% CI, 1.3-32.4]) in patients without TB or other microbiologically confirmed coinfections (n = 159). Six patients had "possible KSHV-inflammatory cytokine syndrome" (KICS): 5 died, representing significantly worse survival (P < .0001), and 1 patient was diagnosed with KSHV-associated multicentric Castleman disease at autopsy. CONCLUSIONS: Given the association of mortality with elevated KSHV-VL in critically ill HIV-infected patients with suspected but not microbiologically confirmed TB, KSHV-VL and KICS criteria may guide diagnostic and therapeutic evaluation.

EPHA2 sequence variants are associated with susceptibility to Kaposi's sarcoma-associated herpesvirus infection and Kaposi's sarcoma prevalence in HIV-infected patients.

BACKGROUND: To determine if variations exist in the KSHV host receptor EPHA2's coding region that affect KSHV infectivity and/or KS prevalence among South African HIV-infected patients. METHODS: A retrospective candidate gene association study was performed on 150 patients which were randomly selected from a total of 756 HIV-infected patients and grouped according to their KS status and KSHV serodiagnosis; namely group 1: KS+/KSHV+; group 2: KS-/KSHV+; group 3: KS-/KSHV-. Peripheral blood DNA was used to extract DNA and PCR amplify and sequence the entire EPHA2 coding region, which was compared to the NCBI reference through multiple alignment. RESULTS: 100% (95% CI 92.9-100%) of the KS positive patients, and 31.6% (95% CI 28.3-35.1%) of the KS negative patients were found to be KSHV seropositive. Aggregate variation across the entire EPHA2 coding region identified an association with KS (OR = 6.6 (95% CI 2.8, 15.9), p = 2.2 × 10-5). This was primarily driven by variation in the functionally important protein tyrosine kinase domain (Pkinase-Tyr; OR = 4.9 (95% CI 1.9, 12.4), p = 0.001) and the sterile-α-motif (SAM; OR = 13.8 (95% CI 1.7, 111.6), p = 0.014). Mutation analysis revealed two novel, non-synonymous heterozygous variants (c.2254 T > C: OR undefined, adj. p = 0.02; and c.2990 G > T: OR undefined, adj. p = 0.04) in Pkinase-Tyr and SAM, respectively, to be statistically associated with KS; and a novel heterozygous transition (c.2727C > T: OR = 6.4 (95% CI 1.4, 28.4), adj. p = 0.03) in Pkinase-Tyr to be statistically associated with KSHV. CONCLUSIONS: Variations in the KSHV entry receptor gene EPHA2 affected susceptibility to KSHV infection and KS development in a South African HIV-infected patient cohort.

The Role of Type 2 Diabetes for the Development of Pathogen-Associated Cancers in the Face of the HIV/AIDS Epidemic.

The contribution of HIV to the development of pathogen-associated cancers has long been recognized, as has the contribution of type 2 diabetes for the development of several types of cancer. While HIV/AIDS-associated immunosuppression reduces immunosurveillance and indirectly contributes favorably to cancerogenesis, diabetes directly increases cancer development due to chronic low-grade inflammation, dysregulated glucose metabolism, hyperactivation of insulin-responsive pathways, and anti-apoptotic signaling. Pathogen-associated cancers contribute significantly to the cancer burden particularly in low- and middle-income countries. In those countries, the incidence of type 2 diabetes has increased alarmingly over the last decades, in part due to rapid changes in diet, lifestyle, and urbanization. It is likely that the HIV/AIDS epidemic and the steadily increasing rate of type 2 diabetes display synergistic effects on oncogenesis. Although this possible link has not been extensively investigated, it might become more important in the years to come not least due to the stimulating effects of antiretroviral therapy on the development of type 2 diabetes. This review provides an overview of the current understanding of pathogen- and diabetes- associated cancers with focus on geographical regions additionally burdened by the HIV/AIDS epidemic. As both HIV and carcinogenic infections as well as the onset of type 2 diabetes involve environmental factors that can be avoided to a certain extent, this review will support the hypothesis that certain malignancies are potentially preventable. Deploying effective infection control strategies together with educational policies on diet and lifestyle may in the long term reduce the burden of preventable cancers which is of particular relevance in low-resource settings.

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions.

Human papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells.IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

Interaction of human tumor viruses with host cell surface receptors and cell entry.

Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.