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).

Clinical Significance of Elevated KSHV Viral Load in HIV-Related Kaposi's Sarcoma Patients in South Africa.

Kaposi's sarcoma (KS) is an AIDS-defining illness caused by Kaposi's sarcoma-associated herpesvirus (KSHV) predominantly in the context of HIV-related immune suppression. We aimed to explore the usefulness of KSHV DNA viral load (VL) measurement in predicting the severity, response to treatment and outcome of KS. We retrospectively assessed a cohort of KS patients (n = 94) receiving treatment at Groote Schuur Hospital, Cape Town, South Africa. Demographic and clinical data, KS staging and response to treatment were extracted from patient files, while long-term survival was ascertained from hospital records. KSHV serology and VL and hIL-6 were determined empirically from patients' blood. All patients were HIV-positive adults, the majority of whom were on HAART at the time of recruitment. KSHV VL was detectable in 65 patients' blood (median: 280.5/106 cells (IQR: 69.7-1727.3)) and was highest in patients with S1 HIV-related systemic disease (median 1066.9/106 cells, IQR: 70.5-11,269.6). KSHV VL was associated with the S1 stage in a binomial regression controlling for confounders (adjusted odds ratio 5.55, 95% CI: 1.28-24.14, p = 0.022). A subset of six patients identified to have extremely high KSHV VLs was predominantly T1 stage with pulmonary KS, and most had died at follow-up. In our cohort, elevated KSHV VL is associated with systemic HIV-related illness in KS disease. Extremely high KSHV VLs warrant further investigation for patients potentially requiring intensive treatment and investigation for progression or diagnosis of concurrent KSHV lytic syndromes.

Protective efficacy of a plant-produced beta variant rSARS-CoV-2 VLP vaccine in golden Syrian hamsters.

In the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.

Metabolic Profiling, Antiviral Activity and the Microbiome of Some Mauritian Soft Corals.

Soft corals, recognized as sessile marine invertebrates, rely mainly on chemical, rather than physical defense, by secreting intricate secondary metabolites with plausible pharmaceutical implication. Their ecological niche encompasses a diverse community of symbiotic microorganisms which potentially contribute to the biosynthesis of these bioactive metabolites. The emergence of new viruses and heightened viral resistance underscores the urgency to explore novel pharmacological reservoirs. Thus, marine organisms, notably soft corals and their symbionts, have drawn substantial attention. In this study, the chemical composition of four Mauritian soft corals: Sinularia polydactya, Cespitularia simplex, Lobophytum patulum, and Lobophytum crassum was investigated using LC-MS techniques. Concurrently, Illumina 16S metagenomic sequencing was used to identify the associated bacterial communities in the named soft corals. The presence of unique biologically important compounds and vast microbial communities found therein was further followed up to assess their antiviral effects against SARS-CoV-2 and HPV pseudovirus infection. Strikingly, among the studied soft corals, L. patulum displayed an expansive repertoire of unique metabolites alongside a heightened bacterial consort. Moreover, L. patulum extracts exerted some promising antiviral activity against SARS-CoV-2 and HPV pseudovirus infection, and our findings suggest that L. patulum may have the potential to serve as a therapeutic agent in the prevention of infectious diseases, thereby warranting further investigation.

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.

A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters.

Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.

TBX3 Promotes Cervical Cancer Proliferation and Migration via HPV E6 and E7 Signaling.

Cervical cancer is a leading cause of cancer-related deaths in women globally and 99% of cases are caused by persistent infection with high-risk strains of the human papillomavirus (HPV). The HPV oncoproteins E6 and E7 establish the cancer phenotype by cooperating with host proteins and identifying them may have important therapeutic benefits. T-box transcription factor 3 (TBX3) is a critical developmental regulator, and when it is overexpressed postnatally, it contributes to several cancers, but little is known about its expression and role in cervical cancer. The current study shows that TBX3 is upregulated in cervical cancer cell lines as well as precancerous and cervical cancer patient tissue and is associated with larger and more invasive tumors. Knockdown and overexpression cell culture models show that TBX3 promotes HPV-positive cell proliferation, migration, and spheroid growth; however, TBX3 inhibits these processes in HPV-negative cells. Importantly, we show that the tumor promoting activity of TBX3 in cervical cancer is dependent on E6/E7. IMPLICATIONS: In summary, our study highlights the importance of TBX3 as a cooperating partner of E6/E7 in HPV-positive cervical cancer and identifies TBX3 as a potential therapeutic target to treat this neoplasm.

Effects of tuberculosis and/or HIV-1 infection on COVID-19 presentation and immune response in Africa.

Few studies from Africa have described the clinical impact of co-infections on SARS-CoV-2 infection. Here, we investigate the presentation and outcome of SARS-CoV-2 infection in an African setting of high HIV-1 and tuberculosis prevalence by an observational case cohort of SARS-CoV-2 patients. A comparator group of non SARS-CoV-2 participants is included. The study includes 104 adults with SARS-CoV-2 infection of whom 29.8% are HIV-1 co-infected. Two or more co-morbidities are present in 57.7% of participants, including HIV-1 (30%) and active tuberculosis (14%). Amongst patients dually infected by tuberculosis and SARS-CoV-2, clinical features can be typical of either SARS-CoV-2 or tuberculosis: lymphopenia is exacerbated, and some markers of inflammation (D-dimer and ferritin) are further elevated (p < 0.05). Amongst HIV-1 co-infected participants those with low CD4 percentage strata exhibit reduced total, but not neutralising, anti-SARS-CoV-2 antibodies. SARS-CoV-2 specific CD8 T cell responses are present in 35.8% participants overall but undetectable in combined HIV-1 and tuberculosis. Death occurred in 30/104 (29%) of all COVID-19 patients and in 6/15 (40%) of patients with coincident SARS-CoV-2 and tuberculosis. This shows that in a high incidence setting, tuberculosis is a common co-morbidity in patients admitted to hospital with COVID-19. The immune response to SARS-CoV-2 is adversely affected by co-existent HIV-1 and tuberculosis.

Activity-Based Proteomic Identification of the S-Thiolation Targets of Ajoene in MDA-MB-231 Breast Cancer Cells.

Garlic is a medicinal plant and spice that has been used for millennia for its health-promoting effects. These medicinal properties are associated with low molecular weight organosulfur compounds, produced following the crushing of garlic cloves. One of these compounds, ajoene, is proposed to act by S-thioallylating cysteine residues on target proteins whose identification in cancer cells holds great promise for understanding mechanistic aspects of ajoene's cancer cell cytotoxicity. To this end, an ajoene analogue (called biotin-ajoene, BA), containing a biotin affinity tag, was designed as an activity-based probe specific for the protein targets of ajoene in MDA-MB-231 breast cancer cells. BA was synthesized via a convergent "click" strategy and found to retain its cytotoxicity against MDA-MB-231 cells compared to ajoene. Widespread biotinylation of proteins was found to occur via disulfide bond formation in a dose-dependent manner, and the biotin-ajoene probe was found to share the same protein targets as its parent compound, ajoene. The biotinylated proteins were affinity-purified from the treated MDA-MB-231 cell lysate using streptavidin-coated magnetic beads followed by an on-bead reduction, alkylation, and digestion to liberate the peptide fragments, which were analyzed by liquid chromatography tandem mass chromatography. A total of 600 protein targets were identified, among which 91% overlapped with proteins with known protein cysteine modification (PCM) sites. The specific sites were enriched for those susceptible to S-glutathionylation (-SSG) (16%), S-sulfhydration (-SSH) (20%), S-sulfenylation (-SOH) (22%), and S-nitrosylation (-SNO) (31%). As target validation, both ajoene and a dansylated ajoene (DP) were found to S-thiolate the pure recombinant forms of glutathione S-transferase pi 1 (GSTP1) and protein disulfide isomerase (PDI), and the ajoene analogue DP was found to be a more potent inhibitor than 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Pathway analysis elucidated that ajoene targets functional and signaling pathways that are implicated in cancer cell survival, specifically cellular processes, metabolism, and genetic information processing pathways. The results of this study provide mechanistic insights into the character of the anti-cancer activity of the natural dietary compound ajoene.

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.

Cervical cancer therapies: Current challenges and future perspectives.

Cervical cancer is the fourth most common female cancer worldwide and results in over 300 000 deaths globally. The causative agent of cervical cancer is persistent infection with high-risk subtypes of the human papillomavirus and the E5, E6 and E7 viral oncoproteins cooperate with host factors to induce and maintain the malignant phenotype. Cervical cancer is a largely preventable disease and early-stage detection is associated with significantly improved survival rates. Indeed, in high-income countries with established vaccination and screening programs it is a rare disease. However, the disease is a killer for women in low- and middle-income countries who, due to limited resources, often present with advanced and untreatable disease. Treatment options include surgical interventions, chemotherapy and/or radiotherapy either alone or in combination. This review describes the initiation and progression of cervical cancer and discusses in depth the advantages and challenges faced by current cervical cancer therapies, followed by a discussion of promising and efficacious new therapies to treat cervical cancer including immunotherapies, targeted therapies, combination therapies, and genetic treatment approaches.

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2-specific T-cells.

BACKGROUND: Rapid tests to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. METHODS: Using a rapid whole blood assay requiring a minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T-cell responses in 31 healthcare workers using flow cytometry. RESULTS: 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T-cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR-negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce interferon (IFN)-γ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, coexpressing IFN-γ and tumour necrosis factor-α and also Granzyme B. CONCLUSIONS: This proof-of-concept study presents a scalable alternative to peripheral blood mononuclear cell-based assays to enumerate and phenotype SARS-CoV-2-responding T-cells, thus representing a practical tool to monitor adaptive immunity due to natural infection or vaccine trials.

Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection.

Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus' interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin's stabilisation and formulation before its development as an alternative prophylactic means.

Advances in Targeting HPV Infection as Potential Alternative Prophylactic Means.

Infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer and other anogenital cancers. The majority of cervical cancer cases occur in low- and middle- income countries (LMIC). Concurrent infection with Human Immunodeficiency Virus (HIV) further increases the risk of HPV infection and exacerbates disease onset and progression. Highly effective prophylactic vaccines do exist to combat HPV infection with the most common oncogenic types, but the accessibility to these in LMIC is severely limited due to cost, difficulties in accessing the target population, cultural issues, and maintenance of a cold chain. Alternative preventive measures against HPV infection that are more accessible and affordable are therefore also needed to control cervical cancer risk. There are several efforts in identifying such alternative prophylactics which target key molecules involved in early HPV infection events. This review summarizes the current knowledge of the initial steps in HPV infection, from host cell-surface engagement to cellular trafficking of the viral genome before arrival in the nucleus. The key molecules that can be potentially targeted are highlighted, and a discussion on their applicability as alternative preventive means against HPV infection, with a focus on LMIC, is presented.

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.

Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate.

Given the complex maturation requirements of viral glycoproteins and the challenge they often pose for expression in plants, the identification of host constraints precluding their efficient production is a priority for the molecular farming of vaccines. Building on previous work to improve viral glycoprotein production in plants, we investigated the production of a soluble SARS-CoV-2 spike comprising the ectopic portion of the glycoprotein. This was successfully transiently expressed in N. benthamiana by co-expressing the human lectin-binding chaperone calreticulin, which substantially increased the accumulation of the glycoprotein. The spike was mostly unprocessed unless the protease furin was co-expressed which resulted in highly efficient processing of the glycoprotein. Co-expression of several broad-spectrum protease inhibitors did not improve accumulation of the protein any further. The protein was successfully purified by affinity chromatography and gel filtration, although the purified product was heterogenous and the yields were low. Immunogenicity of the antigen was tested in BALB/c mice, and cellular and antibody responses were elicited after low dose inoculation with the adjuvanted protein. This work constitutes an important proof-of-concept for host plant engineering in the context of rapid vaccine development for SARS-CoV-2 and other emerging viruses.

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.