Owen Ngalamika

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus causally linked to several malignancies, including lymphomas and solid tumors. Despite its high global prevalence, a comprehensive profile of humoral responses to EBV is yet to be described. Moreover, how HIV-1 co-infection influences this response can reveal new insights into how cancer risk increases in people with HIV-1 (PWH). Here, we employed phage immunoprecipitation and sequencing (PhIP-Seq) using the VirScan phage-display library to map antibody responses against the EBV proteome in 115 adults from sub-Saharan Africa, with and without HIV-1 co-infection (43 HIV-positive and 72 HIV-negative) at peptide-level resolution. We identified EBNA1, EBNA2, EBNA3B, and the viral capsid antigen protein BFRF3 as the most immunodominant EBV proteins. Differential antibody reactivities between EBV Type 1 and Type 2 were observed against EBNA2, with increased antibody recognition near the C-terminal transactivation domain (C-TAD) observed for Type 1. A specific region of EBNA1 containing the nuclear localization sequence (NLS) was the most frequently targeted epitope of the EBV proteome. Our comprehensive and high-resolution analysis identified several immunodominant EBV proteins that are targeted by most individuals regardless of HIV-1 status. HIV-1 co-infected individuals (PWH) had increased antibody recognition against most EBV proteins compared to HIV-uninfected individuals, but the magnitudes of those responses varied across the proteome. Overall, our findings suggest that HIV-1 co-infection alters both the breadth and magnitude of anti-EBV responses, potentially reflecting increased viral reactivation and HIV-driven mechanisms of immunosuppression that may impact the magnitude of antibody responses in EBV. IMPORTANCE: This study employs the first comprehensive high-resolution antibody profiling against Epstein-Barr virus (EBV) among healthy adults and people with HIV-1 (PWH). Our results indicated that HIV-1 has a considerable influence on the anti-EBV repertoire that may provide further evidence of how HIV-1 significantly increases the risk of EBV-associated malignancies such as lymphoma. We showed evidence that HIV-1 co-infection leads to higher exposure to viral antigens, likely due to viral reactivation, reflected by a higher percentage of patients with antibodies reactive to the majority of EBV proteins when compared to people without HIV-1. Since PWH are known to be more susceptible to EBV-associated cancers, whether these changes in humoral immune response can be used as prognostic biomarkers for cancer risks should be further investigated with longitudinal studies.

Angiotensin‐converting enzyme inhibitors (ACEIs) are widely prescribed but are associated with adverse drug reactions (ADR), including angioedema, which is typically reported in 0.1%–0.7%. Data from sub‐Saharan Africa are limited. This study aimed to estimate the incidence and risk factors of enalapril‐associated ADRs in a Zambian cohort, with particular focus on angioedema. We conducted an ambispective cohort study of 400 adults initiating enalapril at two tertiary hospitals in Lusaka, Zambia. Enalapril was the only ACEI available to the participants, and the primary outcome was the occurrence of enalapril‐associated ADRs requiring treatment discontinuation or modification. Kaplan–Meier methods were used to estimate event‐free survival, and Cox proportional hazards regression was used to identify factors associated with ADR occurrence. Angioedema occurred in 19/400 participants (4.8%). Overall, 22% (88/400) of participants experienced at least one ADR impacting treatment. The most frequent ADRs were cough (10.3%) and dizziness (5.0%). In multivariable analysis, male sex was independently associated with a lower risk of ADR (adjusted hazard ratio [aHR] 0.59, 95% CI 0.37–0.93). No significant associations were identified for the high rate of angioedema. The incidence of enalapril‐associated angioedema in this cohort was substantially higher than that reported in global as well as African American populations. These findings have clinical relevance for prescribing and monitoring ACEIs in sub‐Saharan African populations. Further studies are needed to confirm this signal and explore underlying mechanisms.

Introduction: Natural killer/T-cell (NK/T) lymphomas are very rare with poor prognosis. These cancers are more prevalent in Asian and South American populations, are often Epstein-Barr virus positive, and usually involve the nasal cavity or paranasal sinuses. NK/T lymphomas originating from the orbit is an extremely rare occurrence. Case Presentation: Here we report the clinical, radiological, and histopathologic features of an 18-year-old male who presented with a painful right orbital mass associated with ipsilateral headache and complete vision loss in the affected eye, that was histologically diagnosed as an NK/T lymphoma. Despite a favorable response to gemcitabine, dexamethasone, and cisplatin treatment, the patient died as a result of treatment-induced complications. We present the clinical and radiologic parameters, the histological and molecular characterization of the tumor, including treatment and outcome. Conclusion: This case underscores the importance of promptly characterizing atypical orbital masses, maintaining a high index of suspicion for NK/T lymphoma in the differential diagnosis even for young patients, the need for safer targeted treatments especially in resource-limited settings, and the need for close monitoring of side effects during treatment.

Background: Penile squamous cell carcinoma (PSCC) is the most common malignancy of the penis. Considering the increase in incidence of PSCC in many countries, there is a need for better and effective therapies for these patients. The tumor microenvironment may offer insights into a better understanding of the tumor, which may inform on predictive and prognostic targets. In this study, we investigated immune cell infiltration into the stroma of PSCC, and how it may be affected by multiple factors including smoking, HIV infection, and/or HPV infection.Methods: We carried out a prospective analytical cross-sectional study at the University Teaching Hospital in Lusaka, Zambia. Consenting patients with confirmed PSCC, attending the Urology Clinic and scheduled for partial or total penectomy were enrolled into the study. HIV testing by serology, HPV detection and genotyping on fresh tumors by real time PCR, hematoxylin and eosin (H&E) staining and immunohistochemistry staining for CD3⁺ and CD8⁺ cells on formalin-fixed paraffin-embedded tissue, and flow cytometry for immunophenotyping circulating immune cells were done.Results: We enrolled 33 participants into the study. The participants had a mean age of 56 years, the majority (84.8%) were HIV positive, high-risk HPV was detected in 63.6% of the tumors, and 57.6% were positive for both HIV and high-risk HPV. HPV-positive PSCC tumors had a significantly lower proportion of infiltrating lymphocytes in the stroma on H&E staining than HPV-negative tumors [18% vs 53%; p=0.025]. Smokers had a significantly lower number of infiltrating CD8⁺ cells in the stroma than non-smokers [68 vs 99; p=0.035]. No difference in the density of stromal lymphocytes between HPV/HIV co-infected and non-co-infected individuals was observed. There was a statistically significant positive correlation in number of CD3⁺ (⍴=0.38; p=0.027) and CD8⁺ (⍴=0.40; p=0.02) cells in the stroma and intra-tumor. Factors including histological stage, tumor grade, HPV status, and HIV status seem to influence the correlation in number of stroma and intra-tumoral immune cells.Conclusion: Smoking and presence of hrHPV is associated with a lower density of infiltrating lymphocytes in the stroma of PSCC. There is a positive correlation in the number of CD3⁺ and CD8⁺ cells between the stroma and intra-tumoral compartment of PSCC.

Kaposi sarcoma (KS) is an AIDS-defining angio-proliferative malignancy, with the Kaposi sarcoma-associated herpes virus (KSHV) as its etiologic agent. Upon treatment with chemotherapy, a proportion of HIV-associated KS patients experience disease recurrence within a few months of completing treatment. We aimed at determining whether KSHV-specific adaptive immune responses were associated with KS recurrence upon complete remission. We conducted a prospective cohort study. The primary outcome was the recurrence of HIV-associated KS. An immunofluorescence assay was used to determine anti-KSHV antibodies, an enzyme-linked immunospot was conducted for T cell responses, PCR was carried out to determine KSHV status, and flow cytometry was used for CD4 counting and immunophenotyping. KSHV detection in PBMCs was high and not associated with KS recurrence-free survival (p = 0.29). Anti-KSHV antibody titers were high and not associated with recurrence-free survival (p = 0.63). KSHV-specific T cell responses dropped from baseline levels among individuals with recurrence, but the drop was not statistically significant. Individuals experiencing KS recurrence had a significantly higher proportion of T cell subsets expressing PD1, while those with sustained remission had a significant increase in CD4 T cell counts from baseline levels during the follow-up period (p = 0.02). Anti-KSHV antibodies are not a good correlate of protection from KS recurrence. T cells in individuals experiencing KS recurrence hadhigh PD1 expression, while an increase in CD4 counts was associated with sustained KS remission.

Penile squamous cell carcinoma (PSCC) occurs more frequently in some developing countries compared to developed countries. Infection with HIV and/or high-risk human papillomavirus (hrHPV) are risk factors for penile cancer development. The tumor microenvironment of PSCC may predict prognosis and may inform on the best targets for immunotherapy. We evaluated the immune microenvironment of penile tumors histologically, and determined whether and/or how HIV and/or hrHPV infections affect this tumor microenvironment. We conducted a prospective analytical cross-sectional study in which penile cancer tumors from 35 patients presenting at the University Teaching Hospital in Lusaka, Zambia were histologically staged and assessed for presence of tumor infiltrating immune cells and expression of immune checkpoints. Immunohistochemistry was used to evaluate immune checkpoints and infiltrating immune cells, while multiplex real-time polymerase chain reaction was used for hrHPV genotyping. The median age of all participants was 55 years. About 24% had advanced histological stage, 83% were HIV⁺, and 63% had hrHPV detected in their tumors using multiplex real-time polymerase chain reaction. PDL1 expression was significantly higher in HIV^- participants than HIV⁺ participants (p = 0.02). Tumors with multiple hrHPV infections had a significantly higher number of cells expressing TIM3 than those with one hrHPV (p = 0.04). High grade tumors had a significantly higher infiltrate of FoxP3⁺ cells (p = 0.02), CD68⁺ cells (p = 0.01), CD163⁺ cells (p = 0.01), LAG3⁺ cells (p = 0.01), PD1⁺ cells (p = 0.01) and TIM3⁺ cells (p = 0.03) when compared with low grade tumours. There was significant moderate to strong positive correlation of cells expressing PD1 and LAG3 (⍴ = 0.69; p = 0.0001), PD1 and TIM3 (⍴ = 0.49; p = 0.017) and TIM3 and LAG3 PDL1 (⍴ = 0.61; p = 0.001). In conclusion, the tumor microenvironment of penile squamous cell carcinoma seems to be affected by both HIV and HPV infections. TIM3 appears to be a potential therapeutic target in PSCC patients with hrHPV infections.

Seasonal coronaviruses (HCoVs) are known to contribute to cross-reactive antibody (Ab) responses against SARS-CoV-2. While these responses are predictable due to the high homology between SARS-CoV-2 and other CoVs, the impact of these responses on susceptibility to SARS-CoV-2 infection in cancer patients is unclear. To investigate the influence of prior HCoV infection on anti-SARS-CoV-2 Ab responses among COVID-19 asymptomatic individuals with cancer and controls without cancers, we utilized the VirScan technology in which phage immunoprecipitation and sequencing (PhIP-seq) of longitudinal plasma samples was performed to investigate high-resolution (i.e., epitope level) humoral CoV responses. Despite testing positive for anti-SARS-CoV-2 Ab in the plasma, a majority of the participants were asymptomatic for COVID-19 with no prior history of COVID-19 diagnosis. Although the magnitudes of the anti-SARS-CoV-2 Ab responses were lower in individuals with Kaposi sarcoma (KS) compared to non-KS cancer individuals and those without cancer, the HCoV Ab repertoire was similar between individuals with and without cancer independent of age, sex, HIV status, and chemotherapy. The magnitudes of the anti-spike HCoV responses showed a strong positive association with those of the anti-SARS-CoV-2 spike in cancer patients, and only a weak association in non-cancer patients, suggesting that prior infection with HCoVs might play a role in limiting SARS-CoV-2 infection and COVID-19 disease severity.

Background: A proportion of COVID19 survivors may present with long‐COVID, which is persistent symptoms lasting four or more weeks post SARS‐CoV‐2 infection. These symptoms may be mild to severe, and may affect different organ‐systems of the body.Aims: The main objective of this study was to determine the demographic, clinical and immunological factors associated with long COVID.Materials & Methods: We conducted a nested case control study, with a total of 94 study participants initially included, and 64 participants matched for age and sex for biomarker analyses.Results: 32/94 (34.1%) of all the participants had long COVID. Respiratory symptoms were the most common (59.5%) followed by the musculoskeletal symptoms (28.1%). HIV was an independent predictor of long COVID (adjusted odds ratio = 2.7; p = .037). In all the 64 matched cases and controls, IFN‐β was significantly higher among controls than cases. After stratifying by HIV, IL6 was significantly higher among cases than controls in the HIV‐ group (2.06 vs. 0.81 pg/mL; p = .02). On the other hand, IFN‐β was significantly higher among controls than cases in the HIV+ group (251 vs. 0 pg/mL; p = .01).Conclusion: HIV infection is a risk factor for long COVID, and inflammatory markers associated with long COVID may be slightly different for HIV− and HIV+ individuals.