The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacted healthcare, the workforce, and worldwide socioeconomics. Multi-dose mono- or bivalent mRNA vaccine regimens have shown high efficacy in protection against SARS-CoV-2 and its emerging variants with varying degrees of efficacy. Amino acid changes, primarily in the receptor-binding domain (RBD), result in selection for viral infectivity, disease severity, and immune evasion. Therefore, many studies have centered around neutralizing antibodies that target the RBD and their generation achieved through infection or vaccination. Here, we conducted a unique longitudinal study, analyzing the effects of a three-dose mRNA vaccine regimen exclusively using the monovalent BNT162b2 (Pfizer/BioNTech) vaccine, systematically administered to nine previously uninfected (naïve) individuals. We compare changes in humoral antibody responses across the entire SARS-CoV-2 spike glycoprotein (S) using a high-throughput phage display technique (VirScan). Our data demonstrate that two doses of vaccination alone can achieve the broadest and highest magnitudes of anti-S response. Moreover, we present evidence of novel highly boosted non-RBD epitopes that strongly correlate with neutralization and recapitulate independent findings. These vaccine-boosted epitopes could facilitate multi-valent vaccine development and drug discovery.

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: Although antiretroviral therapy (ART) can suppress plasma viral loads in people living with human immunodeficiency virus (HIV), it cannot eliminate HIV-1 proviruses persisting in various tissue reservoirs, and HIV-1 will rebound after ART cessation. We have identified the appendix as a novel HIV tissue reservoir, but nothing is known about the provirus and the cellular tropism in this tissue. Methods: We used single-genome analysis of HIV-1 env DNA and compared the viral sequences from appendix tissues and lymph nodes of the same people living with HIV-1. Sequential multiplex immunofluorescence labeling and RNAscope in situ hybridization on formalin-fixed, paraffin-embedded appendix sections were performed to identify cell types harboring HIV-1 proviral DNA in the appendix, by visualizing viral and cellular biomarkers colocalization. Results: The viral populations from the appendix were less heterogeneous than those from the lymph nodes in aviremic individuals, suggesting there is reduced ART selective pressure or the presence of appendix tropic HIV-1. Furthermore, we demonstrated that follicular dendritic cell (FDCs), rather than CD4+ T cells within the appendix, are the major cell type harboring subtype C HIV-1. Notably, although some subtype C HIV-1 signals could be remaining virions captured by FDCs even in aviremic individuals, HIV-1 proviral DNA was detected in some FDCs in the appendix tissues of aviremic individuals, indicating the presence of infected FDCs. Conclusions: These results highlight the importance of analyzing all potential tissue and cellular tropism across different HIV-1 subtypes, to inform tailored therapeutic strategies for the diverse reservoirs that may differ across subtypes.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival rate of 13.3%. First-line treatment relies on two chemotherapy regimens, FOLFIRINOX (FOLFNX) or gemcitabine plus nab-paclitaxel (GEMPAC). However, direct clinical comparisons between these regimens have yielded inconsistent results across survival and toxicity endpoints, and the molecular basis of heterogeneous treatment responses remains poorly defined. To investigate regimen-specific tumor-cell-intrinsic mechanisms, we performed quantitative proteomic profiling of a primary PDAC-derived MIA PaCa-2 cell line following treatment with FOLFNX or GEMPAC. Differentially expressed proteins were analyzed using Gene Ontology, KEGG, and Ingenuity Pathway Analysis to define pathway-level alterations, and findings were contextualized using TCGA transcriptomic data. Proteomic analyses revealed that FOLFNX and GEMPAC engage in distinct cytotoxic programs. FOLFNX predominantly suppressed ribosome biogenesis and mitochondrial translation, consistent with sustained metabolic and biosynthetic stress, whereas GEMPAC preferentially disrupted mitotic cytokinesis and phosphatidylinositol phosphate biosynthesis, consistent with mitotic failure. Integration with TCGA data showed that FOLFNX-altered proteins aligned with favorable prognostic expression signatures, whereas GEMPAC-associated proteins were enriched among adverse profiles, reflecting engagement of distinct tumor-intrinsic programs. Together, these findings provide mechanistic insight into differential chemotherapy responses and establish a foundation for proteomics-based biomarkers to guide personalized chemotherapy selection in PDAC.

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.

Prostate cancer cells often develop therapy resistance by switching their identity through lineage plasticity and epigenetic reprogramming. A key player in this process is the transcription factor SPDEF, which we previously found is lost during cancer progression, often due to DNA hypermethylation. In this study, we explored how histone modifications—specifically H3K27me3 mediated by PRC2/EZH2—work alongside DNA methylation to silence SPDEF and drive metastasis. By analyzing public ChIP-seq datasets and various prostate cancer cell lines, we mapped the chromatin landscape of the SPDEF gene. We found that cells with high SPDEF levels (like LNCaP) are marked by active H3K4me3 signals, while cells with low SPDEF levels (like PC3 and DU145) show heavy H3K27me3 repression. Clinical data further supported this, showing that EZH2 and KDM5B levels are high in advanced cases (high Gleason scores) and inversely related to SPDEF expression. Our functional experiments revealed that while inhibiting KDM5B didn't restore SPDEF, targeting EZH2 with the inhibitor GSK126 or siRNA effectively reduced H3K27me3 at the SPDEF locus. This treatment successfully turned SPDEF and E-cadherin back on. Most importantly, combining EZH2 and DNMT1 (DNA methylation) inhibitors significantly blocked the ability of cancer cells to migrate and invade. Ultimately, these findings suggest that the EZH2/SPDEF axis is a promising therapeutic target for stopping the spread of advanced prostate cancer