Rosemary Rochford’s research while affiliated with University of Colorado and other places

Introduction Epstein-Barr Virus (EBV) is an oncogenic herpesvirus linked to hematologic malignancies. EBV enters latency within host cells and can undergo lytic activation, resulting in release of infectious virions. Latent EBV mediates malignant transformation, while lytic EBV is a frequent complication in immune suppression settings like post-transplant lymphoproliferative disease (PTLD). Current clinical assays detect only the presence of EBV via qPCR, limiting their diagnostic use. DNA methylation on EBV genomes is essential for interconversion of lytic/latent states, making it a promising virology biomarker. Methods We have developed a high-throughput multiplex PCR profiling assay (iPLEX) to assess quantitative EBV DNA methylation at n=35 CpG dinucleotide sites in liquid biopsies (plasma/serum). We applied this assay to a cohort of n=246 plasma samples collected at the OSU Wexner Medical Center via the Leukemia Tissue Bank Shared Resource. Our cohort consisted of viremic patients with EBV-associated lymphoma or non-malignant EBV activation, including n=118 organ transplant recipients. We complemented our analysis with single molecule readout of EBV plasma methylomes (n=27) using Nanopore sequencing of native DNA methylation. Results Low EBV methylation distinguished lytic from latent EBV (sensitivity: 93.3%, specificity: 97.2%), outperforming clinical EBV qPCR. We revealed distinct EBV methylation in lymphoma subtypes, ranging from intermediate (Hodgkin lymphoma, polymorphic PTLD) to high (Burkitt-, NK/T-cell lymphoma, monomorphic PTLD). Assessment of EBV methylation in treatment follow-up (n=55) highlighted malleable EBV methylation was associated with improved survival under treatment (p=0.0005). We further identified unique EBV methylation dynamics under epigenetic therapies, including DNA methyltransferase (DNMT) inhibitor decitabine. DNA methylation in the EBV protein kinase gene BGLF4 was further associated with response to antiviral ganciclovir (GCV) use. Lastly, assessment of overall methylation patterns revealed four EBV methylation groups. Single molecule methylome analysis via Nanopore sequencing validated our data and identified distinct states of molecular epigenetic heterogeneity in EBV lymphoma and PTLDs. Methylation groups showed highly significant association with overall survival in multiple disease entities, including Hodgkin- and diffuse large B-cell lymphoma (DLBCL). Conclusion Our data reveal the epigenetic EBV landscape in lymphoma and non-malignant viremic conditions. EBV methylation can identify lymphoma through a minimally invasive plasma test for earlier and more reliable diagnosis of EBV-driven malignancy. EBV methylation could guide informed treatment decisions, especially with regard to epigenetic- and antiviral drug use, and aid in personalized care in the future. Citation Format Christoph Weigel, Haley L. Klimaszewski, Cara Noel, Ada C. Sher, Kurtis M. Host, Yue-Zhong Wu, Sarah Y. Schlotter, Eric Brooks, Charles T. Gregory, Esko Kautto, Elshafa H. Ahmed, Rosemary Rochford, James S. Blachly, Mark Lustberg, Timothy Voorhees, Christopher C. Oakes, Robert A. Baiocchi. DNA methylation profiling of Epstein-Barr virus (EBV) in lymphoproliferative disease reveals diagnostic and therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3242.

Burkitt lymphoma (BL) is characterized by elevated levels of the enzyme activation-induced cytidine deaminase (AID), an enzyme critical for MYC translocation that is the hallmark of BL. Both EBV and Plasmodium falciparum malaria are cofactors in the etiology of BL. However, how these 2 pathogens drive BL pathogenesis is not yet understood. In this study, we tested the hypothesis that P. falciparum and EBV synergize to induce dysregulated expression of AID. Using flow cytometry, intracellular AID expression was measured in PBMCs from a cohort of children from Western Kenya with uncomplicated malaria and community controls. Children with uncomplicated malaria had elevated levels of CD19+ AID+ B cells compared to controls. This high level of AID was sustained up to 8 weeks after parasite clearance. Using ImageStream flow cytometry, we found that 52% of AID was localized in the nucleus of CD19+ B cells in children with malaria. To test whether EBV and P. falciparum synergized to drive the expression of AID, we stimulated CD19+ B cells with EBV, CpG (to mimic P. falciparum DNA), or BAFF (induced during P. falciparum infection), or as a combination. Individually, EBV, BAFF and CpG induced AID expression. However, when combined, there was a significant increase of ∼30% in the frequency of CD19+AID+ cells above cells treated with EBV, BAFF, or CpG individually. Collectively, these data suggest that P. falciparum malaria and EBV coinfection result in sustained AID expression, potentially influencing the MYC translocation that is characteristic of BL.

Individuals with AIDS and Kaposi sarcoma (AIDS‐KS) with pulmonary involvement have high‐risk of poor outcomes but diagnosis of pulmonary KS in low‐resource settings is difficult. We aimed to discover plasma proteins that distinguish individuals with pulmonary KS from those without pulmonary involvement. SomaScan proteomics screen measured 7288 plasma proteins in 22 cases and 17 controls selected from 181 participants with HIV‐1 and cutaneous KS who underwent bronchoscopy. Cases had KS in the lower respiratory tract by bronchoscopy. Controls had no KS lesions detected by bronchoscopy. Results of the proteomics screen were confirmed by ELISA measurement of plasma alpha‐1‐antichymotrypsin (SERPINA3) in 18 cases and 13 controls and in an additional 162 individuals with AIDS‐KS who were not included in the case–control analysis. Proteomics identified 12 plasma proteins with differential levels in controls and cases. Plasma alpha‐1‐antichymotrypsin (SERPINA3) complex was consistently higher in cases compared to controls in the proteomics assay. Measurement of plasma alpha‐1‐antichymotrypsin by ELISA confirmed higher levels in cases (median 399.4, IQR 95.77–766.4 μg/ml) versus controls (median 39.98, IQR 31.2–170.2 μg/ml; p = .001). Plasma alpha‐1‐antichymotrypsin correlated with the estimated burden of pulmonary KS in the respiratory tract (r = 0.439; p = .0002) and 234 μg/ml had 51% sensitivity and 94% specificity for detection of pulmonary KS by bronchoscopy. Measurement of plasma alpha‐1‐antichymotrypsin has potential for identifying persons with pulmonary AIDS‐KS and estimating the burden of KS in the lower respiratory tract.

The late René Dubos, a microbiologist and an ecological philosopher, famously coined the phrase, “Think global, act local.” This phrase presents a framework for how we might envision a new direction in education on pathogens across the globe. Infectious diseases can be considered at a global scale, as demonstrated by the recent COVID-19 pandemic, and also at the community scale, as is the case for endemic liver fluke disease in Southeast Asia. While the 30,000 foot view can help us to understand some aspects of a given pathogen and disease, it is critical that we shift our framework, such that local factors affecting disease burden are considered and relevant education is provided to the affected communities. Traditional training in microbiology has focused on knowing the pathogen. To again quote Dubos, “the etiology of disease cannot be entirely explained by the etiology of infection.” If we accept this thesis, then future training in infectious diseases must also encompass knowledge of the environmental and social determinants of health. In this chapter, we will provide a brief historical framework regarding education and research in the field of Tropical Medicine. We will then offer perspectives from the field for contextualizing the current state of training. Finally, we will conclude with ideas on how to create more holistic educational pathways that build interdisciplinary teams able to tackle both infectious disease research and improved wellbeing of affected communities.

This qualitative sub-study investigated household practices affecting orally shed infections using Kaposi’s sarcoma-associated herpesvirus (KSHV) as a focus. Participants enrolled from 50 households in rural south-western Uganda were followed monthly up to three times. At enrolment, in-depth interviews were completed, and venous blood collected. KSHV seropositivity was defined as anti-KSHV antibody detection to any of 25 antigens by multiplex bead-based assay. Mouthwash samples from every visit were tested by qPCR and KSHV shedders defined as individuals with KSHV DNA detected. At least one KSHV seropositive person was in 48/49(98%) households. Among those, 79% had 1+ KSHV shedders including 45% with 1+ always shedders and 92% with 1+ intermittent shedders, not mutually exclusively. All respondents reported feeding infants with pre-masticated hard food/fruits and testing food/tea temperature. Temperature was tested by tasting, pouring tea on their hand, or touching the cup to their cheek. Some cooled food/tea using a utensil or blowing over it. Food sharing amongst children and adults and using the same dish was common practice. To treat colic pain, carers/mothers reported chewing herbs and spitting into the child’s mouth. Feeding and treatment practices did not vary by KSHV status. We identified potential KSHV transmission modes in rural Ugandan households.

Background We report the impact of HIV infection within a household on oral Kaposi’s sarcoma-associated herpesvirus (KSHV) shedding. Methods We enrolled 469 individuals from 90 households. Mouthwash rinse samples collected at three monthly visits were analyzed for KSHV DNA using quantitative polymerase chain reaction (qPCR). Generalized linear mixed effects logistic models were applied to analyze factors associated with KSHV ever shedding, and among shedders, always versus intermittent shedding. Linear mixed effects models were applied to models of KSHV viral loads. Intraclass correlation coefficients (ICCs) were calculated to assess the contribution of household-level factors to variations in shedding probabilities. Hotspot analyses of geospatial feature clusters were calculated using Getis-Ord Gi* statistic and visualized using inverse distance weighted interpolation. Results Analyses included 340 KSHV seropositive individuals, aged 3 + years, with qPCR results from 89 households. Forty households had 1 + persons living with HIV (PLWH), while 49 had none. Among participants, 149(44%) were KSHV ever shedders. Of 140 who shed KSHV at two or more visits, 34(24%) were always shedders. Increasing number of KSHV seropositive household members was significantly associated with ever shedding [Odds ratio(OR) (95% Confidence Interval(95%CI)):1.14(1.03,1.26);p = 0.013]. Among KSHV shedders, a statistically significant age-related trend was identified with 10–19 years being more likely to be always shedders (type III test p = 0.039) and to have higher viral loads (type III test p = 0.027). In addition, higher viral loads were significantly associated with increasing number of household members [coefficient(95%CI):0.06(0.01,0.12);p = 0.042], increasing number of KSHV seropositive members [coefficient(95%CI):0.08(0.01,0.15);p = 0.021], and living in households with 1 + PLWH [coefficient(95%CI):0.51(0.04,0.98);p = 0.033]. Always shedders exhibited higher viral loads than intermittent shedders [coefficient(95%CI):1.62(1.19,2.05);p < 0.001], and viral loads increased with the number of visits where KSHV DNA was detected in saliva (type III test p < 0.001). Household-level factors attributed for 19% of the variability in KSHV shedding (ICC:0.191;p = 0.010). Geospatial analysis indicated overlapping hotspots of households with more KSHV seropositive individuals and KSHV shedders, distinct from areas where PLWH were clustered. Discussion KSHV oral shedding is influenced by multiple factors at the individual, household, and regional levels. To mitigate ongoing KSHV transmission a comprehensive understanding of factors contributing to oral KSHV reactivation and transmission within households is needed.

Multiplex-based serological surveillance is a valuable but underutilized tool to understand gaps in population-level exposure, susceptibility, and immunity to infectious diseases. Assays for which blood samples can be tested for antibodies against several pathogens simultaneously, such as multiplex bead immunoassays, can more efficiently integrate public health surveillance in low- and middle-income countries. On March 7–8, 2023 a group of experts representing research institutions, multilateral organizations, private industry, and country partners met to discuss experiences, identify challenges and solutions, and create a community of practice for integrated, multi-pathogen serosurveillance using multiplex bead assay technologies. Participants were divided into six working groups: 1) supply chain; 2) laboratory assays; 3) seroepidemiology; 4) data analytics; 5) sustainable implementation; and 6) use case scenarios. These working groups discussed experiences, challenges, solutions, and research needs to facilitate integrated, multi-pathogen serosurveillance for public health. Several solutions were proposed to address challenges that cut across working groups.

Background We report the impact of HIV infection within a household on oral Kaposi's sarcoma-associated herpesvirus (KSHV) shedding. Methods We enrolled 469 individuals from 90 households. Mouthwash rinse samples collected at three monthly visits, were analyzed for KSHV DNA using quantitative polymerase chain reaction (qPCR). Generalized linear mixed effects logistic models were applied to analyze factors associated with KSHV ever shedding, and among shedders, always versus intermittent shedding. Linear mixed effects models were applied to models of KSHV viral loads. Intraclass correlation coefficients (ICCs) were calculated to assess the contribution of household-level factors to variations in shedding probabilities. Hotspot analyses of geospatial feature clusters were calculated using Getis-Ord Gi* statistic and visualized using inverse distance weighted interpolation. Results Analyses included 340 KSHV seropositive individuals, aged 3 + years, with qPCR results from 89 households. Forty households had 1 + persons living with HIV (PLWH), while 49 had none. Among participants, 149(44%) were KSHV ever shedders. Of 140 who shed KSHV at two or more visits, 34(24%) were always shedders. Increasing number of KSHV seropositive household members was significantly associated with ever shedding [Odds ratio(OR) (95% Confidence Interval(95%CI)):1.14(1.03,1.26);p = 0.013]. Among KSHV shedders, a statistically significant age-related trend was identified with 10–19 years being more likely to be always shedders (type III test p = 0.039) and to have higher viral loads (type III test p = 0.027). In addition, higher viral loads were significantly associated with increasing number of household members [coefficient(95%CI):0.06(0.01,0.12);p = 0.042], increasing number of KSHV seropositive members [coefficient(95%CI):0.08(0.01,0.15);p = 0.021], and living in households with 1 + PLWH [coefficient(95%CI):0.51(0.04,0.98);p = 0.033]. Always shedders exhibited higher viral loads than intermittent shedders [coefficient(95%CI):1.62(1.19,2.05);p < 0.001], and viral loads increased with the number of visits where KSHV DNA was detected in saliva (type III test p < 0.001). Household-level factors attributed for 19% of the variability in KSHV shedding (ICC:0.191;p = 0.010). Geospatial analysis indicated overlapping hotspots of households with more KSHV seropositive individuals and KSHV shedders, distinct from areas where PLWH were clustered. Discussion KSHV oral shedding is influenced by multiple factors at the individual, household, and regional levels. To mitigate ongoing KSHV transmission a comprehensive understanding of factors contributing to oral KSHV reactivation and transmission within households is needed.

Multiplex-based serological surveillance is a valuable but underutilized tool to understand gaps in population-level exposure, susceptibility, and immunity to infectious diseases. Assays for which blood samples can be tested for antibodies against several pathogens simultaneously, such as multiplex bead immunoassays, can more efficiently integrate public health surveillance in low- and middle-income countries. From March 7-8, 2023, a group of experts representing research institutions, multilateral organizations, private industry, and country partners met to discuss experiences, identify challenges and solutions, and create a community of practice for integrated, multi-pathogen serosurveillance using multiplex bead assay technologies. Participants were divided into six working groups: (1) supply chain; (2) laboratory assays; (3) seroepidemiology; (4) data analytics; (5) sustainable implementation; and (6) use case scenarios. These working groups discussed experiences, challenges, solutions, and research needs to facilitate integrated, multi-pathogen serosurveillance for public health. Several solutions were proposed to address challenges that cut across working groups.

Epstein-Barr virus (EBV) is a potent carcinogen linked to hematologic and solid malignancies, causing significant global morbidity and mortality. Therapy using allogeneic EBV-specific lymphocytes shows promise in certain populations, but the impact of EBV genome variation on these strategies remains unexplored. To address this, we sequenced 217 EBV genomes, including hematologic malignancies from Guatemala, Peru, Malawi, and Taiwan, and analyzed them alongside 1,307 publicly available EBV genomes from cancer, non-malignant diseases, and healthy individuals across Africa, Asia, Europe, North America, and South America. These included the first NK/T-cell lymphoma (NKTCL) EBV genomes reported outside East Asia. Our findings indicate that previously proposed EBV genome variants specific to certain cancer types are more closely tied to geographic origin than cancer histology. This included variants previously reported to be specific to NKTCL but were prevalent in EBV genomes from other cancer types and healthy individuals in East Asia. After controlling for geographic region, we did identify multiple NKTCL-specific variants associated with a 7.8- to 21.9- fold increased risk. We also observed frequent variations in EBV genomes affecting peptide sequences previously reported to bind common MHC alleles. Finally, we found several non-synonymous variants spanning the coding sequences of current vaccine targets BALF4, BKRF2, BLLF1, BXLF2, BZLF1, and BZLF2. These results highlight the need to consider geographic variation in EBV genomes when devising strategies for exploiting adaptive immune responses against EBV-related cancers, ensuring greater global effectiveness and equity in prevention and treatment.