Sydney Merritt

The ongoing Bundibugyo virus disease (BVD) outbreak in the Democratic Republic of the Congo (DRC) is the second-largest Ebola disease outbreak in recorded history, and only the second recorded BVD outbreak in the country. While the DRC has recorded 18 filovirus disease outbreaks, the historical frequency and geographic distribution of human filovirus exposures remain poorly understood. Using a multiplexed multi-filovirus immunoassay, we assessed antibody reactivity against antigens of filoviruses linked to human disease, Bundibugyo virus (BDBV), Ebola virus (EBOV), Marburg virus (MARV), and Sudan virus (SUDV), among eleven cohorts from seven provinces between 2015–2023 (n=5,852). Participants with median fluorescence intensity values exceeding established antigen-specific cutoffs were classified as seroreactive. Seroreactivity against glycoprotein (GP) targets of all four viruses were detected in every cohort, demonstrating evidence of filovirus or an antigenically related exposure well beyond known outbreak regions. Seroreactivity estimates varied by cohort site, with 60.8% of participants (n=175/288) in Mongala Province in April 2016 considered seroreactive against BDBV GP. In this cohort, 4.2%, 29.9%, and 34.7% of participants were seroreactive against EBOV, MARV and SUDV GP, respectively yet only 1.7% were seroreactive against all four GP antigens. In adjusted analyses, site was the primary predictor of seroreactivity, with odds for BDBV seroreactivity 9.1-fold (95%CI 7.0-11.9) and 9.5-fold (95%CI 6.9-13.2) higher in Mongala and Tshuapa provinces than in Kwilu. Similarly, the adjusted odds of MARV and SUDV GP seroreactivity were 4.5-fold and 7.1-fold higher in Mongala, and 3.7-fold and 6.1-fold higher in Tshuapa, than Kwilu. Among 280 individuals sampled longitudinally, BDBV GP detected seroconversion was comparable by site, with 12.8% in Kwilu and 10.4% in Mongala having detected seroreactivity three and five years after initial contact, respectively. Overall, these results indicate widespread, geographically heterogeneous filovirus seroreactivity across sampled cohorts. These findings may reflect zoonotic spillover events, leading to undetected human-to-human filovirus circulation, including paucisymptomatic or asymptomatic infections, or exposure to as-yet unidentified antigenically cross-reactive viruses. These data highlight gaps in understanding the historical and geographic distribution of filovirus exposure in the DRC and support further prospective serological, epidemiological, and molecular investigations, especially given the current outbreak.

Background: Filoviruses, specifically Ebola virus (EBOV), Bundibugyo virus (BDBV), and Marburg virus (MARV), have been the etiologic agents of multiple outbreaks of severe disease in the Democratic Republic of the Congo (DRC). Two EBOV-specific vaccine regimens have been approved for use in outbreak scenarios, including the Ad26.ZEBOV/MVA-BN-Filo (Zabdeno/Mvabea) two-dose vaccine, where the MVA-BN-Filo booster dose contains EBOV, MARV, and Sudan (SUDV) virus glycoproteins (GPs) and the nucleoprotein from Tai Forest virus. While long-term immunogenicity of up to two years has been found in clinical trial settings, limited real-world data exists. Methods: Using a multiplexed immunoassay, samples ( n =698 at baseline) collected in Mbandaka, Equateur province, DRC, between September 2023 and October 2024 were assessed for seroreactivity to six filovirus antigens (1 BDBV, 2 EBOV, 2 MARV, and 1 SUDV) at three distinct periods: baseline/pre-vaccination, 6-8 weeks post-first dose (Zabdeno), at booster administration (Mvabea), and one-year post-boost dose. Results: Substantial filovirus seroreactivity was found at baseline, with 48.7% ( n =340/698) of individuals being seroreactive to at least one virus antigen. High baseline seroreactivity was found against BDBV GP (31.1%, n =217/698), with lower baseline seroreactivity to EBOV GP (15.5%, n =106/698), comparatively. Overall EBOV GP seroreactivity increased to 66.8% ( n =376/563) at one year-post vaccine boost and 57.6% ( n =250/434) of participants were seroreactive. Despite the booster dose inclusion of SUDV GP and MARV GP, there were no substantial increases in reactivity against either antigen at subsequent timepoints. Overall filovirus seroreactivity increased at all timepoints for the entire cohort, although filovirus reactivity decreased over the study period for those who were seroreactive at baseline. Findings: These data highlight detection of significantly elevated seoreactivity to EBOV GP following first-dose administration of Ad26.ZEBOV, which remained relatively stable one-year post-booster dose administration. Additionally, this study identifies high levels of baseline seroreactivity to BDBV GP despite no previous documented outbreaks in this region of DRC. These data suggest that vaccination with the Ad26.ZEBOV-MVA-BN-Filo vaccine induces stable seroreactivity against EBOV GP, but not to other filovirus antigens.

We report general acceptance (79.0%) of Ebola vaccination in the Democratic Republic of the Congo (DRC) between 2023-2024 (n=5,062). Acceptance varied significantly by province with acceptance ranging from 47.6% in Kongo Central to 92.1% in Tshuapa province and was highest among healthcare workers. While Ebola vaccine acceptance and disease knowledge are relatively strong nationally in the DRC, regional differences do exist and may impact future vaccine rollout.

During the 2022 global mpox outbreak, gay, bisexual and other men who have sex with men (gbMSM) were disproportionately affected. However, the true number of cases is likely higher than reported due in part to the stigma associated with mpox. This stigma may have played an even more influential role on health-seeking behaviour in politically conservative regions, such as the Canadian Prairies. The shifting epidemiology may have also led to misdiagnoses during initial months of the outbreaks. Seroprevalence studies can help assess these gaps in surveillance and population-level immunity. To address this, we conducted a cross-sectional seroprevalence study among 275 individuals who identified as potentially eligible to receive the mpox vaccine in the Canadian provinces of Manitoba and Alberta from June 2024 to December 2025 based on provincial vaccination criteria. This eligibility included, but was not exclusively limited to, gbMSM, gender diverse individuals, and sex workers. Dried blood spots were analyzed using the Meso Scale Discovery Orthopoxvirus assay targeting five orthologous mpox virus and vaccinia virus antigens. In total, 14.5% of individuals self-reported receiving the mpox vaccine and of those without reported Orthopoxvirus vaccination, 5.9% were seroreactive to multiple mpox virus antigens. These results suggest the potential for undetected mpox circulation within Manitoba and Alberta. While primarily men who have sex with men were eligible for the mpox vaccine, an expansive range of gender and sexual orientation identities were classified as seroreactive, suggesting a misalignment between those who were seroreactive and vaccine eligibility criteria. This emphasizes how broader mpox vaccine eligibility, not restricted by specific gender and sexuality requirements, may better encompass those at risk of infection.

Multiplex bead-based immunoassays (MIAs) are promising tools for simultaneously detecting humoral immunity to multiple targets, potentially playing a crucial role in serosurveillance and vaccine response assessments. However, evaluation of assay performance is paramount prior to widespread use. This study presents a performance evaluation of a pan-filovirus MIA through characterization of the analytical range for the EBOV glycoprotein (GP) target and assessments of assay precision and antigen discrimination. The precision of the MIA was evaluated by comparing the detection of anti-filovirus antibodies at two independent laboratory sites: the University of Hawai’i, Honolulu (UH), and the Institut National de Recherche Biomédicale (INRB) in Kinshasa, Democratic Republic of the Congo (DRC). Forty-six samples from Yambuku, DRC, including Ebola virus Disease (EVD) survivors and close contacts, were tested at both sites. Additionally, 907 samples were tested in DRC before and after vaccination with a prophylactic EVD vaccine, ERVEBO. Results demonstrated low variability between laboratories, with intra-assay and inter-laboratory coefficients of variation below predefined thresholds for all filovirus targets included in the multiplex panel. Analyte correlation between sites was high (r ² = 0.86–0.92). Longitudinal analysis detected increased EBOV GP reactivity following vaccination, while reactivity to non-vaccine filovirus antigens remained stable, consistent with minimal cross-reactivity in a vaccinated cohort. These findings suggest that this pan-filovirus MIA produces reproducible results across distinct laboratory settings and may serve as a useful tool for comparative serologic investigations, serosurveillance, and evaluation of EBOV vaccine-associated antibody responses.

Background: There are currently no vaccines approved for the prevention or treatment of Orthoebolavirus bundibugyoense (Bundibugyo virus; BDBV). The recombinant vesicular stomatitis virus- Zaire ebolavirus glycoprotein vaccine (rVSV-ZEBOV-GP; ERVEBO) has been widely deployed during Ebola virus disease (EVD) outbreaks caused by Orthoebolavirus zairense (Ebola virus; EBOV). Given the lack of vaccines and medical countermeasures we evaluated development of antibodies to Bundibugyo glycoprotein (GP) following rVSV-ZEBOV-GP vaccination in two EVD outbreak-affected populations in the Democratic Republic of the Congo (DRC). Methods: Between 2018 and 2023, serum samples were collected from vaccine recipients in Mbandaka, Equateur Province (n=482 at baseline), and Beni, North Kivu Province (n=599 at baseline). Antibody reactivity was assessed using a multiplex pan-filovirus immunoassay. We evaluated longitudinal trends in BDBV GP seroreactivity across follow-up visits extending to approximately five years after vaccination. Findings: We collected 2552 samples from 482 participants in Mbandaka and 3297 samples from 599 participants in Beni. BDBV GP responses diverged by location. Baseline BDBV GP seroreactivity differed between sites, with 3.3% of participants reactive in Mbandaka and 10.4% in Beni. In Mbandaka, BDBV GP titers remained unchanged through 6 months post-vaccination but increased markedly between 2.5 and 3.5 years (mean MFI 1,238 to 4,845; p<0.0001), accompanied by a rise in seroreactivity to 35.3%, followed by waning at later visits. In Beni, BDBV GP titers increased rapidly after vaccination, reaching peak seroreactivity at 21 days (52.3%) and 6 months (53.9%), with mean fold increases of 21.9 and 20.1 among baseline-naive participants. Although antibody levels declined after 6 months, EBOV GP and BDBV GP titers remained above baseline, and significant increases in EBOV GP, BDBV GP, and SUDV GP titers were observed between 2.5 and 5 years. Interpretation: We observed detectable BDBV GP seroreactivity in two geographically distinct populations in the DRC. However, there was no consistent evidence showing that rVSV-ZEBOV-GP vaccination was associated with increased seroreactivity to BDBV GP across study populations. Baseline BDBV GP seroreactivity observed prior to vaccination warrants further investigation into the origins and epidemiological significance of pre-existing filovirus-reactive antibodies, including the possibility of prior exposure to related filoviruses, cross-reactive immune responses, high non-specific background reactivity unrelated to filoviruses, or other unrecognized sources of earlier antigenic stimulation. In the absence of licensed BDBV-specific vaccines, these data may inform preparedness planning, evaluation of existing countermeasures, and future vaccine development efforts during current and future BDBV outbreaks. It may further contribute to the study of natural history of filovirus immunity with a direct implication to understanding the possibility of cross-reactive and possible cross-protective responses in humans.

Mpox has evolved into a global public health concern following rapid geographic expansion and altered clinical and epidemiological characteristics. The 2022 outbreak of monkeypox virus (MPXV) marked the first widespread expansion to non-endemic regions, particularly among men who have sex with men (MSM). In the Democratic Republic of the Congo (DRC), where Clade I MPXV is endemic, data on urban exposures are limited. We evaluated serologic evidence of exposure using a four-plex MPXV assay among 1,823 participants from two major DRC cities, Kinshasa and Goma, between April and June 2024. Of these participants, 523 (28.7%) self-identified as MSM, 498 (27.3%) as professional sex workers (SW), 107 (5.9%) as MSM and SW, and 695 (38.1%) as other at-risk individuals. Overall, 2.9% of participants were seroreactive. By subpopulation, 4.8%, 2.1%, and 1.7% of at-risk, MSM, and SW participants were seroreactive ( p = 0.0086). Classification tree modelling identified age as the strongest predictor of reactivity, with higher MPXV seroreactivity in older participants. These data provide evidence of mpox seroreactivity prior to sustained human-to-human transmission in urban settings. Understanding exposure dynamics is critical for anticipating re-emergence of mpox in global cities where interpersonal networks may sustain transmission even in the absence of zoonotic spillover.

Ebola virus disease (EVD), caused by the Ebola virus (EBOV), is characterized by high morbidity and mortality, with 16 distinct EVD outbreaks reported in the Democratic Republic of the Congo (DRC), alone. As part of the formal response to the 2018 outbreaks in Equateur and North Kivu provinces, a recombinant vesicular stomatitis virus-Zaire Ebolavirus envelope glycoprotein vaccine (rVSV-ZEBOV-GP) vaccine was deployed under emergency use. While clinical trials have evaluated vaccine safety and efficacy, there is a paucity of real-world data documenting antibody durability for longer periods post-vaccination. Here, we present serologic data from 1081 individuals in Beni, North Kivu (n = 599) and Mbandaka, Equateur (n = 482) who were vaccinated during the outbreaks–with samples from baseline up to five-years following vaccination. Using a multiplexed immunoassay, we show sustained anti-EBOV GP reactivity: at year-5 collection, 72% of individuals naïve at time of vaccination remained seroreactive to EBOV GP. Stratifying by site, antibody titers remained significantly elevated after baseline across all post-vaccination timepoints in both linear and logistic mixed-effects models. Pre-existing EBOV GP reactivity at baseline was the strongest independent predictor of antibody response in Mbandaka, associated with higher titers and greater odds of seropositivity (OR = 3.87, 95% CI: 2.50-6.01, p-value < 0.001), consistent with a boosting effect among previously exposed individuals. However, this was not replicated in Beni (OR: 0.66, 95% CI: 0.27-1.58, p-value = 0.348). In Mbandaka, among those recipients who reported receiving a booster dose, the odds of seroreactivity were 12.75-fold (p-value < 0.001) and 3.68-fold higher (p-value =0.04) at 4.2 and 5-years post-vaccination, respectively, in comparison to odds of reactivity at three weeks following administration of the initial dose. Occupational groups with zoonotic or community-level exposure had trending lower odds of seroreactivity relative to healthcare workers, most consistently in Beni. Ultimately, these data indicate that five years following administration of the rVSV-ZEBOV-GP vaccine, most vaccinated individuals retain detectable anti-EBOV GP antibodies. While no correlate of protection for EVD is established at this time, sustained IgG seroreactivity to EBOV GP may serve as a marker for future understandings of the durability of and variation in immune responses to this high-consequence pathogen.

Background: Historically, the Democratic Republic of the Congo (DRC) has faced the greatest public health burden from mpox, including more than 70 000 probable cases from 1 January 2024 to 2 February 2025. However, there has been a relative paucity of investigation focused on mpox community engagement in DRC, including assessments of disease knowledge and risk perception. Methods: Given the ongoing Clade I mpox public health emergency of international concern, and the linkage between sustained human-to-human transmission and dense sexual networks, we sought to investigate mpox knowledge and sexual behaviours among key populations. Between 20 March 2024 and 25 August 2024, we recruited 2794 participants distributed across Kinshasa, Kwango and North Kivu provinces, with a focus in urban centres where mpox risk was considered high. Results: Most participants were considered other at-risk populations (948; 33.9%), followed by men who have sex with men (MSM, 828; 29.6%) and sex workers (897; 32.1%). Mpox knowledge, including transmission routes as well as sexual and health-seeking behaviours, was evaluated through questionnaires led by peer educators. Overall, only 6.1% of all participants reported prior mpox knowledge. Among this participant subset, zoonosis (‘direct contact with infected animals’) and ‘people living in high-risk areas’ were the most frequently selected options in regard to mpox transmission and populations at risk, respectively. When considering at-risk behaviours for mpox, those who identified as sex workers reported significantly higher risk sexual activities, including multiple sexual partners (80.3% of sex work participants), engaging in transactional sex (84.7%) and anonymous sex (80.8%) compared with MSM. However, both sex workers (44.8%) and MSM (56.7%) reported the highest health seeking behaviours for a suspected sexually transmitted infection. Conclusions: Our results highlight the need to evaluate knowledge in high-risk communities, especially in an endemic setting. Community engagement, which incorporates both mpox knowledge and risk perception activities, is inclusive of at-risk populations and is needed for ongoing mpox containment and mitigation efforts.