Scientific Publications

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.

Background
Recent outbreaks of Ebola virus disease (EVD) and Marburg virus disease (MVD) in sub-Saharan Africa illustrate the need to better understand animal reservoirs, burden of disease, and human transmission of filoviruses. This protocol outlines a systematic literature review to assess the prevalence of filoviruses that infect humans in sub-Saharan Africa. A secondary aim is to qualitatively describe and evaluate the assays used to assess prevalence.

Methods
The data sources for this systematic review include PubMed, Embase, and Web of Science. Titles, abstracts, and full texts will be reviewed for inclusion by a primary reviewer and then by a team of secondary reviewers, and data will be extracted using a pre-specified and piloted data extraction form. The review will include human cross-sectional studies, cohort studies, and randomized controlled trials conducted in sub-Saharan Africa up until March 13, 2024 that have been published in peer-reviewed scientific journals, with no language restrictions. Prevalence will be stratified by pathogen, population, assay, and sampling methodology and presented in forest plots with estimated prevalence and 95% confidence intervals. If there are enough studies within a stratum, I2 statistics will be calculated (using R statistical software), and data will be pooled if heterogeneity is low. In addition, assays used to detect infection will be evaluated. All studies included in the review will be assessed for quality and risk of bias using the JBI Prevalence Critical Appraisal Tool and for certainty using the GRADE certainty ratings.

Discussion
Accurately measuring the rate of exposure to filoviruses infecting humans in sub-Saharan Africa using prevalence provides an essential understanding of natural history, transmission, and the role of subclinical infection. This systematic review will identify research gaps and provide directions for future research seeking to improve our understanding of filovirus infections. Understanding the natural history, transmission, and the role of subclinical infection is critical for predicting the impact of an intervention on disease burden.

Systematic Review Registration
In accordance with the guidelines outlined in the PRISMA-P methodology, this protocol was registered with PROSPERO on April 7, 2023 (ID: CRD42023415358).

Supplementary Information
The online version contains supplementary material available at 10.1186/s13643-024-02626-w.

Adolescents face a higher risk for HIV, STIs, and unintended pregnancy than any other age group in sub–Saharan Africa, and have unique health care needs as they navigate this period of growth and developmental milestones. We conducted the Youth Friendly Services study among adolescents in Rustenburg, South Africa to address some of these concerns. Participants aged 12–19 were followed quarterly for 12 months, asked at baseline about demographics, their sexual behavior, and tested for HIV, STIs, and pregnancy (girls). Report of sexual activity was not a requirement for enrollment. Assent and parental consent were obtained for participants under 18. Some follow up visits fell during COVID-mandated shutdowns, and we worked with participants to reschedule and extend follow up as appropriate. Here we present data on reported behaviors, participant attrition, risk of HIV, other STI, and pregnancy. From May 2018 to August 2019, we enrolled 223 HIV-negative, non-pregnant adolescents (64% girls). The median age was 17 (IQR: 14–18). Among the 119 (53%) participants who reported being sexually active at baseline, the median age at first sex was 16 years (IQR: 15–17). During follow-up, an additional 16 (7%) participants reported having their first sexual encounter. Among the sexually active participants, the incidence of HIV was 1.5 cases / 100 person-years at risk (PYAR, 95% CI: 0.4–6.0), the incidence of chlamydia was 15.7 cases (95% CI: 10.1–24.4), gonorrhea was 4.7 cases (95% CI: 2.1–10.5), and HSV was 6.3 cases (95% CI: 3.1–12.6); we observed no cases of incident syphilis. The incidence of pregnancy among sexually active girls was 15.0 pregnancies / 100 PYAR (95% CI: 8.5–26.5). Despite small numbers, the incidence of most STIs was significantly higher in females compared to males. We also observed two pregnancies and 5 incident STIs among participants who reported never having had sex, these tended to be younger participants. From March to September 2020, the clinic was shut down for COVID-19, and 53 study visits were postponed. Follow up was concluded in November 2020, a total of 19 participants were lost to follow up, however only one participant dropped off-study during COVID-19 shutdowns. Retention at the final visit was 91.5%. We successfully completed a prospective study of adolescents to learn more about the risks they face as they navigate sexual debut in the context of a program of youth-friendly counseling and services. Among self-reported sexually active participants, we observed a high rate of HIV, STI and pregnancy, however we also observed pregnancy and STIs among those who reported no sexual activity.

Monoclonal antibodies (mAbs) are revolutionizing management of non-communicable diseases in high-income countries and are increasingly being advanced for a range of infectious diseases (IDs). However, access to existing mAbs is limited in low- and middle-income countries (LMICs), and investment in developing fit-for-purpose mAbs for IDs that disproportionately affect LMICs has been limited. Underlying these access barriers are systemic challenges, including a lack of commercial incentives to target LMIC markets and complexity in manufacturing and regulatory processes. Novel strategies are needed to overcome systemic access barriers for mAbs. We outline key areas where new approaches could address these barriers, based on a multistakeholder consultation in March 2023. Three disease-market archetypes are identified to guide thinking about business models tailored to different contexts. New business models are needed to incentivize development and manufacturing of ID mAbs and to ensure mAbs are optimized with a target product profile and cost of goods that enable use in diverse LMIC settings. Lessons can be applied from voluntary licensing strategies and product development partnerships that have shown success in catalysing development and affordable supply for a range of infectious diseases. Technology transfer will be key to expand LMIC research and manufacturing capacity and to enable sustainable and diversified supply. Improved market intelligence, demand aggregation mechanisms, and portfolio-based manufacturing models could be used to de-risk commercial investment and establish a sustainable manufacturing ecosystem for affordable mAbs. Novel regulatory approaches and robust technology transfer may reduce data requirements and timelines for biosimilar approvals. Trailblazer products, with coordinated “end-to-end” support from funders, can demonstrate proof of concept for pathways to accessible mAbs across a broader range of LMICs. Research funders; local, regional, global health agencies; and, private sector partners should commit to implementing innovative partnerships and end-to-end strategies that enable equitable access to mAbs for infectious diseases in LMICs.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor–competitor affinity gap.

Background
Novel HIV pre-exposure prophylaxis (PrEP) methods including a potential future HIV vaccine, will increase prevention options for adolescent girls and young women (AGYW) at high risk of HIV infection in Eastern and Southern Africa, yet data on AGYW’s preferences for various PrEP methods is limited. We investigated preferences for five biomedical PrEP methods (oral, injectable, vaginal ring, implant, HIV vaccine) among 14–24-years-old AGYW in Kampala, Uganda.

Methods
From January to December 2019, we conducted a mixed methods study including 265 high-risk AGYW. After receiving two education sessions on the five PrEP methods, participants were asked about their “most preferred PrEP method.” Multinomial logistic regression (oral PrEP as reference category) was used to determine participant characteristics associated with method preference. Results are presented as adjusted relative risk ratios (aRRR) with 95% confidence intervals (CI). In-depth interviews were conducted with 20 selected participants to examine reasons influencing PrEP preferences and suggestions for method improvements. Transcripts were analyzed thematically.

Results
Participants preferred methods were: HIV vaccine (34.7%), oral PrEP (25.7%), injectable PrEP (24.9%), PrEP implant (13.6%), and vaginal ring (1.1%). Preference for injectable PrEP increased with every year of age (aRRR 1.22; 95% CI 1.04–1.44) and among participants with chlamydia or gonorrhoea (aRRR 2.53; 95% CI 1.08–5.90), while it was lower among participants having sexual partner(s) living with HIV or of unknown HIV status (aRRR 0.30; 95% CI 0.10–0.91). Preference for PrEP implants also increased with age (aRRR 1.42; 95% CI 1.14–1.77) and was strong among participants having ≥10 sexual partners in the past 3 months (aRRR 3.14; 95% CI 1.16–8.55), while it was lower among those with sexual partner(s) living with HIV or of unknown HIV status (aRRR 0.25; 95% CI 0.07–0.92). PrEP method preference was influenced by product attributes and prior experiences with similar product forms commonly used in health care.

Conclusion
AGYW have varied preferences for biomedical PrEP method and those with higher sexual behavioral risk prefer long-acting methods. As we anticipate more available PrEP options, oral PrEP use should be supported among AGYW, especially for those with sexual partners living with HIV or of unknown HIV status.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Tools to evaluate and accelerate tuberculosis (TB) vaccine development are needed to advance global TB control strategies. Validated human infection studies for TB have the potential to facilitate breakthroughs in understanding disease pathogenesis, identify correlates of protection, develop diagnostic tools, and accelerate and de-risk vaccine and drug development. However, key challenges remain for realizing the clinical utility of these models, which require further discussion and alignment among key stakeholders. In March 2023, the Wellcome Trust and the International AIDS Vaccine Initiative convened international experts involved in developing both TB and bacillus Calmette-Guérin (BCG) human infection studies (including mucosal and intradermal challenge routes) to discuss the status of each of the models and the key enablers to move the field forward. This report provides a summary of the presentations and discussion from the meeting. Discussions identified key issues, including demonstrating model validity, to provide confidence for vaccine developers, which may be addressed through demonstration of known vaccine effects (eg, BCG vaccination in specific populations), and by comparing results from field efficacy and human infection studies. The workshop underscored the importance of establishing safe and acceptable studies in high-burden settings, and the need to validate >1 model to allow for different scientific questions to be addressed as well as to provide confidence to vaccine developers and regulators around use of human infection study data in vaccine development and licensure pathways.

Product development is a high-risk undertaking, especially so when investments are prioritized for low- and middle-income countries (LMICs) where markets may be smaller, fragile, and resource-constrained. New HIV prevention technologies, such as the dapivirine vaginal ring (DVR) and long-acting injectable cabotegravir (CAB-LA), are being introduced to these markets with one indication, meeting different needs of groups such as adolescent girls and young women (AGYW) and female sex workers (FSWs) in settings with high HIV burden. However, limited supply and demand have made their uptake a challenge. Economic evaluations conducted before Phase III trials can help optimize the potential public health value proposition of products in early-stage research and development (R&D), targeting investments in the development pathway that result in products likely to be available and taken up. Public investors in the HIV prevention pipeline, in particular those focused on innovative presentations such as multipurpose prevention technologies (MPTs), can leverage early economic evaluations to understand the intrinsic uncertainty in market characterization. In this perspective piece, we reflect on the role of economic evaluations in early product development and on methodological considerations that are central to these analyses. We also discuss methods, in quantitative and qualitative research that can be deployed in early economic evaluations to address uncertainty, with examples applied to the development of future technologies for HIV prevention and MPTs.