Scientific Publications

Background: Human immunodeficiency virus type 1 (HIV-1) is one of the fastest-evolving human pathogens. Understanding HIV-1 transmission, within-host adaptation, and evolutionary dynamics is pivotal for development of interventions and vaccines. HIV-1 infection is generally caused by a single transmitted founder virus (TFV), and TFV sequences are typically obtained using single genome amplification (SGA). However, suboptimal sample quality can cause sequencing failures, representing considerable losses considering the scarcity of acute HIV-1 infection (AHI) samples. Sequencing failures may be mitigated by molecular cloning (MC), which can be less vulnerable to sample quality but more susceptible to polymerase chain reaction (PCR) errors. Here, we explore the feasibility of supplementing SGA with MC data using samples from clinical and research cohorts to determine whether sequence diversity and evolutionary rate estimates are comparable between the techniques. Methods: Plasma samples were selected from participants with documented AHI from an East African research cohort (the International AIDS Vaccine Initiative, 2006-2011) and a clinical cohort from Sweden (1983-2011). SGA and MC sequencing were done on the HIV-1 env V1-V3 region (~940 base pairs). Within-host sequence diversity was determined from maximum likelihood phylogenetic trees, and evolutionary rate by Bayesian phylogenetic analysis. Highlighter plots, Hamming distances, and assessment of star phylogenies were used to quantify TFVs. Results: One hundred participants (median age 30.3 years, 15% female), contributing 350 samples from four longitudinal time points, 10-540 days post-infection, met the inclusion criteria. SGA succeeded on 90% of research cohort and 48% of clinical cohort samples. Comparative analysis of linked SGA and MC data from 10 samples indicated that approximately eight sequences were necessary for diversity estimates. Consistently higher sequence diversity was observed among MC relative to SGA sequences (median [IQR]: 0.009 [0.003, 0.015] and 0.004 [0.001, 0.012] substitutions/site, P = .002), whereas evolutionary rates were comparable between the two methods (0.016 [0.012, 0.019] and 0.011 [0.008, 0.020] substitutions/site/year, P = .232). Five participants with samples obtained within 45 days post-infection were eligible for TFV quantification, and all found to have one TFV using both techniques. Conclusion: MC data is a suitable supplement for SGA-based HIV-1 studies to preserve the value of precious samples for analysis of evolutionary rate, but not for sequence diversity.

Background: Targeted peer mobilisation can improve access to HIV testing and care and may impact onward HIV transmission. We describe a qualitative exploration of the experience with a peer mobilisation training programme for oral HIV self-testing (OST) and referral for acute HIV infection (AHI) testing among gay and bisexual men (GBMSM) and transgender women (TGW) in coastal Kenya. Methods: The training programme covered five modules: 1) safe sex, 2) OST, 3) AHI, 4) HIV partner notification services, and 5) mobilisation skills. Mobilisers attended two training sessions and weekly meetings between March and June 2019. Mobilisers offered OST to GBMSM and TGW peers and extended an AHI referral card for point-of-care HIV-RNA testing when peers reported AHI symptoms. Two focus group discussions with 18 mobilisers and 15 in-depth interviews with mobilised clients who were newly HIV diagnosed were conducted to explore the experiences of the training programme. Results: Mobilisers felt empowered through the training programme, which enhanced their mobilisation skills across two areas: (1) networking skills and (2) client empowerment. Facilitators for HIV testing were confidentiality of the OST, presence of STI symptoms, and building trust between mobilisers and clients. Mobilisers and clients reported challenges as: (1) misconceptions regarding OST and symptoms of AHI, (2) logistical and financial issues, and (3) stigma and security concerns. Discussion: Our training programme facilitated peer mobilisers to extend OSTs among GBMSM and TGW in coastal Kenya while it was more difficult to refer clients directly for AHI testing. Mobilisers felt empowered through enhanced mobilisation skills which helped them to mobilise clients for HIV testing. A targeted training programme was helpful in mobilising peers to take up HIV testing.

Indian HIV-1 subtype C, infecting 2.6 million individuals, demonstrates unique geospatial diversity reflecting distinct evolution and host-pathogen interactions that may instruct the development of region-specific therapeutic strategies. An Indian PLHIV cohort was profiled for immune dysfunction, proviral load, broadly neutralizing antibody sensitivity, and drug resistance mutations in putative CD4+ T cell reservoirs. We demonstrate therapy state specific immune dysfunction, including in ART responding individuals, coincident but not correlated with stable proviral load, apparently enriched in CD4+ T memory subsets. Reservoir derived full length envs displayed distinct neutralization profiles against best-in-class broadly neutralizing antibodies, highlighting the need for a combinatorial approach to target potential breakthrough viruses. Surveillance of the archival repertoire demonstrated the occurrence of drug resistance conferring mutations (>10%) across therapy states, including instances of primary and acquired resistance to recently introduced integrase strand transfer inhibitors. Our data, underlines the need for incorporating reservoir diversity in intervention and management strategies.

Background: Despite national declines in HIV prevalence, adolescent girls and young women (AGYW) in India remain disproportionately vulnerable to sexually transmitted infections (STIs), including HIV. This vulnerability stems from a convergence of biological, social, and structural factors, including early marriage, gendered expectations, poverty, and limited access to sexual and reproductive health (SRH) information and services. While prior research has documented these determinants, few studies explore how they intersect and evolve to shape AGYW's lived experiences of risk, particularly within the general population. Methods: This qualitative study used an adapted socio-ecological framework (which conceptualizes how structural, community, and individual-level factors interact to influence health and vulnerability) to examine the pathways of vulnerability to STIs and HIV among AGYW aged 16-24 in two urban regions: Delhi NCR and Mumbai. Data were collected through 42 in-depth interviews (IDIs), 4 focus group discussions (FGDs), and 18 key informant interviews (KIIs) with health providers, NGO staff, and program implementers. Reflexive thematic analysis was applied, guided by deductive codes from the eco-social model and inductive insights from participants' narratives. Results: Findings show that AGYW's vulnerability is shaped by interconnected macro (e.g., early marriage, patriarchal norms), meso (e.g., school-based silences, provider bias), and micro (e.g., relationship coercion, low self-efficacy) level factors. Although some AGYW had SRH knowledge, stigma, lack of autonomy, and unsupportive environments often constrained its use. Married and low-income AGYW were particularly disadvantaged, while non-governmental organizations (NGOs) played an important but uneven role-offering crucial safe spaces for awareness and support, yet limited by inconsistent coordination and resources. Conclusion: AGYW's vulnerability to STIs/HIV in urban India is produced through dynamic and intersecting structural, institutional, and interpersonal constraints. Addressing these requires integrated, gender-sensitive interventions that promote agency, reduce stigma, and foster collaboration between NGOs and public systems. Programs must go beyond awareness to reshape the environments in which AGYW make sexual health decisions.

The kinetics and identification of targets of Human Immunodeficiency Virus (HIV) infection within mucosae is a valuable tool for the development of new HIV-prevention strategies. Human tissue explants offer an informative model for studying HIV-1 pathogenesis and can support the development of novel HIV prevention interventions. Here, we infected cervical explants from HIV-1-uninfected women undergoing routine surgery with HIVBaL, a lab-adapted virus, and isolates HIV4790 and HIV4791, transmitted/founder (T/F) HIV-1 variants, and monitored the subsequent viral infection and replication using real-time quantitative PCR. The rates of infection and replication of HIV-1BaL exceeded those of both HIV4790 and HIV4791. The two T/F isolates were not significantly different from each other overall in the explant comparison (endo and ecto cervical tissue combined); however, all three viruses demonstrated different tissue tropism. HIV-1BaL and HIV4790 replicated at equivalent levels in endocervical explants, but HIV4790 replicated significantly less well in ectocervical explants. Alternatively, HIV4791 demonstrated inferior replication in endocervical tissues compared to HIVBaL and HIV4790 but improved replication in ectocervical explants compared to HIV4790. Immunofluorescent analysis of the cervical tissues revealed the presence of viable immune cells that are targets of HIV-1 infection, thus validating our ex vivo model in its ability to maintain viable cells in culture for a longer period. This allows for assessing the dynamics of HIV replication in the cervical tissues. Our data suggests that endocervical tissues may be more susceptible to HIV-1 infections than ectocervix, revealing the complex dynamics across different sites of the lower female reproductive tract.

Malaria is a leading cause of disease in developing countries. The licensed malaria vaccines (RTS,S/AS01 and R21/Matrix-M) have shown significant efficacy in human phase 3 trials. Vaccination with radiation-attenuated sporozoites has achieved high levels of protection against malaria in controlled infection studies, although protection was more moderate in clinical trials conducted in malaria-endemic areas. RTS,S/AS01, and R21/Matrix-M contain the repeating NANP motif and the C-terminal domain of the dominant surface circumsporozoite protein (CSP) of Plasmodium falciparum (Pf) sporozoites, but do not include the CSP N-terminal domain or epitopes in the junctional region between the N-terminal domain and the NANP repeats. In pursuit of a second-generation malaria PfCSP vaccine that surpasses the protection elicited by attenuated sporozoites and current subunit vaccines, we developed self-assembling nanoparticle immunogens each displaying one or more of four different classes of PfCSP epitope regions: NANP-repeat epitopes, junctional region-repeat epitopes, and epitopes from the N-terminal and C-terminal domains. In a mouse model of malaria infection, immunization with protein nanoparticles displaying different CSP epitope regions showed a reduction in liver burden ranging from minimal to 90%, with N- and C-terminal domains providing little reduction, but a combination of junctional and NANP repeat epitopes providing a strong reduction. mRNA-delivered nanoparticle and membrane-anchored immunogens displaying both the junctional and NANP repeat epitopes were most effective, exhibiting 99% reduction in liver burden and sterilizing immunity from parasitemia in some mice. The mRNA immunogens represent promising candidates for rapid translation to human challenge studies and could be combined with T cell vaccines to comprise a potential next-generation malaria vaccine.

Durability of T follicular helper (Tfh) cell responses is pivotal for generating high affinity antibodies. We characterized Tfh cell responses to HIV Env immunization longitudinally in non-human primates, analyzing >500,000 CD4+ T cells from 192 lymph node (LN) samples collected over 60 weeks, including >36,000 vaccine-specific Tfh cells. An escalating-dose priming regimen elicited higher and more sustained Tfh cell responses in LNs, compared with conventional bolus immunization. Multiple vaccine-specific germinal center (GC)-Tfh subpopulations, including interleukin (IL)4hi and IL21hi GC-Tfh cells, were continually present. Antigen-specific Tfh clones persisted within GCs for over 6 months, maintaining stable gene expression profiles and showing no signs of exhaustion. Vaccine-specific Tfh proliferation signatures were detectable for 27+ weeks after priming. Tfh subpopulations correlated with HIV Env-specific antibody and neutralization titers. Additionally, substantial Tfh clonal migration occurred between LNs. Thus, complex populations of GC-Tfh can enhance antibody responses and survive for 48 weeks in GC responses without new antigen delivery, with implications for immunization regimens.

Background: A lack of general vaccine confidence has been identified as a potential barrier to the introduction of new tuberculosis (TB) vaccines. In the absence of TB-specific vaccine confidence surveys, analysis of general national vaccine confidence data can provide a useful proxy to determine where demand generation strategies may need to be focused ahead of future TB vaccine introductions. Methods: We analysed 2023 Vaccine Confidence Index (VCI) data from 18 of the 49 countries present on at least one of the three World Health Organisation (WHO) high TB burden lists, and together containing 65% of the global TB burden, to explore overall confidence in vaccines in high TB burden countries. Based on collected answers to three different statements, we categorised responses 1-2 as 'positive' (vaccine confident) and 3-4 as 'negative' (vaccine hesitant) and calculated a total vaccine confidence score using the mean proportion of positive responses across the three statements. Results: In 2023, over 80% of respondents in 14 of the 18 countries analysed, and over 60% of respondents in all 18 countries, believed that 'vaccines are important for people of all ages'. India, accounting for around 30% of global TB cases, demonstrated confidence levels exceeding 90%, as did Vietnam, Ethiopia and Sierra Leone. South Africa, the country with the seventh highest TB burden (280,000 incident cases in 2023), Russia and Cameroon exhibited a relatively low vaccine confidence score of 75.5% or lower, signalling a potential area for concern. These countries may require focused awareness-raising and advocacy efforts prior to the rollout of new TB vaccines, though additional research on TB-specific confidence indicators is needed. Conclusions: This analysis underscores the importance of monitoring vaccine confidence levels to address emerging challenges to maintaining or bolstering the public's trust in vaccination. Our findings could help determine which countries to prioritise for social mobilisation and demand generation efforts to boost vaccine confidence, and thus improve readiness for new TB vaccines.

Background: No vaccine is currently available for Lassa fever, a viral hemorrhagic disease that is estimated to cause thousands of deaths each year in western Africa. A replication-competent recombinant vesicular stomatitis virus-vectored vaccine encoding a Lassa virus (LASV) glycoprotein complex, rVSVΔG-LASV-GPC, has been developed, but data on its safety and immunogenicity are limited. Methods: In this phase 1, double-blind trial conducted in the United States and Liberia, we randomly assigned healthy adults (18 to 50 years of age) to receive rVSVΔG-LASV-GPC or placebo intramuscularly. Participants received a single vaccine dose of 2×104 plaque-forming units (PFU), 2×105 PFU, 2×106 PFU, or 2×107 PFU or placebo or received two vaccine doses of 2×107 PFU or placebo, within a window of 6 to 20 weeks. The side-effect profile was assessed according to the incidence of solicited and unsolicited adverse events (primary end point). Because Lassa fever can cause sensorineural hearing loss, hearing acuity was measured before and after the injection. Secondary end points were levels of binding antibodies against LASV glycoprotein, neutralizing antibodies, and vaccine vector-derived viral RNA and PFU in plasma, urine, and saliva. Results: A total of 114 adults were enrolled. No serious vaccine-related adverse events were reported. The vaccine caused minimal local reactions and dose-dependent, mild-to-severe early-onset systemic reactogenicity events that were transient. No hearing loss was detected. All doses induced robust long-lasting cellular and humoral (binding and neutralizing) responses that cross-reacted against common LASV lineages. No infectious vaccine virus particles were found in plasma, urine, or saliva. Conclusions: The rVSVΔG-LASV-GPC vaccine resulted in transient local and systemic reactogenicity events but no hearing loss or serious adverse events. The vaccine had immunogenicity over a wide dose range in healthy adults in the United States and Liberia. (Funded by the Coalition for Epidemic Preparedness Innovations and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT04794218; Pan African Clinical Trials Registry number, PACTR2021106625781067.).

Current licensure trials of new vaccines to prevent tuberculosis disease use bacteriologically confirmed symptomatic tuberculosis disease as the primary endpoint. Globally, the incidence of symptomatic tuberculosis disease is low, making licensure trials large, long, and expensive. New data suggest that bacteriologically confirmed asymptomatic tuberculosis disease might occur more frequently than symptomatic tuberculosis disease. Therefore, if vaccines have efficacy against asymptomatic disease, tuberculosis vaccine licensure trials could include it in the primary endpoint, potentially leading to smaller or shorter trials. We describe the potential benefits and risks of this inclusion in the primary endpoint of tuberculosis vaccine licensure trials. We also simulate licensure trial endpoint accrual and summarise feedback from anonymous regulators and policy makers on the knowledge needed to consider this proposal and research studies needed to fill these evidence gaps. If bacteriologically confirmed asymptomatic tuberculosis disease could be included in the primary endpoint of tuberculosis disease licensure trials, it could lead to cheaper and more rapid tuberculosis vaccine development.

mRNA vaccines have emerged as an important platform for vaccine development. Unlike protein subunit vaccines, mRNA-expressed antigens can be expressed in either secreted or transmembrane (TM) forms mimicking a viral envelope (Env) protein. Here, we investigated the impact of antigen expression format on the antigenicity profile, glycosylation, and immunogenicity of stabilized HIV Env trimer immunogens expressed from self-replicating RNA (replicon) vaccines. Replicon-encoded trimers in both forms exhibited proper folding, and replicon-expressed secreted trimers exhibited glycosylation patterns largely consistent with recombinant trimer protein, although with enrichment of complex glycans over high mannose at some sites. Both formats were highly immunogenic in mice, eliciting comparable serum antibody and T cell responses. Interestingly, the TM format initiated smaller germinal center (GC) responses, but these GCs were enriched for trimer-binding B cells compared to secreted trimer vaccines. In a B cell receptor knockin adoptive transfer model for assessing germline targeting, the replicon-encoded TM trimer elicited a greater frequency of epitope-targeting antibodies and recruited broadly neutralizing antibody precursor B cells to the GC response more efficiently compared to the replicon-encoded secreted trimer or protein trimer combined with adjuvant. These results indicate that the form of immunogen expression can impact key elements of immune responses to RNA vaccines.

People with HIV (PWH) are understudied in COVID-19 vaccine trials, leaving knowledge gaps on whether the identified immune correlates of protection also hold in PWH. CoVPN 3008 (NCT05168813) enrolled predominantly PWH and reported lower COVID-19 incidence for a Hybrid vs. Vaccine Group (baseline SARS-CoV-2-positive and one mRNA-1273 dose vs. negative and two doses). Using case-cohort sampling, antibody markers at enrolment (M0) and four weeks post-final vaccination (Peak) are assessed as immune correlates of COVID-19. For the Hybrid Group [n = 287 (195 PWH)], all M0 markers inversely correlate with COVID-19 through 230 days post-Peak, with 50% inhibitory dilution BA.4/5 neutralizing antibody titer (nAb-ID50 BA.4/5) the strongest and only independent correlate (HR per 10-fold increase=0.46, 95% CI 0.28, 0.75; P = 0.002). For the Vaccine Group [n = 115 (86 PWH)], Peak nAb-ID50 BA.4/5 correlates with reduced COVID-19 risk (1.9%, 1.1%, and 0.3% at titers 10, 100, and 1000 AU/ml) through 92, but not 165, days post-Peak. Using multivariable Cox analysis of binding and nAb, nAb titers predict COVID-19 in PWH. Two doses of a 100-µg Ancestral strain mRNA vaccine in baseline-SARS-CoV-2-negative individuals elicit sufficient cross-reacting Omicron antibodies to reduce COVID-19 incidence for 90 days post-Peak, but viral evolution and waning antibodies abrogate this protection thereafter.

The S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is highly conserved across coronavirus strains and therefore is a potential pan-coronavirus vaccine target. However, antibodies targeting this region are typically non-neutralizing. We report herein that S2-targeting antibodies from patients who recovered from SARS-CoV-2 infection bound only closely related sarbecovirus subgenus strains and, like most known S2 antibodies, none of these were neutralizing. In contrast, first-exposure, severe acutely infected COVID-19 patients predominantly induced back-boosted antibody-secreting cells imprinted against past common cold coronavirus strain OC43 that were cross-reactive to as many as five subgenera of betacoronavirus strains and gave rise to antibodies that were neutralizing and protective. The antibodies targeted two different sites: one defined by competition with stem helix antibodies, and the second to an underdescribed epitope at the apex of S2. These findings suggest that S2-targeted vaccines could strategically exploit controlled OC43 priming followed by SARS-CoV-2 boosting to enhance the breadth and quality of protective antibody responses.