Scientific Publications

Priming rare subdominant precursor B cells in germinal centers (GCs) is a central goal of vaccination to generate broadly neutralizing antibodies (bnAbs) against HIV. Multivalent immunogen display on protein nanoparticle scaffolds can promote such responses, but it also generates scaffold-specific B cells that could theoretically limit bnAb precursor expansion in GCs. We rationally designed DNA origami-based virus-like particles (DNA-VLPs) displaying a germline-targeting HIV envelope protein immunogen, which elicited no scaffold-specific antibody responses. Compared with a state-of-the-art clinical protein nanoparticle, these DNA-VLPs increased the expansion of epitope-specific GC B cells relative to off-target B cells and enhanced expansion of bnAb-lineage B cells in a humanized mouse model of CD4 binding site priming. Thus, minimizing off-target responses enhances bnAb priming and indicates that DNA-VLPs are a promising vaccine platform.

Introduction: HIV testing is at the cornerstone for interventions aimed at controlling the number of new HIV infections in Sub-Saharan Africa. The current HIV RDTs in use are not able to identify acute HIV infection. Methods: We collaborated with 10 health centers in Uganda, offering HIV voluntary counselling and testing services to obtain 'left-over' EDTA blood samples after HIV RDT. We investigated for HIV RNA using the GeneXpert HIV-1 Viral Load assay among individuals who had non-reactive Determine (HIV negative) or an inconclusive test result (Indeterminate). We used a formula from Brookmeyer and Quinn to estimate HIV incidence. The study started on the 17th February 2021 and ended on the 24th December 2021. Study activities were suspended between June and September 2021, due to travel restrictions instituted by government of Uganda aimed at reducing levels of connectivity between and within communities, to curb the rising number of COVID-19 infections. Results: Fourteen of 4,941 'left-over' plasma samples retested were reactive with a mean pVL of 1.5X106 copies per ml. The overall HIV incidence per 100 person years was 7.4 (95% CI: 4.3-12.5): higher for females at 9.4 (95% CI: 5.2-16.9) compared to 4.2 (95% CI: 1.3-12.9) for males. The HIV Incidence per 100 person years was 7.8 (95% CI: 3.7-16.4) before travel restrictions were instituted and 7.0 (95% CI: 3.3-14.7) after they were lifted. Conclusion: We demonstrated that incident HIV infection can be determined without assembling a longitudinal cohort. GeneXpert HIV-1 Viral Load assay can detect acute infection. There is no evidence that COVID-19 travel restrictions was associated with increased HIV Incidence rates in our communities.

Applying cryoelectron microscopy (cryo-EM) to small protein complexes is usually challenging due to their lack of features for particle alignment. Here, we characterized antibody responses to 21 kDa human immunodeficiency virus (HIV) membrane-proximal external region germline-targeting (MPER-GT) immunogens through cryo-EM by complexing them with 10E8 or Fabs derived from MPER-GT-immunized animals. Distinct antibody-antigen interactions were analyzed using atomic models generated from cryo-EM maps. Mutagenesis screening revealed that off-target monoclonal antibodies (mAbs), which do not compete with 10E8, bind non-MPER epitopes, and the binding of two most dominant epitopes were verified by cryo-EM. The structures of 10E8-class on-target Fabs showed binding patterns that resemble the YxFW motif in the 10E8 heavy chain complementarity-determining region 3 (HCDR3) loop. Additionally, we demonstrate that high-resolution maps can be generated from heterogeneous samples with pooled competing Fabs. Overall, our findings will facilitate the optimization of MPER-GT antigens and push the size limit for cryo-EM-based epitope mapping with smaller antigens and heterogeneous antibody mixes.

Background: Monoclonal antibodies (mAbs) are an increasingly essential class of medicines across many disease areas. In the human body, there are five antibody isotypes, each with potential therapeutic benefits for different disease indications. However, 97% of all clinically approved mAbs are produced as the IgG isotype, largely due to challenges associated with recombinantly producing non-IgG isotypes like IgM or IgA, which have additional N-linked glycan sites and can present as multivalent oligomers. One potential way to circumvent this challenge is to express mAbs in situ using mRNA encapsulated in lipid nanoparticles (LNP), bypassing the need for recombinant protein production. Objective: Here, we demonstrate the feasibility of expressing a mAb as both IgG and IgA in non-human primates (NHPs) using mRNA-LNPs. Methods: We express ePGDM1400v9, a broadly neutralizing mAb targeting human immunodeficiency virus (HIV), in both IgG1 and IgA2 formats by infusing NHPs with LNPs containing the appropriate mRNAs. Results: Though IgA2 expression levels were low, both formats were detectable in serum within one day of LNP infusion in all NHPs, and both were detectable in mucosal secretions of most animals. Importantly, serum mRNA-produced IgG1 and IgA2 retained HIV-neutralizing function. Furthermore, mass spectrometry analysis confirmed that mAbs of either isotype produced in situ exhibited glycosylation patterns highly similar to that of native antibody, which is likely to confer therapeutic advantages. Conclusion: Altogether, this work demonstrates that mRNA-LNPs can be used to express native like mAbs of non-IgG isotypes in primates at detectable levels and enables further development and optimization of non-IgG mAb constructs.

Rhesus macaques (RMs) are a vital model for studying human disease and are invaluable to preclinical vaccine research, particularly for the study of broadly neutralizing antibody responses. Such studies require robust genetic resources for antibody-encoding genes within the immunoglobulin (IG) loci. The complexity of the IG loci has historically made them challenging to characterize accurately. To address this, we developed experimental and computational methodologies to generate a collection of integrated antibody repertoire and long-read genomic sequencing data in 106 Indian-origin RMs. We created a resource of IG heavy- and light-chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs). This includes the curation of 1,095 previously unidentified alleles, unveiling tremendous diversity and expanding existing IG allele sets by 40%. This publicly available, continually updated resource (https://vdjbase.org/reference_book/Rhesus_Macaque) provides the foundation for advancing RM immunogenomics, vaccine discovery, and translational research.

RNA-based vaccines have emerged as a highly effective delivery platform. However, this approach eliminates the possibility for immunogen purification, common in manufacturing of recombinant immunogens. In HIV-1 vaccine design, this is of particular importance because non-native epitopes can compromise the desired immune response, and native immunogen assembly is important for presentation of glycan-based epitopes targeted by broadly neutralizing antibodies. Here, we investigate the assembly and glycosylation of the archetypal trimeric HIV-1 immunogen, BG505, in the soluble single-chain format (NFL.664) that bypasses the need of maturation by furin cleavage. We have investigated the presence of the trimer-associated mannose-patch as oligomannose-type structures at these N-linked glycosylation sites are indicative of native-like glycoprotein structure. Despite the presence of features of native-like glycosylation, both electron microscopy and glycopeptide analysis indicated the presence of a sub-population of non-native material. We also investigated the glycosylation of material derived from cell-types that likely produce immunogens near the site of intramuscular RNA injection. We show that replicon-transformed dendritic and muscle cell lines generate immunogens displaying similar oligomannose-type glycan content, whereas sites presenting complex-type glycosylation differed substantially in the levels of glycan processing. Overall, the control of the immunogen assembly by protein engineering is sufficient to drive native-like glycosylation at the majority of glycosylation sites independent of producer cells. Furthermore, we explored the engineering of RNA immunogens to improve glycan site occupancy. Controlling immunogen assembly at the nucleotide level offers a route to enhanced RNA-based immunogens.

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.