Scientific Publications

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.

Interleukin-21 (IL-21) has been linked with the generation of virus-specific memory CD8+ T cells following acute infection with HIV and reduced exhaustion of CD8+ T cells. IL-21 has also been implicated in the promotion of CD8+ T cell effector functions during viral infection. Little is known about the expression of interleukin-21 receptor (IL-21R) during HIV-1 infection or its role in HIV-1-specific CD8+ T cell maintenance and subsequent viral control.

The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and repertoire sequencing to identify neutralizing antibodies (NAbs) from immunized animals. A panel of mouse NAbs was isolated from mice immunized with a 60-meric I3-01 NP presenting 20 stabilized trimers. Three mouse NAbs potently neutralized BG505.T332N by recognizing a glycan epitope centered in the C3/V4 region on BG505 Env, as revealed by electron microscopy (EM), X-ray crystallography, and epitope mapping. A set of rabbit NAbs was isolated from rabbits immunized with a soluble trimer and a 24-meric ferritin NP presenting 8 trimers. Neutralization assays against BG505.T332N variants confirmed that potent rabbit NAbs targeted previously described glycan holes on BG505 Env and accounted for a significant portion of the autologous NAb response in both the trimer and ferritin NP groups. Last, we examined NAb responses that were induced by non-BG505 Env immunogens. We determined a 3.4-Å-resolution crystal structure for the clade C transmitted/founder (T/F) Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend in gp41. This clade C Env, in a soluble trimer form and in a multivalent form with 8 trimers attached to ferritin NP, and the gp41-stabilized clade A Q482-d12 Env trimer elicited distinct NAb responses in rabbits, with notable differences in neutralization breadth. Although eliciting a broad NAb response remains a major challenge, our study provides valuable information on an HIV-1 vaccine design strategy that combines gp41 stabilization and NP display. Self-assembling protein nanoparticles (NPs) presenting BG505 envelope (Env) trimers can elicit tier 2 HIV-1-neutralizing antibody (NAb) responses more effectively than soluble trimers. In the present study, monoclonal NAbs were isolated from previously immunized mice and rabbits for structural and functional analyses, which revealed that potent mouse NAbs recognize the C3/V4 region and small NP-elicited rabbit NAbs primarily target known glycan holes on BG505 Env. This study validates the gp41 stabilization strategy for HIV-1 Env vaccine design and highlights the challenge in eliciting a broad NAb response.

Beliefs about gender roles and high-risk sexual behaviours underlie the human immunodeficiency virus (HIV) epidemic in South Africa. Yet, there is limited information on the relationships between beliefs about gender roles and risky sexual behaviours. Few studies have explored the association between beliefs about gender roles, high risk sexual behaviour, and health-seeking behaviour among men.

The prevalence of latent tuberculosis infection (LTBI) is vastly higher than that of tuberculosis (TB) disease and this enormous reservoir of individuals with LTBI impacts the global TB control strategy. Adolescents are at greatest risk of TB infection and are thus an ideal target population for a potential effective TB vaccine to be added to the current BCG programme as it could reduce the number of latent infections and consequently the number of adults with TB disease. However, LTBI rates are often unknown for this population. This study aims to estimate the magnitude of LTBI and to determine if Tanzanian adolescents would be a good population for a prevention of TB infection trial.

Evolving diversity in globally circulating HIV-1 subtypes presents a formidable challenge in defining and developing neutralizing antibodies for prevention and treatment. HIV-1 subtype C is responsible for majority of global HIV-1 infections. In the present study, we examined the diversity in genetic signatures and attributes that differentiate region-specific HIV-1 subtype C gp120 sequences associated with virus neutralization outcomes to key bnAbs having distinct epitope specificities. A total of 1814 full length HIV-1 subtype C gp120 sequence from 37 countries were retrieved from Los Alamos National Laboratory HIV database (www.hiv.lanl.gov). The amino acid sequences were assessed for their phylogenetic association, variable loop lengths and prevalence of potential N-linked glycosylation sites (pNLGS). Responses of these sequences to bnAbs were predicted with a machine learning algorithm 'bNAb-ReP' and compared with those reported in the CATNAP database. Subtype C sequences from Asian countries including India differed phylogenetically when compared with that from African countries. Variable loop lengths and charges within Indian and African clusters were also found to be distinct from each other, specifically for V1, V2 and V4 loops. Pairwise analyses at each of the 25 pNLG sites indicated distinct country specific profiles. Highly significant differences (p<0.001***) were observed in prevalence of four pNLGS (N130, N295, N392 and N448) between South Africa and India, having most disease burden associated with subtype C. Our findings highlight that distinctly evolving clusters within global intra-subtype C gp120 sequences are likely to influence the disparate region-specific sensitivity of circulating HIV-1 subtype C to bnAbs.

Pre-existing immunity to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, induced from natural infection or vaccination. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. Here we compare serum antibody and memory B cell responses to coronavirus spike proteins from pre-pandemic and SARS-CoV-2 convalescent donors using binding and functional assays. We show weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we find evidence of pre-existing cross-reactive memory B cells that are activated during SARS-CoV-2 infection. Monoclonal antibodies show varying degrees of cross-reactivity with betacoronaviruses, including SARS-CoV-1 and endemic coronaviruses. We identify one cross-reactive neutralizing antibody specific to the S2 subunit of the S protein. Our results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

The potential use of the broadly neutralizing monoclonal antibodies (bnAbs) towards prophylaxis and treatment to HIV-1 is currently being explored. While a number of promising bnAbs have been discovered and a few of them have progressed towards clinical development, their extent of neutralization coverage with respect to global HIV-1 variants given the existence of genetically distinct subtypes and recombinants circulating globally is not clearly known. In the present study, we examined the variation in the neutralization susceptibility of pseudoviruses expressing 71 full length primary HIV-1 subtype C envs obtained from limited cross-sectional individuals over different time points against four bnAbs that target gp120 with distinct specificities: VRC01, CAP256-VRC26.25, PGDM1400 and PGT121.

The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower.

Algorithms that bridge the gap between syndromic sexually transmitted infection (STI) management and treatment based in realistic diagnostic options and local epidemiology are urgently needed across Africa. Our objective was to develop and validate a risk algorithm for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) diagnosis among symptomatic Rwandan women and to compare risk algorithm performance to the current Rwandan National Criteria for NG/CT diagnosis.

The longstanding inadequacies of syndromic management for genital ulceration and inflammation are well-described. The Rwanda National Guidelines for sexually transmitted infection (STI) syndromic management are not yet informed by the local prevalence and correlates of STI etiologies, a component World Health Organization guidelines stress as critical to optimize locally relevant algorithms.

The stochastic development of broadly neutralizing antibodies (bnAbs) to HIV-1 is influenced by complex viral and host interactions. In this issue of Cell Host & Microbe, Townsley et al. reveal that early B cell and virus interactions during acute infection are predictive for developing bnAb responses later in infection.

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.