Scientific Publications

Lassa fever is a severe, often fatal febrile illness endemic to West Africa caused by Lassa virus (LASV), with different virus lineages predominating across West African countries. The viral nucleoprotein (NP) is a target antigen for serological assays to identify previous exposure to LASV. To our knowledge, there is no commercially available assay that reliably quantifies anti-LASV-NP IgG antibodies in human serum. We report the development and qualification of an ELISA designed to detect and quantify anti-LASV-NP IgG in human serum samples. Following assay optimization, performance was assessed through assay qualification at clinical trial laboratories within Ghana. Assay positivity criteria, lower limit of detection, upper and lower limits of quantification, inter-assay precision, selectivity and dilutional linearity were determined. A new reference standard prepared from pooled sera from donors in endemic Lassa fever regions was established and calibrated to the first WHO international standard for LASV antibodies. One ELISA assay utilizing lineage IV LASV-NP was applicable for detection of anti-LASV-NP IgG antibodies in serum samples from different West African countries where either LASV lineages I, II, III and IV predominate. The ELISA remained selective in hemolysed serum samples with minimal loss of signal across repeated sample freeze-thaw cycles. Crucially, the developed ELISA was fully concordant with a now discontinued commercially available ELISA kit for quantification of anti-LASV-NP antibodies. Our anti-LASV-NP IgG ELISA was shown to reliably measure anti-LASV-NP IgG levels in human serum. Establishing and conducting this assay within West Africa represents an essential step towards strengthening LASV epidemiology research and supporting urgently needed development of a vaccine to prevent Lassa Fever.

The human immune system is a sophisticated network of cells and molecules that plays a vital role in safeguarding the body against a broad range of foreign agents. One of the key components of the immune system is the circulating antibody repertoire, a vast collection of different antibodies that recognize and eliminate foreign agents specifically. The diversity and specificity of the circulating antibody repertoire are essential for effective immunity. In a study of ten healthy donors, we previously demonstrated the accessible human antibody repertoire to be on the order of 1015-1018 distinct members, although an individual will only sample a fraction of that repertoire at a given time point. The mode of generation of the naïve antibody repertoire through random recombination before antigen contact suggests that the composition of the circulating antibody repertoire may drift over time, but the magnitude of this drift is poorly understood. Here, we used high-throughput sequencing to analyze two donors from our original study, approximately four years later. Our results reveal conservation of the size, overall diversity, and gross features of the circulating antibody repertoire, such as V- and J-gene usage and CDRH3 length distribution, over time but suggest substantial changes to fine features of the repertoire, such as combinations of V, J, D gene segments and P and N sequences, which may influence primary responses to newly encountered pathogens as well as to immunization procedures.

Background: The current wide-spread drug and insecticide resistance in malaria parasites and mosquito vectors reinforces the urgent need of innovative interventions such as stage-specific vaccines. Transmission-blocking vaccine (TBV) candidates, including Plasmodium falciparum Pfs48/45 and Pfs230 play an essential role in enabling parasite fertilization within mosquitoes. This study evaluated the genetic diversity and evolutionary dynamics of the Pfs230 gene to provide critical information on its potential as a suitable TBV candidate. Methods: The study utilized genomic data from the MalariaGEN Pf7 database, which included samples collected from four East African countries: Ethiopia, Kenya, Tanzania, and Uganda. Genetic metrics including nucleotide diversity, haplotype diversity (Hd), Tajima's D, and Wright's fixation index (FST) were computed to characterize the genetic diversity of the Pfs230 gene. Results: Of the 1471 sequences retrieved, 1312 passed quality filtering and were retained for downstream analysis. Overall, nucleotide diversity of the Pfs230 gene was low across the four countries (π = 5.9 × 10-4). The Hd was 0.999 with 496 haplotypes among 718 monoclonal sequences, and 8.1% (n = 40) of the haplotypes were shared between two or more populations. The overall non-synonymous to synonymous substitution ratio was 0.43, and the Tajima's D values were negative in all countries, with statistically significant lower values in Kenya (- 2.131; P < 0.01) and Tanzania (- 2.056; P < 0.05), while Ethiopia (- 0.972; P > 0.10) and Uganda (- 0.096; P > 0.10) had relatively higher values. Principal component analysis (PCA) and Wright's fixation index (FST) did not show any population differentiation. Similarly, the phylogenetic analysis indicated limited sequence divergence among populations. Conclusion: Low levels of genetic diversity and differentiation in the parasite populations was observed across the four countries suggesting that the Pfs230 gene is highly conserved, supporting its potential as a promising TBV candidate. Future studies focusing on Pfs230, alongside other key TBV targets will be essential for strengthening evidence-based needs to prioritize and incorporate this gene in the search for malaria vaccines with a broader protection against different parasite stages.

In anticipation of future coronavirus (CoV) pandemics, developing vaccines that elicit broadly neutralizing antibodies (bnAbs) against diverse CoVs is critical. Here, we vaccinated rhesus macaques with the SARS-CoV-2 spike (S)-protein, then boosted with heterologous β-CoV S-proteins to focus responses to common conserved S2 bnAb epitopes. Initial SARS-CoV-2 priming elicited receptor-binding domain (RBD)-focused responses, while MERS-CoV boosting redirected responses toward the S2 region, including the stem-helix bnAb site. Although S2-directed serum cross-neutralization was undetectable and most isolated cross-reactive monoclonal antibodies (mAbs) targeted non-neutralizing epitopes, two S2 stem-helix mAbs were identified from memory B cells. These bnAbs neutralized diverse sarbeco- and merbecoviruses, including MERS-CoV, and conferred robust in vivo protection against SARS-CoV-2 challenge. Structural studies reveal that these macaque bnAbs closely mimic human S2 stem bnAbs induced by infection. These findings provide proof-of-principle for vaccination strategies that elicit broadly protective β-coronavirus responses and highlight non-human primates as a translational model for evaluating S2-targeted immunogens.

Machine learning (ML) has the potential to revolutionize antibody design and selection, but its success depends on access to well-curated datasets of antibody-antigen interactions. We developed a synthetic Fab yeast display library optimized for seamless integration with ML processes, focusing on sequence diversity within the complementary determining region heavy chain CDRH3 loop. The library incorporates key sequence features derived from human B cell repertoires captured in a compact antigen recognition module (ARM) format. Built with the VH1-69 heavy chain and four light chains, the library was evaluated against ten human and murine cell surface antigens, including programmed cell death ligand 1 (PD-L1), T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT), and roundabout guidance receptor 1 (ROBO1). This approach yielded hundreds of antibodies with robust biophysical properties, some of which were validated by flow cytometry and immunohistochemistry. Furthermore, ML analysis identified additional antibodies for ROBO2 and PD-L2 from the aggregate sequencing data. The publicly available dataset establishes an ML-compatible framework designed to accelerate and streamline antibody discovery and development. A record of this paper's transparent peer review process is included in the supplemental information.

The penile epithelium, encompassing multiple anatomical sites, is the primary location of human immunodeficiency virus (HIV) acquisition in heterosexual men. Although the per-contact risk of penile HIV acquisition is generally low, substantial global discrepancies in HIV prevalence still exist, particularly in low-income regions. In uncircumcised men, the immune milieu of the subpreputial space is a key determinant of HIV risk, with inflammation-mediated epithelial disruption and target cell recruitment facilitating viral infection. Specific bacterial components of the penile microbiome cause local inflammation and enhance susceptibility, while penile circumcision reduces HIV risk by both removing susceptible foreskin tissues and reducing the abundance of these bacteria. The penile urethra is also an important site of HIV acquisition, particularly among circumcised men, but determinants of urethral susceptibility remain poorly understood. Penile-vaginal sex induces transient inflammation and epithelial damage at both the subpreputial space and urethra, likely mediated by mechanical effects and/or the sexual exchange of pro-inflammatory bacteria. This review summarizes knowledge regarding the immunological and microbial determinants of penile HIV acquisition risk, highlights biological factors and sexual practices that shape the penile immune milieu, and discusses current advances in microbiome-targeting interventions as potential HIV prevention strategies.