Characterization of vaginal lactobacilli from South African women toward the development of a biotherapeutic to optimize the vaginal microbiome.

HIV remains among the world's most serious healthcare challenges, with adolescent girls and young women in sub-Saharan Africa at particularly high risk of infection. Bacterial vaginosis (BV) is a key risk factor for HIV acquisition; however, current treatment strategies are limited. Optimal vaginal lactobacilli protect against BV and HIV, largely through immunoregulatory and antimicrobial activities mediated in part by lactic acid. Toward the development of a live biotherapeutic for African women, we sampled 181 isolates of vaginal lactobacilli from 25 BV-negative South African women. Fifty isolates were selected for evaluation of inflammatory responses using vaginal epithelial cells, D- and L-lactate, lactic acid production, and culture acidification. Aside from a single Ligilactobacillus salivarius strain, Lactobacillus crispatus isolates acidified the culture media the most and produced the most D- and L-lactic acid. Inflammatory cytokine responses to different strains of lactobacilli were variable, with Lb. crispatus eliciting the lowest levels of cytokine production, while some strains induced substantial inflammatory responses. When all properties were evaluated collectively, Lb. crispatus strains exhibited the most desirable biotherapeutic characteristics. Whole genome sequence analysis of 10 Lb. crispatus isolates led to the identification of putative bacteriocins and intact prophage sequences in all isolates, while no antimicrobial resistance elements were detected. Importantly, the majority of Lb. crispatus isolates were more closely related to one another than to isolates from other geographical regions. This supports the need for live biotherapeutics to be tailored for the population of intended use.IMPORTANCEHIV remains highly prevalent in sub-Saharan Africa, particularly among adolescent girls and young women. Bacterial vaginosis (BV), characterized by the loss of protective lactobacilli, affects approximately one in four women in this region and increases HIV susceptibility. However, effective and durable therapeutics are lacking. Live biotherapeutics containing beneficial lactobacilli represent a promising treatment strategy, yet there are no approved products including strains isolated from African women, despite well-established geographic variation in vaginal microbiome composition. Through the characterization of 50 vaginal Lactobacillus, Limosilactobacillus, and Ligilactobacillus strains from 25 BV-negative South African women, we demonstrate substantial strain-level variation in live biotherapeutic-relevant properties. Some isolates produced minimal lactic acid or induced marked inflammatory responses, highlighting the importance of rigorous strain selection. Notably, whole genome sequencing revealed that South African Lactobacillus crispatus strains had distinct genomes compared to isolates from other regions, providing evidence that vaginal live biotherapeutics should be tailored to the populations of intended use.