The research, led by Ethan Gough and Soumya Basu of the Department of International Health at the Johns Hopkins Bloomberg School of Public Health, together with senior author Subhra Chakraborty and colleagues, drew on stool samples and immune measurements from 27 adult volunteers who participated in a controlled clinical trial. Each volunteer received two doses of ACE527, a live attenuated oral ETEC vaccine candidate, and was subsequently challenged with the virulent ETEC strain H10407, a well-characterized isolate that has long served as a benchmark strain in human challenge studies of enteric vaccines. The trial was registered under ClinicalTrials.gov identifier NCT01060748, and the microbiome analysis was approved by the Johns Hopkins University Institutional Review Board under protocol IRB 20100221.

To characterize the bacterial communities living in the participants’ intestines, the team performed 16S ribosomal RNA sequencing on stool samples collected during the trial. This technique amplifies and sequences a molecular marker present in all bacteria, allowing researchers to identify which microbial taxa are present and in what relative abundances, without needing to culture the organisms in the laboratory. From these data the investigators computed two standard measures of community structure: alpha diversity, which captures how many species are present and how evenly distributed they are within a single sample, and beta diversity, which measures how similar or different microbial communities are between individuals or across time points.

The immune outcomes of interest were drawn from enzyme-linked immunosorbent assay measurements of antibody responses directed against two key ETEC antigens: the B subunit of heat-labile toxin, known as LTB, and colonization factor antigen I, or CFA/I, a surface structure the bacteria use to adhere to the intestinal lining. Antibodies were quantified both in serum, reflecting systemic immunity, and in the antibody-in-lymphocyte-supernatant assay, or ALS, which captures antibodies secreted by circulating plasmablasts and serves as a surrogate for mucosal immune activation in the gut. Both IgG and IgA classes were measured, with IgA of particular interest because it is the dominant antibody isotype at mucosal surfaces and is widely regarded as a correlate of protection against intestinal pathogens.

The statistical analysis went well beyond simple comparisons. The team used regression models to link individual bacterial taxa to immune readouts, applied the Microbiome Regression-Based Kernel Association Test, known as MiRKAT, to test whether overall community composition predicted immune responses, and employed a relaxed least absolute shrinkage and selection operator, or LASSO, approach to identify parsimonious sets of taxa associated with protection. Protection itself was assessed clinically, based on whether volunteers developed severe diarrhea and on whether ETEC could be detected as colonizing the gut after the challenge with H10407.

One of the clearest signals emerged from the alpha diversity analysis. Volunteers with higher inverse-Simpson diversity, a metric that increases both with the number of species present and with their evenness, tended to mount weaker ALS IgA responses against both LTB and CFA/I. In other words, a more diverse gut community was associated with a blunted mucosal antibody response to the vaccine. This finding is intriguing because it runs counter to the common assumption that greater microbial diversity is inherently beneficial; in the specific context of oral live attenuated vaccine immunogenicity, a densely populated and diverse intestinal ecosystem may compete with the vaccine strain or modulate innate immune signaling in ways that dampen the antigen-specific response.

Beta diversity told a different story. The overall composition of the gut community, considered as a whole, correlated with increased serum anti-CFA/I IgA, suggesting that the identity of the microbes present, rather than simply their number, shapes the systemic arm of the antibody response. At the level of individual taxa, the vaccination series was associated with a measurable increase, on the order of 25 to 30 percent, in the relative abundance of several groups, including the Eubacterium brachy group, members of Family XIII AD3011, and Actinomyces. Whether these shifts represent a direct effect of vaccine colonization on the ecosystem or an indirect consequence of the immune activation that vaccination triggers remains an open question, but the consistency of the direction of change across participants suggests a reproducible vaccine-microbiome interaction.

Perhaps the most consequential findings concerned specific bacterial families whose abundance tracked with protection. Members of Anaerovoraceae, Peptostreptococcaceae, Oscillospiraceae, and Veillonellaceae were associated with enhanced immune responses and, importantly, with protection against severe diarrhea and against ETEC colonization following the challenge. In contrast, several other groups, including Ruminococcaceae, Sutterellaceae, Coriobacteria, Clostridia, and Actinobacteria, showed antagonistic associations, being linked in the opposite direction with immune outcomes or protection. These antagonistic taxa are not necessarily harmful in their own right; the associations are statistical relationships that require mechanistic follow-up. Nonetheless, the pattern suggests that the interplay between a person’s resident microbiota and an oral vaccine is not uniform across the microbial community but instead involves specific lineages that either support or hinder the generation of protective immunity.

The implications for vaccine development are considerable. Oral live attenuated vaccines must survive passage through the stomach, establish limited replication in the intestine, and present their antigens to the gut-associated lymphoid tissue, and every one of those steps occurs in the context of a complex microbial ecosystem that can facilitate or obstruct them. If the microbiome modulates take of the vaccine strain, the magnitude of mucosal IgA responses, or the likelihood of sterilizing protection after exposure to wild-type bacteria, then microbiome status may help explain a long-standing puzzle in ETEC vaccine research: why candidate vaccines that perform reasonably well in controlled human challenge studies among adults in industrialized settings often show weaker immunogenicity and efficacy when tested in infants and young children in endemic countries, whose gut communities differ markedly from those of the trial volunteers in Baltimore.

The authors emphasize that the study, funded by the National Institute of Allergy and Infectious Diseases through grants R01AI153399 and R56AI168316 and additionally supported by the PATH agreement GAT.1371-05689-CTA, is an early step in a longer research program. With 27 participants, the analysis is necessarily exploratory, and associations identified through sequencing and regression modeling will need to be validated in larger and more diverse cohorts, ideally including populations in ETEC-endemic regions where the public health burden is greatest. Still, the work offers a concrete roadmap: specific taxonomic markers, measurable by inexpensive 16S sequencing, could eventually help researchers stratify volunteers in vaccine trials, stratify risk in endemic populations, or even guide the rational use of microbiome-directed adjuncts such as targeted prebiotics to maximize the protective potential of future ETEC vaccines. As enteric vaccine science matures, it appears increasingly clear that the answer to whether a vaccine works may lie not only in the vaccine itself but in the trillions of microbes with which it shares the intestine.

Subject of Research: How gut microbiota composition influences immune responses and protection following oral ETEC vaccination and challenge

Article Title: Influence of gut microbiota on immune responses and protection in volunteers receiving the live attenuated oral ETEC vaccine ACE527 followed by virulent ETEC H10407 challenge

Article References: Gough, E., Basu, S., Brubaker, J., DeNeraing, B., Sack, D., Bourgeois, A. L., Walker, R., Harro, C. D., & Chakraborty, S. (2026). Influence of gut microbiota on immune responses and protection in volunteers receiving the live attenuated oral ETEC vaccine ACE527 followed by virulent ETEC H10407 challenge. Gut Pathogens. https://doi.org/10.1186/s13099-026-00878-6

Image Credits: AI Generated

DOI: 10.1186/s13099-026-00878-6

Keywords: ETEC, gut microbiota, ACE527 vaccine, H10407 challenge, IgA response, mucosal immunity, 16S rRNA sequencing, diarrhea, vaccine immunogenicity, microbiome diversity, Influence, microbiota