The COVID-19 pandemic, causing over 6 million deaths, necessitated the development of effective vaccines. mRNA vaccines like BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) demonstrated high efficacy in initial trials, but their protection wanes over time, highlighting the need to understand factors influencing immunogenicity. Age, medications, comorbidities, and immune system disorders affect vaccine response. Emerging evidence suggests the gut microbiome plays a crucial role, impacting the efficacy of various vaccines. The gut microbiota's crosstalk with the immune system is vital for homeostasis; dysbiosis is associated with immune imbalances. Studies using *Bifidobacterium*, *Clostridium*, and butyrate (a *Clostridia* product) demonstrate their immunomodulatory effects, impacting Treg cell activation and anti-inflammatory functions. Animal and clinical studies show the gut microbiome's influence on vaccine immunogenicity; for example, *Bifidobacterium longum* is linked to T-cell immunity against tuberculosis and polio vaccines. The mechanism involves bacterial-derived adjuvants activating immune pathways. Germ-free or antibiotic-treated mice exhibit lower antibody responses, which can be restored by flagellated *Escherichia coli*, illustrating the adjuvant effect of gut bacteria. Factors like age, obesity, and comorbidities reduce vaccine efficacy. Elderly individuals and those with chronic diseases, especially immunocompromised individuals like PLWH, exhibit lower immunogenicity to COVID-19 vaccines. This study aimed to investigate the association between gut microbiome and COVID-19 vaccine immunogenicity in PLWH and healthy controls, potentially identifying poor responders and paving the way for microbiome-based therapies to improve vaccine efficacy.

Extensive research has established the critical role of the gut microbiome in shaping immune responses. Studies have demonstrated the impact of gut microbiota composition on the effectiveness of various vaccines, including those targeting polio, tuberculosis, and influenza. These studies show a correlation between microbial diversity and vaccine efficacy, with reduced diversity often associated with improved immunogenicity. The mechanisms by which the microbiome influences vaccine responses are complex and multifaceted, involving interactions between microbial metabolites, immune cells, and the gut barrier. For instance, microbial-derived metabolites can act as adjuvants, enhancing immune responses, while imbalances in gut microbiota composition can lead to impaired immune responses. Moreover, the importance of the gut barrier integrity in maintaining the balance and effectiveness of the immune response has also been consistently demonstrated in literature.

This open-label, non-randomized clinical trial involved healthy controls (HC) and people living with HIV (PLWH). Fecal samples were collected at baseline, and blood samples were collected at baseline and day 35 post-second vaccination. The BNT162b2 mRNA vaccine was used. 16S rRNA gene sequencing was performed on the fecal samples to analyze gut microbiota composition and diversity. Spike IgG titers and spike-specific CD4⁺ T-cell responses were measured in the blood samples. Alpha diversity (richness and evenness) was assessed using Observed, Shannon, and Simpson indices. Beta diversity was analyzed using Bray-Curtis distance and NMDS ordination. Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify differentially abundant taxa between groups. Correlation analyses (Spearman correlation) and network analysis were performed to investigate relationships between microbiota, immune responses, and clinical factors. Multivariate regression analysis was used to assess the independent effects of alpha-diversity and other factors on spike IgG levels and CD4⁺ T-cell responses. Differential abundance analysis between high and low responders was performed using DESeq2 to determine log fold change.

The study found a significant negative correlation between gut microbial alpha diversity and both spike IgG titers and spike-specific CD4⁺ T-cell responses after vaccination in both the overall cohort and within the HC and PLWH subgroups. High responders (those with high spike IgG titers and CD4⁺ T-cell responses) exhibited lower alpha diversity compared to low responders. Specific bacterial genera were associated with high or low responses. *Agathobacter*, *Lachnopsira*, and Lachnospiraceae FCS020 group were enriched in high responders, while *Butyricimonas*, *Cloacibacillus*, *Intestinimonas*, *Ruminococcaceae* DTU089, and *Paraprevotella* were enriched in low responders. Within the HC group, *Bacteroides* and *Sutterella* were enriched in high responders, while *Alloprevotella*, *Anaerofilum*, *Succinivibrio*, *Moryella*, and some *Ruminococcaceae* were enriched in low responders. In the PLWH group, *Flavonifractor*, *Lachnospira*, and *Oscillibacter* were increased in high responders, while *Butyricimonas* and *Paraprevotella* were decreased. *Hydrogenoanaerobacterium*, *Methanobrevibacter*, *Cloacibacillus*, and *Ruminococcaceae* DTU089 were enriched in low responders in both groups. The study also found a negative association between age and alpha diversity, with younger adults exhibiting lower diversity and higher spike IgG levels. Network analysis revealed positive associations between spike IgG levels and *Lachnospira*, *Faecalibacterium*, and *Bifidobacterium*, while negative associations were observed with *Methanobrevibacter*, *Ruminococcaceae* DTU089, *Paraprevotella*, *Marvinbryantia*, *Cloacibacillus*, and *Succinivibrio*. DESeq2 analysis showed that *Bifidobacterium*, *Faecalibacterium*, *Blautia*, *Catenibacterium*, and *Hungatella* were enriched in high responders, while *Cloacibacillus* was enriched in low responders, independent of age, gender, and disease status. Multivariate regression analysis confirmed a significant independent association between lower alpha diversity and higher spike IgG levels, even after adjusting for age. The study also observed changes in beta diversity and specific bacterial genera according to immune response, and age.

This study's findings strongly support the hypothesis that the gut microbiome plays a crucial role in modulating the immunological response to COVID-19 mRNA vaccination. The negative correlation between gut microbial diversity and vaccine immunogenicity, observed consistently across the study groups, corroborates previous research on the impact of gut microbiota on vaccine efficacy. The identification of specific bacterial taxa associated with either high or low immune responses provides valuable insights into potential mechanisms. The significant association of *Bifidobacterium* and *Faecalibacterium* with higher antibody responses supports the potential of microbiome-based therapies to improve vaccine efficacy. The negative association of age with diversity and vaccine immunogenicity underscores the importance of age-specific interventions. Further research should investigate the causal relationships between specific microbial taxa and immune responses, potentially leading to personalized approaches to vaccination. The mechanism by which the gut microbiota modulates immune responses is likely multifactorial, and understanding these pathways is critical to developing effective strategies to enhance vaccine immunogenicity.

This study provides compelling evidence for the significant role of gut microbiome composition and diversity in shaping the immunological response to COVID-19 mRNA vaccines. The negative correlation between alpha diversity and vaccine immunogenicity, coupled with the identification of specific bacterial genera associated with high or low responses, highlights the potential for microbiome-based strategies to improve vaccine effectiveness. Future research could focus on targeted interventions to modulate the gut microbiome to enhance immune responses to vaccination, potentially leading to improved vaccine efficacy across different populations.

The observational nature of the study limits the ability to establish causal relationships between gut microbiota and vaccine responses. The relatively small sample size, although encompassing both healthy controls and PLWH, might limit the generalizability of findings. The study focused on a single mRNA vaccine, and results may not be generalizable to other vaccine platforms. Longitudinal studies are needed to assess the long-term impact of gut microbiota on vaccine immunity.