Influenza A virus (IAV) poses a significant threat to swine production globally, causing economic losses and impacting animal welfare. Vaccination is a crucial preventative measure, yet variability in vaccine response exists within pig populations. This study aimed to determine whether pre-vaccination fecal microbiota composition influences the intensity of the immune response to an IAV vaccine. Previous research in humans and mice has shown a link between gut microbiota and vaccine response, suggesting the microbiota's role in modulating immunity and potentially influencing vaccine efficacy. In pigs, prior studies have demonstrated correlations between pre-vaccination fecal microbiota and the response to other vaccines, particularly those involving *Mycoplasma hyopneumoniae*. The present study builds upon this existing knowledge by focusing on IAV vaccination in a cohort of Large White pigs, analyzing the fecal microbiota before and after vaccination and assessing the humoral immune response through serum IAV-specific IgG and HAI titers.

The literature demonstrates that both host genetics and environmental factors contribute to the variable responses observed in vaccination against various pathogens in swine. Genetic studies have shown a significant influence of host genetics on antibody response to different vaccines. However, growing evidence indicates the gut microbiota plays a crucial role in modulating the immune response to vaccines across various species. In humans, this has been shown through the adjuvant properties of specific intestinal bacteria for oral vaccines, stabilization of viruses by bacterial components, and the influence of gut microbiota on remote body systems, like the respiratory system, affecting influenza severity and vaccine response. Specific bacterial metabolites and cell wall components have been identified as potential enhancers of vaccine response. This existing research highlights the need to understand the complex interplay between the gut microbiota and the immune system to optimize vaccination strategies.

This study utilized 98 Large White piglets, vaccinated against IAV at 28 days of age with a booster at 49 days. Fecal samples were collected before vaccination (D28) and at various time points after vaccination (D49, D63, and D146) for 16S rRNA gene sequencing to characterize the fecal microbiota. Blood samples were collected concurrently to assess the humoral immune response via serum levels of IAV-specific IgG and HAI titers. The FROGS pipeline was employed for sequence processing and OTU identification. Alpha diversity (richness, Shannon index) and beta diversity (Bray-Curtis distances) were analyzed. Statistical analyses included linear regression models to assess associations between alpha diversity and vaccine response, Adonis models to compare beta diversity between high and low responders, and differential abundance analysis (metagenomeSeq) to identify OTUs differentially abundant between high and low responders. sPLS-DA was used to identify OTUs predictive of vaccine response levels. A PLS-DA was performed to test the prediction capability of a combined set of predictive OTUs. Finally, a PLS analysis was conducted to evaluate predictability on the whole population using a combined set of OTUs.

The study revealed significant associations between pre-vaccination fecal microbiota composition and vaccine response. Higher alpha diversity (richness) at D28 was associated with stronger vaccine responses (IAV-specific IgG and HAI titers) at D56 and D63. Beta diversity analysis at D28 showed significant differences between high and low responders for IAV-specific IgG at D63. 23 OTUs were differentially abundant between these groups at D28. OTUs associated with a strong response were mainly assigned to the genus *Prevotella* and family *Muribaculaceae*. OTUs linked to a weak response included *Helicobacter*, *Bacteroides*, and *Escherichia-Shigella*. sPLS-DA identified a set of 81 OTUs from the D28 microbiota that accurately predicted IAV-specific IgG and HAI titers at all time points (D49, D56, D63, and D146). The predictive performance was better for IAV-specific IgG, especially at D63 and D146. There was no consistent OTU signature associated with high or low vaccine responses across all time points after D28, suggesting early-life microbiota plays a critical role.

The findings demonstrate a strong link between the pre-vaccination fecal microbiota and the subsequent immune response to IAV vaccination in pigs, supporting the hypothesis that gut microbiota influences vaccine efficacy. The positive association of *Prevotella* and *Muribaculaceae* with stronger responses aligns with their known roles in promoting gut health and immune function in other species. Conversely, the negative association of pathogens like *Helicobacter* and *Bacteroides* with weaker responses suggests that dysbiosis might compromise vaccine efficacy. The ability to predict vaccine response based on the pre-vaccination microbiota composition has significant implications for optimizing vaccination strategies and potentially improving herd immunity. The results highlight the importance of maintaining a diverse and healthy gut microbiota in young pigs to enhance vaccine efficacy.

This study establishes a clear link between the composition of the pre-vaccination fecal microbiota and the immune response to IAV vaccination in pigs. Specifically, *Prevotella* and *Muribaculaceae* OTUs were associated with a stronger response, while *Helicobacter* and *Bacteroides* OTUs were linked to weaker responses. A set of 81 OTUs accurately predicted vaccine response across different time points. These findings suggest that interventions aimed at promoting a diverse and balanced gut microbiota in young pigs may enhance vaccine efficacy and contribute to improved herd health. Future research should focus on mechanistic studies to understand how specific bacterial taxa influence the immune response and explore the potential of microbiota-based interventions to improve vaccine outcomes.

The study primarily focused on Large White pigs, limiting the generalizability of findings to other breeds. The observational nature of the study limits the ability to establish causality between microbiota composition and vaccine response. Future studies utilizing fecal microbiota transplantation could provide stronger evidence of causality. The study also relies on 16S rRNA gene sequencing, which may not fully capture the functional diversity of the microbiota. More comprehensive approaches such as metagenomics may provide additional insights.