Vaccination with an HIV T-cell immunogen induces alterations in the mouse gut microbiota

Abstract

The gut microbiota is emerging as a crucial factor modulating vaccine responses; however, few studies have investigated if vaccines, in turn, can alter the microbiota and to what extent such changes may improve vaccine efficacy. To understand the effect of T-cell vaccination on the gut microbiome, we administered an HIV-1 T-cell immunogen (HTI arm) or PBS (control, Mock arm) to C57Bl/6 mice following a heterologous prime-boost scheme. The longitudinal dynamics of the mice gut microbiota was characterized by 16 S ribosomal RNA sequencing in fecal samples collected from cages, as well as from three gut sections (cecum, small and large intestine). Serum and spleen cells were obtained at the last time point of the study to assess immune correlates using IFNγ ELISPOT and cytokine Luminex^® assays. Compared with Mock, HTI-vaccinated mice were enriched in Clostridiales genera (Eubacterium xylanophilum group, Roseburia and Ruminococcus) known as primary contributors of anti-inflammatory metabolites, such as short-chain fatty acids. Such shift was observed after the first HTI dose and remained throughout the study follow-up (18 weeks). However, the enriched Clostridiales genera were different between feces and gut sections. The abundance of bacteria enriched in vaccinated animals positively correlated with HTI-specific T-cell responses and a set of pro-inflammatory cytokines, such as IL-6. This longitudinal analysis indicates that, in mice, T-cell vaccination may promote an increase in gut bacteria known to produce anti-inflammatory molecules, which in turn correlate with proinflammatory cytokines, suggesting an adaptation of the gut microbial milieu to T-cell-induced systemic inflammation.

Fig. 1. Study design and sample collection.

Female and male mice were immunized with an HIV T-cell vaccine (three DNA.HTI primes followed by a ChAd.HTI and MVA.HTI booster; n = 20) or a mock vaccine (PBS; n = 20). Antibiotic conditioning followed by fecal microbiota transfer was performed prior to vaccination to homogenize the intestinal content across all animals. Collection of samples, including stool, luminal content, serum and spleen was completed at the indicated time points. Abbreviations: FMT fecal microbiota transfer, Atb Antibiotic, PBS phosphate-buffered saline. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

Fig. 2. Temporal dynamics in microbiome composition after vaccination.

a Stacked bar chart displaying the relative abundance of bacterial families (top 15 most abundant) at different timepoints in HTI-vaccinated (Immunized) or PBS-vaccinated (Mock) mice. CageID (pooled feces) and individual samples for each cage (intestinal content) are reported in the x-axis. PCoA biplot based on the first and second components (Bray–Curtis distances at genus level) of (b) fecal samples from cages (c) and from gut sections (small intestine, caecum and large intestine). Biplots report genera with the top 5 effects on the community composition, with vector position indicating the direction of the effect. Arbitrary ellipses were drawn to facilitate the interpretation of the figure. PERMANOVA r-squared and p-values are shown. d Alpha diversity based on Shannon entropy index (ASV level) of individual fecal samples obtained at the last point of the study from small intestine, caecum and large intestine. Boxplots are colored by the experimental group. Median values and interquartile ranges are shown in boxplots. p-values from Wilcoxon signed rank tests are reported.

Fig. 3. Discriminative genera between HTI- and PBS-immunized mice following vaccination.

LEfSe analysis comparing the relative abundance of bacterial genera between Immunized and Mock groups during vaccine administration (pooled fecal samples at 3 week, 9 week, 6 week and 15 week) and at end of study at week 15 for individual fecal samples from small intestine, caecum and large intestine. Horizontal bars represent the effect size for each discriminant genus (p < 0.05 and LDA scores > 2.0). The bar length represents the log10 transformed LDA score, indicated by vertical lines.

Fig. 4. Correlation analysis between discriminant bacteria, serum cytokines and immunological responses at the last time point of the study.

Spearman rank correlations between bacterial genera (only bacteria longitudinally enriched in HTI-immunized mice were evaluated), HTI immunogenicity (magnitude and breadth of the response) and serum cytokine levels. Associations are assessed for each gut section (small intestine, caecum and large intestine). Cytokines for each gut section are ordered based on unsupervised hierarchical clustering. Color and size of the square represent the magnitude of the correlation. Significance is indicated by white asterisks (*p < 0.05; **p < 0.01; ***p < 0.001 after Benjamini–Hochberg adjustment for multiple comparisons).