B and T cell responses to the BNT162b2 COVID-19 mRNA vaccine are not impaired in germ-free or antibiotic-treated mice

As reviewed by us recently, an increasing number of studies suggest that the gut microbiota is an important regulator of immune responses to vaccination. Consistent with these data, several recent clinical studies, including three published in Gut, uncover correlations between the composition of the faecal/gut microbiota and antibody responses to different COVID19 vaccines. Additionally, recent antibiotic usage has also been associated with a lower seroconversion rate following BNT162b2 vaccination. These studies suggest that the gut microbiota plays a significant role in regulating optimal immune responses to COVID19 vaccines. Despite these important studies, the causal and mechanistic links between the gut microbiota and responses to COVID19 vaccines remain to be elucidated. To investigate these links, we turned to two wellestablished models used previously to assess the relationship between the gut microbiota and responses to vaccination; antibiotictreated and germfree (GF) mice. We first assessed Spikespecific and receptorbinding domain (RBD)specific IgG responses (online supplemental material 1) in BNT162b2vaccinated antibiotics treated (ABX) mice, relative to vaccinated untreated (No ABX) SPF mice (two 3 μg doses intramuscular injection (i.m.) 2 weeks apart). Despite significant depletion of the gut microbiota at the time of primary vaccination (online supplemental figures S1A), Spike/RBDspecific IgG responses were not significantly different between ABX and No ABX mice before, or after, the secondary vaccination (figure 1AB, online supplemental figures S1B). Next, we assessed T cell cytokine responses following in vitro stimulation with an overlapping Spike peptide pool at 6 weeks postboost. While CD4 T cells demonstrated minimal cytokine secretion (online supplemental figures S1CD), CD8 T cells mounted robust Spikespecific recall responses, however, there was no significant difference in cytokine secretion between ABX and No ABX mice (figure 1CD). We next considered that complete depletion of the gut microbiota in GF mice might have a greater impact on immune responses to the BNT162b2 vaccine. We therefore assessed Spikespecific IgG responses in BNT162b2vaccinated GF mice in comparison to conventional SPF mice and GF mice that were recolonised via a faecal microbiota transplant (FMT). As we observed in ABX mice, total IgG responses to vaccination were not significantly impaired in GF mice (figure 1EF). There was also no significant difference in the number of Spikespecific IgG antibody secreting cells recovered from the spleen and bone marrow at 6 weeks postboost (figure 1GH). Additionally, there was no significant difference in the pseudovirus (WuhanHu1 and Omicron) neutralising capacity of BNT162b2induced antibodies at 2 weeks postboost (online supplemental figures S1EF). Interestingly, there may be some differences in class switching in GF relative to SPF mice, as IgG1 and IgG2c responses were significantly higher and lower, respectively, in GF mice at specific time points postvaccination (online supplemental figures S1GH). At most time points, however, there were no significant differences between GF and SPF mice. The number and proportion of germinal centre B (GCB) and T follicular helper (Tfh) cells in the vaccinedraining iliac lymph node (iliLN) were also not significantly different in GF, compared with SPF mice, at 2 weeks postboost (online supplemental figures S1IL). In the spleens of GF mice, however, there was a significant increase in the frequency and number of total GCB but not Tfh cells, relative to SPF mice (online supplemental figures S1MN). Spikespecific CD8 T cell responses were also assessed by intracellular cytokine staining and were not significantly Letter