SARS-CoV-2 infection and replication take place in the oral cavity and saliva (1). The presence of local immune control is important in limiting the viral infection and transmission. A durable mucosal immunity at this site is therefore a highly desired outcome in a COVID-vaccination. One of the most immunogenic COVID-19 vaccines - the BNT162b2 (Comirnaty^®) mRNA vaccine induces high titres of systemic SARS-CoV-2 spike-specific antibodies (2, 3). In the oral cavity and saliva, it conveys virus-binding antibodies highly correlative to neutralizing capacity. The magnitude of this acquired local immunity however varies among both healthy and immunocompromised vaccinees (4), and the durability of those translocated specific antibodies at the oral mucosa is presently unknown. Recent developments in techniques for microbiome sequencing have enabled a comprehensive analysis of commensal microbiota, with over 700 bacterial species detected in the oral cavity (5). The variants of microorganisms form unique communities, and the oral microbiome composition is believed to be resilient and remain rather stable within individuals longitudinally (6). The normal oral microbiota is continuously in contact with the oral mucosa and plays a role in modulating immune surveillance mechanisms (7). Dysbiotic oral microbiota on the other hand is associated with oral diseases, medical conditions, dietary habits, and lifestyles (8–10). Recently, a negative link between vaccine efficacy and gut microbiota variation linked to BMI and systemic inflammation was reported (11), other studies have reported that decreased seroconversion in BNT162b2 mRNA recipients is associated with antibiotic use (12). Although the gut microbiota is recognized as a key factor in supporting gut homeostasis and health (13), the contribution of oral microbiota to COVID vaccination is still unknown.

In the present study, we hypothesize that oral microbiota composition has a role in the maintenance of the salivary immunity induced by mRNA vaccination. We investigated the salivary microbiota using 16S rRNA sequencing analysis in samples obtained from healthy participants (HC) and people living with HIV (PLHIV). The aim was to characterize salivary microbiota signatures associated with a durable vaccine response capable of persisting at the oral mucosal site.

The microbiota is fundamental for health and the evolution of the immune system with the microbiota is interconnected (13). We here describe the baseline oral microbiome composition with immunogenicity follow-up in a longitudinal cohort of healthy or people living with HIV vaccinated with BNT162b2 mRNA vaccine. We found that numerous features in the oral microbiome, including the diversity, specific bacteria taxa, and functions, correlated significantly with the immunogenicity of mRNA COVID-19 vaccination - in a primary anatomical site that needs immune protection against the SARS-CoV-2 infection (1). Our finding is in line with recent studies indicating that the gut microbiome or antibiotics usage could influence the systemic immunogenicity of the same mRNA vaccine (11) studied in the present study. We found that the durability of the salivary as well as the serum immunity through the mRNA-vaccination (two doses) lasted for at least six months. But it was negatively affected by the presence of a dysbiotic oral microbiome signature in both healthy and HIV participants who participated in our clinical trial (16). Here, we identified that the oral microbiota signature of high responders in HC and PLHIV individuals harboured significantly higher abundances of gram-positive facultative anaerobes saccharolytic bacteria, particularly Corynebacterium spp., Abiotrophia spp., Rothia spp., Gemella spp., and Granulicatella spp. In contrast, the low responders showed significantly higher abundances of gram-negative rod-shaped anaerobic proteolytic bacteria, including Campylobacter spp., Selenomonas spp., Butyrivibrio spp., Leptotrichia spp., Megasphaera spp., and Prevotella spp. Our results further indicate that a combined oral bacterial panel has the highest ability to predict the antibody magnitude and duration in saliva following the mRNA vaccination, which is consistent with the recent gut microbiome study on a one-month follow-up of COVID-19 vaccinees (11). Besides that, our findings also suggest that the durability of vaccine-induced immunity in the oral cavity could be influenced by the baseline oral microbiome of the vaccinees up to six months.

That the oral microbiome community might regulate local vaccine-induced mucosal immunity is intriguing and to our knowledge, similar results have not been reported before for other COVID-vaccines. The findings are of interest for vaccine strategies that aim at improving the mucosal immune memory. Although the precise mechanistic role of oral microbiota in vaccine response is unknown, several potential mechanisms could explain the link between deferentially enriched oral taxa and the persistence of salivary spike-IgG in the vaccinees. While most salivary IgG antibodies are derived from the bloodstream by passive leakage of the periodontal epithelium, there can also be some local production by salivary gland plasma cells (17). Data from molecular studies suggest that extracellular receptors such as toll-like receptor 2 (TLR2) and cytoplasmic receptors such as nucleotide-binding oligomerization domain protein 2 (NOD2) can recognize Gram-positive peptide fragments or metabolites released by bacteria that trigger the NFκB pathway (18). This pathway induces further phosphorylation of tight junction proteins, which promotes the expression of polymeric Ig receptor (pIgR), controlling the rate of production, translocation, and secretion of salivary immunoglobulins. Therefore, the high abundance of Gram-positive facultative bacteria in high responders may explain the durability of salivary spike IgG levels. Previous clinical studies have also shown an inverse relationship between oral diseases such as tooth decay, oral mucositis, and salivary immunoglobulin antibodies (19, 20). Further, pathobionts “formerly known as periodontitis-associated bacteria” and abundant also in patients with edentulism, play unique and synergistic roles in dysbiosis of oral microbiota (21). Intriguingly, such pathobionts, especially the strictly anaerobe LPS-producing rods (Campylobacter spp., Selenomonas spp., Butyrivibrio spp., Leptotrichia spp., Megasphaera spp., and Prevotella spp.) are enriched in the saliva of the low responders. These pathobionts are known for potent proteolytic activity that can break down immunoglobulins, complements, and other innate defence proteins (22), and an inflammation-type dysbiosis has similarly been associates with long COVID (23). Whether oral health-related interventions or inhibition of specific microbial adhesion, pro-inflammatory mechanisms can further improve the duration of protective antibodies in the oral cavity during systemic or even mucosal vaccination, therefore deserves further investigation.

Our study design had considered age, sex, and BMI, which are all associated with vaccine efficacy against SARS-CoV-2 (24), as well as antibiotic use that could reduce COVID-19 vaccine immunogenicity (12). Those with exposure to SARS-CoV-2 infection before and during the study were omitted to reduce the potential bias of natural infection affecting vaccine-related antibody levels. All test platforms and clinical trial related procedures were also highly standardised and monitored (4, 14, 16). The present study is not without limitations, only baseline microbiome timepoint was characterised only, the sample size is relatively small, oral health data are lacking, the microbiome analysis was a targeted approach, and only binding IgG antibodies up to six-months after vaccination were measured.

In conclusion, our study is the first to demonstrate that oral microbiome may have a role in maintaining the long-term antibody persistence in saliva and in blood after COVID-19 mRNA vaccination. Including oral health- or microbiome-targeted interventions to improve the long-term mucosal memory after vaccination should be further explored.

The salivary antibody data were acquired as median fluorescence intensities (MFI) for each sample and antigen. The antigen and assay specific cutoff for positivity was calculated as the mean plus 6x standard deviation (SD) of the intensity signals of the 12 selected negative controls. The inter-assay variability was estimated for Spike-f and S1 as the average percent CV of the 16 control samples included in all 6 assay runs required to test the samples included in the current study. Statistical analyses (except microbiome analysis) were performed with Prism software v.9 (GraphPad) and SPSS version 24.0 (IBM Corp, Armonk, NY, USA) Statistics. Datasets initially underwent a data normality distribution test. Differences between groups of samples were analyzed by Mann-Whitney U test for univariate analysis. Correlations were determined using Spearman rank correlation. Two-sided p values <0.05 were considered significant.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA900274.

The studies involving human participants were reviewed and approved by Swedish Ethical Review Authority. The patients/participants provided their written informed consent to participate in this study.

Conception, planning, funds acquisition: PN, H-GL, PL. SA, MSC. Planning and organised clinical work: PB, PL, PC, PN, SA. Performed laboratory analyses or statistics: MG, KA-M, SN, KH, GG, MS, SR, MA, SM, GB, EP. Conclusion and interpretation of result: MG, KA-M, EP, PN, SA, MSC. Wrote first draft: MG, KA-M, MSC. Reviewed and edited final version: All co-authors. All authors contributed to the article and approved the submitted version.