Transcriptionally active nasopharyngeal commensals and opportunistic microbial dynamics define mild symptoms in the COVID 19 vaccination breakthroughs

Abstract

The development of COVID 19 vaccines as an effort to mitigate the outbreak, has saved millions of lives globally. However, vaccination breakthroughs have continuously challenged the vaccines' effectiveness and provided incentives to explore facets holding potential to alter vaccination-induced immunity and protection from subsequent infection, especially VOCs (Variants Of Concern). We explored the functional dynamics of nasopharyngeal transcriptionally active microbes (TAMs) between vaccination breakthroughs and unvaccinated SARS-CoV-2 infected individuals. Microbial taxonomic communities were differentially altered with skewed enrichment of bacterial class/genera of Firmicutes and Gammaproteobacteria with grossly reduced phylum Bacteroidetes in vaccination breakthrough individuals. The Bacillus genus was abundant in Firmicutes in vaccination breakthrough whereas Prevotella among Bacteroides dominated the unvaccinated. Also, Pseudomonas and Salmonella of Gammaproteobacteria were overrepresented in vaccination breakthrough, whilst unvaccinated showed presence of several genera, Achromobacter, Bordetella, Burkholderia, Neisseria, Hemophilus, Salmonella and Pseudomonas, belonging to Proteobacteria. At species level, the microbiota of vaccination breakthrough exhibited relatively higher abundance of unique commensals, in comparison to potential opportunistic microbes enrichment in unvaccinated patients' microbiota. Functional metabolic pathways like amino acid biosynthesis, sulphate assimilation, fatty acid and beta oxidation, associated with generation of SCFAs (short chain fatty acids), were enriched in vaccination breakthroughs. Majorly, metabolic pathways of LCFAs biosynthesis (long chain fatty acids; oleate, dodecenoate, palmitoleate, gondoate) were found associated with the unvaccinated. Our research highlights that vaccination decreases the microbial diversity in terms of depleting opportunistic pathogens and increasing the preponderance of commensals with respect to unvaccinated patients. Metabolic pathway analysis substantiates the shift in diversity to functionally modulate immune response generation, which may be related to mild clinical manifestations and faster recovery times during vaccination breakthroughs.

Fig 1. Summary of study cohort, dual RNA-seq, functional analysis, inference and clinical sample metadata.

(a) Study design illustrating the patient cohorts, experimental workflow and functional interpretation of the analysis for the differentially abundant microbes. It also highlights the sample cohort of vaccination breakthrough/unvaccinated and the differential disease severity of mild, moderate and severe. (b) Individual plotting of clinical symptoms for unvaccinated and vaccination breakthrough patients with specific details of hospital stay, fever, cough, body ache, shortness of breath, comorbidities and vaccination status. Figure created with Biorender.com.

Fig 2. Microbial abundance, richness and evenness distribution across the vaccination breakthrough and the unvaccinated patients.

Alpha diversity: a) Shannon (Richness), b) Simpson (Evenness), c) Chao-1 (Abundance-based estimator) showing significant changes in microbiome abundance for vaccination breakthrough and unvaccinated patients, with p-values calculated by the Kruskal Wallis test. d) Beta diversity: Principal Coordinate Analysis (PCoA) shows the differential composition of the nasopharyngeal microbes between vaccination breakthrough and the unvaccinated patients. (e) Illustration of the percent differential abundance of phyla and genera across unvaccinated and vaccination breakthrough cases.

Fig 3. Microbial features across unvaccinated and vaccination breakthrough cohorts.

(a) Cladogram (b) Histogram. Differential taxa between two groups selected on the basis of discriminative features by LEfSe analysis (LDA scores >4.0). Green and red indicate taxa enriched in the vaccination breakthrough and the unvaccinated patients, respectively. Depiction of the most abundant microbes at different levels in each group: k, kingdom; p, phylum; f, family; c, class; g, genus; s, species.

Fig 4. Unique and shared microbial species between the vaccination breakthrough and the unvaccinated patients.

(a) Heatmap highlights the sample level dynamics of the common microbial species between the two sub-groups. The sub-clustering of the microbial species has been further highlighted with an encircled box. (b) The microbial class of the differentially abundant unique microbes between the vaccination breakthrough and unvaccinated patients highlight the possible functional dynamics with differential abundance of the commensal and opportunistic microbes.

Fig 5. Summary of the differentially abundant microbial species, their known functional role and understanding the disease trajectory.

Illustrates the differentially abundant microbial species into commensal and opportunistic, uniquely and shared presence in the groups, functional interpretation, and their role towards distinct immune response in the unvaccinated and vaccination breakthrough individuals correlating with the clinical outcome. Figure created with Biorender.com.

Fig 6. Functional metabolic pathways analysis for the differentially abundant microbial species.

(a) Top 29 MetaCyc pathways identified through STAMP analysis which are differentially significantly abundant for the unvaccinated and the vaccination breakthrough patients. (b) Bacterial species contribution from the unvaccinated and vaccination breakthrough cohort leading to enrichment of the top 29 MetaCyc pathways. The color gradient denotes differences in enrichment of pathways based on bacterial species contribution.

Fig 7. Comparison of microbial diversity between the two cohorts and validation of the disease severity associated microbial species.

Alpha diversity comparison (a) Shannon (b) Simpson of COVID 19 sub-phenotypes (mild moderate & severe) with unvaccinated and vaccination breakthrough groups. (c) Principal Coordinate Analysis (PCoA) plot representing the beta diversity of bacterial species across mild, moderate, severe, unvaccinated and vaccination breakthrough groups. (d-f) Comparison of three differentially abundant bacterial species (Halomonas sp) in mild, (V. Parvula) in moderate and (L. Buccalis) in severe with unvaccinated and vaccination breakthrough cases, respectively.

Fig 8. Summarizes differential abundance of commensals and opportunistic microbes modulating disease trajectory in vaccination breakthroughs and unvaccinated infections of SARS-CoV-2.

Figure created with Biorender.com.