Intertwined Dysregulation of Ribosomal Proteins and Immune Response Delineates SARS-CoV-2 Vaccination Breakthroughs

Abstract

Globally, COVID-19 vaccines have emerged as a boon, especially during the severe pandemic phases to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, saving millions of lives. However, mixed responses to vaccination with breakthrough challenges provided a rationale to explore the immune responses generated postvaccination, which plausibly alter the subsequent course of infection. In this regard, we comprehensively profiled the nasopharyngeal transcriptomic signature of double-dose-vaccinated individuals with breakthrough infections in comparison to unvaccinated infected persons. The vaccinated individuals demonstrated a gross downregulation of ribosomal proteins along with immune response genes and transcription/translational machinery that methodically modulated the entire innate immune landscape toward immune tolerance, a feature of innate immune memory. This coordinated response was orchestrated through 17 transcription factors captured as differentially expressed in the vaccination breakthroughs, including epigenetic modulators of CHD1 and LMNB1 and several immune response effectors, with ELF1 emerging as one of the important transcriptional regulators of the antiviral innate immune response. Deconvolution algorithm using bulk gene expression data revealed decreased T-cell populations with higher expression of memory B cells in the vaccination breakthroughs. Thus, vaccination might synergize the innate immune response with humoral and T-cell correlates of protection to more rapidly clear SARS-CoV-2 infections and reduce symptoms within a shorter span of time. An important feature invariably noted after secondary vaccination is downregulation of ribosomal proteins, which might plausibly be an important factor arising from epigenetic reprogramming leading to innate immune tolerance. IMPORTANCE The development of multiple vaccines against SARS-CoV-2 infection is an unprecedented milestone achieved globally. Immunization of the mass population is a rigorous process for getting the pandemic under control, yet continuous challenges are being faced, one of them being breakthrough infections. This is the first study wherein the vaccination breakthrough cases of COVD-19 relative to unvaccinated infected individuals have been explored. In the context of vaccination, how do innate and adaptive immune responses correspond to SARS-CoV-2 infection? How do these responses culminate in a milder observable phenotype with shorter hospital stay in vaccination breakthrough cases compared with the unvaccinated? We identified a subdued transcriptional landscape in vaccination breakthroughs with decreased expression of a large set of immune and ribosomal proteins genes. We propose a module of innate immune memory, i.e., immune tolerance, which plausibly helps to explain the observed mild phenotype and fast recovery in vaccination breakthroughs.

Keywords: COVID-19; immune tolerance; milder disease severity; ribosomal proteins; transcription factors; vaccination breakthroughs.

FIG 1

Overview of study design and experimental workflow. (a) Study design illustrating sample collection, patient cohorts, and experimental workflow toward RT-PCR, SARS-CoV-2 genome sequencing, and human host RNA-seq. Highlights the sample cohorts of vaccination breakthrough/unvaccinated and the differential disease severity of mild, moderate and severe. (b) Transcriptomic data analysis followed by screening of differentially expressed genes, downstream functional analyses, and visualizations toward inferences drawn.

FIG 2

Clinical parameters for the vaccination breakthrough and unvaccinated cohorts. (a) Spider plot capturing various disease symptoms of fever, shortness of breath, body ache, headache, sore throat, and cough between the vaccination breakthrough and the unvaccinated individuals. (b to e) Individual clinical variables have been plotted for C_T value of SARS-CoV-2 RdRp gene (b), SpO₂ level (c), number of patients requiring respiratory support (RS) (d), and duration of hospital stay (e), with statistical significance measured using Mann-Whitney U test. ns, not significant.

FIG 3

Differential expression, functional enrichment, and interactome analysis in the vaccination breakthrough and unvaccinated cohorts. (a) Volcano plot representing differentially expressed genes (DEGs) highlights genes with log₂ fold change of ±62 and adjusted P value of <0.05. (b) Classification of DEGs under different functional categories. (c) Dot plot visualization of enriched pathways from the DEGs. (d) PPi enrichment network visualization among the DEGs showing the cluster similarities of enriched terms, taking 10 terms per cluster into consideration. Cluster annotations are shown in color code. (e) Module detection from the PPi interactome using MCODE clustering. Circles represent all the protein nodes. Nodes in each subgraph are colored differently for specific modules.

FIG 4

Coexpression and functional elucidation of transcription factors (TFs). (a) Illustration of TF regulation of different components of DEGs from the vaccination breakthrough and unvaccinated cohorts. (b) Number of target genes associated with TFs from the DEGs. (c) Heat map representing Pearson correlation coefficient between differentially expressed TFs and target genes. (d) Pathway enrichment of TFs regulating the ribosomal and the immune components together as well as differentially associated with ribosomal and immune response genes.

FIG 5

Profiles of immune cell subtype distribution pattern in the vaccination breakthrough and unvaccinated cohorts. (a) Bar plot visualization for the relative proportions of 7 immune cells across all the sample. (b) Box plot of seven specific immune cells with differential infiltrated fraction. (c to e) Box plots for differential infiltrated fraction of dendritic cells and macrophage subtypes (c), B-cell subtypes (d), and CD4⁺ and CD8⁺ T-cell subtypes (e). (f) Correlation heat map of 42 selective marker genes specific to cell types with 17 TFs showing possible correlation of TFs with cell type.

FIG 6

Transcriptomic profile comparison and validation of the specific immune genes for functional concordance across the two study cohorts. (a) Venn diagram representing summary of shared/unique DEGs, presence of immune response, and ribosomal genes across the two-cohort group comparison between the vaccination breakthrough and disease severity subphenotype classification. (b) Heat map representing expression profile (using log₂ fold change) of key marker genes for severity subphenotype classification from previous study cohort. (c) Box plot of key gene expression showing significant presence.

FIG 7

Schematic presentation of summary, possible mechanism for vaccination breakthroughs and milder symptoms. (Top) Concordance of findings from COVID-19 vaccination breakthrough with yellow fever and smallpox vaccination studies reflecting consensus presence of deregulated ribosomal genes. (Bottom) Working hypothesis proposing a possible mechanism for vaccination-induced effect on host immune response associated with vaccination breakthroughs.

FIG 8

Summary of the study highlights. Vaccination breakthroughs demonstrate trained innate immunity with humoral and T-cell correlates of protection to more rapidly clear SARS-CoV-2 infections, leading to fast recovery.