On the origin of Omicron's unique Spike gene insertion

The emergence of a heavily mutated SARS-CoV-2 variant (Omicron; B.1.1.529/BA.1/BA.2) and its rapid spread globally created public health alarms. Characterizing the mutational profile of Omicron is necessary to interpret its shared or distinctive clinical phenotypes with other SARS-CoV-2 variants. We compared the mutations of Omicron with prior variants of concern (Alpha, Beta, Gamma, Delta), variants of interest (Lambda, Mu, Eta, Iota and Kappa), and [~]1500 SARS-CoV-2 lineages constituting [~]5.8 million SARS-CoV-2 genomes. Omicrons Spike protein has 26 amino acid mutations (23 substitutions, two deletions and one insertion) that are distinct compared to other variants of concern. Whereas the substitution and deletion mutations have appeared in previous SARS-CoV-2 lineages, the insertion mutation (ins214EPE) has not been previously observed in any other SARS-CoV-2 lineage. Here, we discuss various mechanisms through which the nucleotide sequence encoding for ins214EPE could have been acquired and highlight the plausibility of template switching via either the human transcriptome or prior viral genomes. Analysis of homology of the inserted nucleotide sequence and flanking regions suggests that this template switching event could have involved the genomes of SARS-CoV-2 variants (e.g. B.1.1 strain), other human coronaviruses that infect the same host cells as SARS-CoV-2 (e.g. HCoV-OC43 or HCoV-229E), or a human transcript expressed in a host cell that was infected by the Omicron precursor. Whether ins214EPE impacts the epidemiological or clinical properties of Omicron (e.g. transmissibility) warrants further investigation. There is also a need to understand whether human host cells are being exploited by SARS-CoV-2 as an evolutionary sandbox for inter-viral or host-virus genomic interplay to produce new SARS-CoV-2 variants.

Genomic diversification of long polynucleotide fragments is a signature of emerging SARS-CoV-2 variants of concern

Highly transmissible or immuno-evasive SARS-CoV-2 variants have intermittently emerged and outcompeted previously circulating strains, resulting in repeated COVID-19 surges, reinfections, and breakthrough infections in vaccinated individuals. With over 5 million SARS-CoV-2 genomes sequenced globally over the last 2 years, there is unprecedented data to decipher how competitive viral evolution results in the emergence of fitter SARS-CoV-2 variants. Much attention has been directed to studying how specific mutations in the Spike protein impact its binding to the ACE2 receptor or viral neutralization by antibodies, but there is limited knowledge of a genomic signature that is shared primarily by the sequential dominant variants. Here we introduce a methodology to quantify the genome-wide distinctiveness of polynucleotide fragments of various lengths (3-to 240-mers) that constitute SARS-CoV-2 sequences (freely available at https://academia.nferx.com/GENI). Compared to standard phylogenetic distance metrics and overall mutational load, the quantification of distinctive 9-mer polynucleotides provides a higher resolution of separation between VOCs (Reference = 89, IQR: 65-108; Alpha = 166, IQR: 150-182; Beta 130, IQR: 113-147; Gamma = 165, IQR: 152-180; Delta = 234, IQR: 216-253; and Omicron = 294, IQR: 287-315). Omicrons exceptionally high genomic distinctiveness may confer a competitive advantage over both prior VOCs (including Delta) and the recently emerged and highly mutated B.1.640.2 (IHU) lineage. Expanding on this analysis, evaluation of genomic distinctiveness weighted by intra-lineage 9-mer conservation for 1,363 lineages annotated in GISAID highlights that genomic distinctiveness has increased over time (R2=0.37) and that VOCs score significantly higher than contemporary non-VOC lineages, with Omicron among the most distinctive lineages observed till date. This study demonstrates the value of characterizing new SARS-CoV-2 variants by their genome-wide polynucleotide distinctiveness and emphasizes the need to go beyond a narrow set of mutations at known functionally or antigenically salient sites on the Spike protein. The consistently higher distinctiveness of each emerging VOC compared to prior VOCs suggests that real-time monitoring of genomic distinctiveness would aid in more rapid assessment of viral fitness.

High diversity in Delta variant across countries revealed via genome-wide analysis of SARS-CoV-2 beyond the Spike protein

The highly contagious Delta variant of SARS-CoV-2 has emerged as the new dominant global strain, and reports of reduced effectiveness of COVID-19 vaccines against the Delta variant are highly concerning. While there has been extensive focus on understanding the amino acid mutations in the Delta variant s Spike protein, the mutational landscape of the rest of the SARS-CoV-2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS-CoV-2 proteins from nearly 2 million SARS-CoV-2 genomes from 176 countries/territories. Six highly-prevalent missense mutations in the viral life cycle-associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant (cosine similarity: meanAlpha = 0.94, S.D.Alpha = 0.05; meanDelta = 0.86, S.D.Delta = 0.1; Cohen s dAlpha-Delta = 1.17, p-value < 0.001). Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of targetable amino acid mutations in the Delta variant s proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.

COVID-19 vaccines dampen genomic diversity of SARS-CoV-2: Unvaccinated patients exhibit more antigenic mutational variance

Variants of SARS-CoV-2 are evolving under a combination of immune selective pressure in infected hosts and natural genetic drift, raising a global alarm regarding the durability of COVID-19 vaccines. Here, we conducted longitudinal analysis over 1.8 million SARS-CoV-2 genomes from 183 countries or territories to capture vaccination-associated viral evolutionary patterns. To augment this macroscale analysis, we performed viral genome sequencing in 23 vaccine breakthrough COVID-19 patients and 30 unvaccinated COVID-19 patients for whom we also conducted machine-augmented curation of the electronic health records (EHRs). Strikingly, we find the diversity of the SARS-CoV-2 lineages is declining at the country-level with increased rate of mass vaccination (n = 25 countries, mean correlation coefficient = -0.72, S.D. = 0.20). Given that the COVID-19 vaccines leverage B-cell and T-cell epitopes, analysis of mutation rates shows neutralizing B-cell epitopes to be particularly more mutated than comparable amino acid clusters (4.3-fold, p < 0.001). Prospective validation of these macroscale evolutionary patterns using clinically annotated SARS-CoV-2 whole genome sequences confirms that vaccine breakthrough patients indeed harbor viruses with significantly lower diversity in known B cell epitopes compared to unvaccinated COVID-19 patients (2.3-fold, 95% C.I. 1.4-3.7). Incidentally, in these study cohorts, vaccinated breakthrough patients also displayed fewer COVID-associated complications and pre-existing conditions relative to unvaccinated COVID-19 patients. This study presents the first known evidence that COVID-19 vaccines are fundamentally restricting the evolutionary and antigenic escape pathways accessible to SARS-CoV-2. The societal benefit of mass vaccination may consequently go far beyond the widely reported mitigation of SARS-CoV-2 infection risk and amelioration of community transmission, to include stemming of rampant viral evolution.

Antigenic minimalism of SARS-CoV-2 is linked to surges in COVID-19 community transmission and vaccine breakthrough infections

The raging COVID-19 pandemic in India and reports of "vaccine breakthrough infections" globally have raised alarm mandating the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 1.57 million SARS-CoV-2 genomes from 187 countries/territories and performed whole-genome viral sequencing from 53 COVID-19 patients, including 20 vaccine breakthrough infections. We identified 89 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a three-month window. Deletions in the Spike protein N-terminal domain (NTD) are highly enriched for these surge-associated mutations (Odds Ratio = 41.8, 95% CI: 6.36-1758, p-value = 7.7e-05). In the recent COVID-19 surge in India, an NTD deletion ({Delta}F157/R158) increased over 10-fold in prevalence from February 2021 (1.1%) to April 2021 (15%). During the recent surge in Chile, an NTD deletion ({Delta}246-253) increased rapidly over 30-fold in prevalence from January 2021 (0.86%) to April 2021 (33%). Strikingly, these simultaneously emerging deletions associated with surges in different parts of the world both occur at an antigenic supersite that is targeted by neutralizing antibodies. Finally, we generated clinically annotated SARS-CoV-2 whole genome sequences and identified deletions within this NTD antigenic supersite in a patient with vaccine breakthrough infection ({Delta}156-164) and other deletions from unvaccinated severe COVID-19 patients that could represent emerging deletion-prone regions. Overall, the expanding repertoire of Spike protein deletions throughout the pandemic and their association with case surges and vaccine breakthrough infections point to antigenic minimalism as an emerging evolutionary strategy for SARS-CoV-2 to evade immune responses. This study highlights the urgent need to sequence SARS-CoV-2 genomes at a larger scale globally and to mandate a public health policy for transparent reporting of relevant clinical annotations (e.g. vaccination status) in order to aid the development of comprehensive therapeutic strategies.

Female-male differences in COVID vaccine adverse events have precedence in seasonal flu shots: a potential link to sex-associated baseline gene expression patterns

Nearly 150 million doses of FDA-authorized COVID vaccines have been administered in the United States. Sex-based differences of adverse events remain poorly understood, mandating the need for real-world investigation from Electronic Health Records (EHRs) and broader epidemiological data sets. Based on an augmented curation of EHR clinical notes of 31,064 COVID-vaccinated individuals (19,321 females and 11,743 males) in the Mayo Clinic, we find that nausea and vomiting were documented significantly more frequently in females than males after both vaccine doses (nausea: RRDose 1 = 1.67, pDose 1 <0.001, RRDose 2 = 2.2, pDose 1 < 0.001; vomiting: RRDose 1 = 1.58, pDose 1 < 0.001, RRDose 2 = 1.88, pDose 1 = 3.4x10-2). Conversely, fever, fatigue, and lymphadenopathy were more common in males after the first dose vaccination (fever RR = 0.62; p = 8.65x10-3; fatigue RR = 0.86, p = 2.89x10-2; lymphadenopathy RR = 0.61, p = 3.45x10-3). Analysis of the Vaccine Adverse Events Reporting System (VAERS) database further confirms that nausea comprises a larger fraction of total reports among females than males (RR: 1.58; p<0.001), while fever comprises a larger fraction of total reports among males than females (RR: 0.84; p<0.001). Importantly, increased reporting of nausea and fever among females and males, respectively, is also observed for prior influenza vaccines in the VAERS database, establishing that these differences are not unique to the recently developed COVID-19 vaccines. Investigating the mechanistic basis underlying these clinical findings, an analysis of bulk RNA-sequencing data from 12,158 human blood samples (8626 female, 3532 male) reveals 85 genes that are not only significantly different in their gene expression between females and males at baseline, but also have established literature-based associations to COVID-19 as well as the vaccine-related adverse events of clinical consequence. The NLRP3 inflammasome and the NR3C1 glucocorticoid receptor emerge as particularly promising baseline links to sex-associated vaccine adverse events, warranting targeted investigation of these signaling pathways and associated cell types. From a public health standpoint, our clinical findings shall aid in educating patients on the sex-associated risks they should expect for COVID-19 vaccines and also promote better clinical management of vaccine-associated adverse events.