Structural topological analysis of spike proteins of SARS-CoV-2 variants of concern highlight distinctive amino acid substitution patterns

Since the onset of pandemic in 2019, SARS-CoV-2 has diverged into numerous variants driven by antigenic and infectivity-oriented selection. Some variants have accumulated fitness-enhancing mutations, evaded immunity and spread despite global vaccination campaigns. The spike (S) glycoprotein of SARS-CoV-2 demonstrated the greatest immunogenicity and amino acid substitution diversity owing to its importance in the interaction with human angiotensin receptor 2 (hACE2). The S protein consistently emerges as an amino acid substitution (AAS) hotspot in all six lineages, however, in Omicron this enrichment is significantly higher. This study attempts to design and validate a method of mapping S-protein substitution profile across variants to identify the conserved and AAS regions. A substitution matrix was created based on publicly available databases, and the substitution localization was illustrated on a cryo-electron microscopy generated S-protein model. Our analyses indicated that the diversity of N-terminal (NTD) and receptor-binding (RBD) domains exceeded that of any other regions but still contained extended low substitution density regions particularly considering significantly broader substitution profiles of Omicron BA.2 and BA.4/5. Finally, the substitution matrix was compared to a random sample alignment of variant sequences, revealing discrepancies. Therefore, it was suggested to improve matrix accuracy by processing a large number of S-protein sequences using an automated algorithm. Several critical immunogenic and receptor-interacting residues were identified in the conserved regions within NTD and RBD. In conclusion, the structural and topological analysis of S proteins of SARS-CoV-2 variants highlight distinctive amino acid substitution patterns which may be foundational in predicting future variants.     

Severity of the Omicron SARS-CoV-2 variant compared with the previous lineages: A systematic review

The Omicron variant was first detected in October 2021, which evolved from the original SARS-CoV-2 strain and was found to possess many mutations. Immune evasion was one of the notable consequences of these mutations. Despite Omicron exhibiting increased transmissibility, the rates of hospitalizations and deaths among patients infected with this variant were substantially lower when compared to other strains. However, concluding that the Omicron variant is less severe than other variants of SARS-CoV-2 requires consideration of multiple factors, including the vaccination status of infected patients as well as any previous infections with other variants. This review compiled data about any reported indicators of severity in Omicron-infected patients, including studies comparing Omicron with other variants while adjusting for confounders. A comprehensive search was conducted using different databases to target any studies about Omicron. In total, 62 studies met our inclusion criteria and were included in this study. Many studies reported a significantly reduced risk of hospitalization, ICU admission, need for oxygenation/ventilation, and death in Omicron-infected patients compared to patients infected with other variants, such as Delta. Some studies, however, reported comparable severity in Omicron infected patients as to other variants emphasizing a substantial risk for severe illness. Furthermore, the COVID-19 vaccines were less effective against Omicron relative to previous lineages, except after receiving the booster dose. One study recommended vaccination during pregnancy, which may help prevent future cases of severe SARS-CoV-2 pneumonia in neonates and young infants due to the transfer of humoral response from the mother.     

Humoral and Cellular Immune Responses of COVID-19 vaccines against SARS-Cov-2 Omicron variant: a systemic review

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has undergone multiple mutations since its emergence, and its latest variant, Omicron (B.1.1.529), is the most contagious variant of concern (VOC) which poses a major and imminent threat to public health. Since firstly reported by World Health Organization (WHO) in November 2021, Omicron variant has been spreading rapidly and has become the dominant variant in many countries worldwide. Omicron is the most mutated variant so far, containing 60 mutations in its genome, including 37 mutations in the S-protein. Since all current COVID-19 vaccines in use were developed based on ancestral SARS-CoV-2 strains, whether they are protective against Omicron is a critical question which has been the center of study currently. In this article, we systemically reviewed the studies regarding the effectiveness of 2- or 3-dose vaccines delivered in either homologous or heterologous manner. The humoral and cellular immune responses elicited by various vaccine regimens to protect against Omicron variant are discussed. Current understanding of the molecular basis underlying immune escape of Omicron was also analyzed. These studies indicate that two doses of vaccination are insufficient to elicit neutralizing antibody responses against Omicron variant. Nevertheless, Omicron-specific humoral immune responses can be enhanced by booster dose of almost all type vaccines in certain degree, and heterologous vaccination strategy may represent a better choice than homogenous regimens. Intriguingly, results of studies indicate that all current vaccines are still able to elicit robust T cell response against Omicron. Future focus should be the development of Omicron variant vaccine, which may induce potent humoral as well as cellular immune responses simultaneously against all known variants of the SARS-CoV-2 virus.     

Clinical characteristics of 310 SARS-CoV-2 Omicron variant patients and comparison with Delta and Beta variant patients in China

Although the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread, data on the clinical characteristics of infected patients are limited. In this study, the demographic, clinical characteristics, and laboratory data of 310 SARS-CoV-2 Omicron variant patients treated at Haihe Hospital of Tianjin were collected and analyzed. Information on these patients was compared to 96 patients with the Delta variant of concern (VOC) and 326 patients with the Beta VOC during the previous coronavirus disease 2019 (COVID-19) outbreak in Harbin. Of the 310 patients infected with the Omicron variant, the median age was 35 years. Most patients were clinically classified as mild (57.74%), and the most common symptoms were cough (48.71%), fever (39.35%), and sore throat (38.26%). The results for different vaccination groups in the Omicron group showed that the median of “SARS-CoV-2 specific IgG” after 2 or 3 doses of vaccination was higher than the unvaccinated group (all Ps < 0.05). Older age was associated with a higher proportion of moderate cases and lower asymptomatic and mild cases based on clinical classifications. Compared to the Delta and Beta groups, the median age of the Omicron group was younger. The total number of asymptomatic patients and mild patients in the Omicron virus group was higher than the Delta and Beta groups (60.97% vs. 54.17% vs. 47.55%). This study presented the clinical characteristics of the first group of patients infected with the Omicron variant in Tianjin, China, and compared their clinical features with patients infected by the Delta and Beta variants, which would increase our understanding of the characteristics of SARS-CoV-2 Omicron variant.     

Evidence for a mouse origin of the SARS-CoV-2 Omicron variant

The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting a possibility of host-jumping. The molecular spectrum of mutations (i.e., the relative frequency of the 12 types of base substitutions) acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients but resembled the spectra associated with virus evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.     

In-silico genomic landscape characterization and evolution of SARS-CoV-2 variants isolated in India shows significant drift with high frequency of mutations

In-silico studies on SARS-CoV-2 genome are considered important to identify the significant pattern of variations and its possible effects on the structural and functional characteristics of the virus. The current study determined such genetic variations and their possible impact among SARS-CoV-2 variants isolated in India. A total of 546 SARS-CoV-2 genomic sequences (India) were retrieved from the gene bank (NCBI) and subjected to alignment against the Wuhan variant (NC_045512.2), the corresponding amino acid changes were analyzed using NCBI Protein-BLAST. These 546 variants revealed 841 mutations; most of these were non-synonymous 464/841 (55.1%), there was no identical variant compared to the original strain. All genes; coding and non-coding showed nucleotide changes, most of the structural genes showed frequent nonsynonymous mutations. The most affected genes were ORF1a/b followed by the S gene which showed 515/841 (61.2%) and 120/841 (14.3%) mutations, respectively. The most frequent non-synonymous mutation 486/546 (89.01%) occurred in the S gene (structural gene) at position 23,403 where A changed to G leading to the replacement of aspartic acid by glycine in position (D614G). Interestingly, four variants also showed deletion. The variants MT800923 and MT800925 showed 12 consecutive nucleotide deletion in position 21982–21993 resulting in 4 consecutive amino acid deletions that were leucine, glycine, valine, and tyrosine in positions 141, 142, 143, and 144 respectively. The present study exhibited a higher mutations rate per variant compared to other studies carried out in India.     

Ancestral SARS-CoV-2, but not Omicron,replicates less efficiently in primary pediatric nasal epithelial cells

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air–liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Children typically experience more mild symptoms of COVID-19 when compared to adults; why is this? This study uses nasal epithelial cells from children and adults to show that the ancestral SARS-CoV-2 and Delta, but not the Omicron variant, replicate less efficiently in pediatric nasal epithelial cells.     

Genetic and Structural Analysis of SARS-CoV-2 Spike Protein for Universal Epitope Selection

Evaluation of immunogenic epitopes for universal vaccine development in the face of ongoing SARS-CoV-2 evolution remains a challenge. Herein, we investigate the genetic and structural conservation of an immunogenically relevant epitope (C662–C671) of spike (S) protein across SARS-CoV-2 variants to determine its potential utility as a broad-spectrum vaccine candidate against coronavirus diseases. Comparative sequence analysis, structural assessment, and molecular dynamics simulations of C662–C671 epitope were performed. Mathematical tools were employed to determine its mutational cost. We found that the amino acid sequence of C662–C671 epitope is entirely conserved across the observed major variants of SARS-CoV-2 in addition to SARS-CoV. Its conformation and accessibility are predicted to be conserved, even in the highly mutated Omicron variant. Costly mutational rate in the context of energy expenditure in genome replication and translation can explain this strict conservation. These observations may herald an approach to developing vaccine candidates for universal protection against emergent variants of coronavirus.     

The Global Landscape of SARS-CoV-2 Genomes,Variants, and Haplotypes in 2019nCoVR

On January 22, 2020, China National Center for Bioinformation (CNCB) released the 2019 Novel Coronavirus Resource (2019nCoVR), an open-access information resource for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 2019nCoVR features a comprehensive integration of sequence and clinical information for all publicly available SARS-CoV-2 isolates, which are manually curated with value-added annotations and quality evaluated by an automated in-house pipeline. Of particular note, 2019nCoVR offers systematic analyses to generate a dynamic landscape of SARS-CoV-2 genomic variations at a global scale. It provides all identified variants and their detailed statistics for each virus isolate, and congregates the quality score, functional annotation, and population frequency for each variant. Spatiotemporal change for each variant can be visualized and historical viral haplotype network maps for the course of the outbreak are also generated based on all complete and high-quality genomes available. Moreover, 2019nCoVR provides a full collection of SARS-CoV-2 relevant literature on the coronavirus disease 2019 (COVID-19), including published papers from PubMed as well as preprints from services such as bioRxiv and medRxiv through Europe PMC. Furthermore, by linking with relevant databases in CNCB, 2019nCoVR offers data submission services for raw sequence reads and assembled genomes, and data sharing with NCBI. Collectively, SARS-CoV-2 is updated daily to collect the latest information on genome sequences, variants, haplotypes, and literature for a timely reflection, making 2019nCoVR a valuable resource for the global research community. 2019nCoVR is accessible at https://bigd.big.ac.cn/ncov/.     

Vaccine effectiveness against SARS-CoV-2 infection or COVID-19 hospitalization with the Alpha,Delta, or Omicron SARS-CoV-2 variant: A nationwide Danish cohort study

BackgroundThe continued occurrence of more contagious Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants and waning immunity over time require ongoing reevaluation of the vaccine effectiveness (VE). This study aimed to estimate the effectiveness in 2 age groups (12 to 59 and 60 years or above) of 2 or 3 vaccine doses (BNT162b2 mRNA or mRNA-1273) by time since vaccination against SARS-CoV-2 infection and Coronavirus Disease 2019 (COVID-19) hospitalization in an Alpha-, Delta-, or Omicron-dominated period.Methods and findingsA Danish nationwide cohort study design was used to estimate VE against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha, Delta, or Omicron variant. Information was obtained from nationwide registries and linked using a unique personal identification number. The study included all previously uninfected residents in Denmark aged 12 years or above (18 years or above for the analysis of 3 doses) in the Alpha (February 20 to June 15, 2021), Delta (July 4 to November 20, 2021), and Omicron (December 21, 2021 to January 31, 2022) dominated periods. VE estimates including 95% confidence intervals (CIs) were calculated (1-hazard ratio∙100) using Cox proportional hazard regression models with underlying calendar time and adjustments for age, sex, comorbidity, and geographical region. Vaccination status was included as a time-varying exposure. In the oldest age group, VE against infection after 2 doses was 90.7% (95% CI: 88.2; 92.7) for the Alpha variant, 82.3% (95% CI: 75.5; 87.2) for the Delta variant, and 39.9% (95% CI: 26.3; 50.9) for the Omicron variant 14 to 30 days since vaccination. The VE waned over time and was 73.2% (Alpha, 95% CI: 57.1; 83.3), 50.0% (Delta, 95% CI: 46.7; 53.0), and 4.4% (Omicron, 95% CI: −0.1; 8.7) >120 days since vaccination. Higher estimates were observed after the third dose with VE estimates against infection of 86.1% (Delta, 95% CI: 83.3; 88.4) and 57.7% (Omicron, 95% CI: 55.9; 59.5) 14 to 30 days since vaccination. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 98.1% or above for the Alpha and Delta variants. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 95.5% or above for the Omicron variant. The main limitation of this study is the nonrandomized study design including potential differences between the unvaccinated (reference group) and vaccinated individuals.ConclusionsTwo vaccine doses provided high protection against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha and Delta variants with protection, notably against infection, waning over time. Two vaccine doses provided only limited and short-lived protection against SARS-CoV-2 infection with Omicron. However, the protection against COVID-19 hospitalization following Omicron SARS-CoV-2 infection was higher. The third vaccine dose substantially increased the level and duration of protection against infection with the Omicron variant and provided a high level of sustained protection against COVID-19 hospitalization among the +60-year-olds.

Mie Agermose Gram and colleagues estimate vaccine effectiveness against infection and COVID-19 hospitalization with the Alpha, Delta or Omicron variant in Denmark.     

Mutational sensitivity of D614G in spike protein of SARS-CoV-2 in Jordan

BackgroundSpike protein is the surface glycoprotein of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) necessary for the entry of the virus via the transmembrane receptors of the human respiratory cells causing COVID-19 disease.AimHere, we aimed to predict the three-dimensional monomer structure of spike protein of SARS-CoV-2 from 20 Jordanian nasopharyngeal samples and to determine the percentage of single amino acid variants (SAV) in the spike protein of SARS-CoV-2.MethodsThe output of the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) found four single amino acid variants in the spike gene.ResultsThe first variant represented by 5% of samples that showed tyrosine deletion at Y144 located in the N terminal domain. The second and the dominant variant, represented by 62%, showed aspartate a coil amino acid substitution to glycine an extracellular amino acid at D614G located in the spike recognition binding site. The third variant, represented by 5%, showed aspartate substitution to tyrosine at D1139Y, and the fourth variant, represented by 5% glycine substitution to serine at G1167S.ConclusionOur results have shown low mutational sensitivity in all variants except to D614G the one with the most likely neutral mutational sensitivity that all variants might not explicitly affect the function of spike glycoprotein. However, D614G might change the viral conformational plasticity and hence a potential viral fitness gain but one must be cautious about drawing any concrete conclusions about the severity of symptoms and viral transmission from genomic data only.General significanceStudying mutations such as D614G in deep is essential to control the pandemic in terms of immune systems, antibodies, or even vaccines.     

Mutations in spike protein T cell epitopes of SARS-COV-2 variants: Plausible influence on vaccine efficacy

With emerging SARS-CoV-2 variants, vaccines approved so far are under scrutiny for long term effectiveness against the circulating strains. There is a prevalent obsession with humoral immunity as in vitro studies have indicated diminished effects of vaccine-induced neutralizing antibodies. However, this need not clinically translate to vaccine resistance as immune response against all forms of present vaccine preparations is T dependent unlike that against native viral particles which can induce T independent immune responses. Thus, we focused on this major correlate of protection against infections, T cell response. Using bioinformatics tools, we analyzed SARS-CoV-2 Spike protein T cell epitopes and their diversity across Delta plus/B.1.617.2.1, Gamma/P.1 (variant of concern), B.1.1.429, Zeta/P.2 and Mink cluster 5/B.1.1.298 variants as well as Omicron/B.1.1.529 (variant of concern). We also compared HLA restriction profiles of the mutant epitopes with that of the native epitopes (from Wuhan_hu_1 strain, used in vaccine formulations). Our observations show ~90% conservation of CD4+ and CD8+ epitopes across Delta plus/B.1.617.2.1, Gamma/P.1 (variant of concern), B.1.1.429, Zeta/P.2 and Mink cluster 5/B.1.1.298. For the Omicron/B.1.1.529 variant, ~75% of CD4+ and ~ 87% CD8+ epitopes were conserved. Majority of the mutated CD4+ and CD8+ epitopes of this variant were predicted to retain the HLA restriction pattern as their native epitopes. The results of our bioinformatics analysis suggest largely conserved T cell responses across the studied variants, ability of T cells to tackle new SARS-CoV-2 variants and aid in protection from COVID-19 post vaccination. In conclusion, the results suggest that current vaccines may not be rendered completely ineffective against new variants.     

Hetero-bivalent nanobodies provide broad-spectrum protection against SARS-CoV-2 variants of concern including Omicron

SARS-CoV-2 variants with adaptive mutations have continued to emerge, causing fresh waves of infection even amongst vaccinated population. The development of broad-spectrum antivirals is thus urgently needed. We previously developed two hetero-bivalent nanobodies (Nbs), aRBD-2-5 and aRBD-2-7, with potent neutralization activity against the wild-type (WT) Wuhan isolated SARS-CoV-2, by fusing aRBD-2 with aRBD-5 and aRBD-7, respectively. Here, we resolved the crystal structures of these Nbs in complex with the receptor-binding domain (RBD) of the spike protein, and found that aRBD-2 contacts with highly-conserved RBD residues and retains binding to the RBD of the Alpha, Beta, Gamma, Delta, Delta plus, Kappa, Lambda, Omicron BA.1, and BA.2 variants. In contrast, aRBD-5 and aRBD-7 bind to less-conserved RBD epitopes non-overlapping with the epitope of aRBD-2, and do not show apparent binding to the RBD of some variants. However, when fused with aRBD-2, they effectively enhance the overall binding affinity. Consistently, aRBD-2-5-Fc and aRBD-2-7-Fc potently neutralized all of the tested authentic or pseudotyped viruses, including WT, Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.1.1 and BA.2. Furthermore, aRBD-2-5-Fc provided prophylactic protection against the WT and mouse-adapted SARS-CoV-2 in mice, and conferred protection against the Omicron BA.1 variant in hamsters prophylactically and therapeutically, indicating that aRBD-2-5-Fc could potentially benefit the prevention and treatment of COVID-19 caused by the emerging variants of concern. Our strategy provides new solutions in the development of broad-spectrum therapeutic antibodies for COVID-19.Subject terms: X-ray crystallography, Innate immunity     

COVID-19 infection and transmission includes complex sequence diversity

SARS-CoV-2 whole genome sequencing has played an important role in documenting the emergence of polymorphisms in the viral genome and its continuing evolution during the COVID-19 pandemic. Here we present data from over 360 patients to characterize the complex sequence diversity of individual infections identified during multiple variant surges (e.g., Alpha and Delta). Across our survey, we observed significantly increasing SARS-CoV-2 sequence diversity during the pandemic and frequent occurrence of multiple biallelic sequence polymorphisms in all infections. This sequence polymorphism shows that SARS-CoV-2 infections are heterogeneous mixtures. Convention for reporting microbial pathogens guides investigators to report a majority consensus sequence. In our study, we found that this approach would under-report sequence variation in all samples tested. As we find that this sequence heterogeneity is efficiently transmitted from donors to recipients, our findings illustrate that infection complexity must be monitored and reported more completely to understand SARS-CoV-2 infection and transmission dynamics. Many of the nucleotide changes that would not be reported in a majority consensus sequence have now been observed as lineage defining SNPs in Omicron BA.1 and/or BA.2 variants. This suggests that minority alleles in earlier SARS-CoV-2 infections may play an important role in the continuing evolution of new variants of concern.     

Targeting ACLY efficiently inhibits SARS-CoV-2 replication

The Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the biggest public health challenge the world has witnessed in the past decades. SARS-CoV-2 undergoes constant mutations and new variants of concerns (VOCs) with altered transmissibility, virulence, and/or susceptibility to vaccines and therapeutics continue to emerge. Detailed analysis of host factors involved in virus replication may help to identify novel treatment targets. In this study, we dissected the metabolome derived from COVID-19 patients to identify key host factors that are required for efficient SARS-CoV-2 replication. Through a series of metabolomic analyses, in vitro, and in vivo investigations, we identified ATP citrate lyase (ACLY) as a novel host factor required for efficient replication of SARS-CoV-2 wild-type and variants, including Omicron. ACLY should be further explored as a novel intervention target for COVID-19.     

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M^pro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the M^pro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the M^pro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M^pros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant M^pro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and β M^pro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.