A fourth dose of the inactivated SARS-CoV-2 vaccine redistributes humoral immunity to the N-terminal domain.

The effectiveness of a 3rd dose of SARS-CoV-2 vaccines waned quickly in the Omicron-predominant period. In response to fast-waning immunity and the threat of Omicron variant of concern (VOC) to healthcare workers (HCWs), we conduct a non-randomized trial (ChiCTR2200055564) in which 38 HCWs volunteer to receive a homologous booster of inactivated vaccines (BBIBP-CorV) 6 months after the 3rd dose. The primary and secondary outcomes are neutralizing antibodies (NAbs) and the receptor-binding domain (RBD)-directed antibodies, respectively. The 4th dose recalls waned immunity while having distinct effects on humoral responses to different antigens. The peak antibody response to the RBD induced by the 4th dose is inferior to that after the 3rd dose, whereas responses to the N-terminal domain (NTD) of spike protein are further strengthened significantly. Accordingly, the 4th dose further elevates the peak level of NAbs against ancestral SARS-CoV-2 and Omicron BA.2, but not BA.1 which has more NTD mutations. No severe adverse events related to vaccination are recorded during the trial. Here, we show that redistribution of immune focus after repeated vaccinations may modulate cross-protective immune responses against different VOCs.

Trem2 deficiency impairs recovery and phagocytosis and dysregulates myeloid gene expression during virus-induced demyelination.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (Trem2) plays a protective role in neurodegenerative diseases. By contrast, Trem2 functions can exacerbate tissue damage during respiratory viral or liver infections. We, therefore, investigated the role of Trem2 in a viral encephalomyelitis model associated with prominent Th1 mediated antiviral immunity leading to demyelination. METHODS: Wild-type (WT) and Trem2 deficient (Trem2-/-) mice were infected with a sublethal glia tropic murine coronavirus (MHV-JHM) intracranially. Disease progression and survival were monitored daily. Leukocyte accumulation and pathological features including demyelination and axonal damage in spinal cords (SC) were determined by flow cytometry and tissue section immunofluorescence analysis. Expression of select inflammatory cytokines and chemokines was measured by RT-PCR and global myeloid cell gene expression in SC-derived microglia and infiltrated bone-marrow-derived macrophages (BMDM) were determined using the Nanostring nCounter platform. RESULTS: BMDM recruited to SCs in response to infection highly upregulated Trem2 mRNA compared to microglia coincident with viral control. Trem2 deficiency did not alter disease onset or severity, but impaired clinical recovery after onset of demyelination. Disease progression in Trem2-/- mice could not be attributed to altered virus control or an elevated proinflammatory response. A prominent difference was increased degenerated myelin not associated with the myeloid cell markers IBA1 and/or CD68. Gene expression profiles of SC-derived microglia and BMDM further revealed that Trem2 deficiency resulted in impaired upregulation of phagocytosis associated genes Lpl and Cd36 in microglia, but a more complex pattern in BMDM. CONCLUSIONS: Trem2 deficiency during viral-induced demyelination dysregulates expression of other select genes regulating phagocytic pathways and lipid metabolism, with distinct effects on microglia and BMDM. The ultimate failure to remove damaged myelin is reminiscent of toxin or autoimmune cell-induced demyelination models and supports that Trem2 function is regulated by sensing tissue damage including a dysregulated lipid environment in very distinct inflammatory environments.

In silico structural inhibition of ACE-2 binding site of SARS-CoV-2 and SARS-CoV-2 omicron spike protein by lectin antiviral dyad system to treat COVID-19.

Spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds angiotensin-converting enzyme-2 (ACE-2) receptors via its receptor-binding domain (RBD) and mediates virus-to-host cell fusion. Recently emerged omicron variant of SARS-CoV-2 possesses around 30 mutations in spike protein where N501Y tremendously increases viral infectivity and transmission. Lectins interact with glycoproteins and mediate innate immunity displaying antiviral, antibacterial, and anticarcinogenic properties. In this study, we analyzed the potential of lectin, and lectin-antibody (spike-specific) complex to inhibit the ACE-2 binding site of wild and N501Y mutated spike protein by utilizing in silico molecular docking and simulation approach. Docking of lectin at reported ACE-2 binding spike-RBD residues displayed the ZDock scores of 1907 for wild and 1750 for N501Y mutated spike-RBD. Binding of lectin with antibody to form proposed dyad complex gave ZDock score of 1174 revealing stable binding. Docking of dyad complex with wild and N501Y mutated spike-RBD, at lectin and antibody individually, showed high efficiency binding hence, effective structural inhibition of spike-RBD. MD simulation of 100 ns of each complex proved high stability of complexes with RMSD values ranging from 0.2 to 1.5 nm. Consistent interactions of lead ACE-2 binding spike residues with lectin during simulation disclosed efficient structural inhibition by lectin against formation of spike RBD-ACE-2 complex. Hence, lectins along with their ability to induce innate immunity against spike glycoprotein can structurally inhibit the spike-RBD when given as lectin-antibody dyad system and thus can be developed into a dual effect treatment against COVID-19. Moreover, the high binding specificity of this system with spike-RBD can be exploited for development of diagnostic and drug-delivery systems.

A comparative study of spike protein of SARS-CoV-2 and its variant Omicron (B.1.1.529) on some immune characteristics.

The emergence of Omicron variant raises great concerns because of its rapid transmissibility and its numerous mutations in spike protein (S-protein). S-protein can act as a pathogen-associated molecular pattern and complement activator as well as antigen. We compared some immune characteristics of trimer S-proteins for wild type (WT-S) and B.1.1.529 Omicron (Omicron-S) to investigate whether the mutations have affected its pathogenicity and antigenic shift. The results indicated that WT-S and Omicron-S directly activated nuclear factor-κB (NF-κB) and induced the release of pro-inflammatory cytokines in macrophages, but the actions of Omicron-S were weaker. These inflammatory reactions could be abrogated by a Toll-like receptor 4 antagonist TAK-242. Two S-proteins failed to induce the production of antiviral molecular interferon-ß. In contrast to pro-inflammatory effects, the ability of two S-proteins to activate complement was comparable. We also compared the binding ability of two S-proteins to a high-titer anti-WT-receptor-binding domain antibody. The data showed that WT-S strongly bound to this antibody, while Omicron-S was completely off-target. Collectively, the mutations of Omicron have a great impact on the pro-inflammatory ability and epitopes of S-protein, but little effect on its ability to activate complement. Addressing these issues can be helpful for more adequate understanding of the pathogenicity of Omicron and the vaccine breakthrough infection.

Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants.

The emergence and global spread of the SARS-CoV-2 Omicron variants, which carry an unprecedented number of mutations, raise serious concerns due to the reduced efficacy of current vaccines and resistance to therapeutic antibodies. Here, we report the generation and characterization of two potent human monoclonal antibodies, NA8 and NE12, against the receptor-binding domain of the SARS-CoV-2 spike protein. NA8 interacts with a highly conserved region and has a breadth of neutralization with picomolar potency against the Beta variant and the Omicron BA.1 and BA.2 sublineages and nanomolar potency against BA.2.12.1 and BA.4. Combination of NA8 and NE12 retains potent neutralizing activity against the major SARS-CoV-2 variants of concern. Cryo-EM analysis provides the structural basis for the broad and complementary neutralizing activity of these two antibodies. We confirm the in vivo protective and therapeutic efficacies of NA8 and NE12 in the hamster model. These results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants.

The effect of Omicron breakthrough infection and extended BNT162b2 booster dosing on neutralization breadth against SARS-CoV-2 variants of concern.

COVID-19 vaccines are playing a vital role in controlling the COVID-19 pandemic. As SARS-CoV-2 variants encoding mutations in the surface glycoprotein, Spike, continue to emerge, there is increased need to identify immunogens and vaccination regimens that provide the broadest and most durable immune responses. We compared the magnitude and breadth of the neutralizing antibody response, as well as levels of Spike-reactive memory B cells, in individuals receiving a second dose of BNT162b2 at a short (3-4 week) or extended interval (8-12 weeks) and following a third vaccination approximately 6-8 months later. We show that whilst an extended interval between the first two vaccinations can greatly increase the breadth of the immune response and generate a higher proportion of Spike reactive memory B cells, a third vaccination leads to similar levels between the two groups. Furthermore, we show that the third vaccine dose enhances neutralization activity against omicron lineage members BA.1, BA.2 and BA.4/BA.5 and this is further increased following breakthrough infection during the UK omicron wave. These findings are relevant for vaccination strategies in populations where COVID-19 vaccine coverage remains low.

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force.

Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection.

Identification of three conserved linear B cell epitopes on the SARS-CoV-2 spike protein.

Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1-S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 286TDAVDCALDPLS297, S19.2 464FERDISTEIYQA475, and S49.4 1202ELGKYEQYIKWP1213 were identified as B- cell linear epitopes for the first time. Alanine scans showed that the D467, I468, E471, Q474, and A475 of the epitope S19.2 and K1205, Q1208, and Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by three doses of BNT162b2 vaccine or BA.1 infection.

Distinct SARS-CoV-2 Omicron sublineages have evolved showing increased fitness and immune evasion than the original Omicron variant BA.1. Here, we report the neutralization activity of sera from BNT162b2 vaccinated individuals or unimmunized Omicron BA.1-infected individuals against Omicron sublineages and "Deltacron" variant (XD). BNT162b2 post-dose 3 immune sera neutralized USA-WA1/2020, Omicron BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and XD-spike SARS-CoV-2s with geometric mean titres (GMTs) of 1335, 393, 298, 315, 216, 103, and 301, respectively; thus, BA.4/5 SARS-CoV-2 spike variant showed the highest propensity to evade vaccine neutralization compared to the original Omicron variants BA.1. BA.1-convalescent sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron-spike SARS-CoV-2s with GMTs of 15, 430, 110, 109, 102, 25, and 284, respectively. The unique mutation F486V in the BA.4/5 spike contributes to the increased evasion of antibody neutralization by sublineage BA.4/5. The low neutralization titres of vaccinated sera or convalescent sera from BA.1 infected individuals against the emerging and rapidly spreading Omicron BA.4/5 variants provide important results for consideration in the selection of an updated vaccine in the current Omicron wave.Trial registration: ClinicalTrials.gov; identifier: NCT04368728.

Neutralizing antibodies to SARS-CoV-2 variants of concern including Delta and Omicron in subjects receiving mRNA-1273, BNT162b2, and Ad26.COV2.S vaccines.

SARS-CoV-2 vaccines have contributed to the control of COVID-19 in some parts of the world. However, the constant emergence of variants of concern (VOCs) challenges the effectiveness of SARS-CoV-2 vaccines over time. In particular, Omicron contains a high number of mutations in the spike (S) protein gene, on which most vaccines were developed. In this study, we quantitated neutralizing antibodies in vaccine recipients at various times postvaccination using S protein-based pseudoviruses derived from wild type (WT) SARS-CoV-2 and five VOCs including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). We found that two-dose mRNA-1273 and BNT162b2 vaccines elicited robust neutralizing antibodies against WT, Alpha, Beta, Gamma, and Delta, but wanned after 6 months with a faster decline observed for BNT162b2. Both mRNA-1273 and BNT162b2 elicited weak neutralizing antibodies against Omicron. One dose of Ad26.COV2.S vaccine induced weaker neutralizing antibodies against WT and most VOCs than mRNA-1273 and BNT162b2 did but moderate neutralizing antibodies against Delta and Omicron, which lasted for 6 months. These results support current recommendations of the Centers for Disease Control and Prevention for a booster 5 months after full immunization with an mRNA-based vaccine and the use of an mRNA-based vaccine 2 months after Ad26.COV2.S vaccination.

The mutation features and geographical distributions of the surface glycoprotein (S gene) in SARS-CoV-2 strains: A comparative analysis of the early and current strains.

The surface glycoprotein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was used to develop coronavirus disease 2019 (COVID-19) vaccines. However, SARS-CoV-2, especially the S protein, has undergone rapid evolution and mutation, which has remained to be determined. Here, we analyzed and compared the early (12 237) and the current (more than 10 million) SARS-CoV-2 strains to identify the mutation features and geographical distribution of the S gene and S protein. Results showed that in the early strains, most of the loci were with relative low mutation frequency except S: 23403 (4486 strains), while in the current strains, there was a surge in the mutation strains and frequency, with S: 23403 constantly being the highest one, but tremendously increased to approximately 1050 times. Furthermore, D614 (S: 23403) was one of the most highly frequent mutations in the S protein of Omicron as of March 2022, and most of the mutant strains were still from the United States, and the United Kingdom. Further analysis demonstrated that in the receptor-binding domain, most of the loci with low mutation frequency in the early strains, while S: 22995 was nowadays the most prevalent loci with 3 122 491 strains in the current strains. Overall, we compare the mutation features of the S region in SARS-CoV-2 strains between the early and the current stains, providing insight into further studies in concert with emerging SARS-CoV-2 variants for COVID-19 vaccines.

The influence of new SARS-CoV-2 variant Omicron (B.1.1.529) on vaccine efficacy, its correlation to Delta variants: A computational approach.

The newly discovered COVID variant B.1.1.529 in Botswana has more than 30 mutations in spike and many other in non-spike proteins, far more than any other SARS-CoV-2 variant accepted as a variant of concern by the WHO and officially named Omicron, and has sparked concern among scientists and the general public. Our findings provide insights into structural modification caused by the mutations in the Omicrons receptor-binding domain and look into the effects on interaction with the hosts neutralizing antibodies CR3022, B38, CB6, P2B-2F6, and REGN, as well as ACE2R using an in silico approach. Computational analysis revealed that the Omicron variant has a higher binding affinity for the human ACE2 receptor than the wild and Delta (AY.1 and AY.2 strains), but lower than the Delta AY.3 strain. MD simulation and docking analysis suggest that the omicron and Delta AY.3 were found to have relatively unstable RBD structures and hampered interactions with antibodies more than wild and Delta (AY.1 and AY.2), which may lead to relatively more pathogenicity and antibody escape. In addition, we observed lower binding affinity of Omicron for human monoclonal antibodies (CR3022, B38, CB6, and P2B2F6) when compared to wild and Delta (AY.1 & AY.2). However, the binding affinity of Omicron RBD variants for CR3022, B38, and P2B2F6 antibodies is lower as compared to Delta AY.3, which might promote immune evasion and reinfection and needs further experimental investigation.

Potential linear B-cells epitope change to a helix structure in the spike of Omicron 21L or BA.2 predicts increased SARS-CoV-2 antibodies evasion.

The world health organization has announced that SARS-CoV-2 Omicron variant (B.1.1.529), including the three versions; 21K (BA.1), 21L (BA.2) and 21M (BA.3) as a variant of concern (VOC) on November 2022. In this study, we used the specialized computational platforms to predict the stability and flexibility of the spike protein of Omicron. The aim of this study was to investigate the expected effect of Omicron spike mutations on its physiochemical properties. Findings of this study revealed 16 stabilizing mutations that might explain a newly gained environmental stability. We expect the new mutations to play a crucial role in changing the physiochemical properties of epitopes of the spike protein. The notable finding of SuerPose work was the potential linear B-cells epitope G252 → S255 that has been changed in the spike protein of the Omicron 21L to a helix structure which might confer an escape from human monoclonal antibodies.

Clinical efficacy and in vitro neutralization capacity of monoclonal antibodies for severe acute respiratory syndrome coronavirus 2 delta and omicron variants.

We aimed to provide in vitro data on the neutralization capacity of different monoclonal antibody (mAb) preparations against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta and omicron variant, respectively, and describe the in vivo RNA kinetics of coronavirus disease 2019 (COVID-19) patients treated with the respective mAbs. Virus neutralization assays were performed to assess the neutralizing effect of the mAb formulations casirivimab/imdevimab and sotrovimab on the SARS-CoV-2 delta and omicron variant. Additionally, respiratory tract SARS-CoV-2 RNA kinetics are provided for 25 COVID-19 patients infected with either delta variant (n = 18) or omicron variant (n = 7) treated with the respective mAb formulations during their hospital stay. In the virus neutralization assay, sotrovimab exhibits neutralizing capacity at therapeutically achievable concentrations against the SARS-CoV-2 delta and omicron variant. In contrast, casivirimab/imdevimab had neutralizing capacity against the delta variant but failed neutralization against the omicron variant except for a very high concentration above the currently recommended therapeutic dosage. In patients with delta variant infections treated with casivirimab/imdevimab, we observed a rapid decrease of respiratory viral RNA at day 3 after mAb therapy. In contrast, no such prompt decline was observed in patients with delta variant or omicron variant infections receiving sotrovimab.

Preserved recognition of Omicron spike following COVID-19 messenger RNA vaccination in pregnancy.

BACKGROUND: SARS-CoV-2 infection is associated with enhanced disease severity in pregnant women. Despite the potential of COVID-19 vaccines to reduce severe disease, vaccine uptake remained relatively low among pregnant women. Just as coordinated messaging from the Centers for Disease Control and Prevention and leading obstetrics organizations began to increase vaccine confidence in this vulnerable group, the evolution of SARS-CoV-2 variants of concerns, including the Omicron variant, raised new concerns about vaccine efficacy because of their ability to escape vaccine-induced neutralizing antibodies. Early data point to a milder disease course following infection with the Omicron variant in vaccinated individuals. Thus, these data suggest that alternate vaccine-induced immunity beyond neutralization may continue to attenuate Omicron variant-induced disease, such as Fc-mediated antibody activity. OBJECTIVE: This study aimed to test whether vaccine-induced antibodies raised during pregnancy continue to bind to and leverage Fc receptors to protect against variants of concern including the Omicron variant. STUDY DESIGN: The receptor binding domain or whole spike-specific antibody isotype binding titers and Fc gamma receptor binding directed toward variants of concern, including the Omicron variant, were analyzed in pregnant women after receiving the full dose regimen of either the Pfizer/BioNTech BNT62b2 (n=10) or Moderna mRNA-1273 (n=10) vaccination using a multiplexing Luminex assay. RESULTS: Reduced isotype recognition of the Omicron receptor binding domain was observed following administration of either vaccine with relatively preserved, albeit reduced, recognition of the whole Omicron spike by immunoglobulin M and G antibodies. Despite the near complete loss of Fc receptor binding to the Omicron receptor binding domain, Fc receptor binding to the Omicron spike was more variable but largely preserved. CONCLUSION: Reduced binding titers to the Omicron receptor binding domain aligns with the observed loss of neutralizing activity. Despite the loss of neutralization, preserved, albeit reduced, Omicron spike recognition and Fc receptor binding potentially continue to attenuate disease severity in pregnant women.

Vive la Résistance: T-cell Immunity in the Protection against SARS-CoV-2 Infection.

SUMMARY: Fahrner and colleagues investigated the immune response of patients with cancer and cancer-free individuals to SARS-CoV-2 and found that a propensity toward an IL5-predominant Th2/Tc2 response was predictive of susceptibility to infection. The results of this study also suggest that a cellular response against the Spike 1 protein receptor binding domain (S1-RBD) region of the SARS-CoV-2 proteome contributes to protection and that mutations in this region may drive viral evolution and immune escape. See related article by Fahrner et al., p. 958 (8).

SARS-COV-2 RBD (Receptor binding domain) mutations and variants (A sectional-analytical study).

An essential step in SARS-CoV-2 infection is binding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the ACE2 receptor on the surface of host cells. Therefore, variation in this region can have crucial effects on clinical outcomes and the emergence of variants of concern (VOCs) and variants of interest (VOIs). In this cross-sectional descriptive study, 54 patients with SARS-COV-2 infection were enrolled. After collecting samples and identifying the virus using the One-Step Real-Time qRT-PCR technique and confirming the viral infection, the region containing the RBD region for detection of any mutations was amplified using the Nested-PCR method. Finally, to identify probable mutations, the Nested-PCR product was sequenced. Our data show that the most mutant strains in circulation in our population are the delta variant (90.74%), alpha variant (5.56%), and omicron variant (3.70%), respectively. Pangolin Lineages strains were B.1.1.7(Alpha variant), B.1.617.2(Delta variant) and B.1.1.529(Omicron variant). Also, the mutation profile of variants suggests that N501Y, T478K, and D614G amino acid substitutions, are the significant mutations in the alpha and delta variants that are common with the Omicron variant. The highest frequency of clinical signs in the patients were: lung involvement (42.59%); fever, chills (40.74%); body pain (15%), and other signs (1.67%). Our data revealed that SARS-COV-2 RBD region variation results in substituting essential amino acids and the emergence of the new variant. We can consider it as a predictor for monitoring the emergence of variants of concerns and viral outcomes.

Omicron variant receptor-binding domain phylogenetics and molecular dynamics.

BACKGROUND: We investigated the evolutionary relationships, mutations, antigenic epitopes, and structural dynamics of the receptor-binding domain (RBD) of SARS-CoV-2, Omicron and other recently evolved variants. METHODS: The RBD of SARS-CoV-2 and its Omicron, Alpha, Beta, Gamma, Delta, and Mu variants were subjected to pairwise sequence matrix evaluation, antigenic epitope prediction, and phylogenetic relationship and structural dynamics analyses. RESULTS: The Omicron RBD contained 13-15 amino acid mutations, of which 12 were new and three conserved with other variants. In addition, two mutations found in the Alpha, Beta, Gamma, and Mu variants were not found in the Omicron RBD. The ultrametric clustering unweighted pair group method with arithmetic mean identified Omicron as a novel monophyletic class, but the neighbor-joining method clustered Omicron with Alpha and Delta variants. In the SARS-CoV-2 RBD, five main antigenic epitopes were predicted, and these epitopes were conserved across all SARS-CoV-2 variants tested. Surprisingly, the additional mutations in the Omicron variant increased the size of the expected antigenic sites in two of these antigenic epitopes. Molecular dynamics (MD) simulations revealed higher root-mean-square deviation in the Omicron RBD, greater residue fluctuation at residues 32-42 and 140-160, and increased solvent-accessible surface area. CONCLUSIONS: The Omicron RBD mutations indicate the variant is within a new phylogenetic class of SARS-CoV-2 and significantly impact RBD structure, conformation, and molecular dynamics. However, conserved anticipated antigenic sites may imply partial changes in receptor affinity and response to immune reactions. Omicron RBD binding with the angiotensin-converting enzyme 2 receptor was suggested to be weaker than the original SARS-CoV-2 binding in MD simulations.

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

Development of a Monoclonal Antibody PMab-292 Against Ferret Podoplanin.

Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs and evaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs that recognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, are limited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab-292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for CHO/ferPDPN was 3.4 × 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman's capsule in the kidney using immunohistochemistry. Hence, these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.