Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.

Replicating-Competent VSV-Vectored Pseudotyped Viruses.

Vesicular stomatitis virus (VSV) is prototype virus in the family of Rhabdoviridae. Reverse genetic platform has enabled the genetic manipulation of VSV as a powerful live viral vector. Replicating-competent VSV is constructed by replacing the original VSV glycoprotein gene with heterologous envelope genes. The resulting recombinant viruses are able to replicate in permissive cells and incorporate the foreign envelope proteins on the surface of the viral particle without changing the bullet-shape morphology. Correspondingly, the cell tropism of replicating-competent VSV is determined by the foreign envelope proteins. Replicating-competent VSVs have been successfully used for selecting critical viral receptors or host factors, screening mutants that escape therapeutic antibodies, and developing VSV-based live viral vaccines.

Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

Evolution of Anti-SARS-CoV-2 Therapeutic Antibodies.

Since the first COVID-19 reports back in December of 2019, this viral infection caused by SARS-CoV-2 has claimed millions of lives. To control the COVID-19 pandemic, the Food and Drug Administration (FDA) and/or European Agency of Medicines (EMA) have granted Emergency Use Authorization (EUA) to nine therapeutic antibodies. Nonetheless, the natural evolution of SARS-CoV-2 has generated numerous variants of concern (VOCs) that have challenged the efficacy of the EUA antibodies. Here, we review the most relevant characteristics of these therapeutic antibodies, including timeline of approval, neutralization profile against the VOCs, selection methods of their variable regions, somatic mutations, HCDR3 and LCDR3 features, isotype, Fc modifications used in the therapeutic format, and epitope recognized on the receptor-binding domain (RBD) of SARS-CoV-2. One of the conclusions of the review is that the EUA therapeutic antibodies that still retain efficacy against new VOCs bind an epitope formed by conserved residues that seem to be evolutionarily conserved as thus, critical for the RBD:hACE-2 interaction. The information reviewed here should help to design new and more efficacious antibodies to prevent and/or treat COVID-19, as well as other infectious diseases.

SARS-CoV-2 infection in cancer patients on active therapy after the booster dose of mRNA vaccines.

INTRODUCTION: The protective role against SARS-CoV-2 infection by the third booster dose of mRNA vaccines in cancer patients with solid malignancies is presently unknown. We prospectively investigated the occurrence of COVID-19 in cancer patients on active therapy after the booster vaccine dose. METHODS: Cancer patients on treatment at the Center for Immuno-Oncology (CIO) of the University Hospital of Siena, Italy, and health care workers at CIO who had received a booster third dose of mRNA vaccine entered a systematic follow-up monitoring period to prospectively assess their potential risk of SARS-CoV-2 infection. Serological and microneutralization assay were utilized to assess levels of anti-spike IgG, and of neutralizing antibodies to the SARS-CoV-2 Wild Type, Delta and Omicron variants, respectively, after the booster dose and after negativization of the nasopharyngeal swab for those who had developed COVID-19. RESULTS: Ninety cancer patients with solid tumors on active treatment (Cohort 1) and 30 health care workers (Cohort 2) underwent a booster third dose of mRNA vaccine. After the booster dose, the median value of anti-spike IgG was higher (p = 0.009) in patients than in healthy subjects. Remarkably, 11/90 (12%) patients and 11/30 (37%) healthy subjects tested positive to SARS-CoV-2 infection during the monitoring period. Similar levels of anti-spike IgG and of neutralizing antibodies against all the investigated variants, with geometric mean titers of neutralizing antibodies against the Omicron being the lowest were detected after the booster dose and after COVID-19 in both Cohorts. CONCLUSIONS: The occurrence of SARS-CoV-2 infection we observed in a sizable proportion of booster-dosed cancer patients and in healthy subjects during the Omicron outbreak indicates that highly specific vaccines against SARS-CoV-2 variants are urgently required.

The SARS-CoV-2 Delta-Omicron Recombinant Lineage (XD) Exhibits Immune-Escape Properties Similar to the Omicron (BA.1) Variant.

Recently, a recombinant SARS-CoV-2 lineage, XD, emerged that harbors a spike gene that is largely derived from the Omicron variant BA.1 in the genetic background of the Delta variant. This finding raised concerns that the recombinant virus might exhibit altered biological properties as compared to the parental viruses and might pose an elevated threat to human health. Here, using pseudotyped particles, we show that ACE2 binding and cell tropism of XD mimics that of BA.1. Further, XD and BA.1 displayed comparable sensitivity to neutralization by antibodies induced upon vaccination with BNT162b2/Comirnaty (BNT) or BNT vaccination followed by breakthrough infection. Our findings reveal important biological commonalities between XD and Omicron BA.1 host cell entry and its inhibition by antibodies.

In Silico Genome Analysis Reveals the Evolution and Potential Impact of SARS-CoV-2 Omicron Structural Changes on Host Immune Evasion and Antiviral Therapeutics.

New variants of SARS-CoV-2 continue to evolve. The novel SARS-CoV-2 variant of concern (VOC) B.1.1.529 (Omicron) was particularly menacing due to the presence of numerous consequential mutations. In this study, we reviewed about 12 million SARS-CoV-2 genomic and associated metadata using extensive bioinformatic approaches to understand how evolutionary and mutational changes affect Omicron variant properties. Subsampled global data based analysis of molecular clock in the phylogenetic tree showed 29.56 substitutions per year as the evolutionary rate of five VOCs. We observed extensive mutational changes in the spike structural protein of the Omicron variant. A total of 20% of 7230 amino acid and structural changes exclusive to Omicron's spike protein were detected in the receptor binding domain (RBD), suggesting differential selection pressures exerted during evolution. Analyzing key drug targets revealed mutation-derived differential binding affinities between Delta and Omicron variants. Nine single-RBD substitutions were detected within the binding site of approved therapeutic monoclonal antibodies. T-cell epitope prediction revealed eight immunologically important functional hotspots in three conserved non-structural proteins. A universal vaccine based on these regions may likely protect against all these SARS-CoV-2 variants. We observed key structural changes in the spike protein, which decreased binding affinities, indicating that these changes may help the virus escape host cellular immunity. These findings emphasize the need for continuous genomic surveillance of SARS-CoV-2 to better understand how novel mutations may impact viral spread and disease outcome.

Computational modeling of the effect of five mutations on the structure of the ACE2 receptor and their correlation with infectivity and virulence of some emerged variants of SARS-CoV-2 suggests mechanisms of binding affinity dysregulation.

Interactions between the human angiotensin-converting enzyme 2 (ACE2) and the RBD region of the SARS-CoV-2 Spike protein are critical for virus entry into the host cell. The objective of this work was to identify some of the most relevant SARS-CoV-2 Spike variants that emerged during the pandemic and evaluate their binding affinity with human variants of ACE2 since some ACE2 variants can enhance or reduce the affinity of the interaction between the ACE2 and S proteins. However, no information has been sought to extrapolate to different variants of SARS-CoV-2. Therefore, to understand the impact on the affinity of the interaction between ACE2 protein variants and SARS-CoV-2 protein S variants, molecular docking was used in this study to predict the effects of five mutations of ACE2 when they interact with Alpha, Beta, Delta, Omicron variants and a hypothetical variant, which present mutations in the RBD region of the SARS-CoV-2 Spike protein. Our results suggest that these variants could alter the interaction of the Spike and the human ACE2 protein, losing or creating new inter-protein contacts, enhancing viral fitness by improving binding affinity, and leading to an increase in infectivity, virulence, and transmission. This investigation highlighted that the S19P mutation of ACE2 decreases the binding affinity between the ACE2 and Spike proteins in the presence of the Beta variant and the wild-type variant of SARS-CoV-2 isolated in Wuhan-2019. The R115Q mutation of ACE2 lowers the binding affinity of these two proteins in the presence of the Beta and Delta variants. Similarly, the K26R mutation lowers the affinity of the interaction between the ACE2 and Spike proteins in the presence of the Alpha variant. This decrease in binding affinity is probably due to the lack of interaction between some of the key residues of the interaction complex between the ACE2 protein and the RBD region of the SARS-CoV-2 Spike protein. Therefore, ACE2 mutations appear in the presence of these variants, they could suggest an intrinsic resistance to COVID-19 disease. On the other hand, our results suggested that the K26R, M332L, and K341R mutations of ACE2 expressively showed the affinity between the ACE2 and Spike proteins in the Alpha, Beta, and Delta variants. Consequently, these ACE2 mutations in the presence of the Alpha, Beta, and delta variants of SARS-CoV-2 could be more infectious and virulent in human cells compared to the SARS-CoV-2 isolated in Wuhan-2019 and it could have a negative prognosis of the disease. Finally, the Omicron variant in interaction with ACE2 WT, S19P, R115Q, M332L, and K341R mutations of ACE2 showed a significant decrease in binding affinity. This could be consistent that the Omicron variant causes less severe symptoms than previous variants. On the other hand, our results suggested Omicron in the complex with K26R, the binding affinity is increased between ACE2/RBD, which could indicate a negative prognosis of the disease in people with these allelic conditions.

Comparative Analysis of Antibody Titers against the Spike Protein of SARS-CoV-2 Variants in Infected Patient Cohorts and Diverse Vaccination Regimes.

The presence of neutralizing antibodies against SARS-CoV-2 correlates with protection against infection and severe COVID-19 disease courses. Understanding the dynamics of antibody development against the SARS-CoV-2 virus is important for recommendations on vaccination strategies and on control of the COVID-19 pandemic. This study investigates the dynamics and extent of α-Spike-Ab development by different vaccines manufactured by Johnson & Johnson, AstraZeneca, Pfizer-BioNTech and Moderna. On day 1 after vaccination, we observed a temporal low-grade inflammatory response. α-Spike-Ab titers were reduced after six months of vaccination with mRNA vaccines and increased 14 days after booster vaccinations to a maximum that exceeded titers from mild and critical COVID-19 and Long-COVID patients. Within the group of critical COVID-19 patients, we observed a trend for lower α-Spike-Ab titers in the group of patients who survived COVID-19. This trend accompanied higher numbers of pro-B cells, fewer mature B cells and a higher frequency of T follicular helper cells. Finally, we present data demonstrating that past infection with mild COVID-19 does not lead to long-term increased Ab titers and that even the group of previously infected SARS-CoV-2 patients benefit from a vaccination six months after the infection.

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 universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells.

New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.

Dynamic SARS-CoV-2 emergence algorithm for rationally-designed logical next-generation vaccines.

SARS-CoV-2 worldwide spread and evolution has resulted in variants containing mutations resulting in immune evasive epitopes that decrease vaccine efficacy. We acquired SARS-CoV-2 positive clinical samples and compared the worldwide emerged spike mutations from Variants of Concern/Interest, and developed an algorithm for monitoring the evolution of SARS-CoV-2 in the context of vaccines and monoclonal antibodies. The algorithm partitions logarithmic-transformed prevalence data monthly and Pearson's correlation determines exponential emergence of amino acid substitutions (AAS) and lineages. The SARS-CoV-2 genome evaluation indicated 49 mutations, with 44 resulting in AAS. Nine of the ten most worldwide prevalent (>70%) spike protein changes have Pearson's coefficient r > 0.9. The tenth, D614G, has a prevalence >99% and r-value of 0.67. The resulting algorithm is based on the patterns these ten substitutions elucidated. The strong positive correlation of the emerged spike protein changes and algorithmic predictive value can be harnessed in designing vaccines with relevant immunogenic epitopes. Monitoring, next-generation vaccine design, and mAb clinical efficacy must keep up with SARS-CoV-2 evolution, as the virus is predicted to remain endemic.

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.

Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus-host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Δfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development.

Improved Neutralisation of the SARS-CoV-2 Omicron Variant following a Booster Dose of Pfizer-BioNTech (BNT162b2) COVID-19 Vaccine.

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired over 30 mutations in the spike protein (with 15 in the receptor-binding domain), raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three, and six months post-two doses of Pfizer-BioNTech BNT162b2 had a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres were boosted. Despite this increase, neutralising antibody titres were reduced fourfold for Omicron compared to lineage A.2.2 SARS-CoV-2.

Coronavirus Vaccination: Spike Antibody Levels in Health Workers after Six Months-A Cross-Sectional Study.

Healthcare workers bear a high risk of infection during epidemics and pandemics such as the current SARS-CoV-2 pandemic. Various new vaccines have been approved. We investigated the influence of the time elapsed since vaccination, as well as of vaccination schema, on health workers' spike antibody levels following their second vaccination. Blood samples were obtained from employees working at a German hospital between August 2021 and December 2021 on average half a year (range 130-280 days) after their second vaccination. Levels of SARS-CoV-2-IgG antibodies (spike and nucleocapsid protein) were qualitatively detected via chemiluminescent immunoassays (CLIAs). A previous infection with SARS-CoV-2 was an exclusion criterion. In total, 545 persons were included in this cross-sectional study. Most participants (97.8%) showed elevated anti-spike concentrations. Anti-spike levels differed significantly among vaccination schemas. Repeated vector vaccinations resulted in lower protective antibody levels. Higher age levels, immunosuppression and a longer time period since the second vaccination resulted in lower anti-spike levels. Women's antibody levels were higher, but not significantly. Since anti-spike levels drop after vaccination, further boosters are required to increase immunoreactivity. If two vector vaccines have been administered, it is possible that an mRNA booster might increase the anti-spike level.

Enhanced antibody responses in fully vaccinated individuals against pan-SARS-CoV-2 variants following Omicron breakthrough infection.

Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.

Immune response to SARS-CoV-2 Omicron variant in patients and vaccinees following homologous and heterologous vaccinations.

The SARS-CoV-2 Omicron variant has rapidly replaced the Delta variant of concern. This new variant harbors worrisome mutations on the spike protein, which are able to escape the immunity elicited by vaccination and/or natural infection. To evaluate the impact and susceptibility of different serum samples to the Omicron variant BA.1, samples from COVID-19 patients and vaccinated individuals were tested for their ability to bind and neutralize the original SARS-CoV-2 virus and the Omicron variant BA.1. COVID-19 patients show the most drastic reduction in Omicron-specific antibody response in comparison with the response to the wild-type virus. Antibodies elicited by a triple homologous/heterologous vaccination regimen or following natural SARS-CoV-2 infection combined with a two-dose vaccine course, result in highest neutralization capacity against the Omicron variant BA.1. Overall, these findings confirm that vaccination of COVID-19 survivors and booster dose to vaccinees with mRNA vaccines is the correct strategy to enhance the antibody cross-protection against Omicron variant BA.1.

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.

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients.

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.