SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

Viral codon optimization on SARS-CoV-2 Spike boosts immunity in the development of COVID-19 mRNA vaccines.

Life-long persistent herpesviruses carry "trans-inducers" to overcome the unusual codon usage of their glycoproteins for efficient expression. Strikingly, this "trans-inducibility" can be achieved by simply changing the codon-usage of acute virus glycoproteins to that of persistent herpesvirus glycoproteins with herpesviral trans-inducer. Here, we apply the "persistent viral codon-usage-trans-inducer" principle to SARS-CoV-2 Spike mRNA vaccine platform, in which the codon-usage of Spike is changed to that of Herpes Simplex Virus-1 (HSV-1) glycoprotein B (gB) with its "trans-inducer" ICP27. The HSVgB-ICP27-codon-optimized Spike mRNA vaccine induced markedly high antigen expression and stability, total IgG, neutralizing antibody, and T cell response, ultimately enhancing protection against lethal SARS-CoV-2 challenge. Moreover, the HSVgB- codon-optimized Delta (B.1.617.2) strain Spike mRNA vaccine provided significant enhancements in antigen expression and long-term protection against SARS-CoV-2 challenge. Thus, we report a novel persistent viral codon-usage-trans-inducer mRNA vaccine platform for enhanced antigen expression and long-term protection against lethal viral infection.

Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant, BA.2.86, has emerged and spread to numerous countries worldwide, raising alarm because its spike protein contains 34 additional mutations compared with its BA.2 predecessor1. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was no more resistant to human sera than the currently dominant XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from people who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. Although BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the 'inner face' of the RBD that is exposed only when this domain is in the 'up' position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.

Mapping SARS-CoV-2 antigenic relationships and serological responses.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, multiple variants escaping preexisting immunity emerged, causing reinfections of previously exposed individuals. Here, we used antigenic cartography to analyze patterns of cross-reactivity among 21 variants and 15 groups of human sera obtained after primary infection with 10 different variants or after messenger RNA (mRNA)-1273 or mRNA-1273.351 vaccination. We found antigenic differences among pre-Omicron variants caused by substitutions at spike-protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months after a second dose. We found changes in immunodominance of different spike regions, depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine-strain selection.

Analytical Ultracentrifugation Detects Quaternary Rearrangements and Antibody-Induced Conformational Selection of the SARS-CoV-2 Spike Trimer.

Analytical ultracentrifugation (AUC) analysis shows that the SARS-CoV-2 trimeric Spike (S) protein adopts different quaternary conformations in solution. The relative abundance of the "open" and "close" conformations is temperature-dependent, and samples with different storage temperature history have different open/close distributions. Neutralizing antibodies (NAbs) targeting the S receptor binding domain (RBD) do not alter the conformer populations; by contrast, a NAb targeting a cryptic conformational epitope skews the Spike trimer toward an open conformation. The results highlight AUC, which is typically applied for molecular mass determination of biomolecules as a powerful tool for detecting functionally relevant quaternary protein conformations.

Humoral immunity to an endemic coronavirus is associated with postacute sequelae of COVID-19 in individuals with rheumatic diseases.

Beyond the acute illness caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) infection, about one-fifth of infections result in long-term persistence of symptoms despite the apparent clearance of infection. Insights into the mechanisms that underlie postacute sequelae of COVID-19 (PASC) will be critical for the prevention and clinical management of long-term complications of COVID-19. Several hypotheses have been proposed that may account for the development of PASC, including persistence of virus and dysregulation of immune responses. Among the immunological changes noted in PASC, alterations in humoral immunity have been observed in some patient subsets. To begin to determine whether SARS-CoV-2- or other pathogen-specific humoral immune responses evolve uniquely in PASC, we performed comprehensive antibody profiling against SARS-CoV-2, a panel of endemic pathogens, and a panel of routine vaccine antigens using systems serology in two cohorts of patients with preexisting systemic autoimmune rheumatic disease (SARD) who either developed or did not develop PASC. A distinct qualitative shift observed in Fcγ receptor (FcγR) binding was observed in individuals with PASC. Specifically, individuals with PASC harbored weaker FcγR-binding anti-SARS-CoV-2 antibodies and stronger FcγR-binding antibody responses against the endemic coronavirus OC43. Individuals with PASC developed an OC43 S2-specific antibody response with stronger FcγR binding, linked to cross-reactivity across SARS-CoV-2 and common coronaviruses. These findings identify previous coronavirus imprinting as a potential marker for the development of PASC in individuals with SARDs.

Despite delayed kinetics, people living with HIV achieve equivalent antibody function after SARS-CoV-2 infection or vaccination.

The kinetics of Fc-mediated functions following SARS-CoV-2 infection or vaccination in people living with HIV (PLWH) are not known. We compared SARS-CoV-2 spike-specific Fc functions, binding, and neutralization in PLWH and people without HIV (PWOH) during acute infection (without prior vaccination) with either the D614G or Beta variants of SARS-CoV-2, or vaccination with ChAdOx1 nCoV-19. Antiretroviral treatment (ART)-naïve PLWH had significantly lower levels of IgG binding, neutralization, and antibody-dependent cellular phagocytosis (ADCP) compared with PLWH on ART. The magnitude of antibody-dependent cellular cytotoxicity (ADCC), complement deposition (ADCD), and cellular trogocytosis (ADCT) was differentially triggered by D614G and Beta. The kinetics of spike IgG-binding antibodies, ADCC, and ADCD were similar, irrespective of the infecting variant between PWOH and PLWH overall. However, compared with PWOH, PLWH infected with D614G had delayed neutralization and ADCP. Furthermore, Beta infection resulted in delayed ADCT, regardless of HIV status. Despite these delays, we observed improved coordination between binding and neutralizing responses and Fc functions in PLWH. In contrast to D614G infection, binding responses in PLWH following ChAdOx-1 nCoV-19 vaccination were delayed, while neutralization and ADCP had similar timing of onset, but lower magnitude, and ADCC was significantly higher than in PWOH. Overall, despite delayed and differential kinetics, PLWH on ART develop comparable responses to PWOH, supporting the prioritization of ART rollout and SARS-CoV-2 vaccination in PLWH.

An ACE2-Based Bimodular Fusion Protein Enables Reorientation of Endogenous Anti-Epstein-Barr Virus Antibodies Toward SARS-CoV-2 Spike.

The use of soluble recombinant angiotensin-converting enzyme 2 (rACE2) as a decoy capable of blocking SARS-CoV-2 entry into cells has been envisaged as a therapeutic strategy to reduce viral loads in patients with severe COVID-19. We engineered a novel form of rACE2, fused to the Epstein-Barr virus antigen P18F3 (rACE2-P18F3), to reorient a preexisting humoral response toward Epstein-Barr virus against SARS-CoV-2 particles. Recombinant ACE2-P18F3 was able to bind to the SARS-CoV-2 spike protein, neutralize viral entry into cells, and promote the phagocytosis of spheres coated with different spike variants by monocytic cells. The results position rACE2-P18F3 as a promising therapeutic candidate to universally block coronavirus cell entry and clear viral particles.

A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection.

Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic1-4. Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01-peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01-mediated pre-existing immunity.

Protective immunity induced by an inhaled SARS-CoV-2 subunit vaccine.

Targeting the site of infection is a promising strategy for improving vaccine effectivity. To date, licensed COVID-19 vaccines have been administered intramuscularly despite the fact that SARS-CoV-2 is a respiratory virus. Here, we aim to induce local protective mucosal immune responses with an inhaled subunit vaccine candidate, ISR52, based on the SARS-CoV-2 Spike S1 protein. When tested in a lethal challenge hACE2 transgenic SARS-CoV-2 mouse model, intranasal and intratracheal administration of ISR52 provided superior protection against severe infection, compared to the subcutaneous injection of the vaccine. Interestingly for a protein-based vaccine, inhaled ISR52 elicited both CD4 and CD8 T-cell Spike-specific responses that were maintained for at least 6 months in wild-type mice. Induced IgG and IgA responses cross-reacting with several SARS- CoV-2 variants of concern were detected in the lung and in serum and protected animals displayed neutralizing antibodies. Based on our results, we are developing ISR52 as a dry powder formulation for inhalation, that does not require cold-chain distribution or the use of needle administration, for evaluation in a Phase I/II clinical trial.

New antibodies that the coronavirus can't elude.

Researchers aim to make monoclonal antibodies that mutations in SARS-CoV-2 won't thwart.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Searching for common ground.

The conserved coronavirus fusion peptide is a target of broadly neutralizing antibodies.

Immunity to Seasonal Coronavirus Spike Proteins Does Not Protect from SARS-CoV-2 Challenge in a Mouse Model but Has No Detrimental Effect on Protection Mediated by COVID-19 mRNA Vaccination.

Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E, or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. IMPORTANCE The impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.

Critical review of conformational B-cell epitope prediction methods.

Accurate in silico prediction of conformational B-cell epitopes would lead to major improvements in disease diagnostics, drug design and vaccine development. A variety of computational methods, mainly based on machine learning approaches, have been developed in the last decades to tackle this challenging problem. Here, we rigorously benchmarked nine state-of-the-art conformational B-cell epitope prediction webservers, including generic and antibody-specific methods, on a dataset of over 250 antibody-antigen structures. The results of our assessment and statistical analyses show that all the methods achieve very low performances, and some do not perform better than randomly generated patches of surface residues. In addition, we also found that commonly used consensus strategies that combine the results from multiple webservers are at best only marginally better than random. Finally, we applied all the predictors to the SARS-CoV-2 spike protein as an independent case study, and showed that they perform poorly in general, which largely recapitulates our benchmarking conclusions. We hope that these results will lead to greater caution when using these tools until the biases and issues that limit current methods have been addressed, promote the use of state-of-the-art evaluation methodologies in future publications and suggest new strategies to improve the performance of conformational B-cell epitope prediction methods.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2.

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.

Emergence of Delta and Omicron variants carrying resistance-associated mutations in immunocompromised patients undergoing sotrovimab treatment with long-term viral excretion.

OBJECTIVES: To monitor the early emergence of genetic mutations related to reduced susceptibility to monoclonal anti-body (mAb)-based treatment in immunocompromised patients with long-term viral excretion using whole-genome sequencing at a tertiary university hospital in Barcelona, Spain. METHODS: Serial severe acute respiratory syndrome coronavirus 2-positive samples (mid-December 2021-mid-March 2022) from eight immunosuppressed, fully vaccinated patients (for solid-organ transplantation or haematologic malignancies) with long-term viral excretion despite undergoing mAb therapy (sotrovimab) for coronavirus disease 2019 were selected. Whole-genome sequencing was performed following the ARTIC, version 4.1, protocol on the MiSeq platform. Mutations in the coding sequence of the spike protein with a frequency of ≥5% were studied. RESULTS: A total of 37 samples from the studied cases were analysed. All the cases, except one, were confirmed to have the Omicron variant BA.1; one had Delta (AY.100). Thirty-four different mutations were detected within the receptor-binding domain of the spike protein in 62.5% of patients, eight of which were not lineage related and located in the sotrovimab target epitope (P337L, E340D, E340R, E340K, E340V, E340Q, R346T and K356T). Except for P337L, all changes showed a significant increase in frequency or fixation after the administration of sotrovimab. Some of them have been associated with either reduced susceptibility to mAb therapy, such as those at position 340, or the acquisition of a new glycosylation site (346 and 356 positions). CONCLUSIONS: This study highlights the importance of monitoring for early in vivo selection of mutations associated with reduced susceptibility to mAb therapy, especially in immunocompromised patients receiving anti-viral drugs, whose immune response is not able to control viral replication, resulting in long-term viral shedding, and those receiving selective evolution pressure. Virologic surveillance of genetically resistant viruses to available anti-viral therapies is considered a priority for both patients and the community.

Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution.

Continuous evolution of Omicron has led to a rapid and simultaneous emergence of numerous variants that display growth advantages over BA.5 (ref. 1). Despite their divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots. The driving force and destination of such sudden convergent evolution and its effect on humoral immunity remain unclear. Here we demonstrate that these convergent mutations can cause evasion of neutralizing antibody drugs and convalescent plasma, including those from BA.5 breakthrough infection, while maintaining sufficient ACE2-binding capability. BQ.1.1.10 (BQ.1.1 + Y144del), BA.4.6.3, XBB and CH.1.1 are the most antibody-evasive strains tested. To delineate the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies isolated from individuals who had BA.2 and BA.5 breakthrough infections2,3. Owing to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection reduced the diversity of the neutralizing antibody binding sites and increased proportions of non-neutralizing antibody clones, which, in turn, focused humoral immune pressure and promoted convergent evolution in the RBD. Moreover, we show that the convergent RBD mutations could be accurately inferred by deep mutational scanning profiles4,5, and the evolution trends of BA.2.75 and BA.5 subvariants could be well foreseen through constructed convergent pseudovirus mutants. These results suggest that current herd immunity and BA.5 vaccine boosters may not efficiently prevent the infection of Omicron convergent variants.