Antigenicity assessment of SARS-CoV-2 saltation variant BA.2.87.1.

The recent emergence of a SARS-CoV-2 saltation variant, BA.2.87.1, which features 65 spike mutations relative to BA.2, has attracted worldwide attention. In this study, we elucidate the antigenic characteristics and immune evasion capability of BA.2.87.1. Our findings reveal that BA.2.87.1 is more susceptible to XBB-induced humoral immunity compared to JN.1. Notably, BA.2.87.1 lacks critical escaping mutations in the receptor binding domain (RBD) thus allowing various classes of neutralizing antibodies (NAbs) that were escaped by XBB or BA.2.86 subvariants to neutralize BA.2.87.1, although the deletions in the N-terminal domain (NTD), specifically 15-23del and 136-146del, compensate for the resistance to humoral immunity. Interestingly, several neutralizing antibody drugs have been found to restore their efficacy against BA.2.87.1, including SA58, REGN-10933 and COV2-2196. Hence, our results suggest that BA.2.87.1 may not become widespread until it acquires multiple RBD mutations to achieve sufficient immune evasion comparable to that of JN.1.

Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages.

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.

Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting.

The continuing emergence of SARS-CoV-2 variants highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by vaccination based on the ancestral (hereafter referred to as WT) strain would compromise the antibody response to Omicron-based boosters1-5. Vaccination strategies to counter immune imprinting are critically needed. Here we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. In mice, the efficacy of single Omicron boosting is heavily limited when using variants that are antigenically distinct from WT-such as the XBB variant-and this concerning situation could be mitigated by a second Omicron booster. Similarly, in humans, repeated Omicron infections could alleviate WT vaccination-induced immune imprinting and generate broad neutralization responses in both plasma and nasal mucosa. Notably, deep mutational scanning-based epitope characterization of 781 receptor-binding domain (RBD)-targeting monoclonal antibodies isolated from repeated Omicron infection revealed that double Omicron exposure could induce a large proportion of matured Omicron-specific antibodies that have distinct RBD epitopes to WT-induced antibodies. Consequently, immune imprinting was largely mitigated, and the bias towards non-neutralizing epitopes observed in single Omicron exposures was restored. On the basis of the deep mutational scanning profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated that these mutations could further boost the immune-evasion capability of XBB.1.5 while maintaining high ACE2-binding affinity. Our findings suggest that the WT component should be abandoned when updating COVID-19 vaccines, and individuals without prior Omicron exposure should receive two updated vaccine boosters.

Convergent evolution of SARS-CoV-2 XBB lineages on receptor-binding domain 455-456 synergistically enhances antibody evasion and ACE2 binding.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) XBB lineages have achieved dominance worldwide and keep on evolving. Convergent evolution of XBB lineages on the receptor-binding domain (RBD) L455F and F456L is observed, resulting in variants with substantial growth advantages, such as EG.5, FL.1.5.1, XBB.1.5.70, and HK.3. Here, we show that neutralizing antibody (NAb) evasion drives the convergent evolution of F456L, while the epistatic shift caused by F456L enables the subsequent convergence of L455F through ACE2 binding enhancement and further immune evasion. L455F and F456L evade RBD-targeting Class 1 public NAbs, reducing the neutralization efficacy of XBB breakthrough infection (BTI) and reinfection convalescent plasma. Importantly, L455F single substitution significantly dampens receptor binding; however, the combination of L455F and F456L forms an adjacent residue flipping, which leads to enhanced NAbs resistance and ACE2 binding affinity. The perturbed receptor-binding mode leads to the exceptional ACE2 binding and NAb evasion, as revealed by structural analyses. Our results indicate the evolution flexibility contributed by epistasis cannot be underestimated, and the evolution potential of SARS-CoV-2 RBD remains high.

Single-cell bisulfite-free 5mC and 5hmC sequencing with high sensitivity and scalability.

Existing single-cell bisulfite-based DNA methylation analysis is limited by low DNA recovery, and the measurement of 5hmC at single-base resolution remains challenging. Here, we present a bisulfite-free single-cell whole-genome 5mC and 5hmC profiling technique, named Cabernet, which can characterize 5mC and 5hmC at single-base resolution with high genomic coverage. Cabernet utilizes Tn5 transposome for DNA fragmentation, which enables the discrimination between different alleles for measuring hemi-methylation status. Using Cabernet, we revealed the 5mC, hemi-5mC and 5hmC dynamics during early mouse embryo development, uncovering genomic regions exclusively governed by active or passive demethylation. We show that hemi-methylation status can be used to distinguish between pre- and post-replication cells, enabling more efficient cell grouping when integrated with 5mC profiles. The property of Tn5 naturally enables Cabernet to achieve high-throughput single-cell methylome profiling, where we probed mouse cortical neurons and embryonic day 7.5 (E7.5) embryos, and constructed the library for thousands of single cells at high efficiency, demonstrating its potential for analyzing complex tissues at substantially low cost. Together, we present a way of high-throughput methylome and hydroxymethylome detection at single-cell resolution, enabling efficient analysis of the epigenetic status of biological systems with complicated nature such as neurons and cancer cells.

Immune evasion and ACE2 binding affinity contribute to SARS-CoV-2 evolution.

Mutations in the SARS-CoV-2 genome could confer resistance to pre-existing antibodies and/or increased transmissibility. The recently emerged Omicron subvariants exhibit a strong tendency for immune evasion, suggesting adaptive evolution. However, because previous studies have been limited to specific lineages or subsets of mutations, the overall evolutionary trajectory of SARS-CoV-2 and the underlying driving forces are still not fully understood. Here we analysed all open-access SARS-CoV-2 genomes (up to November 2022) and correlated the mutation incidence and fitness changes with the impacts of mutations on immune evasion and ACE2 binding affinity. Our results show that the Omicron lineage had an accelerated mutation rate in the RBD region, while the mutation incidence in other genomic regions did not change dramatically over time. Mutations in the RBD region exhibited a lineage-specific pattern and tended to become more aggregated over time, and the mutation incidence was positively correlated with the strength of antibody pressure. Additionally, mutation incidence was positively correlated with changes in ACE2 binding affinity, but with a lower correlation coefficient than with immune evasion. In contrast, the effect of mutations on fitness was more closely correlated with changes in ACE2 binding affinity than with immune evasion. Our findings suggest that immune evasion and ACE2 binding affinity play significant and diverse roles in the evolution of SARS-CoV-2.

Post-exposure prophylaxis with SA58 (anti-SARS-COV-2 monoclonal antibody) nasal spray for the prevention of symptomatic COVID-19 in healthy adult workers: a randomized, single-blind, placebo-controlled clinical study.

Monoclonal antibodies (mAbs) and the post-exposure prophylaxis (PEP) with mAbs represent a very important public health strategy against coronavirus disease 2019 (COVID-19). This study has assessed a new Anti-SARS-COV-2 mAb (SA58) Nasal Spray for PEP against COVID-19 in healthy adults aged 18 years and older within three days of exposure to a SARS-CoV-2 infected individual. Recruited participants were randomized in a ratio of 3:1 to receive SA58 or placebo. Primary endpoints were laboratory-confirmed symptomatic COVID-19 within the study period. A total of 1222 participants were randomized and dosed (SA58, n = 901; placebo, n = 321). Median of follow-up was 2.25 and 2.79 days for SA58 and placebo, respectively. Adverse events occurred in 221 of 901 (25%) and 72 of 321 (22%) participants with SA58 and placebo, respectively. All adverse events were mild in severity. Laboratory-confirmed symptomatic COVID-19 developed in 7 of 824 participants (0.22 per 100 person-days) in the SA58 group vs. 14 of 299 (1.17 per 100 person-days) in the placebo group, resulting in an estimated efficacy of 80.82% (95%CI 52.41%-92.27%). There were 32 SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) positives (1.04 per 100 person-days) in the SA58 group vs. 32 (2.80 per 100 person-days) in the placebo group, resulting in an estimated efficacy of 61.83% (95%CI 37.50%-76.69%). A total of 21 RT-PCR positive samples were sequenced and all were the Omicron variant BF.7. In conclusion, SA58 Nasal Spray showed favourable efficacy and safety in preventing symptomatic COVID-19 or SARS-CoV-2 infection in adults who had exposure to SARS-CoV-2 within 72 h.

Safety and Effectiveness of SA58 Nasal Spray Against COVID-19 Infection in Medical Personnel: An Open-Label, Blank-Controlled Study - Hohhot City, Inner Mongolia Autonomous Region, China, 2022.

What is already known about this topic?: The active ingredient of the SA58 Nasal Spray is a broad-spectrum neutralizing antibody with a high neutralizing capacity against different Omicron sub-variants in vitro studies. What is added by this report?: This study demonstrated the safety and effectiveness of SA58 Nasal Spray against coronavirus disease 2019 (COVID-19) infection in medical personnel for the first time. What are the implications for public health practice?: This study provides an effective approach for the public to reduce their risk of COVID-19 infection. The findings of this research have the potential to significantly reduce the risk of infection and limit human-to-human transmission in the event of a COVID-19 outbreak.

MR-BOIL: Causal inference in one-sample Mendelian randomization for binary outcome with integrated likelihood method.

Mendelian randomization is a statistical method for inferring the causal relationship between exposures and outcomes using an economics-derived instrumental variable approach. The research results are relatively complete when both exposures and outcomes are continuous variables. However, due to the noncollapsing nature of the logistic model, the existing methods inherited from the linear model for exploring binary outcome cannot take the effect of confounding factors into account, which leads to biased estimate of the causal effect. In this article, we propose an integrated likelihood method MR-BOIL to investigate causal relationships for binary outcomes by treating confounders as latent variables in one-sample Mendelian randomization. Under the assumption of a joint normal distribution of the confounders, we use expectation maximization algorithm to estimate the causal effect. Extensive simulations demonstrate that the estimator of MR-BOIL is asymptotically unbiased and that our method improves statistical power without inflating type I error rate. We then apply this method to analyze the data from Atherosclerosis Risk in Communications Study. The results show that MR-BOIL can better identify plausible causal relationships with high reliability, compared with the unreliable results of existing methods. MR-BOIL is implemented in R and the corresponding R code is provided for free download.

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.

Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages have escaped most receptor-binding domain (RBD)-targeting therapeutic neutralizing antibodies (NAbs), which proves that previous NAb drug screening strategies are deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following these criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.

Characterization of the enhanced infectivity and antibody evasion of Omicron BA.2.75.

Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a growth advantage over circulating BA.2.38, BA.2.76, and BA.5 in India. However, the underlying mechanisms for enhanced infectivity, especially compared with BA.5, remain unclear. Here, we show that BA.2.75 exhibits substantially higher affinity for host receptor angiotensin-converting enzyme 2 (ACE2) than BA.5 and other variants. Structural analyses of BA.2.75 spike shows its decreased thermostability and increased frequency of the receptor binding domain (RBD) in the "up" conformation under acidic conditions, suggesting enhanced low-pH-endosomal cell entry. Relative to BA.4/BA.5, BA.2.75 exhibits reduced evasion of humoral immunity from BA.1/BA.2 breakthrough-infection convalescent plasma but greater evasion of Delta breakthrough-infection convalescent plasma. BA.5 breakthrough-infection plasma also exhibits weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75's distinct neutralizing antibody (NAb) escape pattern. Antibody therapeutics Evusheld and Bebtelovimab remain effective against BA.2.75. These results suggest BA.2.75 may prevail after BA.4/BA.5, and its increased receptor-binding capability could support further immune-evasive mutations.