An optimized thermodynamics integration protocol for identifying beneficial mutations in antibody design.

Accurate identification of beneficial mutations is central to antibody design. Many knowledge-based (KB) computational approaches have been developed to predict beneficial mutations, but their accuracy leaves room for improvement. Thermodynamic integration (TI) is an alchemical free energy algorithm that offers an alternative technique for identifying beneficial mutations, but its performance has not been evaluated. In this study, we developed an efficient TI protocol with high accuracy for predicting binding free energy changes of antibody mutations. The improved TI method outperforms KB methods at identifying both beneficial and deleterious mutations. We observed that KB methods have higher accuracies in predicting deleterious mutations than beneficial mutations. A pipeline using KB methods to efficiently exclude deleterious mutations and TI to accurately identify beneficial mutations was developed for high-throughput mutation scanning. The pipeline was applied to optimize the binding affinity of a broadly sarbecovirus neutralizing antibody 10-40 against the circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant. Three identified beneficial mutations show strong synergy and improve both binding affinity and neutralization potency of antibody 10-40. Molecular dynamics simulation revealed that the three mutations improve the binding affinity of antibody 10-40 through the stabilization of an altered binding mode with increased polar and hydrophobic interactions. Above all, this study presents an accurate and efficient TI-based approach for optimizing antibodies and other biomolecules.

A multi-specific, multi-affinity antibody platform neutralizes sarbecoviruses and confers protection against SARS-CoV-2 in vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Impaired potency of neutralizing antibodies against cell-cell fusion mediated by SARS-CoV-2.

The SARS-CoV-2 Omicron subvariants have dominated the pandemic due to their high transmissibility and immune evasion conferred by the spike mutations. The Omicron subvariants can spread by cell-free virus infection and cell-cell fusion, the latter of which is more effective but has not been extensively investigated. In this study, we developed a simple and high-throughput assay that provides a rapid readout to quantify cell-cell fusion mediated by the SARS-CoV-2 spike proteins without using live or pseudotyped virus. This assay can be used to identify variants of concern and to screen for prophylactic and therapeutic agents. We further evaluated a panel of monoclonal antibodies (mAbs) and vaccinee sera against D614G and Omicron subvariants, finding that cell-cell fusion is substantially more resistant to mAb and serum inhibition than cell-free virus infection. Such results have important implications for the development of vaccines and antiviral antibody drugs against cell-cell fusion induced by SARS-CoV-2 spikes.

Changes in serum neutralizing antibody potency and breadth post SARS-CoV-2 mRNA vaccine boost

A better understanding of the durability and breadth of serum neutralizing antibody responses against multiple SARS-CoV-2 variants elicited by Covid-19 vaccines is crucial in addressing the current pandemic. In this study, we quantified the decay of serum neutralization antibodies (nAbs) after second and third doses of the original Covid-19 mRNA vaccine. Using an authentic virus neutralization assay, we found that decay half-lives of WA1- and Delta-nAbs were both ∼60 days post second and third vaccine dose. Unexpectedly, the durability of serum antibodies that neutralize three different Omicron subvariants (BA.1.1, BA.5, BA.2.12.1) was substantially better, with half-lives of ≥ 6 months. A booster dose of the original Covid-19 vaccine was also found to broaden antibody responses against SARS-CoV and four other sarbecoviruses, in addition to multiple SARS-CoV-2 strains. These findings suggest that repeated vaccinations with the Covid-19 vaccine may confer a degree of protection against future spillover of sarbecoviruses from animal reservoirs. Graphical abstract

Changes in serum-neutralizing antibody potency and breadth post-SARS-CoV-2 mRNA vaccine boost.

A better understanding of the durability and breadth of serum-neutralizing antibody responses against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants elicited by COVID-19 vaccines is crucial in addressing the current pandemic. In this study, we quantified the decay of serum neutralization antibodies (nAbs) after second and third doses of the original COVID-19 mRNA vaccine. Using an authentic virus-neutralization assay, we found that decay half-lives of WA1- and Delta-nAbs were both ∼60 days after second and third vaccine dose. Unexpectedly, the durability of serum antibodies that neutralize three different Omicron subvariants (BA.1.1, BA.5, BA.2.12.1) was substantially better, with half-lives of ≥6 months. A booster dose of the original COVID-19 vaccine was also found to broaden antibody responses against SARS-CoV and four other sarbecoviruses, in addition to multiple SARS-CoV-2 strains. These findings suggest that repeated vaccinations with the COVID-19 vaccine may confer a degree of protection against future spillover of sarbecoviruses from animal reservoirs.

Structural insights into broadly neutralizing antibodies against SARS-CoV-2 elicited by hybrid immunity.

Increasing spread by SARS-CoV-2 Omicron variants challenges existing vaccines and broadly reactive neutralizing antibodies (bNAbs) against COVID-19. Here we determine the diversity, potency, breadth and structural insights of bNAbs derived from memory B cells of BNT162b2-vaccinee after homogeneous Omicron BA.1 breakthrough infection. The infection activates diverse memory B cell clonotypes for generating potent class I/II or III bNAbs with new epitopes mapped to receptor-binding domain (RBD). The top eight bNAbs neutralize wildtype and BA.1 potently but display divergent IgH/IgL sequences and neuralization profiles against other variants of concern (VOCs). Two of them (P2D9 and P3E6) belonging to class III NAbs display comparable potency against BA.4/BA.5, although structural analysis reveals distinct modes of action. P3E6 neutralizes all variants tested through a unique bivalent interaction with two RBDs. Our findings provide new insights into hybrid immunity on BNT162b2-induced diverse memory B cells in response to Omicron breakthrough infection for generating diverse bNAbs with distinct structural basis.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.

Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants.

The BQ and XBB subvariants of SARS-CoV-2 Omicron are now rapidly expanding, possibly due to altered antibody evasion properties deriving from their additional spike mutations. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals boosted with a WA1/BA.5 bivalent mRNA vaccine. Titers against BQ and XBB subvariants were lower by 13- to 81-fold and 66- to 155-fold, respectively, far beyond what had been observed to date. Monoclonal antibodies capable of neutralizing the original Omicron variant were largely inactive against these new subvariants, and the responsible individual spike mutations were identified. These subvariants were found to have similar ACE2-binding affinities as their predecessors. Together, our findings indicate that BQ and XBB subvariants present serious threats to current COVID-19 vaccines, render inactive all authorized antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.

Quantifying the impact of immune history and variant on SARS-CoV-2 viral kinetics and infection rebound: A retrospective cohort study.

Background: The combined impact of immunity and SARS-CoV-2 variants on viral kinetics during infections has been unclear. Methods: We characterized 1,280 infections from the National Basketball Association occupational health cohort identified between June 2020 and January 2022 using serial RT-qPCR testing. Logistic regression and semi-mechanistic viral RNA kinetics models were used to quantify the effect of age, variant, symptom status, infection history, vaccination status and antibody titer to the founder SARS-CoV-2 strain on the duration of potential infectiousness and overall viral kinetics. The frequency of viral rebounds was quantified under multiple cycle threshold (Ct) value-based definitions. Results: Among individuals detected partway through their infection, 51.0% (95% credible interval [CrI]: 48.3-53.6%) remained potentially infectious (Ct <30) 5 days post detection, with small differences across variants and vaccination status. Only seven viral rebounds (0.7%; N=999) were observed, with rebound defined as 3+days with Ct <30 following an initial clearance of 3+days with Ct ≥30. High antibody titers against the founder SARS-CoV-2 strain predicted lower peak viral loads and shorter durations of infection. Among Omicron BA.1 infections, boosted individuals had lower pre-booster antibody titers and longer clearance times than non-boosted individuals. Conclusions: SARS-CoV-2 viral kinetics are partly determined by immunity and variant but dominated by individual-level variation. Since booster vaccination protects against infection, longer clearance times for BA.1-infected, boosted individuals may reflect a less effective immune response, more common in older individuals, that increases infection risk and reduces viral RNA clearance rate. The shifting landscape of viral kinetics underscores the need for continued monitoring to optimize isolation policies and to contextualize the health impacts of therapeutics and vaccines. Funding: Supported in part by CDC contract #200-2016-91779, a sponsored research agreement to Yale University from the National Basketball Association contract #21-003529, and the National Basketball Players Association.

Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2.75 emerged recently and appears to be spreading. It has nine mutations in spike compared with the currently circulating BA.2, raising concerns that it may further evade vaccine-elicited and therapeutic antibodies. We found BA.2.75 to be moderately more neutralization resistant to sera from vaccinated/boosted individuals than BA.2 (1.8-fold), similar to BA.2.12.1 (1.1-fold), but more neutralization sensitive than BA.4/5 (0.6-fold). Relative to BA.2, BA.2.75 showed heightened resistance to class 1 and class 3 monoclonal antibodies targeting the spike-receptor-binding domain while gaining sensitivity to class 2 antibodies. Resistance was largely conferred by G446S and R460K mutations. BA.2.75 was slightly resistant (3.7-fold) to bebtelovimab, a therapeutic antibody with potent activity against all Omicron subvariants. BA.2.75 also exhibited a higher binding affinity to host receptor ACE2 than other Omicron subvariants. BA.2.75 provides further insight into SARS-CoV-2 evolution as it gains transmissibility while incrementally evading antibody neutralization.

Mapping monoclonal anti-SARS-CoV-2 antibody repertoires against diverse coronavirus antigens.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged continuously, challenging the effectiveness of vaccines, diagnostics, and treatments. Moreover, the possibility of the appearance of a new betacoronavirus with high transmissibility and high fatality is reason for concern. In this study, we used a natively paired yeast display technology, combined with next-generation sequencing (NGS) and massive bioinformatic analysis to perform a comprehensive study of subdomain specificity of natural human antibodies from two convalescent donors. Using this screening technology, we mapped the cross-reactive responses of antibodies generated by the two donors against SARS-CoV-2 variants and other betacoronaviruses. We tested the neutralization potency of a set of the cross-reactive antibodies generated in this study and observed that most of the antibodies produced by these patients were non-neutralizing. We performed a comparison of the specific and non-specific antibodies by somatic hypermutation in a repertoire-scale for the two individuals and observed that the degree of somatic hypermutation was unique for each patient. The data from this study provide functional insights into cross-reactive antibodies that can assist in the development of strategies against emerging SARS-CoV-2 variants and divergent betacoronaviruses.

Mapping monoclonal anti-SARS-CoV-2 antibody repertoires against diverse coronavirus antigens

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged continuously, challenging the effectiveness of vaccines, diagnostics, and treatments. Moreover, the possibility of the appearance of a new betacoronavirus with high transmissibility and high fatality is reason for concern. In this study, we used a natively paired yeast display technology, combined with next-generation sequencing (NGS) and massive bioinformatic analysis to perform a comprehensive study of subdomain specificity of natural human antibodies from two convalescent donors. Using this screening technology, we mapped the cross-reactive responses of antibodies generated by the two donors against SARS-CoV-2 variants and other betacoronaviruses. We tested the neutralization potency of a set of the cross-reactive antibodies generated in this study and observed that most of the antibodies produced by these patients were non-neutralizing. We performed a comparison of the specific and non-specific antibodies by somatic hypermutation in a repertoire-scale for the two individuals and observed that the degree of somatic hypermutation was unique for each patient. The data from this study provide functional insights into cross-reactive antibodies that can assist in the development of strategies against emerging SARS-CoV-2 variants and divergent betacoronaviruses.

Genomic Epidemiology and Serology Associated with a SARS-CoV-2 R.1 Variant Outbreak in New Jersey.

Examining the neutralizing capacity of monoclonal antibodies (MAbs) used to treat COVID-19, as well as antibodies recovered from unvaccinated, previously vaccinated, and infected individuals, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) remains critical to study. Here, we report on a SARS-CoV-2 nosocomial outbreak caused by the SARS-CoV-2 R.1 variant harboring the E484K mutation in a 281-bed psychiatric facility in New Jersey among unvaccinated inpatients and health care professionals (HCPs). A total of 81 inpatients and HCPs tested positive for SARS-Cov-2 by reverse transcription (RT)-PCR from 29 October 9 to 30 November 2020. The R.1 variant exhibits partial or complete resistance to two MAbs in clinical use, as well as 2 receptor binding domain MAbs and 4 N-terminal domain (NTD) MAbs. NTD MAbs against pseudovirus harboring single characteristic R.1 mutations highlight the role of S255F in loss of activity. Additionally, we note dampened neutralization capacity by plasma from individuals with previous SARS-CoV-2 infection or sera from vaccinated individuals. The relative resistance of the R.1 variant is likely lower than that of B.1.351 and closer to that of P.1 and B.1.526. The R.1 lineage has been reported in 47 states in the United States and 40 countries. Although high proportions exhibited symptoms (26% and 61% among patients and HCPs, respectively) and relative antibody resistance, we detected only 10 R.1 variants from over 2,900 samples (~0.34%) collected from January to October 2021. Among 3 vaccinated individuals previously infected with R.1, we observed robust neutralizing antibody responses against SARS-CoV-2 wild type and VOCs. IMPORTANCE The neutralizing capacities of monoclonal antibodies used to treat COVID-19 and of those recovered from previously infected and vaccinated individuals against SARS-CoV-2 variants of concern (VOCs) remain important questions. We report on a nosocomial outbreak caused by a SARS-CoV-2 R.1 variant harboring an E484K mutation among 81 unvaccinated inpatients and health care professionals. We note high attack rates with symptoms in nearly 50% of infected individuals, in sharp contrast to an unrelated institutional outbreak caused by the R.1 variant among a vaccinated population. We found little evidence of significant community spillover. This variant exhibits partial or complete resistance to two monoclonal antibodies in clinical use and dampened the neutralization capacity of convalescent-phase plasma from individuals with previous infection or sera from vaccinated individuals. Among three vaccinated individuals previously infected with R.1, we observed robust neutralizing antibody responses against SARS-CoV-2 wild type and VOCs. These findings underscore the importance of vaccination for prevention of symptomatic COVID-19 disease.

Functional map of SARS-CoV-2 3CL protease reveals tolerant and immutable sites.

The SARS-CoV-2 3CL protease (3CLpro) is an attractive therapeutic target, as it is essential to the virus and highly conserved among coronaviruses. However, our current understanding of its tolerance to mutations is limited. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the 3CLpro and validate a subset of our results within authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein. Yet, we also identify specific residues that appear immutable, suggesting that these may be targets for future 3CLpro inhibitors. Finally, we utilize our screening as a basis to identify E166V as a resistance-conferring mutation against the clinically used 3CLpro inhibitor, nirmatrelvir. Collectively, the functional map presented herein may serve as a guide to better understand the biological properties of the 3CLpro and for drug development against coronaviruses.