Cross-reactive sarbecovirus antibodies induced by mosaic RBD nanoparticles

Bacterial outer membrane vesicles (OMVs) represent a promising and versatile platform for vaccine delivery. Their inherent self-adjuvant properties and the ability to be adorned with a wide range of heterogeneous antigens position them as a powerful tool in the fight against infectious diseases. Escherichia coli Nissle 1917 (EcN) stands out as a highly valuable probiotic strain because of its long history of safe use and proven clinical benefits in humans. The EcN strain was genetically engineered to derive OMVs displaying receptor binding domain (RBD) of SARS-CoV-2 spike protein on their surface. Although some research groups have previously expressed the SARS-CoV-2 viral spike protein or the RBD in E. coli, particularly in EcN, this study shows a maiden effort to utilize the gene encoding native RBD. The probiotic EcN exhibited a significant level of native RBD expression, demonstrating a more efficient codon usage pattern compared to commonly used bacterial expression systems such as BL21, and DH5α. EcN was engineered to display the native form of viral RBD on the surface using the Lpp-OmpA system. Cell fractionation studies clearly indicated the presence of RBD in the membrane fraction. OMVs displaying RBD on their surface were isolated using ultracentrifugation and the presence of RBD in the OMVs was confirmed by western blot followed by immunofluorescence analyses. Due to their preferential uptake by antigen presenting cells, OMVs derived from EcN bearing native form of RBD hold promise as a potential COVID-19 vaccine candidate.

ABSTRACTSARS-CoV-2 continues adapting to human hosts during the current worldwide pandemic since 2019. This virus evolves through multiple means, such as single nucleotide mutations and structural variations, which has brought great difficulty to disease prevention and control of COVID-19. Structural variation, including multiple nucleotide changes like insertions and deletions, has a greater impact relative to single nucleotide mutation on both genome structures and protein functions. In this study, we found that deletion occurred frequently in not only SARS-CoV-2 but also in other SARS-related coronaviruses. These deletions showed obvious location bias and formed 45 recurrent deletion regions in the viral genome. Some of these deletions showed proliferation advantages, including four high-frequency deletions (nsp6 Δ106-109, S Δ69-70, S Δ144, and Δ28271) that were detected in around 50% of SARS-CoV-2 genomes and other 19 median-frequency deletions. In addition, the association between deletions and the WHO reported variants of concern (VOC) and variants of interest (VOI) of SARS-CoV-2 indicated that these variants had a unique combination of deletion patterns. In the spike (S) protein, the deletions in SARS-CoV-2 were mainly in the N-terminal domain. Some deletions, such as S Δ144/145 and S Δ243-244, have been confirmed to block the binding sites of neutralizing antibodies. Overall, this study revealed a conservative regional pattern and the potential effect of some deletions in SARS-CoV-2 over the whole genome, providing important evidence for potential epidemic control and vaccine development.IMPORTANCE Mutations in SARS-CoV-2 were studied extensively, while only the structure variations on the spike protein were discussed well in previous studies. To study the role of structural variations in virus evolution, we described the distribution of structure variations on the whole genome. Conserved patterns were found of deletions among SARS-CoV-2, SARS-CoV-2-like, and SARS-CoV-like viruses. There were 45 recurrent deletion regions (RDRs) in SARS-CoV-2 generated through the integration of deleted positions. In these regions, four high-frequency deletions parallelly appeared in multiple strains. Furthermore, in the spike protein, the deletions in SARS-CoV-2 were mainly in the N-terminal domain, blocking the binding sites of some neutralizing antibodies, while the structural variations in SARS-related coronavirus were mainly in the N-terminal domain and receptor binding domain. The receptor binding domain is highly related to hosting recognition. The deletions in the receptor binding domain may play a role in host adaption.

The aim of the study is to synthesize a recombinant protein in Escherichia coli cells that carries the receptor-binding domain (RBD) of Spike protein of SARS CoV-2, with antiviral activity comparable to the activity of RBD obtained in a eukaryotic cells (eRBD). 6His-RBDshort (24.4 kDa) and 6His-RBDlong (33.7 kDa) proteins were expressed in Escherichia coli strain BL21 (DE3). Chromatographic purification of the proteins was carried out on WorkBeads 40 Ni-NTA and WorkBeads 40S sorbents followed by multistage refolding. Enzyme immunoassay was performed using GamP2C5 and GamXRH19 humanized single-domain monoclonal antibodies specific for SARS-CoV-2 Spike protein RBD. Antiviral activity against the SARS-CoV-2 virus was studied using Vero E6 cells. 6His-RBDshort recombinant protein was synthesized in Escherichia coli cells, including the Spike protein RBM (receptor-binding motif) of SARS-CoV-2 virus (330–527 а.а.). Two-stage chromatographic purification of 6His-RBDshort recombinant protein was performed, followed by refolding. Enzyme immunoassay demonstrated effective interaction of 6His-RBDshort recombinant protein with virus neutralizing antibodies, comparable to eRBD. The study of antiviral activity showed inhibition of SARS-CoV-2 virus reproduction after treatment of Vero E6 cells with 6His-RBDshort (45.1%) and eRBD (42.8%) proteins. The 6His-RBDlong recombinant protein obtained in the same work, which included a longer fragment of RBD, did not interact with virus neutralizing antibodies and did not inhibit the replication of the SARS-CoV-2 virus. After conducting additional studies, the developed 6His-RBDshort recombinant protein can be considered a promising drug for therapeutic use as an ACE2 receptor blocker.

Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis: The case of RBD of SARS-CoV-2 spike protein and its antibody

Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.

The SARS-CoV-2 virion responsible for the current world-wide pandemic COVID-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD) within its distal, prefusion S1 domain. The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-CoV-2 Spike protein in its static prefusion state based on two recent and independent experimental structure publications. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called “glue” points) between residues in the Up and Down state protomers. The S2 proximal, fusion domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer’s intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. This included, for example, a key RBD residue ARG357 in the Up-Down interaction and a three residue sequence ALA520-PRO521-ALA522, associated with a turn structure in the RBD of the Up state protomer, acting as a stabilizing interaction with the NTD of its neighbor protomer. Additionally, our intra chain dominant energy mappings within each protomer, identified a significant “glue” point or possible “latch” for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the amino acid R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-CoV-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. We conducted preliminary molecular dynamics simulations across 0.1 μ seconds to see if this latch provided structural stability and indeed found that a single point mutation (Q564G) resulted in the latch releasing transforming the protomer from the Down to the Up state conformation. Full trimeric Spike protein studies of the same mutation across all protomers, however, did not exhibit latch release demonstrating the critical importance of interchain interactions across the S1 domain, including RBD-NTD neighboring chain interactions. Therapies aimed at disrupting these noncovalent interactions could be a viable route for the physico-chemical mitigation of this deadly virion.

Objective(s):The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), giving rise to the coronavirus disease 2019 (COVID-19), has become a danger to wellbeing worldwide. Thus, finding efficient and safe vaccines for COVID-19 is of great importance. As a basic step amid contamination, SARS-CoV-2 employs the receptor-binding domain (RBD) of the spike protein to lock in with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells. SARS-CoV-2 receptor-binding domain (RBD) is the main human antibody target for developing vaccines and virus inhibitors, as well as neutralizing antibodies. A bacterial procedure was developed for the expression and purification of the SARS-CoV-2 spike protein receptor-binding domain.Materials and Methods:In this research study, RBD was expressed by Escherichia coli and purified with Ni-NTA chromatography. Then it was affirmed by the western blot test. The immunogenicity and protective efficacy of RBD recombinant protein were assessed on BALB/c mice. Additionally, RBD recombinant protein was tested by ELISA utilizing sera of COVID-19 healing patients contaminated with SARS-CoV-2 wild type and Delta variation. Results:Indirect ELISA was able to detect the protein RBD in serum of the immunized mouse expressed in E. coli. The inactive SARS-CoV2 was detected by antibodies within the serum of immunized mice. Serum antibodies from individuals recovered from Covid19 reacted to the expressed protein.Conclusion:Our findings showed that RBD is of great importance in vaccine design and it can be used to develop recombinant vaccines through induction of antibodies against RBD.

Coronavirus disease 2019 (COVID-19) is arguably the most socially and economically disruptive pandemic since the 1918 influenza pandemic. Although pediatric COVID-19 shares features with the adult disease, there are several differences. Children produce virus in amounts at least equal to adults, if not higher,1 and can transmit the virus, just as adults can.2 School-aged children are generally less-severely affected than infants or adults, but some children without significant underlying disease become ill or die in a disease process analogous to the one most commonly seen in adults: severe pulmonary disease and respiratory failure.3–6 Children and adults appear to have different humoral immune responses to COVID-19.7A small fraction of children with COVID-19 experience a hyperinflammatory process,8–13 termed multisystem inflammatory syndrome in children (MIS-C) in the United States, with features distinct from Kawasaki disease.14 This case definition includes ≥2 of these symptoms: rash, conjunctivitis, or mucocutaneous inflammation; hypotension; cardiac disease; coagulopathy; or acute gastrointestinal problems.15 MIS-C, which is not correlated with viral load levels, typically appears some time after initial infection.13 MIS-C’s incidence is difficult to determine, given the high rate of asymptomatic infection.12Adults with COVID-19 also can experience inflammatory disorders: coagulopathies, vasculitis, cardiomyositis, and neuroinflammatory processes.16–18 That the most effective therapy to reduce mortality in adults with severe COVID-19 yet established is an immune suppressant, dexamethasone,19 reveals the importance of inflammatory and immune-mediated pathologies.In this issue of Pediatrics, Rostad et al20 showed that children hospitalized with MIS-C have significantly higher concentrations of antibodies against the receptor-binding domain (RBD), a part of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S). RBD is the part of S mediating virus binding to its receptor, angiotensin-converting enzyme 2, on host cells. This finding is reinforced by other research, which showed that anti-RBD antibody concentrations were higher in children with severe MIS-C than in children with mild MIS-C or patients not meeting MIS-C definitions.1 The children also had high SARS-CoV-2 neutralization titers, consistent with the observations that anti-RBD antibodies can effectively neutralize the virus.21 The patients with MIS-C described in Rostad et al20 also tended to have higher antibody levels against the entire spike protein and the viral nucleocapsid protein, which is not exposed on the surface of the virion. The anti-RBD antibody levels correlated with the erythrocyte sedimentation rate, suggesting that higher anti-RBD antibodies are associated with a more proinflammatory state.The finding that patients with MIS-C have higher anti-RBD antibodies is interesting and potentially important because, despite case definitions, MIS-C may be difficult to diagnose. If high levels of anti-RBD antibodies are associated with MIS-C, quantitative assays for anti-RBD antibodies might enable more-accurate MIS-C diagnosis or predict patients at higher risk for MIS-C, potentially enabling early interventions.So far, authors of reports describing associations of high anti-RBD IgG with MIS-C have looked at anti-RBD IgG bulk properties. However, all antibodies may not have equal activity to cause, or equal propensities to be associated with, MIS-C. Detailed study of the antibodies, including mapping binding sites within RBD, may yield improved understanding of pathogenesis. Immune responses to COVID-19 are heterogeneous. Neutralizing antibody concentrations vary widely; some patients do not develop detectable titers.22 Patients who recover exhibit different antibody responses from those who die.23 It may be helpful to modulate immune responses to achieve ideal levels of activity generally, such as dexamethasone for severe disease, or specifically, if anti-RBD immune responses prove problematic.Beyond potential use in diagnosing MIS-C, the findings may have implications for pathogenesis, therapy, and vaccine development. In producing immune responses against COVID-19, there may be a response that optimally addresses threats posed by infection without initiating harmful hyperactive immune responses. The response against RBD may reflect general characteristics of immune responses against SARS-CoV-2, or there may be something particularly problematic about immune responses directed against RBD, per se, an idea reinforced by the finding that RBD antibodies are correlated with inflammatory markers.Another implication may be that if anti-RBD antibodies are associated with MIS-C, it might be desirable to screen convalescent sera for anti–SARS-CoV-2 neutralizing activity and anti-RBD activity, selecting units with good neutralizing activity but with lower anti-RBD activity. This may also be a consideration for anti–SARS-CoV-2 therapeutic monoclonal antibody development.Some investigators have hypothesized that children may have milder COVID-19 disease because of cross-reactive immunity to other coronaviruses,24,25 but it is also conceivable that such previous coronavirus exposures might have increased risks of an inflammatory response or worse disease, as seen with dengue hemorrhagic fever.26,27With their findings, Rostad et al20 may also have implications for vaccine development. Close attention to vaccines eliciting anti-RBD antibodies may be advisable, if dysregulated or aberrant responses against RBD or parts of RBD contribute to hyperinflammation. Many candidate vaccines aim to elicit responses against the entire S, including RBD. Some aim to specifically elicit antibodies against RBD.28 Although a COVID-19 vaccine is urgently needed, leading vaccinologists have cautioned against deploying vaccines without thorough safety evaluations, recalling unfortunate past tragedies involving candidate vaccines, with vaccination yielding increased morbidity and mortality when vaccine recipients were later infected with circulating virus.29–32 If strong anti-RBD responses are associated with an increased risk of inflammatory disorders, it may then be advantageous to develop vaccines that, while eliciting excellent anti–SARS-CoV-2 neutralizing activity, preferentially avoid eliciting strong anti-RDB immune responses.How could aberrant immune responses promote MIS-C? Hypothetical mechanisms include antibody-dependent enhancement, direct cytotoxicity, immune complexes, and macrophage hyperinflammatory responses,33 perhaps enabled by substantial differences in humoral responses.7 Another theoretical possibility would be innate activities of anti-RBD antibodies. Antibodies with catalytic activity (abzymes) have been described and can be significant in autoimmune diseases.34,35 Antibodies against proteins that bind enzymes can themselves have weak catalytic activity; an antibody against a protein (RBD) that binds an enzyme (angiotensin-converting enzyme 2) could plausibly have catalytic activity, with physiologic consequences.36MIS-C and COVID-19 inflammatory disorders in children are relatively uncommon but can be serious. Better diagnostics and improved management will be important as more children are infected. Greater understanding of MIS-C pathogenesis may help optimize convalescent plasma therapy and inform vaccine development.

The changes in charge distribution caused by mutations in the spike protein may play a crucial role in balancing infectivity and immune evasion during the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To explore how charge increments in spike protein variants influence viral evolution, a statistical analysis was conducted on 57 SARS-CoV-2 variants, examining relationships between charge distribution, lineage divergence, angiotensin-converting enzyme 2 (ACE2) affinity, immune evasion, and receptor-binding domain (RBD) expression. A phylogenetic tree was also reconstructed using only the charge properties of mutation sites. Results indicated that with increasing lineage divergence, overall positive charge initially rose sharply and then more gradually. Partitioning the spike protein into three domains—the RBD, the N-terminal flanking region (B-RBD), and the C-terminal flanking region (A-RBD)—revealed distinct patterns: positive charge increased in the RBD and A-RBD, whereas the B-RBD accumulated negative charge. Charge increments were negatively associated with ACE2 affinity and RBD expression but positively correlated with immune evasion. The k-mer-based tree derived from charge-reduced sequences showed a topology consistent with the whole-genome tree. These findings suggest that charge distribution in spike proteins is closely linked to viral evolution, with the opposing trends in the RBD and B-RBD potentially reflecting a balance between infectivity and immune escape.

GB-2 blocking the interaction between ACE2 and wild type and mutation of spike protein of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI- cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI- cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.

Substitutions that fix between SARS-CoV-2 variants can transform the mutational landscape of future evolution via epistasis. For example, large epistatic shifts in mutational effects caused by N501Y underlied the original emergence of Omicron, but whether such epistatic saltations continue to define ongoing SARS-CoV-2 evolution remains unclear. We conducted deep mutational scans to measure the impacts of all single amino acid mutations and single-codon deletions in the spike receptor-binding domain (RBD) on ACE2-binding affinity and protein expression in the recent Omicron BQ.1.1 and XBB.1.5 variants, and we compared mutational patterns to earlier viral strains that we have previously profiled. As with previous deep mutational scans, we find many mutations that are tolerated or even enhance binding to ACE2 receptor. The tolerance of sites to single-codon deletion largely conforms with tolerance to amino acid mutation. Though deletions in the RBD have not yet been seen in dominant lineages, we observe tolerated deletions including at positions that exhibit indel variation across broader sarbecovirus evolution and in emerging SARS-CoV-2 variants of interest, most notably the well-tolerated Δ483 deletion in BA.2.86. The substitutions that distinguish recent viral variants have not induced as dramatic of epistatic perturbations as N501Y, but we identify ongoing epistatic drift in SARS-CoV-2 variants, including interaction between R493Q reversions and mutations at positions 453, 455, and 456, including F456L that defines the XBB.1.5-derived EG.5 lineage. Our results highlight ongoing drift in the effects of mutations due to epistasis, which may continue to direct SARS-CoV-2 evolution into new regions of sequence space.

SARS-CoV-2 is under strong evolutionary selection to acquire mutations in its spike protein that reduce neutralization by human polyclonal antibodies. Here we use pseudovirus-based deep mutational scanning to measure how mutations to the spike from the recent KP.3.1.1 SARS-CoV-2 strain affect cell entry, binding to ACE2 receptor, RBD up/down motion, and neutralization by human sera and clinically relevant antibodies. The spike mutations that most affect serum antibody neutralization sometimes differ between sera collected before versus after recent vaccination or infection, indicating these exposures shift the neutralization immunodominance hierarchy. The sites where mutations cause the greatest reduction in neutralization by post-vaccination or infection sera include receptor-binding domain (RBD) sites 475, 478 and 487, all of which have mutated in recent SARS-CoV-2 variants. Multiple mutations outside the RBD affect sera neutralization as strongly as any RBD mutations by modulating RBD up/down movement. Some sites that affect RBD up/down movement have mutated in recent SARS-CoV-2 variants. Finally, we measure how spike mutations affect neutralization by three clinically relevant SARS-CoV-2 antibodies: VYD222, BD55-1205, and SA55. Overall, these results illuminate the current constraints and pressures shaping SARS-CoV-2 evolution, and can help with efforts to forecast possible future antigenic changes that may impact vaccines or clinical antibodies.

SARS-CoV-2 variants raise concern because of their high transmissibility and their ability to evade neutralizing antibodies elicited by prior infection or by vaccination. Here, we compared the neutralizing abilities of sera from 70 unvaccinated COVID-19 patients infected before the emergence of variants of concern (VOCs) and of 16 vaccine breakthrough infection (BTI) cases infected with Gamma or Delta against the ancestral B.1 strain, the Gamma, Delta and Omicron BA.1 VOCs using live virus. We further determined antibody levels against the Nucleocapsid (N) and full Spike proteins, the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the Spike protein. Convalescent sera featured considerable variability in the neutralization of B.1 and in the cross-neutralization of different strains. Their neutralizing capacity moderately correlated with antibody levels against the Spike protein and the RBD. All but one convalescent serum failed to neutralize Omicron BA.1. Overall, convalescent sera from patients with moderate disease had higher antibody levels and displayed a higher neutralizing ability against all strains than patients with mild or severe forms of the disease. The sera from BTI cases fell into one of two categories: half the sera had a high neutralizing activity against the ancestral B.1 strain as well as against the infecting strain, while the other half had no or a very low neutralizing activity against all strains. Although antibody levels against the spike protein and the RBD were lower in BTI sera than in unvaccinated convalescent sera, most neutralizing sera also retained partial neutralizing activity against Omicron BA.1, suggestive of a better cross-neutralization and higher affinity of vaccine-elicited antibodies over virus-induced antibodies. Accordingly, the IC50: antibody level ratios were comparable for BTI and convalescent sera, but remained lower in the neutralizing convalescent sera from patients with moderate disease than in BTI sera. The neutralizing activity of BTI sera was strongly correlated with antibodies against the Spike protein and the RBD. Together, these findings highlight qualitative differences in antibody responses elicited by infection in vaccinated and unvaccinated individuals. They further indicate that breakthrough infection with a pre-Omicron variant boosts immunity and induces cross-neutralizing antibodies against different strains, including Omicron BA.1.

Severe acute respiratory syndrome (SARS) and novel coronavirus disease (COVID-19) are caused by two closely related beta-coronaviruses, SARS-CoV and SARS-CoV-2, respectively. The envelopes surrounding these viruses are decorated with spike proteins, whose receptor binding domains (RBDs) initiate invasion by binding to the human angiotensin-converting enzyme 2 (ACE2). Subtle changes at the interface with ACE2 seem to be responsible for the enhanced affinity for the receptor of the SARS-CoV-2 RBD compared to SARS-CoV RBD. Here, we use Elastic Network Models (ENMs) to study the response of the viral RBDs and ACE2 upon dissassembly of the complexes. We identify a dominant detachment mode, in which the RBD rotates away from the surface of ACE2, while the receptor undergoes a conformational transition which stretches the active-site cleft. Using the Structural Perturbation Method, we determine the network of residues, referred to as the Allostery Wiring Diagram (AWD), which drives the large-scale motion activated by the detachment of the complex. The AWD for SARS-CoV and SARS-CoV-2 are remarkably similar, showing a network that spans the interface of the complex and reaches the active site of ACE2, thus establishing an allosteric connection between RBD binding and receptor catalytic function. Informed in part by the AWD, we used Molecular Dynamics simulations to probe the effect of interfacial mutations in which SARS-CoV-2 residues are replaced by their SARS-CoV counterparts. We focused on a conserved glycine (G502 in SARS-CoV-2, G488 in SARS-CoV) because it belongs to a region that initiates the dissociation of the complex along the dominant detachment mode, and is prominent in the AWD. Molecular Dynamics simulations of SARS-CoV-2 wild-type and G502P mutant show that the affinity for the human receptor of the mutant is drastically diminished. Our results suggest that in addition to residues that are in direct contact with the interface those involved in long range allosteric communication are also a determinant of the stability of the RBD-ACE2 complex.