Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology.

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

Neutralization, effector function and immune imprinting of Omicron variants.

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody1. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab2, S2X2593 and S2H974. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Structural basis of malaria RIFIN binding by LILRB1-containing antibodies.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to ß2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.

Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies.

Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant1, which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b22. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

Increased breadth and neutralization of antibodies against SARS-CoV-2 variants after infection and vaccination: A serosurveillance study in pediatric patients of Southern Switzerland.

Little information is available about the nature of the immune response in children after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. The aim of this study is to define the seroprevalence and the features of the antibody response in children of Southern Switzerland during the different waves of Coronavirus Disease 2019 (COVID-19) pandemic. By analyzing 756 sera collected from children aged 0 to 16 years admitted to the Institute of Pediatrics of Southern Switzerland during the prepandemic period (before March 2020) and the first four pandemic waves (between March 2020 and June 2022), we investigated binding titers, cross-reactivity, and neutralizing properties of the serum antibodies against SARS-CoV-2 variants. Seroprevalence varied from 6% during the first wave to 14% and 17% during the second and third waves, respectively, peaking at 39% during the fourth wave. The 96 seropositive cases were mostly asymptomatic (42.7%) or showed mild (20.8%) to moderate (32.3%) symptoms. Moderate symptoms and close contact with COVID-19-positive individuals were associated with a higher infection risk (P < 0.001). The antibody response was mainly driven by IgG directed to the receptor-binding domain (RBD) of Wuhan-1 SARS-CoV-2 Spike (S). Children infected in the first three waves produced antibodies with up to 11-fold and 5.5-fold reduction in binding and neutralizing titers, respectively, against different SARS-CoV-2 variants, including Beta, Delta, and Omicron BA.1, BA.2, and BA.5. Such reductions were less pronounced in children infected during the fourth wave, who showed the highest frequency and titers of neutralizing antibodies against the same variants. Compared to infection, vaccination with a Wuhan-1-based messenger RNA (mRNA) vaccine induced higher and heterogenous levels of antibodies cross-reacting to the different SARS-CoV-2 variants analyzed.   Conclusions: Despite the high burden of COVID-19 in Southern Switzerland, we observed an initial low seroprevalence of SARS-CoV-2 in children, which increased in the later waves. The antibody response was poor in the first three waves and improved in the fourth wave, when children produced higher levels of neutralizing antibodies after vaccination or infection with Delta and/or Omicron variants. What is Known: • Children were marginally affected by the initial SARS-CoV-2 variants. • The number of infected and hospitalized children increased after the appearance of the Omicron variants. What is New: • Seroprevalence of SARS-CoV-2 in children of Southern Switzerland increased overtime. • Children produced higher levels of neutralizing antibodies after vaccination or infection with Delta and/or Omicron variants in the fourth wave compared to children infected in the first three waves.

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity, and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month time frame. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both prefusion and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sublineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants.

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

Poor neutralization and rapid decay of antibodies to SARS-CoV-2 variants in vaccinated dialysis patients.

Patients on dialysis are at risk of severe course of SARS-CoV-2 infection. Understanding the neutralizing activity and coverage of SARS-CoV-2 variants of vaccine-elicited antibodies is required to guide prophylactic and therapeutic COVID-19 interventions in this frail population. By analyzing plasma samples from 130 hemodialysis and 13 peritoneal dialysis patients after two doses of BNT162b2 or mRNA-1273 vaccines, we found that 35% of the patients had low-level or undetectable IgG antibodies to SARS-CoV-2 Spike (S). Neutralizing antibodies against the vaccine-matched SARS-CoV-2 and Delta variant were low or undetectable in 49% and 77% of patients, respectively, and were further reduced against other emerging variants. The fraction of non-responding patients was higher in SARS-CoV-2-naïve hemodialysis patients immunized with BNT162b2 (66%) than those immunized with mRNA-1273 (23%). The reduced neutralizing activity correlated with low antibody avidity. Patients followed up to 7 months after vaccination showed a rapid decay of the antibody response with an average 21- and 10-fold reduction of neutralizing antibodies to vaccine-matched SARS-CoV-2 and Delta variant, which increased the fraction of non-responders to 84% and 90%, respectively. These data indicate that dialysis patients should be prioritized for additional vaccination boosts. Nevertheless, their antibody response to SARS-CoV-2 must be continuously monitored to adopt the best prophylactic and therapeutic strategy.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages carry distinct spike mutations resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters elicit plasma-neutralizing antibodies against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5, and that breakthrough infections, but not vaccination alone, induce neutralizing antibodies in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1, BA.2, and BA.4/5 receptor-binding domains, whereas Omicron primary infections elicit B cells of narrow specificity up to 6 months after infection. Although most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant-neutralizing antibody that is a strong candidate for clinical development.

Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice.

We used human monoclonal antibodies (humAbs) to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate these antibodies as a safe preventive and therapeutic substitute for hyperimmune sera to treat tetanus in mice. By screening memory B cells from immune donors, we selected 2 tetanus neurotoxin-specific mAbs with exceptionally high neutralizing activities and extensively characterized them both structurally and functionally. We found that these antibodies interfered with the binding and translocation of the neurotoxin into neurons by interacting with 2 epitopes, whose identification pinpoints crucial events in the cellular pathogenesis of tetanus. Our observations explain the neutralization ability of these antibodies, which we found to be exceptionally potent in preventing experimental tetanus when injected into mice long before the toxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential for therapeutic use via intrathecal injection. As such, we believe these humAbs, as well as their Fab derivatives, meet the requirements to be considered for prophylactic and therapeutic use in human tetanus and are ready for clinical trials.

Risk assessment and seroprevalence of SARS-CoV-2 infection in healthcare workers of COVID-19 and non-COVID-19 hospitals in Southern Switzerland.

BACKGROUND: Hospital healthcare workers (HCW), in particular those involved in the clinical care of COVID-19 cases, are presumably exposed to a higher risk of acquiring the disease than the general population. METHODS: Between April 16 and 30, 2020 we conducted a prospective, SARS-CoV-2 seroprevalence study in HCWs in Southern Switzerland. Participants were hospital personnel with varying COVID-19 exposure risk depending on job function and working site. They provided personal information (including age, sex, occupation, and medical history) and self-reported COVID-19 symptoms. Odds ratio (OR) of seropositivity to IgG antibodies was estimated by univariate and multivariate logistic regressions. FINDINGS: Among 4726 participants, IgG antibodies to SARS-CoV-2 were detected in 9.6% of the HCWs. Seropositivity was higher among HCWs working on COVID-19 wards (14.1% (11.9-16.5)) compared to other hospital areas at medium (10.7% (7.6-14.6)) or low risk exposure (7.3% (6.4-8.3)). OR for high vs. medium wards risk exposure was 1.42 (0.91-2.22), P = 0.119, and 1.98 (1.55-2.53), P<0.001 for high vs. low wards risk exposure. The same was for true for doctors and nurses (10.1% (9.0-11.3)) compared to other employees at medium (7.1% (4.8-10.0)) or low risk exposure (6.6% (5.0-8.4)). OR for high vs. medium profession risk exposure was 1.37 (0.89-2.11), P = 0.149, and 1.75 (1.28-2.40), P = 0.001 for high vs. low profession risk exposure. Moreover, seropositivity was higher among HCWs who had household exposure to COVID-19 cases compared to those without (18.7% (15.3-22.5) vs. 7.7% (6.9-8.6), OR 2.80 (2.14-3.67), P<0.001). INTERPRETATION: SARS-CoV-2 antibodies are detectable in up to 10% of HCWs from acute care hospitals in a region with high incidence of COVID-19 in the weeks preceding the study. HCWs with exposure to COVID-19 patients have only a slightly higher absolute risk of seropositivity compared to those without, suggesting that the use of PPE and other measures aiming at reducing nosocomial viral transmission are effective. Household contact with known COVID-19 cases represents the highest risk of seropositivity. FUNDING: Henry Krenter Foundation, Ente Ospedaliero Cantonale and Vir Biotechnology.

SARS-CoV-2 immune evasion by variant B.1.427/B.1.429.

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.

SARS-CoV-2 immune evasion by the B.1.427/B.1.429 variant of concern.

A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies.