High genetic barrier to escape from human polyclonal SARS-CoV-2 neutralizing antibodies

The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in SARS-CoV-2 convalescent and vaccinated individuals are key determinants of neutralization breadth and, consequently, the genetic barrier to viral escape. Using chimeric viruses and antibody-selected viral mutants, we show that multiple neutralizing epitopes, within and outside the viral receptor binding domain (RBD), are variably targeted by polyclonal plasma antibodies and coincide with sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic polymutant spike proteins that resisted polyclonal antibody neutralization to a similar degree as currently circulating variants of concern (VOC). Importantly, by aggregating VOC-associated and plasma-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in SARS-CoV-2 spike are sufficient to confer near-complete resistance to the polyclonal neutralizing antibodies generated by convalescents and mRNA vaccine recipients. Strikingly however, plasma from individuals who had been infected and subsequently received mRNA vaccination, neutralized this highly resistant SARS-CoV-2 polymutant, and also neutralized diverse sarbecoviruses. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against future sarbecovirus pandemics.

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Rapid seroconversion and persistent functional IgG antibodies in severe COVID-19 patients correlates with an IL-12p70 and IL-33 signature

Scientific Reports ◽

10.1038/s41598-021-83019-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ariel Munitz ◽

L. Edry-Botzer ◽

M. Itan ◽

R. Tur-Kaspa ◽

D. Dicker ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Neutralizing Antibodies ◽

Host Response ◽

Humoral Response ◽

Rapid Onset ◽

Response Analysis ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Viral Receptor ◽

Iga Antibodies

AbstractDespite ongoing efforts to characterize the host response toward SARS-CoV-2, a major gap in our knowledge still exists regarding the magnitude and duration of the humoral response. Analysis of the antibody response in mild versus moderate/severe patients, using our new developed quantitative electrochemiluminescent assay for detecting IgM/IgA/IgG antibodies toward SARS-CoV-2 antigens, revealed a rapid onset of IgG/IgA antibodies, specifically in moderate/severe patients. IgM antibodies against the viral receptor binding domain, but not against nucleocapsid protein, were detected at early stages of the disease. Furthermore, we observed a marked reduction in IgM/IgA antibodies over-time. Adapting our assay for ACE2 binding-competition, demonstrated that the presence of potentially neutralizing antibodies is corelated with IgG/IgA. Finally, analysis of the cytokine profile in COVID-19 patients revealed unique correlation of an IL-12p70/IL33 and IgG seroconversion, which correlated with disease severity. In summary, our comprehensive analysis has major implications on the understanding and monitoring of SARS-CoV-2 infections.

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Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

10.1101/2021.02.03.429355 ◽

2021 ◽

Author(s):

Carl Graham ◽

Jeffrey Seow ◽

Isabella Huettner ◽

Hataf Khan ◽

Neophytos Kouphou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Infectious Virus ◽

Antigenic Drift ◽

Host Cells ◽

Receptor Binding Domain ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

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An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity

10.1101/2021.04.02.438288 ◽

2021 ◽

Cited By ~ 1

Author(s):

Chihiro Motozono ◽

Mako Toyoda ◽

Jiri Zahradnik ◽

Terumasa Ikeda ◽

Akatsuki Saito ◽

...

Keyword(s):

Receptor Binding ◽

Cellular Immunity ◽

Human Leukocyte ◽

Spike Protein ◽

Viral Infectivity ◽

Receptor Binding Domain ◽

Viral Receptor ◽

Leukocyte Antigen ◽

Immune Resistance ◽

Naturally Occurring

SummaryDuring the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic.Graphical Abstract

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Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

Nature Immunology ◽

10.1038/s41590-021-01068-z ◽

2021 ◽

Author(s):

Vincent Dussupt ◽

Rajeshwer S. Sankhala ◽

Letzibeth Mendez-Rivera ◽

Samantha M. Townsley ◽

Fabian Schmidt ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Low Dose ◽

Viral Escape ◽

Spike Protein ◽

Receptor Binding Domain ◽

Viral Inactivation ◽

Angiotensin Converting Enzyme 2 ◽

Therapeutic Monoclonal Antibodies ◽

Potent Protection

AbstractPrevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

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Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies

10.1101/2020.08.30.273920 ◽

2020 ◽

Cited By ~ 8

Author(s):

Christopher O. Barnes ◽

Claudia A. Jette ◽

Morgan E. Abernathy ◽

Kim-Marie A. Dam ◽

Shannon R. Esswein ◽

...

Keyword(s):

Immune Responses ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Health Crisis ◽

Naturally Occurring ◽

Insight Into

AbstractThe COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1–5 show therapeutic promise and are being evaluated clincally6–8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to “up” RBDs, (2) ACE2-blocking hNAbs that bind both “up” and “down” RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize “up” and “down” RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only “up” RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent “down” RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.

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Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

10.1101/2021.03.15.435528 ◽

2021 ◽

Author(s):

Alexandra C Walls ◽

Marcos C Miranda ◽

Minh N Pham ◽

Alexandra Schaefer ◽

Allison Greaney ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Polyclonal Antibodies ◽

Clinical Stage ◽

Binding Domain ◽

Single Shot ◽

Receptor Binding Domain ◽

Negative Consequences ◽

Neutralizing Activity

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

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mRNA Vaccine-Induced Antibodies More Effective than Natural Immunity in Neutralizing SARS-CoV-2 and its High Affinity Variants

10.21203/rs.3.rs-659065/v1 ◽

2021 ◽

Author(s):

Yunkai Yu ◽

Dominic Esposito ◽

Zhigang Kang ◽

Jianming Lu ◽

Alan Remaley ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Similar Degree ◽

Natural Immunity ◽

Receptor Binding Domain ◽

Blood Samples ◽

High Affinity ◽

Functional Assays ◽

Highly Effective ◽

Antibody Levels ◽

Neutralization Ability

Abstract Several variants of SARS-CoV-2 have emerged. Those with mutations in the angiotensin-converting enzyme (ACE2) receptor binding domain (RBD) are associated with increased transmission and severity. In this study, we developed both antibody quantification and functional assays. Analyses of both COVID-19 convalescent and diagnostic cohorts strongly support the use of RBD antibody levels as an excellent surrogate to biochemical neutralization activities. Data further revealed that the samples from mRNA vaccinated individuals had a median of 17 times higher RBD antibody levels and a similar degree of increased neutralization activities against RBD-ACE2 binding than those from natural infections. Our data showed that N501Y RBD had 5-fold higher ACE2 binding than the original variant. While antisera from naturally infected subjects had substantially reduced neutralization ability against N501Y RBD, all blood samples from vaccinated individuals were highly effective in neutralizing it. Thus, our data indicates that mRNA vaccination is far more effective than natural immunity in generating highly effective neutralizing antibodies. It further suggests a potential need to maintain high RBD antibody levels to control the more infectious SARS-CoV-2 variants.

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Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

10.1101/2020.07.21.214759 ◽

2020 ◽

Cited By ~ 19

Author(s):

Yiska Weisblum ◽

Fabian Schmidt ◽

Fengwen Zhang ◽

Justin DaSilva ◽

Daniel Poston ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Receptor Binding Domain ◽

Reporter Virus ◽

Low Frequencies ◽

Protein Variants ◽

Prior Infection ◽

Neutralizing Epitopes

AbstractNeutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

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Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity

10.1101/2021.03.11.435037 ◽

2021 ◽

Author(s):

Sophie Gobeil ◽

Katarzyna Janowska ◽

Shana McDowell ◽

Katayoun Mansouri ◽

Robert Parks ◽

...

Keyword(s):

South Africa ◽

United Kingdom ◽

Neutralizing Antibodies ◽

Viral Escape ◽

Human Populations ◽

S Protein ◽

The United Kingdom ◽

Protein Mutations ◽

Structural Impacts ◽

Neutralizing Epitopes

New SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes.

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Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

10.1101/2021.04.06.438709 ◽

2021 ◽

Author(s):

Tyler N Starr ◽

Nadine Czudnochowski ◽

Fabrizia Zatta ◽

Young-Jun Park ◽

Zhuoming Liu ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Binding Domain ◽

Viral Escape ◽

Binding Motif ◽

Receptor Binding Domain ◽

Viral Neutralization ◽

Parental Antibody

An ideal anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse SARS-related coronaviruses, and be highly protective through viral neutralization and effector functions. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics 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), including S309, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

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