Comprehensive mapping of binding hot spots of SARS-CoV-2 RBD-specific neutralizing antibodies for tracking immune escape variants

Abstract Background The receptor-binding domain (RBD) variants of SARS-CoV-2 could impair antibody-mediated neutralization of the virus by host immunity; thus, prospective surveillance of antibody escape mutants and understanding the evolution of RBD are urgently needed. Methods Using the single B cell cloning technology, we isolated and characterized 93 RBD-specific antibodies from the memory B cells of four COVID-19 convalescent individuals in the early stage of the pandemic. Then, global RBD alanine scanning with a panel of 19 selected neutralizing antibodies (NAbs), including several broadly reactive NAbs, was performed. Furthermore, we assessed the impact of single natural mutation or co-mutations of concern at key positions of RBD on the neutralization escape and ACE2 binding function by recombinant proteins and pseudoviruses. Results Thirty-three amino acid positions within four independent antigenic sites (1 to 4) of RBD were identified as valuable indicators of antigenic changes in the RBD. The comprehensive escape mutation map not only confirms the widely circulating strains carrying important immune escape RBD mutations such as K417N, E484K, and L452R, but also facilitates the discovery of new immune escape-enabling mutations such as F486L, N450K, F490S, and R346S. Of note, these escape mutations could not affect the ACE2 binding affinity of RBD, among which L452R even enhanced binding. Furthermore, we showed that RBD co-mutations K417N, E484K, and N501Y present in B.1.351 appear more resistant to NAbs and human convalescent plasma from the early stage of the pandemic, possibly due to an additive effect. Conversely, double mutations E484Q and L452R present in B.1.617.1 variant show partial antibody evasion with no evidence for an additive effect. Conclusions Our study provides a global view of the determinants for neutralizing antibody recognition, antigenic conservation, and RBD conformation. The in-depth escape maps may have value for prospective surveillance of SARS-CoV-2 immune escape variants. Special attention should be paid to the accumulation of co-mutations at distinct major antigenic sites. Finally, the new broadly reactive NAbs described here represent new potential opportunities for the prevention and treatment of COVID-19.

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Immune escape mutations selected by neutralizing antibodies in natural HIV-1 infection can alter coreceptor usage repertoire of the transmitted/founder virus

10.1101/2021.11.10.467978 ◽

2021 ◽

Author(s):

Manukumar Honnayakanahalli Marichannegowda ◽

Hongshuo Song

Keyword(s):

Neutralizing Antibodies ◽

Immune Escape ◽

Escape Mutation ◽

Coreceptor Usage ◽

Founder Virus ◽

The Impact ◽

Glycan Site ◽

Hiv 1 ◽

Transmitted Founder Virus

The ability of HIV-1 to evade neutralizing antibodies (NAbs) in vivo is well demonstrated, but the impact of NAb escape mutations on HIV-1 phenotype other than immune escape itself has rarely been studied. Here, we show that immune escape mutations selected by V3-glycan specific NAbs in vivo can alter the coreceptor usage repertoire of the transmitted/founder (T/F) HIV-1. In a participant developed V3-glycan NAb response, naturally selected mutations at the V3 N301 and N332 glycan sites abrogated CCR8 usage while conferred APJ usage on the cognate T/F strain. Mutations at the N301 glycan also impaired CCR3 usage and partially compromised the efficiency in using CCR5, which could be fully restored by a single escape mutation at the N332 glycan site. Our study demonstrates the link between NAb escape and coreceptor usage alteration in natural HIV-1 infection and indicates that NAb response could drive virus entry tropism evolution in vivo.

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Immunogenicity of heterologous BNT162b2 booster in fully vaccinated individuals with CoronaVac against SARS-CoV-2 variants Delta and Omicron: the Dominican Republic Experience

10.1101/2021.12.27.21268459 ◽

2021 ◽

Author(s):

Eddy Perez-Then ◽

Carolina Lucas ◽

Valter Silva Monteiro ◽

Marija Miric ◽

Vivian Brache ◽

...

Keyword(s):

Dominican Republic ◽

Dose Regimen ◽

Neutralizing Antibodies ◽

Immune Escape ◽

Neutralizing Antibody ◽

Fold Increase ◽

Neutralization Activity ◽

Recent Emergence ◽

Virus Specific Antibody ◽

The Impact

The recent emergence of the SARS-CoV-2 Omicron variant is raising concerns because of its increased transmissibility and by its numerous spike mutations with potential to evade neutralizing antibodies elicited by COVID-19 vaccines. The Dominican Republic was among the first countries in recommending the administration of a third dose COVID-19 vaccine to address potential waning immunity and reduced effectiveness against variants. Here, we evaluated the effects of a heterologous BNT162b2 mRNA vaccine booster on the humoral immunity of participants that had received a two-dose regimen of CoronaVac, an inactivated vaccine used globally. We found that heterologous CoronaVac prime followed by BNT162b2 booster regimen induces elevated virus-specific antibody levels and potent neutralization activity against the ancestral virus and Delta variant, resembling the titers obtained after two doses of mRNA vaccines. While neutralization of Omicron was undetectable in participants that had received a two-dose regimen of CoronaVac vaccine, BNT162b2 booster resulted in a 1.4-fold increase in neutralization activity against Omicron, compared to two-dose mRNA vaccine. Despite this increase, neutralizing antibody titers were reduced by 6.3-fold and 2.7-fold for Omicron compared to ancestral and Delta variant, respectively. Surprisingly, previous SARS-CoV-2 infection did not affect the neutralizing titers for Omicron in participants that received the heterologous regimen. Our findings have immediate implications for multiples countries that previously used a two-dose regimen of CoronaVac and reinforce the notion that the Omicron variant is associated with immune escape from vaccines or infection-induced immunity, highlighting the global need for vaccine boosters to combat the impact of emerging variants.

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Extended antibody-framework-to-antigen distance observed exclusively with broad HIV-1-neutralizing antibodies recognizing glycan-dense surfaces

Nature Communications ◽

10.1038/s41467-021-26579-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Myungjin Lee ◽

Anita Changela ◽

Jason Gorman ◽

Reda Rawi ◽

Tatsiana Bylund ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Data Bank ◽

The Body ◽

Protein Antigen ◽

Antibody Recognition ◽

Immunodeficiency Virus ◽

Dynamics Simulations ◽

The Impact ◽

Hiv 1

AbstractAntibody-Framework-to-Antigen Distance (AFAD) – the distance between the body of an antibody and a protein antigen – is an important parameter governing antibody recognition. Here, we quantify AFAD for ~2,000 non-redundant antibody-protein-antigen complexes in the Protein Data Bank. AFADs showed a gaussian distribution with mean of 16.3 Å and standard deviation (σ) of 2.4 Å. Notably, antibody-antigen complexes with extended AFADs (>3σ) were exclusively human immunodeficiency virus-type 1 (HIV-1)-neutralizing antibodies. High correlation (R2 = 0.8110) was observed between AFADs and glycan coverage, as assessed by molecular dynamics simulations of the HIV-1-envelope trimer. Especially long AFADs were observed for antibodies targeting the glycosylated trimer apex, and we tested the impact of introducing an apex-glycan hole (N160K); the cryo-EM structure of the glycan hole-targeting HIV-1-neutralizing antibody 2909 in complex with an N160K-envelope trimer revealed a substantially shorter AFAD. Overall, extended AFADs exclusively recognized densely glycosylated surfaces, with the introduction of a glycan hole enabling closer recognition.

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Neutralization of the emerging SARS-CoV-2 variant Lambda by antibodies elicited by inactivated virus and mRNA vaccines

10.21203/rs.3.rs-782704/v1 ◽

2021 ◽

Author(s):

Mónica Acevedo ◽

Luis Alonso-Palomares ◽

Marco Montes de Oca ◽

Andrés Bustamante ◽

Aldo Gaggero ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Immune Escape ◽

Neutralizing Antibody ◽

Neutralization Capacity ◽

Antigenic Sites ◽

Antibody Titers ◽

Different Levels

Abstract Here, we used pseudotyped viruses to characterize the neutralization capacity of antibodies elicited by the CoronaVac and BNT162b2 vaccines against the emerging variant of interest Lambda. We observed that BNT162b2 elicits higher neutralizing antibody titers than CoronaVac, ranging from 5.8-fold for the ancestral spike to 9.4-fold for the Lambda variant. Neutralization against D614G, Alpha, Gamma, and Lambda variants was reduced between 1.78 to 3.05-fold for CoronaVac and 1.10 to 1.87-fold for BNT162b2. Structural analyses of the Lambda spike show significant changes in antigenic sites including the 246–252 deletion in an antigenic supersite at the NTD loop and, L452Q/F490S within the RBD that may account for immune escape. Our analysis of pseudotyped viruses also suggests increased infectivity driven by the Lambda spike. Together, our data indicate that inactivated virus and mRNA vaccines elicit different levels of neutralizing antibodies with different potency to neutralize SARS-CoV-2 variants, including the emergent variant Lambda.

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Epitope-Specific Serological Assays for RSV: Conformation Matters

Vaccines ◽

10.3390/vaccines7010023 ◽

2019 ◽

Vol 7 (1) ◽

pp. 23 ◽

Cited By ~ 11

Author(s):

Emily Phung ◽

Lauren Chang ◽

Kaitlyn Morabito ◽

Masaru Kanekiyo ◽

Man Chen ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Antigenic Site ◽

Immune Monitoring ◽

Effective Vaccine ◽

F Protein ◽

Correlates Of Protection ◽

Vaccine Trials ◽

Antigenic Sites ◽

Syncytial Virus ◽

The Impact

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in children and older adults. An effective vaccine must elicit neutralizing antibodies targeting the RSV fusion (F) protein, which exists in two major conformations, pre-fusion (pre-F) and post-fusion (post-F). Although 50% of the surface is shared, pre-F contains highly neutralization-sensitive antigenic sites not present on post-F. Recent advancement of several subunit F-based vaccine trials has spurred interest in quantifying and understanding the protective potential of antibodies directed to individual antigenic sites. Monoclonal antibody competition ELISAs are being used to measure these endpoints, but the impact of F conformation and competition from antibodies binding to adjacent antigenic sites has not been thoroughly investigated. Since this information is critical for interpreting clinical trial outcomes and defining serological correlates of protection, we optimized assays to evaluate D25-competing antibodies (DCA) to antigenic site Ø on pre-F, and compared readouts of palivizumab-competing antibodies (PCA) to site II on both pre-F and post-F. We show that antibodies to adjacent antigenic sites can contribute to DCA and PCA readouts, and that cross-competition from non-targeted sites is especially confounding when PCA is measured using a post-F substrate. While measuring DCA and PCA levels may be useful to delineate the role of antibodies targeting the apex and side of the F protein, respectively, the assay limitations and caveats should be considered when conducting immune monitoring during vaccine trials and defining correlates of protection.

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Contribution of endogenous and exogenous antibodies to clearance of SARS-CoV-2 during convalescent plasma therapy

10.1101/2021.12.09.21267513 ◽

2021 ◽

Author(s):

Maddalena Marconato ◽

Irene A. Abela ◽

Anthony Hauser ◽

Magdalena Schwarzmüller ◽

Rheliana Katzensteiner ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Early Stage ◽

Antibody Therapy ◽

Viral Clearance ◽

Therapeutic Effects ◽

Efficacy Evaluation ◽

Plasma Therapy ◽

Early Stages ◽

Rapid Clearance ◽

The Impact

AbstractNeutralizing antibodies are considered a key correlate of protection by current SARS-CoV-2 vaccines. The ability of antibody-based therapies, including convalescent plasma, to affect established disease remains to be elucidated. Only few monoclonal therapies and only when used at a very early stage of infection have shown efficacy. Here, we conducted a proof-of-principle study of convalescent plasma therapy in a phase I trial in 30 COVID-19 patients including immunocompromised individuals hospitalized early after onset of symptoms. A comprehensive longitudinal monitoring of the virologic, serologic, and disease status of recipients in conjunction with detailed post-hoc seroprofiling of transfused convalescent plasma, allowed deciphering of parameters on which plasma therapy efficacy depends. Plasma therapy was safe and had a significant effect on viral clearance depending on neutralizing and spike SARS-CoV-2 antibody levels in the supplied convalescent plasma. Endogenous immunity had strong effects on virus control. Lack of endogenous neutralizing activity at baseline was associated with a higher risk of systemic viremia. The onset of endogenous neutralization had a noticeable effect on viral clearance but, importantly, even after adjusting for their endogenous neutralization status recipients benefitted from therapy with high neutralizing antibody containing plasma.In summary, our data demonstrate a clear impact of exogenous antibody therapy on the rapid clearance of viremia in the early stages of infection and provide directions for improved efficacy evaluation of current and future SARS-CoV-2 therapies beyond antibody-based interventions. Incorporating an assessment of the endogenous immune response and its dynamic interplay with viral production is critical for determining therapeutic effects.One Sentence SummaryThis study demonstrates the impact of exogenous antibody therapy by convalescent plasma containing high neutralizing titers on the rapid clearance of viremia in the early stages of SARS-CoV-2 infection.

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ADCC-mediating non-neutralizing antibodies can exert immune pressure in early HIV-1 infection

PLoS Pathogens ◽

10.1371/journal.ppat.1010046 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010046

Author(s):

Dieter Mielke ◽

Gama Bandawe ◽

Jie Zheng ◽

Jennifer Jones ◽

Melissa-Rose Abrahams ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Immune Escape ◽

Viral Evolution ◽

Neutralizing Antibody ◽

Protective Role ◽

Viral Escape ◽

Immune Pressure ◽

Control Virus ◽

Kinetics Of ◽

Hiv 1

Despite antibody-dependent cellular cytotoxicity (ADCC) responses being implicated in protection from HIV-1 infection, there is limited evidence that they control virus replication. The high mutability of HIV-1 enables the virus to rapidly adapt, and thus evidence of viral escape is a very sensitive approach to demonstrate the importance of this response. To enable us to deconvolute ADCC escape from neutralizing antibody (nAb) escape, we identified individuals soon after infection with detectable ADCC responses, but no nAb responses. We evaluated the kinetics of ADCC and nAb responses, and viral escape, in five recently HIV-1-infected individuals. In one individual we detected viruses that escaped from ADCC responses but were sensitive to nAbs. In the remaining four participants, we did not find evidence of viral evolution exclusively associated with ADCC-mediating non-neutralizing Abs (nnAbs). However, in all individuals escape from nAbs was rapid, occurred at very low titers, and in three of five cases we found evidence of viral escape before detectable nAb responses. These data show that ADCC-mediating nnAbs can drive immune escape in early infection, but that nAbs were far more effective. This suggests that if ADCC responses have a protective role, their impact is limited after systemic virus dissemination.

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507. Activation of Macrophages Enhances Susceptibility to SARS-CoV-2 Antibody-Dependent Enhancement and Promotes Damage to Downstream Epithelial Cells

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.701 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S319-S319

Author(s):

Jennifer K DeMarco ◽

William E Severson ◽

Daniel R DeMarco ◽

Jon Gabbard ◽

Kenneth E Palmer

Keyword(s):

Epithelial Cells ◽

Cell Viability ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Raw 264.7 Cells ◽

E Coli ◽

Respiratory Epithelia ◽

Peripheral Monocyte ◽

Time Of Infection ◽

The Impact

Abstract Background The distinct shift in peripheral monocyte activation and infiltration of these cells into the respiratory tract observed in severe cases of COVID-19 suggests that like SARS-CoV-1, the acute respiratory distress syndrome (ARDs) observed in SARS-CoV-2 infections may result from damage to the respiratory epithelia by improperly activated macrophages (MPs). In this study, we examined the ability of non-neutralizing antibodies to sensitize MPs to killing by SARS-CoV-2, as well as the impact of these cells on downstream epithelial cells. Methods Raw 264.7 cells were seeded into 96-well plates at a density of 1x104/well and incubated overnight in the presence or absence of heat-inactivated LPS derived from either E. coli (EC) or S. enteritidis (Sal). Cells were then treated with non-neutralizing antibodies or vehicle control at the time of infection with SARS-CoV-2. Viability was assessed 48 hours post-infection by luminescence following the addition of CellTiter-Glo® (Promega). Results While no decrease in cell viability was observed with SARS-CoV-2 alone, the presence of non-neutralizing antibodies against either the nucleocapsid or spike protein of SARS-CoV-2 decreased cell survival to 35.98% and 53.67% of the cell control, respectively (p< 0.0001 and p=0.0003). Activation of MPs with Sal-derived LPS sensitized MPs to viral killing, even in the absence of non-neutralizing antibody (20.12% viability, p< 0.0001). This was not observed in MPs activated by EC LPS. MP activation by both Sal and EC LPS further enhanced viral killing in the presence of anti-nucleocapsid, reducing cell viability to 12.21% (0.0001) and 6.46% (p< 0.0001). Finally, supernatants collected from naïve MPs subjected to ADE markedly increased the susceptibility of Vero E6 cells to SARS-CoV-2 nearly 9.8-fold (p< 0.0001). Conclusion Here we demonstrate that naïve MPs, normally resistant to infection by SARS-CoV-2, are rendered susceptible to viral killing by activation and the presence of non-neutralizing antibodies to SARS-CoV-2. Furthermore, MPs secrete an as yet, unknown factor that enhances the susceptibility of Vero E6 to SARS-CoV-2. Taken together, these data suggest that MPs play an important role in determining the severity of SARS-CoV-2 infection. Disclosures All Authors: No reported disclosures

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Glycan-masking spike antigen in NTD and RBD elicits broadly neutralizing antibodies against SARS-CoV-2 variants

10.1101/2021.11.01.466834 ◽

2021 ◽

Author(s):

Wei-Shuo Lin ◽

I-Chen Chen ◽

Hui-Chen Chen ◽

Yi-Chien Lee ◽

Suh-Chin Wu

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Fold Increase ◽

Vaccine Antigen ◽

Broadly Neutralizing Antibodies ◽

Neutralization Titer ◽

Cell Responses ◽

Folded Structure ◽

Antibody Titers ◽

The Impact

Glycan-masking the vaccine antigen by mutating the undesired antigenic sites with an additional N-linked glycosylation motif can refocus B-cell responses to desired/undesired epitopes, without affecting the antigen overall-folded structure. This study examine the impact of glycan-masking mutants of the N-terminal domain (NTD) and receptor-binding domain (RBD) of SARS-CoV-2, and found that the antigenic design of the S protein increases the neutralizing antibody titers against the Wuhan-Hu-1 ancestral strain and the recently emerged SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Our results demonstrated that the use of glycan-masking Ad-S-R158N-Y160T in the NTD elicited a 2.8-fold, 6.5-fold, and 4.6-fold increase in the IC-50 NT titer against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants, respectively. Glycan-masking of Ad-S-D428N in the RBD resulted in a 3.0-fold and 2.0-fold increase in the IC50 neutralization titer against the Alpha (B.1.1.7) and Beta (B.1.351) variants, respectively. The use of glycan-masking in Ad-S-R158N-Y160T and Ad-S-D428N antigen design may help develop universal COVID-19 vaccines against current and future emerging SARS-CoV-2 variants.

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Structural and computational insights into the SARS-CoV-2 Omicron RBD-ACE2 interaction

10.1101/2022.01.03.474855 ◽

2022 ◽

Author(s):

xinquan wang ◽

Tong Wang ◽

Jiwan Ge ◽

Linqi Zhang ◽

Jun Lan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Immune Escape ◽

Binding Free Energy ◽

Viral Fitness ◽

Dynamics Simulation ◽

Energy Calculation ◽

Structural Comparison ◽

Antibody Recognition ◽

Markov State Model ◽

Local Conformation

Since SARS-CoV-2 Omicron variant (B.1.1.529) was reported in November 2021, it has quickly spread to many countries and outcompeted the globally dominant Delta variant in several countries. The Omicron variant contains the largest number of mutations to date, with 32 mutations located at spike (S) glycoprotein, which raised great concern for its enhanced viral fitness and immune escape[1-4]. In this study, we reported the crystal structure of the receptor binding domain (RBD) of Omicron variant S glycoprotein bound to human ACE2 at a resolution of 2.6 angstrom. Structural comparison, molecular dynamics simulation and binding free energy calculation collectively identified four key mutations (S477N, G496S, Q498R and N501Y) for the enhanced binding of ACE2 by the Omicron RBD compared to the WT RBD. Representative states of the WT and Omicron RBD-ACE2 systems were identified by Markov State Model, which provides a dynamic explanation for the enhanced binding of Omicron RBD. The effects of the mutations in the RBD for antibody recognition were analyzed, especially for the S371L/S373P/S375F substitutions significantly changing the local conformation of the residing loop to deactivate several class IV neutralizing antibodies.

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