Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

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Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Science ◽

10.1126/science.abe3354 ◽

2020 ◽

Vol 370 (6519) ◽

pp. 950-957 ◽

Cited By ~ 21

Author(s):

M. Alejandra Tortorici ◽

Martina Beltramello ◽

Florian A. Lempp ◽

Dora Pinto ◽

Ha V. Dang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Viral Escape ◽

Angiotensin Converting Enzyme 2 ◽

Effector Functions ◽

Human Antibodies ◽

Binding Domains ◽

Isolation And Characterization ◽

Cryo Electron Microscopy ◽

Escape Mutants

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo–electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.

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Mutations in two SARS-CoV-2 variants of concern reflect two distinct strategies of antibody escape

10.1101/2021.07.23.453327 ◽

2021 ◽

Author(s):

Sebastian Fiedler ◽

Viola Denninger ◽

Alexey S. Morgunov ◽

Alison Ilsley ◽

Roland Worth ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Cell Receptor ◽

Converting Enzyme ◽

Wild Type ◽

Angiotensin Converting Enzyme 2 ◽

Binding Domains ◽

Host Cell Receptor

Understanding the factors that contribute to antibody escape of SARS-CoV-2 and its variants is key for the development of drugs and vaccines that provide broad protection against a variety of virus variants. Using microfluidic diffusional sizing, we determined the dissociation constant ((KD)) for the interaction between receptor binding domains (RBDs) of SARS-CoV-2 in its original version (WT) as well as alpha and beta variants with the host-cell receptor angiotensin converting enzyme 2 (ACE2). For RBD-alpha, the ACE2-binding affinity was increased by a factor of ten when compared with RBD-WT, while ACE2-binding of RBD-beta was largely unaffected. However, when challenged with a neutralizing antibody that binds to both RBD-WT and RBD-alpha with low nanomolar (KD) values, RBD-beta displayed no binding, suggesting a substantial epitope change. In SARS-CoV-2 convalescent sera, RBD-binding antibodies showed low nanomolar affinities to both wild-type and variant RBD proteins—strikingly, the concentration of antibodies binding to RBD-beta was half that of RBD-WT and RBD-alpha, again indicating considerable epitope changes in the beta variant. Our data therefore suggests that one factor contributing to the higher transmissibility and antibody evasion of SARS-CoV-2 alpha and beta is a larger fraction of viruses that can form a complex with ACE2. However, the two variants employ different mechanisms to achieve this goal. While SARS-CoV-2 alpha RBD binds with greater affinity to ACE2 and is thus more difficult to displace from the receptor by neutralizing antibodies, RBD-beta is less accessible to antibodies due to epitope changes which increases the chances of ACE2-binding and infection.

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

Science ◽

10.1126/science.abi6226 ◽

2021 ◽

Vol 373 (6555) ◽

pp. eabi6226

Author(s):

Sophie M.-C. Gobeil ◽

Katarzyna Janowska ◽

Shana McDowell ◽

Katayoun Mansouri ◽

Robert Parks ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Interspecies Transmission ◽

Human Populations ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Cryo Electron Microscopy ◽

Up States ◽

Antibody Resistance ◽

Computational Analyses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo–electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)–up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.

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In Vivo Demonstration of the Superior Replication and Infectivity of Genotype 2.1 with Respect to Genotype 3.4 of Classical Swine Fever Virus by Dual Infections

Pathogens ◽

10.3390/pathogens9040261 ◽

2020 ◽

Vol 9 (4) ◽

pp. 261 ◽

Cited By ~ 2

Author(s):

Yu-Liang Huang ◽

Kuo-Jung Tsai ◽

Ming-Chung Deng ◽

Hsin-Meng Liu ◽

Chin-Cheng Huang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Classical Swine Fever ◽

Dual Infection ◽

Maternal Antibodies ◽

Febrile Reaction ◽

Wild Type ◽

Viral Loads ◽

Virus Genotypes ◽

Modified Live Vaccine

In Taiwan, the prevalent CSFV population has shifted from the historical genotype 3.4 (94.4 strain) to the newly invading genotype 2.1 (TD/96 strain) since 1996. This study analyzed the competition between these two virus genotypes in dual infection pigs with equal and different virus populations and with maternally derived neutralizing antibodies induced by a third genotype of modified live vaccine (MLV), to simulate that occurring in natural situations in the field. Experimentally, under various dual infection conditions, with or without the presence of maternal antibodies, with various specimens from blood, oral and fecal swabs, and internal organs at various time points, the TD/96 had consistently 1.51−3.08 log higher loads than those of 94.4. A second passage of competition in the same animals further widened the lead of TD/96 as indicated by viral loads. The maternally derived antibodies provided partial protection to both wild type CSFVs and was correlated with lower clinical scores, febrile reaction, and animal mortality. In the presence of maternal antibodies, pigs could be infected by both wild type CSFVs, with TD/96 dominating. These findings partially explain the CSFV shift observed, furthering our understanding of CSFV pathogenesis in the field, and are helpful for the control of CSF.

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Infection of wild-type mice by SARS-CoV-2 B.1.351 variant indicates a possible novel cross-species transmission route

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00848-1 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Ting Pan ◽

Ran Chen ◽

Xin He ◽

Yaochang Yuan ◽

Xiaohui Deng ◽

...

Keyword(s):

Human Populations ◽

Transmission Route ◽

Wild Type ◽

Angiotensin Converting Enzyme 2 ◽

Genetic Modifications ◽

Infection Models ◽

Contact Transmission ◽

Wild House Mice ◽

Natural Situation ◽

High Binding Affinity

AbstractCOVID-19 is identified as a zoonotic disease caused by SARS-CoV-2, which also can cross-transmit to many animals but not mice. Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection. Although neither is the natural situation, they are currently utilized to establish mouse infection models. Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice. The SARS-CoV-2 (B.1.351) replicated efficiently and induced significant pathological changes in lungs and tracheas, accompanied by elevated proinflammatory cytokines in the lungs and sera. Mechanistically, the receptor-binding domain (RBD) of SARS-CoV-2 (B.1.351) spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2 (mACE2), allowing the mice highly susceptible to SARS-CoV-2 (B.1.351) infection. Our work suggests that SARS-CoV-2 (B.1.351) expands the host range and therefore increases its transmission route without adapted mutation. As the wild house mice live with human populations quite closely, this possible transmission route could be potentially risky. In addition, because SARS-CoV-2 (B.1.351) is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated, the SARS-CoV-2 (B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.

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N439K variant in spike protein may alter the infection efficiency and antigenicity of SARS-CoV-2 based on molecular dynamics simulation

10.1101/2020.11.21.392407 ◽

2020 ◽

Author(s):

Wenyang Zhou ◽

Chang Xu ◽

Pingping Wang ◽

Meng Luo ◽

Zhaochun Xu ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Protein Interactions ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Salt Bridge ◽

Dynamics Simulation ◽

Wild Type ◽

Protein Protein Interactions ◽

Angiotensin Converting Enzyme 2

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing an outbreak of coronavirus disease 2019 (COVID-19), has been undergoing various mutations. The analysis of the structural and energetic effects of mutations on protein-protein interactions between the receptor binding domain (RBD) of SARS-CoV-2 and angiotensin converting enzyme 2 (ACE2) or neutralizing monoclonal antibodies will be beneficial for epidemic surveillance, diagnosis, and optimization of neutralizing agents. According to the molecular dynamics simulation, a key mutation N439K in the SARS-CoV-2 RBD region created a new salt bridge which resulted in greater electrostatic complementarity. Furthermore, the N439K-mutated RBD bound hACE2 with a higher affinity than wild-type, which may lead to more infectious. In addition, the N439K-mutated RBD was markedly resistant to the SARS-CoV-2 neutralizing antibody REGN10987, which may lead to the failure of neutralization. These findings would offer guidance on the development of neutralizing antibodies and the prevention of COVID-19.

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CD4 T-Cell-Independent Antibody Response Reduces Enterovirus 71 Lethality in Mice by Decreasing Tissue Viral Loads

Clinical and Developmental Immunology ◽

10.1155/2012/580696 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Li-Chiu Wang ◽

Chia-Min Kao ◽

Pin Ling ◽

Ih-Jen Su ◽

Tung-Miao Chang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antibody Response ◽

Viral Infection ◽

Neutralizing Antibodies ◽

Enterovirus 71 ◽

Cd4 T Cell ◽

Wild Type ◽

Viral Loads ◽

Deficient Mice

Enterovirus 71 (EV71) has induced fatal encephalitis in hundreds of thousands of infants and young children in the Asia-Pacific region since the past decade. Lymphocyte and antibody responses have been suspected to aggravate EV71-induced neurological symptoms, so anti-inflammatory agents have been used to treat patients with neurological symptoms. In the present study, we found that mice deficient in CD4+T cells were resistant to EV71 infection as wild-type mice, whereas mice deficient in B cells were highly susceptible to viral infection. Compensation of CD4 T-cell function by other immune cells was not likely, because wild-type mice depleted of CD4+T cells were also resistant to viral infection. Infected CD4 T-cell-deficient mice produced virus-specific neutralizing antibodies, IgM and IgG. Moreover, adoptive transfer of the virus-specific antibody produced by infected CD4 T-cell-deficient mice protected B-cell-deficient mice from infection by reducing tissue viral loads. Collectively, our results show that the CD4 T-cell-independent antibody response promotes the survival of EV71-infected mice and suggest great potential for the use of vaccines and neutralizing antibodies to reduce fatal symptoms in patients.

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SARS-CoV-2 recruits a haem metabolite to evade antibody immunity

10.1101/2021.01.21.21249203 ◽

2021 ◽

Author(s):

Annachiara Rosa ◽

Valerie E. Pye ◽

Carl Graham ◽

Luke Muir ◽

Jeffrey Seow ◽

...

Keyword(s):

Electron Microscopy ◽

Humoral Immunity ◽

Conformational Changes ◽

Neutralizing Antibodies ◽

Viral Antigen ◽

Antibody Binding ◽

X Ray ◽

X Ray Crystallography ◽

Flexible Loop ◽

Cryo Electron Microscopy

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

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Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

PLoS Pathogens ◽

10.1371/journal.ppat.1009501 ◽

2021 ◽

Vol 17 (4) ◽

pp. e1009501

Author(s):

Huihui Mou ◽

Brian D. Quinlan ◽

Haiyong Peng ◽

Guanqun Liu ◽

Yan Guo ◽

...

Keyword(s):

Infectious Virus ◽

Wild Type ◽

Viral Receptor ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Binding Domains ◽

Protein Receptor ◽

Horseshoe Bat ◽

High Level ◽

Horseshoe Bats

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

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Structural Insights of SARS-CoV-2 Spike Protein from Delta and Omicron Variants

10.1101/2021.12.08.471777 ◽

2021 ◽

Author(s):

Ali Sadek ◽

David Zaha ◽

Mahmoud Salama Ahmed

Keyword(s):

Viral Entry ◽

Structural Changes ◽

Structural Basis ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

X Ray ◽

X Ray Crystallography ◽

Binding Domains ◽

Structural Insights ◽

Sheer Number

Given the continuing heavy toll of the COVID-19 pandemic and the emergence of the Delta (B.1.617.2) and Omicron (B.1.1.529) variants, the WHO declared both as variants of concern (VOC). There are valid concerns that the latest Omicron variant might have increased infectivity and pathogenicity. In addition, the sheer number of S protein mutations in the Omicron variant raise concerns of potential immune evasion and resistance to therapeutics such as monoclonal antibodies. However, structural insights that underpin the potential increased pathogenicity are unknown. Here we adopted an artificial intelligence (AI)-based approach to predict the structural changes induced by mutations of the Delta and Omicron variants in the spike (S) protein using Alphafold. This was followed by docking the human angiotensin-converting enzyme 2 (ACE2) with the predicted S proteins for Wuhan-Hu-1, Delta, and Omicron variants. Our in-silico structural analysis indicates that S protein for Omicron variant has a higher binding affinity to ACE-2 receptor, compared to Wuhan-Hu-1 and Delta variants. In addition, the recognition sites of the receptor binding domains for Delta and Omicron variants showed lower electronegativity compared to Wuhan-Hu-1. Importantly, further molecular insights revealed significant changes induced at fusion protein (FP) site, which may mediate enhanced viral entry. These results represent the first computational analysis of structural changes associated with Omicron variant using Alphafold, Collectively, our results highlight potential structural basis for enhanced pathogenicity of the Omicron variant, however further validation using X-ray crystallography and cryo-EM are warranted.

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