Antibody recognition of the Pneumovirus fusion protein trimer interface.

Human metapneumovirus (hMPV) is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory tract infection in infants, the elderly, and immunocompromised patients. However, there remain no licensed vaccines or specific treatments for hMPV infection. Although the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological properties of hMPV F remain poorly understood. To further define the humoral immune response to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and MPV465. Both mAbs are neutralizing in vitro and were determined to target a unique antigenic site using competitive biolayer interferometry. We determined both MPV458 and MPV465 have higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV F protein, suggesting significant breathing of the hMPV F protein must occur for host immune recognition of the novel epitope. In addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. The data presented identifies a novel epitope on the hMPV F protein for epitope-based vaccine design, and illustrates a new mechanism for human antibody neutralization of viral glycoproteins.

The neutralizing breadth of antibodies targeting diverse conserved epitopes between SARS-CoV and SARS-CoV-2.

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

Shared N417-Dependent Epitope on the SARS-CoV-2 Omicron, Beta, and Delta Plus Variants.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, several variants of concern (VOCs) have arisen which are defined by multiple mutations in their spike proteins. These VOCs have shown variable escape from antibody responses and have been shown to trigger qualitatively different antibody responses during infection. By studying plasma from individuals infected with either the original D614G, Beta, or Delta variants, we showed that the Beta and Delta variants elicit antibody responses that are overall more cross-reactive than those triggered by D614G. Patterns of cross-reactivity varied, and the Beta and Delta variants did not elicit cross-reactive responses to each other. However, Beta-elicited plasma was highly cross-reactive against Delta Plus (Delta+), which differs from Delta by a single K417N mutation in the receptor binding domain, suggesting that the plasma response targets the N417 residue. To probe this further, we isolated monoclonal antibodies from a Beta-infected individual with plasma responses against Beta, Delta+, and Omicron, which all possess the N417 residue. We isolated an N417-dependent antibody, 084-7D, which showed similar neutralization breadth to the plasma. The 084-7D MAb utilized the IGHV3-23*01 germ line gene and had somatic hypermutations similar to those of previously described public antibodies which target the 417 residue. Thus, we have identified a novel antibody which targets a shared epitope found on three distinct VOCs, enabling their cross-neutralization. Understanding antibodies targeting escape mutations, such as K417N, which repeatedly emerge through convergent evolution in SARS-CoV-2 variants, may aid in the development of next-generation antibody therapeutics and vaccines. IMPORTANCE The evolution of SARS-CoV-2 has resulted in variants of concern (VOCs) with distinct spike mutations conferring various immune escape profiles. These variable mutations also influence the cross-reactivity of the antibody response mounted by individuals infected with each of these variants. This study sought to understand the antibody responses elicited by different SARS-CoV-2 variants and to define shared epitopes. We show that Beta and Delta infections resulted in antibody responses that were more cross-reactive than the original D614G variant, but they had differing patterns of cross-reactivity. We further isolated an antibody from Beta infection which targeted the N417 site, enabling cross-neutralization of Beta, Delta+, and Omicron, all of which possess this residue. The discovery of antibodies which target escape mutations common to multiple variants highlights conserved epitopes to target in future vaccines and therapeutics.

Targeting intra-viral conserved nucleocapsid (N) proteins as novel vaccines against SARS-CoVs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic of the Coronavirus disease in late 2019 (COVID-19). Vaccine development efforts have predominantly been aimed at 'Extra-viral' Spike (S) protein as vaccine vehicles, but there are concerns regarding 'viral immune escape' since multiple mutations may enable the mutated virus strains to escape from immunity against S protein. The 'Intra-viral' Nucleocapsid (N-protein) is relatively conserved among mutant strains of coronaviruses during spread and evolution. Herein, we demonstrate novel vaccine candidates against SARS-CoV-2 by using the whole conserved N-protein or its fragment/peptides. Using ELISA assay, we showed that high titers of specific anti-N antibodies (IgG, IgG1, IgG2a, IgM) were maintained for a reasonably long duration (> 5 months), suggesting that N-protein is an excellent immunogen to stimulate host immune system and robust B-cell activation. We synthesized three peptides located at the conserved regions of N-protein among CoVs. One peptide showed as a good immunogen for vaccination as well. Cytokine arrays on post-vaccination mouse sera showed progressive up-regulation of various cytokines such as IFN-γ and CCL5, suggesting that TH1 associated responses are also stimulated. Furthermore, vaccinated mice exhibited an elevated memory T cells population. Here, we propose an unconventional vaccine strategy targeting the conserved N-protein as an alternative vaccine target for coronaviruses. Moreover, we generated a mouse monoclonal antibody specifically against an epitope shared between SARS-CoV and SARS-CoV-2, and we are currently developing the First-in-Class humanized anti-N-protein antibody to potentially treat patients infected by various CoVs in the future.

A broad and potent neutralization epitope in SARS-related coronaviruses.

Many neutralizing antibodies (nAbs) elicited to ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through natural infection and vaccination have reduced effectiveness to SARS-CoV-2 variants. Here, we show that therapeutic antibody ADG20 is able to neutralize SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize a broad range of VOCs, albeit with reduced potency against Omicron. Thus, this conserved and vulnerable site can be exploited for the design of universal vaccines and therapeutic antibodies.

Immunoglobin G Sero-Dynamics Aided Host Specific Linear Epitope Identification and Differentiation of Infected from Vaccinated Hosts.

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.

Correlates of protection against SARS-CoV-2 infection and COVID-19 disease.

Antibodies against epitopes in S1 give the most accurate CoP against infection by the SARS-CoV-2 coronavirus. Measurement of those antibodies by neutralization or binding assays both have predictive value, with binding antibody titers giving the highest statistical correlation. However, the protective functions of antibodies are multiple. Antibodies with multiple functions other than neutralization influence efficacy. The role of cellular responses can be discerned with respect to CD4⁺ T cells and their augmentation of antibodies, and with respect to CD8⁺ cells with regard to control of viral replication, particularly in the presence of insufficient antibody. More information is needed on mucosal responses.

Risk of rapid evolutionary escape from biomedical interventions targeting SARS-CoV-2 spike protein.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells in vitro. This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.

Reverse vaccinology approach for multi-epitope centered vaccine design against delta variant of the SARS-CoV-2.

The ongoing COVID-19 outbreak, initially identified in Wuhan, China, has impacted people all over the globe and new variants of concern continue to threaten hundreds of thousands of people. The delta variant (first reported in India) is currently classified as one of the most contagious variants of SARS-CoV-2. It is estimated that the transmission rate of delta variant is 225% times faster than the alpha variant, and it is causing havoc worldwide (especially in the USA, UK, and South Asia). The mutations found in the spike protein of delta variant make it more infective than other variants in addition to ruining the global efficacy of available vaccines. In the current study, an in silico reverse vaccinology approach was applied for multi-epitope vaccine construction against the spike protein of delta variant, which could induce an immune response against COVID-19 infection. Non-toxic, highly conserved, non-allergenic and highly antigenic B-cell, HTL, and CTL epitopes were identified to minimize adverse effects and maximize the efficacy of chimeric vaccines that could be developed from these epitopes. Finally, V1 vaccine construct model was shortlisted and 3D modeling was performed by refinement, docking against HLAs and TLR4 protein, simulation and in silico expression. In silico evaluation showed that the designed chimeric vaccine could elicit an immune response (i.e., cell-mediated and humoral) identified through immune simulation. This study could add to the efforts of overcoming global burden of COVID-19 particularly the variants of concern.

Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is continuously evolving, and this poses a major threat to antibody therapies and currently authorized Coronavirus Disease 2019 (COVID-19) vaccines. It is therefore of utmost importance to investigate and predict the putative mutations on the spike protein that confer immune evasion. Antibodies are key components of the human immune system's response to SARS-CoV-2, and the spike protein is a prime target of neutralizing antibodies (nAbs) as it plays critical roles in host cell recognition, fusion, and virus entry. The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations. In this review, we discuss (1) the emerging SARS-CoV-2 variants; (2) the structural basis for antibody-mediated neutralization of SARS-CoV-2 and nAb classification; and (3) identification of the RBD escape mutations for several antibodies that resist antibody binding and neutralization. These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines.

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2.

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.

Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern.

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization.

SignificanceSARS-CoV-2 continues to evolve through emerging variants, more frequently observed with higher transmissibility. Despite the wide application of vaccines and antibodies, the selection pressure on the Spike protein may lead to further evolution of variants that include mutations that can evade immune response. To catch up with the virus's evolution, we introduced a deep learning approach to redesign the complementarity-determining regions (CDRs) to target multiple virus variants and obtained an antibody that broadly neutralizes SARS-CoV-2 variants.

CRESSP: a comprehensive pipeline for prediction of immunopathogenic SARS-CoV-2 epitopes using structural properties of proteins.

The development of autoimmune diseases following SARS-CoV-2 infection, including multisystem inflammatory syndrome, has been reported, and several mechanisms have been suggested, including molecular mimicry. We developed a scalable, comparative immunoinformatics pipeline called cross-reactive-epitope-search-using-structural-properties-of-proteins (CRESSP) to identify cross-reactive epitopes between a collection of SARS-CoV-2 proteomes and the human proteome using the structural properties of the proteins. Overall, by searching 4 911 245 proteins from 196 352 SARS-CoV-2 genomes, we identified 133 and 648 human proteins harboring potential cross-reactive B-cell and CD8+ T-cell epitopes, respectively. To demonstrate the robustness of our pipeline, we predicted the cross-reactive epitopes of coronavirus spike proteins, which were recognized by known cross-neutralizing antibodies. Using single-cell expression data, we identified PARP14 as a potential target of intermolecular epitope spreading between the virus and human proteins. Finally, we developed a web application (https://ahs2202.github.io/3M/) to interactively visualize our results. We also made our pipeline available as an open-source CRESSP package (https://pypi.org/project/cressp/), which can analyze any two proteomes of interest to identify potentially cross-reactive epitopes between the proteomes. Overall, our immunoinformatic resources provide a foundation for the investigation of molecular mimicry in the pathogenesis of autoimmune and chronic inflammatory diseases following COVID-19.

Identification of a Novel Neutralizing Epitope on the N-Terminal Domain of the Human Coronavirus 229E Spike Protein.

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. IMPORTANCE Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

In-Silico Analysis of Monoclonal Antibodies against SARS-CoV-2 Omicron.

Omicron was designated by the WHO as a VOC on 26 November 2021, only 4 days after its sequence was first submitted. However, the impact of Omicron on current antibodies and vaccines remains unknown and evaluations are still a few weeks away. We analysed the mutations in the Omicron variant against epitopes. In our database, 132 epitopes of the 120 antibodies are classified into five groups, namely NTD, RBD-1, RBD-2, RBD-3, and RBD-4. The Omicron mutations impact all epitopes in NTD, RBD-1, RBD-2, and RBD-3, with no antibody epitopes spared by these mutations. Only four out of 120 antibodies may confer full resistance to mutations in the Omicron spike, since all antibodies in these three groups contain one or more epitopes that are affected by these mutations. Of all antibodies under EUA, the neutralisation potential of Etesevimab, Bamlanivimab, Casirivimab, Imdevima, Cilgavimab, Tixagevimab, Sotrovimab, and Regdanvimab might be dampened to varying degrees. Our analysis suggests the impact of Omicron on current therapeutic antibodies by the Omicron spike mutations may also apply to current COVID-19 vaccines.

Analysis of the Neutralizing Activity of Antibodies Targeting Open or Closed SARS-CoV-2 Spike Protein Conformations.

SARS-CoV-2 infection elicits a polyclonal neutralizing antibody (nAb) response that primarily targets the spike protein, but it is still unclear which nAbs are immunodominant and what distinguishes them from subdominant nAbs. This information would however be crucial to predict the evolutionary trajectory of the virus and design future vaccines. To shed light on this issue, we gathered 83 structures of nAbs in complex with spike protein domains. We analyzed in silico the ability of these nAbs to bind the full spike protein trimer in open and closed conformations, and predicted the change in binding affinity of the most frequently observed spike protein variants in the circulating strains. This led us to define four nAb classes with distinct variant escape fractions. By comparing these fractions with those measured from plasma of infected patients, we showed that the class of nAbs that most contributes to the immune response is able to bind the spike protein in its closed conformation. Although this class of nAbs only partially inhibits the spike protein binding to the host's angiotensin converting enzyme 2 (ACE2), it has been suggested to lock the closed pre-fusion spike protein conformation and therefore prevent its transition to an open state. Furthermore, comparison of our predictions with mRNA-1273 vaccinated patient plasma measurements suggests that spike proteins contained in vaccines elicit a different nAb class than the one elicited by natural SARS-CoV-2 infection and suggests the design of highly stable closed-form spike proteins as next-generation vaccine immunogens.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants.

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.