A dataset comprised of binding interactions for 104,972 antibodies against a SARS-CoV-2 peptide.

The dataset presented here contains quantitative binding scores of scFv-format antibodies against a SARS-CoV-2 target peptide collected via an AlphaSeq assay that can be used in the development and benchmarking of machine learning models. Starting from three seed sequences identified from a phage display campaign using a human naïve library, four sets of 29,900 antibodies were designed in silico by creating all k = 1 mutations and random k = 2 and k = 3 mutations throughout the complementary-determining regions (CDRs). Of the 119,600 designs, 104,972 were successfully built in to the AlphaSeq library and target binding was subsequently measured with 71,384 designs resulting in a predicted affinity value for at least one of the triplicate measurements. Data include antibodies with predicted affinity measurements ranging from 37 pM to 22 mM. To our knowledge, this dataset is the largest, publicly available dataset that contains antibody sequences, antigen sequence and quantitative measurements of binding scores and provides an opportunity to serve as a benchmark to evaluate antibody-specific representation models for machine learning.

Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2.

Using the regioselective cyanobenzothiazole condensation reaction with an N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the novel severe acute respiratory syndrome virus 2 (SARS-CoV-2) Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and angiotensin-converting enzyme 2 (ACE2), the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 µM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 µM. The current study demonstrates that two kinetically controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals.

Isolation of a human SARS-CoV-2 neutralizing antibody from a synthetic phage library and its conversion to fluorescent biosensors.

Since late 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant spread of COVID-19 have given rise to a worldwide health crisis that is posing great challenges to public health and clinical treatment, in addition to serving as a formidable threat to the global economy. To obtain an effective tool to prevent and diagnose viral infections, we attempted to obtain human antibody fragments that can effectively neutralize viral infection and be utilized for rapid virus detection. To this end, several human monoclonal antibodies were isolated by bio-panning a phage-displayed human antibody library, Tomlinson I. The selected clones were demonstrated to bind to the S1 domain of the spike glycoprotein of SARS-CoV-2. Moreover, clone A7 in Fab and IgG formats were found to effectively neutralize the binding of S protein to angiotensin-converting enzyme 2 in the low nM range. In addition, this clone was successfully converted to quench-based fluorescent immunosensors (Quenchbodies) that allowed antigen detection within a few minutes, with the help of a handy fluorometer.

Peptide-Antibody Fusions Engineered by Phage Display Exhibit an Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants.

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naïve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide-IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Peptide Binder with High-Affinity for the SARS-CoV-2 Spike Receptor-Binding Domain.

Rapid antigen detection tests are urgently needed for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The discovery of a binder with high affinity and selectivity for the biomarkers presented by SARS-CoV-2 is crucial to the development of the rapid antigen detection method. We utilized the surface biopanning to identify a peptide binder R1 from a phage-displayed peptide library consisting of 109 independent phage recombinants. The R1 peptide exhibited high-affinity for specific binding with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with a dissociation constant KD of (7.5 ± 1.9) × 10-10 M, which maintained high binding affinity with the RBD derived from Gamma, Lambda, Delta, and Omicron variants. The composition and sequence dependence of binding characteristics in R1-RBD interactions was revealed by the binding affinity fluctuations between RBD and the scrambled sequences or single-site mutants of R1. The R1-functionalized gold nanoparticles possessed concentration-dependent response to RBD and selectivity over bovine serum albumin and human serum albumin. The peptide binder R1 shows the potential to be used for constructing a rapid detection method for the early-stage diagnostics for SARS-CoV-2.

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis.

The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

SARS-CoV-2 reactive and neutralizing antibodies discovered by single-cell sequencing of plasma cells and mammalian display.

Characterization of COVID-19 antibodies has largely focused on memory B cells; however, it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in resolving SARS-CoV-2 infection. Little is known about the specificity of plasma cells, largely because plasma cells lack surface antibody expression, thereby complicating their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and mammalian display to interrogate the specificity of plasma cells from 16 convalescent patients. Single-cell sequencing allows us to profile antibody repertoire features and identify expanded clonal lineages. Mammalian display screening is used to reveal that 43 antibodies (of 132 candidates) derived from expanded plasma cell lineages are specific to SARS-CoV-2 antigens, including antibodies with high affinity to the SARS-CoV-2 receptor-binding domain (RBD) that exhibit potent neutralization and broad binding to the RBD of SARS-CoV-2 variants (of concern/interest).

Generation and utility of a single-chain fragment variable monoclonal antibody platform against a baculovirus expressed recombinant receptor binding domain of SARS-CoV-2 spike protein.

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation.

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.

A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein.

Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8+ T cell responses in HLA-A transgenic mice.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8+ T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8+ T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8+ T cell responses in COVID-19 patients.

A Collection of Designed Peptides to Target SARS-CoV-2 Spike RBD-ACE2 Interaction.

The angiotensin-converting enzyme 2 (ACE2) is the receptor used by SARS-CoV and SARS-CoV-2 coronaviruses to attach to cells via the receptor-binding domain (RBD) of their viral spike protein. Since the start of the COVID-19 pandemic, several structures of protein complexes involving ACE2 and RBD as well as monoclonal antibodies and nanobodies have become available. We have leveraged the structural data to design peptides to target the interaction between the RBD of SARS-CoV-2 and ACE2 and SARS-CoV and ACE2, as contrasting exemplar, as well as the dimerization surface of ACE2 monomers. The peptides were modelled using our original method: PiPreD that uses native elements of the interaction between the targeted protein and cognate partner(s) that are subsequently included in the designed peptides. These peptides recapitulate stretches of residues present in the native interface plus novel and highly diverse conformations surrogating key interactions at the interface. To facilitate the access to this information we have created a freely available and dedicated web-based repository, PepI-Covid19 database, providing convenient access to this wealth of information to the scientific community with the view of maximizing its potential impact in the development of novel therapeutic and diagnostic agents.

Neutralizing Antibodies to SARS-CoV-2 Selected from a Human Antibody Library Constructed Decades Ago.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.

Prospects of Neutralizing Nanobodies Against SARS-CoV-2.

Since December 2019, the SARS-CoV-2 has erupted on a large scale worldwide and spread rapidly. Passive immunization of antibody-related molecules provides opportunities for prevention and treatment of high-risk patients and children. Nanobodies (Nbs) have many strong physical and chemical properties. They can be atomized, administered by inhalation, and can be directly applied to the infected site, with fast onset, high local drug concentration/high bioavailability, and high patient compliance (no needles). It has very attractive potential in the treatment of respiratory viruses. Rapid and low-cost development of Nbs targeting SARS-CoV-2 can quickly be achieved. Nbs against SARS-CoV-2 mutant strains also can be utilized quickly to prevent the virus from escaping. It provides important technical supports for the treatment of the SARS-CoV-2 and has the potential to become an essential medicine in the toolbox against the SARS-CoV-2.

Protocol for constructing large size human antibody heavy chain variable domain (VH) library and selection of SARS-CoV-2 neutralizing antibody domains.

This protocol is a comprehensive guide to phage display-based selection of virus neutralizing VH antibody domains. It details three optimized parts including (1) construction of a large-sized (theoretically > 1011) naïve human antibody heavy chain domain library, (2) SARS-CoV-2 antigen expression and stable cell line construction, and (3) library panning for selection of SARS-CoV-2-specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

Application of spectral library prediction for parallel reaction monitoring of viral peptides.

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.

SARS-CoV-2 Proteome Harbors Peptides Which Are Able to Trigger Autoimmunity Responses: Implications for Infection, Vaccination, and Population Coverage.

Autoimmune diseases (ADs) could occur due to infectious diseases and vaccination programs. Since millions of people are expected to be infected with SARS-CoV-2 and vaccinated against it, autoimmune consequences seem inevitable. Therefore, we have investigated the whole proteome of the SARS-CoV-2 for its ability to trigger ADs. In this regard, the entire proteome of the SARS-CoV-2 was chopped into more than 48000 peptides. The produced peptides were searched against the entire human proteome to find shared peptides with similar experimentally confirmed T-cell and B-cell epitopes. The obtained peptides were checked for their ability to bind to HLA molecules. The possible population coverage was calculated for the most potent peptides. The obtained results indicated that the SARS-CoV-2 and human proteomes share 23 peptides originated from ORF1ab polyprotein, nonstructural protein NS7a, Surface glycoprotein, and Envelope protein of SARS-CoV-2. Among these peptides, 21 peptides had experimentally confirmed equivalent epitopes. Amongst, only nine peptides were predicted to bind to HLAs with known global allele frequency data, and three peptides were able to bind to experimentally confirmed HLAs of equivalent epitopes. Given the HLAs which have already been reported to be associated with ADs, the ESGLKTIL, RYPANSIV, NVAITRAK, and RRARSVAS were determined to be the most harmful peptides of the SARS-CoV-2 proteome. It would be expected that the COVID-19 pandemic and the vaccination against this pathogen could significantly increase the ADs incidences, especially in populations harboring HLA-B*08:01, HLA-A*024:02, HLA-A*11:01 and HLA-B*27:05. The Southeast Asia, East Asia, and Oceania are at higher risk of AD development.