Innovations and trends in antibody repertoire analysis.

Monoclonal antibodies have revolutionized the treatment of human diseases, which has made them the fastest-growing class of therapeutics, with global sales expected to reach $346.6 billion USD by 2028. Advances in antibody engineering and development have led to the creation of increasingly sophisticated antibody-based therapeutics (e.g. bispecific antibodies and chimeric antigen receptor T cells). However, approaches for antibody discovery have remained comparatively grounded in conventional yet reliable in vitro assays. Breakthrough developments in high-throughput single B-cell sequencing and immunoglobulin proteomic serology, however, have enabled the identification of high-affinity antibodies directly from endogenous B cells or circulating immunoglobulin produced in vivo. Moreover, advances in artificial intelligence offer vast potential for antibody discovery and design with large-scale repertoire datasets positioned as the optimal source of training data for such applications. We highlight advances and recent trends in how these technologies are being applied to antibody repertoire analysis.

Graphene Field Effect Biosensor for Concurrent and Specific Detection of SARS-CoV-2 and Influenza.

The SARS-CoV-2 pandemic has highlighted the need for devices capable of carrying out rapid differential detection of viruses that may manifest similar physiological symptoms yet demand tailored treatment plans. Seasonal influenza may be exacerbated by COVID-19 infections, increasing the burden on healthcare systems. In this work, we demonstrate a technology based on liquid-gated graphene field-effect transistors (GFETs), for rapid and ultraprecise sensing and differentiation of influenza and SARS-CoV-2 surface protein. Most distinctively, the device consists of 4 onboard GFETs arranged in a quadruple architecture, where each quarter is functionalized individually (with either antibodies or chemically passivated control) but measured jointly. The sensor platform was tested against a range of concentrations of viral surface proteins from both viruses with the lowest tested and detected concentration at ∼50 ag/mL, or 88 zM for COVID-19 and 227 zM for Flu, which is 5-fold lower than the values reported previously on a similar platform. Unlike the classic real-time polymerase chain reaction test, which has a turnaround time of a few hours, the graphene technology presents an ultrafast response time of ∼10 s even in complex and clinically relevant media such as saliva. Thus, we have developed a multianalyte, highly sensitive, and fault-tolerant technology for rapid diagnostic of contemporary, emerging, and future pandemics.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there have been demands on the testing infrastructure that have strained testing capacity. As a simplification of method, we confirm the efficacy of RNA extraction-free RT-qPCR and saline as an alternative patient sample storage buffer. In addition, amongst potential reagent shortages, it has sometimes been difficult to obtain inactivated viral particles. We have therefore also characterized armored SARS-CoV-2 RNA from Asuragen as an alternative diagnostic standard to ATCC genomic SARS-CoV-2 RNA and heat inactivated virions and provide guidelines for its use in RT-qPCR.

Convalescent plasma anti-SARS-CoV-2 spike protein ectodomain and receptor-binding domain IgG correlate with virus neutralization.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the relationship among anti-spike ectodomain (anti-ECD), anti-receptor-binding domain (anti-RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by 2 in vitro assays using convalescent plasma samples from 68 patients with COVID-19. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titers. The probability of a VN titer of ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers of ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers of ≥1:160, and all of them had anti-RBD titers of ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.

Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma.

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma.

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq 1 ) to the spike ectodomain (S-ECD 2 ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å 2 ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning 3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.