Screening Antibodies Raised against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

Isolation of monoclonal antibodies from anti-synthetase syndrome patients and affinity maturation by recombination of independent somatic variants.

The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.

A mixture of functionally oligoclonal humanized monoclonal antibodies that neutralize Clostridium difficile TcdA and TcdB with high levels of in vitro potency shows in vivo protection in a hamster infection model.

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.