Longitudinal kinetics of RBD+ antibodies in COVID-19 recovered patients over 14 months.

We describe the longitudinal kinetics of the serological response in COVID-19 recovered patients over a period of 14 months. The antibody kinetics in a cohort of 192 recovered patients, including 66 patients for whom follow-up serum samples were obtained at two to four clinic visits, revealed that RBD-specific antibodies decayed over the 14 months following the onset of symptoms. The decay rate was associated with the robustness of the response in that antibody levels that were initially highly elevated after the onset of symptoms subsequently decayed more rapidly. An exploration of the differences in the longitudinal kinetics between recovered patients and naïve vaccinees who had received two doses of the BNT162b2 vaccine showed a significantly faster decay in the naïve vaccinees, indicating that serological memory following natural infection is more robust than that following to vaccination. Our data highlighting the differences between serological memory induced by natural infection vs. vaccination contributed to the decision-making process in Israel regarding the necessity for a third vaccination dose.

PASA: Proteomic analysis of serum antibodies web server.

MOTIVATION: A comprehensive characterization of the humoral response towards a specific antigen requires quantification of the B-cell receptor repertoire by next-generation sequencing (BCR-Seq), as well as the analysis of serum antibodies against this antigen, using proteomics. The proteomic analysis is challenging since it necessitates the mapping of antigen-specific peptides to individual B-cell clones. RESULTS: The PASA web server provides a robust computational platform for the analysis and integration of data obtained from proteomics of serum antibodies. PASA maps peptides derived from antibodies raised against a specific antigen to corresponding antibody sequences. It then analyzes and integrates proteomics and BCR-Seq data, thus providing a comprehensive characterization of the humoral response. The PASA web server is freely available at https://pasa.tau.ac.il and open to all users without a login requirement.

Production of F(ab')2 from Monoclonal and Polyclonal Antibodies.

Antibodies are widely used in therapeutic, diagnostic, and research applications, and antibody derivatives such as F(ab')2 fragments are used when only a particular antibody region is required. F(ab')2 can be produced through antibody engineering, but some applications require F(ab')2 produced from an original formulated antibody or directly from a polyclonal antibody pool. The cysteine protease immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes digests immunoglobulin G (IgG) specifically and efficiently to produce F(ab')2 . Here we detail the production and purification of recombinant IdeS; its utilization to digest monoclonal or polyclonal antibodies to F(ab')2 fragments; and F(ab')2 purification through consecutive affinity chromatography steps. The resultant F(ab')2 exhibit high purity, retain antigen-binding functionality, and are readily utilizable in various downstream applications. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Production and purification of F(ab')2 fragments from monoclonal and polyclonal antibodies using IdeS Alternate Protocol: Purification of polyclonal antigen-specific F(ab')2 fragments from human serum or secretions Support Protocol: Production and purification of IdeS.

Molecular Landscape of Anti-Drug Antibodies Reveals the Mechanism of the Immune Response Following Treatment With TNFα Antagonists.

Drugs formulated from monoclonal antibodies (mAbs) are clinically effective in various diseases. Repeated administration of mAbs, however, elicits an immune response in the form of anti-drug-antibodies (ADA), thereby reducing the drug's efficacy. Notwithstanding their importance, the molecular landscape of ADA and the mechanisms involved in their formation are not fully understood. Using a newly developed quantitative bio-immunoassay, we found that ADA concentrations specific to TNFα antagonists can exceed extreme concentrations of 1 mg/ml with a wide range of neutralization capacity. Our data further suggest a preferential use of the λ light chain in a subset of neutralizing ADA. Moreover, we show that administration of TNFα antagonists result in a vaccine-like response whereby ADA formation is governed by the extrafollicular T cell-independent immune response. Our bio-immunoassay coupled with insights on the nature of the immune response can be leveraged to improve mAb immunogenicity assessment and facilitate improvement in therapeutic intervention strategies.

The negative effect of starvation and the positive effect of mild thermal stress on thermal tolerance of the red flour beetle, Tribolium castaneum.

The thermal tolerance of a terrestrial insect species can vary as a result of differences in population origin, developmental stage, age, and sex, as well as via phenotypic plasticity induced in response to changes in the abiotic environment. Here, we studied the effects of both starvation and mild cold and heat shocks on the thermal tolerance of the red flour beetle, Tribolium castaneum. Starvation led to impaired cold tolerance, measured as chill coma recovery time, and this effect, which was stronger in males than females, persisted for longer than 2 days but less than 7 days. Heat tolerance, measured as heat knockdown time, was not affected by starvation. Our results highlight the difficulty faced by insects when encountering multiple stressors simultaneously and indicate physiological trade-offs. Both mild cold and heat shocks led to improved heat tolerance in both sexes. It could be that both mild shocks lead to the expression of heat shock proteins, enhancing heat tolerance in the short run. Cold tolerance was not affected by previous mild cold shock, suggesting that such a cold shock, as a single event, causes little stress and hence elicits only weak physiological reaction. However, previous mild heat stress led to improved cold tolerance but only in males. Our results point to both hardening and cross-tolerance between cold and heat shocks.

The Effects of Temperature and Diet during Development, Adulthood, and Mating on Reproduction in the Red Flour Beetle.

The effects of different temperatures and diets experienced during distinct life stages are not necessarily similar. The silver-spoon hypothesis predicts that developing under favorable conditions will always lead to better performing adults under all adult conditions. The environment-matching hypothesis suggests that a match between developmental and adult conditions will lead to the best performing adults. Similar to the latter hypothesis, the beneficial-acclimation hypothesis suggests that either developing or acclimating as adults to the test temperature will improve later performance under such temperature. We disentangled here between the effect of growth, adult, and mating conditions (temperature and diet) on reproduction in the red flour beetle (Tribolium castaneum), in reference to the reproduction success rate, the number of viable offspring produced, and the mean offspring mass 13 days after mating. The most influential stage affecting reproduction differed between the diet and temperature experiments: adult temperature vs. parental growth diet. Generally, a yeast-rich diet or warmer temperature improved reproduction, supporting the silver-spoon hypothesis. However, interactions between life stages made the results more complex, also fitting the environment-matching hypothesis. Warm growth temperature positively affected reproduction success, but only when adults were kept under the same warm temperature. When the parental growth and adult diets matched, the mean offspring mass was greater than in a mismatch between the two. Additionally, a match between warm adult temperature and warm offspring growth temperature led to the largest offspring mass. These findings support the environment-matching hypothesis. Our results provide evidence for all these hypotheses and demonstrate that parental effects and plasticity may be induced by temperature and diet.