Objective·To screen the human-mouse chimeric antibodies that neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and analyse the key amino acid site changes in their light ...chain.Methods·Antibody heavy chain gene-humanized mice were immunized with the spike protein of SARS-CoV-2. Enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay were performed to analyse the antibody titer in serum level. Flow cytometry was used to sort plasma cells and germinal center (GC) B cells from the mouse spleen and lymph nodes, and the proportions of different B cells were analysed. RNA was extracted to construct a mouse-origin antibody light chain gene library, which was paired with the knocked-in human-origin antibody heavy chain gene for antibody expression. ELISA was employed to screen for high-affinity chimeric antibodies. Amino acid changes in the light chain of high-affinity chimeric antibodies were analysed by comparing them with mouse-origin germline antibody light chain g
Breakthrough infection of SARS-CoV-2 is a serious challenge, as increased infections were documented in fully-vaccinated individuals. Recipients with poor antibody response are highly vulnerable to ...reinfection, whereas those with strong antibody responses achieve sterilizing immunity. Thus far, biomarkers associated with levels of vaccine-elicited antibody response are still lacking. Here, we studied the antibody response of age- and gender-controlled healthy cohort, who received inactivated SARS-CoV-2 vaccines and profiled the B cell receptor repertoires in longitudinally consecutive samples. Upon vaccination, all vaccinated individuals displayed a convergent antibody response with shared common antibody clones and public neutralizing antibodies. Strikingly, poor vaccine-responders are distinguishable from strong vaccine-responders by a biased V-usage before vaccination and IgG to IgM mRNA ratio. These findings reveal molecular signatures associated with the different levels of vaccine-induced antibody response, which could be further developed into biomarkers for the design of vaccination strategies.
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide severe coronavirus disease 2019 (COVID-19) pandemic since December 2019. There is a great demand for ...effective therapies for the prevention and treatment of COVID-19. Developing therapeutic neutralizing antibodies (NAbs), which could block viral infection, is such a promising approach, as NAbs have been successfully applied to the treatment of other viral infections. The recent advances of antibody technology have greatly accelerated the discovery of SARS-CoV-2 NAbs, and many of which are now actively tested in clinical trials. Here, we review the approaches applied for SARS-CoV-2 NAb development, and discuss the emerging technologies underlining the antibody discovery. We further summarize the common features of these antibodies including the shared neutralizing epitopes and sequence features.
Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations ...intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.
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•AID activity on single-strand DNA per se shapes the CDR-preferential SHM profile•Non-coding function of DNA at the mesoscale level directs AID deamination in vivo•Flexibility of single-strand DNA substrate contributes to AID substrate preference•DNA alteration can make a cold FR hot
Somatic hypermutation in antibody-coding sequences preferentially occurs within specific regions; this study uncovers that the intrinsic single-stranded DNA flexibility within the regions confers the specificity.
Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, ...we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.
Insertions and deletions (indels) are low-frequency deleterious genomic DNA alterations. Despite their rarity, indels are common, and insertions leading to long complementarity-determining region 3 ...(CDR3) are vital for antigen-binding functions in broadly neutralizing and polyreactive antibodies targeting viruses. Because of challenges in detecting indels, the mechanism that generates indels during immunoglobulin diversification processes remains poorly understood. We carried out ultra-deep profiling of indels and systematically dissected the underlying mechanisms using
mouse models. We found that activation-induced cytidine deaminase-dependent ±1-base pair (bp) indels are the most prevalent indel events, biasing deleterious outcomes, whereas longer in-frame indels, especially insertions that can extend the CDR3 length, are rare outcomes. The ±1-bp indels are channeled by base excision repair, but longer indels require additional DNA-processing factors. Ectopic expression of a DNA exonuclease or perturbation of the balance of DNA polymerases can increase the frequency of longer indels, thus paving the way for models that can generate antibodies with long CDR3. Our study reveals the mechanisms that generate beneficial and deleterious indels during the process of antibody somatic hypermutation and has implications in understanding the detrimental genomic alterations in various conditions, including tumorigenesis.
Based on fuzzy control theory, a new method for 3- Degree-of-freedom (DOF) parallel robot control is presented in this paper. Take example for DELTA robot; input the established physical model in ...Pro/E into simulation software ADAMS to establish its mechanical system model by seamless interface software MECH/Pro, where DELTA robot can do kinematics and dynamics simulation. Build the fuzzy controller and control system model by the use of MATLAB software. Realize co-simulation in accordance with a given target trajectory by the use of ADAMS/MATLAB software. The results analysis shows that this control method has solved coordinate control for the 3-DOF parallel robot so that each output is fed back to the three drive rod, effectively improving the control precision, tracing trajectory in real time and providing a method of control for multi-DOF parallel robot.
By analyzing the mechanism of Delta parallel robot and setting up coordinate system on both static platform and moving platform, then the constraint equation and Jacob matrix can be gained. Combined ...with the multi-body of Delta parallel robot built with Pro/E, and then the model is transferred to ADAMS by MECHANISM/Pro. The inverse solution and spline curves and spline function can be obtained through simulation at first, by using the result of spline curves and spline function can obtain the direct solution. This way is much easier, faster and accurately than the ways of numerical solution and analytical method. It is proved that the results can be put into practice.
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