Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

Immunoglobulin gene analysis as a tool for investigating human immune responses.

The human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self-reactive B cells removed. Later antigen-dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases, and cancer. It can also be used to identify antigen-specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub-speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.

BRepertoire: a user-friendly web server for analysing antibody repertoire data.

Antibody repertoire analysis by high throughput sequencing is now widely used, but a persisting challenge is enabling immunologists to explore their data to discover discriminating repertoire features for their own particular investigations. Computational methods are necessary for large-scale evaluation of antibody properties. We have developed BRepertoire, a suite of user-friendly web-based software tools for large-scale statistical analyses of repertoire data. The software is able to use data preprocessed by IMGT, and performs statistical and comparative analyses with versatile plotting options. BRepertoire has been designed to operate in various modes, for example analysing sequence-specific V(D)J gene usage, discerning physico-chemical properties of the CDR regions and clustering of clonotypes. Those analyses are performed on the fly by a number of R packages and are deployed by a shiny web platform. The user can download the analysed data in different table formats and save the generated plots as image files ready for publication. We believe BRepertoire to be a versatile analytical tool that complements experimental studies of immune repertoires. To illustrate the server's functionality, we show use cases including differential gene usage in a vaccination dataset and analysis of CDR3H properties in old and young individuals. The server is accessible under http://mabra.biomed.kcl.ac.uk/BRepertoire.

Promiscuous antibodies characterised by their physico-chemical properties: From sequence to structure and back.

Human B cells produce antibodies, which bind to their cognate antigen based on distinct molecular properties of the antibody CDR loop. We have analysed a set of 10 antibodies showing a clear difference in their binding properties to a panel of antigens, resulting in two subsets of antibodies with a distinct binding phenotype. We call the observed binding multiplicity 'promiscuous' and selected physico-chemical CDRH3 characteristics and conformational preferences may characterise these promiscuous antibodies. To classify CDRH3 physico-chemical properties playing a role in their binding properties, we used statistical analyses of the sequences annotated by Kidera factors. To characterise structure-function requirements for antigen binding multiplicity we employed Molecular Modelling and Monte Carlo based coarse-grained simulations. The ability to predict the molecular causes of promiscuous, multi-binding behaviour would greatly improve the efficiency of the therapeutic antibody discovery process.

Transitional B Cells in Early Human B Cell Development - Time to Revisit the Paradigm?

The B cell repertoire is generated in the adult bone marrow by an ordered series of gene rearrangement processes that result in massive diversity of immunoglobulin (Ig) genes and consequently an equally large number of potential specificities for antigen. As the process is essentially random, the cells exhibiting excess reactivity with self-antigens are generated and need to be removed from the repertoire before the cells are fully mature. Some of the cells are deleted, and some will undergo receptor editing to see if changing the light chain can rescue an autoreactive antibody. As a consequence, the binding properties of the B cell receptor are changed as development progresses through pre-B ≫ immature ≫ transitional ≫ naïve phenotypes. Using long-read, high-throughput, sequencing we have produced a unique set of sequences from these four cell types in human bone marrow and matched peripheral blood, and our results describe the effects of tolerance selection on the B cell repertoire at the Ig gene level. Most strong effects of selection are seen within the heavy chain repertoire and can be seen both in gene usage and in CDRH3 characteristics. Age-related changes are small, and only the size of the CDRH3 shows constant and significant change in these data. The paucity of significant changes in either kappa or lambda light chain repertoires implies that either the heavy chain has more influence over autoreactivity than light chain and/or that switching between kappa and lambda light chains, as opposed to switching within the light chain loci, may effect a more successful autoreactive rescue by receptor editing. Our results show that the transitional cell population contains cells other than those that are part of the pre-B ≫ immature ≫ transitional ≫ naïve development pathway, since the population often shows a repertoire that is outside the trajectory of gene loss/gain between pre-B and naïve stages.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic resistance in tuberculosis.

BACKGROUND: Drug-resistant tuberculosis (TB), especially multidrug-resistant (MDR, resistance to rifampicin and isoniazid) disease, is associated with a worse patient outcome. Drug resistance diagnosed using microbiological culture takes days to weeks, as TB bacteria grow slowly. Rapid molecular tests for drug resistance detection (1 day) are commercially available and may promote faster initiation of appropriate treatment. OBJECTIVES: To (1) conduct a systematic review of evidence regarding diagnostic accuracy of molecular genetic tests for drug resistance, (2) conduct a health-economic evaluation of screening and diagnostic strategies, including comparison of alternative models of service provision and assessment of the value of targeting rapid testing at high-risk subgroups, and (3) construct a transmission-dynamic mathematical model that translates the estimates of diagnostic accuracy into estimates of clinical impact. REVIEW METHODS AND DATA SOURCES: A standardised search strategy identified relevant studies from EMBASE, PubMed, MEDLINE, Bioscience Information Service (BIOSIS), System for Information on Grey Literature in Europe Social Policy & Practice (SIGLE) and Web of Science, published between 1 January 2000 and 15 August 2013. Additional 'grey' sources were included. Quality was assessed using quality assessment of diagnostic accuracy studies version 2 (QUADAS-2). For each diagnostic strategy and population subgroup, a care pathway was constructed to specify which medical treatments and health services that individuals would receive from presentation to the point where they either did or did not complete TB treatment successfully. A total cost was estimated from a health service perspective for each care pathway, and the health impact was estimated in terms of the mean discounted quality-adjusted life-years (QALYs) lost as a result of disease and treatment. Costs and QALYs were both discounted at 3.5% per year. An integrated transmission-dynamic and economic model was used to evaluate the cost-effectiveness of introducing rapid molecular testing (in addition to culture and drug sensitivity testing). Probabilistic sensitivity analysis was performed to evaluate the impact on cost-effectiveness of diagnostic and treatment time delays, diagnosis and treatment costs, and associated QALYs. RESULTS: A total of 8922 titles and abstracts were identified, with 557 papers being potentially eligible. Of these, 56 studies contained sufficient test information for analysis. All three commercial tests performed well when detecting drug resistance in clinical samples, although with evidence of heterogeneity between studies. Pooled sensitivity for GenoType® MTBDRplus (Hain Lifescience, Nehren, Germany) (isoniazid and rifampicin resistance), INNO-LiPA Rif.TB® (Fujirebio Europe, Ghent, Belgium) (rifampicin resistance) and Xpert® MTB/RIF (Cepheid Inc., Sunnyvale, CA, USA) (rifampicin resistance) was 83.4%, 94.6%, 95.4% and 96.8%, respectively; equivalent pooled specificity was 99.6%, 98.2%, 99.7% and 98.4%, respectively. Results of the transmission model suggest that all of the rapid assays considered here, if added to the current diagnostic pathway, would be cost-saving and achieve a reduction in expected QALY loss compared with current practice. GenoType MTBDRplus appeared to be the most cost-effective of the rapid tests in the South Asian population, although results were similar for GeneXpert. In all other scenarios GeneXpert appeared to be the most cost-effective strategy. CONCLUSIONS: Rapid molecular tests for rifampicin and isoniazid resistance were sensitive and specific. They may also be cost-effective when added to culture drug susceptibility testing in the UK. There is global interest in point-of-care testing and further work is needed to review the performance of emerging tests and the wider health-economic impact of decentralised testing in clinics and primary care, as well as non-health-care settings, such as shelters and prisons. STUDY REGISTRATION: This study is registered as PROSPERO CRD42011001537. FUNDING: The National Institute for Health Research Health Technology Assessment programme.