EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem

Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coliAssisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag–Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein–protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.

The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21

Humoral immune responses require germinal centres (GC) for antibody affinity maturation. Within GC, B cell proliferation and mutation are segregated from affinity-based positive selection in the dark zone (DZ) and light zone (LZ) substructures, respectively. While IL-21 is known to be important in affinity maturation and GC maintenance, here we show it is required for both establishing normal zone representation and preventing the accumulation of cells in the G1 cell cycle stage in the GC LZ. Cell cycle progression of DZ B cells is unaffected by IL-21 availability, as is the zone phenotype of the most highly proliferative GC B cells. Collectively, this study characterises the development of GC zones as a function of time and B cell proliferation and identifies IL-21 as an important regulator of these processes. These data help explain the requirement for IL-21 in normal antibody affinity maturation.

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti–SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti–receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect–based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor

Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.

Programming isotype specific plasma cell differentiation

Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell dependent immunization and within established steady-state immunity. Using integrated single cell strategies, we reveal conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation modulating programs dictated by IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC inhibitory IgG1+ and inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady-state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.

Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2

Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potential beneficial as well as detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be re-activated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Understanding the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be deconvoluted by surface-based assays like enzyme-linked immunosorbent assays (ELISAs) which are routinely used to assess cross-reactivity. Here, we have used microfluidic antibody-affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory re-activation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS CoV-2.

Coordinated changes in glycosylation regulate the germinal centre through CD22

Germinal centres (GC) are sites of B-cell expansion and selection, which are essential for antibody affinity maturation. Compared to naive follicular B-cells, GC B-cells have several notable changes in their cell surface glycans. While these changes are routinely used to identify the GC, functional roles for these changes have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a reduction in the glycan ligands for CD22, which is an inhibitory co-receptor of the B-cell receptor (BCR). To test a functional role for downregulated CD22 ligands in the GC, we generated a mouse model that maintains CD22 ligands on GC B-cells. With this model, we demonstrate that glycan remodeling is crucial for proper GC B-cell response, including plasma cell output and affinity maturation of antibodies. The defect we observe in this model is dependent on CD22, highlighting that coordinated downregulation of CD22 ligands on B cells plays a critical function in the GC. Collectively, our study uncovers a crucial role for glycan remodeling and CD22 in B-cell fitness in the GC.