B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

Signal Enhancement in Oriented Immunosorbent Assays: A Balance between Accessibility of Antigen Binding Sites and Avidity

The sensitivity of immunoassays was reported to be increased by the orientation of antibodies. We investigated how the size and valence of antigens and orientation and valence of antibodies contribute to the analytical sensitivity of ELISA. Antigens differing in size and number of epitopes were compared using oriented and non-oriented ELISAs: the orientation of antibodies was obtained coating half-fragment antibodies on maleimide microplates, while, in non-oriented ELISA, whole antibodies were randomly physisorbed. The oriented assay performed better than the non-oriented one at each concentration (0.4–3.3 ng/mL) of a small monomeric antigen (cardiac Troponin I, 24 kDa, Rh 3 nm). No significant differences were observed with a large monovalent antigen (prostate-specific antigen-alpha(1) antichymotrypsin, 90 kDa, Rh > 3 nm), since its steric hindrance overcame the increased availability of antigen binding sites given by orientation. Large multivalent antigens (ferritin, 280 kDa, Rh 6 nm; α-fetoprotein, >70 kDa, Rh > 3.3 nm) performed better in non-oriented assays. In this case, the repeated epitopes on the surface of the antigens favored the engagement of both antigen binding sites of the whole IgG, thus suggesting that avidity represented the leading force in this experimental setting. In conclusion, the design of high-sensitivity ELISAs should consider the dimension and valency of antigens in addition to the affinity and avidity of antibodies.

Induced Polyspecificity of Human Secretory Immunoglobulin A Antibodies: Is It Possible to Improve Their Ability to Bind Pathogens?

<b><i>Introduction:</i></b> As has been shown previously, various protein-modifying agents can change the antigen-binding properties of immunoglobulins. However, induced polyspecificity of human secretory immunoglobulin A (sIgA) has not been previously characterized in detail. <b><i>Methods:</i></b> In the present study, human secretory immunoglobulin A (IgA) was exposed to buffers with acidic pH, to free heme, or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens was compared using Western blotting and enzyme-linked immunosorbent assay. The ability of these agents to modulate the antigen-binding properties of human sIgA toward a wide range of pathogen peptides was investigated using an epitope microarray. <b><i>Results:</i></b> We have shown that acidic pH, heme, and pro-oxidative ferrous ions influenced the binding of secretory IgA in opposite directions (either increasing or decreasing); however, the strongest effect was observed when using buffers with low pH. This fraction had the highest number of affected reactivities; most of them were increased and most of the new ones were toward common pathogens. <b><i>Conclusions:</i></b> Thus, it was shown that all investigated treatments can alter to some degree the antigen-binding of secretory IgA, but acidic pH has the most potentially beneficial effect by increasing binding to a largest number of common pathogens’ antigens.

Recombinant neutralizing secretory IgA antibodies for preventing mucosal carriage and transmission of SARS-CoV-2

Passive delivery of antibodies to mucosal sites might be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. However, monoclonal IgG antibody therapies, currently used for treatment of severe infections, are unlikely to prove useful in mucosal sites where SARS-CoV-2 resides and replicates in early infection. Here, we investigated the feasibility of producing neutralising monoclonal IgA antibodies against SARS-COV-2. We identified two class-switched mAbs that express well as monomeric and secretory IgA variants with retained antigen binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralisation activities than IgG mAbs and were able to reduce SARS-CoV-2 infection in an in vivo murine model. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.

Autologous enzyme-linked immunosorbent facilitated antigen binding detects IgE-blocking activity based on direct competition between allergen-specific IgE and non-IgE

Aim: To measure IgE-blocking activity induced by allergen immunotherapy (AIT) by an enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay based on autologous immunoglobulin competition. Methods: The developed ELIFAB assay was used to investigate the kinetics of IgE-blocking activity in 87 patients at multiple AIT treatment time points, in comparison to the changes in IgG4. Results: High ELIFAB response was observed until 2.5 months of AIT, then significantly decreased after 4 months and remained suppressed during the 3-year AIT period. After treatment cessation, the ELIFAB response was maintained at the level seen at the 4–6 month treatment time point, similar to IgG4, indicating sustained IgE-blocking activity related to IgG4. Conclusion: This ELIFAB assay measures the IgE-blocking activity for autologous allergen-specific IgE and non-IgE during and after immunotherapy. It is suited for measuring the sustained IgE-blocking activity induced by AIT.

TCR-BERT: learning the grammar of T-cell receptors for flexible antigen- binding analyses

The T-cell receptor (TCR) allows T-cells to recognize and respond to antigens presented by infected and diseased cells. However, due to TCRs' staggering diversity and the complex binding dynamics underlying TCR antigen recognition, it is challenging to predict which antigens a given TCR may bind to. Here, we present TCR-BERT, a deep learning model that applies self-supervised transfer learning to this problem. TCR-BERT leverages unlabeled TCR sequences to learn a general, versatile representation of TCR sequences, enabling numerous downstream applications. We demonstrate that TCR-BERT can be used to build state-of-the-art TCR-antigen binding predictors with improved generalizability compared to prior methods. TCR-BERT simultaneously facilitates clustering sequences likely to share antigen specificities. It also facilitates computational approaches to challenging, unsolved problems such as designing novel TCR sequences with engineered binding affinities. Importantly, TCR-BERT enables all these advances by focusing on residues with known biological significance. TCR-BERT can be a useful tool for T-cell scientists, enabling greater understanding and more diverse applications, and provides a conceptual framework for leveraging unlabeled data to improve machine learning on biological sequences.

Retention of Activity by Antibodies Immobilized on Gold Nanoparticles of Different Sizes: Fluorometric Method of Determination and Comparative Evaluation

Antibody–nanoparticle conjugates are widely used analytical reagents. An informative parameter reflecting the conjugates’ properties is the number of antibodies per nanoparticle that retain their antigen-binding ability. Estimation of this parameter is characterized by a lack of simple, reproducible methods. The proposed method is based on the registration of fluorescence of tryptophan residues contained in proteins and combines sequential measurements of first the immobilized antibody number and then the bound protein antigen number. Requirements for the measurement procedure have been determined to ensure reliable and accurate results. Using the developed technique, preparations of spherical gold nanoparticles obtained by the most common method of citrate reduction of gold salts (the Turkevich–Frens method) and varying in average diameter from 15 to 55 nm have been characterized. It was shown that the number of antibodies (immunoglobulins G) bound by one nanoparticle ranged from 30 to 194 during adsorptive unoriented monolayer immobilization. C-reactive protein was considered as the model antigen. The percentage of antibody valences that retained their antigen-binding properties in the conjugate increased from 17 to 34% with an increase in the diameter of gold nanoparticles. The proposed method and the results of the study provide tools to assess the capabilities of the preparations of gold nanoparticles and their conjugates as well as the expediency of seeking the best techniques for various practical purposes.