Using antibody synergy to engineer a high potency biologic cocktail against C. difficile

We applied a mathematical framework originally used to model the effects of multiple inhibitors on enzyme activity to guide the development a therapeutic antibody cocktail, LMN-201, to prevent and treat C. difficile infection (CDI). CDI causes hundreds of thousands of cases of severe, often recurrent diarrhea and colitis in the United States annually and is associated with significant morbidity and mortality worldwide. Current therapies for preventing recurrent CDI are only partially successful, and there are no options available to prevent initial bouts of CDI in at-risk populations. Almost all antibody therapies have been developed and administered as monotherapies. Antibody cocktails are relatively rare even though they have the potential to greatly increase efficacy. One reason for this is our limited understanding of how antibody interactions can enhance potency, which makes it difficult to identify and develop antibodies that can be assembled into optimally effective cocktails. In contrast to the view that antibody synergies depend on unusual instances of cooperativity or allostery, we show that synergistic efficacy requires nothing more than that the antibodies bind independently to distinct epitopes on a common target. Therefore, synergy may be achieved much more readily than is generally appreciated. Due to synergy the LMN-201 antibody cocktail, which targets the C. difficile exotoxin B (TcdB), is 300- to 3000-fold more potent at neutralizing the most clinically prevalent TcdB toxin types than bezlotoxumab, the only monoclonal antibody currently approved for treatment or prevention of CDI. The efficacy of LMN-201 is further enhanced by inclusion of a phage-derived endolysin that destroys the C. difficile bacterium, and which therefore has a complementary mechanism of action to the antibody cocktail. These observations may serve as a paradigm for the development of high potency biologic cocktails against targets that have proven challenging for single-agent therapies.

Predictions of the SARS-CoV-2 Omicron Variant (B.1.1.529) Spike Protein Receptor-Binding Domain Structure and Neutralizing Antibody Interactions

The genome of the SARS-CoV-2 Omicron variant (B.1.1.529) was released on November 22, 2021, which has caused a flurry of media attention due the large number of mutations it contains. These raw data have spurred questions around vaccine efficacy. Given that neither the structural information nor the experimentally-derived antibody interaction of this variant are available, we have turned to predictive computational methods to model the mutated structure of the spike protein’s receptor binding domain and posit potential changes to vaccine efficacy. In this study, we predict some structural changes in the receptor-binding domain that may reduce antibody interaction, but no drastic changes that would completely evade existing neutralizing antibodies (and therefore current vaccines).

More than meets the Kappa for Antibody Superantigen Protein L (PpL)

Immunoglobulin superantigens play an important role in the affinity purification of antibodies and underlie the microbiota-immune axis at mucosal areas Focussing on the Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG), and the Finegoldia Protein L (PpL) that were previously thought to bind to only specific regions of human antibodies, a systematic and holistic analysis of the antibody regions using 63 antibody permutations involving six Vκ and seven VH region IgG1 revealed showed novel PpL-antibody interactions. While SpA and SpG showed relatively consistent interactions with the antibodies, our findings showed PpL binding to certain VH-Vκ2, 5 and 6 interactions had contribution by other antibody regions. The findings of this have implications on PpL-based affinity antibody purifications and antibody design as well as provides novel insights to PpL-based microbiota-immune axis effects.

Binding Strength and Hydrogen Bond Numbers between COVID-19 RBD and HVR of Antibody

The global battle against the COVID-19 pandemic relies strongly on the human defense of antibody, which is assumed to bind the antigen’s receptor binding domain (RBD) with its hypervariable region (HVR). Due to the similarity to other viruses such as SARS, however, our understanding of the antibody-virus interaction has been largely limited to the genomic sequencing, which poses serious challenges to containment and rapid serum testing. Based on the physical/chemical nature of the interaction, infrared spectroscopy was employed to reveal the binding disparity, the real cause of the antibody-virus specificity at the molecular level, which is inconceivable to be investigated otherwise. Temperature dependence was discovered in the absorption value from the 1550 cm−1 absorption band, attributed to the hydrogen bonds by carboxyl/amino groups, binding the SARS-CoV-2 spike protein and closely resembled SARS-CoV-2 or SARS-CoV-1 antibodies. The infrared absorption intensity, associated with the number of hydrogen bonds, was found to increase sharply between 27 °C and 31 °C, with the relative absorbance matching the hydrogen bonding numbers of the two antibody types (19 vs. 12) at 37 °C. Meanwhile, the ratio of bonds at 27 °C, calculated by thermodynamic exponentials, produces at least 5% inaccuracy. Beyond genomic sequencing, the temperature dependence, as well as the bond number match at 37 °C between relative absorbance and the hydrogen bonding numbers of the two antibody types, is not only of clinical significance in particular but also as a sample for the physical/chemical understanding of vaccine–antibody interactions in general.

SARS-CoV-2 receptor binding mutations and antibody contact sites

Background SARS-CoV-2 mutations can impact infectivity, viral load, and overall morbidity/mortality during infection. In this analysis, we look at the mutational landscape of the SARS-CoV-2 receptor binding domain, a structure that is antigenic and allows for viral binding to the host. Methods We develop a bioinformatics platform and analyze 104193 GISAID sequences acquired on October 15th, 2020 with a majority of sequences (96%) containing point mutations. Results We report high frequency mutations with improved binding affinity to ACE2 including S477N, N439K, V367F, and N501Y and address the potential impact of RBD mutations on antibody binding. The high frequency S477N mutation is present in 6.7% of all SARS-CoV-2 sequences, co-occurs with D614G, and is currently present in 14 countries. To address RBD-antibody interactions we take a subset of human derived antibodies and define their interacting residues using PDBsum. Conclusions Our analysis shows that RBD mutations were found in approximately 9% of our dataset, with some mutations improving RBD-ACE2 interactions. We also show that antibody mediated immunity against SARS-CoV-2 enlists broad coverage of the RBD, with multiple antibodies targeting a variety of RBD regions. These data suggest that it is unlikely for neutralization/RBD antibody binding to be significantly impacted, as a whole, in the presence of RBD point mutations that conserve the RBD structure. Statement of significance SARS-CoV2 is responsible for the current COVID-19 pandemic. In this work we developed a MATLAB program to analyze SARS-CoV-2 RBD mutations and conducted a thorough analysis of all SARS-CoV-2 RBD mutations using the GISAID database. We found four high frequency variants with improved binding to ACE2—S477N, N439K, V367F, and N501Y and cross-referenced antibody interaction data with RBD mutations.

Electrochemical Immunosensors for Quantification of Procalcitonin: Progress and Prospects

Human procalcitonin (PCT) is a peptide precursor of the calcium-regulating hormone calcitonin. Traditionally, PCT has been used as a biomarker for severe bacterial infections and sepsis. It has also been recently identified as a potential marker for COVID-19. Normally, serum PCT is intracellularly cleaved to calcitonin, which lowers the levels of PCT (<0.01 ng/mL). In severe infectious diseases and sepsis, serum PCT levels increase above 100 ng/mL in response to pro-inflammatory stimulation. Development of sensors for specific quantification of PCT has resulted in considerable improvement in the sensitivity, linear range and rapid response. Among the various sensing strategies, electrochemical platforms have been extensively investigated owing to their cost-effectiveness, ease of fabrication and portability. Sandwich-type electrochemical immunoassays based on the specific antigen–antibody interactions with an electrochemical transducer and use of nanointerfaces has augmented the electrochemical response of the sensors towards PCT. Identification of a superior combination of electrode material and nanointerface, and translation of the sensing platform into flexible and disposable substrates are under active investigation towards development of a point-of-care device for PCT detection. This review provides an overview of the existing detection strategies and limitations of PCT electrochemical immunosensors, and the emerging directions to address these lacunae.

Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice

Complement impacts innate and adaptive immunity. Using a model in which the human KEL glycoprotein is expressed on murine red blood cells (RBCs), we have shown that polyclonal immunoprophylaxis (KELIg) prevents alloimmunization to transfused RBCs when a recipient is in their baseline state of heath but with immunoprophylaxis failure occurring in the presence of a viral-like stimulus. As complement can be detected on antibody coated KEL RBCs following transfusion, we hypothesized that recipient complement synergizes with viral-like inflammation to reduce immunoprophylaxis efficacy. Indeed, we found recipient C3 and C1q were critical to immunoprophylaxis failure in the setting of a viral-like stimulus, with no anti-KEL IgG alloantibodies generated in C3-/- or C1q-/- mice following KELIg treatment and KEL RBC transfusion. Differences in RBC uptake were noted in mice lacking C3, with lower consumption by splenic and peripheral blood inflammatory monocytes. Finally, no alloantibodies were detected in the setting of a viral-like stimulus following KELIg treatment and KEL RBC transfusion in mice lacking complement receptors (CR1/2-/-), narrowing key cells for immunoprophylaxis failure to those expressing these complement receptors. In-vitro studies showed complement fixed opsonized RBCs were significantly less likely to bind to B-cells from CR1/2-/- than wild type mice, potentially implicating lowered B-cell activation threshold in the presence of complement as being responsible for these findings. We thus propose a two-hit model for inflammation-induced immunoprophylaxis failure, where the first “hit” is recipient inflammation and the second “hit” is complement production/sensing. These results may have translational relevance to antigen-antibody interactions in humans.

Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

Interactions between rheumatoid arthritis antibodies are associated with the response to anti-tumor necrosis factor therapy

Background Blocking of the Tumor Necrosis Factor (TNF) activity is a successful therapeutic approach for 50–60% of rheumatoid arthritis (RA) patients. However, there are yet no biomarkers to stratify patients for anti-TNF therapy. Rheumatoid factor (RF) and anti-cyclic-citrullinated antibodies (anti-CCP) have been evaluated as biomarkers of response but the results have shown limited consistency. Anti-carbamylated protein (anti-CarP) and anti-peptidylarginine deiminase type 4 (anti-PAD4) antibodies have been much less studied. Despite being linked to common immune processes, the interaction between these markers has not been evaluated yet. Our aim was to analyze the interaction between these four antibodies in relation to the response to anti-TNF therapy. Methods For this objective, a prospective cohort of n = 80 RA patients starting anti-TNF therapy was recruited. Serum determinations at baseline were performed for RF, anti-CCP, anti-CarP and anti-PAD4 antibodies using enzyme-linked immunosorbent assays (ELISA). The clinical response to anti-TNF therapy was determined at week 12 using the change in DAS28 score. Association was performed using multivariate linear regression adjusting for baseline DAS28, sex and age. Results The interaction between pairs of antibodies was tested by the addition of an interaction term. We found two highly significant antibody interactions associated with treatment response: anti-CarP with anti-PAD4 (p = 0.0062), and anti-CCP with RF (p = 0.00068). The latter antibody interaction was replicated in an independent retrospective cohort of RA patients (n = 199, p = 0.04). Conclusions The results of this study suggest that antibody interaction effects are important factors in the response to anti-TNF therapy in RA.

Towards Understanding ChAdOx1 nCov-19 Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT)

BackgroundSARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that—like autoimmune heparin-induced thrombocytopenia—is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies.MethodsWe investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using dynamic light scattering, 3D-super-resolution microscopy, and electron microscopy. Mass spectrometry was used to analyze vaccine composition. We investigated the mechanism for early post-vaccine inflammatory reactions as potential co-stimulant for anti-PF4 immune response. Finally, we evaluated VITT antibodies for inducing release of procoagulant DNA-containing neutrophil extracellular traps (NETs), and measured DNase activity in VITT patient serum.ResultsBiophysical analyses showed formation of complexes between PF4 and vaccine constituents, including virus proteins that were recognized by VITT antibodies. EDTA, a vaccine constituent, increased microvascular leakage in mice allowing for circulation of virus- and virus-producing cell culture-derived proteins. Antibodies in normal sera cross-reacted with human proteins in the vaccine and likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. Polyphosphates and DNA enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven procoagulant NETs formation, while DNase activity was reduced in VITT sera, with granulocyte-rich cerebral vein thrombosis observed in a VITT patient.ConclusionsChAdOx1 nCoV-19 vaccine constituents (i) form antigenic complexes with PF4, (ii) EDTA increases microvascular permeability, and (iii) vaccine components cause acute inflammatory reactions. Antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. High-titer anti-PF4 antibodies activate platelets and induce neutrophil activation and NETs formation, fueling the VITT prothrombotic response.