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Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

Maintaining high-affinity antibodies after vaccination may be important for long-lasting immunity to malaria, but data on induction and kinetics of affinity is lacking. In a phase 1 malaria vaccine trial, antibody affinity increased following a second vaccination but declined substantially over 12 months, suggesting poor maintenance of high-affinity antibodies. Clinical Trials Registration Australian New Zealand Clinical Trials Registry ACTRN12607000552482. [ABSTRACT FROM AUTHOR]

Purposes: STAT1 is a transduction and transcriptional regulator that functions within the classical JAK/STAT pathway. In addition to chronic mucocutaneous candidiasis, bacterial infections are a common occurrence in patients with STAT1 gain-of-function (GOF) mutations. These patients often exhibit skewing of B cell subsets; however, the impact of STAT1-GOF mutations on B cell-mediated humoral immunity remains largely unexplored. It is also unclear whether these patients with IgG within normal range require regular intravenous immunoglobulin (IVIG) therapy. Methods: Eleven patients (harboring nine different STAT1-GOF mutations) were enrolled. Reporter assays and immunoblot analyses were performed to confirm STAT1 mutations. Flow cytometry, deep sequencing, ELISA, and ELISpot were conducted to assess the impact of STAT1-GOF on humoral immunity. Results: All patients exhibited increased levels of phospho-STAT1 and total STAT1 protein, with two patients carrying novel mutations. In vitro assays showed that these two novel mutations were GOF mutations. Three patients with normal total IgG levels received regular IVIG infusions, resulting in effective control of bacterial infections. Four cases showed impaired affinity and specificity of pertussis toxin-specific antibodies, accompanied by reduced generation of class-switched memory B cells. Patients also had a disrupted immunoglobulin heavy chain (IGH) repertoire, coupled with a marked reduction in the somatic hypermutation frequency of switched Ig transcripts. Conclusion: STAT1-GOF mutations disrupt B cell compartments and skew IGH characteristics, resulting in impaired affinity and antigen-specificity of antibodies and recurrent bacterial infections. Regular IVIG therapy can control these infections in patients, even those with normal total IgG levels. [ABSTRACT FROM AUTHOR]

Background: Patients with autoimmune/inflammatory conditions on anti- CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARSCoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. Methods: We performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV- 2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidicbased immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30). Results: Rituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7-- 38.8) nM vs. 4.6 (2.3--44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximabtreated patients [median (range) KD: 1.05 (0.45--1.84) nM vs. 20.25 (13.2--38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV- 2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination. Discussion: Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response. [ABSTRACT FROM AUTHOR]

BackgroundPatients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients.MethodsWe performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidic-based immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30).ResultsRituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7–38.8) nM vs. 4.6 (2.3–44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45–1.84) nM vs. 20.25 (13.2–38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination.DiscussionOur results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.

COVID-19, caused by SARS-CoV-2, and meningococcal disease, caused by Neisseria meningitidis , are relevant infectious diseases, preventable through vaccination. Outer membrane vesicles (OMVs), released from Gram-negative bacteria, such as N. meningitidis , present adjuvant characteristics and may confer protection against meningococcal disease. Here, we evaluated in mice the humoral and cellular immune response to different doses of receptor binding domain (RBD) of SARS-CoV-2 adjuvanted by N. meningitidis C:2a:P1.5 OMVs and aluminum hydroxide, as a combined preparation for these pathogens. The immunization induced IgG antibodies of high avidity for RBD and OMVs, besides IgG that recognized the Omicron BA.2 variant of SARS-CoV-2 with intermediary avidity. Cellular immunity showed IFN-γ and IL-4 secretion in response to RBD and OMV stimuli, demonstrating immunologic memory and a mixed Th1/Th2 response. Offspring presented transferred IgG of similar levels and avidity as their mothers. Humoral immunity did not point to the superiority of any RBD dose, but the group immunized with a lower antigenic dose (0.5 μg) had the better cellular response. Overall, OMVs enhanced RBD immunogenicity and conferred an immune response directed to N. meningitidis too.

Monoclonal antibody (mAbs) therapeutics cannot evade the occurrence of adverse effects. Thus, mAbs are commonly triggering immune responses corresponding to the expression of antidrug antibodies. Antidrug antibodies can neutralize mAbs, leading to their inhibition and hasten clearance, which dramatically hampers their therapeutic effects. Therefore, studies concerning the affinity between a mAbs and the corresponding antidrug antibody are demonstrating a growing interest to further understand the outcome of biotherapeutics after administration. We describe a novel analytical approach for the determination of the dissociation constant ( K d ) between a mAb and an antidrug antibody using capillary electrophoresis coupled to mass spectrometry (CE-MS/MS). The CE-MS/MS method employs a competitive assay, followed by the quantification of the residual amount of the active mAbs. The methodology allowed for the measurement of K d using serum samples without the implementation of immobilization to achieve protein-protein interaction. This characteristic enabled us to generate the interaction in conditions reflecting the physiological environment. A mathematical treatment was developed to calculate K d from MS/MS data, taking into account the stoichiometry of the mAbs/antidrug antibody complex and the bivalent properties of the two immunoglobulins. Prior to CE-MS/MS analysis, the interaction of the two proteins was studied by using mass photometry (MP) to determine equilibrium conditions and complexation stoichiometry. CE-MS/MS was used to investigate the interaction between infliximab and a neutralizing anti-infliximab antibody. Results allowed to measure a K d of 14.4 ± 2.9 nM. MS instrumentation sensitivity and specificity showed to be relevant to achieve accurate and robust K d measurements for strong interactions in the nanomolar range.

The high binding affinity of antibodies toward their cognate targets is key to eliciting effective immune responses, as well as to the use of antibodies as research and therapeutic tools. Here, we propose ANTIPASTI, a convolutional neural network model that achieves state-of-the-art performance in the prediction of antibody binding affinity using as input a representation of antibody-antigen structures in terms of normal mode correlation maps derived from elastic network models. This representation captures not only structural features but energetic patterns of local and global residue fluctuations. The learnt representations are interpretable: they reveal similarities of binding patterns among antibodies targeting the same antigen type, and can be used to quantify the importance of antibody regions contributing to binding affinity. Our results show the importance of the antigen imprint in the normal mode landscape, and the dominance of cooperative effects and long-range correlations between antibody regions to determine binding affinity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study aims to assess the effectiveness of mCSM-AB2, a graph-based signature machine learning method, for affinity engineering of the humanized single-chain Fv anti-CD147 (HuScFvM6-1B9). In parallel, molecular dynamics (MD) simulations were used to gain valuable insights into the dynamics and affinity of the HuScFvM6-1B9-CD147 complex. The result analysis involved integrating free energy changes calculated from the mCSM-AB2 with binding free energy predictions from MD simulations. The simulated structures of the modified HuScFvM6-1B9-CD147 domain 1 complex from MD simulations were used to highlight critical residues participating in the binding surface. Interestingly, alterations in the pattern of amino acids of HuScFvM6-1B9 at the complementarity determining regions interacting with the 31EDLGS35 epitope were observed, particularly in mutants that lost binding activity. The predicted mutants of HuScFvM6-1B9 were subsequently engineered and expressed in E. coli for subsequent binding property validation. Compared to WT HuScFvM6-1B9, the mutant HuScFvM6-1B9 L1:N32Y exhibited a 1.66-fold increase in binding affinity, with a K D of 1.75 × 10 -8  M. While mCSM-AB2 demonstrates insignificant improvement in predicting binding affinity enhancements, it excels at predicting negative effects, aligning well with experimental validation. In addition to binding free energies, total entropy was considered to explain the discrepancy between mCSM-AB2 predictions and experimental results. This study provides guidelines and identifies the limitations of mCSM-AB2 and MD simulations in antibody engineering., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chatchai Tayapiwatana reports financial support was provided by Chiang Mai University. Chatchai Tayapiwatana reports financial support was provided by National Research Council of Thailand. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In vivo in infection, virions are constantly produced and die rapidly. In contrast, most antibody binding assays do not include such features. Motivated by this, we considered virions with n = 100 binding sites in simple mathematical models with and without the production of virions. In the absence of viral production, at steady state, the distribution of virions by the number of sites bound is given by a binomial distribution, with the proportion being a simple function of antibody affinity (K on /K off ) and concentration; this generalizes to a multinomial distribution in the case of two or more kinds of antibodies. In the presence of viral production, the role of affinity is replaced by an infection analog of affinity (IAA), with IAA = K on /(K off  + d v  + r), where d v is the virus decay rate and r is the infection growth rate. Because in vivo d v can be large, the amount of binding as well as the effect of K off on binding are substantially reduced. When neutralization is added, the effect of K off is similarly small which may help explain the relatively high K off reported for many antibodies. We next show that the n+2-dimensional model used for neutralization can be simplified to a 2-dimensional model. This provides some justification for the simple models that have been used in practice. A corollary of our results is that an unexpectedly large effect of K off in vivo may point to mechanisms of neutralization beyond stoichiometry. Our results suggest reporting K on and K off separately, rather than focusing on affinity, until the situation is better resolved both experimentally and theoretically., (© 2024. The Author(s), under exclusive licence to the Society for Mathematical Biology.)

Pathogens constantly evolve and can develop mutations that evade host immunity and treatment. Addressing these escape mechanisms requires targeting evolutionarily conserved vulnerabilities, as mutations in these regions often impose fitness costs. We introduce adaptive multi-epitope targeting with enhanced avidity (AMETA), a modular and multivalent nanobody platform that conjugates potent bispecific nanobodies to a human immunoglobulin M (IgM) scaffold. AMETA can display 20+ nanobodies, enabling superior avidity binding to multiple conserved and neutralizing epitopes. By leveraging multi-epitope SARS-CoV-2 nanobodies and structure-guided design, AMETA constructs exponentially enhance antiviral potency, surpassing monomeric nanobodies by over a million-fold. These constructs demonstrate ultrapotent, broad, and durable efficacy against pathogenic sarbecoviruses, including Omicron sublineages, with robust preclinical results. Structural analysis through cryoelectron microscopy and modeling has uncovered multiple antiviral mechanisms within a single construct. At picomolar to nanomolar concentrations, AMETA efficiently induces inter-spike and inter-virus cross-linking, promoting spike post-fusion and striking viral disarmament. AMETA's modularity enables rapid, cost-effective production and adaptation to evolving pathogens., Competing Interests: Declaration of interests Y.X. and Y.S. are co-inventors on a provisional patent of AMETA technology filed by Icahn School of Medicine at Mount Sinai. The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories, and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, and AstraZeneca. A.G.-S. is an inventor of patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. Y.X. and Y.S. are co-inventors on a patent of Nbs filed by Univ. of Pittsburgh. Y.S. is a co-founder of Antenna Biotech Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for the development of next generation therapeutics. This study delves into the refinement and engineering of complementarity-determining regions (CDRs) through in silico affinity maturation followed by binding validation using isothermal titration calorimetry (ITC) and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Protein-protein docking analysis identified 32 single mutants of which nine mutants were selected for molecular dynamics (MD) simulations. Subsequently, biophysical ITC studies provided insights into binding affinity, and consistent with in silico findings, six mutations that demonstrated better binding affinity than native nanobody were further tested in vitro for neutralization activity against SARS-CoV-2 pseudovirus. Leu106Thr mutant was found to be most effective in virus-neutralization with IC 50 values of ∼0.03 μM, as compared to the native nanobody (IC 50 ∼0.77 μM). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens., Competing Interests: Declaration of competing interest We declare that we have no conflicts of interest in the authorship or publication of this contribution., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study aims to analyze if the results from different serological assays, used alone or combined, could match the outcome of challenge infection with foot-and-mouth disease virus (FMDV) after vaccination in cattle. Day-of-challenge sera from animals that had been vaccinated 21 days before with monovalent formulations containing inactivated A Iran 96 or A Iran 99 virus strains were used. Challenge and serology were performed with A22 Iraq strain. IgG1 titers and total-IgG avidity indexes were significantly higher in protected animals (p < 0.01) while IgG2-titers were not related to protection (p > 0.05). An IgG1 avidity ELISA was developed to analyze in one step, IgG1 levels and avidity. This assay estimated protection with 96 % accuracy. A strong agreement with challenge results was achieved (K = 0.85), suggesting a role of high-affinity IgG1 in protection against FMDV. These results support the assessment of the single dilution IgG1-Avidity ELISA to predict cross-protection in FMDV-vaccinated cattle., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nancy Cardoso reports financial support and travel were provided by Global Foot and Mouth Disease Research Alliance. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Congenital cytomegalovirus (cCMV) is a common congenital viral infection. Testing for cCMV usually begins with assessing maternal CMV serology, specifically IgM and IgG antibodies. A negative maternal CMV IgM suggests a low risk of recent maternal CMV infection, thereby suggesting a low risk of cCMV in the fetus. Consequently, cCMV is often ruled out when maternal CMV IgM is negative., Methods: In our perinatal autopsy and placental pathology database, we identified 5 cases of cCMV despite negative maternal CMV IgM results in the second trimester., Results: In all 5 cases, fetal abnormalities were first detected by ultrasound in the second trimester, prompting maternal CMV testing. Since second trimester maternal CMV IgM was negative in all cases, cCMV was considered unlikely, thus precluding further prenatal CMV testing in 4 of these cases. The diagnosis of cCMV was subsequently made through placental and/or autopsy examinations. Following this diagnosis, retrospective CMV serology and IgG avidity testing was performed on stored frozen first-trimester maternal blood samples in 3 cases. Among these, the first-trimester samples in 2 cases were IgG+, IgM+, and exhibited low IgG avidity, suggesting a primary maternal CMV infection around the time of conception. In the third case, both first and second-trimester maternal blood samples were IgG+, IgM-, and showed high IgG avidity, suggesting a non-primary maternal CMV infection (i.e., reactivation or reinfection of CMV)., Conclusion: A negative maternal CMV IgM in the second trimester cannot exclude cCMV infection. While CMV IgG avidity testing and analysis of stored frozen first-trimester maternal blood samples provide valuable insights, they have limitations. CMV PCR performed on amniotic fluid is a useful prenatal diagnostic tool. For cases of unexplained fetal abnormalities or death, autopsy and placental examination are recommended., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In this study, we have demonstrated a complementary-determining region (CDR) grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the CDR/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of CDR with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties., (Copyright © 2024 The Society for Biotechnology, Japan. All rights reserved.)

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most common tick-borne illness in the United States. Despite the rise in Lyme disease incidence, there is no vaccine against B. burgdorferi approved for human use. Little is known about the immune correlates of protection needed to prevent Lyme disease. In this work, a mouse model was used to characterize the immune response and compare the protection provided by two USDA-approved vaccines for use in canines: Duramune (bacterin vaccine) and Vanguard crLyme (subunit vaccine composed of two outer surface proteins, OspA and OspC). C3H/HeNCrl mice were immunized with two doses of either Duramune or Vanguard, and immune responses and protection against B. burgdorferi were assessed in short (35 days) and long-term (120 days) studies. Flow cytometry, ELISPOT detection of antibody-producing cells, and antibody affinity studies were performed to identify correlates of vaccine-mediated protection. Both vaccines induced humoral responses, with high IgG titers against B. burgdorferi. However, the levels of anti-B. burgdorferi antibodies decayed over time in Vanguard-vaccinated mice. While both vaccines triggered the production of antibodies against both OspA and OspC, antibody levels against these proteins were also lower in Vanguard-vaccinated mice 120 days post-vaccination. Both vaccines only provided partial protection against B. burgdorferi at the dose used in this model. The protection provided by Duramune was superior to Vanguard 120 days post-vaccination, and was characterized by higher antibody titers, higher abundance of long-lived plasma cells, and higher avidity antibodies than Vanguard. Overall, these studies provide insights into the importance of the humoral memory response to veterinary vaccines against Lyme disease and will help inform the development of future human vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The antibody response to vaccination and infection is a key component of the immune response to pathogens. Sequencing of peripheral B cells may not represent the complete B cell receptor repertoire. Here we present a method for sequencing human plasma-derived polyclonal IgG using a combination of mass spectrometry and B-cell sequencing. We investigate the IgG response to the Moderna Spikevax COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generate 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo protein sequencing, exhibit similar or higher binding affinities than the original natural polyclonal antibody. Neutralization tests reveal that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies and the potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Directly examining the circulating IgG pool is crucial due to potential misrepresentations by B-cell analysis alone., (© 2024. The Author(s).)

Monoclonal antibodies identified using display technologies like phage display occasionally suffers from a lack of affinity making it unsuitable for application. This drawback is circumvented with the application of affinity maturation. Affinity maturation is an essential step in the natural evolution of antibodies in the immune system. The evolution of molecular based methods has seen the development of various mutagenesis approaches. This allows for the natural evolutionary process during somatic hypermutation to be replicated in the laboratories for affinity maturation to fine-tune the affinity and selectivity of antibodies. In this review, we will discuss affinity maturation strategies for mAbs generated through phage display systems. The review will highlight various in vitro stochastic and non-stochastic affinity maturation approaches that includes but are not limited to random mutagenesis, site-directed mutagenesis, and gene synthesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Three glycosylated proteins were prepared from 2′-fucosyllactose (2′-FL) with α-lactalbumins, β-lactoglobulin and whey protein isolate, respectively, and their protein structures, glycosylation sites, digestion and transport capacities, and antibody binding capacities were determined. The findings indicate that following heating at 68 °C for 90 min, 2′-FL can form stable bonds with amino acid residues in milk proteins, leading to enhanced antioxidant properties, reduced hydrophobicity, and modified secondary structure content of the proteins. The glycosylation sites were mainly located in the amino acid sequence of β-lactoglobulin (K47, K91 and K135) and significantly reduce the affinity of its specific antibodies. 2′-FL glycosylation did not have a significant effect on the digestibility of cow's milk proteins, but it could significantly reduce the affinity of their specific antibody, indicating that it could be used in producing hypoallergenic dairy product. [Display omitted] • 2′-FL cross-link with cow's milk protein under mild conditions that simulate actual dairy processing conditions. • Cross-linking of oligosaccharides does not increase cytotoxicity and does not impede the digestion and absorption of milk proteins. • The glycosylation sites partially overlapped with the cow's milk allergenic epitopes and significantly reduced the antigenicity of the cow's milk proteins. [ABSTRACT FROM AUTHOR]

Ricin is a highly potent toxin that has been used in various attempts at bioterrorism worldwide. Although a vaccine for preventing ricin poisoning (RiVax™) is in clinical development, there are currently no commercially available prophylaxis or treatments for ricin intoxication. Numerous studies have highlighted the potential of passive immunotherapy using anti-ricin monoclonal antibodies (mAbs) and have shown promising results in preclinical models. In this article, we describe the neutralizing and protective efficacy of a new generation of high-affinity anti-ricin mAbs, which bind and neutralize very efficiently both ricin isoforms D and E in vitro through cytotoxicity cell assays. In vivo, protection assay revealed that one of these mAbs (RicE5) conferred over 90% survival in a murine model challenged intranasally with a 5 LD 50 of ricin and treated by intravenous administration of the mAbs 6 h post-intoxication. Notably, a 35% survival rate was observed even when treatment was administered 24 h post-exposure. Moreover, all surviving mice exhibited long-term immunity to high ricin doses. These findings offer promising results for the clinical development of a therapeutic candidate against ricin intoxication and may also pave the way for novel vaccination strategies against ricin or other toxins.

Bats are reservoirs of many zoonotic viruses that are fatal in humans but do not cause disease in bats. Moreover, bats generate low neutralizing antibody titers in response to experimental viral infection, although more robust antibody responses have been observed in wild-caught bats during times of food stress. Here, we compared the antibody titers and B cell receptor (BCR) diversity of Jamaican fruit bats (Artibeus jamaicensis; JFBs) and BALB/c mice generated in response to T-dependent and T-independent antigens. We then manipulated the diet of JFBs and challenged them with H18N11 influenza A-like virus or a replication incompetent Nipah virus VSV (Nipah-riVSV). Under standard housing conditions, JFBs generated a lower avidity antibody response and possessed more BCR mRNA diversity compared to BALB/c mice. However, withholding protein from JFBs improved serum neutralization in response to Nipah-riVSV and improved serum antibody titers specific to H18 but reduced BCR mRNA diversity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Crowley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

High affinity is crucial for the efficacy and specificity of antibody. Due to involving high-throughput screens, biological experiments for antibody affinity maturation are time-consuming and have a low success rate. Precise computational-assisted antibody design promises to accelerate this process, but there is still a lack of effective computational methods capable of pinpointing beneficial mutations within the complementarity-determining region (CDR) of antibodies. Moreover, random mutations often lead to challenges in antibody expression and immunogenicity. In this study, to enhance the affinity of a human antibody against avian influenza virus, a CDR library was constructed and evolutionary information was acquired through sequence alignment to restrict the mutation positions and types. Concurrently, a statistical potential methodology was developed based on amino acid interactions between antibodies and antigens to calculate potential affinity-enhanced antibodies, which were further subjected to molecular dynamics simulations. Subsequently, experimental validation confirmed that a point mutation enhancing 2.5-fold affinity was obtained from 10 designs, resulting in the antibody affinity of 2 nM. A predictive model for antibody-antigen interactions based on the binding interface was also developed, achieving an Area Under the Curve (AUC) of 0.83 and a precision of 0.89 on the test set. Lastly, a novel approach involving combinations of affinity-enhancing mutations and an iterative mutation optimization scheme similar to the Monte Carlo method were proposed. This study presents computational methods that rapidly and accurately enhance antibody affinity, addressing issues related to antibody expression and immunogenicity., (© The Author(s) 2024. Published by Oxford University Press.)

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections is challenging with current serology assays and is further complicated by the marked decrease in routine viral testing practices as viral transmission increased during Omicron. Here, we provide proof-of-principle that high-avidity anti-nucleocapsid (N) antibodies detects reinfections after a single infection with higher specificity (85%; 95% confidence interval [95% CI], 80%-90%) compared to anti-N antibody levels (72%; 95% CI, 66%-79%) in a vaccinated cohort. This method could be used to retroactively investigate the epidemiology and incremental long-term health consequences of SARS-CoV-2 reinfections., Competing Interests: Potential conflicts of interest . B. A. R. has received honoraria for participation in live meetings from Sanofi Pasteur France and Canada related to pertussis and RSV, but not for this work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

Aflatoxin B1 (AFB 1 ) accumulates in crops, where it poses a threat to human health. To detect AFB 1 , anti-AFB 1 monoclonal antibodies have been developed and are widely used. While the sensitivity and specificity of these antibodies have been extensively studied, information regarding the atomic-level docking of AFB 1 (and its derivatives) with these antibodies is limited. Such information is crucial for understanding the key interactions that are required for high affinity and specificity in aflatoxin binding. First, a 3D comparative model of anti-AFB 1 antibody (Ab-4B5G6) was predicted from the sequence using RosettaAntibody. We then utilized RosettaLigand to dock AFB 1 onto ten homology models, producing a total of 10,000 binding modes. Interestingly, the best-scoring mode predicted strong interactions involving four sites within the heavy chain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding interactions exclusively involve the variable domain of the heavy chain. The best-scoring mode with AFB 1 was also obtained through AF multimer combined with RosettaLigand, and two interactions at TRP and HIS were consistent with those found by Rosetta antibody-ligand computational simulation. The role of tryptophan in π interactions in antibodies was confirmed through mutation experiments, and the resulting mutant (W99A) exhibited a >1000-fold reduction in binding affinity for AFB 1 and analogs, indicating the effect of tryptophan on the stability of CDR-H3 region. Additionally, we evaluated the binding of two glycolic acid-derived molecular derivatives (with impaired hydrogen bonding potential), and these derivatives (AFB 2 -GA and AFG 2 -GA) demonstrated a very weak binding affinity for Ab-4B5G6. The heavy chain was successfully isolated, and its sensitivity and specificity were consistent with those of the intact antibody. The homology models of variable heavy (VH) single-domain antibodies were established by RosettaAntibody, and the docking analysis revealed the same residues, including Ala, His, and Trp. Compared to the potential binding mode of fragment variable (FV) region, the results from a model of VH indicated that there are seven models involved in hydrophobic interaction with TYR32, which is usually referred to as polar amino acid and has both hydrophobic and hydrophilic features depending on the circumstances. Our work encompasses the entire process of Rosetta antibody-ligand computational simulation, highlighting the significance of variable heavy domain structural design in enhancing molecular interactions.

Increasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential in in silico affinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC 50 values of the designed antibody mutants are decreased by up to 17 fold, and K D values by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks., (© 2024. The Author(s).)

Immunoglobulin G (IgG) purification is a critical process for evaluating its role in autoimmune diseases, which are defined by the occurrence of autoantibodies. Affinity chromatography with protein G is widely considered to be the optimal technique for laboratory-scale purification. However, this technique has some limitations, including the exposure of IgG to low pH, which can compromise the quality of the purified IgG. Here, we show that alternative methods for IgG purification are possible while maintaining the quality of IgG. Different techniques for IgG purification from serum were evaluated and compared with protein G-based approaches: Melon Gel, caprylic acid-ammonium sulfate (CAAS) precipitation, anion-exchange chromatography with diethylamino ethyl (DEAE) following ammonium sulfate (AS) precipitation, and AS precipitation alone. The results demonstrated that the purification yield of these techniques surpassed that of protein G. However, differences in the purity of IgG were observed using GeLC-MS/MS. The avidity of purified IgG against selected targets (SARS-CoV-2 and topoisomerase-I) was similar between purified IgG obtained using all techniques and unpurified sera. Our work provides valuable insights for future studies of IgG function by recommending alternative purification methods that offer advantages in terms of yield, time efficiency, cost-effectiveness, and milder pH conditions than protein G.

Immunoassay relies on antibodies, but traditional antibodies such as monoclonal antibody (mAb) require animal immunization and complex procedures. Single-chain variable fragment (scFv) becomes a potential alternative to mAb with advantages of the low cost, rapid and easy prepared. In the present study, we prepared scFvs against dihydroartemisinin (DHA) based on E. coli and HEK293T cell expression system, named MBP-scFv and scFv-Fc, respectively. Their properties were compared with the parent mAb. The calculated affinity constants of mAb, MBP-scFv and scFv-Fc were 2.1 × 10 8  L mol -1 , 2.2 × 10 7  L mol -1 and 1.6 × 10 8  L mol -1 , respectively. The half inhibitory concentration (IC 50 ) of mAb, MBP-scFv and scFv-Fc were 1.16 ng mL -1 , 2.15 ng mL -1 and 6.57 ng mL -1 , respectively. Both the scFv showed less sensitive than the mAb based on the IC 50 . The cross-reactivities of MBP-scFv for artemisinin and artesunate exhibited similarities to the mAb, yet the cross-reactivities of scFv-Fc for these compounds exceeded those of the mAb significantly. The stability of the scFvs was ascertained to be maintained for over 5 days at room temperature, and for more than a month at both 4 °C and - 20 °C. After that, the indirect competitive enzyme-linked immunosorbent assays (icELISAs) based on the scFv from E. coli were used to detect the DHA content in eight drug samples, and the result was consistent with ultra-performance liquid chromatography simultaneously. Although scFv can be used for quantitative determination of drugs, but it still cannot completely replace mAb in immunoassay without evolution and modification., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gα i - or β-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stephanie M. Anbuhl reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. Angela I. Schriek reports financial support was provided by Aids Fund. Steven W. de Taeye reports financial support was provided by Health Holland. Tom N. Grossmann reports was provided by European Research Council. Saskia Neubacher reports financial support was provided by Horizon 2020 European Innovation Council Transition Open Program. Raimond Heukers reports a relationship with QVQ holding BV that includes: board membership, employment, and equity or stocks. Stephanie M. Anbuhl reports a relationship with QVQ Holding BV that includes: employment. Tom N. Grossmann reports a relationship with Incircular B.V. that includes: board membership, consulting or advisory, and equity or stocks. Saskia Neubacher reports a relationship with Incircular B.V. that includes: board membership, consulting or advisory, and equity or stocks. Tom N. Grossmann has patent #WO2019203645A1 pending to Stichting VU. Xavier Dervillez has patent #WO2017202776A1 pending to Luxembourg Institute of Health. Saskia A. Neubacher has patent #WO2019203645A1 pending to Stichting VU. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic, which caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lead to a crisis with devastating disasters to global public economy and health. Several studies suggest that the SARS-CoV-2 nucleocapsid protein (N protein) is one of uppermost structural constituents of SARS-CoV-2 and is relatively conserved which could become a specific diagnostic marker. In this study, eight single domain antibodies recognized the N protein specifically which were named pN01-pN08 were screened using human phage display library. According to multiple sequence alignment and molecular docking analyses, the interaction mechanism between antibody and N protein was predicted. ELISA results indicated pN01-pN08 with high affinity to protein N. To improve their efficacy, two fusion proteins were prepared and their affinity was tested. These finding showed that fusion proteins had higher affinity than single domain antibodies and will be used as diagnosis for the pandemic of SARS-CoV-2., Competing Interests: The authors declare that they have no competing interests., (© 2024 Yang et al.)

The aggregation of α-Synuclein (αSyn) is strongly linked to neuronal death in Parkinson's disease and other synucleinopathies. The spreading of aggregated αSyn between neurons is at least partly dependent on electrostatic interactions between positively charged stretches on αSyn fibrils and the negatively charged heparan sulphate proteoglycans on the cell surface. To date there is still no therapeutic option available that could halt the progression of Parkinson's disease and one of the major limitations is likely the relatively low proportion of αSyn aggregates accessible to drugs in the extracellular space. Here, we investigated whether a negatively charged peptide tail fused to the αSyn aggregate-specific antibodies SynO2 and 9E4 could enhance the antibodies' avidity to αSyn aggregates in order to improve their potential therapeutic effect through inhibiting cell-to-cell spreading and enhancing the clearance of extracellular aggregates. We performed ELISAs to test the avidity to αSyn aggregates of both monovalent and bivalent antibody formats with and without the peptide tail. Our results show that the addition of the negatively charged peptide tail decreased the binding strength of both antibodies to αSyn aggregates at physiological salt conditions, which can likely be explained by intermolecular repulsions between the tail and the negatively charged C-terminus of αSyn. Additionally, the tail might interact with the paratopes of the SynO2 antibody abolishing its binding to αSyn aggregates. Conclusively, our peptide tail did not fulfil the required characteristics to improve the antibodies' binding to αSyn aggregates. Fine-tuning the design of the peptide tail to avoid its interaction with the antibodies' CDR and to better mimic relevant characteristics of heparan sulphates for αSyn aggregate binding may help overcome the limitations observed in this study., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Petersen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The prediction of binding affinity changes caused by missense mutations can elucidate antigen-antibody interactions. A few accessible structure-based online computational tools have been proposed. However, selecting suitable software for particular research is challenging, especially research on the SARS-CoV-2 spike protein with antibodies. Therefore, benchmarking of the mutation-diverse SARS-CoV-2 datasets is critical. Here, we collected the datasets including 1216 variants about the changes in binding affinity of antigens from 22 complexes for SARS-CoV-2 S proteins and 22 monoclonal antibodies as well as applied them to evaluate the performance of seven binding affinity prediction tools. The tested tools' Pearson correlations between predicted and measured changes in binding affinity were between -0.158 and 0.657, while accuracy in classification tasks on predicting increasing or decreasing affinity ranged from 0.444 to 0.834. These tools performed relatively better on predicting single mutations, especially at epitope sites, whereas poor performance on extremely decreasing affinity. The tested tools were relatively insensitive to the experimental techniques used to obtain structures of complexes. In summary, we constructed a list of datasets and evaluated a range of structure-based online prediction tools that will explicate relevant processes of antigen-antibody interactions and enhance the computational design of therapeutic monoclonal antibodies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Affinity maturation increases antigen-binding affinity and specificity of antibodies by somatic hypermutation. Various monoclonal antibodies against (4-hydroxy-3-nitrophenyl)acetyl (NP) were obtained during affinity maturation. Among them, highly matured anti-NP antibodies, such as E11 and E3, possess Cys96 H and Cys100 H in the complementarity-determining region 3 of the heavy chain, which would form a disulfide bond. In this study, we evaluated the effects of disulfide bonds on antigen binding by generating single-chain Fv (scFv) antibodies of E11 and its mutants, E11&#95;C96K H /C100E H and E11&#95;C96K H /C100Q H , and determined their antigen-binding thermodynamics and kinetics. The binding affinities of the Cys mutants were lower than that of E11 scFv, indicating that the disulfide bond contributed to antigen binding, especially for stable complex formation. This was also supported by the decreased affinity of E11 scFv in the presence of a reducing agent. The crystal structures of NP-free and NP-bound E11 scFvs were determined at high resolution, showing the existence of a disulfide bond between Cys96 H and Cys100 H , and the antigen recognition mechanism, which could be compared with those of other anti-NP antibodies, such as germline-type N1G9 and matured-type C6, as reported previously. These structures could explain the molecular basis of changes in antigen-binding affinity and thermal stability in the absence or presence of antigens. Small-angle X-ray scattering further showed a local conformational change in E11 scFv upon antigen binding in solution., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The main social impact of toxoplasmosis stems from its ability to be vertically transmitted. Postnatally acquired infection is generally asymptomatic in approximately 70-90% of cases, making diagnosis often dependent on laboratory tests using serological methods to search for anti-T. gondii antibodies. This study aimed to investigate the ability of the VIDAS TOXO IgG avidity and VIDAS TOXO IgM assays to confirm recent toxoplasmosis. In total, 341 pregnant women with suspected acute toxoplasmosis were systematically monitored in the Program for Control of Congenital Toxoplasmosis in Minas Gerais State, Brazil. We conducted an observational analytical-descriptive cross-sectional study and grouped according to clinical and laboratory criteria as having acute or chronic toxoplasmosis. The VIDAS TOXO IgG avidity and VIDAS TOXO IgM assays were evaluated to investigate the capacity to identify acute infection. IgG avidity showed good performance in identifying acute toxoplasmosis when the IgG avidity index was lower than or equal to 0.1. Values greater than or equal to 3.16 according to the TOXO IgM kit were associated with a greater chance of acute infection. These results may contribute to a more adequate diagnosis of acute gestational toxoplasmosis and, consequently, the avoidance of inadequate or unnecessary treatments.

Antibodies play a pivotal role in immune defense and serve as key therapeutic agents. The process of affinity maturation, wherein antibodies evolve through somatic mutations to achieve heightened specificity and affinity to target antigens, is crucial for effective immune response. Despite their significance, assessing antibody-antigen binding affinity remains challenging due to limitations in conventional wet lab techniques. To address this, we introduce AntiFormer, a graph-based large language model designed to predict antibody binding affinity. AntiFormer incorporates sequence information into a graph-based framework, allowing for precise prediction of binding affinity. Through extensive evaluations, AntiFormer demonstrates superior performance compared with existing methods, offering accurate predictions with reduced computational time. Application of AntiFormer to severe acute respiratory syndrome coronavirus 2 patient samples reveals antibodies with strong neutralizing capabilities, providing insights for therapeutic development and vaccination strategies. Furthermore, analysis of individual samples following influenza vaccination elucidates differences in antibody response between young and older adults. AntiFormer identifies specific clonotypes with enhanced binding affinity post-vaccination, particularly in young individuals, suggesting age-related variations in immune response dynamics. Moreover, our findings underscore the importance of large clonotype category in driving affinity maturation and immune modulation. Overall, AntiFormer is a promising approach to accelerate antibody-based diagnostics and therapeutics, bridging the gap between traditional methods and complex antibody maturation processes., (© The Author(s) 2024. Published by Oxford University Press.)

Large language models trained on sequence information alone can learn high-level principles of protein design. However, beyond sequence, the three-dimensional structures of proteins determine their specific function, activity, and evolvability. Here, we show that a general protein language model augmented with protein structure backbone coordinates can guide evolution for diverse proteins without the need to model individual functional tasks. We also demonstrate that ESM-IF1, which was only trained on single-chain structures, can be extended to engineer protein complexes. Using this approach, we screened about 30 variants of two therapeutic clinical antibodies used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We achieved up to 25-fold improvement in neutralization and 37-fold improvement in affinity against antibody-escaped viral variants of concern BQ.1.1 and XBB.1.5, respectively. These findings highlight the advantage of integrating structural information to identify efficient protein evolution trajectories without requiring any task-specific training data.

Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella -specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 10 4 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources., (© 2024 Wiley Periodicals LLC.)

Compared with naïve B cells, the B cell receptor (BCR) signal in germinal center (GC) B cells is attenuated; however, the significance of this signaling attenuation has not been well defined. Here, to investigate the role of attenuation of BCR signaling, we employed a Csk mutant mouse model in which Csk deficiency in GC B cells resulted in augmentation of net BCR signaling with no apparent effect on antigen presentation. We found that Csk is required for GC maintenance and efficient antibody affinity maturation. Mechanistically, ROS-induced apoptosis was exacerbated concomitantly with mitochondrial dysfunction in Csk-deficient GC B cells. Hence, our data suggest that attenuation of the BCR signal restrains hyper-ROS production, thereby protecting GC B cells from apoptosis and contributing to efficient affinity maturation., (© 2024 Inoue et al.)

Toxoplasma gondii is a neurotropic protozoan parasite that affects neuronal processing in the brain. This study aimed to investigate the prevalence of T. gondii infection in psychiatric disorder patients. We also investigated the potential association between sociodemographic, clinical manifestation, and behavior of Toxoplasma-seropositive patients with psychiatric disorders. Commercial ELISAs (IgG, IgM, and IgG avidity) using serum and PCR using buffy coat were performed on samples from 54 individuals in each of the following groups: patients diagnosed with depressive disorder, bipolar disorder, and schizophrenia, as well as psychiatrically healthy subjects (control group). They were recruited from the Hospital Universiti Sains Malaysia in Kelantan, Malaysia. Of 54 patients with depressive disorder, 24/54 (44.4 %) were seropositive for IgG, and four (16.7 %) were IgG+/IgM+. Among the latter, a high avidity index indicating a past infection was observed in half of the samples (50.0 %), and the other half (50.0 %) showed a low avidity index, indicating a possible recent infection. Meanwhile, 30/54 (55.6 %) patients with bipolar disorder were seropositive for IgG+, five (16.7 %) were IgG+/IgM+, and four of them had a high avidity index, and one had a low avidity index. Patients with schizophrenia showed 29/54 (53.7 %) seropositive for IgG, two of them (6.9 %) were IgG+/IgM+; one of latter had a high avidity index, and one had a low avidity index. Of 54 people in the control group, 37.0 % (20/54) were seropositive for T. gondii IgG antibodies. However, no significant difference was observed in seroprevalence between the control group and each patient group. No PCR-positive results were documented. A Chi-Square and multiple logistic regression showed that age (p = 0.031), close contact with cats/pets (p = 0.033) and contact with soil (p = 0.012) were significantly associated with Toxoplasma seropositivity in patients with psychiatric disorders. Additional research is needed to elucidate the causal relationships and underlying mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: To study the role of Toxoplasma IgG avidity in evaluating the stage of systemic infection during manifestation as toxoplasma retinochoroiditis and its clinical implications in eastern India., Methods: Retrospective chart review of Toxoplasma retinochoroiditis cases with Toxoplasma serology for IgG, IgM, and IgG avidity., Results: Included in this study were 17 eyes of 17 patients who had active retinitis located in the macula (14), mid-periphery (2), or periphery (1). They were either primary lesions (12) or reactivations (5). All the cases had Toxoplasma IgG positive; one case had IgM positivity, while all the cases had high IgG avidity values. IgG avidity had a positive correlation with the duration of symptoms., Conclusion: We observed high IgG avidity values in active retinochoroiditis in both primary ocular Toxoplasmosis and reactivation subgroups. These results indicate a late ocular manifestation after initial systemic infection with a possible incubation period ranging from 5 weeks to 5 months., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Background: The value of the widely applied maternal cytomegalovirus (CMV) serological testing approach in predicting intrauterine transmission in highly seroprevalent regions remains unknown., Methods: A nested case-control study was conducted based on a maternal-child cohort study. Newborns with congenital CMV (cCMV) infection were included, and each of them was matched to 3 newborns without cCMV infection. Retrospective samples were tested for immunoglobulin G (IgG) avidity and immunoglobulin M (IgM) antibodies in maternal serum and CMV DNA in maternal blood and urine to analyze their associations with cCMV infection., Results: Forty-eight newborns with cCMV infection and 144 matched newborns without infection were included in the study. Maternal IgM antibodies and IgG avidity during pregnancy were not statistically associated with intrauterine transmission. The presence of CMV DNAemia indicated a higher risk of cCMV infection, with odds ratio values of 5.7, 6.5, and 13.0 in early, middle, and late pregnancy, respectively. However, the difference in CMV shedding rates in transmitters and nontransmitters was not significant in urine., Conclusions: The value of current maternal CMV serological testing in regions with high seropositivity rates is very limited and should be reconsidered. The detection of DNAemia would be helpful in assessing the risk of intrauterine transmission., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment., Methods and Results: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20., Conclusion: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The most common application of phage-display technology is the discovery of peptides or proteins that specifically bind some molecule or other substance of interest-for example, antibodies that specifically bind an antigen. The discovery process starts with a library encompassing a very large array of proteins or peptides with a great diversity of binding specificities-for example, single-chain antibodies with a great diversity of antigen-binding sites. Each member of the array is displayed on the surface of hundreds to billions of identical virus particles (virions) belonging to a single-phage clone; the library as a whole comprises millions to billions of such clones, all mixed together in a single vessel. Affinity selection is the process by which a molecule or substance of interest-generically called the selector-is used to select very rare clones in the library displaying proteins or peptides that happen to bind the selector with high affinity and selectivity. Here, I explain general principles guiding a successful affinity-selection project-principles grounded in phage biology, kinetics of reversible binding, technological advances, and the practical experience of thousands of investigators around the globe., (© 2024 Cold Spring Harbor Laboratory Press.)

As the demand for immunotherapy to treat and manage cancers, infectious diseases and other disorders grows, a comprehensive understanding of amino acids and their intricate role in antibody engineering has become a prime requirement. Naturally produced antibodies may not have the most suitable amino acids at the complementarity determining regions (CDR) and framework regions, for therapeutic purposes. Therefore, to enhance the binding affinity and therapeutic properties of an antibody, the specific impact of certain amino acids on the antibody's architecture must be thoroughly studied. In antibody engineering, it is crucial to identify the key amino acid residues that significantly contribute to improving antibody properties. Therapeutic antibodies with higher binding affinity and improved functionality can be achieved through modifications or substitutions with highly suitable amino acid residues. Here, we have indicated the frequency of amino acids and their association with the binding free energy in CDRs. The review also analyzes the experimental outcome of two studies that reveal the frequency of amino acids in CDRs and provides their significant correlation between the outcomes. Additionally, it discusses the various bond interactions within the antibody structure and antigen binding. A detailed understanding of these amino acid properties should assist in the analysis of antibody sequences and structures needed for designing and enhancing the overall performance of therapeutic antibodies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

SARS-CoV-2 has rampantly spread around the globe and continues to cause unprecedented loss through ongoing waves of (re)infection. Increasing our understanding of the protection against infection with SARS-CoV-2 is critical to ending the pandemic. Serological assays have been widely used to assess immune responses, but secretory antibodies, the essential first line of defense, have been studied to only a limited extent. Of particular interest and importance are neutralizing antibodies, which block the binding of the spike protein of SARS-CoV-2 to the human receptor angiotensin-converting enzyme-2 (ACE2) and thus are essential for immune defense. Here, we employed Microfluidic Diffusional Sizing (MDS), an immobilization-free technology, to characterize neutralizing antibody affinity to SARS-CoV-2 spike receptor-binding domain (RBD) and spike trimer in saliva. Affinity measurement was obtained through a contrived sample and buffer using recombinant SARS-CoV-2 RBD and monoclonal antibody. Limited saliva samples demonstrated that MDS applies to saliva neutralizing antibody measurement. The ability to disrupt a complex of ACE2-Fc and spike trimer is shown. Using a quantitative assay on the patient sample, we determined the affinity and binding site concentration of the neutralizing antibodies., (© 2024. The Author(s).)

Deep learning has achieved impressive results in various fields such as computer vision and natural language processing, making it a powerful tool in biology. Its applications now encompass cellular image classification, genomic studies and drug discovery. While drug development traditionally focused deep learning applications on small molecules, recent innovations have incorporated it in the discovery and development of biological molecules, particularly antibodies. Researchers have devised novel techniques to streamline antibody development, combining in vitro and in silico methods. In particular, computational power expedites lead candidate generation, scaling and potential antibody development against complex antigens. This survey highlights significant advancements in protein design and optimization, specifically focusing on antibodies. This includes various aspects such as design, folding, antibody-antigen interactions docking and affinity maturation., (© The Author(s) 2024. Published by Oxford University Press.)

The optimization of therapeutic antibodies through traditional techniques, such as candidate screening via hybridoma or phage display, is resource-intensive and time-consuming. In recent years, computational and artificial intelligence-based methods have been actively developed to accelerate and improve the development of therapeutic antibodies. In this study, we developed an end-to-end sequence-based deep learning model, termed AttABseq, for the predictions of the antigen-antibody binding affinity changes connected with antibody mutations. AttABseq is a highly efficient and generic attention-based model by utilizing diverse antigen-antibody complex sequences as the input to predict the binding affinity changes of residue mutations. The assessment on the three benchmark datasets illustrates that AttABseq is 120% more accurate than other sequence-based models in terms of the Pearson correlation coefficient between the predicted and experimental binding affinity changes. Moreover, AttABseq also either outperforms or competes favorably with the structure-based approaches. Furthermore, AttABseq consistently demonstrates robust predictive capabilities across a diverse array of conditions, underscoring its remarkable capacity for generalization across a wide spectrum of antigen-antibody complexes. It imposes no constraints on the quantity of altered residues, rendering it particularly applicable in scenarios where crystallographic structures remain unavailable. The attention-based interpretability analysis indicates that the causal effects of point mutations on antibody-antigen binding affinity changes can be visualized at the residue level, which might assist automated antibody sequence optimization. We believe that AttABseq provides a fiercely competitive answer to therapeutic antibody optimization., (© The Author(s) 2024. Published by Oxford University Press.)

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility., Competing Interests: Declaration of interests G.R.S. sits on the GSK Vaccines Scientific Advisory Board, consults for AstraZeneca, and is a founder member of RQ Biotechnology. D.I.S. consults for AstraZeneca. Oxford University holds intellectual property related to SARS-CoV-2 mAbs discovered in G.R.S.’s laboratory. S.J.D. is a scientific advisor to the Scottish Parliament on COVID-19., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)