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Anelloviruses are nonpathogenic viruses that comprise a major portion of the human virome. Despite being ubiquitous in the human population, anelloviruses (ANVs) remain poorly understood. Basic features of the virus, such as the identity of its capsid protein and the structure of the viral particle, have been unclear until now. Here, we use cryogenic electron microscopy to describe the first structure of an ANV-like particle. The particle, formed by 60 jelly roll domain-containing ANV capsid proteins, forms an icosahedral particle core from which spike domains extend to form a salient part of the particle surface. The spike domains come together around the 5-fold symmetry axis to form crown-like features. The base of the spike domain, the P1 subdomain, shares some sequence conservation between ANV strains while a hypervariable region, forming the P2 subdomain, is at the spike domain apex. We propose that this structure renders the particle less susceptible to antibody neutralization by hiding vulnerable conserved domains while exposing highly diverse epitopes as immunological decoys, thereby contributing to the immune evasion properties of anelloviruses. These results shed light on the structure of anelloviruses and provide a framework to understand their interactions with the immune system., (© 2024. The Author(s).)

Porcine epidemic diarrhea virus (PEDV) is a serious disease that poses a significant threat to the pig industry. This study focused on analyzing the Spike protein of PEDV, which harbors crucial antigenic determinants, in identifying dominant epitopes. Immunoinformatics tools were used to screen for B-cell, CD4+ and CD8+ predominance epitopes. These epitopes were then connected to the N-terminal of ferritin to form a self-assembled nanoparticle vaccine. Various physical and chemical properties of the candidate vaccine were analyzed, including secondary structure prediction, tertiary structure modeling, molecular docking, immune response simulation and computer cloning. The results demonstrated that the candidate vaccine was antigenic, soluble, stable, non-allergic, and formed a stable complex with the target receptor TLR-3. Immune simulation analysis showed that the candidate vaccine effectively stimulated both cellular and humoral reactions, leading to increased related cytokines production. Furthermore, efficient and stable expression of the candidate vaccine was achieved through reverse translation in the Escherichia coli K12 expression system following codon optimization and in silico cloning. The developed nanoparticle candidate vaccine in this study holds promise as an effective PEDV vaccine candidate, offering a new approach for the research, development and improvement of vaccines targeting porcine enteric diarrhea coronavirus., (© 2024. The Author(s).)

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium., (© 2024. The Author(s).)

Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations., (© 2024. The Author(s).)

Human immunodeficiency virus type 1 (HIV-1) vaccine immunogens capable of inducing broadly neutralizing antibodies (bNAbs) remain obscure. HIV-1 evades immune responses through enormous diversity and hides its conserved vulnerable epitopes on the envelope glycoprotein (Env) by displaying an extensive immunodominant glycan shield. In elite HIV-1 viremic controllers, glycan-dependent bNAbs targeting conserved Env epitopes have been isolated and are utilized as vaccine design templates. However, immunological tolerance mechanisms limit the development of these antibodies in the general population. The well characterized bNAbs monoclonal variants frequently exhibit extensive levels of somatic hypermutation, a long third heavy chain complementary determining region, or a short third light chain complementarity determining region, and some exhibit poly-reactivity to autoantigens. This review elaborates on the obstacles to engaging and manipulating the Env glycoprotein as an effective immunogen and describes an alternative reverse vaccinology approach to develop a novel category of bNAb-epitope-derived non-cognate immunogens for HIV-1 vaccine design., (© 2024. The Author(s).)

Objective: Rheumatoid arthritis (RA) is characterised by the presence of autoantibodies, among which those targeting the constant region of immunoglobulin G (IgG), called rheumatoid factors (RF). Despite this link, RFs can also be found in other disorders and the healthy population, which hampers its use as a diagnostic tool. We recently showed that a subset of RA-derived RFs target a distinct epitope on the IgG-Fc, a feature that is currently not used in the clinic., Methods: We determined immunoglobulin M (IgM)-RF levels specific against an RA-associated epitope (using our engineered next-generation RF antigen 'T3-17') in a prospective cohort of 475 patients with seropositive (for IgM-RF or aCCP) arthralgia that were followed for 5 years or until the development of arthritis., Results: The presence of RFs targeting T3-17 was more strongly associated with progression to arthritis in comparison to traditional RF measurements. Within the group of patients positive for T3-17 RF the risk of arthritis development was increased as compared with wild-type RF, HR=3.2 (95% CI 2.4 to 4.3) vs HR=2.2 (95% CI 1.7 to 3.0). Predictive power of T3-17 RF was improved in combination with aCCP titres, HR=6.4 (4.7-8.7) vs HR=5.1 (3.9-6.8). This combination performed better than aCCP detection on its own., Conclusion: The detection of disease-specific RF is feasible and seems to improve the diagnostic power of RF and should be considered to be implemented in the clinic., Competing Interests: Competing interests: DvS, GW and TR are inventors on a patent application based on the use of bioengineered IgG targets for the characterisation of rheumatoid factor reactivity patterns., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC's LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance., (© 2024. The Author(s).)

We describe the molecular-level composition of polyclonal immunoglobulin G (IgG) anti-spike antibodies from ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccination, or their combination ("hybrid immunity") at monoclonal resolution. Infection primarily triggers S2/N-terminal domain (NTD)-reactive antibodies, whereas vaccination mainly induces anti-receptor-binding domain (RBD) antibodies. This imprint persists after secondary exposures wherein >60% of ensuing hybrid immunity derives from the original IgG pool. Monoclonal constituents of the original IgG pool can increase breadth, affinity, and prevalence upon secondary exposures, as exemplified by the plasma antibody SC27. Following a breakthrough infection, vaccine-induced SC27 gained neutralization breadth and potency against SARS-CoV-2 variants and zoonotic viruses (half-maximal inhibitory concentration [IC 50 ] ∼0.1-1.75 nM) and increased its binding affinity to the protective RBD class 1/4 epitope (dissociation constant [K D ] < 5 pM). According to polyclonal escape analysis, SC27-like binding patterns are common in SARS-CoV-2 hybrid immunity. Our findings provide a detailed molecular definition of immunological imprinting and show that vaccination can produce class 1/4 (SC27-like) IgG antibodies circulating in the blood., Competing Interests: Declaration of interests R.S.B. is a member of advisory boards for Vaxart, Takeda, and Invivyd and has collaborative projects with Gilead, J&J, and HilleVax, focused on unrelated projects. S.R.L. and R.S.B. are co-inventors of methods and uses of mouse-adapted SARS-CoV-2 viruses (US patent US11225508B1). J.D.B. is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. J.D.B. and B.D. are inventors on Fred Hutchinson Cancer Center-licensed patents related to viral deep mutational scanning. R.S.B., G.G., W.N.V., J.J.L., and G.C.I. are inventors on a provisional US patent application for mAb SC27 and other new antibodies described in this manuscript, entitled “Broadly neutralizing human monoclonal antibodies that target the SARS-CoV-2 receptor binding domain (RBD)” (63/491,270)., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused significant disruptions in porcine breeding and raised concerns about potential human infection. The nucleocapsid (N) protein of SADS-CoV plays a vital role in viral assembly and replication, but its structure and functions remain poorly understood. This study utilized biochemistry, X-ray crystallography, and immunization techniques to investigate the N protein's structure and function in SADS-CoV. Our findings revealed distinct domains within the N protein, including an RNA-binding domain, two disordered domains, and a dimerization domain. Through biochemical assays, we confirmed that the N-terminal domain functions as an RNA-binding domain, and the C-terminal domain is involved in dimerization, with the crystal structure analysis providing visual evidence of dimer formation. Immunization experiments demonstrated that the disordered domain 2 elicited a significant antibody response. These identified domains and their interactions are crucial for viral assembly. This comprehensive understanding of the N protein in SADS-CoV enhances our knowledge of its assembly and replication mechanisms, enabling the development of targeted interventions and therapeutic strategies., Importance: SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs., Competing Interests: The authors declare no conflict of interest.

Both Old World and New World hantaviruses are transmitted through rodents and can lead to hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome in humans without the availability of specific therapeutics. The square-shaped surface spikes of hantaviruses consist of four Gn-Gc heterodimers that are pivotal for viral entry into host cells and serve as targets for the immune system. Previously, a human-derived neutralizing monoclonal antibody, AH100, demonstrated specific neutralization against the Old World hantavirus, Hantaan virus. However, the precise mode binding of this neutralizing monoclonal antibody remains unclear. In the present study, we determined the structure of the Hantaan virus Gn-AH100 antigen-binding fragment complex and identified its epitope. Crystallography revealed that AH100 targeted the epitopes on domain A and b-ribbon and E3-like domain. Epitope mapping onto a model of the higher order (Gn-Gc) 4 spike revealed its localization between neighboring Gn protomers, distinguishing this epitope as a unique site compared to the previously reported monoclonal antibodies. This study provides crucial insights into the structural basis of hantavirus neutralizing antibody epitopes, thereby facilitating the development of therapeutic antibodies.IMPORTANCEHantaan virus (HTNV) poses a significant threat to humans by causing hemorrhagic fever with renal syndrome with high mortality rates. In the absence of FDA-approved drugs or vaccines, it is urgent to develop specific therapeutics. Here, we elucidated the epitope of a human-derived neutralizing antibody, AH100, by determining the HTNV glycoprotein Gn-AH100 antigen-binding fragment (Fab) complex structure. Our findings revealed that the epitopes situated on the domain A and b-ribbon and E3-like domain of the HTNV Gn head. By modeling the complex structure in the viral lattice, we propose that AH100 neutralizes the virus by impeding conformational changes of Gn protomer, which is crucial for viral entry. Additionally, sequence analysis of all reported natural isolates indicated the absence of mutations in epitope residues, suggesting the potential neutralization ability of AH100 in diverse isolates. Therefore, our results provide novel insights into the epitope and the molecular basis of AH100 neutralization., Competing Interests: The authors declare no conflict of interest.

Acinetobacter baumannii is a gram-negative bacillus prevalent in nature, capable of thriving under various environmental conditions. As an opportunistic pathogen, it frequently causes nosocomial infections such as urinary tract infections, bacteremia, and pneumonia, contributing to increased morbidity and mortality in clinical settings. Consequently, developing novel vaccines against Acinetobacter baumannii is of utmost importance. In our study, we identified 10 highly conserved antigenic proteins from the NCBI and UniProt databases for epitope mapping. We subsequently screened and selected 8 CTL, HTL, and LBL epitopes, integrating them into three distinct vaccines constructed with adjuvants. Following comprehensive evaluations of immunological and physicochemical parameters, we conducted molecular docking and molecular dynamics simulations to assess the efficacy and stability of these vaccines. Our findings indicate that all three multi-epitope mRNA vaccines designed against Acinetobacter baumannii are promising; however, further animal studies are required to confirm their reliability and effectiveness., (© 2024. The Author(s).)

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes severe viral hemorrhagic fever and thrombocytopenia syndrome with a fatality rate of up to 30%. No licensed vaccines or therapeutics are currently available for humans. Here, we develop seven monoclonal antibodies (mAbs) against SFTSV surface glycoprotein Gn. Mechanistic studies show that three neutralizing mAbs (S2A5, S1G3, and S1H7) block multiple steps during SFTSV infection, including viral attachment and membrane fusion, whereas another neutralizing mAb (B1G11) primarily inhibits the viral attachment step. Epitope binning and X-ray crystallographic analyses reveal four distinct antigenic sites on Gn, three of which have not previously been reported, corresponding to domain I, domain II, and spanning domain I and domain II. One of the most potent neutralizing mAbs, S2A5, binds to a conserved epitope on Gn domain I and broadly neutralizes infection of six SFTSV strains corresponding to genotypes A to F. A single dose treatment of S2A5 affords both pre- and post-exposure protection of mice against lethal SFTSV challenge without apparent weight loss. Our results support the importance of glycoprotein Gn for eliciting a robust humoral response and pave a path for developing prophylactic and therapeutic antibodies against SFTSV infection., (© 2024. The Author(s).)

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na 7 PMo 11 O 39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na 7 PMo 11 O 39 NPs. The Au/Na 7 PMo 11 O 39 -CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na 7 PMo 11 O 39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 10 7 -2.81 × 10 8 particles/mL, with a limit of detection (LOD) of 5.82 × 10 7 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening., 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.)

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory disease in humans. High fatality rates and continued infectiousness remain a pressing concern for global health preparedness. Antibodies targeted at the receptor-binding domain (RBD) are major countermeasures against human viral infection. Here, we report four potent nanobodies against MERS-CoV, which are isolated from alpaca, and especially the potency of Nb14 is highest in the pseudotyped virus assay. Structural studies show that Nb14 framework regions (FRs) are mainly involved in interactions targeting a novel epitope, which is entirely distinct from all previously reported antibodies, and disrupt the protein-carbohydrate interaction between residue W535 of RBD and hDPP4 N229-linked carbohydrate moiety (hDPP4-N229-glycan). Different from Nb14, Nb9 targets the cryptic face of RBD, which is distinctive from the hDPP4 binding site and the Nb14 epitope, and it induces the β5-β6 loop to inflect towards a shallow groove of the RBD and dampens the accommodation of a short helix of hDPP4. The particularly striking epitopes endow the two Nbs administrate synergistically in the pseudotyped MERS-CoV assays. These results not only character unprecedented epitopes for antibody recognition but also provide promising agents for prophylaxis and therapy of MERS-CoV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ma 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 COVID-19 pandemic continues to cause infections and deaths, which are attributable to the SARS-CoV-2 Omicron variant of concern (VOC). Moderna's response to the declining protective efficacies of current SARS-CoV-2 vaccines against Omicron was to develop a bivalent booster vaccine based on the Spike (S) protein from the Wuhan and Omicron BA.4/BA.5 strains. This approach, while commendable, is unfeasible in light of rapidly emerging mutated viral strains. PubMed and Google Scholar were systematically reviewed for peer-reviewed papers up to January 2024. Articles included focused on specific themes such as the clinical history of recombinant protein vaccine development against different diseases, including COVID-19, the production of recombinant protein vaccines using different host expression systems, aspects to consider in recombinant protein vaccine development, and overcoming problems associated with large-scale recombinant protein vaccine production. In silico approaches to identify conserved and immunogenic epitopes could provide broad protection against SARS-CoV-2 VOCs but require validation in animal models. The recombinant protein vaccine development platform has shown a successful history in clinical development. Recombinant protein vaccines incorporating conserved epitopes may utilize a number of expression systems, such as yeast (Saccharomyces cerevisiae), baculovirus-insect cells (Sf9 cells), and Escherichia coli (E. coli). Current multi-epitope subunit vaccines against SARS-CoV-2 utilizing synthetic peptides are unfeasible for large-scale immunizations. Recombinant protein vaccines based on conserved and immunogenic proteins produced using E. coli offer high production yields, convenient purification, and cost-effective production of large-scale vaccine quantities capable of protecting against the SARS-CoV-2 D614G strain and its VOCs., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Candida albicans can cause superficial or systemic infections in humans, particularly in immunocompromised individuals. Vaccination strategies targeting specific antigens of C. albicans have shown promise in providing protection against invasive candidiasis. This study aimed to evaluate the immuno-protective capacity of a KLH conjugated complex peptide, 3P-KLH, containing epitopes from C. albicans antigens Als3, Hwp1, and Met6 in a murine model of hematogenously induced candidiasis. Mice immunized with 3P-KLH raised a specific antibody response, and protection against C. albicans infection was assessed. Immunized mice exhibited significantly lower fungal load in their kidneys compared to the control group. Moreover, 37.5 % of immunized mice survived 21 days after the infection, while all control animals died within the first nine days. These findings suggest that the 3P-KLH complex peptide, targeting C. albicans key antigens, elicits a protective immune response and reduces the severity of systemic Candida infection. In addition, the high binding affinity of the selected epitopes with MHC II alleles further supports the potential immunogenicity of this peptide in humans. This research provides insights into the development of novel immunotherapeutic approaches against invasive candidiasis., 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 long-term value of efficient antigen discovery includes gaining insights into the variety of potential cancer neoantigens, effective vaccines lacking adverse effects, and adaptive immune receptor (IR) targets for blocking adaptive IR-antigen interactions in autoimmunity. While the preceding goals have been partially addressed via big data approaches to HLA (human leukocyte antigen)-epitope binding, there has been little such progress in the big data setting for adaptive IR-epitope binding. This delay in progress for the latter is likely due to, among other things, the much more complicated adaptive IR repertoire in an individual compared to individual HLA alleles. Thus, results described here represent the application of an algorithm for efficient assessment of immunoglobulin heavy chain complementarity determining region-3 (IGH CDR3)-gliadin epitope interactions, with a focus on epitopes known to be associated with an immune response in celiac disease. The hydrophobic, chemical complementarity between celiac case IGH CDR3s and known celiac epitopes was found to be greater in comparison to the hydrophobic, chemical complementarity between the same celiac case IGH CDR3s and a series of control epitopes. Thus, the approaches indicated here likely offer guidance for the development of conveniently applied algorithms for antigen verification and discovery., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences. The model always ranks the binding of the cognate sequence in the top 100 sequences, and for 6 of the 8 mAbs, the cognate sequence ranks in the top ten. Practically, this approach has potential utility in selecting highly specific mAbs for therapeutics or diagnostics. More fundamentally, this demonstrates that very sparse random sampling of a large amino acid sequence spaces is sufficient to generate comprehensive models predictive of highly specific molecular recognition., (© 2024. The Author(s).)

Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development., (© 2024. The Author(s).)

Artificial Intelligence (AI) techniques have made great advances in assisting antibody design. However, antibody design still heavily relies on isolating antigen-specific antibodies from serum, which is a resource-intensive and time-consuming process. To address this issue, we propose a Pre-trained Antibody generative large Language Model (PALM-H3) for the de novo generation of artificial antibodies heavy chain complementarity-determining region 3 (CDRH3) with desired antigen-binding specificity, reducing the reliance on natural antibodies. We also build a high-precision model antigen-antibody binder (A2binder) that pairs antigen epitope sequences with antibody sequences to predict binding specificity and affinity. PALM-H3-generated antibodies exhibit binding ability to SARS-CoV-2 antigens, including the emerging XBB variant, as confirmed through in-silico analysis and in-vitro assays. The in-vitro assays validate that PALM-H3-generated antibodies achieve high binding affinity and potent neutralization capability against spike proteins of SARS-CoV-2 wild-type, Alpha, Delta, and the emerging XBB variant. Meanwhile, A2binder demonstrates exceptional predictive performance on binding specificity for various epitopes and variants. Furthermore, by incorporating the attention mechanism inherent in the Roformer architecture into the PALM-H3 model, we improve its interpretability, providing crucial insights into the fundamental principles of antibody design., (© 2024. The Author(s).)

The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes., Competing Interests: K. Kiyotani is a scientific advisor of Cancer Precision Medicine, Inc. No disclosures were reported by the other authors. We have confirmed that our adherence to all PLOS ONE policies on data and material sharing remains unchanged., (Copyright: © 2024 Ito 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.)

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine., Competing Interests: Declaration of interests Ted Ross is an inventor on influenza vaccines., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aims: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development., Methods and Results: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P < 0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P < 0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera., Conclusions: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

The SARS-CoV-2 virus responsible for the COVID-19 global pandemic has exhibited a striking capacity for viral evolution that drives continued evasion from vaccine and infection-induced immune responses. Mutations in the receptor binding domain of the S1 subunit of the spike glycoprotein have led to considerable escape from antibody responses, reducing the efficacy of vaccines and monoclonal antibody (mAb) therapies. Therefore, there is a need to interrogate more constrained regions of spike, such as the S2 subdomain. Here, we present a collection of S2 mAbs from two SARS-CoV-2 convalescent individuals that target multiple regions in S2, including regions outside of those commonly reported. One of the S2 mAbs, C20.119, which bound to a highly conserved epitope in the fusion peptide, was able to broadly neutralize across SARS-CoV-2 variants, SARS-CoV-1, and closely related zoonotic sarbecoviruses. The majority of the mAbs were non-neutralizing; however, many of them could mediate antibody-dependent cellular cytotoxicity (ADCC) at levels similar to the S1-targeting mAb S309 that was previously authorized for treatment of SARS-CoV-2 infections. Several of the mAbs with ADCC function also bound to spike trimers from other human coronaviruses (HCoVs), such as MERS-CoV and HCoV-HKU1. Our findings suggest S2 mAbs can target diverse epitopes in S2, including functional mAbs with HCoV and sarbecovirus breadth that likely target functionally constrained regions of spike. These mAbs could be developed for potential future pandemics, while also providing insight into ideal epitopes for eliciting a broad HCoV response., Competing Interests: J.O. is a consultant for Aerium Therapeutics, Inc. H.Y.C reported consulting with Ellume, Merck, Abbvie, Pfizer, Medscape, Vindico, and the Bill and Melinda Gates Foundation. She has received research funding from Gates Ventures, and support and reagents from Ellume and Cepheid outside of the submitted work. J.O. and J.G are listed on a patent application (22-173-US-PSP2) and license agreement with Aerium Therapeutics, Inc for SARS-CoV-2 antibodies not described in this manuscript., (Copyright: © 2024 Guenthoer 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.)

Helicobacter pylori (H. pylori) is responsible for various chronic or acute diseases, such as stomach ulcers, dyspepsia, peptic ulcers, gastroesophageal reflux, gastritis, lymphoma, and stomach cancers. Although specific drugs are available to treat the bacterium's harmful effects, there is an urgent need to develop a preventive or therapeutic vaccine. Therefore, the current study aims to create a multi-epitope vaccine against H. pylori using lipid nanoparticles. Five epitopes from five target proteins of H. pylori, namely, Urease, CagA, HopE, SabA, and BabA, were used. Immunogenicity, MHC (Major Histocompatibility Complex) bonding, allergenicity, toxicity, physicochemical analysis, and global population coverage of the entire epitopes and final construct were carefully examined. The study involved using various bioinformatic web tools to accomplish the following tasks: modeling the three-dimensional structure of a set of epitopes and the final construct and docking them with Toll-Like Receptor 4 (TLR4). In the experimental phase, the final multi-epitope construct was synthesized using the solid phase method, and it was then enclosed in lipid nanoparticles. After synthesizing the construct, its loading, average size distribution, and nanoliposome shape were checked using Nanodrop at 280 nm, dynamic light scattering (DLS), and atomic force microscope (AFM). The designed vaccine has been confirmed to be non-toxic and anti-allergic. It can bind with different MHC alleles at a rate of 99.05%. The construct loading was determined to be about 91%, with an average size of 54 nm. Spherical shapes were also observed in the AFM images. Further laboratory tests are necessary to confirm the safety and immunogenicity of the multi-epitope vaccine., (© 2024. The Author(s).)

Bovine betacoronavirus (BoCoV) is a pneumoenteric pathogen of cattle that is closely related to human coronavirus OC43. Vaccines are administered to protect against diseases caused by BoCoV, but knowledge gaps exist with regard to correlates of protection and the effect of immune evasion on driving evolution. In this study, immune epitopes were mapped onto BoCoV structural proteins, including spike and haemagglutinin esterase (HE), and then supported with targeted gene sequencing of Irish clinical isolates and selective pressure analysis. Increased prevalence of diversifying selection and amino acid changes in some mapped immune epitopes suggests that immune escape is selecting for non-synonymous mutations arising in these regions. Selection analysis and sequencing provided increased support for neutralising antibody (nAb) epitopes compared to others, suggesting that nAbs are an important arm of the immune response to BoCoV. Phylogenetic analysis of spike and HE sequences showed that Irish isolates from this study were in the European clade, except for one HE sequence that sat in the Asian/American clade, while the spike gene of this sample was in the European clade. Recombination between a European and an Asian/American isolate would give rise to such a sequence. This study has gathered evidence suggesting that pressure to evade the nAb response is contributing to BoCoV evolution.

Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl 2 ). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl 2 . The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection 1,2 , yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases., (© 2024. The Author(s).)

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury 1-4 . Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8 + T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Recent studies show an important role for non-neutralizing anti-spike antibodies, including monoclonal antibodies (mAbs), in robustly protecting against SARS-CoV-2 infection. These mAbs use Fc-mediated functions such as complement activation, phagocytosis, and cellular cytotoxicity. There is an untapped potential for using non-neutralizing mAbs in durable antibody treatments; because of their available conserved epitopes, they may not be as sensitive to virus mutations as neutralizing mAbs. Here, we discuss evidence of non-neutralizing mAb-mediated protection against SARS-CoV-2 infection. We explore how non-neutralizing mAb Fc-mediated functions can be enhanced via novel antibody-engineering techniques. Important questions remain to be answered regarding the characteristics of protective non-neutralizing mAbs, including the models and assays used for study, the risks of ensuing detrimental inflammation, as well as the durability and mechanisms of protection., Competing Interests: Declaration of interests A.I. and P.N. have a patent pending for anti-SARS-CoV-2 mAbs., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum's life cycle., Methods: We analysed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Database. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis., Findings: Among the ten antigens analysed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP1 19 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission., Interpretation: These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum. Based on our results, we recommend prioritising conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives., Funding: Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680)., Competing Interests: Declaration of interests All authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

The Duffy-binding protein (DBP) is a promising antigen for a malaria vaccine that would protect against clinical symptoms caused by Plasmodium vivax infection. Region II of DBP (DBP-II) contains the receptor-binding domain that engages host red blood cells, but DBP-II vaccines elicit many non-neutralizing antibodies that bind distal to the receptor-binding surface. Here, we engineered a truncated DBP-II immunogen that focuses the immune response to the receptor-binding surface. This immunogen contains the receptor-binding subdomain S1S2 and lacks the immunodominant subdomain S3. Structure-based computational design of S1S2 identified combinatorial amino acid changes that stabilized the isolated S1S2 without perturbing neutralizing epitopes. This immunogen elicited DBP-II-specific antibodies in immunized mice that were significantly enriched for blocking activity compared to the native DBP-II antigen. This generalizable design process successfully stabilized an integral core fragment of a protein and focused the immune response to desired epitopes to create a promising new antigen for malaria vaccine development., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

To identify the key amino acids (AAs) affecting the allergenicity of hemocyanin (HC) allergens from Chinese mitten crabs, in this study, two epitopes, P1-SHFTGSKSNPEQR and P2-LSPGANTITR were employed and four potential key AAs (P1: F3 and N9 and P2: N6 and R10) were predicted. Mast cell and mouse models revealed that four mutants induced lower levels of immunoglobulin E (IgE) and Th2 type cytokines (15.47-49.89 %), proving that F3, N9, N6, and R10 were the key AAs of two epitopes. Mutants reduce allergic responses via the Th2 pathway. However, the roles of every key AA affecting allergenicity were different (P1-F3 > N9 and P2-N6 > R10). In addition, lower transport and higher efflux were observed in the mutants during transport absorption by Caco-2 cells. The allergenicity of HC was stronger when the transport absorption efficiency of epitopes and mutants was higher and their efflux was lower. Our study provides a novel method for revealing the allergenic molecular mechanisms of food allergens., Competing Interests: Declaration of competing interest The authors attested that there were no conflicts of interest concerning this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rotavirus, a primary contributor to severe cases of infantile gastroenteritis on a global scale, results in significant morbidity and mortality in the under-five population, particularly in middle to low-income countries, including India. WHO-approved live-attenuated vaccines are linked to a heightened susceptibility to intussusception and exhibit low efficacy, primarily attributed to the high genetic diversity of rotavirus, varying over time and across different geographic regions. Herein, molecular data on Indian rotavirus A (RVA) has been reviewed through phylogenetic analysis, revealing G1P[8] to be the prevalent strain of RVA in India. The conserved capsid protein sequences of VP7, VP4 and VP6 were used to examine helper T lymphocyte, cytotoxic T lymphocyte and linear B-cell epitopes. Twenty epitopes were identified after evaluation of factors such as antigenicity, non-allergenicity, non-toxicity, and stability. These epitopes were then interconnected using suitable linkers and an N-terminal beta defensin adjuvant. The in silico designed vaccine exhibited structural stability and interactions with integrins (αvβ3 and αIIbβ3) and toll-like receptors (TLR2 and TLR4) indicated by docking and normal mode analyses. The immune simulation profile of the designed RVA multiepitope vaccine exhibited its potential to trigger humoral as well as cell-mediated immunity, indicating that it is a promising immunogen. These computational findings indicate potential efficacy of the designed vaccine against rotavirus infection., 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 Ltd. All rights reserved.)

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8 + cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C 8 Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C 8 Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C 8 Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17- DFFTAP -22 sequence is important for the recognition by C 8 Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C 8 Mab-2.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains Rotarix TM and RotaTeq TM , multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect., Competing Interests: Declaration of competing interest Author and co-author declare no potential conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Akabane virus (AKAV) is characterized by abortion, stillbirth, premature birth, and congenital deformities in livestock and is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa. Gc protein is the major neutralizing target of AKAV and is often considered as an immunogen to prepare neutralizing antibodies. In this study, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain). Western blot (WB) and indirect immunofluorescence assay (IFA) analysis proved that the mAbs can react with both the truncated recombinant AKAV Gc protein and the natural Gc protein produced in the AKAV-infected cells. Further research demonstrated that these mAbs possess neutralizing activity. We next defined a neutralizing epitope 1134 SVQSFDGKL 1142 by screening a panel of overlapping peptides spanning the truncated Gc protein (aa991∼1232) using the generated neutralizing mAbs. Bioinformatic analysis shows that the neutralizing epitope is highly conserved across different genotypes of AKAV. The newly produced neutralizing mAbs and the identified neutralizing epitope in this study enrich the antigenic epitope information of the AKAV Gc protein and could have potential applications in the development of antigen and antibody detection systems that are specific to AKAV., 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. Published by Elsevier B.V.)

In order to develop a safe and effective broad-spectrum vaccine for foot-and-mouth disease (FMDV), here, we developed a recombinant FMD multiple-epitope trivalent vaccine based on three distinct topotypes of FMDV. Potency of the vaccine was evaluated by immune efficacy in pigs. The results showed that the vaccine with no less than 25 μg of antigen elicited FMDV serotype O specific antibodies and neutralization antibodies by primary-booster regime, and offered immune protection to pigs. More importantly, the vaccine elicited not only the same level of neutralization antibodies against the three distinct topotypes of FMDV, but also provided complete protection in pigs from the three corresponding virus challenge. None of the fully protected pigs were able to generate anti-3ABC antibodies throughout the experiment, which implied the vaccine can offer sterilizing immunity. The vaccine elicited lasting-long high-level antibodies and effectively protected pigs from virulent challenge within six months of immunization. Therefore, we consider that this vaccine may be used in the future for the prevention and control of FMD., 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 Inc. All rights reserved.)

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa is associated with different infections such as urinary tract infections (UTIs). The increased antibiotic resistance reaches the need to develop vaccine against the infections. In the present study, bioinformatics approaches were applied to design a novel multi-epitope of PcrV and OmpE from P. aeruginosa . The raised antibody against the multi-epitope was evaluated and challenge experiment was done to evaluate the efficacy of the multi-epitope. The results of epitope mapping of B-cells indicated 8 regions for PcrV and OmpE. The predicted 3D structure showed C-score = -1 and Z-score = -8.12. Molecular docking indicated high interaction between residues of Toll-like receptor 2 (TLR2) and TLR4 with the multi-epitope. The results of in silico simulation of the immune responses showed elevated levels of B-cell, T-cell, and memory cells. PcrV, OmpE, and the multi-epitope were expressed in pET28a- E. coli BL21 (DE3) and purified by Nickel columns. Our findings indicated that the sera collected from immunized rabbits with the multi-epitope reacted with the multi-epitope, PcrV, and OmpE in western blot. According to the ELISA results, the antibody developed against the multi-epitope showed cross-reactivity with individual proteins PcrV and OmpE. The level of antibody raised against the multi-epitope was significantly higher than the antibody reacted with PcrV or OmpE alone in ELISA. The challenge results confirmed that the load of bacteria was decreased in immunized rabbits as compared to the control. The results present the multi-epitope composed of PcrV and OmpE as a promising candidate against P. aeruginosa . Further evaluations are under investigation in animal model.Communicated by Ramaswamy H. Sarma.

Wilms' tumor (WT1), a transcription factor highly expressed in various leukemias and solid tumors, is a highly specific intracellular tumor antigen, requiring presentation through complexation with HLA-restricted peptides.. WT1-derived epitopes are able to assemble with MHC-I and thereby be recognized by T cell receptors (TCR). Identification of new targetable epitopes derived from WT1 on solid tumors is a challenge, but meaningful for the development of therapeutics that could in this way target intracellular oncogenic proteins. In this study, we developed and comprehensively describe methods to validate the formation of the complex of WT1 126-134 and HLA-A2. Subsequently, we developed an antibody fragment able to recognize the extracellular complex on the surface of cancer cells. The single chain variable fragment (scFv) of an established TCR-mimic antibody, specifically recognizing the WT1-derived peptide presented by the HLA-A2 complex, was expressed, purified, and functionally validated using a T2 cell antigen presentation model. Furthermore, we evaluated the potential of the WT1-derived peptide as a targetable extracellular antigen in multiple solid tumor cell lines. Our study describes methodology for the evaluation of WT1-derived peptides as tumor-specific antigen on solid tumors, and may facilitate the selection of potential candidates for future immunotherapy targeting WT1 epitopes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sandro Matosevic reports financial support was provided by National Institutes of Health. 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 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Chlamydiosis is a widespread ailment affecting humans, livestock, and wildlife, caused by C. abortus, a member of the Chlamydia genus. This disease leads to reproductive disorders in bovines and poses a zoonotic risk, resulting in adverse outcomes such as abortion, stillbirths, weak offspring, endometritis, repeat breeding, and perinatal mortality. However, current chlamydiosis vaccines have limitations in terms of safety, efficacy, and stability, necessitating the development of effective and safe alternatives. In this study, our objective was to design a multi-epitope vaccine (MEV) targeting all strains of C. abortus using bioinformatics and immunoinformatics approaches. We identified highly antigenic and non-allergic proteins (yidC, yajC, secY, CAB503, and CAB746) using VaxiJen and AlgPred tools. Physicochemical analyses and secondary structure predictions confirmed protein stability through ProtParam and SOPMA methods. Furthermore, we employed IEDB-AR, NETMHCpan, and ToxinPred2 tools to predict cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes, resulting in the identification of conserved epitopes for further analysis. The MEV construct, consisting of 545 amino acids, incorporated the adjuvant Beta defensin-3, along with 9 CTL epitopes and 21 HTL epitopes linked by EAAAK, KK, and AAY linkers. We assessed the safety and immunogenicity of the vaccine through comprehensive evaluations of antigenicity, toxicity, allergenicity, and physicochemical properties. Structural stability and quality were examined using 3D modeling via the ab initio approach with the Robetta platform. Molecular docking analysis explored the compatibility of the MEV with Toll-like receptor 9 (TLR9) using ClusPro, while molecular dynamics simulation with the DESMOND Maestro software predicted the stability and flexibility of the docked complex. Despite promising in silico findings, further wet lab investigations are crucial to validate the safety and efficacy of the MEV. Successful development and validation of this MEV hold significant potential in combatting chlamydiosis in both animal and human populations.Communicated by Ramaswamy H. Sarma.

Chikungunya virus (CHIKV), a type A virus borne by mosquitoes that can cause major clinical manifestations including rash, fever and debilitating arthritis, grown into a reemerging serious public health issue. Currently, there is no licensed therapy or vaccine available for CHIKV, although the most promising form of treatment appears to be immunotherapy. Neutralizing antibodies for CHIKV can provide high protection for all CHIKV strains, as well as other alphaviruses. Development of a protective vaccine may be an effective strategy to prevent the outbreak of CHIKV and provide protection for travelers. In this study, we designed a multi-epitope vaccine with a 543-amino-acid structure based on the E1, E2 and capsid proteins of CHIKV, including 6 CTL epitopes, 6 HTL epitopes, 12 linear B epitopes, along with the adjuvant β-defensin III. All T-cell epitopes were docked with their corresponding MHC alleles to validate their effect on inducing immune responses, and the vaccine's sequence was proven to have acceptable physicochemical properties. Further, the developed vaccine was docked with TLR3 and TLR8, both of which play an important role in recognizing RNA viruses. Basic analyses of the docked complexes and molecular dynamic simulations revealed that the vaccine interacted strongly with TLRs. Immunological simulations indicated that the vaccine could induce both cellular and humoral immunity. Hopefully, this proposed vaccine structure can serve as a viable candidate against CHIKV infection.Communicated by Ramaswamy H. Sarma.

A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines., (© 2024. The Author(s).)

Background: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire., Objective: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2., Methods: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis., Results: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants., Conclusions: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics., Competing Interests: Disclosure statement Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) (award nos. R01AI077653-13 [to A.P., M.D.C., and M.C.] and R01AI155668 and R21AI123307 [to S.A.S.]). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The research was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES102906 [to G.A.M.]). The structural results shown in this report are derived from data collected at Southeast Regional Collaborative Access Team (SER-CAT; 22ID) beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract nos. DE-AC02-06CH11357 and W-31-109-Eng-38). This work was partially supported by an ASPIRE III grant from the Office of the Vice President of Research at the University of South Carolina. This research is funded by the above mentioned NIH/NIAID award to InBio. Disclosure of potential conflict of interest: A. Pomés is an employee of InBio; and is the contact principal investigator of the NIH R01 award that provided funding for the study. M.D. Chapman is an employee of InBio; has a financial interest in InBio; and is a co-investigator on the NIH R01 award. A. Ball is an employee of InBio. The hIgE mAbs and some of the allergens described herein were produced by InBio. InBio has a license agreement with the VUMC for commercialization of hIgE mAbs for research and diagnostic purposes. The hIgE mAbs covered by this agreement are available from InBio (www.inbio.com). S.A. Smith is an inventor on US patent 10908168-B2 for generation of hIgE mAbs; has received patent royalties; and has related patents pending. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Hepatitis E virus (HEV) is a foodborne virus transmitted through the faecal-oral route that causes viral hepatitis in humans worldwide. Ever since its discovery as a zoonotic agent, HEV was isolated from several species with an expanding range of hosts. HEV possesses several features of other RNA viruses but also has certain HEV-specific traits that make its viral-host interactions inimitable. HEV leads to severe morbidity and mortality in immunocompromised people and pregnant women across the world. The situation in underdeveloped countries is even more alarming. Even after creating a menace across the world, we still lack an effective vaccine against HEV. Till date, there is only one licensed vaccine for HEV available only in China. The development of an anti-HEV vaccine that can reduce HEV-induced morbidity and mortality is required. Live attenuated and killed vaccines against HEV are not accessible due to the lack of a tolerant cell culture system, slow viral replication kinetics and varying growth conditions. Thus, the main focus for anti-HEV vaccine development is now on the molecular approaches. In the current study, we have designed a multi-epitope vaccine against HEV through a reverse vaccinology approach. For the first time, we have used viral ORF3, capsid protein and polyprotein altogether for epitope prediction. These are crucial for viral replication and persistence and are major vaccine targets against HEV. The proposed in silico vaccine construct comprises of highly immunogenic and antigenic T-cell and B-cell epitopes of HEV proteins. The construct is capable of inducing an effective and long-lasting host immune response as evident from the simulation results. In addition, the construct is stable, non-allergic and antigenic for the host. Altogether, our findings suggest that the in silico vaccine construct may be useful as a vaccine candidate for preventing HEV infections., (© 2024 John Wiley & Sons Ltd.)