Epitope Mapping Research Articles

Unveiling the functional epitopes of cobra venom cytotoxin by immunoinformatics and epitope-omic analyses

Approximate 70% of cobra venom is composed of cytotoxin (CTX), which is responsible for the dermonecrotic symptoms of cobra envenomation. However, CTX is generally low in immunogenicity, and the antivenom is ineffective in attenuating its in vivo toxicity. Furthermore, little is known about its epitope properties for empirical antivenom therapy. This study aimed to determine the epitope sequences of CTX using the immunoinformatic analyses and epitope-omics profiling. A conserved CTX was used in this study to determine its T-cell and B-cell epitope sequences using immunoinformatic tools and molecular docking simulation with different Human Leukocyte Antigens (HLAs). The potential T-cell and B-cell epitopes were 'KLVPLFY,' 'CPAGKNLCY,' 'MFMVSTPTK,' and 'DVCPKNSLL.' Molecular docking simulations disclosed that the HLA-B62 supertype exhibited the greatest binding affinity towards cobra venom cytotoxin. The namely L7, G18, K19, N20, M25, K33, V43, C44, K46, N47, and S48 of CTX exhibited prominent intermolecular interactions with HLA-B62. The multi-enzymatic-limited-digestion/liquid chromatography-mass spectrometry (MELD/LC–MS) also revealed three potential epitope sequences as 'LVPLFYK,' 'MFMVS,' and ‘TVPVKR’. From different epitope mapping approaches, we concluded four potential epitope sites of CTX as ‘KLVPLFYK’, ‘AGKNL’, ‘MFMVSTPKVPV’ and ‘DVCPKNSLL’. Site-directed mutagenesis of these epitopes confirmed their locations at the functional loops of CTX. These epitope sequences are crucial to CTX’s structural folding and cytotoxicity. The results concluded the epitopes that resided within the functional loops constituted potential targets to fabricate synthetic epitopes for CTX-targeted antivenom production.

Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus (ASFV) is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects. To date, no licensed prophylactic vaccine exists. Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens. As such, there is a considerable requirement to understand the functional monoclonal antibodies (mAbs) and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines, therapeutics, and diagnostics. In this study, we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs, further screened a specific ASFV major capsid protein (p72) single-chain antibody and fused with an IgG Fc fragment (scFv-83-Fc), which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain. Using the scFv-83-Fc mAb, we selected a conserved epitope peptide (221MTGYKH226) of p72 retrieved from a phage-displayed random peptide library. Moreover, flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs, which indicated its potential application in vaccine and diagnostic reagent development. Overall, this study provided a valuable platform for identifying targets for ASFV vaccine development, as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

Identification of vaccine candidates against rhodococcus equi by combining pangenome analysis with a reverse vaccinology approach

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that can cause life-threatening infections. The rapid evolution of multidrug-resistant R. equi and the fact that there is no currently licensed effective vaccine against R. equi warrant the need for vaccine development. Reverse vaccinology (RV), which involves screening a pathogen’s entire genome and proteome using various web-based prediction tools, is considered one of the most effective approaches for identifying vaccine candidates. Here, we performed a pangenome analysis to determine the core proteins of R. equi. We then used the RV approach to examine the subcellular localization, host and gut flora homology, antigenicity, transmembrane helices, physicochemical properties, and immunogenicity of the core proteins to select potential vaccine candidates. The vaccine candidates were then subjected to epitope mapping to predict the exposed antigenic epitopes that possess the ability to bind with major histocompatibility complex I/II (MHC I/II) molecules. These vaccine candidates and epitopes will form a library of elements for the development of a polyvalent or universal vaccine against R. equi. Sixteen R. equi complete proteomes were found to contain 6,238 protein families, and the core proteins consisted of 3,969 protein families (∼63.63% of the pangenome), reflecting a low degree of intraspecies genomic variability. From the pool of core proteins, 483 nonhost homologous membrane and extracellular proteins were screened, and 12 vaccine candidates were finally identified according to their antigenicity, physicochemical properties and other factors. These included four cell wall/membrane/envelope biogenesis proteins; four amino acid transport and metabolism proteins; one cell cycle control, cell division and chromosome partitioning protein; one carbohydrate transport and metabolism protein; one secondary metabolite biosynthesis, transport and catabolism protein; and one defense mechanism protein. All 12 vaccine candidates have an experimentally validated 3D structure available in the protein data bank (PDB). Epitope mapping of the candidates showed that 16 MHC I epitopes and 13 MHC II epitopes with the strongest immunogenicity were exposed on the protein surface, indicating that they could be used to develop a polypeptide vaccine. Thus, we utilized an analytical strategy that combines pangenome analysis and RV to generate a peptide antigen library that simplifies the development of multivalent or universal vaccines against R. equi and can be applied to the development of other vaccines.

Epitope mapping of Acinetobacter baumannii outer membrane protein W (OmpW) and laboratory study of an OmpW-derivative peptide

Outer membrane protein W (OmpW) is a less-known A. baumannii antigen with potential immunogenic properties. The epitopes of this protein are not well-identified yet. Therefore, in the present study, B- and T-cell epitopes of A. baumannii OmpW were found using comprehensive in silico and partially in vitro studies. The T-cell (both class-I and class-II) and B-cell (both linear and conformational) epitopes were predicted and screened through many bioinformatics approaches including the prediction of IFN-γ production, immunogenicity, toxicity, allergenicity, human similarity, and clustering. A single 15-mer epitopic peptide containing a linear B-cell and both classes of T-cell epitopes were found and used for further assays. For in vitro assays, patient- and healthy control-derived peripheral blood mononuclear cells were stimulated with the 15-mer peptide, Phytohemagglutinin, or medium alone, and cell proliferation and IFN-γ production assays were performed. The bioinformatics studies led to mapping OmpW epitopes and introducing a 15-mer peptide. In vitro assays to some extent showed its potency in cell proliferation but not in IFN-γ induction, although the responses were not very expressive and faced some questions/limitations. In general, in the current study, we mapped the most immunogenic epitopes of OmpW that may be used for future studies and also assayed one of these epitopes in vitro, which was shown to have an immunogenicity potential. However, the induced immune responses were not strong which suggests that the present peptide needs a series of biotechnological manipulations to be used as a potential vaccine candidate. More studies in this field are recommended.

DEVELOPMENT OF A SELECTIVE CD16A-BASED NK CELL ENGAGER UTILIZING ANTIBODIES TARGETING A SINGLE AMINO ACID VARIATION

Abstract Background and Significance Natural killer (NK) cells play a vital role in the human innate immune system and are being explored as a promising approach for cancer immunotherapy. Of particular interest are NK cell engagers that can target and activate NK cells to attack cancer cells. In this study, we developed novel NK cell engagers by targeting the NK cell activating receptor CD16a using antibodies that selectively distinguish between CD16a on NK cells and CD16b on granulocytes, which are highly homologous to each other. Methods and Results To generate antibodies with high developability, we employed a rational design approach to construct large yeast display libraries of human antibodies. This approach was based on the analysis of a deep sequencing dataset of human antibodies from over 500 individuals, which allowed us to determine the natural amino acid usage patterns of human antibody CDRs and mimic human antibody repertoires. Through screening these libraries, we discovered two classes of antibody clones that selectively recognize CD16a without cross-reactivity to CD16b. Epitope mapping revealed that a single amino acid difference confers over 10,000-fold selectivity for one class of antibody clones, while for the other class a second unique epitope on CD16a was identified. To evaluate the activity of these antibody clones, we produced bispecific antibody clones with one arm targeting CD16a and the other arm targeting a tumor-associated antigen (TAA). Our results demonstrated potent tumor cell-dependent activation of NK cells and effective killing of tumor cells. Several of these antibodies had greatly enhanced resistance to human IgG inhibition in killing target cells. Significantly, our anti-CD16a antibody clones exhibited superior performance compared to leading reference anti-CD16a clones in two distinct NK cell engager formats. This included higher affinity for CD16a, higher thermostability, and more potent killing activity both in the absence and presence of 10 mg/mL human IgGs as competitors. Conclusion Our findings indicate that anti-CD16a antibody-based NK cell engagers have significant potential for cancer immunotherapies.

Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein-Ligand Interactions from a Single NMR Sample.

We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (∼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.

Development of a multi-epitope vaccine candidate against Pseudomonas aeruginosa causing urinary tract infection and evaluation of its immunoreactivity in a rabbit model

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

Anti-nanodisc antibodies specifically capture nanodiscs and facilitate molecular interaction kinetics studies for membrane protein

Nanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. A nanodisc is designed as a vehicle for membrane proteins that provide a native-like phospholipid environment and better thermostability in a detergent-free buffer. This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix-4 structure of MSP. Further, we performed kinetics binding analysis between adenosine A2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs.

Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Structure of a transmission blocking antibody in complex with Outer surface protein A from the Lyme disease spirochete, Borreliella burgdorferi.

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA β-strands 19, 20, and 21, and the loops between β-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines.

Soluble prefusion-closed HIV-envelope trimers with glycan-covered bases

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N-linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers-even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response.

Selection for immune evasion in SARS-CoV-2 revealed by high-resolution epitope mapping and sequence analysis

Here, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses. Applying a high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses allowed identification of B cell epitopes and relate them to their evolutionary and structural properties. We identify hotspots of pre-existing immunity and identify cross-reactive epitopes that contribute to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves, and SARS-CoV-2 variants. Lastly, we show that mutations in spike and nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts.

Linear epitope identification of monoclonal antibodies against the duck Tembusu virus NS1

Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition.

A survey of ORF8 sequence and immunoinformatics features during alpha, delta, and wild type peaks of the SARS-CoV-2 pandemic in Iran.

The Coronavirus disease 2019 (COVID-19) pandemic influences all around the world. The SARS-CoV-2 ORF8 accessory gene represents multiple functions in virus-host interaction. The current study aimed to compare the ORF8 substitutions and epitope features of these substitutions in the various SARS-CoV-2 outbreaks including delta, alpha, and wild type variants in Iran from 2020 to 2022. In addition, we evaluate B cell, HLA I and II epitopes, by in-silico approach to ORF8 binding site prediction. The samples were collected from patients diagnosed with SARS-CoV-2 infection via a real-time PCR assay. Then, a conventional PCR was carried out for ORF8 mutations analysis and further Sanger sequencing. Possible important alterations in epitope features of the ORF8 were evaluated by epitope mapping. B cell, HLA class I and II epitopes, evaluated by online databases ABCpred, NetMHCpan-4.1, and NetMHCIIpan-3.2, respectively. The current study results could not represent novel variations in seven full-length ORF8 sequences or major ORF8 deletions in 80 evaluated samples. In addition, we could not find any ORF8 A382 during each outbreak of variants. Epitope mapping represents differences between the Alpha and other variants, especially in B cell potential epitopes and HLA I. The immunoinformatic evaluation of ORF8 suggested epitopes represent major differences for the Alpha variant in comparison with other variants. In addition, having mild pathogenesis of the Omicron variant does not seem to be associated with ORF8 alteration by phylogenetic evaluation. Future in-vitro studies for a clear conclusion about the epitope features of ORF8 are required.

Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an N-glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibited robust tolerance to various buffer conditions and was employed in a column format that can be readily adapted into a typical HDX-MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.

Molecular Simulation of B-Cell Epitope Mapping from Nipah Virus Attachment Protein to Construct Peptide-Based Vaccine Candidate: A Reverse Vaccinology Approach

Molecular Simulation of B-Cell Epitope Mapping from Nipah Virus Attachment Protein to Construct Peptide-Based Vaccine Candidate: A Reverse Vaccinology Approach

Identification of novel B cell epitopes in Fiber-2 protein of duck adenovirus 3 and their application

Duck adenovirus 3 (DAdV-3), a newly emerged duck adenovirus, has resulted in significant economic losses to the duck industry across China since 2014. However, little is known about the B cell epitopes in major antigen of DAdV-3 and the serological approach for detection of DAdV-3 is not available. In this study, four monoclonal antibodies (mAbs) specific to Fiber-2 protein of DAdV-3 were first generated and designated as 2G10, 3D9, 5E6, and 6B12. Indirect immunofluorescence assay (IFA) showed that all of the mAbs reacted with the Fiber-2. Moreover, mAbs 2G10, 5E6, and 6B12 demonstrated good activity with Fiber-2 in Western blot. Notably, the Fiber-2 could be immunoprecipitated efficiently by mAb 3D9. Epitope mapping revealed that mAbs 2G10, 3D9, 5E6, and 6B12 recognized 397-429aa, 463-481aa, 67-99aa, and 1-66aa of Fiber-2, respectively. Besides, a novel sandwich ELISA for efficient detection of DAdV-3 was developed based on mAb 3D9 and horseradish peroxidase (HRP) conjugated mAb 3D9. The sandwich ELISA only reacted with DAdV-3 but not with other duck-associated viruses. The limit of detection of the ELISA was 6.25 × 103 TCID50/mL. Overall, the mAbs generated laid the foundation for elucidating the critical role of Fiber-2 in mediating infection and pathogenesis, and the sandwich ELISA approach established here provided efficient and rapid serological diagnostic tool for DAdV-3.

Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C44Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3-10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3-10 and HSC-3 was 1.6 × 10-7 M and 1.7 × 10-7 M, respectively. Furthermore, C44Mab-18 detected CD44v3-10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

Analysis of Adeno-Associated Virus Serotype 8 (AAV8)-antibody complexes using epitope mapping by molecular imprinting leads to the identification of Fab peptides that potentially evade AAV8 neutralisation

Gene therapy is a promising approach for treating genetic disorders by delivering therapeutic genes to replace or correct malfunctioning genes. However, the introduced gene therapy vector can trigger an immune response, leading to reduced efficacy and potential harm to the patient. To improve the efficiency and safety of gene therapy, preventing the immune response to the vector is crucial. This can be achieved through the use of immunosuppressive drugs, vector engineering to evade the immune system, or delivery methods that bypass the immune system altogether. By reducing the immune response, gene therapy can deliver therapeutic genes more effectively and potentially cure genetic diseases. In this study, a novel molecular imprinting technique, combined with mass-spectrometry and bioinformatics, was used to identify four antigen-binding fragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodies capable of binding to AAV. The identified Fab peptides were shown to prevent AAV8's binding to antibodies, demonstrating their potential to improve gene therapy efficiency by preventing the immune response.

Epitope mapping and a cocktail of monoclonal antibodies to achieve full detection coverage of potato virus Y.

Epitope mapping and a cocktail of monoclonal antibodies to achieve full detection coverage of potato virus Y.