Main Epitope Research Articles

Amino Acid Substitutions in the Fusion Protein of Respiratory Syncytial Virus in Fukushima, Japan, During 2008–2023 and Their Effects

Abstract Background Amino acid (AA) substitutions in the fusion (F) protein of respiratory syncytial virus (RSV) and their effects on antibody susceptibility remain unclear. We analyzed AA substitutions in the main neutralizing epitopes of the F protein. Methods We analyzed F protein genes of 236 RSV strains isolated from children hospitalized with RSV infection in Fukushima, Japan (June 2008–February 2023). AA substitutions in antigenic sites II, V, and Ø were detected, and their effects on antibody susceptibility and viral replication were evaluated. Results Site II: The K272M (RSV-A) and the K272E (RSV-B) substitutions in strains from palivizumab-treated children reduced antibody susceptibility. Site V: In RSV-A, >50% of strains isolated since 2022 harbored the V178I substitution; however, this did not change antibody susceptibility. In RSV-B, L172Q/S173L mutant strains became predominant around 2016, leading to reduced antibody susceptibility. Site Ø: No AA substitutions were detected in RSV-A. In RSV-B, the I206M/Q209R mutant strain became predominant around 2018, leading to improved antibody susceptibility and replicative ability. However, none of the substitutions reduced antibody susceptibility. Conclusions The RSV F protein in Fukushima has naturally undergone AA substitutions with corresponding changes in antibody susceptibility, including unique regional patterns. Monitoring substitutions and antibody susceptibility is essential.

Mutations in the main antigenic sites of VP7 and VP8* from G3P[8] rotavirus a strains circulating in Brazil may impact immune evasion to rotavirus vaccination.

In the post-rotavirus (RVA) vaccination era, uncommon and zoonotic strains have emerged as causative agents of acute gastroenteritis in humans, including the equine-like G3P[8] strains. First identified in 2013, this strain has quickly spread worldwide, reaching the position of the most prevalent genotype in many countries, including Brazil. Here, we report full genotype characterization and phylogenetic analysis of two equine-like G3P[8] strains detected in Goiás, a state in the Cerrado biome of the Brazilian Midwestern region, during the year of 2019. The strains were detected in different socioeconomic and demographic contexts: GO-MR from an asymptomatic adult living in a rural traditional community and GO-H5 from a symptomatic child from the state capital, with access to safe drinking water and essential sanitation services. These strains also displayed different backbone constellations considering the NSP2 gene segment (G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for GO-MR and G3-P[8]-I2-R2-C2-M2-A2-N1-T2-E2-H2 for GO-H5). Furthermore, significant mutations in the main epitope sites of the VP7 and VP8* proteins of the detected strains, and other Brazilian G3P[8] viruses, were found with the comparison to RV1 and RV5 vaccine proteins, indicating a potential ability of these viruses to evade vaccine protection, which may contribute to their prevalence both nationally and globally. In summary, this study corroborates the genetic diversity of equine-like G3P[8] DS-1-like strains circulating worldwide, highlights the epidemiological importance of adults as reservoirs of RVA and shows the substantial differences between these emerging strains and the currently used anti-RVA vaccines, which may partially explain their predominance due to potential evasion of vaccine-induced protection.

Prevalent and persistent new-onset autoantibodies in mild to severe COVID-19

Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients at five time points over a 16-month period in 2020 and 2021 using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months. We found an increased prevalence of new-onset autoantibodies after severe COVID-19 and demonstrated associations between distinct new-onset autoantibodies and neuropsychiatric symptoms post-COVID-19. Using epitope mapping, we determined the main epitopes of selected new-onset autoantibodies, validated them in independent cohorts of neuro-COVID and pre-pandemic healthy controls, and identified sequence similarities suggestive of molecular mimicry between main epitopes and the conserved fusion peptide of the SARS-CoV-2 Spike glycoprotein. Our work describes the complexity and dynamics of the autoantibody repertoire emerging with COVID-19 and supports the need for continued analysis of the new-onset autoantibody repertoire to elucidate the mechanisms of the post-COVID-19 condition.

Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides.

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Рекомбинантный гликопротеин вируса бешенства (rRVG) для серологической диагностики

This paper presents the results of developing a method for obtaining a recombinant rabies virus glycoprotein (RVG) for serological diagnostics. We performed a bioinformatics analysis of the genomes of rabies virus strains and epizootic isolates, determined the main immunogenic epitopes and other structures that affect the choice of glycoengineering strategy for protein expression. Recombinant DNA was constructed based on fragments of the rabies virus G gene. Based on the expression strain E. coli BL21(DE3), a producer strain was obtained that allows expressing a mature recombinant product with a molecular weight of ≈ 28 kDa. The purified protein had pronounced antigenic activity against specific antisera. The presented data open the possibility of using coliexpression to produce a recombinant analogue of glycoprotein G of the rabies virus. The resulting RVG is also of interest as the basis of a candidate recombinant rabies vaccine.

Effect of Antigen Valency on Autoreactive B-Cell Targeting.

Many autoimmune diseases are characterized by B cells that mistakenly recognize autoantigens and produce antibodies toward self-proteins. Current therapies aim to suppress the immune system, which is associated with adverse effects. An attractive and more specific approach is to target the autoreactive B cells selectively through their unique B-cell receptor (BCR) using an autoantigen coupled to an effector molecule able to modulate the B-cell activity. The cellular response upon antigen binding, such as receptor internalization, impacts the choice of effector molecule. In this study, we systematically investigated how a panel of well-defined mono-, di-, tetra-, and octavalent peptide antigens affects the binding, activation, and internalization of the BCR. To test our constructs, we used a B-cell line expressing a BCR against citrullinated antigens, the main autoimmune epitope in rheumatoid arthritis. We found that the dimeric antigen construct has superior targeting properties compared to those of its monomeric and multimeric counterparts, indicating that it can serve as a basis for future antigen-specific targeting studies for the treatment of RA.

Ferret model to mimic the sequential exposure of humans to historical H3N2 influenza viruses.

Mutations accumulate in influenza A virus proteins, especially in the main epitopes on the virus surface glycoprotein hemagglutinin (HA). For influenza A(H3N2) viruses, in particular, the antigenicity of their HA has altered since their emergence in 1968, requiring changes of vaccine strains every few years. Most adults have been exposed to several antigenically divergent H3N2 viruses through infection and/or vaccination, and those exposures affect the immune responses of those individuals. However, animal models reflecting this 'immune history' in humans are lacking and naïve animals are generally used for vaccination and virus challenge studies. Here, we describe a ferret model to mimic the serial exposure of humans to antigenically different historical H3HA proteins. In this model, ferrets were sequentially immunized with adjuvanted recombinant H3HA proteins from two or three different H3HA antigenic clusters in chronological order, and serum neutralizing antibody titers were examined against the homologous virus and viruses from different antigenic clusters. For ferrets immunized with a single HA antigen, serum neutralizing antibody titers were elevated specifically against the homologous virus. However, after immunization with the second or third antigenically distinct HA antigen in chronological order, the ferrets showed an increase in more broadly cross-reactive neutralizing titers against the antigenically distinct viruses and against the homologous virus. Sequentially immunized animals challenged with an antigenically advanced H3N2 virus showed attenuated virus growth and less body temperature increase compared with naïve animals. These results suggest that sequential exposure to antigenically different HAs elicits broader neutralizing activity in sera and enhances immune responses against more antigenically distinct viruses Our findings may partly explain why adults who have been exposed to antigenically divergent HAs are less likely to be infected with influenza virus and have severe symptoms than children.

Characteristics and Functions of HEV Proteins.

Hepatitis E virus (HEV) is a non-enveloped virus containing a single-stranded, positive-sense RNA genome of 7.2kb, which consists of a 5' non-coding region, three open reading frames (ORFs), and a 3' non-coding region. ORF1 is diverse between genotypes and encodes the nonstructural proteins, which include the enzymes needed for virus replication. In addition to its role in virus replication, the function of ORF1 is relevant to viral adaption in culture and may also relate to virus infection and HEV pathogenicity. ORF2 protein is the capsid protein, which is about 660 amino acids in length. It not only protects the integrity of the viral genome, but is also involved in many important physiological activities, such as virus assembly, infection, host interaction, and innate immune response. The main immune epitopes, especially neutralizing epitopes, are located on ORF2 protein, which is a candidate antigen for vaccine development. ORF3 protein is a phosphoprotein of 113 or 114 amino acids with a molecular weight of 13kDa with multiple functions that can also induce strong immune reactivity. A novel ORF4 has been identified only in genotype 1 HEV and its translation promotes viral replication.

Phosphorylcholine Monoclonal Antibody Therapy Decreases Intraplaque Angiogenesis and Intraplaque Hemorrhage in Murine Vein Grafts.

Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease.

HK is the apple of FXI's eye

HK is the apple of FXI's eye

Discovery of Chemokine CXCL12 Inhibitors by Tandem Application of Virtual Screening and NMR Spectrometry.

The CXC chemokine ligand CXCL12 and its receptor CXCR4 play critical roles in stem-cell homing, infectious diseases, and cancer, which led the CXCL12/CXCR4 signaling axis to attract much attention in drug discovery. CXCR4 is regarded as the primary target while CXCL12 is considered too small to be a druggable target. In this paper, we employed virtual screening approaches and ligand-based NMR screening methods from a SPECS library and in-house natural products to discover new CXCR12 inhibitors. Four natural triterpene saponins were confirmed, and the triterpene sapogenin was identified as the main binding epitope by saturation transfer difference-nuclear magnetic resonance and molecular docking studies. The pentacyclic triterpene scaffold and its elucidated structure-activity relationships provide a new and valuable research direction for the development of novel CXCL12 inhibitors.

Rational designing of peptide-ligand conjugates-based immunotherapy for the treatment of complicated malaria

AimsMalaria deaths occur primarily due to complicated malaria associated with the sequestration of Plasmodium falciparum-infected erythrocyte (PfIE) in the capillary microvasculature. This study aims to design peptide ligand conjugates (PLCs) for treating complicated malaria using various in silico techniques. The PLC includes a natural ligand for the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1): expressed explicitly on the surface of PfIE, and a highly immunogenic peptide derived from the commonly used peptide vaccines in malaria-endemic countries. The ligand is predicted to prevent the sequestration of PfIE, and the peptide is predicted to eliminate PfIE from circulation by the pre-existing vaccine-induced immunity. Main methodsThe epitope identification from the vaccines and the analysis of physicochemical properties, antigenicity, allergenicity, and toxicity were performed using SVMTriP, ProtParam, VaxiJen, AllerTop, and ToxinPred servers, respectively. The high throughput virtual screening (HTVS) and drug-like properties analysis of natural compound ligands were carried out by Schrodinger-2021 software. The molecular dynamics simulations were performed through the WebGro server. Key findingsHTVS revealed three bioactive natural ligands for PfEMP1 from (NPASS) database. Three super immunogenic peptides were identified from malaria-endemic countries' commonly used peptide vaccines. Finally, Nine PLCs were designed with different combinations of peptides and ligands with the suitable non-cleavable triazole linker. SignificanceAntimalarials have been losing efficacy in a time when malaria deaths in 2020 significantly increased than in 2019. In this scenario, further research on the designed PLCs may offer some innovative immune therapeutics for complicated malaria with minimum possibilities of drug resistance.

Genetic deletions and high diversity of Plasmodium falciparum histidine-rich proteins 2 and 3 genes in parasite populations in Ghana.

Rapid diagnostic tests (RDTs) are used to diagnose malaria in Ghana and other malaria endemic countries. Plasmodium falciparum histidine-rich protein 2 (PFHRP2) based RDTs are widely used, however the occurrence of deletions of the pfhrp2 gene in some parasites have resulted in false negative test results. Monoclonal antibodies of PFHRP2 cross reacts with PFHRP3 because they share structural similarities and this complements the detection of the parasites by RDT. These two genes were investigated in Ghanaian P. falciparum parasite population to detect deletions and the polymorphisms in exon 2 of the pfhrp2 and pfhrp3 genes. Parasite isolates (2,540) from children ≤ 12 years with uncomplicated malaria from 2015 to 2020 transmission seasons were used. Both genes were amplified using nested PCR and negative results indicated the presence of the deletion of genes. Amplified genes were sequenced for the detection of the amino acid repeats. Deletions were observed in 30.7% (780/2,540) and 17.2% (438/2,540) of the samples for pfhrp2 and pfhrp3 respectively with increasing trends over the three time periods (χ2 -10.305, p = 0.001). A total of 1,632 amplicons were sequenced for each gene, analysis was done on 1,124 and 1,307 good quality sequences for pfhrp2 and pfhrp3 respectively. Pfhrp2 repeat polymorphisms were dominantly of types 2 (AHHAHHAAD) and 7 (AHHAAD) with large numbers of variants. A novel variant of type 14 (AHHANHATD) was seen for pfhrp2. For the pfhrp3 repeat types, 16 (AHHAAN), 17 (AHHDG) and 18 (AHHDD) were the dominant types observed. Variants of type 16 (AHHAAH) and (AHHASH) were also dominant. Repeat types 1, 2, 3, 4, 5, 6, 7, 8, 11, 13, 15, 16, and 19 were observed be shared by both genes. The haplotype diversity of both genes ranged between 0.872 and 1 indicating high diversity of the polymorphisms in the isolates. The implication of the findings of the frequencies of the pfhrp2 and pfhrp3 deletions as well as the variants of the main epitopes of the monoclonal antibodies for the RDT (types 2 and 7) in our isolates is an indication of decreased sensitivity of the RDTs in diagnosing malaria infections in Ghana.

Autoanticorps anti ARN polymérase III, antigènes cibles et méthodes d’identification

AbstractRNA polymerases are nuclear enzymes essential for the transcription of RNAs. They are composed of multiple subunits, sometime shared between the three RNA polymerases or exhibiting antigenic homologies. Anti-RNA polymerase autoantibodies were described in the early 1980s, in patients suffering from various connective tissue diseases. In the mid-1990s, anti-RNA polymerase III antibodies (ARA) were demonstrated as specifically associated with diffuse systemic sclerosis (dSSc). Presence of ARA is associated with a rapid and severe evolution of dSSc associating particular complication risks, among which, hypertensive renal crisis of poor prognosis. ARA identification is then particularly useful for the management of patients suffering of SSc, particularly at the onset of the disease, justifying of specific medical care. However, ARA screening might only be engaged on specific medical prescription because fluorescent aspects on HEp2 cells are not sufficiently evocative and screening ENA tests do not associate this specificity. Elisa and related tests use the main antigenic epitope RPC155 of the RNA polymerase III. The immunodots combine epitopes derived from the RPC11 subunit of RNA polymerase III, with RPC155. The interest of combining these 2 antigenic subunits in the immunodot approach will be discussed.

Characterization of a new NSP2-deletion NADC34-Like Porcine Reproductive and Respiratory Syndrome Virus in China

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS), which has caused huge economic losses to the pig industry worldwide. PRRSV NADC34-Like PRRSV 2020-Acheng-1 strain, which caused high morbidity and high mortality were isolated from dead piglets (high-throughput sequencing to show that only PRRSV and TGEV) on a farm in northeastern China. The full-length genome sequence of 2020-Acheng-1 shares 95.6% nucleotide homology with NADC34 PRRSV without any gene insertion, but has a unique 17 amino acid (469aa to 486aa) deletion in Nsp2 compared with all NADC34-Like strains in NCBI and there are unique 100 amino acid deletions. In addition, difference degree of changes in signal peptide, trans-membrane region (TM), main neutralizing epitope (PNE), non-neutralizing epitope and N-glycosylation site were observed in GP5 of 2020-Acheng-1 and other PRRSV-2 strains, we only found a change in the fifteenth amino acid of signal peptide of in GP5 of 2020-Acheng-1 with NADC34 strains. Recombination analysis showed that 2020-Acheng-1 strain did not have any recombination events with representative PRRSV-2 strains in China. This study provided valuable evidence for understanding the role of NADC34-Like strain that impact on pathogenicity.

Structural Dynamics of the SARS-CoV-2 Spike Protein: A 2-Year Retrospective Analysis of SARS-CoV-2 Variants (from Alpha to Omicron) Reveals an Early Divergence between Conserved and Variable Epitopes.

We analyzed the epitope evolution of the spike protein in 1,860,489 SARS-CoV-2 genomes. The structural dynamics of these epitopes was determined by molecular modeling approaches. The D614G mutation, selected in the first months of the pandemic, is still present in currently circulating SARS-CoV-2 strains. This mutation facilitates the conformational change leading to the demasking of the ACE2 binding domain. D614G also abrogated the binding of facilitating antibodies to a linear epitope common to SARS-CoV-1 and SARS-CoV-2. The main neutralizing epitope of the N-terminal domain (NTD) of the spike protein showed extensive structural variability in SARS-CoV-2 variants, especially Delta and Omicron. This epitope is located on the flat surface of the NTD, a large electropositive area which binds to electronegatively charged lipid rafts of host cells. A facilitating epitope located on the lower part of the NTD appeared to be highly conserved among most SARS-CoV-2 variants, which may represent a risk of antibody-dependent enhancement (ADE). Overall, this retrospective analysis revealed an early divergence between conserved (facilitating) and variable (neutralizing) epitopes of the spike protein. These data aid in the designing of new antiviral strategies that could help to control COVID-19 infection by mimicking neutralizing antibodies or by blocking facilitating antibodies.

Omicron variant receptor-binding domain phylogenetics and molecular dynamics

BackgroundWe investigated the evolutionary relationships, mutations, antigenic epitopes, and structural dynamics of the receptor-binding domain (RBD) of SARS-CoV-2, Omicron and other recently evolved variants. MethodsThe RBD of SARS-CoV-2 and its Omicron, Alpha, Beta, Gamma, Delta, and Mu variants were subjected to pairwise sequence matrix evaluation, antigenic epitope prediction, and phylogenetic relationship and structural dynamics analyses. ResultsThe Omicron RBD contained 13–15 amino acid mutations, of which 12 were new and three conserved with other variants. In addition, two mutations found in the Alpha, Beta, Gamma, and Mu variants were not found in the Omicron RBD. The ultrametric clustering unweighted pair group method with arithmetic mean identified Omicron as a novel monophyletic class, but the neighbor-joining method clustered Omicron with Alpha and Delta variants. In the SARS-CoV-2 RBD, five main antigenic epitopes were predicted, and these epitopes were conserved across all SARS-CoV-2 variants tested. Surprisingly, the additional mutations in the Omicron variant increased the size of the expected antigenic sites in two of these antigenic epitopes. Molecular dynamics (MD) simulations revealed higher root-mean-square deviation in the Omicron RBD, greater residue fluctuation at residues 32–42 and 140–160, and increased solvent-accessible surface area. ConclusionsThe Omicron RBD mutations indicate the variant is within a new phylogenetic class of SARS-CoV-2 and significantly impact RBD structure, conformation, and molecular dynamics. However, conserved anticipated antigenic sites may imply partial changes in receptor affinity and response to immune reactions. Omicron RBD binding with the angiotensin-converting enzyme 2 receptor was suggested to be weaker than the original SARS-CoV-2 binding in MD simulations.

Screening and identification of B cell epitope of the nucleocapsid protein in SARS-CoV-2 using the monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease (COVID-19). It is confirmed that nucleocapsid (N) protein is closely related to viral pathogenesis, modulation of host immune response, RNA transcription, and replication and virus packaging. Therefore, the N protein is a preponderant antigen target for virus detection. The codon-optimized N gene was designed according to the encoding characteristics of insect cells and inserted into pFastBacTM1 vector with 6 × His-tag-fused N protein for expression in insect sf21 cells. Six anti–N mAbs (4G3, 5B3, 12B6, 18C7-A2, 21H10-A3, 21H10-E9) were prepared by recombinant N protein. The mAbs showed high titers, antibody affinity, and reactivity with the SARS-CoV-2 N protein. Then, fourteen overlapped peptides that covered the intact N protein were synthesized (N1-N14). Peptide N14 was identified as the main linear B-cell epitope region via peptide-ELISA and dot-blot assay, and this region was truncated gradually until mapping the peptide 401-DFSKQLQQ-408. Simultaneously, compared with the sequence of variants of concern (VOCs) and variants of interest (VOIs) strains among the several countries, epitope 401-DFSKQLQQ-408 is very conservative among them. The findings provide new guidance for the design and detection of COVID-19 targets.Key points• The N protein was optimized according to the insect cell codon preference and was highly expressed.• The monoclonal antibodies prepared in this study were shown high antibody titers and high affinity.• Monoclonal antibodies were used to map the epitope 401–408 amino acids of N protein for the first time in this study.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11769-6.

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms.

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.

Epitope Mapping of an Anti-CD20 Monoclonal Antibody (C20Mab-60) Using the HisMAP Method.

CD20 is one of the B-lymphocyte antigens and an effective target for the detection and treatment of B cell lymphomas; specific and sensitive monoclonal antibodies (mAbs) are required thus for their diagnosis. Recently, we developed a novel anti-CD20 mAb (clone C20Mab-60), which is not only useful for flow cytometry but also for Western blot and immunohistochemical analyses. However, the epitope of C20Mab-60 has not been determined. To clarify the binding region of mAbs against their target molecules, it is essential to understand the pharmacological function of each mAb. In this study, we aimed to identify the epitope of C20Mab-60 for CD20 using the novel histidine tag (His-tag) insertion for epitope mapping (HisMAP) method. We first established an anti-His-tag mAb, HisMab-1 (mouse IgG2b, kappa), by immunizing mice with recombinant proteins containing an N-terminal His-tag. Although HisMab-1 detected the 4x, 5x, and 6xHis tag-inserted CD20 proteins using flow cytometry, 5xHis tag was selected. While HisMab-1 recognized all the 5xHis tag-inserted CD20 from the 142nd to the 183rd amino acid (aa), C20Mab-60 did not react with the 5xHis tag-inserted CD20 from the 171st to the 174th aa. These results indicate that the main epitope of C20Mab-60 for CD20 is a peptide from 171st to 174th aa of CD20. HisMAP method could be advantageous in the determination of the critical epitope of functional mAbs against many target molecules.