Specific Epitopes Research Articles

Immunization Effects of a Novel α-Synuclein-Based Peptide Epitope Vaccine in Parkinson's Disease-Associated Pathology.

Parkinson's disease (PD) is a chronic neurodegenerative disease that affects the central nervous system, specifically the motor system. It is mainly caused by the loss of dopamine due to the accumulation of α-synuclein (α-syn) protein in the striatum and substantia nigra pars compacta (SNpc). Previous studies have reported that immunization may be a potential preventive strategy for neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Therefore, the aim of the study was to design an α-syn specific epitope vaccine and investigate its effect in PD-related pathophysiology using an α-syn-induced mouse model. We used an in silico model to identify and design a non-toxic α-syn-based peptide epitope vaccine and, to overcome poor immunogenicity, the vaccine was coupled with immunogenic carrier proteins, i.e., ovalbumin (OVA) and keyhole limpet haemocyanin (KLH). Our results showed that vaccinated PD mouse models, especially with vaccines with carrier proteins, improved in motor functions compared with the non-vaccinated PD model. Additionally, the vaccinated groups showed increased immunoglobulin G (IgG) levels in the spleen and plasma as well as decreased interleukin-10 (IL-10) levels in the plasma. Furthermore, vaccinated groups, especially OVA and KLH groups, showed decrease in α-syn levels and increased dopamine-related markers, i.e., tyrosine hydroxylase (TH), vesicle monoamine transporter 2 (VMAT2), and dopamine transporter (DAT), and autophagy activities in the striatum and SNpc. Lastly, our data showed decreased neuroinflammation by reducing the activation of microglia and astrocytes and pro-inflammatory cytokines in the immunized groups, especially with OVA and KLH carrier proteins. Overall, these results suggest that vaccination, especially with immunogenic carrier proteins, is effective in reducing the accumulation of α-syn aggregates in the brain and ameliorate PD-related pathophysiology. Hence, further development of this approach might have a potential role in preventing the development of PD.

Assessment of some immunological markers in patients and vaccinated individuals with COVID-19 in Kirkuk city

Abstract Background: Infection with coronavirus disease-2019 (COVID-19) can trigger both innate and adaptive immune responses, resulting in large inflammatory reactions later in the disease. The initiation of immunological responses entails a complicated interaction between innate immune components, which quickly respond in a nonspecific manner, and specialized components of the immune system can recognize specific epitopes of antigens. Objective: The objective of this study was to assess some co-stimulating molecules in patients with COVID-19 (hospitalized and nonhospitalized) and vaccinated individuals compared with a control group in Kirkuk city. Materials and Methods: The immunological markers under study in which our methods tried to estimate them are CD28, CD80, and CD86. From 90 individuals of patients with COVID-19, vaccinated persons, and control group blood samples were collected and centrifuged to get the serum to carry out the immunological analysis. Through using nasopharyngeal swabs that were collected from non-hospitalized patients (patients out of the hospital), coronavirus infection was confirmed by polymerase chain reaction (PCR). Additionally, PCR tests were run on the control group to make sure they were not infected with COVID-19. Results: For the vaccinated group especially in comparison to COVID-19 patients, the revealed significant differences in the immunological markers among tested groups with respect to the CD28 test with (P value > 0.0001) and CD80 test with (P value > 0.0001), as well as the CD86 test appears to show a significant difference with (P value > 0.0001). Conclusion: This study revealed that, compared to patients with COVID-19 who were not given the vaccine, the vaccine had a role on those who received it and significantly increased some immunological markers..

Routes to standardisation for tau protein measurement in CSF

AbstractBackgroundCSF biomarkers are the target of several commercially available in vitro diagnostic (IVD) kits allowing an early diagnosis of Alzheimer’s disease (AD) and eventually the monitoring of treatment effects in clinical trials. Tau hyper‐phosphorylation is one of the main hallmarks of AD and its measurement provides higher accuracy to stratify patients at an early‐stage.Due to the rapid advancements in proteomics, standardisation of measurements can be achieved through the development of reference measurement procedures (RMPs) and certified reference materials (CRMs).MethodSI‐traceable protein and peptide primary calibrators were developed for t‐tau and p‐tau: mass fraction was determined by amino acid analysis and purity was assessed by LC‐MS at high resolution. CSF pools at different tau concentration were used for the development and validation of LC‐IDMS candidate RMP and their commutability was assessed.ResultAn LC‐IDMS candidate reference method was develop and validated in CSF for t‐tau using a SI‐traceable candidate protein primary calibrator. Three different CSF pools (low, medium, high) were successfully quantified with a relative expanded uncertainty below 10%. The commutability of 13 matrix‐based CRMs was assessed involving 8 immunoassays from 5 IVD providers by measuring 40 single CSF donations.According to the AT(N) classification, high levels of p‐tau(181) are highly correlated to tau pathology and commercially available immunoassays are able to target this specific epitope. We conducted a feasibility study on the development of a candidate reference method for p‐tau(181) in CSF relying on a SI‐traceable peptide primary calibrator and an antibody‐free sample preparation workflow.New assays targeting p‐tau(217) in CSF and plasma were also developed, making it necessary the organisation of standardisation initiatives. We sourced and characterised multiple phosphopeptide primary calibrators as well as a GSK3beta‐phosphorylated protein primary calibrator for the development of an LC‐MS RMP including an immunoprecipitation step in the sample preparation workflow.Conclusiontau is considered as a priority for the IFCC WG on NDD and the development of SI traceable RMPs and CRMs is essential to underpin a worldwide standardisation of results. This will help IVD providers to address the traceability requirements of the IVDR thus facilitating regulatory approvals of the kits.

Cryptic-site-specific antibodies to the SARS-CoV-2 receptor binding domain can retain functional binding affinity to spike variants.

Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

Isatuximab Plus Pomalidomide and Dexamethasone in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM) in Real-Life Context in France: IMAGE Subgroup Analysis Based on Prior Lines of Therapy and Refractory Status in Anti-CD38-Naïve Population

Introduction: Isatuximab, a CD38 monoclonal antibody (mAb), targets a specific CD38 epitope to induce myeloma cell death. Based on the results of the Phase 3 ICARIA-MM study, isatuximab (Isa) plus pomalidomide (P) and dexamethasone (d; Isa-Pd) is approved in adult patients (pts) with relapsed/refractory multiple myeloma (RRMM) who have received ≥2 prior therapies. Prior to Isa-Pd regulatory approval, Isa was available in France under early access programs (EAPs). IMAGE is a non-interventional, retrospective cohort study of pts treated with Isa-Pd under the French EAPs. In the IMAGE overall effectiveness population (median follow-up 14.2 months [mo]), median progression-free survival (mPFS) was 12.4 mo; overall response rate (ORR) was 46.3%; 27.9% of pts achieved a very good partial response (VGPR) and best response was VGPR for 60.3% of pts. In the current myeloma landscape, not all pts with RRMM have received a prior anti-CD38 mAb, particularly where access is difficult; thus, the real-world effectiveness of Isa in daratumumab (dara)-naïve pts needs to be assessed in light of the Phase 3 APOLLO and ICARIA-MM trials investigating dara-Pd and Isa-Pd in dara-naïve populations. Effectiveness results from the IMAGE subgroup analysis of dara-exposed and -refractory pts have been presented previously. Here, we describe results for dara-naïve pts based on prior lines of therapy (LOT) and lenalidomide (len)-refractory status. Methods: Data were collected from medical records of adult pts with RRMM who received ≥1 dose of Isa under the EAPs from 29 Jul 2019-1 Sep 2020. The effectiveness analysis was restricted to pts with ≥1 year of follow-up after Isa initiation. PFS was defined as the time from Isa-Pd initiation to date of disease progression or death. VGPR was defined as a ≥90% reduction in serum or urine M-protein, or in the difference between involved and non-involved free light chain. The description of adverse events (AEs; verbatim) was recorded as a reason for treatment temporary or permanent discontinuation or dose reduction. Data were recorded per the subject's medical file. No specific grading was performed. Results: The safety population included 299 pts who received ≥1 Isa dose under the EAPs; of these, 294 received ≥1 Isa dose and met all inclusion/exclusion criteria, thus comprising the effectiveness population. Within the overall effectiveness population, 216 (73.5%) pts were dara-naïve; among them, 13.4%, 58.8%, and 27.8% received 1, 2, and ≥3 prior LOT, respectively; 72.2% were len-refractory (median 2 prior LOT); and 54.2% were len-refractory at last prior LOT. At a median follow-up of 14.2 mo, mPFS (95% CI) in dara-naïve pts was not reached (NR; 14.6-NR), 15.0 mo (10.6-NR), and 14.8 mo (7.6-NR) with 1, 2, and ≥3 prior LOT, respectively; 13.2 mo (9.0-18.6) in those refractory to len; and 13.2 (9.0-NR) in those refractory to len at last prior LOT. ORR (of which, VGPR) was 41.4% (17.2%), 52.0% (34.6%), and 50.0% (31.7%) in pts with 1, 2, and ≥3 prior LOT, respectively; 49.4% (29.5%) in len-refractory pts; and 50.4% (29.9%) in len-refractory at last prior LOT pts (Table). At least 1 AE was reported in 53 (24.4%) pts in the dara-naïve safety population (n=217): 7 (24.1%), 32 (25.2%), and 14 (23.0%) in the 1, 2, and ≥3 prior LOT subgroups, respectively; 41 (26.1%) in the len-refractory subgroup; and 32 (27.4%) in the len-refractory at last prior LOT subgroup. AEs led to permanent Isa discontinuation in 3 (2.4%) pts with 2 prior LOT; 3 (1.9%) len-refractory pts; and 2 (1.7%) len-refractory at last prior LOT pts. The most common AEs were blood and lymphatic system disorders, occurring in 39 (18.0%) pts. Infections and infestations were reported in 2 (0.9%) pts: 1 had received 1 prior LOT and 1 had 2 prior LOT; both pts were len-refractory at last prior LOT. Conclusions: In the dara-naïve populations from APOLLO (dara-Pd) and ICARIA-MM (Isa-Pd), mPFS was 12.4 and 11.1 mo, respectively. This subgroup analysis of IMAGE provides real-world evidence of Isa effectiveness in dara-naïve subgroups: Isa-Pd demonstrated a high mPFS of 16.6 mo in dara-naïve pts and meaningful mPFS at first and subsequent relapse, including in difficult-to-treat len-refractory pts; mPFS was generally comparable to that of the overall effectiveness population. No new safety signals were observed compared with ICARIA-MM. However, the incidence of some AEs like infections was low, possibly due to the retrospective nature of the study. Funding: Sanofi.

Domain Specificity of Anti-Prothrombin Antibodies That Express Lupus Anticoagulant Activity

Background: The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the persistent presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies directed against negatively charged phospholipids or phospholipid-binding proteins. Some of these autoantibodies exert lupus anticoagulant (LAC) activity, a laboratory phenomenon characterized by the prolongation of phospholipid-dependent coagulation tests. Recently, our group identified that LAC-positive anti-prothrombin antibodies activate platelets via the platelet receptor, FcγRIIA. However, a lot is still unclear regarding the mechanism by which these antibodies cause LAC and why some, but not all, antibodies in this heterogeneous group express LAC activity. Furthermore, experimental data on the binding site of these pathogenic anti-prothrombin antibodies are lacking. Aims: In this study we aimed to investigate the binding domain of LAC-positive monoclonal anti-prothrombin antibodies and APS patient derived anti-prothrombin antibodies. Method: Recombinant fragments of prothrombin were designed and produced in Chinese hamster ovary cells. Prothrombin fragment 1, fragment 2 and fragment 1.2 were coated on ELISA plates to study epitope specificity. Two LAC-positive monoclonal anti-prothrombin antibodies (3B1 and 28F4), two LAC-negative anti-prothrombin monoclonal antibodies (11H2 and 9C6), isolated anti-prothrombin antibodies derived from APS patients and plasma from APS patients were studied. A modified dilute Russell's viper venom time (DRVVT) test was used to evaluate whether the recombinant prothrombin constructs could neutralize the effect of LAC-positive monoclonal and patient derived anti-prothrombin antibodies. Furthermore, a fragment 1.2 nanobody was produced by immunizing Llama's followed by phage display. The ability of this nanobody to compete with LAC-positive monoclonal and patient derived anti-prothrombin antibodies for the prothrombin binding site was tested using a modified DRVVT test. Results: LAC-positive monoclonal anti-prothrombin antibodies recognized prothrombin fragment 2. Interestingly, LAC-negative anti-prothrombin antibodies bound exclusively to prothrombin fragment 1. In addition, LAC-ratios of patient samples correlated significantly with anti-fragment 2 antibody levels ( p<0.05, ρ=0.33), but not with anti-fragment 1 antibody levels. Furthermore, incubation with the prothrombin F1.2 nanobody almost completely reversed the prolonged DRVVT clotting time in plasma containing monoclonal and patient derived LAC-positive anti-prothrombin antibodies, but not in plasma containing LAC-positive anti-β2GP1 antibodies. This neutralising effect was also observed when the monoclonal anti-prothrombin antibody was incubated with prothrombin fragment 2. In contrast, the LAC-activity of anti-prothrombin antibodies was not inhibited when incubated with prothrombin fragment 1. Conclusion: Our data show that LAC-positive anti-prothrombin antibodies react with fragment 2 of prothrombin. Furthermore, a novel nanobody directed against prothrombin fragment 1.2 and prothrombin fragment 2 were able to neutralize a prothrombin-dependent LAC.

Prevalence, Domain Specificity, and Residue Specificity of IgG, IgM, and IgA Anti-β2GPI Antibodies in Tetra-Positive Antiphospholipid Syndrome Patients: Identification of Two Non-Overlapping Epitopes in Domain I of β2GPI

Background. Risk stratification of antiphospholipid syndrome (APS) patients involves several laboratory tests, including lupus anticoagulant (LAC), anti-cardiolipin antibodies (anti-CL), anti-β2GPI antibodies (anti-β2GPI), and, more recently, anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT). Patients with positive results for all four tests, known as tetra-positive APS patients, are considered at high risk for thrombotic recurrence. β2GPI is a protein composed of five domains: DI, DII, DIII, DIV, and DV, arranged in a flexible elongated structure. Previous studies have demonstrated that DI is frequently targeted by IgG in thrombotic APS patients. However, the prevalence, domain specificity, and residue specificity of the three different isotypes of anti-β2GPI antibodies in tetra-positive APS patients remain unknown. Objective. To establish the prevalence, domain specificity, and residue specificity of IgG, IgM, and IgA anti-β2GPI antibodies in tetra-positive APS patients. Methods. Plasma samples from 32 clinically confirmed tetra-positive APS patients and 8 healthy controls were analyzed for levels of IgG, IgM, and IgA using an enzyme-linked immunosorbent assay (ELISA) with immobilized β2GPI as the antigen. Domain and residue mapping were investigated using five deleted domain variants and eight single-point mutations. Mutants lacking specific disulfide bonds were used to assess whether the epitopes targeted by the antibodies are linear or conformational. Results. Among the 32 patients tested, 31 (96%) had IgG, 18 (56%) had IgM, and 18 (56%) had IgA anti-β2GPI antibodies. Ten patients (31%) had all three isotypes, 7 (22%) had IgG and IgM, and 8 (25%) had IgG and IgA. Six patients (19%) had only IgG, 1 had only IgM, and none had only IgA. The removal of DI from the β2GPI molecule led to an almost complete loss of binding (~80%) for the majority of IgG antibodies. There was a lesser but still significant reduction in binding when DII and DV were removed. However, no loss of binding was observed with variants lacking DIII and DIV. Notably, IgA antibodies behaved similarly to IgG but differently from IgM, as their reactivity towards DI and DV was significantly attenuated. To investigate the contribution of specific residues to binding, charged residues K19, E23, E26, E27, R39, R43, K44, and K59 in DI were individually replaced with alanine (A). The most important residue for IgG and IgA antibodies was R43, followed by R39 and K44. In contrast, most residues contributed to optimal binding for IgM antibodies, suggesting a less specific epitope definition. Interestingly, the mutations R39A and K44A often led to loss of binding in conjunction with the mutation R43A, but rarely simultaneously, suggesting the existence of two distinct epitopes for anti-DI antibodies-one located north and one south of R43. Finally, IgG, IgM, and IgA antibodies did not react against the mutant C32S/C60S but showed good reactivity against the mutant C288S/C326S, indicating that epitopes in DI are conformational and require the integrity of the disulfide bond 32-60. Conclusions. In tetra-positive APS patients, IgG anti-β2GPI antibodies are the most prevalent isotype, followed by IgA and IgM. The IgG and IgA isotypes exhibit a pronounced preference for DI, while the IgM isotype demonstrates lower specificity, which aligns with IgM's role as initial responders in infections. Notably, our research revealed that anti-DI antibodies are diverse, as they recognize epitopes within DI that are closely related but physically distinct. Ongoing studies aim to further characterize these antibody subtypes and understand their significance in APS. This knowledge may inform improved diagnostic and therapeutic approaches for APS patients.

Mapping IgA Epitope and Cross-Reactivity between Severe Acute Respiratory Syndrome-Associated Coronavirus 2 and DENV.

The newly introduced COVID-19 vaccines have reduced disease severity and hospitalizations. However, they do not significantly prevent infection or transmission. In the same context, measuring IgM and IgG antibody levels is important, but it does not provide information about the status of the mucosal immune response. This article describes a comprehensive mapping of IgA epitopes of the S protein, its cross-reactivity, and the development of an ELISA-peptide assay. IgA epitope mapping was conducted using SPOT synthesis and sera from RT-qPCR COVID-19-positive patients. Specific and cross-reacting epitopes were identified, and an evolutionary analysis from the early Wuhan strain to the Omicron variant was performed using bioinformatics tools and a microarray of peptides. The selected epitopes were chemically synthesized and evaluated using ELISA-IgA. A total of 40 IgA epitopes were identified with 23 in S1 and 17 in the S2 subunit. Among these, at least 23 epitopes showed cross-reactivity with DENV and other organisms and 24 showed cross-reactivity with other associated coronaviruses. Three MAP4 polypeptides were validated by ELISA, demonstrating a sensitivity of 90-99.96% and a specificity of 100%. Among the six IgA-RBD epitopes, only the SC/18 epitope of the Omicron variants (BA.2 and BA.2.12.1) presented a single IgA epitope. This research unveiled the IgA epitome of the S protein and identified many epitopes that exhibit cross-reactivity with DENV and other coronaviruses. The S protein of variants from Wuhan to Omicron retains many conserved IgA epitopes except for one epitope (#SCov/18). The cross-reactivity with DENV suggests limitations in using the whole S protein or the S1/S2/RBD segment for IgA serological diagnostic tests for COVID-19. The expression of these identified specific epitopes as diagnostic biomarkers could facilitate monitoring mucosal immunity to COVID-19, potentially leading to more accurate diagnoses and alternative mucosal vaccines.

T cells specific to multiple Bet v 1 peptides are highly cross-reactive toward the corresponding peptides from the homologous group of tree pollens.

Allergens from Fagales trees frequently cause spring allergy in Europe, North America, and some parts of Asia. The definition of the birch homologous group, which includes birch (Bet v), oak (Que a), alder (Aln g), hazel (Cor a), hornbeam (Car b), beech (Fag s), and chestnut (Cas s), is based on high allergen sequence identity and extensive IgE cross-reactivity. Clinical effect was seen during the alder/hazel, birch, and oak pollen seasons after treatment with tree SLIT-tablets containing only birch allergen extract. Here, we characterize T-cell reactivity with respect to epitope specificities and cross-reactivity toward various Bet v 1 family members, (PR-10/group 1 major allergens). This cross-reactivity may be part of the immunological basis of clinical effect or cross-protection when exposed to birch homologous tree species. T-cell lines were generated from 29 birch-allergic individuals through stimulation of peripheral blood mononuclear cells (PBMCs) with birch/Bet v or oak/Que a allergen extracts. T-cell responses to allergen extracts, purified group 1 allergens, and overlapping 20-mer peptides (Bet v 1, Aln g 1, Cor a 1, and Que a 1) were investigated by T-cell proliferation and cytokine production. Cross-reactivity was evaluated based on Pearson's correlations of response strength and further investigated by flow cytometry using tetramer staining for homologous peptide pairs. T-cell reactivity toward extracts and group 1 allergens from across the birch homologous group was observed for birch/Bet v as well as oak/Que a T-cell lines. T-cell lines responded to multiple Bet v 1 homologous peptides from Aln g 1 and Cor a 1 and a subset of Que a 1 peptides. Significant Pearson's correlations between frequently recognized peptides derived from Bet v 1 and the corresponding peptides derived from alder, hazel, and oak strongly supported the T-cell cross-reactivity toward these allergens. Cross-reactivity between birch and birch homologous peptides was confirmed by pMHCII tetramer staining. T cells from birch tree pollen allergic individuals respond to multiple trees within the birch homologous group in accordance with the level of sequence homology between Bet v 1 family members, (PR-10 allergens) from these allergen sources, confirming the basis for clinical cross-protection.

Putative novel outer membrane antigens multi-epitope DNA vaccine candidates identified by Immunoinformatic approaches to control Acinetobacter baumannii

Multi-epitope polypeptide vaccines, a fusion protein, often have a string-of-beads system composed of various specific peptide epitopes, potential adjuvants, and linkers. When choosing the sequence of various segments and linkers, many alternatives are available. These variables can influence the vaccine’s effectiveness through their effects on physicochemical properties and polypeptide tertiary structure.The most conserved antigens were discovered using BLASTn. To forecast the proteins’ subcellular distribution, PSORTb 3.0.2 was used. Vaxign was used for the preliminary screening and antigenicity assessment. Protein solubility was also predicted using the ccSOL omics. Using PRED-TMBB, it was anticipated that the protein would localize across membranes. The IEDB and BepiPred-2.0 databases were used to predict the immunogenicity of B cell epitopes. A multi-epitope construct was developed and analyzed to evaluate. Twenty epitopes from A. baumannii’s outer membrane protein (omp) were included in the vaccination. TLR4 agonist explosibility was investigated. The physicochemical characteristics, secondary and tertiary structures, and B-cell epitopes of vaccine constructs were assessed. Additionally, docking and MD experiments were used to examine the relationship between TLR4 and its agonist.Thirteen antigens were discovered, and eight of the 13 chosen proteins were predicted to be surface proteins. The 34 kDa outer membrane protein, Omp38, Omp W, CarO, putative porin, OmpA, were chosen as having the right antigenicity (≥0.5). FhuE and CdiA were eliminated from further study because of their low antigenicity. The vaccine design was developed by combining the most effective 10 B-cell and 10 MHC-I/MHCII combined coverage epitopes. The molecular formula of the vaccine was determined to be C1718H2615N507O630S17. The vaccine form has a molecular weight of 40,996.70 Da and 47 negatively charged residues (Asp + Glu), whereas 28 positively charged residues (Arg + Lys). The estimated half-life was 7.2 hours (mammalian reticulocytes, in vitro), > 20 hours (yeast, in vivo) and > 10 hours (Escherichia coli, in vivo) for the vaccine. The multi-epitope vaccine insertion is carried via the expression vector pcDNA3.1 (+).The multi-epitope vaccine may stimulate humoral and cellular immune responses, according to our findings, and it may be a candidate for an A. baumannii vaccine.

Deciphering the antigen specificities of antibodies by clustering their complementarity determining region sequences.

Determining antigen and epitope specificity is an essential step in the discovery of therapeutic antibodies as well as in the analysis adaptive immune responses to disease or vaccination. Despite extensive efforts, deciphering antigen specificity solely from BCR amino acid sequence remains a challenging task, requiring a combination of experimental and computational approaches. Here, we describe and experimentally validate a simple and straightforward approach for grouping antibodies that share antigen and epitope specificities based on their CDR sequence similarity. This approach allows us to identify the specificities of a large number of antibodies whose antigen targets are unknown, using a small fraction of antibodies with well-annotated binding specificities.

Novel and unique rheumatoid factors cross-react with viral epitopes in COVID-19

Rheumatoid factors (RFs), polyreactive antibodies canonically known to bind two conformational epitopes of IgG Fc, are a hallmark of rheumatoid arthritis but also can arise in other inflammatory conditions and infections. Also, infections may contribute to the development of rheumatoid arthritis and other autoimmune diseases. Recently, RFs only in rheumatoid arthritis were found to bind novel linear IgG epitopes as well as thousands of other rheumatoid arthritis autoantigens. Specific epitopes recognized by infection-induced polyreactive RFs remain undefined but could provide insights into loss of immune tolerance. Here, we identified novel linear IgG epitopes bound by RFs in COVID-19 but not rheumatoid arthritis or other conditions. The main COVID-19 RF was polyreactive, binding two IgG and multiple viral peptides with a tripeptide motif, as well as IgG Fc and SARS-CoV-2 spike proteins. In contrast, a rheumatoid arthritis-specific RF recognized IgG Fc, but not tripeptide motif-containing peptides or spike. Thus, RFs have disease-specific IgG reactivity and distinct polyreactivities that reflect the broader immune response. Moreover, the polyreactivity of a virus-induced RF appears to be attributable to a very short peptide motif. These findings refine our understanding of RFs and provide new insights into how viral infections may contribute to autoimmunity.

A quantitative comparison of antibodies against epitope tags for immunofluorescence detection.

Epitope tags recognized by specific antibodies have been widely used over the last few decades, notably to localize tagged proteins within cells by immunofluorescence. The diversity of tags and antibodies usually prevents a side-by-side comparison of the efficiency with which each antibody recognizes its cognate tag. We expressed chimeric proteins, each composed of an invariant domain (IL2Ra) associated with a specific epitope tag. Double immunofluorescence allowed us to quantify in parallel the reference signal generated by the anti-IL2Ra antibody and the signal generated by the anti-epitope tag antibody. Since all antibodies used in this study were recombinant antibodies fused to the same mouse Fc domain, the generated signals were directly comparable. Three groups of tags/antibodies were revealed: 'good' antibodies generated high signals even when used at a low concentration (50 ng·mL-1 ), 'fair' antibodies generated a high signal only at high concentrations (5000 ng·mL-1 ), and 'mediocre' antibodies generated positive but weak signals. Except for an anti-myc antibody, similar results were obtained when cells were fixed in paraformaldehyde or methanol. These results provide a side-by-side quantitative evaluation of different tag/antibody pairs. This information will be useful to optimize the choice of epitope tags and to choose optimal antibodies.

Pooled Analysis on Bispecific Antibody-Related Toxicities in Multiple Myeloma

Background: Multiple myeloma (MM) is a hematologic malignancy caused by the uncontrolled proliferation of malignant plasma cells in the bone marrow. Bispecific antibodies (BsAbs) are a new novel class of drugs currently assessed in the setting of relapsed/refractory MM (RRMM), with impressive outcomes. BsAbs are designed to direct against specific antigenic epitopes on the surface of malignant plasma cells, with two main categories: B-cell maturation antigen (BCMA) or other (non-BCMA) antigens which include G-protein coupled receptor family C group 5 member D (GPRC5D) and Fc Receptor-Like 5 (FcRH5). Methods: We conducted a pooled analysis of the available literature on BsAbs reported in the form of abstract or full peer-reviewed publications, published until June 2023, using PubMed, Scopus, Google Scholar and Embase databases. BsAbs were classified into two groups: BCMA and non-BCMA-directed agents. Studies on BsAbs used in combination with other plasma-cell directed agents, and studies reporting outcomes of patients who had prior exposure of other BsAbs were excluded. BsAb included in our study were the following: Teclistamab, Elranatamab, REGN-5458, AMG420, AMG701, CC-93269, TNB-383B, Linvoseltamab, Talquetamab, and Cevostamab. We collected safety data including cytokine release syndrome (CRS), neurotoxicity, infections rates and hematologic toxicities. We used T-test statistics to determine significance (p-value<0.05) and comparisons between the BCMA and non-BCMA groups. Results: A total of 1926 patients with RRMM treated with BsAbs were included in this analysis. Among them, 1288 (66.7%) received a BCMA-directed BsAbs, while 638 (33.1%) received a non-BCMA-directed bsAb. Patient baseline characteristics including prior lines of therapy (LOT), and refractoriness status were comparable between the two groups. The median age of the entire cohort was 63 years and had a median of 5 prior LOT. Median follow-up duration was 5.35 and 5.6 months for the patients treated with BCMA agents and non-BCMA agents, respectively. CRS was reported in 59.6% of all patients, infections in 50%, neutropenia in 41.86%, anemia in 37.32%, and lymphopenia in 36.41%. For grade 3-4 adverse events (AEs), the percentages were as follows: CRS 22.9%, infections 21.46%, neutropenia 38.12%, and anemia 25.37%. Non-BCMA-targeting BsAbs showed no significant difference in all grade and type hematologic toxicities (neutropenia, anemia, lymphopenia, leukopenia, and thrombocytopenia combined) compared to BCMA-targeting BsAbs (p=0.57). However, a significant difference was observed when comparing BCMA-targeting to non-BCMA-targeting BsAbs in terms of combined grade 3-4 hematologic toxicity (p=0.0002). In addition, the group of patients who received BCMA-targeting BsAbs had significantly worse hypogammaglobulinemia compared to patients treated with non-BCMA BsAbs (p=0.006). The overall CRS rate (all grades) was significantly higher in non-BCMA BsAbs (p<0.0001), however, both groups experienced similar grade 3-4 CRS rates. Neurotoxicity rates were also similar between the two groups (p=0.067). Conclusion: The use of BsAbs in MM has demonstrated remarkable efficacy; however, these have been linked to a unique adverse event profile. Our results showed that non-BCMA bsAb were associated less hematotoxicity (combined grade 3-4 events and hypogammaglobulinemia), whereas BCMA bsAb were associated with less CRS rates. This is important information for treatment selection and mitigation strategy development aiming to optimize patient outcomes.

Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes.

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy.

Virus-like particle vaccines with epitopes from porcine epidemic virus and transmissible gastroenteritis virus incorporated into self-assembling ADDomer platform provide clinical immune responses in piglets.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are major intestinal coronaviruses that cause vomiting, diarrhea, dehydration, and mortality in piglets. These viruses coexist and lead to significant economic losses in the swine industry. Virus-like particles (VLPs) have emerged as promising alternatives to conventional inactivated vaccines due to their exceptional safety, efficacy, and ability to provide multi-disease protection with a single dose. Our study focused on specific antigenic epitopes from the PEDV S protein (SS2 and 2C10 regions) and the TGEV S protein (A and D sites) as target candidates. These epitopes were integrated into the ADDomer framework, and we successfully generated recombinant proteins AD, AD-P, AD-T, and AD-PT using the baculovirus expression vector system (BEVS). By meticulously optimizing conditions in High Five cells, we successfully expressed and purified the recombinant proteins. Subsequently, we developed the recombinant ADDomer-VLP vaccine and conducted a comprehensive evaluation of its efficacy in piglets. Following ultrafiltration concentration and sucrose gradient centrifugation purification, the recombinant proteins self-assembled into VLPs as observed by transmission electron microscopy (TEM). Administration of the vaccine did not result in any adverse reactions in the immunized piglets. Additionally, no significant instances of fever were detected in any of the experimental groups, and there were no notable changes in average daily weight gain compared to the control group that received PBS. The recombinant ADDomer-VLP vaccines demonstrated strong immunogenicity, effectively stimulating the production of neutralizing antibodies against both PEDV and TGEV. Moreover, the recombinant ADDomer-VLP vaccine induced elevated levels of IFN-γ, IL-2, and IL-4, and enhanced cytotoxic T lymphocyte (CTL) activity in the peripheral blood of piglets. These recombinant VLPs have demonstrated the ability to induce strong cellular and humoral immune responses in piglets, making them an incredibly promising platform for the rapid and simplified development of epitope vaccines.

Features and protective efficacy of human monoclonal antibodies targeting Mycobacterium tuberculosis arabinomannan.

A better understanding of the epitopes most relevant for antibody-mediated protection against tuberculosis (TB) remains a major knowledge gap. We have shown that human polyclonal IgG to the Mycobacterium tuberculosis (Mtb) surface glycan arabinomannan (AM) and related lipoarabinomannan (LAM) is protective against TB. To investigate the impact of AM epitope recognition and Fc-gamma receptor (FcgR)-binding on antibody functions against Mtb, we isolated a high-affinity human monoclonal antibody (mAb; P1AM25) to AM and show its binding to oligosaccharide (OS) motifs we previously found to be associated with in vitro functions of human polyclonal anti-AM IgG. Human IgG1 P1AM25, but not two other high-affinity human IgG1 anti-AM mAbs reactive with different AM OS motifs, enhanced Mtb phagocytosis by macrophages and reduced intracellular growth in an FcgR-dependent manner. P1AM25 in murine IgG2a, but neither murine IgG1 nor a non-FcgR-binding IgG, given intraperitoneally prior to and after aerosolized Mtb infection was protective in C57BL/6 mice. Moreover, we demonstrate the protective efficacy of human IgG1 P1AM25 in passive transfer with Mtb-infected FcgR-humanized mice. These data enhance our knowledge of the important interplay between both antibody epitope specificity and Fc effector functions in the defense against Mtb and could inform development strategies of vaccines against TB.

Determining epitope specificity of T-cell receptors with transformers.

T-cell receptors (TCRs) on T cells recognize and bind to epitopes presented by the major histocompatibility complex in case of an infection or cancer. However, the high diversity of TCRs, as well as their unique and complex binding mechanisms underlying epitope recognition, make it difficult to predict the binding between TCRs and epitopes. Here, we present the utility of transformers, a deep learning strategy that incorporates an attention mechanism that learns the informative features, and show that these models pre-trained on a large set of protein sequences outperform current strategies. We compared three pre-trained auto-encoder transformer models (ProtBERT, ProtAlbert, and ProtElectra) and one pre-trained auto-regressive transformer model (ProtXLNet) to predict the binding specificity of TCRs to 25 epitopes from the VDJdb database (human and murine). Two additional modifications were performed to incorporate gene usage of the TCRs in the four transformer models. Of all 12 transformer implementations (four models with three different modifications), a modified version of the ProtXLNet model could predict TCR-epitope pairs with the highest accuracy (weighted F1 score 0.55 simultaneously considering all 25 epitopes). The modification included additional features representing the gene names for the TCRs. We also showed that the basic implementation of transformers outperformed the previously available methods, i.e. TCRGP, TCRdist, and DeepTCR, developed for the same biological problem, especially for the hard-to-classify labels. We show that the proficiency of transformers in attention learning can be made operational in a complex biological setting like TCR binding prediction. Further ingenuity in utilizing the full potential of transformers, either through attention head visualization or introducing additional features, can extend T-cell research avenues. Data and code are available on https://github.com/InduKhatri/tcrformer.

Developing a vaccine against velogenic sub-genotype seven of Newcastle disease virus based on virus-like particles

In the present study, for the first time, we released and assembled the particles of three major structural proteins of velogenic NDV (M, HN, and F glycoproteins) as a NDV-VLPs. The ElISA result of the cytokines of splenocyte suspension cells showed that IL2, IL10, TNF-α, and IFN- ˠ titers were significantly higher (p ≤ 0.05) in mice that were immunized only with NDV-VLPs three times with a 10-day interval, in comparison to those that were immunized with NDV-VLPs twice in a 10-day interval and received a B1 live vaccine boost on the third interval. Flow cytometry results showed that CD8 + titers in the group that only received NDV-VLP was higher than other group. However, serum ELISA results did not show a significantly (p ≥ 0.05) higher NDV antibody titer in NDV-VLPs immunized mice compared to the boosted group. Besides, HI results of SPF chickens vaccinated with NDV-VLPs and boosted with B1 live vaccine were significantly (p ≤ 0.05) higher than those that only received NDV-VLPs. Interestingly, after challenging with NDV sub-genotype VII, all the chickens that were solely vaccinated with NDV-VLPs remained alive (six out of six), whereas two out of six chickens that were vaccinated with NDV-VLPs and also received the B1 live vaccine boost died. In conclusion, our results strongly indicated that the T-cell immune response in an NDV host is more important than the B-cell response. Also, the results of the present study revealed that to completely protect chickens against velogenic NDV strains, a vaccine comprising specific epitopes of velogenic strain is needed.

Antibodies elicited by Plasmodium falciparum circumsporozoite proteins lacking sequentially deleted C-terminal amino acids reveal mouse strain and epitopes specific differences

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.