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PurposeMutations in TP53 induce autoantibody immune responses in a subset of cancer patients, which have been proposed as biomarkers for early detection. Here, we investigate the association of p53-specific autoantibodies with multiple tumor subtypes and determine the association with p53 mutation status and epitope specificity.Experimental designIgG p53 autoantibodies (p53-AAb), were quantified in 412 serum samples using a programmable ELISA assay from patients with serous ovarian, pancreatic adenocarcinoma, and breast cancer. To determine if patients generated mutation-specific autoantibodies we designed a panel of the most relevant 51 p53 point mutant proteins, to be displayed on custom programmable protein microarrays. To determine the epitope specificity we displayed 12 overlapping tiling fragments and 38 N- and C-terminal deletions spanning the length of the wild-type p53 protein.ResultsWe detected p53-AAb with sensitivities of 58.8% (ovarian), 22% (pancreatic), 32% (triple negative breast cancer), and 10.2% (HER2+ breast cancer) at 94% specificity. Sera with p53-AAb contained broadly reactive autoantibodies to 51 displayed p53 mutant proteins, demonstrating a polyclonal response to common epitopes. All p53-AAb displayed broad polyclonal immune response to both continuous and discontinuous epitopes at the N- and C-terminus as well as the DNA-binding domain.Conclusion and clinical relevanceIn this comprehensive analysis, mutations in tumor p53 induce strong, polyclonal autoantibodies with broadly reactive epitope specificity.

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

Background: In Burkina Faso, red blood cell (RBC) transfusion remains the crucial anaemia treatment following chronic renal failure (CRF) as erythropoietin and its analogues are unavailable. However, blood group matching beyond the ABO and Rhesus is not common in Burkina Faso. Thus, alloimmunisation is a potential issue for transfused patients. Objective: Our study aimed to identify anti-erythrocyte antibodies in multi-transfused CRF patients at the Yalgado Ouedraogo Teaching Hospital, Ouagadougou, Burkina Faso. Methods: This cross-sectional study, conducted from October 2018 to November 2019, included CRF patients who had received at least two RBC units. We screened patients for the presence of RBC antibodies using three commercial Cells panels and identified antibody specificities for positive screenings using 11 Cells panels for an indirect antiglobulin test (IAT) in a low ionic strength microcolumn gel-card system. Results: Two hundred and thirty-five patients (45.1% female; average age: 41.5 years) were included. The median number of blood units received per patient was 10 (interquartile range: 5–20). The overall alloimmunisation rate was 5.9% (14/235). Antibodies identified included: anti-D (1 case), anti-C (1 case), anti-D+C (4 cases), anti-CW (1 case), anti-E (1 case), anti-S (1 case) and anti-Lea (1 case). In four positive patients, the specificity of the antibodies was indeterminate. No risk factors were associated with alloimmunisation. Conclusion: In Burkina Faso, screening for RBC alloantibodies should be mandated for patients at risk. The high rate of indeterminate antibodies suggests the need to develop a local RBC antibody panel adapted to the local population. [ABSTRACT FROM AUTHOR]

Background and Objectives: Alloimmunization is a very common clinical problem in transfusion-dependent patients but rarely detected in Bangladesh. The risk of alloimmunization is especially high in patients who receive repeated transfusion, i.e., thalassemia patients. The objective of this study was to investigate the seroprevalence and specificity of red blood cell antibodies in multitransfused thalassemic patients. Methods: This cross-sectional study was conducted at a tertiary care referral center from January 1, 2013, to March 31, 2016. Blood specimens were sent from different facilities and also from daycare transfusion center of the same when cross-match difficulties occur. When the Coomb's test showed positive result, then Rhesus, Kell, Kid, Duffy, and Lewis blood group typing were done according to the patients' need. Serum was used to identify the presence of alloantibody by the indirect Coomb's test with known cell panels. An autocontrol, positive and negative control for each specimen was incorporated to detect autoantibody and correctness of the procedure done. We performed a simple percentage to evaluate the analysis. Results: Of 1286 cases, alloantibody was detected in 334 (25.97%) cases. Among the 334 cases, 80 (23.95%) cases were followed up by antibody identification. The remaining 254 cases were referred cases with insufficient history and were dropped from the study. The male-to-female ratio was 1:2.2. The age group of 21–30 years showed the maximum antibody specificity (7.48%). Among the 80 samples with alloantibodies, Rh antibodies were noted in 80% (64) samples. Other antibodies detected belong to Kell, Kid, Duffy, and Lewis systems. Conclusion: Multitransfused (thalassemic) patients are more vulnerable to develop alloantibody. Therefore, routine antibody screening of donors and patients and the provision of antigen negative blood is recommended.

The specificity of antibodies (Ab) is essential for the precise recognition of foreign or dangerous molecules. We have shown that mice infected with non-pathogenic Lactate Dehydrogenase Elevating Virus (LDV) inoculated with human growth hormone (hGH) or Ovalbumin (OVA), exhibit modified specificity of anti-hGH or anti-OVA Ab associated with the secretion of IFN-γ, IL-13, and IL-17. Cytokines are directly or indirectly involved in the isotypes, specificity, and affinity of Ab. Accordingly, here we investigated the effect of IL-17 neutralization on Ab specificities to OVA and Diphtheria Toxoid (DTx) in a mouse model of viral infection. Thereby, we employed an anti-cytokine "auto-vaccination" with an OVA/IL-17A complex or a Monoclonal Ab (MAb) anti-IL-17A (MM17/F3). Competitive ELISA assays were used to estimate the quality of the humoral immune response and the amount of Abs to conformational versus linear antigenic determinants. Results indicated that the OVA/IL-17A complex increased Abs levels to conformational epitopes of OVA, while LDV prolonged antibodies for a longer period. Mice treated with MM17F3 MAb showed an increase in Abs to conformational epitopes of OVA. A similar effect, confirmed by a competitive Western-blot assay, was produced by LDV. Moreover, an increased level of IgM, IgG1, and IgG2a was found in infected animals. Similarly, MAb anti-IL-17A treatment increased the proportion of Ab to conformational epitopes of DTx in uninfected mice, while LDV decreased this parameter. In conclusion, our findings demonstrate a correlation between IL-17A neutralization and a change in Ab specificity to OVA or DTx, presenting a novel strategy for obtaining Abs with higher specificity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Immunoglobulin A (IgA) is notable for its broad specificity toward multiple bacteria. Phosphorylcholine (PC) plays a role in the infection of pathogenic bacteria carrying PC and in the induction of IgA responses in the host immune system. The commercially available mouse monoclonal IgA, TEPC15-IgA, is a distinctive antibody with specificity for PC, warranting further exploration of its response to PC-bearing enteric bacteria. In this study, using 17 different enteric bacteria, including 3 aerobic and 14 anerobic bacteria that could be cultured in vitro, we confirmed that TEPC15-IgA recognizes 4 bacterial species: Lactobacillus taiwanensis, Limosilactobacillus frumenti, Streptococcus infantis, and Escherichia coli, although reactivity varied. Interestingly, TEPC15-IgA did not react with four of six Lactobacillus species used. Moreover, distinct target molecules associated with PC in L. taiwanensis and L. frumenti were evident, differing in molecular weight. These findings suggest that the natural generation of PC-specific IgA could prevent PC-mediated infections and potentially facilitate the formation of a microflora rich in indigenous bacteria with PC, particularly in the gastrointestinal tract.

Objective: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii , and explore its preliminary applications., Methods: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA)., Results: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells., Conclusions: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells 1 . Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies 2 . However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs 2-5 . Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies., (© 2024. The Author(s).)

Constant domains in antibody molecules at the level of the Fab (C H 1 and C L ) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharides from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extended to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by C H 1 domains., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made. [ABSTRACT FROM AUTHOR]

Detection of HLA‐specific memory B cells can provide additional information on sensitization of alloantigen‐exposed individuals and refine immunological risk assessment. We have recently developed an assay enabling profiling of memory B cell‐derived HLA antibodies using luminex single antigen bead (SAB) assay. Here, we compared the performance of the SAB kits from two vendors for memory B cell‐derived HLA antibody detection. IgG was isolated from culture supernatants of polyclonally activated B cells from alloantigen‐exposed (n = 7) or nonexposed (n = 5) individuals, using our previously established method. Eluates containing isolated IgG from culture supernatants were tested for the presence of HLA antibodies using luminex SAB analysis from both One Lambda and Lifecodes (Immucor). In contrast to Lifecodes, high mean fluorescence intensity (MFI) signals were found for negative control beads in One Lambda (median MFI for class I:1730 and for class II:728), accompanied by high MFI values for self HLA‐coated beads, especially for HLA‐C. Despite high background in the One Lambda assays, 91% concordance for HLA class I and 85% concordance for HLA class II were found between the specificities detected using SAB kits from the two vendors. Our results show that HLA‐specific memory B cells can be profiled using kits from both vendors. However, when analyzing One Lambda results one should be aware of the restrictions related to nonspecific binding particularly in HLA‐C‐coated beads, and pay attention to self HLA‐coated beads in order to accurately identify the reactivities leading to the definition of the actual HLA antibody specificities. [ABSTRACT FROM AUTHOR]

The enormous diversity of antibody specificities is generated by random rearrangement of immunoglobulin gene segments and is important for general protection against pathogens. Since random rearrangement harbors the risk of producing self‐destructive antibodies, it is assumed that autoreactive antibody specificities are removed during early B‐cell development leading to a peripheral compartment devoid of autoreactivity. Here, we immunized wild‐type mice with insulin as a common self‐antigen and monitored diabetes symptoms as a measure for autoimmune disease. Our results show that autoreactive anti‐insulin IgM and IgG antibodies associated with autoimmune diabetes can readily be generated in wild‐type animals. Surprisingly, recall immunizations induced increased titers of high‐affinity insulin‐specific IgM, which prevented autoimmune diabetes. We refer to this phenomenon as adaptive tolerance, in which high‐affinity memory IgM prevents autoimmune destruction by competing with self‐destructive antibodies. Together, this study suggests that B‐cell tolerance is not defined by the absolute elimination of autoreactive specificities, as harmful autoantibody responses can be generated in wild‐type animals. In contrast, inducible generation of autoantigen‐specific affinity‐matured IgM acts as a protective mechanism preventing self‐destruction. Synopsis: Autoantibodies that target self‐molecules are involved in autoimmune diseases. Here, a novel class of autoreactive IgM is found to be capable of protecting its cognate autoantigen from degradation. Antibody production depends on the antigen valency regardless of self or foreign.Soluble autoantigens are able to modulate autoantibody responses towards IgM.Affinity‐matured monospecific autoreactive IgM (PR‐IgM) protects its cognate autoantigen from degradation (adaptive tolerance). [ABSTRACT FROM AUTHOR]

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay's specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay's specificity and build confidence in a study's conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study's results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results. [ABSTRACT FROM AUTHOR]

The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self‐reactivity. At the molecular level this is regulated by signaling from membrane Ig and the BAFF‐receptor that sustain a pro‐survival program of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here, we show that the polypyrimidine tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high‐density of binding to 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the maturation of quiescent B cells by regulating gene expression at the posttranscriptional level. [ABSTRACT FROM AUTHOR]

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specificmemory B cells encoding Abs that mediate ADCC froma subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activitywhen compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise fromunique B-cell lineages in the same individual. Further, thesemAbsmediate potent plasma IgG-specificADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual's recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86-0.93), whereas responses to six antigens accurately estimated an individual's malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.

Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bNAbs). Such bNAbs target HIV's trimeric, membrane-embedded envelope glycoprotein spikes (mEnv). Soluble Env (sEnv) trimers have been used as vaccines, but engineering sEnvs for stability, multivalency, and desired antigenicity is problematic and deletes key neutralizing epitopes on glycoprotein 41 (gp41) while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. mEnv trimers were fixed, purified, and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter, 133 nm) in correct orientation. These particles were recognized by HIV bNAbs and not non-NAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provides clues on how NAb responses can be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enables rational immunization strategies to elicit HIV bNAbs using multimerized mEnv. IMPORTANCE A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane-embedded envelope glycoprotein spikes (mEnv) that HIV uses to enter cells. Due to HIV Env's low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties, including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane-embedded targets of therapeutic interest. [ABSTRACT FROM AUTHOR]

Background: Early childhood influenza infections imprint influenza-specific immune memory, with most studies evaluating antibody specificity. In this study, we examined how infection versus inactivated influenza vaccination (IIV) establish pediatric CD4 T-cell mediated immunity to influenza and whether this poises the immune system to respond differently to IIV the following year.Methods: We tracked influenza-specific CD4 T-cell responses in 16 H3N2 infected and 28 IIV immunized children following both initial exposure and after cohorts were revaccinated with IIV the following fall. PBMCs were stimulated with peptide pools encompassing the translated regions of the H3 HA and NP proteins and were then stained to assess CD4 T-cell specificity and function.Results: Compared to IIV, infection primed a greater magnitude CD4 T-cell response specific for the infecting HA and NP proteins, with more robust NP-specific immunity persisting through year 2. Post infection, CD4 T cells preferentially produced combinations of cytokines that included interferon-γ. Interestingly, age-specific patterns in CD4 T-cell reactivity demonstrated the impact of multiple influenza exposures over time.Conclusions: These data indicate that infection and vaccination differentially prime influenza-specific CD4 T-cell responses in early childhood, with these differences contributing to the lasting immunologic imprinting established following early influenza infection.Clinical Trials Registration: NCT02559505. [ABSTRACT FROM AUTHOR]

Objectives: Current inactivated influenza vaccines provide suboptimal protection against antigenic drift, and repeated annual vaccinations shape antibody specificity but the effect on protection from infection is not well understood. Methods: We studied the effects of cumulative and staggered vaccinations in mice to determine the effect of influenza vaccination on protection from infection and immune quality. Results: We found that the timing of vaccination and antigenic change impacted the quality of immune responses. When mice received two different H3N2 strains (A/Hong Kong/4801/2014 and A/Singapore/INFIMH‐16‐0019/2016) by staggered timing of vaccination, there were higher H3HA antibody and B‐cell memory responses than four cumulative vaccinations or when two vaccinations were successive. Interestingly, after challenge with a lethal‐drifted H3N2 virus (A/Hong Kong/1/1968), mice with staggered vaccination were unable to produce high titres of antibodies specific to the challenge strain compared to other vaccination regimens because of high levels of vaccine‐specific cross‐reactive antibodies. All vaccination regimens resulted in protection, in terms of viral loads and survival, from lethal challenge, while lung IL‐6 and inflammation were lowest in staggered or cumulative vaccination groups, indicating further advantage. Conclusion: Our findings help justify influenza vaccination policies that currently recommend repeat vaccination in infants and annual seasonal vaccination, with no evidence for impaired immunity by repeated seasonal vaccination. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "The impact of ageing on the distribution of preformed anti-HLA and anti-MICA antibody specificities in recipients from eastern China prior to initial HSCT." The correction updates the article title to "The impact of ageing on the distribution of preformed anti-HLA and anti-MICA antibody specificities in recipients from eastern China prior to initial HSCT." The original article has been updated accordingly. The correction notice also includes the names of the authors involved in the study. [Extracted from the article]

UnlabelledThe serologic hallmark of primary biliary cirrhosis (PBC), the antimitochondrial response to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), has unique features, including continuous high titers of immunoglobulin M (IgM) and IgG reactivity throughout all stages of disease, capable not only of target enzyme inhibition, but also crossreactive with chemical xenobiotics that share molecular homology with the inner lipoyl domain of PDC-E2; such chemicals have been proposed as potential etiological agents. We used flow cytometry and enzyme-linked immunospot assay (ELISPOT) to examine B-cell subsets in 59 subjects, including 28 with PBC, 13 with primary sclerosing cholangitis (PSC), and 18 healthy controls. Strikingly, in PBC, although there were no significant differences in B-cell phenotype subpopulations, 10% of the total IgG and IgA plasmablast population and 23% of the IgM plasmablast population were uniquely reactive with PDC-E2, detected in the CXCR7+ CCR10low plasmablast population. In contrast, plasmablast reactivity to a control antigen, tetanus toxoid, was minimal and similar in all groups. Additionally, we isolated plasmablast-derived polyclonal antibodies and compared reactivity with plasma-derived antibodies and noted a distinct noncirculating tissue source of xenobiotic crossreacting antibodies. The high levels of autoantigen specific peripheral plasmablasts indicate recent activation of naive or memory B cells and a continuous and robust activation. The presence of CXCR7+ CCR10low PDC-E2-specific ASCs suggests a mechanistic basis for the migration of circulating antigen specific plasmablasts to the mucosal epithelial ligands CXCL12 and CCL28.ConclusionOur findings suggest a sustained rigorous B-cell response in PBC, likely activated and perpetuated by cognate autoantigen.

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5-25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL-GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.

Mosquito salivary glands have important roles in blood feeding and pathogen transmission. However, the biological relevance of many salivary components has yet to be determined. Aegyptin, a secreted salivary protein from Aedes aegypti, binds collagen and inhibits platelet aggregation and adhesion. We used a transgenic approach to study the relevance of Aegyptin in mosquito blood feeding. Aedes aegypti manipulated genetically to express gene-specific inverted-repeat RNA sequences exhibited significant reductions in Aegyptin mRNA accumulation (85-87%) and protein levels (>80-fold) in female mosquito salivary glands. Transgenic mosquitoes had longer probing times (78-300 s, P < 0.0001) when feeding on mice compared with controls (15-56 s), feeding success was reduced, and those feeding took smaller blood meals. However, no differences in feeding success or blood meal size were found in membrane feeding experiments using defibrinated human blood. Salivary gland extracts from transgenic mosquitoes failed to inhibit collagen-induced platelet aggregation in vitro. Reductions of Aegyptin did not affect salivary ADP-induced platelet aggregation inhibition or disturb anticlotting activities. Our results demonstrate the relevance of Aegyptin for A. aegypti blood feeding, providing further support for the hypothesis that platelet aggregation inhibition is a vital salivary function in blood feeding arthropods. It has been suggested that the multiple mosquito salivary components mediating platelet aggregation (i.e., Aegyptin, apyrase, D7) represent functional redundancy. Our findings do not support this hypothesis; instead, they indicate that multiple salivary components work synergistically and are necessary to achieve maximum blood feeding efficiency.

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3(zp3-Cre)) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3(zp3-Cre) mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3(zp3-Cre) mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3(zp3-Cre) mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-β1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3(zp3-Cre) mice. hORMDL3(zp3-Cre) mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-β1, ADAM8).

The Lyn tyrosine kinase regulates inhibitory signaling in B and myeloid cells: loss of Lyn results in a lupus-like autoimmune disease with hyperactive B cells and myeloproliferation. We have characterized the relative contribution of Lyn-regulated signaling pathways in B cells specifically to the development of autoimmunity by crossing the novel lyn(flox/flox) animals with mice carrying the Cre recombinase under the control of the Cd79a promoter, resulting in deletion of Lyn in B cells. The specific deletion of Lyn in B cells is sufficient for the development of immune complex-mediated glomerulonephritis. The B cell-specific Lyn-deficient mice have no defects in early bone marrow B cell development but have reduced numbers of mature B cells with poor germinal centers, as well as increased numbers of plasma and B1a cells, similar to the lyn(-/-) animals. Within 8 mo of life, B cell-specific Lyn mutant mice develop high titers of IgG anti-Smith Ag ribonucleoprotein and anti-dsDNA autoantibodies, which deposit in their kidneys, resulting in glomerulonephritis. B cell-specific Lyn mutant mice also develop myeloproliferation, similar to the lyn(-/-) animals. The additional deletion of MyD88 in B cells, achieved by crossing lyn(flox/flox)Cd79a-cre mice with myd88(flox/flox) animals, reversed the autoimmune phenotype observed in B cell-specific Lyn-deficient mice by blocking production of class-switched pathogenic IgG autoantibodies. Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease.

Age-related increase in L-type Ca(2+) channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca(2+) influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age-related cognitive deficits. Here, using specific antibodies against Cav 1.2 and Cav 1.3 subunits of LTCCs, we systematically re-examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Cav 1.2 and Cav 1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Cav 1.2 in the CA1 and CA3 regions and of Cav 1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Cav 1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Cav 1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age-related cognitive deficits cannot be attributed to a global change in L-type channel expression nor to the level of phosphorylation of Cav 1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age-related increase in L-type Ca(2+) channel activity in CA1 pyramidal neurons.

The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.

Lack of a universal site-specific conjugation methodology for antibodies limits their potential to be developed as tumor-specific imaging agents or targeted therapeutics. A potential mechanism for site-specific conjugation involves utilization of the conserved N-glycosylation site in the CH2 domain. We sought to develop an antibody with an altered azido-sugar at this site whereby site-specific label could be added. The HB8059 hybridoma was cultured with peracetylated N-azidoacetlymannosamine (Ac4ManNAz). The resulting azido-sugar antibody was conjugated to phosphine-polyethylene glycol (PEG3)-biotin via a modified Staudinger reaction. Biochemical and functional characterization of the biotinylated antibody was performed. The azido-sugar antibody was also labeled with DyLight-650-Phosphine and injected into mice harboring pancreatic cancer xenografts. The tumors were dissected and imaged utilizing an IVIS fluorescent camera. The antibody was successfully produced in 100 μM Ac4ManNAz. The biotinylated antibody demonstrated a 50 kDa heavy and 25 kDa light chain on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but demonstrated a single band at 50 kDa on western blot. Treatment with a N-linked glycosidase extinguished the band. Flow cytometry demonstrated antigen-specific binding of CA19-9-positive cells and the antibody localized to the antigen-positive tumor in vivo. We successfully produced an antibody with an azido-sugar at the conserved CH2 glycosylation site. We were able to utilize this azide to label the antibody with biotin or fluorescent label and demonstrate that the label is added in a site-specific manner to the heavy chain, N-linked glycosylation site. Finally, we demonstrated functionality of our antibody for in vitro and in vivo targeting of pancreatic cancer cells.

Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor-binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor-binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell-activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α-driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE.

Irisin is a muscle factor discovered in 2012 that plays an important role in many tissues, including bone. Eight years since its discovery, there are still many controversies regarding its molecular biology, detection, and effects on bone. This article summarizes the points raised to date, and discusses the mechanisms by which irisin regulates bone cells. The information reviewed here provides a useful foundation for future research. • Only acute exercise can temporarily increase plasma irisin levels in humans • Low antibody specificity in detection of irisin content and protein loss during sample pretreatment cause inaccurate results • The non-canonical start codon of irisin in humans has not been confirmed by research • Irisin has different effects on bone formation and resorption depending on its concentration and exposure time [ABSTRACT FROM AUTHOR]

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping. Author summary: The interplay between circulating virus variants and broadly cross neutralizing polyclonal antibodies developed in a subset of elite neutralizers is widely believed to provide strategies for rational immunogen design. In the present study, we studied the structural, antigenic and immunogenic properties of a novel soluble trimeric protein with near native pre-fusion conformation prepared using the primary sequence of an HIV-1 clade C env isolated from the broadly cross neutralizing plasma of an elite neutralizer. This novel SOSIP Env trimer demonstrated comparable antigenic, structural and immunogenic properties that favoured several ongoing subunit vaccine design efforts. Interestingly, ns-EMPEM analysis suggested the novel clade C SOSIP induced a comparable neutralizing antibody specificity in rabbits to one that was elicited during the course of natural infection. A better understanding of how vaccine-induced polyclonal neutralizing antibody responses compares to responses that developed in natural infection will improve our knowledge in designing better vaccine design strategies. [ABSTRACT FROM AUTHOR]

Introduction: As soon as the association of lupus anticoagulant (LAC) and anticardiolipin antibodies (aCL) with thrombosis and miscarriages was described in the 1980s, the definition of the antiphospholipid syndrome (APS) became a need. Early descriptions of the disease by members of the Graham Hughes team included broad categories and unexplained laboratory inclusions. Over time, new clinical and experimental data refined the criteria, especially the obstetric manifestations, as well as the laboratory criteria. Areas covered: The authors performed a review of the literature using the PubMed database, and the following keywords were used: 'antiphospholipid antibody', 'antiphospholipid syndrome', and 'criteria of antiphospholipid'. The history of antiphospholipid criteria, clinical and experimental advancements, and other expert opinions were included in this paper. Expert opinion: It has been 14 years since an international congress on antiphospholipid antibodies has generated new classification based on the recent extensive research performed in the field. Currently, there is a need to update the international APS classification taking into consideration the inclusion of new clinical criteria such as aPL-related nephropathy as well as new standardized antibody specificities (e.g., anti-phosphatidylserine/prothrombin antibodies) with the adoption of a standardized scoring system that can stratify APS patients. [ABSTRACT FROM AUTHOR]

The four dengue virus serotypes (DENV1-4) infect several hundred million people each year living in tropical and sub-tropical regions. Clinical development of DENV vaccines is difficult because immunity to a single serotype increases risk of severe disease during a second infection with a new serotype. Leading vaccines are based on tetravalent formulations to induce simultaneous and balanced protective immunity to all 4 serotypes. TAK-003 is a tetravalent live attenuated dengue vaccine candidate developed by Takeda Vaccines Inc, which is currently being evaluated in phase 3 efficacy trials. Here, we use antibody depletion methods and chimeric, epitope transplant DENVs to characterize the specificity of neutralizing antibodies in dengue-naïve adults and non-human primates immunized with TAK-003. Our results demonstrate that TAK-003 induced high levels of DENV2 neutralizing antibodies that recognized unique (type-specific) epitopes on DENV2. In contrast, most vaccinated subjects developed lower levels of DENV1, DENV3 and DENV4 neutralizing antibodies that mainly targeted epitopes that were conserved (cross-reactive) between serotypes. Trial Registration: ClinicalTrials.gov NCT02425098. Author summary: The licensed tetravalent dengue vaccine Dengvaxia is indicated for individuals with previous exposure to dengue. In subjects with no past dengue infection, vaccine trials showed low efficacy against some serotypes and increased risk of severe disease upon post-vaccination infection. The development of tetravalent dengue vaccines has been guided by neutralizing antibodies to each serotype as a measure of safe and effective immunity. However, the absolute level of neutralizing antibodies to each serotype has proven to be an unreliable correlate of protection. Recent studies suggest that a better correlate may be levels of antibodies to epitopes that are unique to each serotype and are independently stimulated by each vaccine component, rather than total quantity of neutralizing antibodies. Here, we mapped the antibody specificity induced by the Takeda tetravalent dengue vaccine TAK-003 in monkeys and humans with no prior immunity to dengue. The vaccine induces high levels of dengue serotype 2 specific neutralizing antibodies that map to known protective epitopes. In contrast, the dengue serotype 1, 3 and 4 specific responses are lower and predominantly consist of cross-reactive antibodies binding to antigenic regions conserved between serotypes. It remains to be determined whether these cross-reactive antibodies, most likely induced by the serotype 2 component, contribute to long-term protection after vaccination. [ABSTRACT FROM AUTHOR]

Appropriate hapten design and synthesis have been identified as critical steps to generate high-performance immunoreagents and to develop sensitive and selective immunoanalytical methods. Antibodies and immunoassays for the major mycotoxin zearalenone have been reported and marketed. However, zearalenone haptens have mostly been prepared by the oxime active ester technique, and hapten characterization has generally been poor or non-existent. In the present study, novel haptens of zearalenone with longer linkers and with alternative tethering sites have been designed for immunizing and assay conjugate preparation. All of these molecules were purified and spectroscopically verified, and a structure-activity relationship evaluation was carried out. This approach revealed that the hapten with the linker at the carbonyl group generated antibodies with a higher affinity than the hapten functionalized at the phenyl moiety. Antibodies produced with the latter hapten, on the other hand, showed lower cross-reactivity values to the major zearalenone metabolites. Finally, similar immunoassay sensitivity was achieved with all of the antibodies when heterologous haptens were employed. Furthermore, by altering the structure of the competing antigen, the immunoassay selectivity was modified. These results demonstrate that immunochemical methods for zearalenone rapid analysis can still be improved in terms of sensitivity and selectivity.

Aberrant changes in post-translational modifications (PTMs) such as phosphate groups underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often require PTM-specific monoclonal antibodies (mAbs), which are challenging to generate using traditional antibody-selection methods. Here we outline a general strategy for producing synthetic, PTM-specific mAbs by engineering a motif-specific hot spot into an antibody scaffold. Inspired by a natural phosphate-binding motif, we designed and selected mAb scaffolds with hot spots specific for phosphoserine, phosphothreonine or phosphotyrosine. Crystal structures of the phospho-specific mAbs revealed two distinct modes of phosphoresidue recognition. Our data suggest that each hot spot functions independently of the surrounding scaffold, as phage display antibody libraries using these scaffolds yielded >50 phospho- and target-specific mAbs against 70% of target peptides. Our motif-specific scaffold strategy may provide a general solution for rapid, robust development of anti-PTM mAbs for signaling, diagnostic and therapeutic applications.

Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl β-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a (5)Ile→Ala and (12)Ile→Ala double mutant. Single alanine replacement at (5)Ile, (12)Ile, and (15)Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at (5)Ile, (12)Ile, and (15)Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the (5)Ile and (12)Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl β-sheet structural conformation in anti-PDC-E2 recognition.

Autism spectrum disorders (ASDs) are neurodevelopmental in origin, affecting an estimated 1 in 88 children in the United States. We previously described ASD-specific maternal autoantibodies that recognize fetal brain antigens. Herein, we demonstrate that lactate dehydrogenase A and B (LDH), cypin, stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2) and Y-box-binding protein to comprise the seven primary antigens of maternal autoantibody-related (MAR) autism. Exclusive reactivity to specific antigen combinations was noted in 23% of mothers of ASD children and only 1% of controls. ASD children from mothers with specific reactivity to LDH, STIP1 and CRMP1 and/or cypin (7% vs 0% in controls; P

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde-acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.

Paper-based diagnostics are leading the field of low-cost, point of care analytical techniques. However, large scale testing facilities such as hospitals are still primarily using the gel column agglutination technique. This is because paper-based systems are single use tests that are generally more time consuming and less automatable than traditional methods. Here, we present a novel, rapid and scalable, paper-based blood typing method that can produce test results in under ten seconds. We believe this is the fastest blood typing test that is appropriate for large scale automation. The test consists of placing a drop of antibody solution on paper, followed by a drop of blood on the same locus, and measuring the evolution of blood stain area as a function of time. Positive reactions for both forward and reverse tests have significantly slower growth rates and smaller final stain sizes when compared to negatives. We analyse the effect paper type, red blood cell concentration, antibody specificity (A, B and D) and antibody dilution have on the sensitivity and reproducibility of the technique. A high sensitivity is found in papers with a low density and thickness. The optimum red blood cell concentration is determined from a balance between wicking rate, strength of reaction and optical contrast. A and B antibodies give more sensitive results than D; however, the D antigen can still be successfully identified. This technique has the potential to significantly cut down the time and cost of blood typing tests and enable design of a new high throughput and fully automatable system. [ABSTRACT FROM AUTHOR]

Ipilimumab (IPI) can enhance immunity to the cancer-testis antigen NY-ESO-1. A clinical trial was designed to assess safety, immunogenicity, and clinical responses with IPI + NY-ESO-1 vaccines and effects on the tumor microenvironment (TME). Patients with measurable NY-ESO-1+ tumors were enrolled among three arms: A) IPI + NY-ESO-1 protein + poly-ICLC (pICLC) + incomplete Freund's adjuvant (IFA); B) IPI + NY-ESO-1 overlapping long peptides (OLP) + pICLC + IFA; and C) IPI + NY-ESO-1 OLP + pICLC. Clinical responses were assessed by irRC. T cell and Ab responses were assessed by ex vivo IFN-gamma ELIspot and ELISA. Tumor biopsies pre- and post-treatment were evaluated for immune infiltrates. Eight patients were enrolled: 5, 2, and 1 in Arms A-C, respectively. There were no DLTs. Best clinical responses were SD (4) and PD (4). T-cell and antibody (Ab) responses to NY-ESO-1 were detected in 6 (75%) and 7 (88%) patients, respectively, and were associated with SD. The breadth of Ab responses was greater for patients with SD than PD (p = .036). For five patients evaluable in the TME, treatment was associated with increases in proliferating (Ki67+) CD8+ T cells and decreases in RORyt+ CD4+ T cells. T cell densities increased for those with SD. Detection of T cell responses to NY-ESO-1 ex vivo in most patients suggests that IPI may have enhanced those responses. Proliferating intratumoral CD8+ T cells increased after vaccination plus IPI suggesting favorable impact of IPI plus NY-ESO-1 vaccines on the TME. [ABSTRACT FROM AUTHOR]

Background: The Scianna (SC) blood group system comprises seven antigens. They reside on the erythroblast membrane‐associated glycoprotein (ERMAP). The ERMAP and RHCE genes are juxtaposed to each other on chromosome 1. We report a novel SC antigen. Study Design and Methods: Blood samples came from a patient and his two sisters in Saudi Arabia. To investigate the antibody specificity we used the column agglutination technique and soluble recombinant ERMAP protein. The significance of anti‐SCAR was evaluated by the transfusion history and a monocyte monolayer assay. We determined the genomic sequence of ERMAP and RHCE genes. Results: The patientʼs serum showed an antibody of titer 8 against a high‐prevalence antigen. The soluble recombinant ERMAP protein inhibited the antibody. The propositus genotyped homozygous for an ERMAP:c.424C>G variant, for which his sisters were heterozygous. The c.424C>G variant occurred in the SC*01 allele in one haplotype with the RHCE*03 (RHCE*cE) allele. No signs of hemolysis occurred following an incompatible blood transfusion. The monocyte monolayer assay was negative. Conclusions: We characterized a high‐prevalence antigen, with the proposed name "SCAR," which is the eighth antigen of the Scianna blood group system (proposed designation 013.008). Individuals homozygous for ERMAP:p.(Gln142Glu) protein variant can produce anti‐SCAR. Although we did not observe any sign of hemolysis at this time, the anti‐SCAR prompted a change of the treatment regimen. A review of the known reports indicated that all SC alloantibodies of sufficient titer should be considered capable of causing hemolysis. [ABSTRACT FROM AUTHOR]

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate highquality conformational antibodies in a systematic and sitespecific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aβ fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aβ42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinicalstage Aβ antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aβ aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinitymaturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers. [ABSTRACT FROM AUTHOR]

Background: There is insufficient evidence to assess the risk of the production of clinically important alloimmune irregular red blood cell (RBC) antibodies in first-time pregnant women. Methods: Using the microcolumn gel antiglobulin method, 18,010 Chinese women with a history of pregnancy and pregnant women were screened for irregular RBC antibodies, and for those with positive test results, antibody specificity was determined. The detection rate and specificity of irregular RBC antibodies in women with a history of multiple pregnancies (two or more) and first-time pregnant women were determined. Results: In addition to 25 patients who passively acquired anti-D antibodies via an intravenous anti-D immunoglobulin injection, irregular RBC antibodies were detected in 121 (0.67%) of the 18,010 women. Irregular RBC antibodies were detected in 93 (0.71%) of the 13,027 women with a history of multiple pregnancies, and antibody specificity was distributed mainly in the Rh, MNSs, Lewis, and Kidd blood group systems; irregular RBC antibodies were detected in 28 (0.56%) of the 4983 first-time pregnant women, and the antibody specificity was distributed mainly in the MNSs, Rh, and Lewis blood group systems. The difference in the percentage of patients with irregular RBC antibodies between the two groups was insignificant ( χ 2 = 1.248, P > 0.05). Of the 121 women with irregular RBC antibodies, nine had anti-Mur antibodies, and one had anti-Di a antibodies; these antibodies are clinically important but easily missed because the antigenic profile of the reagent RBCs that are commonly used in antibody screens does not include the antigens that are recognized by these antibodies. Conclusion: Irregular RBC antibody detection is clinically important for both pregnant women with a history of multiple pregnancies and first-time pregnant women. Mur and Di a should be included in the antigenic profile of reagent RBCs that are used for performing antibody screens in the Chinese population., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Shujie Wu et al.)

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.