Dependent Epitopes Research Articles

APNmAb005, an anti‐tau antibody targeting synaptic tau oligomers, in Phase 1 for treatment of Alzheimer’s Disease and primary tauopathies

AbstractBackgroundAccumulation of tau oligomers at the synapses in Alzheimer’s disease (AD) disrupts neuronal activities and leads to neurodegeneration. Tau immunotherapy has been actively pursued as a treatment for tauopathies in the past decade. Despite several failed Phase 2 trials of N‐terminus binding antibodies a variety of tau epitopes are being investigated at different stages of clinical trials. APNmAb005 is a humanized monoclonal antibody designed to block synaptic toxicity elicited by tau oligomers.MethodAPNmAb005 was selected as a clinical candidate from APRINOIA’s antibody discovery platform based on its unique binding properties to pathological tau in vitro immunoassays as well in vivo efficacy studies in rTg4510 mice. Humanized APNmAb005 is in IgG4 backbone and the production was scaled up successfully with good stability and solubility to support preclinical and clinical development. GLP safety study was conducted in cynomolgus monkeys. A Phase 1a randomized, double‐blind, placebo‐controlled study of APNmAb005 in healthy subjects is underway.ResultEpitope‐mapping study reveals that APNmAb005 binds to a conformation‐dependent epitope in the mid‐region, which results in a preferable recognition of pathological tau in early Braak stage (III‐IV) AD brains as well as tau aggregates in astrocytes and oligodendrocytes in brains from progressive supranuclear palsy (PSP) patients but not monomeric tau in normal brains. APNmAb005 blocks in vitro tau seeding at nanomolar concentrations and rescues neuronal loss in rTG4510 mice. Four‐week repeat dosing toxicology study shows no observed adverse effects.ConclusionAPNmAb005 is a conformation selective antibody that binds to tau oligomers and aggregates preferentiually in synapses in pathological brain tissues and inhibits tau spreading. The mid‐region conformation dependent epitope is distinct from all other tau antibodies currently in clinical development. APNmAb005 offers a unique tau immunotherapy for neurodegenerative disorders and is currently in Phase 1 to evaluate its safety and tolerability.

Two cases of cutaneous-type pemphigus vulgaris and a case of pemphigus foliaceus without mucosal involvement despite high anti-desmoglein 3 autoantibody levels.

Pemphigus is an autoimmune blistering disease with two major subtypes, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Although most patients with PV show oral lesions, cutaneous type PV (C-PV) is a rare subtype clinically characterized by predominant cutaneous involvement with no or subtle mucosal lesions. Patients with PF present with only skin involvement; they do not have mucosal lesions. Serologically, autoantibodies against desmoglein (Dsg) 3 and Dsg1 are observed in C-PV whereas PF is associated with anti-Dsg1 antibodies only. Herein, we describe three cases of pemphigus presenting with predominant skin lesions and no mucosal involvement despite high anti-Dsg 3 autoantibody levels in chemiluminescent enzyme immune assays (CLEIAs). In addition, anti-Dsg 1 autoantibodies were positive in patients 2 and 3, but negative in patient 1 based on CLEIAs. Histological examination of the skin showed suprabasal acantholysis in patients 1 and 2, and blister formation in the upper epidermis in patient 3. Histopathology of the oral membrane in patients 1 and 2 showed subtle acantholysis in the suprabasal layer. Thus, we diagnosed patients 1 and 2 as having cutaneous type PV and patient 3 as having PF. Ethylenediaminetetraacetic acid-treated enzyme-linked immunosorbent assay demonstrated a low proportion of anti-Dsg3 autoantibodies recognizing Ca2+ -dependent epitopes, antibodies against which are thought to be the main contributor to acantholysis. Thus, along with Dsg1 antibodies, weak anti-Dsg3 antibodies could induce acantholysis in the skin, but they are insufficient to induce mucosal lesions.

Using <i>Bacillus subtilis</i> as an oral vaccine carrier against <i>Streptococcus suis</i>

Due to the progressive growth of the bacteria caused by the widespread use of antibiotics, treatment of streptococcosis is becoming increasingly difficult. Reliable vaccination against Streptococcus suis is necessary. Modern molecular diagnostic and genetic engineering capabilities create prospects for direct cloning of the protective epitopes of the Lmb gene of the local S. suis strain into the proposed delivery system of the pig immune system antigen. Among oral vaccine carriers, Bacillus subtilis is recognized as a relatively environmentally friendly carrier with an efficient protein secretion system and adaptive metabolism capable of spore production under relatively harsh conditions. This spore property can be used to increase the stability and reusability of vaccines. The possibility of using the protective Lmb epitopes of S. suis in B. subtilis as a carrier of an oral recombinant vaccine against Streptococcus suis was studied. The nucleotide sequences of S. suis were obtained from the GenBank database after a preliminary analysis of literature data on the known protective antigens of S. suis of various serotypes. Nucleotide sequence analysis was performed using Unipro UGENE v. 43.0. The Immune Epitope Database (IEDB) was used to search for T (CTL and Th) and B dependent epitopes of the Lmb gene. A computer-designed vaccine in which localization of CTL, B, and Th epitopes is predicted is described. The results of cloning the sequence of the antigenically active epitope of the S. suis Lmb protein in B. subtilis for subsequent oral administration and study of changes in immunological reactions and adverse reactions in animals are described. The possibility to clone the epitopes of recombinant S. suis Lmb protein into the pBE-S polylinker vector was revealed. In the long term, it seems possible to create a new inexpensive and easy-to-use vaccine against S. suis that does not require injection.

β1-adrenergic Receptor Solubilized in the Form of Nanodiscs: Screening of Various Amphipatic Polymers

The development of a reliable and easily used diagnostic test for measuring autoantibodies to ?1-adrenergic receptor (?1ADR Ab) in patient blood is an unmet clinical need. The enzyme-linked immunosorbent assay (ELISA) is considered as the most appropriate method for this task. In ELISA, the use of peptides corresponding to various fragments of amino acid sequence of ?1ADR as antigens leads to inadequate results as β1ADR Ab appear to recognize conformationally dependent epitopes that are generated during the formation of unique tertiary structure of the receptor. Isolation of ?1ADR preserving the native conformation and functional characteristics is a quite challenging task. A promising approach to address this task is the use of amphipatic polymers capable of forming nanodiscs, it permits to successfully solubilize membrane proteins. In order to obtain the preparations of solubilized β1ADR that can be used as antigens in ELISA we have tested 17 various amphipatic polymers. The best relative solubilization values (RSV) were obtained using UltrasoluteTM Amphipol 17 (87%) and 18 (62%), as well as by AASTY 11-45 (76%), 11-50 (77%) and 6-50 (78.5%).

Analysis of two cooperating antibodies unveils immune pressure imposed on HIV Env to elicit a V3-glycan supersite broadly neutralizing antibody lineage.

Elicitation of broadly neutralizing antibodies (bnAbs) is a goal of vaccine design as a strategy for targeting highly divergent strains of HIV-1. Current HIV-1 vaccine design efforts seek to elicit bnAbs by first eliciting their precursors through prime-boost regimens. This requires an understanding of the co-evolution between viruses and antibodies. Towards this goal, we have analyzed two cooperating antibodies, DH475 and DH272, which exerted pressure on the HIV population in an infected donor, called CH848, to evolve in such a way that it became sensitive to the V3-glycan supersite DH270 bnAb lineage. We obtained a 2.90Å crystal structure of DH475 in complex with the Man9 glycan and a negative stain EM model of DH272 in complex with the HIV-1 spike trimer, Env. Coupled with additional modeling studies and biochemical data, our studies reveal that DH475 contacts a V3- and V4-glycan dependent epitope accessible on an open or shed Env and that DH272 makes critical contacts with the V1V2 and V3 loops on HIV-1 Env. Using these data, we suggest a prime-boost regimen that may facilitate the initiation of DH270-like bnAb precursors.

The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual \u201cwindow of treatment success\u201d in RA patients

BackgroundThere is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry.ResultsHnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CNDL-Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CNDL-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.

The D3-GPC2-PBD ADC is Potently Efficacious Against Neuroblastoma and SCLC Via Engagement of a Conformational GPC2 Epitope

The signaling co-receptor heparan sulfate proteoglycan GPC2 is a MYCN-activated, differentially-expressed cell surface oncoprotein and candidate immunotherapeutic target in neuroblastoma. Here we build on GPC2’s attributes as a robust target for immune-based therapies by finding it is highly expressed on the most clinically aggressive human neuroblastomas and small cell lung cancers (SCLCs), with highly enriched expression in the tumor stem cell compartment. A GPC2 directed antibody-drug conjugate (ADC; D3-GPC2-PBD) induces robust and durable tumor regressions in a panel of neuroblastoma and SCLC preclinical models with diverse oncogenic drivers via induction of DNA damage and apoptosis, as well as potent bystander cell killing. Finally, we illustrate that this ADC binds a tumor specific, conformation dependent epitope of the core human and murine GPC2 proteins explaining its tumor specificity and therapeutic tolerability. These studies provide the preclinical data to support the clinical translation of ADCs targeting GPC2.

Dissection of \u03b14\u03b27 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-\u03b27 antibodies

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of β7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of β7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4β7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α4β7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).

Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

AbstractThe development of neutralizing anti-FVIII antibodies (inhibitors) is a major complication of FVIII protein replacement therapy in patients with hemophilia A (HA). Although multiple lines of evidence indicate that the immune response against FVIII is CD4 T-cell–dependent and many FVIII-derived CD4 epitopes have already been discovered, the role of T follicular helper (TFH) cells in FVIII inhibitor development is unknown. TFH cells, a newly identified subset of CD4 T cells, are characterized by expression of the B-cell follicle-homing receptor CXCR5 and PD-1. In this study, we show for the first time that IV FVIII immunization induces activation and accumulation and/or expansion of PD-1+CXCR5+ TFH cells in the spleen of FVIII-deficient (FVIIInull) mice. FVIII inhibitor-producing mice showed increased germinal center (GC) formation and increased GC TFH cells in response to FVIII immunization. Emergence of TFH cells correlated with titers of anti-FVIII inhibitors. Rechallenge with FVIII antigen elicited recall responses of TFH cells. In vitro FVIII restimulation resulted in antigen-specific proliferation of splenic CD4+ T cells from FVIII-primed FVIIInull mice, and the proliferating cells expressed the TFH hallmark transcription factor BCL6. CXCR5+/+ TFH-cell–specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5+/+ TFH cells for the generation of FVIII-neutralizing antibodies. Together, our results demonstrate that the induction of activated TFH cells in FVIIInull mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH-cell activation may be a promising strategy for preventing anti-FVIII inhibitor formation in patients with HA.

RHD del28Phe (DMW) encoded by a novel in‐frame deletion resulting in reduced D antigen expression

Red blood cells (RBCs) of variant Rh antigens, including weak D or partial D phenotypes, may cause anti-D immunization following transfusion or pregnancy.1 Partial D individuals are at risk for producing allo-anti-D against the missing epitopes when they are exposed to normal D-positive cells, with the potential for eliciting hemolytic transfusion reactions and HDFN.1, 2 We have identified a novel RHD allele variant in a pregnant Caucasian woman with a considerably reduced D expression on her red blood cells (RBCs). Rh phenotyping was performed by standard gel card matrix techniques (Micro Typing ID System, Bio-Rad Medical Diagnostic GmbH), including monoclonal (Diaclon ABO/Rh, ABO/D and Rh-subgroups + K), and human antibodies (ABO/Rh and Rh-subgroups + Cw + K). The presence of maternal irregular antibodies was investigated by indirect antiglobulin test prenatally and 6 weeks after delivery. A commercial partial RHD typing set (Bio-Rad Medical Diagnostic GmbH) was used for further characterization. Extended D epitope mapping was done employing 50 different human monoclonal anti-D as previously described.3 The D antigen density was determined by flow cytometry using five primary anti-Ds: Brad-3, P3x290, P3x241, P3x249, and ESD1. RHD was genotyped using RHD detection tests (RBC-Ready Gene CDE and RBC-Ready Gene Zygofast, Inno-train Diagnostik GmbH). DNA sequencing of RHD Exons 1 to 10 and flanking intronic regions was performed.4 RHD mRNA isoforms were analyzed. The identified sequence was deposited under EMBL Accession number LR214928. The designation DMW refers to the identification of the allele by the authors Matzhold and Wagner. A direct antiglobulin test of the newborn was performed. Standard serology determined a C + c + D + E–e + phenotype, with weak positive reactions for the D antigen (3+) in both the monoclonal and human gel card. Accordingly, the RBCs of the proposita were found to have a reduced D antigen density of 1.001 D sites per cell. The commercial monoclonal anti-D panel resulted in at least 2+ positive reactions with all the anti-D, except anti-D LHM59/19 (IgG) recognizing EpD8.1, where only a marginal positive reaction (+/−) was observed. The extended D epitope mapping demonstrated a near-normal, albeit weakened, epitope profile. Consistently, LHM59/19 reacted very weakly (1+). Several maternal antibody screening tests were negative and no allo-anti-D was detected in the proposita's serum. Sequencing analysis revealed the presence of a novel RHD allele (DMW), characterized by a deletion of TCT at nucleotide position 83 (c.83delTCT, p.28delPhe) in Exon 1 (Fig. 1). The major RHD cDNA isoform confirmed the del28Phe alteration, resulting in a shortened protein of 416 instead of 417 amino acids. Neonatal cord blood typing revealed blood group O, D-positive, and a negative direct antiglobulin test. The novel in-frame deletion is associated with a considerably reduced expression of D antigen and a variant RhD phenotype. The mutation occurs in close proximity to the first exofacial loop of the protein which is mainly encoded by sequences of Exon 1. Structural changes caused by the deletion may reach the surface of the protein in this region. Consistent with the substantially weakened agglutination reactions observed with anti-D LHM59/19, the presence of an altered Loop 1–dependent epitope 8.15 appears to be possible. Although all the anti-Ds we used to examine the epitope pattern reacted positive with the proposita's RBCs, the presence of a qualitatively altered D antigen may not be excluded. The primigravida had not received prenatal or postnatal RhIG prophylaxis. Though pregnant with a D-positive fetus, no immunization was observed; however, whether this novel D variant permits anti-D immunization remains unclear. Hence, D-negative transfusion in carriers and prophylactic use of RhIG in pregnancy is recommended. DMW expands the exceptionally rare RHD in-frame deletions reported and should be regarded as a putative partial D allele. The authors thank Anja Stoisser, Alma Mekic, Karin Mallick, and Maria Luise Stubenrauch for their technical assistance. Camilla Drexler is acknowledged for writing assistance and language editing. The authors have disclosed no conflicts of interest. The following Web-based resource was used: http://www.ebi.ac.uk/ena/data/view/LR214928. Accessed January 3, 2019.

Identification of specific T lymphocytes against to an IFNg inducible lysosomal thiol reductase (GILT)-dependent, class II MHC-associated epitope in GILT−/− mice

Abstract IFNγ inducible lysosomal thiol reductase (GILT) is the only thiol reductase localized in lysosomes and late endosomes, with its optimal activity at acidic pH. GILT is constitutively expressed in professional APC, further upregulated by IFNγ, and participates in Ag processing by catalyzing disulfide (S-S) bond reduction in proteins thus facilitating unfolding of native S-S bond containing Ag for further processing. It was reported that GILT−/− mice mounted significantly reduced T cell proliferative responses to S-S bond-containing protein Ags in vitro, but responded normally to a non-S-S bond containing protein. In addition, GILT−/− APC showed a selective deficiency in presenting some cysteine-containing epitopes within a model protein Ag, hen egg white lysozyme. Using HPLC-MS/MS, we first identified one group of unique GILT-dependent, class II MHC-associated peptides from B6 mice, but not from GILT−/− mice. Binding of these GILT dependent, class II MHC-associated peptides to I-Ab proteins was confirmed in vitro. Furthermore, T cells specific to one of the unique GILT-dependent peptides, BP7, was detected in GILT−/− mice, but not found in B6 mice. BP7 peptide specific T cell hybridoma clones were established as well. Taken together, our results indicate that GILT−/− mice have a deficiency in generation of certain T cell epitopes due to loss of GILT activity. Missing of GILT dependent epitopes might lead to survival of the T cells specific to these GILT-dependent epitopes (escaping from the negative selection) in thymus of GILT−/− mice. We are in progress of cloning the BP7 specific TCRs from T cell hybridoma clones. The potential role of the BP7 specific T lymphocytes in autoimmunity will be examined in BP7 specific T cell retrogenic mice.

Detection Methods for Autoantibodies in Suspected Autoimmune Encephalitis.

This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis (AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis.

Plasmodium vivax Pv12 B-cell epitopes and HLA-DRβ1*-dependent T-cell epitopes in vitro antigenicity

Malaria is an infectious disease caused by parasites from the genus Plasmodium (P. falciparum and P. vivax are responsible for 90% of all clinical cases); it is widely distributed throughout the world’s tropical and subtropical regions. The P. vivax Pv12 protein is involved in invasion, is expressed on merozoite surface and has been recognised by antibodies from individuals exposed to the disease. In this study, B- and T-cell epitopes from Pv12 were predicted and characterised to advance in the design of a peptide-based vaccine against malaria.For evaluating the humoral response of individuals exposed to natural P. vivax infection from two endemic areas in Colombia, BepiPred-1.0 software was used for selecting B-cell epitopes. B-cell epitope 39038 displayed the greatest recognition by naturally-acquired antibodies and induced an IgG2/IgG4 response. NetMHCIIpan-3.1 prediction software was used for selecting peptides having high affinity binding for HLA-DRβ1* allele lineages and this was confirmed by in-vitro binding assays. T-epitopes 39113 and 39117 triggered a memory T-cell response (Stimulation Index≥2) and significant cytokine production.Combining in-silico, in-vitro and functional assays, two Pv12 protein regions (containing peptides 39038, 39040, 39113 and 39117) have thus been characterised as promising vaccine candidates against P. vivax malaria.

Identification of a gamma IFN inducible lysosomal thiol reductase (GILT)-dependent MHC-II associated epitope that induces T cell responses in GILT−/− mice

Abstract Gamma-IFN inducible lysosomal thiol reductase (GILT) is currently the only identified thiol reductase that has optimal activity at acidic pH and localizes to lysosomes. GILT is constitutively expressed in antigen presenting cells (APC), is inducible by gamma-IFN, and participates in Ag processing by catalyzing the reduction of disulfide (S-S) bonds, thus facilitating the unfolding of native S-S bond containing Ag for further processing. Previous studies reported that T cells from GILT knock-out (GILT−/−) mice mounted a significantly reduced proliferative response to S-S containing protein Ag in vitro, GILT−/− APCs have a selective deficiency in presentation of some cysteine-containing epitopes from hen egg lysozyme, and GILT−/− mice exhibited a change in the pathogenic epitope of experimental autoimmune encephalomyelitis. Using MS/MS analysis, we identified a set of unique GILT-dependent, MHC-II associated-peptides in WT vs. GILT−/− mice, which we then validate by generating T cells specific to a unique GILT-dependent epitope from GILT−/− but not WT B6 mice. Our data suggests that GILT−/− mice are deficient in the generation of specific T cell epitopes due to a loss in GILT activity. This is consistent with our another on-going study where we find that GILT−/− mice failed to generate an Ab response to insulin due to inefficient processing of an S-S containing epitope in insulin. We hypothesize that in GILT−/− mice, GILT inactivation causes a change in the epitope repertoire that may lead to survival (escape from negative selection) of the T cells specific to GILT dependent epitopes in the thymus. The presence of these auto-reactive T cells might have implications for autoimmunity and autoimmune diseases.

264 Patients with pemphigus foliaceus may retain antibody reactivity against calcium dependent epitopes of desmoglein 1 in remission

264 Patients with pemphigus foliaceus may retain antibody reactivity against calcium dependent epitopes of desmoglein 1 in remission

Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

Construction of bacterial ghosts for transfer and expression of a chimeric hepatitis C virus gene in macrophages

The bacterial ghost (BG) production is a field of biotechnology for applications in vaccine and drug delivery. We assessed the capacity of BG for delivery of a recombinant gene encoded for both cell mediated and antibody dependent epitopes of hepatitis C virus (HCV) into murine macrophages. Escherichia coli (E. coli) cells were transformed with the lysis plasmid (pHH43). To produce chimeric gene, NS3 (non-structural protein 3) and core regions of HCV genome were fused together by splicing by overlap extension (SOEing) PCR and were cloned into plasmid pEGFP-C1. Bacterial ghosts were loaded with recombinant pEGFP-C1 and then were transferred to murine macrophages (RAW 264.7). To investigate plasmid transfection and chimeric mRNA transcription, fluorescent microscopy and RT-PCR were used. In vitro studies indicated that bacterial ghosts loaded with pEGFP-C1 plasmid were efficiently taken up by murine macrophages and indicated a high transfection rate (62%), as shown by fluorescent microscopy. RT-PCR from extracted intracellular mRNAs for chimeric Core-NS3 gene showed a specific 607bp fragment of the gene. The sequence analysis of purified PCR products demonstrated the expected unique mRNA sequence. We constructed a chimeric HCV gene containing both cell mediated and antibody dependent epitopes with a significant expression in murine macrophages delivered by bacterial ghost.

Beyond the Bethesda Assay- Towards Individualized Interpretation of Inhibitor Patients’ FVIII Brand Specific Profiles

Background: Patients with hemophilia A (HA) and inhibitory antibodies are typically treated with bypassing agents if the inhibitor titer is >5 Bethesda units (BU). However, some patients with elevated inhibitor titers are treated with FVIII therapy in conjunction with immune tolerance regimens or in combination with bypassing agents. The Bethesda assay detects the inhibitory capacity for FVIII neutralization after 2 hours’ incubation of patient’s plasma mixed with pooled normal plasma (PNP). Nonetheless the inhibitory titer does not express the kinetic behavior of the inhibitor throughout the 2 hours of incubation. Furthermore, standard plasma derived FVIII is used for the Bethesda assay, whereas many patients are currently treated with recombinant FVIII products. Patients with severe HA who develop inhibitors typically have a polyclonal response. In vitro responses to monoclonal specific anti- epitope antibodies showed that group A anti-A2 antibodies ( high titer, type I antibodies) rapidly and completely inhibit FVIII and are not affected by the presence of Von Willebrand factor (VWF). In contrast, many anti- C1 and C2 antibodies compete with VWF for binding to FVIII. As patients’ response to various FVIII sources (eg: with/ without VWF) may be affected by their unique B cell epitope profile, personalized individually tailored FVIII brand specific therapy may be considered.Aims : We aimed to define our inhibitor patients’ individual response to various FVIII sources by assessing the kinetics of FVIII inhibition and by patients’ FVIII epitope specificity.Methods: Plasma was obtained from HA patients with inhibitors. FVIII neutralization profile was determined against PNP, recombinant full length FVIII (rFVIII= Kogenate- FS, Bayer), plasma derived Von Willebrand containing FVIII (pdFVIII = Optivate, BPL) and correlated to the inhibitory (BU) titer. To test FVIII neutralization profile, patients’ platelet poor plasma was either incubated with PNP or spiked with 2U/ml rFVIII/pdFVIII and sequential measurements of residual FVIII activity was recorded over 120 minutes. FVIII activity was measured by one stage aPTT assay. All inhibitor assays were performed on the same sample simultaneously. Results were compared to the standard Bethesda assay.Domain-specific anti-FVIII antibody mapping was carried out by direct ELISA using purified single human domain hybrid FVIII protein. Patient plasma was serially diluted down the ELISA plate. Domain predominance was determined by comparison of resultant ELISA curves. In addition the presence of anti-A2 group A antibodies in the polyclonal patient plasma was assessed by competition ELISA.Results: Our patient cohort was heterogeneous, consisting of patients aged 3-70 years with inhibitor titers of 0.5-131 BU. No obvious correlation was found between kinetic profiles and the inhibitory BU titer. For each patient, variability of inhibitor activity against different FVIII sources was noted. In individual patients, discrepant inhibitory kinetics were identified when commercial FVIII (rFVIII or pd-FVIII) was added, compared to PNP, utilized for standard determination of inhibitory titer in BU/ml. Among patients with similar BU titer, different FVIII neutralization profile was noted. Furthermore, the source of FVIII yielded individual neutralization curves that did not correlate with the common gold standard of inhibitory (BU) titer (Figure). Correlation between patients’ epitopes and their neutralization profile was difficult to assess as in most patients no single predominant inhibitory epitope could be defined. However, in patients with C2 predominance antibodies improved neutralization was detected with pd -FVIII as compared to rFVIII source. High titer patient plasmas that contained anti-A2 group A antibodies inhibited FVIII completely at the first time point regardless of the VWF content of the FVIII source.Conclusion: The Bethesda assay neither predicts inhibitory kinetics nor defines patients’ response to various FVIII sources. Our combination of assays may identify the best potential FVIII source for treatment of patients with inhibitory antibodies. We suggest that FVIII source dependent neutralization kinetics and epitope mapping may be applied as additional tools for future individual therapy tailoring. Larger sample sizes and repeated samples over time will be required to validate these results. [Display omitted] DisclosuresDunn:CSL Behring,: Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Pfiser: Membership on an entity’s Board of Directors or advisory committees. Meeks:Grifols: Membership on an entity’s Board of Directors or advisory committees; Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Consultancy; Baxter: Membership on an entity’s Board of Directors or advisory committees. Kenet:BPL: Research Funding; Opko Biologics (Prolor Biotech): Consultancy, Research Funding; Novonordisk: Honoraria; Bayer: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Sanofi Aventis: Membership on an entity’s Board of Directors or advisory committees; Baxter: Research Funding.

P010 : COMPARISON OF TREATMENTS OF THE SERUM FOR ELIMINATING INHIBITORY FACTORS IN THE LUMINEX SINGLE ANTIGEN ASSAY

False-low and false-negative reactions may occur in the Luminex single-antigen bead assay, particularly when the tested serum is fresh. The purpose of this study was to compare our current method for eliminating inhibitory factors, i.e., heat 56 °C, 1 min (H-1 min), with the methods that most of the laboratories worldwide appear to use, i.e., treatment of the serum with DTT or EDTA. Two fresh sera with PRA I >80% (serum #1 and #2) and two fresh sera with PRA II >80% (serum #3 and #4), both with at least one reaction with >15,000 MFI, were tested with LABScreen® Single Antigen Class I and Class II (One Lambda), respectively. Serum treatments: H-1 min: the sera, already in Luminex plate, were heated, in a thermocycler, at 56 °C for 1 min; DTT: final concentration 5 mM, 37 °C, 30 min; EDTA: 10 mM. Differences ⩾50% in MFIs between treated and native sera were considered relevant. The different treatments did not significantly affect the reactions on negative nor positive control beads. Serum #1: 11 beads had higher MFIs in sera treated with H-1 min, DTT or EDTA and all these reactions could be explained by one antibody against an epitope shared by HLA-A2, -68, -69, -24, B-57 and B-58 molecules. Serum #2: no effect of any treatment was observed. Serum #3: six beads (five of them with DQB1 ∗ 03) had higher MFIs in H-1 min and EDTA treated sera (MFIs in native, H-1 min, DTT and EDTA treated sera: 13,854, 22,116, 16,494 and 23,744 respectively). Serum # 4: 18 beads had higher MFIs in sera treated with H-1 min, DTT or EDTA and, surprisingly, six reactions (all with beads with DQA1 ∗ 01/DQB1 ∗ 06) were practically abolished in EDTA-treated serum, being restored by the addition of calcium to the EDTA-treaded serum. Heat-1 min, DTT and EDTA treatments were in general very similar regarding their ability to eliminate the inhibitory factors of the serum. We see the following advantages of H-1 min over DTT or EDTA treatments: (a) the same treatment can be used if we want to test for IgM antibodies, which would not be possible with the DTT treatment; and (b) there is no interference with calcium-dependent epitopes, which can happen with EDTA. To the best of our knowledge, this is the first report of a calcium-dependent anti-HLA antibody or, better yet, of an antibody directed against a Ca[2+]-dependent conformational epitope on an HLA molecule.

Long‐term suppression of EAE relapses by pharmacological impairment of epitope spreading

Immune events sustaining dendritic cell (DC)-dependent epitope spreading (ES) are of key relevance to the development of relapses during multiple sclerosis (MS). Although no drugs are currently available to target ES, its inhibition would represent a major advancement in MS therapy. Inhibitors of the enzyme PARP-1 afford protection in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). These drugs epigenetically impair antigen presentation by DCs, but whether these drugs affect ES is unknown. Here, we investigated whether short-term treatments with these compounds would impair ES, thereby preventing EAE relapses. We used a model of relapsing EAE in SJL mice and also adopted in vivo and ex vivo models of DC-dependent T-cell polarization. The effect of PARP-1 inhibitors on ES was evaluated at the humoral and cellular level. Short-term treatments with PARP-1 inhibitors during the acute phase of relapsing EAE of mice induced, at later times, more tolerogenic DCs, increased numbers of Treg cells and impairment of ES at the humoral and cellular level. These effects are followed by long-lasting reduction of relapse severity and incidence, although drug treatment had been discontinued for several weeks. PARP-1 inhibitors also induced tolerogenic DCs and increased Treg cells number and function in a model of ovalbumin immunization. Our data emphasize the therapeutic potential of PARP-1 inhibitors in the treatment of relapsing-remitting MS and additional ES-driven autoimmune disorders.