Monoclonal antibody therapy that targets phospholipid-binding protein delays lupus activity in MRL/lpr mice.

Systemic lupus erythematosus is an autoimmune syndrome characterized by the development of autoantibodies to a wide range of antigens. Together with B cells, respective self-reactive T cells have an important contribution in disease progression as being responsible for inflammatory cytokines secretion, B cell activation and promoting amplification of the autoimmune response. Annexin A1 is expressed by many cell types and binds to phospholipids in a Ca2+ -dependent manner. Abnormal expression of annexin A1 was found on activated B and T cells in both murine and human autoimmunity suggesting its potential role as a therapeutic target. In the present study, we have investigated the possibility to suppress autoimmune manifestation in spontaneous mouse model of lupus using anti-annexin A1 antibody. Groups of lupus-prone MRL/lpr mice were treated with the anti-annexin A1 monoclonal antibody, and the disease activity and survival of the animals were following up. Flow cytometry, ELISA assays, and histological and immunofluorescence kidney analyses were used to determine the levels of Annexin A1 expression, cytokines, anti-dsDNA antibodies and kidney injuries. The administration of this monoclonal antibody to MRL/lpr mice resulted in suppression of IgG anti-dsDNA antibody production, modulated IL-10 secretion, decreased disease activity and prolonged survival compared with the control group.

DNA and protein-generated chimeric molecules for delivery of influenza viral epitopes in mouse and humanized NSG transfer models.

Purified subunit viral antigens are weakly immunogenic and stimulate only the antibody but not the T cell-mediated immune response. An alternative approach to inducing protective immunity with small viral peptides may be the targeting of viral epitopes to immunocompetent cells by DNA and protein-engineered vaccines. This review will focus on DNA and protein-generated chimeric molecules carrying engineered fragments specific for activating cell surface co-receptors for inducing protective antiviral immunity. Adjuvanted protein-based vaccine or DNA constructs encoding simultaneously T- and B-cell peptide epitopes from influenza viral hemagglutinin, and scFvs specific for costimulatory immune cell receptors may induce a significant increase of anti-influenza antibody levels and strong CTL activity against virus-infected cells in a manner that mimics the natural infection. Here we summarize the development of several DNA and protein chimeric constructs carrying influenza virus HA317-41 fragment. The generated engineered molecules were used for immunization in intact murine and experimentally humanized NSG mouse models.

Elimination of autoreactive B cells in humanized SCID mouse model of SLE.

Although the exact etiology of systemic lupus erythematosus (SLE) remains elusive, B-cell hyperactivity and production of autoantibodies directed to components of the cell nucleus are a well-established pathogenetic mechanism of the disease. Therefore, the targeted inhibition of DNA-specific B cells is a logical therapeutic approach. The complement receptor type 1 (CR1, CD35) has been shown to suppress human B-cell activation and proliferation after co-cross-linking with the BCR, and may serve as a mediator for negative signal delivery. In order to evaluate this therapeutic approach in a human-like system, we used immune-restricted SCID mice transferred with PBMCs from SLE patients. The tolerance of these humanized SCID mice to native DNA was re-established after administration of a chimeric molecule consisting of a CR1-specific mAb coupled to the decapeptide DWEYSVWLSN that mimics dsDNA. The generated protein-engineered chimera was able to co-cross-link selectively native DNA-specific BCR with the B-cell inhibitory receptor CR1, thus delivering a strong inhibitory signal.

Simultaneous determination of ochratoxin A and enterotoxin A in milk by magnetic nanoparticles based fluorescent immunoassay.

Ochratoxin A (OTA) and staphylococcus enterotoxin A (SEA) are highly toxic contaminants and have induced human health problems. They commonly occur in milk and milk products. A competitive fluorescent immunoassay was developed for rapid and simultaneous determination of these toxins in milk samples. The procedure was based on the competitive immunoreactions between antigens in sample and antigen-fluorescent dye conjugates with immobilised antibodies on magnetic nanoparticles (MNPs). Each monoclonal antibody specifically recognises its corresponding toxin (antigen), and there is no cross-reactivity in the assay. First, monoclonal antibodies against OTA and SEA were produced. The activity of the obtained antibodies was determined by fluorescent-linked immunosorbent assay. Then, the monoclonal antibodies were immobilised on MNPs. The amounts of immobilised anti-OTA antibody and anti-SEA antibody were determined to be 20 and 22 µg mL-1, respectively. The antigen-fluorescent dye conjugates OTA-OVA-ATTO620 and SEA-FITC were prepared. The optimal amount of immobilised antibodies for competitive immunoassay was determined. It was found that the linear range of OTA in buffer was larger (0.001-100 ng mL-1) than the linear range of SEA (0.001-20 ng mL-1). The results for simultaneous determination of OTA and SEA in sixfold diluted milk were almost the same in buffer; the linear range for OTA was from 0.005  to 100 ng mL-1 and for SEA from 0.005  to 20 ng mL-1. The detection limit for both OTA and SEA in milk was 0.004 ng mL-1. The developed method took half the time of the individual assays (20 min). The assay was evaluated using spiked milk samples. The influences of somatic cell count, fat, pH and protein concentration in milk on immunoassay were studied. In summary, this developed immunoassay could provide an effective and rapid approach for detecting multi-toxins in milk samples.

Anti-ANX A1 Antibody Therapy in MRL/lpr Murine Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease characterized by dysfunction of immune regulation, overproduction of inflammatory cytokines and attack on normal tissues by self-reactive cells and antibodies. The main role in the pathogenesis plays the autoreactive tandem of B-T cells, responsible for lupus progression and acceleration. Both activated B and T cells express a phospholipid binding protein Annexin A1 and abnormal levels of the protein were found in murine and human autoimmune syndromes, potentiating its role as a therapeutic target. Here, using anti-annexin A1 antibody we explore its property to modulate the autoimmune response in MRL/lpr mouse model of lupus. Anti-ANX A1 antibody was tested in vitro using spleen cells from MRL/lpr mice to determine the effect on lymphocyte activation, plasma cells differentiation, apoptosis and proliferation by flow cytometry and ELISpot assays. Subsequently, several groups of young (disease-free) and old (sick) MRL/lpr mice were treated with the antibody to determine the levels of panel auto-antibodies and cytokines, T cell arrest and migration. Treatment of splenocytes with anti-ANX A1 antibody inhibited T-cell activation and proliferation, suppressed anti-dsDNA antibody-producing plasma cells and affected B cell apoptosis. Administration of the antibody to MRL/lpr mice resulted to decreased autoantibody levels to various lupus antigens, suppressed T cell migration from lymph nodes and increased the levels of IL4 mRNA compared to the control group. Anti-ANX A1 antibody therapy suppresses B and T cell over-activation and down- modulates disease activity.

Defective oogenesis in mice with pristane-induced model of systemic lupus.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by generation of autoantibodies and severe damage of various organs. The hormonal changes associated with pregnancy and especially estrogen might lead to damage of reproductive function and ovarian quality. We employed a pristane-induced lupus model of Balb/c mice which resembles human lupus in an attempt to follow oogenesis disruption during the disease progression. The integrity of cytoskeletal and chromatin structures was estimated in oocytes derived by hormonally stimulated ovulation in lupus mice and the results were compared with those from healthy mice. Chromatin, tubulin and actin structures in oocytes were detected by Hoechst 33258, anti-alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. All available meiotic spindles were analyzed - in immature (metaphase I) and mature oocytes (metaphase II). The total number of mature oocytes obtained from lupus mice was lower compared to healthy controls. The maturation rate was 9.8 % for lupus mice, 12.7 % for 7-month old controls, and 14.3 % for the young control mice (4 weeks old). Another major difference between the studied groups was the higher percentage of defective metaphase I spindles registered in oocytes derived from lupus mice (60 % normal spindles), while for the young and older controls this proportion was 86 % and 81 %, respectively. No such difference was registered for metaphase II spindles. For both metaphase I and metaphase II oocytes, the proportions of normal actin cap and chromosomal condensation were similar between the experimental groups.

Entangling COVID-19 associated thrombosis into a secondary antiphospholipid antibody syndrome: Diagnostic and therapeutic perspectives (Review).

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) is a novel ß coronavirus that is the etiological agent of the pandemic coronavirus disease 2019 (COVID­19) that at the time of writing (June 16, 2020) has infected almost 6 million people with some 450,000 deaths. These numbers are still rising daily. Most (some 80%) cases of COVID­19 infection are asymptomatic, a substantial number of cases (15%) require hospitalization and an additional fraction of patients (5%) need recovery in intensive care units. Mortality for COVID­19 infection appears to occur globally between 0.1 and 0.5% of infected patients although the frequency of lethality is significantly augmented in the elderly and in patients with other comorbidities. The development of acute respiratory distress syndrome and episodes of thromboembolism that may lead to disseminated intravascular coagulation (DIC) represent the primary causes of lethality during COVID­19 infection. Increasing evidence suggests that thrombotic diathesis is due to multiple derangements of the coagulation system including marked elevation of D­dimer that correlate negatively with survival. We propose here that the thromboembolic events and eventually the development of DIC provoked by SARS­CoV­2 infection may represent a secondary anti­phospholipid antibody syndrome (APS). We will apply both Baconian inductivism and Cartesian deductivism to prove that secondary APS is likely responsible for coagulopathy during the course of COVID­19 infection. Diagnostic and therapeutic implications of this are also discussed.

Intravenous immunoglobulin up-regulates the expression of the inhibitory FcgammaIIB receptor on B cells.

Intravenous immunoglobulin (IVIg) preparations are known to modulate autoimmune/inflammatory diseases through several F(ab')(2)- and Fc-dependent mechanisms. In this study, we show that the in vitro and the in vivo exposure of B lymphocytes from lupus-prone and from healthy mice to IVIg results in an increased expression of their surface inhibitory FcgammaIIB receptors. Further, this exposure enhanced the ability of a chimeric antibody, cross-linking FcgammaRIIB and immunoglobulin receptors on DNA-specific B lymphocytes, to suppress IgG anti-DNA antibody production. F(ab')(2) fragments of IVIg had a similar activity as the intact preparation, whereas Fc fragments had no effect. This study describes a novel approach with clinical relevance for modulating B lymphocyte activity.

Targeting of Influenza Viral Epitopes to Antigen-Presenting Cells by Genetically Engineered Chimeric Molecules in a Humanized NOD SCID Gamma Transfer Model.

Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcγRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals.

Annexin A1 as a target for managing murine pristane-induced systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a polygenic pathological disorder which involves multiple organs. Self-specific B cells play a main role in the lupus pathogenesis by generating autoantibodies as well as by serving as important autoantigen-presenting cells. Autoreactive T lymphocytes, on the other hand, are responsible for B cell activation and proliferation, and cytokine production. Therefore, both factors promote the idea that a down-modulation of activated self-reactive T and B cells involved in the pathogenic immune response is a reasonable approach for SLE therapy. Annexin A1 (ANX A1) is expressed by many cell types and binds to phospholipids in a Ca2+ dependent manner. Abnormal expression of ANX A1 was found on activated B and T cells in both murine and human autoimmunity, suggesting its potential role as a therapeutic target. While its role on T lymphocytes is through formyl peptide receptor-like molecules (FPRL), and the formed ANX A1/FPRL pathway modulates T cell receptor signalling, there is still no fool-proof data available for the role of ANX A1 in B cells. We employed a lupus model of Balb/c mice with pristane-induced SLE which very closely resembles human lupus. In the present study, we investigated the possibility to modulate the autoimmune response in a pristane-induced mouse model of SLE using an anti- ANX A1 antibody. Administration of this monoclonal antibody resulted in the inhibition of T-cell activation and proliferation, suppression of IgG anti-dsDNA antibody-secreting plasma cells and of proteinuria, decreased disease activity and prolonged survival compared to control group.

Built-in adjuvanticity of genetically and protein-engineered chimeric molecules for targeting of influenza A peptide epitopes.

Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.

Selective silencing of autoreactive B lymphocytes-Following the Nature's way.

A novel approach for the selective silencing of targeted autoreactive B lymphocytes is reviewed that mimics the physiological mechanisms for suppressing B cell activity. It is based on the use of bi- or tri-specific chimeric antibodies that cross-link BCRs with a pre-selected antigen-binding specificity with one or more inhibitory types of receptors on the surface of the same disease-associated B lymphocyte. The effect of these engineered antibodies was proved to be specific as they only suppressed the production of the targeted pathological antibodies while sparing those with other specificities. The administration of the chimeric molecules to lupus-prone MRL/lpr mice resulted in decreased levels of disease-associated IgG autoantibodies and of proteinuria, in the prevention of cutaneous lesions, in decreased sizes of the lymphoid organs and in prolonged survival. These results prove that it is indeed possible to selectively silence unwanted B lymphocytes as well as to significantly delay the natural course of a spontaneous antibody-mediated autoimmune disease.

Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice.

The pathological DNA-specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross-links their surface immunoglobulins with the inhibitory FcgammaIIb surface receptors. A hybrid molecule was constructed by coupling the DNA-mimicking DWEYSVWLSN peptide to a monoclonal anti-mouse FcgammaRIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/lpr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti-DNA but not of anti-diphtheria IgG antibody-producing cells. Intravenous infusions with the DNA-peptide antibody chimera to 7-wk-old animals prevented the appearance of IgG anti-DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA-peptide chimeric antibody to 18 wk-old mice with full-blown disease resulted in the maintenance of a flat level of IgG anti-DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis. The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease-associated B cells in autoimmune diseases.

Serum IgM, IgG and IgA block by F(ab')-dependent mechanism the binding of natural IgG autoantibodies from therapeutic immunoglobulin preparations to self-antigens.

Natural polyreactive IgG autoantibodies are present in the plasma of healthy individuals and as a result in pooled therapeutic intravenous immunoglobulin (i.v.Ig) preparations. The spectrum of self-antigens to which these autoantibodies bind, their fate after intravenous infusion and their biological activity are not well understood. The identity of serum proteins that mask binding of natural autoantibodies to self-proteins is a matter of controversy. The spectrum of native serum proteins bound by i.v.Ig was analyzed by two-dimensional electrophoresis. The reactivity of i.v.Ig was directed mainly to circulating immunoglobulins. The binding of the IgG autoantibodies from i.v.Ig to native human liver antigens was blocked not only by a F(ab')2-dependent mechanism by circulating IgM and IgG (as has been previously suggested), but also by serum IgA. This control of anti-self reactivity may be inefficient in some autoimmune diseases.

Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter.

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.