CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in the murine model of the acute respiratory distress syndrome

Introduction: The acute respiratory distress syndrome (ARDS), secondary to viral pneumonitis, is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019)—ongoing SARS-CoV-2 infection— reached more than 0.7 billion registered cases.Methods: Recently, we elaborated a non-surgical and reproducible method of the unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice–a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data read that two C–C chemokine receptor 5 (CCR5) ligands, macrophage inflammatory proteins (MIPs) MIP-1α/CCL3 and MIP-1β/CCL4, are upregulated in this DAD model up to three orders of magnitude compared to the background level.Results: Here, we showed that a nonpeptide compound TAK-779, an antagonist of CCR5/CXCR3, readily prevents DAD in the lung with a single injection of 2.5 mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung and mononuclear infiltration of the wall and lumen of the alveoli in the TAK-779-treated animals. Administration of TAK-779 decreased the 3–5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1α/β, MCP-1, and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779-treated animals.Discussion: Our pre-clinical data suggest that TAK-779 is more effective than the administration of dexamethasone or the anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors for the treatment of virus-induced hyperinflammation syndromes, including COVID-19

Towards effective COVID\u201119 vaccines: Updates, perspectives and challenges (Review)

In the current context of the pandemic triggered by SARS-COV-2, the immunization of the population through vaccination is recognized as a public health priority. In the case of SARS\u2011COV\u20112, the genetic sequencing was done quickly, in one month. Since then, worldwide research has focused on obtaining a vaccine. This has a major economic impact because new technological platforms and advanced genetic engineering procedures are required to obtain a COVID\u201119 vaccine. The most difficult scientific challenge for this future vaccine obtained in the laboratory is the proof of clinical safety and efficacy. The biggest challenge of manufacturing is the construction and validation of production platforms capable of making the vaccine on a large scale

Genetically engineered CD80–pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide–MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols

Diagnostics of autoimmune neurodegeneration using fluorescent probing

© 2018, The Author(s). The discovery of antibody-mediated catalysis was a breakthrough that showed antibody function is not limited to specific binding interactions, and that immunoglobulins (Igs) may also chemically transform their target antigens. Recently, so-called “natural catalytic antibodies” have been intimately linked with several pathologies, where they either protect the organism or contribute to the development of autoimmune abnormalities. Previously, we showed that myelin-reactive autoantibodies from patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE) exhibit the ability to recognize and hydrolyse distinct epitopes within myelin basic protein (MBP). Further, the antibody-mediated cleavage of encephalitogenic MBP peptide 81–103, flanked by two fluorescent proteins, can serve as a novel biomarker for MS. Here, we report the next generation of this biomarker, based on the antibody-mediated degradation of a novel chemically synthesized FRET substrate, comprising the fluorophore Cy5 and the quencher QXL680, interconnected by the MBP peptide 81–99: Cy5-MBP81–99-QXL680. This substrate is degraded upon incubation with either purified antibodies from MS patients but not healthy donors or purified antibodies and splenocytes from EAE but not from non-immunized mice. Data presented herein suggest the elaboration of potential specific, rapid, and sensitive diagnostic criteria of active progressive MS

Exposure to the Epstein-Barr viral antigen latent membrane protein 1 induces myelin-reactive antibodies in vivo

© 2017 Lomakin, Arapidi, Chernov, Ziganshin, Tcyganov, Lyadova, Butenko, Osetrova, Ponomarenko, Telegin, Govorun, Gabibov and Belogurov. Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading

CD206-Targeted Liposomal Myelin Basic Protein Peptides in Patients with Multiple Sclerosis Resistant to First-Line Disease-Modifying Therapies: A First-in-Human, Proof-of-Concept Dose-Escalation Study

© 2016 The Author(s)Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP46–62, MBP124–139 and MBP147–170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS

CD206-Targeted Liposomal Myelin Basic Protein Peptides in Patients with Multiple Sclerosis Resistant to First-Line Disease-Modifying Therapies: A First-in-Human, Proof-of-Concept Dose-Escalation Study

© 2016, The Author(s).Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP46–62, MBP124–139 and MBP147–170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS

Роль иммунных реакций в патогенезе оптической нейропатии при нормотензивной глаукоме

PURPOSE: To analyze molecular and immunological mechanisms of optic neuropathy development in patients with normal tension glaucoma. METHODS: We have been studying serologic indicators of autoantibodies (AB) in patients with normal tension glaucoma (NTG, n=31), which were compared to corresponding parameters in patients with primary open-angle glaucoma (POAG, n=30). The control group consisted of 25 somatically healthy individuals without ophthalmic pathology and clinical symptoms of systemic autoimmune diseases. For the immunological part of the research we used a wide range of antigens: ENO-1, MBP, NSE, Tβ4, α-crystallin, rhodopsin, GAPDH, actin, α-fodrin. The antibody formation in blood serum was determined with the use of the enzyme-linked immunosorbent assay (ELISA). Antibodies concentration in the blood serum was reflected in a spectrophotometric indicator measured in standard units of optical density. RESULTS: Systemic immune disorders in patients with both forms of glaucoma were revealed. In the NTG group the level of AB to rhodopsin decreased from 1.13±0.13 (Mean±SD) to 0.91±0.19 (p=0.00002, pЦЕЛЬ. Изучение роли иммунных реакций в механизмах оптической нейропатии при нормотензивной глаукоме. МЕТОДЫ. Изучали серологические показатели аутоантител (АТ) в крови пациентов с нормотензивной глаукомой (НТГ, n=31); которые сопоставляли с данными при первичной открытоугольной глаукоме (ПОУГ, n=30). Контрольную группу составили 25 соматически здоровых лиц без офтальмопатологии и клинических признаков системных аутоиммунных заболеваний. Для иммунологических исследований использовали широкий спектр аутоантигенов: ENO-1, MBP, NSE, Tβ4, α-кристаллин, родопсин, GAPDH, актин, α-фодрин. Реакции антителообразования в сыворотке крови оценивали методом иммуноферментного анализа. Концентрацию антител в сыворотке отражал спектрофотометрический показатель, который выражался в условных единицах оптической плотности. РЕЗУЛЬТАТЫ. Выявили нарушения системного иммунитета при обеих формах глаукомы. При НТГ снижался уровень АТ к родопсину с 1,13±0,13 (Mean±SD) до 0,91±0,19 (p=0,00002,

A novel expression cassette delivers efficient production of exclusively tetrameric human butyrylcholinesterase with improved pharmacokinetics for protection against organophosphate poisoning

© 2015 Published by Elsevier B.V. Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t1/2;≈2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via "self-processing" viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t1/2; = 32 ± 1.2 h, MRT = 43 ± 2 h). 3D Fluorescent visualization and distribution of 125I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE-/- mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical