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BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.
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Endopeptidasas , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas , Ratones Endogámicos BALB C , Animales , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Ratones , Endopeptidasas/farmacología , Endopeptidasas/administración & dosificación , Bacterias Gramnegativas/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/administración & dosificación , Ratas , Masculino , Ingeniería de Proteínas/métodosRESUMEN
Seasonal influenza remains a serious global health problem, leading to high mortality rates among the elderly and individuals with comorbidities. Vaccination is generally accepted as the most effective strategy for influenza prevention. While current influenza vaccines are effective, they still have limitations, including narrow specificity for certain serological variants, which may result in a mismatch between vaccine antigens and circulating strains. Additionally, the rapid variability of the virus poses challenges in providing extended protection beyond a single season. Therefore, mRNA technology is particularly promising for influenza prevention, as it enables the rapid development of multivalent vaccines and allows for quick updates of their antigenic composition. mRNA vaccines have already proven successful in preventing COVID-19 by eliciting rapid cellular and humoral immune responses. In this study, we present the development of a trivalent mRNA vaccine candidate, evaluate its immunogenicity using the hemagglutination inhibition assay, ELISA, and assess its efficacy in animals. We demonstrate the higher immunogenicity of the mRNA vaccine candidate compared to the inactivated split influenza vaccine and its enhanced ability to generate a cross-specific humoral immune response. These findings highlight the potential mRNA technology in overcoming current limitations of influenza vaccines and hold promise for ensuring greater efficacy in preventing seasonal influenza outbreaks.
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Inmunidad Humoral , Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Vacunas de ARNm , Animales , Femenino , Humanos , Ratones , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunidad Humoral/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones Endogámicos BALB C , Vacunas de ARNm/administración & dosificación , Vacunas de ARNm/química , Vacunas de ARNm/genética , Vacunas de ARNm/inmunología , Estaciones del Año , Factores de Tiempo , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virologíaRESUMEN
SARS-CoV-2 variants have evolved over time in recent years, demonstrating immune evasion of vaccine-induced neutralizing antibodies directed against the original S protein. Updated S-targeted vaccines provide a high level of protection against circulating variants of SARS-CoV-2, but this protection declines over time due to ongoing virus evolution. To achieve a broader protection, novel vaccine candidates involving additional antigens with low mutation rates are currently needed. Based on our recently studied mRNA lipid nanoparticle (mRNA-LNP) platform, we have generated mRNA-LNP encoding SARS-CoV-2 structural proteins M, N, S from different virus variants and studied their immunogenicity separately or in combination in vivo. As a result, all mRNA-LNP vaccine compositions encoding the S and N proteins induced excellent titers of RBD- and N-specific binding antibodies. The T cell responses were mainly specific CD4+ T cell lymphocytes producing IL-2 and TNF-alpha. mRNA-LNP encoding the M protein did not show a high immunogenicity. High neutralizing activity was detected in the sera of mice vaccinated with mRNA-LNP encoding S protein (alone or in combinations) against closely related strains, but was undetectable or significantly lower against an evolutionarily distant variant. Our data showed that the addition of mRNAs encoding S and M antigens to mRNA-N in the vaccine composition enhanced the immunogenicity of mRNA-N and induced a more robust immune response to the N protein. Based on our results, we suggested that the S protein plays a key role in enhancing the immune response to the N protein when they are both encoded in the mRNA-LNP vaccine.
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The spread of COVID-19 continues due to genetic variation in SARS-CoV-2. Highly mutated variants of SARS-CoV-2 have an increased transmissibility and immune evasion. Due to the emergence of various new variants of the virus, there is an urgent need to develop broadly effective specific drugs for therapeutic strategies for the prevention and treatment of COVID-19. Molnupiravir (EIDD-2801, MK-4482), is an orally bioavailable ribonucleoside analogue of ß-D-N4-hydroxycytidine (NHC), has demonstrated efficacy against SARS-CoV-2 and was recently approved for COVID-19 treatment. To improve antiviral potency of NHC, we developed a panel of NHC conjugates with lipophilic vectors and ester derivatives with amino- and carboxylic-acids. Most of the synthesized compounds had comparable or higher (2-20 times) antiviral activity than EIDD-2801, against different lineages of SARS-CoV-2, MERS-CoV, seasonal coronaviruses OC43 and 229E, as well as bovine coronavirus. For further studies, we assessed the most promising compound in terms of activity, simplicity and cost of synthesis - NHC conjugate with phenylpropionic acid (SN_9). SN_9 has shown high efficacy in prophylactic, therapeutic and transmission models of COVID-19 infection in hamsters. Importantly, SN_9 profoundly inhibited virus replication in the lower respiratory tract of hamsters and transgenic mice infected with the Omicron sublineages XBB.1.9.1, XBB.1.16 and EG.5.1.1. These data indicate that SN_9 represents a promising antiviral drug candidate for COVID-19 treatment, and NHC modification strategies deserve further investigation as an approach to develop prodrugs against various coronaviruses.
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COVID-19 , Citidina/análogos & derivados , Hidroxilaminas , SARS-CoV-2 , Ratones , Animales , Bovinos , Humanos , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19RESUMEN
BACKGROUND: Riboflavin (vitamin B2) is one of the most important water-soluble vitamins and a coenzyme involved in many biochemical processes. It has previously been shown that adjuvant therapy with flavin mononucleotide (a water-soluble form of riboflavin) correlates with normalization of clinically relevant immune markers in patients with COVID-19, but the mechanism of this effect remains unclear. Here, the antiviral and anti-inflammatory effects of riboflavin were investigated to elucidate the molecular mechanisms underlying the riboflavin-induced effects. METHODS: Riboflavin was evaluated for recombinant SARS-CoV-2 PLpro inhibition in an enzyme kinetic assay and for direct inhibition of SARS-CoV-2 replication in Vero E6 cells, as well as for anti-inflammatory activity in polysaccharide-induced inflammation models, including endothelial cells in vitro and acute lung inflammation in vivo. RESULTS: For the first time, the ability of riboflavin at high concentrations (above 50 µM) to inhibit SARS-CoV-2 PLpro protease in vitro was demonstrated; however, no inhibition of viral replication in Vero E6 cells in vitro was found. At the same time, riboflavin exerted a pronounced anti-inflammatory effect in the polysaccharide-induced inflammation model, both in vitro, preventing polysaccharide-induced cell death, and in vivo, reducing inflammatory markers (IL-1ß, IL-6, and TNF-α) and normalizing lung histology. CONCLUSIONS: It is concluded that riboflavin reveals anti-inflammatory rather than antiviral activity for SARS-CoV-2 infection. GENERAL SIGNIFICANCE: Riboflavin could be suggested as a promising compound for the therapy of inflammatory diseases of broad origin.
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COVID-19 , Células Endoteliales , Humanos , Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Antivirales/farmacología , Riboflavina/farmacología , Polisacáridos , AguaRESUMEN
The spread of COVID-19 continues, expressed by periodic wave-like increases in morbidity and mortality. The reason for the periodic increases in morbidity is the emergence and spread of novel genetic variants of SARS-CoV-2. A decrease in the efficacy of monoclonal antibodies (mAbs) has been reported, especially against Omicron subvariants. There have been reports of a decrease in the efficacy of specific antiviral drugs as a result of mutations in the genes of non-structural proteins. This indicates the urgent need for practical healthcare to constantly monitor pathogen variability and its effect on the efficacy of preventive and therapeutic drugs. As part of this study, we report the results of the continuous monitoring of COVID-19 in Moscow using genetic and virological methods. As a result of this monitoring, we determined the dominant genetic variants and identified the variants that are most widespread, not only in Moscow, but also in other countries. A collection of viruses from more than 500 SARS-CoV-2 isolates has been obtained and characterized. The genetic lines XBB.1.9.1, XBB.1.9.3, XBB.1.5, XBB.1.16, XBB.2.4, BQ.1.1.45, CH.1.1, and CL.1, representing the greatest concern, were identified among the dominant variants. We studied the in vitro efficacy of mAbs Tixagevimab + Cilgavimab (Evusheld), Sotrovimab, Regdanvimab, Casirivimab + Imdevimab (Ronapreve), and Bebtelovimab, as well as the specific antiviral drugs Remdesivir, Molnupiravir, and Nirmatrelvir, against these genetic lines. At the current stage of the COVID-19 pandemic, the use of mAbs developed against early SARS-CoV-2 variants has little prospect. Specific antiviral drugs retain their activity, but further monitoring is needed to assess the risk of their efficacy being reduced and adjust recommendations for their use.
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Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 µM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05-0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.
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Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Ratas , Animales , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Triptófano , Receptores Nicotínicos/metabolismo , Antiinflamatorios/farmacología , Analgésicos/farmacología , Hiperalgesia , Antiinflamatorios no EsteroideosRESUMEN
A highly effective humoral immune response induced by the Sputnik V vaccine was demonstrated in independent studies, as well as in large-scale post-vaccination follow-up studies. However, the shifts in the cell-mediated immunity induced by Sputnik V vaccination are still under investigation. This study was aimed at estimating the impact of Sputnik V on activating and inhibitory receptors, activation and proliferative senescence markers in NK and T lymphocytes. The effects of Sputnik V were evaluated by the comparison of PBMC samples prior to vaccination, and then three days and three weeks following the second (boost) dose. The prime-boost format of Sputnik V vaccination induced a contraction in the T cell fraction of senescent CD57+ cells and a decrease in HLA-DR-expressing T cells. The proportion of NKG2A+ T cells was down-regulated after vaccination, whereas the PD-1 level was not affected significantly. A temporal increase in activation levels of NK cells and NKT-like cells was recorded, dependent on whether the individuals had COVID-19 prior to vaccination. A short-term elevation of the activating NKG2D and CD16 was observed in NK cells. Overall, the findings of the study are in favor of the Sputnik V vaccine not provoking a dramatic phenotypic rearrangement in T and NK cells, although it induces their slight temporal non-specific activation.
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The spread of COVID-19 infection continues due to the emergence of multiple transmissible and immune-evasive variants of the SARS-CoV-2 virus. Although various vaccines have been developed and several drugs have been approved for the treatment of COVID-19, the development of new drugs to combat COVID-19 is still necessary. In this work, new 5'-O-ester derivatives of N4-hydroxycytidine based on carboxylic acids were developed and synthesized by Steglich esterification. The antiviral activity of the compounds was assessed in vitro-inhibiting the cytopathic effect of HCoV-229E, and three variants of SARS-CoV-2, on huh-7 and Vero E6 cells. Data have shown that most synthesized derivatives exhibit high activity against coronaviruses. In addition, the relationship between the chemical structure of the compounds and their antiviral effect has been established. The obtained results show that the most active compound was conjugate SN_22 based on 3-methyl phenoxyacetic acid. The results of this study indicate the potential advantage of the chemical strategies used to modify NHC as a promising avenue to be explored in vivo, which could lead to the development of drugs with improved pharmacological properties that potently inhibit SARS-CoV-2.
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WHO has declared the outbreak of monkeypox as a public health emergency of international concern. In less than three months, monkeypox was detected in more than 30 000 people and spread to more than 80 countries around the world. It is believed that the immunity formed to smallpox vaccine can protect from monkeypox infection with high efficiency. The widespread use of Vaccinia virus has not been carried out since the 1980s, which raises the question of the level of residual immunity among the population and the identification of groups requiring priority vaccination. We conducted a cross-sectional serological study of remaining immunity among Moscow residents. To do this, a collection of blood serum samples of age group over 30 years old was formed, an in-house ELISA test system was developed, and a virus neutralization protocol was set up. Serum samples were examined for the presence of IgG antibodies against Vaccinia virus (n=2908), as well as for the ability to neutralize plaque formation with a Vaccinia virus MNIIVP-10 strain (n=299). The results indicate the presence of neutralizing antibody titer of 1/20 or more in 33.3 to 53.2% of people older than 45 years. Among people 30-45 years old who probably have not been vaccinated, the proportion with virus neutralizing antibodies ranged from 3.2 to 6.7%. Despite the higher level of antibodies in age group older than 66 years, the proportion of positive samples in this group was slightly lower than in people aged 46-65 years. The results indicate the priority of vaccination in groups younger than 45, and possibly older than 66 years to ensure the protection of the population in case of spread of monkeypox among Moscow residents. The herd immunity level needed to stop the circulation of the virus should be at least 50.25 - 65.28%.
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Enfermedades Transmisibles , Mpox , Orthopoxvirus , Humanos , Adulto , Persona de Mediana Edad , Monkeypox virus , Estudios Transversales , Moscú/epidemiología , Virus Vaccinia , Anticuerpos NeutralizantesRESUMEN
Despite the widespread use of the COVID-19 vaccines, the search for effective antiviral drugs for the treatment of patients infected with SARS-CoV-2 is still relevant. Genetic variability leads to the continued circulation of new variants of concern (VOC). There is a significant decrease in the effectiveness of antibody-based therapy, which raises concerns about the development of new antiviral drugs with a high spectrum of activity against VOCs. We synthesized new analogs of uracil derivatives where uracil was substituted at the N1 and N3 positions. Antiviral activity was studied in Vero E6 cells against VOC, including currently widely circulating SARS-CoV-2 Omicron. All synthesized compounds of the panel showed a wide antiviral effect. In addition, we determined that these compounds inhibit the activity of recombinant SARS-CoV-2 RdRp. Our study suggests that these non-nucleoside uracil-based analogs may be of future use as a treatment for patients infected with circulating SARS-CoV-2 variants.
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Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Vacunas contra la COVID-19 , Humanos , Uracilo/farmacologíaRESUMEN
Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.
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The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell-cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA2s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA2s on ß-coronaviruses. Thus, snake PLA2s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells.
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Fosfolipasas A2/farmacología , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Venenos de Víboras/farmacología , Acoplamiento Viral/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Afinidad de Anticuerpos/efectos de los fármacos , Antivirales/farmacología , Fusión Celular , Línea Celular , Chlorocebus aethiops , Efecto Citopatogénico Viral/efectos de los fármacos , Células HEK293 , Humanos , Modelos Moleculares , Dominios Proteicos/efectos de los fármacos , Resonancia por Plasmón de Superficie , Células Vero , Venenos de Víboras/enzimología , Tratamiento Farmacológico de COVID-19RESUMEN
The search for effective methods to detect patients who excrete a viable virus is one of the urgent tasks of modern biomedicine. In the present study, we examined the diagnostic value of two antigen tests, BIOCREDIT COVID-19 Ag (RapiGEN Inc., Anyang, Korea) and SGTI-flex COVID-19 Ag (Sugentech Inc., Cheongju, Korea), for their diagnostic value in identifying patients who excrete viable SARS-CoV-2. As part of the study, we examined samples from 106 patients who had just been admitted to the hospital and who had undergone quantitative RT-PCR and assessment of viability of SARS-CoV-2 using cell culture. Assessment of the tests' value for detecting samples containing viable virus showed high sensitivity for both tests. Sensitivity was 78.6% (95% CI, from 49.2% to 95.3%) for SGTI-flex COVID-19 Ag and 100% (95% CI, from 76.8% to 100%) for Biocredit COVID-19 Ag. The specificity of rapid tests was significantly higher than that of RT-PCR and was 66.3% (95% CI, from 55.7% to 75.8%) and 67.4% (95% CI, from 56.8% to 76.8%) for SGTI-flex COVID-19 Ag and Biocredit COVID-19 Ag versus 30.4% (95% CI, from 21.3% to 40.9%) obtained for PCR. Thus, for tasks of identifying viable SARS-CoV-2 during screening of conditionally healthy people, as well as monitoring those quarantined, rapid tests show significantly better results.
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Antígenos Virales/análisis , Antígenos Virales/inmunología , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Prueba de Ácido Nucleico para COVID-19 , Femenino , Humanos , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Moscú , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Since the beginning of the 2021 year, all the main six vaccines against COVID-19 have been used in mass vaccination companies around the world. Virus neutralization and epidemiological efficacy drop obtained for several vaccines against the B.1.1.7, B.1.351 P.1, and B.1.617 genotypes are of concern. There is a growing number of reports on mutations in receptor-binding domain (RBD) increasing the transmissibility of the virus and escaping the neutralizing effect of antibodies. The Sputnik V vaccine is currently approved for use in more than 66 countries but its activity against variants of concern (VOC) is not extensively studied yet. Virus-neutralizing activity (VNA) of sera obtained from people vaccinated with Sputnik V in relation to internationally relevant genetic lineages B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.3 and Moscow endemic variants B.1.1.141 (T385I) and B.1.1.317 (S477N, A522S) with mutations in the RBD domain has been assessed. The data obtained indicate no significant differences in VNA against B.1.1.7, B.1.617.3 and local genetic lineages B.1.1.141 (T385I), B.1.1.317 (S477N, A522S) with RBD mutations. For the B.1.351, P.1, and B.1.617.2 statistically significant 3.1-, 2.8-, and 2.5-fold, respectively, VNA reduction was observed. Notably, this decrease is lower than that reported in publications for other vaccines. However, a direct comparative study is necessary for a conclusion. Thus, sera from "Sputnik V"-vaccinated retain neutralizing activity against VOC B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.3 as well as local genetic lineages B.1.1.141 and B.1.1.317 circulating in Moscow.
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In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients' tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.
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Anticuerpos Monoclonales/metabolismo , Antígenos Virales/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos Virales/inmunología , COVID-19/patología , COVID-19/virología , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Hibridomas/citología , Hibridomas/metabolismo , Inmunohistoquímica , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Immune response during sepsis is characterized by hyper-inflammation followed by immunosuppression. The crucial role of macrophages is well-known for both septic stages, since they are involved in immune homeostasis and inflammation, their dysfunction being implicated in immunosuppression. The cholinergic anti-inflammatory pathway mediated by macrophage α7 nicotinic acetylcholine receptor (nAChR) represents possible drug target. Although α7 nAChR activation on macrophages reduces the production of proinflammatory cytokines, the role of these receptors in immunological changes at the cellular level is not fully understood. Using α7 nAChR selective agonist PNU 282,987, we investigated the influence of α7 nAChR activation on the expression of cytokines and, for the first time, of the macrophage membrane markers: cluster of differentiation 14 (CD14), human leukocyte antigen-DR (HLA-DR), CD11b, and CD54. Application of PNU 282,987 to THP-1MÏ (THP-1 derived macrophages) cells led to inward ion currents and Ca2+ increase in cytoplasm showing the presence of functionally active α7 nAChR. Production of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 was estimated in classically activated macrophages (M1) and treatment with PNU 282,987 diminished IL-10 expression. α7 nAChR activation on THP-1MÏ, THP-1M1, and monocyte-derived macrophages (MDMs) increased the expression of HLA-DR, CD54, and CD11b molecules, but decreased CD14 receptor expression, these effects being blocked by alpha (α)-bungarotoxin. Thus, PNU 282,987 enhances the macrophage-mediated immunity via α7 nAChR by regulating expression of their membrane receptors and of cytokines, both playing an important role in preventing immunosuppressive states.
Asunto(s)
Inmunidad Adaptativa/fisiología , Antígenos HLA-DR/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Macrófagos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Inmunidad Adaptativa/efectos de los fármacos , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Citocinas/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Interleucina-10/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Células THP-1 , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/inmunologíaRESUMEN
BACKGROUND: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. METHODS: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. RESULTS: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.
RESUMEN
Many peptide ligands of nicotinic acetylcholine receptors (nAChRs) contain a large number of positively charged amino acid residues, a striking example being conotoxins RgIA and GeXIVA from marine mollusk venom, with an arginine content of >30%. To determine whether peptides built exclusively from arginine residues will interact with different nAChR subtypes or with their structural homologs such as the acetylcholine-binding protein and ligand-binding domain of the nAChR α9 subunit, we synthesized a series of R3, R6, R8, and R16 oligoarginines and investigated their activity by competition with radioiodinated α-bungarotoxin, two-electrode voltage-clamp electrophysiology, and calcium imaging. R6 and longer peptides inhibited muscle-type nAChRs, α7 nAChRs, and α3ß2 nAChRs in the micromolar range. The most efficient inhibition of ion currents was detected for muscle nAChR by R16 (IC50 = 157 nM) and for the α9α10 subtype by R8 and R16 (IC50 = 44 and 120 nM, respectively). Since the R8 affinity for other tested nAChRs was 100-fold lower, R8 appears to be a selective antagonist of α9α10 nAChR. For R8, the electrophysiological and competition experiments indicated the existence of two distinct binding sites on α9α10 nAChR. Since modified oligoarginines and other cationic molecules are widely used as cell-penetrating peptides, we studied several cationic polymers and demonstrated their nAChR inhibitory activity. SIGNIFICANT STATEMENT: By using radioligand analysis, electrophysiology, and calcium imaging, we found that oligoarginine peptides are a new group of inhibitors for muscle nicotinic acetylcholine receptors (nAChRs) and some neuronal nAChRs, the most active being those with 16 and 8 Arg residues. Such compounds and other cationic polymers are cell-penetrating tools for drug delivery, and we also demonstrated the inhibition of nAChRs for several of the latter. Possible positive and negative consequences of such an action should be taken into account.