Mechanisms of Toll-like receptor tolerance induced by microbial ligands

Some microorganisms can develop tolerance. On the one hand, it allows pathogenic microbes to escape immune surveillance, on the other hand, it provides the possibility to microbiota representatives to colonize different biotopes and build a symbiotic relationship with the host. Complex regulatory interactions between innate and adaptive immune systems as well as stimulation by antigens help microbes control and maintain immunological tolerance. An important role in this process belongs to innate immune cells, which recognize microbial components through pattern-recognition receptors. Toll-like receptors (TLRs) represent the main class of these receptors. Despite the universality of the activated signaling pathways, different cellular responses are induced by interaction of TLRs with microbiota representatives and pathogenic microbes, and they vary during acute and chronic infection. The research on mechanisms underlying the development of TLR tolerance is significant, as the above receptors are involved in a wide range of infectious and noninfectious diseases; they also play an important role in development of allergic diseases, autoimmune diseases, and cancers. The knowledge of TLR tolerance mechanisms can be critically important for development of TLR ligand-based therapeutic agents for treatment and prevention of multiple diseases

Abstract P-44: SARS-CoV-2 Inactivation by Ultraviolet Light Does Not Violate Virus Morphology, Antigenic and Immunogenic Properties

Background: Vaccination is the most effective tool for reducing the morbidity and mortality of COVID-19. Particular interest is the development of whole-virion inactivated vaccines, since such vaccines include a complete set of viral structural proteins. One of the requirements for the whole-virion vaccines is the guarantee of complete virus inactivation while maintaining the native conformation of protective antigens. The aim of this study is to evaluate the effect of ultraviolet (UV) treatment on the morphology, antigenic and immunogenic properties of the SARS-CoV-2. Methods: SARS-CoV-2, strain "Dubrovka" (GenBank No.: MW514307.1), grown in Vero CCL81 cell culture (ATCC). Viral reproduction was monitored by real-time qRT-PCR, ELISA and virus titration in Vero cells. Virus was inactivated by treating with ultraviolet light for 5 minutes using a standard biosafety cabinet UV irradiator. Virus inactivation control was performed by three "blind" passages in Vero cells. Clarified viral material was concentrated on Amicon MWCO 100 kDa (Millipore) columns. Negatively stained with uranyl acetate viral preparation was examined by transmission electron microscopy (TEM). Mice were immunized with a UV-inactivated virus subcutaneously in two variants (5 animals per group) - with and without Freund's adjuvant, twice with an interval between immunizations of 2 weeks. The titer of virus-neutralizing antibodies in mouse sera was determined in Vero cell culture. Results: Preparation of the SARS-CoV-2 coronavirus with a titer of 7.5 lg TCID50/ml and concentration of viral RNA of 8.5 lg copies/ml was obtained in Vero cells. After UV-treatment the presence in the prepapation of SARS-CoV-2 antigen was confirmed by ELISA with a set of COVID-19 convalescent sera. The particles with coronavirus morphodiagnostic signs were imaged by TEM - rounded shape with characteristic spikes of 12-15 nm on the envelope, the diameter of the virion was 90-110 nm. Neutralizing antibodies were detected in the sera of all immunized mice, whereas in animals of the control group neutralizing antibodies were not detected. Neutralizing antibodies titer was significantly higher in animals immunized by a virus with Freund's adjuvant - on average 448, than without adjuvant - 64 (p<0.01). Conclusion: Treatment of SARS-CoV-2 by UV light completely inactivates its infectivity, while retaining the typical coronavirus virions morphology, antigenic properties, and ability to induce in mice a synthesis of neutralizing antibodies

Live attenuated COVID-19 vaccines: approaches to development and prospects for clinical use

Although WHO declared an end to the pandemic, COVID-19 remains a significant public health concern worldwide. Modern vaccines often induce either only humoral or only cellular immunity. Furthermore, new emergent epidemiologically significant SARS-CoV-2 variants and their spread considerably reduce the effectiveness of preventive vaccination. Therefore, there is an urgent need to improve the existing vaccines against COVID-19. One of the promising approaches to the solution of the problem is creation of a "universal" vaccine that would have a cross-protective activity against different antigenic variants of the virus. In this respect, the development of live attenuated vaccine is of special interest, as it can activate not only humoral, but also cell-mediated components of immunity, providing long-term immune response and cross-protection against different variants of the virus. This review highlights the existing approaches to producing attenuated SARS-CoV-2 strains and gives an assessment of their prospects for clinical use. Some researchers use methods of genetic engineering and reverse genetics such as site-directed mutagenesis and codon deoptimization for virus attenuation. Others tend to use traditional approaches focusing on producing virus mutants through extended passaging in cell culture under selective conditions. The gained experience demonstrates great prospects for development of highly effective live-attenuated vaccine against COVID-19

Opportunistic microbiota of breast milk and antimicrobial activity of milk whey at different periods of lactation

Object of study. The evaluation of the interaction between breast milk opportunistic microorganisms abundance and the milk whey antimicrobial activity at different periods of lactation. Materials and methods. 100 samples of breast milk from healthy breastfeeding mothers were inoculated on solid selective media, and then pure cultures of microorganisms were identified by MALDI-TOF mass spectrometry. The antimicrobial activity of the whey against a model culture of Candida albicans was evaluated by spectrophotometry. Results. 270 isolates represented by 36 species of 13 genera of opportunistic bacteria were obtained. None of the 100 samples contained opportunistic fungi. Staphylococci (7 species) and streptococci (11 species) were predominant. The most common were staphylococci S. epidermidis (70.2%) and S. aureus (20.8%), and streptococci S. mitis (27.7%) and S. oralis (21.8%). The total contamination (median) of opportunistic bacteria in the colostrum was 79 103 CFU/ml, transitional milk 4 103 CFU/ml, mature milk 5 102 CFU/ml. The antimicrobial activity of colostrum whey was 87.489.4%; transitional milk 88.2%; mature milk 63.481.9%. The total contamination had a high inverse correlation with the lactation period (r = 0.806) and a high positive correlation with the antimicrobial activity of whey (r = 0.699). Meanwhile, a significant decrease in contamination was noted after 1 month from the beginning of lactation, while a significant decrease in antimicrobial activity was observed after 8 months. Conclusions. The decrease of the breast milk contamination by opportunistic bacteria during the lactation period was found to be primary compared to the decrease of the whey antimicrobial activity. Thus, changes in the whey antimicrobial protection factors occur in response to changes of the opportunistic microbiota abundance

Biological properties of domestic strain vRub-Ant of rubella virus

Introduction. Rubella is a mild infectious disease affecting mainly children and is caused by the rubella virus, part of the Matonoviridae family, genus Rubivirus. Rubella causes congenital rubella syndrome (CRS) and is the main cause of developmental abnormalities, especially blindness and deafness. There is no specific treatment for rubella and CRS. In order to avoid possible complications from rubella infection, a live attenuated rubella vaccine based on the foreign strain of Wistar RA 27/3 rubella virus is used. However, the actual, more effective and preferred vaccine strain the rubella virus for the Russian Federation is considered to be a viral strain of rubella circulating on its territory. The aim of the study was to study the biological properties of the developed domestic cold-adapted strain vRub-Ant circulating in the territory of the Russian Federation. Materials and methods. Following cell cultures were used in the study human embryo lung diploid cell strain LECH-3, transferable cell line from embryonic kidney cells of green monkeys Vero CCL-81 and Vero ECC, human mesenchymal stem cells, human peripheral blood mononuclear cells (PBMC). Cell cultures were grown on a DMEM/F12 nutrient medium with the addition of 5% fetal bovine serum. Swabs from the pharynx and nasal passages from a child with rubella were used as clinical virus-containing material. Monoclonal anti-idiotypic antibodies m(anti-ID)Ab were used to assess the expression level of alpha/beta and gamma interferon receptors (/ and IFN-R)Ab, imitating the biological effects of alpha/beta and gamma interferons (/ and IFN) of humans. The cultural, virological, immunochemical and serological research methods were applied in the study. Results. Attenuation of the vRub-Ant clinical isolate of rubella virus was carried out for 20 consecutive passages on LECH-3 diploid cells at a reduced temperature of 30C. The main biological markers of attenuation were determined to be ts and ca phenotypes. The avirulence of the attenuated viral strain (att-phenotype) was assessed by the level of expression of / and IFN-R. A lower level of / and IFN-R expression was found on the membranes of human PBMC induced by the vaccine strain vRub-Ant in comparison with the parent wild variant of the rubella virus. This trait,the att phenotype, is characteristic of attenuated viral strains. It has been shown that the vaccine strain vRub-Ant has lost neurotropism and was unable to bind to the membrane receptors of the brain (MRB) of guinea pig embryos, unlike its parent rubella virus strain. The high immunogenicity of the domestic cold-adapted strain vRub-Ant was confirmed by high titers of neutralizing rubella antibodies observed in guinea pigs immunized subcutaneously with one vaccination dose of the virus. Conclusion. A domestic attenuated vaccine strain vRub-Ant of the rubella virus that has the main biological markers of attenuation (ts-ca and att phenotypes) has been developed. The vaccine strain vRub-Ant induces a high levels of neutralizing antibodies in guinea pigs following the immunization with a single vaccination dose of the vaccine. The viral strain vRub-Ant has lost its tropism to the MRB of guinea pig embryos, unlike its parent variant

Molecular epidemiological study of clinical cases of acute hepatitis E in Belarus

Relevance. The frequency of occurrence of anamnestic antibodies to the hepatitis E virus (HEV) in the general population of the Republic of Belarus is 7.3%, which is clearly not consistent with the low incidence of hepatitis E (HE). Most of primary HEV infections remain undiagnosed. The intensive epidemic process of HEV in the Belarusian population is hidden. Conducting epidemiological studies, including genotyping of HEV sequences isolated on the territory of the republic, makes it possible to more accurately characterize the sources of HEV infection and the mechanisms of its transmission. Aim molecular epidemiological study of two cases of acute hepatitis E detected in patients from Belarus. Materials and methods. During 20212022, samples of biological material were obtained from two patients undergoing treatment with an established diagnosis of acute hepatitis E. Serum samples were tested to detect antibodies to HEV using enzyme immunoassay, HEV RNA was detected in fecal samples using nested RT-PCR. The nucleotide sequence was determined by an automatic sequencer using the Sanger method. Analysis of nucleotide sequences, their genotyping, and calculation of evolutionary distances were performed using MEGA X software. Results. The HEV sequence isolated from a pregnant woman who had an epidemiological episode of alimentary contact with raw pork meat is clustered into a common phylogenetic clade with HEV sequence obtained from the patient from Belarus with a history of kidney transplantation and HEV sequences isolated from a domestic pigs. The HEV sequence isolated from a patient with a history of travel to Pakistan belongs to the HEV genotype 1 and joins a clade of HEV sequences isolated in Pakistan, India, Nepal and Mongolia

Virus-inhibitory activity of the antigen complex of opportunistic pathogenic bacteria against SARS-CoV-2

Introduction. The antigen complex of opportunistic pathogenic bacteria (ACOPB) has a protective effect against avian influenza viruses, herpes virus type 2, and other viruses that cause acute respiratory viral infections. In the context of the COVID-19 pandemic, an important task is to find out whether ACOPB has a protective effect against SARS-CoV-2. The purpose of the study was to evaluate in vitro the ACOPB virus-inhibitory activity against the Dubrovka laboratory strain of SARS-CoV-2. Materials and methods. The study was performed using Vero cell line CCL-81, human peripheral blood mononuclear cells (PBMCs), mouse monoclonal anti-idiotypic antibodies structurally mimicking biological effects of human interferons (IFNs), the Dubrovka laboratory strain of SARS-CoV-2. The infectivity of the virus was assessed by two methods: by virus titration using cell cultures and the limiting dilution method when the results are assessed by a cytopathic effect; the second method was a plaque assay. The in vitro virus inhibition test was performed using the cell culture susceptible to SARS-CoV-2; the mixture containing a specific dose of the virus and a two-fold dilution of ACOPB was transferred to the cell culture after the ACOPB medication had interacted with the virus at 4C for 2 hours. The ACOPB virus-inhibitory activity against SARS-CoV-2 was assessed by the functional activity of / and IFN receptors (RIFN) in human PBMCs induced in vitro by ACOPB and the ACOPB mixture with the specific dose of SARS-CoV-2. The RIFN expression level was measured by the indirect membrane immunofluorescence test. Results. Hemagglutination assay using chicken, mouse, guinea pig, and human red blood cells was performed for detection of the SARS-CoV-2 inhibitory protein. The lysate of Vero CCL-81 cells infected with SARS-CoV-2 Dubrovka demonstrated the highest hemagglutination activity with guinea pig red blood cells and low titers of hemagglutination in the virus-containing fluid. The virus inhibition test in the Vero CCL-81 cell culture demonstrated that ACOPB inhibited 10 doses of SARS-CoV-2 Dubrovka with the titer 1 : 32, providing 100% protection of the cell culture for 8 days (the monitoring period). ACOPB induced / and RIFN expression on membranes of human PBMCs in in vitro cultures and decreased RIFN / and expression after its interaction with SARS-CoV-2 Dubrovka. Conclusion. The experimental studies including the virus inhibition test in the cell culture susceptible to SARS-CoV-2 Dubrovka and the indirect membrane immunofluorescence assay using monoclonal anti-idiotypic antibodies mimicking IFN-like properties demonstrated that ACOPB had both an immunomodulatory and a virus-inhibitory effect

Unique Profile of Proinflammatory Cytokines in Plasma of Drug-Naïve Individuals with Advanced HIV/TB Co-Infection

HIV-1 infection is characterized by aberrant immune activation, and infection with M. tuberculosis by an unbalanced production of proinflammatory cytokines. The expression of these cytokines in HIV-1/TB coinfection is still understudied. Here, we aimed to compare the production of proinflammatory cytokines in drug-naive patients coinfected with HIV-1 and M. tuberculosis (HIV/TB) compared to patients with respective monoinfections. Plasma samples of patients with HIV/TB coinfection (n = 36), HIV-1 monoinfection (n = 36), and TB monoinfection (n = 35) and healthy donors (n = 36) were examined for the levels of eight proinflammatory cytokines. Their levels were significantly increased in all patient groups compared to healthy donors. At the same time, a drastic decrease in the plasma levels of IFN-γ, TNF-α, Il-1β, IL-15, and IL-17 was detected in patients with HIV/TB coinfection compared to patients with HIV-1 or TB monoinfections. The plasma levels of IL-17 characterized the TB severity: in HIV/TB-coinfected patients with disseminated TB, plasma levels of IL-17 were eight times lower than in patients with less severe TB forms (infiltrative TB or TB of intrathoracic lymph nodes; p p < 0.0001). Thus, on the contrary to the patients with HIV-1 or TB monoinfections, HIV/TB-coinfected patients had suppressed production of most of the proinflammatory cytokines associated with antimicrobial immune response, specifically of T-cells involved in the containment of both infections. At the same time, they demonstrated an expansion of proinflammatory cytokines known to originate from both hematopoietic and nonhematopoietic cells, and manifest tissue inflammation. In HIV-1/TB coinfection, this leads to the disruption of granuloma formation, contributing to bacterial dissemination and enhancing morbidity and mortality

Phage phiKZ—The First of Giants

The paper covers the history of the discovery and description of phiKZ, the first known giant bacteriophage active on Pseudomonas aeruginosa. It also describes its unique features, especially the characteristic manner of DNA packing in the head around a cylinder-shaped structure (“inner body”), which probably governs an ordered and tight packaging of the phage genome. Important properties of phiKZ-like phages include a wide range of lytic activity and the blue opalescence of their negative colonies, and provide a background for the search and discovery of new P. aeruginosa giant phages. The importance of the phiKZ species and of other giant phage species in practical phage therapy is noted given their broad use in commercial phage preparations

Phage phiKZ—The First of Giants

The paper covers the history of the discovery and description of phiKZ, the first known giant bacteriophage active on Pseudomonas aeruginosa. It also describes its unique features, especially the characteristic manner of DNA packing in the head around a cylinder-shaped structure (&ldquo;inner body&rdquo;), which probably governs an ordered and tight packaging of the phage genome. Important properties of phiKZ-like phages include a wide range of lytic activity and the blue opalescence of their negative colonies, and provide a background for the search and discovery of new P. aeruginosa giant phages. The importance of the phiKZ species and of other giant phage species in practical phage therapy is noted given their broad use in commercial phage preparations