Brucellosis is a zoonotic disease caused by Brucella. In the Russian Federation, a live vaccine based on the attenuated strain B. abortus 19BA is used to prevent brucellosis in humans. However, pronounced reactogenicity and the likelihood of developing an infectious process after immunization makes the creation of a safe subunit vaccine an urgent task. For this purpose, we created 47 recombinant brucellosis proteins and assessed their vaccine potential on a mouse cell culture. The aim of the study was to evaluate the immunogenic properties of recombinant Brucella proteins on cell culture and serum of BALB/c mice immunized with a Brucella vaccine strain. Using reverse vaccinology methods, candidate brucellosis proteins were identified, and technology for their production and purification was developed. Forty-seven single and fused recombinant proteins were obtained in the E. coli system of optimal concentration and purity for further use in immune assays. To assess the immunogenicity of recombinant proteins, BALB/c mice were immunized with the B. abortus 19BA vaccine strain, and the blood serum and spleens of the animals were collected on the 30th day of infection. The proteins were used as specific inducers for stimulating lymphocytes in in vitro experiments and antigens for determining serum antibody titers in immune and intact mice. The levels of IFN-γ and IL-4 cytokines in lymphocyte cell supernatants, as well as serum-specific antibody titers, were analyzed by enzyme immunoassay. The ability of 47 recombinant brucellosis proteins to induce the formation of humoral and cellular immune responses was assessed. As a result, 41 proteins activating the synthesis of IFN-γ and eight proteins to which IgG antibodies are formed were identified. Brucellosis proteins capable of inducing a pronounced immune response in immunocompetent cells have been obtained and identified. These proteins can be considered as possible candidates for inclusion in vector, DNA, and subunit vaccines against brucellosis in humans.

The aim of the work was to evaluate the ability of monoclonal antibodies to inhibit the interaction of the receptor binding domain (RBD) in S protein of SARS-CoV-2 virus variants, Wuhan-Hu-1 and BQ 1.1, with the angiotensin-converting receptor 2 (ACE2). Materials and methods. In this study, recombinant RBDs of Wuhan-Hu-1 and BQ 1.1 variants were used as antigens. For mouse monoclonal antibody (mMCA) production, hybridomas were cultured in vivo in BALB/c mice. mMCAs were isolated from ascitic fluid by ammonium sulfate treatment followed by purification through column affinity chromatography with Protein G Sepharose sorbent. The specific activity of mMCAs was assessed by immunoblot with recombinant RBD of Wuhan-Hu-1 variant. To identify the most promising mMCA, the neutralizing activity of mMCA was evaluated by enzyme-linked immunosorbent assay (ELISA) via immobilizing RBD on the surface of a microplate and using ACE2 in the form of horseradish peroxidase conjugate. Recombinant antigens were produced in ExpiCHO-S cell line (Gibco, USA). Results and discussion. Three mMCAs have been described as a result of the study: 5C3, 3F11, 1E6. All antibodies belong to immunoglobulins of subclass G and specifically interact with the RBD in S protein of SARS-CoV-2 virus. The most effective inhibition of the interaction between ACE2 and the RBD of BQ 1.1 strain was observed for murine MCA 3F11 (65 %), while the interaction with the RBD of Wuhan-Hu-1 strain was inhibited by mMCA 5C3 (91 %). The identified characteristics allow for considering the antibodies as potential candidates for the development of antibody-based therapeutics, thus expanding the possibilities of therapy for SARS-CoV-2 virus infection.

Live tularemia vaccine is one of the most effective bacterial vaccines. However, it also has high residual virulence for laboratory animals and may cause adverse reactions in individuals with compromised immune systems. The development of a safe and effective tularemia vaccine is impeded by insufficient understanding of the protection correlates. The aim of this work is to review the literature on the development of post-vaccinal immune responses to live tularemia vaccines and recombinant vaccine candidate strains and to determine the immunological correlates of protection in the formation of specific immunity to Francisella tularensis. This review describes the main aspects of the development of innate and adaptive immune responses to the administration of live tularemia vaccines based on attenuated strains of F. tularensis 15 NIIEG and F. tularensis LVS in humans and in experimental tularemia infection in a mouse model. Studying the mechanisms of adaptive immunity and identifying immunological correlates of protection in experimental tularemia in a murine model is crucial for researching new vaccine strains and improving laboratory methods for assessing the T-cell component of immunity. The main focus is on the study of cellular mechanisms underlying the formation of protective immunity in experimental tularemia, the determination of immunological criteria for its evaluation and the role of identified indicators in long-term protection after the end of the active phase of the immune response induced by immunization with vaccines based on attenuated F. tularensis strains. We discuss the effects of vaccination on the differentiation, functional activity, and duration of specific central and effector CD4+ and CD8+ memory T-cells circulation in humans and mice.

The new coronavirus infection COVID-19 is an acute viral disease that affects primarily the upper respiratory tract. The etiological agent of COVID-19 is the SARS-CoV-2 RNA virus (Coronaviridae family, Betacoronavirus genus, Sarbecovirus subgenus). We have developed a high-affinity human monoclonal antibody, called C6D7-RBD, which is specific to the S protein receptor-binding domain (RBD) from the SARS-CoV-2 Wuhan-Hu-1 strain and exhibits virus-neutralizing activity in a test with recombinant antigens: angiotensin-converting enzyme 2 (ACE2) and RBD.

Introduction: Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies. Methods: Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay. Results: We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusion: The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure.

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease. CD4 + and CD8 + T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (T CM ) and effector memory (T EM ) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG. Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. T EM cells were identified as CD3 + CD4 + CD44 + CD62L - and CD3 + CD8 + CD44 + CD62L - , T CM cells as CD3 + CD4 + CD44 + CD62L + and CD3 + CD8 + CD44 + CD62L + , respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa. Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4 + and CD8 + memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8 + T CM and CD8 + T EM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis .

Оценка эффективности различных адъювантов при получении мышиных моноклональных антител к рецептор-связывающему домену S-белка SARS

Monoclonal antibodies are an established treatment for many illnesses, including cancer, toxicity and infectious diseases. The goal of this work was to optimize the conditions for obtaining hybridomas secreting human monoclonal IgG antibodies. A new protocol has been developed for efficient electrofusion of human B lymphocytes with partner cells. Electrofusion parameters were optimized, and the preferred partner cell line and ratio of cells involved in the fusion were selected. Two myeloma cell lines, K6H6/B5 and SHM-D33, were tested in detail, and the highest fusion efficiency was observed for K6H6/B5. Three hybridomas that secreted fully human monoclonal IgG antibodies were obtained using the optimized electrofusion protocol; the secretion of antibodies was observed for 1 month, which indicates the stability of the clones and the absence of chromosome segregation.

Токсин-нейтрализующая активность моноклональных антител против летального токсина Bacillus anthracisФедеральное бюджетное учреждение науки «Государственный научный центр

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors' blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

Neutralization of the lethal toxin of Bacillus anthracis is an important topic of both fundamental medicine and practical health care, regarding the fight against highly dangerous infections. We have generated a neutralizing monoclonal antibody 1E10 against the lethal toxin of Bacillus anthracis and described the stages of receptor interaction between the protective antigen (PA) and the surface of eukaryotic cells, the formation of PA oligomers, assembly of the lethal toxin (LT), and its translocation by endocytosis into the eukaryotic cell, followed by the formation of a true pore and the release of LT into the cell cytosol. The antibody was shown to act selectively at the stage of interaction between Bacillus anthracis and the eukaryotic cell, and the mechanism of toxin-neutralizing activity of the 1E10 antibody was revealed. The interaction between the 1E10 monoclonal antibody and PA was found to lead to inhibition of the enzymatic activity of the lethal factor (LF), most likely due to a disruption of true pore formation by PA, which blocks the release of LF into the cytosol.

Francisella tularensis is an intracellular bacterium that causes tularemia. Progress in creating a safe and effective vaccine for the prevention of tularemia is challenging due to a lack of knowledge about immunological parameters indicative of protective adaptive immunity. Objective of the research was to assess the effect of modifications of the F. tularensis 15 NIIEG genome on the immunogenic and protective properties of F. tularensis 15/23-1ΔrecA and F. tularensis 15/23-1/sodBΔrecA strains. Materials and methods. Multi-parameter flow cytometry and the measurement of secreted cytokines were used to characterize the responses of mouse spleen lymphocytes in response to re-stimulation of F. tularensis with acid-insoluble complex (AIC) in vitro. Also, the titers of specific antibodies to F. tularensis lipopolysaccharide in blood serum were analyzed by enzyme-linked immunosorbent assay. Results and discussion. It has been shown that immunization with the studied strains led to a significant increase in CD4+ and/or CD8+ T cells capable of expressing functional markers: CD69, CD25 and/or CD28; an increase in the subpopulation of T-helpers synthesizing IFN-γ. In the body of immune mice, a pool of B-lymphocytes was formed, capable of secreting IFN-γ in response to their stimulation with AIC. Immunization with the strain 15/23-1/sodBΔrecA provided 70% protection in mice from intranasal infection with a virulent strain of F. tularensis SchuS4. More pronounced protective properties were associated with the activation of not only B-lymphocytes and T-helpers, but also with the simultaneous activation of cytotoxic T-lymphocytes.

Vaccine prevention of anthrax in Russia is carried out according to epidemiological indications 1 time per year with the use of live anthrax vaccine. The main problem is the lack of a method to control the immunological efficacy of vaccination. The article presents data that analyzes the anthrax human humoral and cellular immunity among donors vaccinated 10–12 months ago with live anthrax vaccine. It was shown that in the blood serum of the most vaccinated donors did not detect antibodies to the protective antigen (PA). However, in all vaccinated patients (with the exception of one donor) T and B lymphocytes circulated in the blood enhanced proliferative activity ant the expression of activation receptors on their cell surface in response to PA in vitro. The obtained data suggested that predominantly specific cellular immunity is persisted in the long-term after vaccination against anthrax.