Laboratory of Immunochemistry

CD45 phosphatase controls the phosphorylation level of a number of receptors and signaling molecules in lymphocytes and is thus an important regulator of the activation process of lymphocytes. However, what controls the activity of CD45 itself remains unclear to date. The so-called "CD45 associated protein" (CD45-AP) can claim to be a regulator. This protein forms a supramolecular complex not only with CD45, but also interacts with the CD4 coreceptor and Lck kinase. There are good reasons to believe that the CD45-AP protein performs some important but as yet unknown functions in the cell. First of all, the association of CD45-AP with CD45, CD4 and Lck is obviously not accidental, but reflects the close interactions between these molecules. Secondly, it is known that CD45-AP protein can be phosphorylated, and this is an indication of its possible regulatory role of CD45-AP protein. Thirdly, the degree of its phosphorylation depends on the activation of the cell. The aim of the project is to clarify the function of the CD45-AP molecule. The CD45-AP protein does not belong to any known protein family and has no homologues. The domain organization of this protein is unknown, and the function has not been determined, therefore, completely new scientific tasks will have to be solved in this study.

The aim of the project is not only to develop a specific treatment for birch pollen allergy, but also, for the first time in the world, to lay the foundation for prevention and immunity strategies to prevent the development of allergies during school age.

Monoclonal antibodies have proven effective in the treatment of a number of oncological and autoimmune diseases. Currently, therapeutic antibodies of human origin are obtained by phage display or using hybridomic technology using mice transgenic by human Ig. However, these approaches are either time-consuming or difficult to access, which limits the range of antibodies generated. The problem of rapid production of therapeutic antibodies can be solved if an effective way of immortalizing human B lymphocytes in vitro is found. It is known that human T-lymphocyte clones are easily obtained in the presence of interleukin 2 and antigen-presenting cells loaded with the target antigen. Attempts to force human B lymphocytes to proliferate in vitro culture have been made for a long time, for example, using the Epstein-Barr virus in combination with ligands of toll-like receptors TLR-7 and TLR-9 (Lanzavecchia 2018). Despite some progress in this direction, the proliferative potential of human B cells remains limited. Recently, Kwakkenbos et al. (2016) demonstrated that interleukin 21 (IL21) in combination with the CD40L molecule expressed on the surface of feeder cells induces powerful proliferation of human B cells. In our preliminary experiments, we found that under these conditions, B cells actively divide, secrete Ig and acquire the phenotype of activated lymphocytes, but by about day 10, the proliferation potential decreases and lymphocytes die by apoptosis. It can be assumed that the combined effect of stimulation and anti-apoptotic reprogramming will overcome the proliferative barrier. To obtain overexpression of the BCL-6 and BCL-XL anti-apoptotic genes, we will use tape and/or retroviral delivery. The novelty of the project lies in the fact that in addition to the known stimuli IL21 and CD40L, we will use the BAFF factor (B-cell activating factor), which selectively increases the survival of plasmoblasts. In addition, we will create a seedless B-lymphocyte activation system. To do this, a recombinant CD40L protein will be obtained, which is fused with the Fc fragment of Ig through an isoleucine zipper (Fig. 1, zajavka.pdf). This will make it possible to obtain the CD40L molecule in hexameric form, which is necessary for its effective action outside the feeder cells. The chimeric CD40L-Fc sorbed onto plastic in the presence of soluble IL21 will stimulate human B lymphocytes. The absence of feeder cells will simplify the stimulation procedure and facilitate the early selection of the most affine clones. In this way, we will be able to combine stimulation with the cloning and testing of antigen-specific B lymphocytes. To obtain antigen-specific immortalized B clones from the blood of volunteers immunized with one of the approved vaccines or who have had an infectious disease, plasmoblasts will be isolated, which will be stimulated and reprogrammed as described above. The antigenic specificity of B-cell clones will be characterized in enzyme immunoassay, Western Blot and ELISpot tests with vaccine antigen. Using high-throughput sequencing, we will compare the repertoires of variable Ig genes before and after stimulation. This will answer the question of whether IL21/CD40L stimulation is accompanied by the process of hypermutations in the Ig genes. In addition, complete DNA sequences of conjugated light and heavy Ig chains will be determined in selected B-cell clones by Sanger sequencing. Using these sequences, it will be possible to produce antigen-specific recombinant antibodies. Three laboratories specializing in immunology, molecular biology and functional genomics will participate in the study. The result of the work will be the creation of a new method of stimulation and immortalization of human B lymphocytes, which will make it easy in the future to obtain therapeutically suitable antibodies of completely human origin.

At the moment, therapeutic antibodies to all known strains of coronavirus, including omicron, have been obtained, and the main task is to obtain universal antibodies that will recognize all possible variants of the virus. The S-protein, which is necessary for the virus to enter human cells, is constantly being replaced, and thus the virus escapes destruction by antibodies, so the main goal is to obtain antibodies to precisely those sections of the S-protein that remain common to all variants of the virus. It can be expected that drugs based on such antibodies will be able to successfully prevent infection or severe disease in people from high-risk groups: the elderly, cancer patients, people with various immunodeficiency, etc.

To predict the further spread of COVID-19, knowledge about the duration of immunological memory for coronavirus antigens is necessary. The effectiveness of the vaccines being developed will also be determined by the resistance of immunological memory against SARS-CoV-2. Acquired immunity consists of three components: humoral, as well as T- and B-cell memory. All three components are involved in the creation and maintenance of immunological protection against SARS-CoV-2. The published data mainly relate to humoral immunological memory for SARS-CoV-2. They indicate that antibody-level memory persists for at least several months. It has also been shown that the T-cell response to SARS-CoV-2 is quite productive. The least studied issue remains the formation and duration of B-cell memory after a coronavirus infection. A number of publications have described memory B cells that form shortly after COVID-19, but in these works memory B cells were used exclusively as a source of Ig genes for the subsequent production of therapeutic monoclonal antibodies. The issues of formation and duration of B-cell memory were not considered in these works. Recently, we conducted a study of B-cell immunity in the acute phase of SARS-CoV-2 infection. The study included more than 100 patients with COVID-19. We found that memory B cells are actively formed during the acute phase of SARS-CoV-2 infection. It has also been shown that memory B cells, when stimulated, are able to differentiate into antibody-secreting cells that produce virus-binding and virus-neutralizing antibodies. As far as we know, this is the first study in which the quantitative determination of SARS-CoV-2 specific antibody-secreting cells was performed using the ELISpot method. The data we have obtained are a good starting point for studying long-term B-cell memory after a coronavirus infection. The project will study the dynamics of memory B cells in patients who have been ill with COVID-19 for 3 years. First of all, the study will include former patients for whom memory B cells have already been identified during the acute phase of COVID-19. Testing of memory B cells will be carried out using the ELISpot method developed by us using the recombinant surface Spike protein from the SARS-CoV-2 virus as an antigen. Since memory B cells are resting lymphocytes, to stimulate antibody production, we will activate B cells in vitro for 7 days using a toll-like receptor agonist 9 or a combination of IL-21/CD40L. In parallel, we will also determine the virus-binding and virus-neutralizing activity of serum antibodies. Testing of virus-neutralizing activity will be carried out using a biosafety system based on a lentivirus pseudotyped with S protein from SARS-CoV-2. We will compare the dynamics of virus-specific memory B cells with the kinetics of humoral immunological memory. We will stain antigen-specific memory B cells with coronavirus Spike protein conjugated with phycoerythrin. Antigen-specific lymphocytes will be sorted and Ig genes will be sequenced from single cells. This will allow us to determine the dynamics of the B-cell receptor repertoire. The project will be implemented in close cooperation with the FNCC FMBA clinic, which has now been completely redesigned into an infection center for the treatment of COVID-19. The obtained data on B-cell immunological memory will help in building an epidemiological model of coronavirus infection, as well as to answer the question of the possibility of reinfection with coronavirus. The results of the project will also be important for evaluating the effectiveness of the vaccines being developed against SARS-CoV-2.