ИСТИНА

To predict the further spread of COVID-19, knowledge of the duration of immunological memory to coronavirus antigens is needed. The effectiveness of vaccines under development 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 creating and maintaining immunological protection against SARS-CoV-2. The published data mainly relate to the humoral immunological memory to SARS-CoV-2. They indicate that memory at the antibody level persists for at least several months. It has also been shown that the T-cell response to SARS-CoV-2 is also productive. The least studied is the question of the formation and duration of B-cell memory after a coronavirus infection. A number of publications have described memory B cells that are formed shortly after COVID-19, however, 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 the formation, as well as the duration of B-cell memory, were not considered in these works. We recently conducted a study of B-cell immunity in the acute phase of SARS-CoV-2 infection. The study included more than 100 COVID-19 patients. We have 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 [Byazrova et al., Pattern of circulating SARS-CoV-2-specific antibody-secreting and memory B cell generation in patients with acute COVID-19. The article is under review in the journal Clinical & Translational Immunology]. To the best of our knowledge, this is the first study to quantify SARS-CoV-2 specific antibody-secreting cells by ELISpot. Our data are a good starting point for studying long-term B-cell memory after coronavirus infection. The overall design of the project is shown in Fig. 1 (see file Zajavka.pdf). In our project, for 3 years, the dynamics of memory B-cells will be studied in patients who have recovered from COVID-19. The study will primarily include former patients for whom memory B cells have already been identified during the acute phase of COVID-19. Memory B cells will be tested 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 9 agonist 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 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 the coronavirus Spike protein conjugated with phycoerythrin. The antigen-specific lymphocytes will be sorted and the 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 carried out in close cooperation with the FNCC FMBA clinic, which has now been completely redesigned into an infectious disease center for the treatment of COVID-19. The data we have obtained on B-cell immunological memory will help in building an epidemiological model of coronavirus infection, as well as to answer the question about the possibility of reinfection with coronavirus.

Будет оптимизирован разработанный нами ранее тест ELISpot для определения SARS-CoV-2-специфичных В-клеток памяти. Будут получены данные о сохранении В-клеточной памяти против коронавирусных антигенов на временном отрезке в три года. В-клетки памяти будут определены двумя независимыми методами: определение антитело-секретирующих клеток с помощью метода ELISpot, а также определение RBD-связывающих В-клеток с помощью проточной цитометрии. Будет проведено сравнение продолжительности В-клеточной памяти с динамикой гуморальной иммунологической памяти. Будут секвенированы гены Ig из единичных В-клеток памяти. Это позволит оценить динамику репертуара антигенных рецепторов В-клеток памяти. В целом результаты проекта помогут в построении эпидемиологической модели короновирусной инфекции. Полученные результаты помогут ответить на вопрос о возможной реинфекции коронавирусом. Результаты проекта будут также важны для оценки эффективности разрабатываемых вакцин против SARS-CoV-2.