Features of the functioning of B-2 lymphocytes in patients with common variable immune deficiency in post-vaccination immunity to SARS-CoV-2

Introduction

Inborn errors of immunity (primary immunodeficiencies (PIDs)), being genetic disorders of the immune system [1][2], are considered to be one of the important problems of modern clinical immunology. More than half of all PIDs are due to antibody formation defects, among which common variable immunodeficiency (CVID) dominates [3-5]. During the SARS-CoV-2 pandemic, the problems of controlling the infection course in patients with PIDs acquired special relevance [6]. In particular, patients with CVID (PIDs associated with impaired formation of a complete humoral immune response and high susceptibility to infections) require increased attention when choosing treatment tactics in order to minimize the consequences of infection [7]. Meanwhile, as the pandemic progressed, it became apparent that mortality among patients with PIDs did not differ from that in the general population, and congenital defects caused by the dysregulation of the immune response served as a risk factor for severe COVID-19. Among humoral PIDs, CVID appeared to be the most vulnerable precisely from these positions: in this variant of PIDs, the main cause of death was a violation of immune regulation, and not infection [8-10]. In addition, it is noted that in another variant of humoral PIDs (X-linked agammaglobulinemia), the likelihood of a severe clinical course of COVID-19 is lower than in CVID due to the absence of B lymphocytes and, accordingly, the absence of the contribution of this cell population to the development of "cytokine storm” [11]. In this regard, the issue of COVID-19 vaccination in a cohort of patients with CVID has acquired particular relevance.

The research was aimed at characterizing B2 lymphocytes in patients with CVID in the process of forming a post-vaccination response to a peptide vaccine against SARS-CoV-2.

Materials and methods

The research included 10 patients with CVID (the mean age was 42±17 years); the diagnosis was verified in accordance with the ESID criteria and federal clinical guidelines for the diagnosis of patients with humoral PIDs. All patients received IVIg maintenance at a dose of 0.4 g/kg of body weight monthly. For the immunoprophylaxis of COVID-19, the recombinant peptide EpiVacCorona vaccine was used, consisting of conserved S-protein epitopes conjugated to a carrier protein (N-protein fragment) of SARS-CoV-2 adsorbed on aluminum hydroxide (registration certificate number LP-006504). The vaccine was administered according to the instructions intramuscularly at a dose of 0.5 ml twice with an interval of 21 days. The parameters of the immune system of patients were assessed before vaccination, 21 days after the first dose, 21 days after the second injection, and 30 days after this period. Phenotypic characterization of peripheral blood B cells was performed by flow cytometry (Cytomics FC 500, USA). When analyzing the parameters of B lymphocytes, the relative (in relation to the total number of peripheral blood lymphocytes) content of B2 lymphocytes (CD3^–CD19⁺CD5^-) and their subpopulations was defined: naive B lymphocytes (CD19⁺CD27^–), switched memory B cells (CD19⁺CD27⁺IgD^–IgM^–), unswitched memory B cells (CD19⁺CD27⁺IgD⁺IgM⁺), regulatory B cells (CD19⁺CD24⁺CD38⁺), and plasmablasts (CD19⁺CD27⁺CD38⁺). The monoclonal antibodies with multicolored labeling manufactured by Beckman Coulter (USA) were used in the research. In order to assess the content of specific antibodies to SARS-CoV-2 in the blood serum, enzyme immunoassay and SARS-CoV-2-IgG test systems, quantitative-ELISA-BEST (JSC Vector-Best), N-CoV-2-IgG PS test systems (Saint Petersburg Pasteur Institute) and SARS-CoV-2-IgG-Vector test systems (The State Research Center of Virology and Biotechnology Vector) were used. Immediately before vaccination and at each subsequent visit during dynamic monitoring of patients, a study was made of the biological material of the upper respiratory tract (pharyngeal and nasal swab) using a kit of reagents COVID-2019 Amp for the detection of SARS-CoV-2 RNA by means of real-time PCR. In order to determine the control values, all of the listed parameters of the immune system were evaluated during the examination of 10 practically healthy blood donors. Statistical processing of the obtained results was performed using the STATISTICA 10.0 program (USA). The results were described by means of calculating the median (Me) and the interquantile range of values between 25 and 75 percentiles, which was presented in the text as Me [LQ; UQ]. The analyzed indicators did not have a normal distribution (the Shapiro-Wilk test was used). The Wilcoxon test was used for the comparative analysis of the groups. Differences were considered significant at p<0.05.

Results

Analysis of the data on the characteristics of the specific humoral immune response in patients of the observation group showed that none of the parameters for assessing specific antibodies (to the peptide antigens of the vaccine, to the N- and S-proteins of SARS-CoV-2) gave positive results. At the same time, the dynamics of the parameters of B2-lymphocytes functioning in response to the introduction of peptide antigens (Table) were recorded.

It should be noted that in patients with CVID before immunization, when comparing the results of immunophenotyping of B lymphocytes to the corresponding parameters of the healthy group, a decrease in the relative content of B2 lymphocytes with phenotypes of both unswitched and switched memory B cells was revealed, as well as an almost four-fold decrease in circulating plasmablasts. These changes served as one of the diagnostic criteria for CVID. At the same time, in the observed group of patients, the relative number of the total pool of B2 lymphocytes, as well as naive B cells, was within the reference values; however, the proportion of regulatory B cells significantly exceeded the corresponding parameter of the comparison group (Table).

In response to the introduction of the first dose of the vaccine after 21 days, patients with CVID showed a statistically significant increase in circulating unswitched memory B cells and, to a greater extent, switched memory B cells, as well as plasmablasts, compared with the initial state, with a two-fold decrease in the proportion of regulatory B lymphocytes (Table).

The spectrum of changes in the B2 lymphocytes subpopulation composition three weeks after the second injection of the vaccine was more diverse and included an increase in the relative number of the total pool of B2 lymphocytes, naive B cells, and memory B cells with a switch in the synthesis of immunoglobulins and plasmablasts, and a progressive decrease in the proportion of regulatory B cells (Table). One month after this observation period (72 days after triggering the post-vaccination immune response), the differences in comparison with the initial parameters consisted in an increase in the number of switched memory B cells, plasmablasts, and a decrease in the number of regulatory B cells. Comparison of data on the B2 lymphocytes subpopulation composition in patients with CVID in this period with the control values of practically healthy people demonstrated differences in the form of a decrease in the relative number of unswitched memory B cells, as in the period before vaccination. At the same time, the content of switched memory B cells exceeded that of the control group, and the number of plasmablasts and regulatory B cells met the control criteria.

It should be noted that all research participants, with the exception of one patient, who received the full course of the EpiVacCorona vaccine series, had no clinical manifestation of respiratory infections during the follow-up year. One patient experienced a novel coronavirus infection after close family contact one week after the second dose of the vaccine. After six weeks from the clinical manifestation of COVID-19, specific antibodies to SARS-CoV-2 class M antigens were detected in the patient: the positive rate of IgM equaled 4.73%, and the specific IgG value was equal to 43 BAU/ml. After four months, the corresponding parameters were 3.55% for IgM and 487 BAU/ml for IgG.

Discussion

Research data are consistent with the published studies confirming the feasibility of attempts to influence the immune system of patients with CVID through RNA vaccines [12]. In contrast to the above examples, in this research, for the prevention of COVID-19, a recombinant vaccine was used, containing, among other things, a peptide component of the N-protein, suggesting the involvement of a cellular link in the development of post-vaccination immunity, which is important in the case of the antiviral immunity development under conditions of a genetic disorder in antibody response. ELISA diagnostics of specific antibodies in the post-vaccination immune response showed negative results in the research. These data did not contradict the data of other studies describing the possibility of a specific response to SARS-CoV-2 infection in patients with CVID, in contrast to post-vaccination humoral immunity [13]. During the research, the dynamics of B2 lymphocytes involvement in response to the peptide vaccine was also traced. The reaction of B cells to vaccination was manifested by an increase in the number of circulating memory B2 lymphocytes, including those with a phenotypic variant of immunoglobulins switched for the synthesis of various isotypes. The probability of an increase in plasmablasts, the immediate precursors of antibody production, in response to the introduction of the vaccine was demonstrated. The research also revealed the fact that the percentage of switched memory B cells in patients could change over time. The research conclusions coincide with the recommendations of a number of authors on the need to continue assessing the immunophenotypic characteristics of B cells after the diagnosis of CVID is established due to the possible variability of memory cell switching [14][15]. It is logical to assume that pre-stimulation with peptide antigens during the vaccination period of a study participant who fell ill with COVID-19 was important in the direction of differentiation of B2 lymphocytes under conditions of subsequent contact with the virus, and provided the possibility of developing a specific humoral immune response in a patient with CVID. The research conclusions echo the opinion of Quinti, Locatelli, and Carsetti that the involvement of innate immune response factors to viral expansion leads to the triggering of regulatory mechanisms that induce a full-fledged response even in conditions of initial immune system dysfunction. According to these authors, the fact that the level of specific antibodies in those patients who recovered after vaccination significantly increases proves the fact of the maturation of memory B cells in patients with CVID and during the post-vaccination response [16].

Conclusion

During the development of a post-vaccination response to the peptide vaccine against SARS-CoV-2 in patients with humoral PIDs (CVID), the subpopulation composition of B lymphocytes is transformed by means of an increase in the number of circulating memory B2 lymphocytes, including those with the phenotypic variant of switched to the synthesis of various classes of immunoglobulins.