The association of HLA class II gene polymorphism with cellular and humoral factors of vaccine-induced anti-plague immunity

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Introduction

Plague is a particularly dangerous zoonotic, naturally occurring infectious disease caused by Yersinia pestis. In the past, this disease has been the cause of many large-scale epidemics, and it continues to pose a serious public health problem today. Every year, cases of human plague are reported worldwide, some of which are fatal [1–3].

Natural foci of plague are present on virtually all continents, with the exception of Antarctica and Australia [1, 2]. There are 11 foci in Russia, 2 of which exhibit high epizootic activity (the Gorno-Altaisk high-altitude and the Tuva mountain foci). In the village of Kosh-Agach, within the Gorno-Altaisk natural plague focus, 1 case of human bubonic plague has been reported over the past 10 years [4].

Specific prevention (vaccination) of at-risk populations is one of the key components of a comprehensive system of anti-epidemic measures in natural plague foci. In Russia, the primary means of specific prevention is the live plague vaccine (LPV) produced by the Stavropol Research Institute of Plague Control under Rospotrebnadzor, which consists of a lyophilized live culture of the vaccine strain of the plague bacterium Y. pestis EV from the NIIEG lineage and induces a robust and effective post-vaccination immune response lasting up to 1 year [5].

The immune response to plague is based on a cascade of humoral and cellular reactions aimed at presenting Y. pestis to immunocompetent cells, the synthesis of specific antibodies by B-lymphocytes, the activation of T-lymphocytes followed by stimulation of the bactericidal activity of macrophages, and increased production of reactive oxygen species and nitroxyl radicals [6]. Thus, type 1 T-helper cells (Th1) produce cytokines (interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-β), which activate macrophages, granulocytes, and endothelial cells, and also induce the expression of the major histocompatibility complex (MHC) class II, thereby disrupting the survival mechanisms of intracellular pathogens and facilitating their destruction. At the same time, type 2 T-helper cells (Th2), on the contrary, support humoral immunity by producing the cytokines IL-4, IL-5, IL-10, and IL-13, which stimulate B-cell differentiation and antibody synthesis, thereby helping to counteract extracellular pathogens [7]. Therefore, to establish effective protective immunity against plague, the participation of both the cellular and humoral components of the host’s immune system is necessary [6].

IFN-γ is a cytokine primarily produced by Th1 cells, natural killer (NK) cells, cytotoxic T lymphocytes, and dendritic cells, and plays a central role in the development of the Th1-type cellular immune response. It enhances phagocytosis, the bactericidal activity of macrophages, the cytotoxicity of CD8⁺ cells, antigen presentation to T lymphocytes, increases HLA expression, suppresses Th2 activity, and more [8]. IL-4, in contrast, is produced primarily by Th2 cells and plays a key role in the development of humoral immunity and the Th2 response, often counteracting the effects of IFN-γ [9]. Its functions include increasing HLA expression, enhancing B-lymphocyte proliferation and differentiation into plasma cells, alternative activation of macrophages, and others. Thus, these two cytokines regulate different branches of the immune response, and their interaction determines the direction and effectiveness against various threats, including plague.

HLA-II is a component of the HLA complex and plays a key role in the formation and regulation of the immune response, including against plague. Its molecules present extracellular antigens to T-helper cells (CD4⁺), ensure the specificity of the immune response through a unique set of alleles, and regulate the balance between immune activation and tolerance. HLA genes are located on the short arm of chromosome 6, with HLA-DP, HLA-DQ, and HLA-DR as the key loci; their polymorphism forms the basis of individual and population resistance to pathogens but may also be a source of risk for the development of various autoimmune disorders [10–12].

There are few scientific studies examining the relationship between the development of anti-plague immunity and HLA [13–19]. Many aspects of the interaction between the immune system and its individual components with Y. pestis, as well as the overall mechanisms of plague pathogenesis and immunogenesis, remain unclear.

The aim of this study is to investigate the relationship between HLA-II genes and markers of the humoral and cellular components of vaccine-induced immunity in people vaccinated against Y. pestis living in the natural foci of the plague in Siberia.

Materials and methods

This study included 902 generally healthy volunteers residing in areas with active natural foci of plague — the Tuva Mountain region (514 individuals, Khandagait village, Ovur Kozhuun, Republic of Tuva) and the Gorno-Altai high-mountain region (388 individuals, Kosh-Agach village, Kosh-Agach District, Republic of Altai). The inclusion criteria for participants in the study were a history of plague vaccination (no later than 6 months prior) and an age of at least 18 years.

Vaccination against plague for the local population of the villages of Kosh-Agach and Khandagayta was carried out in accordance with the Calendar of Preventive Vaccinations for Epidemic Indications by medical workers of the Kosh-Agach District Hospital and the Ovur Central District Hospital, administered once via the intradermal route using a commercial live attenuated plague vaccine.

Biological material (peripheral venous blood) was collected using vacuum blood collection systems into two tubes containing a coagulation activator and K₂EDTA (Vacutest Kima). Serum was obtained from the blood with the coagulation activator, allowed to stand for at least 30 minutes, and then centrifuged at 3000 rpm.

Quantitative determination of the levels of the major immunoglobulin classes (G, M, A, and E) in blood serum was performed using a solid-phase enzyme-linked immunosorbent assay (ELISA) with the commercial test systems Immunoscreen-G, M, A-ELISA-BEST and Total IgE–ELISA-BEST (Vector-Best CJSC) in accordance with the manufacturer’s instructions. Immunoglobulin concentrations were expressed in mg/mL (G, M, A) and IU/mL (E). To determine the titer of specific class G immunoglobulins to the F1 capsular antigen of the plague bacillus, the ELISA-At-F1 Yersinia pestis test system (Russian Anti-Plague Institute Microb) was used. Results were recorded using a StatFax 4200 reader (Awareness Technology).

Lymphocyte phenotyping was performed by flow cytometry using certified monoclonal antibodies (MCA, Becton Dickinson) in the following panel: CD3-PE-Cy7, CD4-PerCP, CD8-APC-Cy7, CD16-PE, CD19-FITC. A mixture of MAb was prepared in advance. Blood containing K2EDTA anticoagulant and the MAb mixture was mixed on a vortex mixer and incubated at 4°C in the dark for 30 minutes. Erythrocyte lysis was performed using BD FACSTM Lysing Solution (Becton Dickinson). The samples were then centrifuged and washed twice with buffered saline. Analysis of the mAb samples was performed on a BD FACSCanto II flow cytometer (Becton Dickinson) using the BD Diva 6.0 software. At least 10,000 cells were analyzed in each sample. To study the cellular component, the following lymphocyte subpopulations were identified: T lymphocytes (CD3⁺), T helper cells (CD3⁺CD4⁺), cytotoxic T lymphocytes (CD3⁺CD8⁺), NK cells (CD3^–CD16⁺), and B lymphocytes (CD3^–CD19⁺). The immunoregulatory index (IRI) was calculated as the ratio of CD3⁺CD4⁺ to CD3⁺CD8⁺.

HLA-II typing was performed using commercial reagent kits for genotyping the DRB1, DQB1, and DQA1 genes (DNA-Technology) via real-time polymerase chain reaction (PCR). DNA was first extracted from peripheral blood cells using K₂EDTA anticoagulant with the Proba-Rapid-Genetics reagent kits (DNA-Technology) according to the instructions for use. Results were detected on the DTprime” thermocycler (DNA-Technology) using RealTime_PCR v. 7.3 software.

Statistical data analysis was performed using Statistica v. 13.0 (TIBCO Software Inc.) and Excel 2013 (Microsoft Corp.) and the nonparametric Mann–Whitney and Kruskal–Wallis tests for paired and multiple comparisons, respectively, since the data samples did not follow a normal distribution and their variances were not equal. The results were presented as the median (Me) and interquartile range [Q₁; Q₃]. Differences were considered statistically significant p <  0.05.

Results

The results of the survey (Fig. 1) showed that the group of volunteers consisted of Tuvans, Kazakhs, Altai people, and Russians. Women made up the majority of the group. Most of the volunteers (90%) were between the ages of 26 and 60. The group included medical professionals, civil servants, teachers, workers, and unemployed individuals.

 

Fig. 1. Characteristics of the surveyed people vaccinated with live plague vaccine by gender (a), age (b), ethnicity (c), and occupation (d).

 

During HLA-II typing of volunteers at the HLA-DRB1 locus, the range of distribution across allelic groups (Fig. 2 a) was 0.6–14.1% — the most common group was the *13 allelic variant (n = 254), while the smallest group consisted of volunteers with the *16 (n = 11). For HLA-DQA1, 280 individuals had the *01:01 allele, 261 had *01:02, 178 had *01:03, 178 had *02:01, 333 had *03:01, 48 had *04:01, 507 had *05:01, and 19 had *06:01 (Fig. 2, b). For the HLA-DQB1 locus, the distribution was as follows: *02 (n = 316), *03:01 (n = 405), *03:02 (n = 124), *03:03 (n = 122), *03:04 (n = 1), *03:05 (n = 17), *04:01/*04:02 (n = 79), *05:01 (n = 175), *05:02/*05:04 (n = 68), *05:03 (n = 61), *06:01 (n = 73), and *06:02–8 (n = 363) (Fig. 2, c).

 

Fig. 2. Allele frequency distribution of the HLA-II genes DRB1 (a), DQA1 (b), and DQB1 (c) in individuals vaccinated with a live plague vaccine

 

Homozygosity has been identified as a potentially negative factor in the production of specific antibodies, the recognition of a wide range of pathogens, immunological surveillance, and the development of various diseases [20–22], for the HLA-DRB1 gene in 67 (7.4%) individuals across 11 allelic groups (*01, *03, *04, *07, *08, *09, *11, *12, *13, *14, and *15); HLA-DQA1 — in 138 (15.3%) volunteers across 6 alleles (*01:01, *01:02, *01:03, *02:01, *03:01, and *05:01); DQB1 — in 82 (9.1%) across 7 alleles (*02, *03:02, *03:03, *03:05, *04:01/*04:02, *06:01, and *06:02-8). No homozygotes for alleles *10 and *16 at the HLA-DRB1 locus, *04:01 and *06:01 (HLA-DQA1), or *03:01, *03:04, *05:01, *05:02/*05:04, and *05:03 (HLA-DQB1) were not identified among the study participants.

Table 1 presents the results of a comparative analysis of the main immunoglobulin classes (E, M, G, and A), as well as the titers of specific antibodies to the Y. pestis F1 capsular antigen in the blood serum of vaccinated Y. pestis volunteers with different HLA-II gene alleles. Significant (p <  0.05) intergroup differences were identified in the concentrations of all immunoglobulin classes studied and in the titers of specific IgG antibodies to the F1 antigen of Y. pestis among carriers of specific HLA-II alleles. For example, significant differences in IgE levels were observed in 30 of the 33 allelic variants studied (p <  0.05). The highest median levels were found in groups of volunteers with DQA1*01:03 (24 IU/mL), DRB1*12 (21 IU/mL), *13 (23 IU/mL), and *14 (21 IU/mL), and DQB1*03:05 (45 IU/mL) and *06:02-8 (24 IU/mL). Moreover, in the case of DQB1*03:05, the level is exceptionally high and significant, indicating a possible association with a more pronounced Th2-mediated (atopic) immune response to vaccination with live plague vaccine.

 

Table 1. Intergroup differences in immunoglobulin levels among individuals vaccinated with live plague vaccine with different HLA-II alleles, Me [Q₁; Q₃]

Locus	Allele groups	IgE, IU/mL	IgМ, mg/mL	IgG, mg/mL	IgA, mg/mL	IgG titer to the F1 antigen of Y. pestis
Reference values		<  100	0,4–2,3	7–16	0,7–4,0	> 1:80
DRB1	*01	15.0 [2.0; 152.6]+	1.9 [0.6; 3.5]+	12.7 [5.0; 26.8]+	1.9 [0.8; 4.9]+	1:101 (0–1:320)
	*03	12.5 [2.0; 79.0]+	1.5 [0.6; 3.4]+	12.5 [4.4; 26.0]+	1.6 [0.7; 3.4]+	1:80 (0–1:320)
	*04	11.0 [0.1; 100.0]+	1.9 [0.7; 4.1]+	14.0 [4.7; 27.0]+	2.1 [0.8; 4.9]+	1:142 (0–1:320)
	*07	15.0 [2.0; 122.5]+	1.8 [0.5; 3.3]+	12.8 [5.7; 23.1]+	1.9 [0.8; 3.8]+	1:135 (0–1:320)+
	*08	15.5 [2.0; 235.0]+	1.8 [0.6; 4.0]+	10.7 [4.3; 25.8]+	1.9 [0.8; 4.5]+	1:125 (0–1:320)+
	*09	10.0 [0.7; 50.0]+	1.5 [0.5; 4.5]+	13.6 [5.6; 29.4]+	2.1 [0.9; 4.9]+	1:188 (0–1:640)+
	*10	10.6 [0.4; 70.0]+	1.3 [0.4; 2.8]+	9.4 [4.2; 19.0]+	1.6 [0.6; 3.7]+	1:138 (0–1:320)
	*11	20.0 [3.0; 110.0]+	1.5 [0.5; 3.2]+	12.9 [5.0; 26.2]+	1.9 [0.7; 4.5]	1:149 (0–1:320)
	*12	21.0 [1.8; 315.0]+	1.5 [0.5; 2.9]+	10.4 [4.0; 24.2]+	2.2 [0.7; 4.7]+	1:133 (0–1:320)
	*13	23.0 [1.0; 196.0]+	1.8 [0.7; 3.9]+	11.0 [4.8; 26.8]+	1.9 [0.8; 4.5]+	1:163 (0–1:320)+
	*14	21.0 [2.0; 190.0]+	1.7 [0.6; 3.7]+	14.0 [4.4; 28.8]+	1.9 [0.8; 4.4]+	1:169 (0–1:320)+
	*15	17.5 [0; 170.0]+	1.6 [0.6; 3.3]+	12.7 [5; 27.5]+	1.9 [0.7; 4.3]+	1:162 (0–1:640)+
	*16	14.5 [2.0; 26.5]+	1.7 [0.5; 2.8]+	8.8 [5.4; 17.4]	2.2 [0.5; 3.0]	1:160 (0–1:160)
DQA1	*01:01	16.8 [1.5; 166.0]+	1.7 [0.6; 3.3]+	12.3 [4.7; 26.2]+	1.9 [0.8; 4.2]	1:101 (0–1:320)
	*01:02	19.8 [1.0; 147.5]+	1.7 [0.7; 4.2]+	12.0 [4.8; 26.8]+	1.9 [0.7; 4.4]	1:101 (0–1:640)
	*01:03	24.0 [1.7; 380.0]+	1.7 [0.7; 3.3]+	11.2 [5.0; 26.8]+	1.9 [0.8; 4.7]	1:80 (0–1:320)
	*02:01	14.0 [1.0; 145.0]+	1.8 [0.5; 3.5]+	12.5 [5.6; 22.2]+	1.9 [0.7; 3.8]+	1:80 (0–1:320)
	*03:01	10.0 [0.7; 81.0]+	1.7 [0.6; 4.1]+	14.0 [5.0; 27.5]+	1.9 [0.8; 4.7]	1:80 (0–1:320)
	*04:01	20.0 [3.0; 196.0]	1.8 [0.9; 5.0]+	8.8 [4.4; 16.0]+	2.3 [1.1; 5.7]+	1:101 (0–1:320)
	*05:01	15.0 [1.9; 145.0]+	1.6 [0.6; 3.3]+	11.8 [4.4; 26.2]+	1.9 [0.7; 4.4]+	1:80 (0–1:320)
	*06:01	25.0 [1.0; 60.0]	0.8 [0.2; 2.3]+	12.7 [6.3; 34.8]+	1.4 [0.7; 3.8]+	1:91 (0–1:320)
DQB1	*02	13.0 [2.0; 110.0]+	1.7 [0.6; 3.3]+	12.7 [5.2; 23.2]+	1.7 [0.7; 3.6]+	1:80 (0–1:320)+
	*03:01	15.0 [1.0; 110.0]+	1.6 [0.5; 3.5]+	12.5 [4.4; 27.2]+	1.9 [0.7; 4.6]+	1:80 (0–1:320)+
	*03:02	9.0 [0.6; 80.0]+	1.7 [0.6; 4.7]+	14.0 [5.0; 27.0]+	1.9 [0.9; 4.5]	1:80 (0–1:320)+
	*03:03	10.0 [0.7; 50.0]+	1.4 [0.5; 3.5]+	12.7 [5.0; 28.8]+	2.1 [0.9; 4.7]+	1:101 (0–1:640)
	*03:05	45.0 [8.0; 117.0]+	1.7 [0.6; 5.6]	11.4 [4.6; 18.0]	2.5 [1.1; 8.2]+	1:101 (1:80–1:320)+
	*04:01/*04:02	21.5 [2.9; 190.0]+	1.8 [0.9; 4.1]+	9.5 [4.6; 21.6]+	2.0 [1.1; 4.9]+	1:101 (0–1:320)
	*05:01	15.0 [1.0; 155.0]+	1.8 [0.6; 3.5]	13.2 [5.0; 26.2]+	1.9 [0.8; 4.6]+	1:101 (0–1:320)
	*05:02/*05:04	20.5 [0; 84.0]	1.4 [0.6; 3.1]+	10.2 [4.4; 23.4]	1.9 [0.7; 3.5]+	1:101 (0–1:640)
	*05:03	18.5 [2.4; 190.0]+	1.7 [0.5; 3.3]	11.9 [4.5; 26.4]+	2.0 [0.4; 4.0]	1:101 (0–1:320)
	*06:01	20.0 [0.5; 555.0]+	1.8 [0.7; 2.9]	11.4 [5.7; 28.8]	1.9 [0.8; 4.7]	1:80 (0–1:640)+
	*06:02-8	24.0 [1.2; 192.0]+	1.7 [0.7; 3.9]+	11.6 [4.8; 26.8]+	1.9 [0.7; 4.4]+	1:101 (0–1:480)+

Note. ⁺p <  0.05 compared to the values for the other allelic groups.

 

Statistically significant differences in IgM concentrations were observed across 21 allele groups (p <  0.05; Table 1). The lowest median value (0.8 mg/mL) was recorded for DQA1*06:01, whereas for most other alleles, median IgM levels ranged from 1.3 to 1.9 mg/mL, which may reflect a relative attenuation of the primary humoral response in DQA1*06:01 carriers, including against the live plague vaccine.

IgG concentrations differed significantly across the 28 allele groups (p <  0.05). The lowest medians were observed in DQA1*04:01 (8.8 mg/mL), DRB1*10 (9.4 mg/mL), and DQB1*04:01/*04:02 (9.5 mg/mL), which potentially indicates a less pronounced secondary humoral response and a general tendency toward lower antibody production in carriers of these alleles following vaccination with live plague vaccine.

IgA levels are stable and within the reference range for most groups. Significant changes in levels were detected less frequently, in only 26 alleles (p <  0.05; Table 1). The highest level was observed in DQB1*03:05 (2.5 mg/mL), indicating a possible enhancement of secretory humoral immunity in this group following vaccination with live plague vaccine.

Elevated median titers of specific IgG antibodies to the F1 antigen were observed for 15 alleles. The highest values were observed in volunteers with allele groups DRB1*09 (1:188), *13 (1:163), *14 (1:169), and *15 (1:162). Alleles DRB1*07, *08, *09, *13, *14, and *15, as well as variants DQB1*02, *03:01, *03:02, *03:05, *06:01, and DQB1*06:02-8, may be associated with more pronounced specific post-vaccination humoral immunity.

A comparative analysis of the median values of major lymphocyte subpopulations revealed statistically significant differences (p <  0.05) in almost all HLA-II allele groups (Table 2). In a certain subset of the samples, the median CD3⁺CD4⁺ cell counts were at the lower limit of the reference range or even below it, which may indicate that vaccination with live plague vaccine has a general effect on reducing the proportion of helper T cells.

 

Table 2. Intergroup differences in subpopulation composition among individuals vaccinated with live plague vaccine with different HLA-II alleles, Me [Q₁; Q₃]

Locus	Allele groups	CD3+- lymphocytes, %	CD3+CD4+- lymphocytes, %	CD3+CD8+- lymphocytes, %	IRI	CD16+- lymphocytes, %	CD19+- lymphocytes, %
Reference values		58–76	36–55	17–37	1.2–2.6	6–26	7–17
DRB1	*01	70.7 [50.7; 79.4]	39.8 [27.0; 48.0]+	26.0 [16.7; 37.0]+	1.4 [0.8; 2.5]+	13.0 [6.3; 29.2]	8.9 [5.1; 14.2]+
	*03	70.7 [58.4; 79.5]+	36.0 [25.3; 47.3]+	30.0 [20.5; 40.0]+	1.1 [0.6; 2.3]+	11.9 [3.3; 25.1]+	10.9 [5.5; 17.5]+
	*04	71.0 [54.0; 79.7]+	37.2 [25.9; 47.0]+	27.8 [19.6; 38.3]+	1.3 [0.7; 2.1]+	13.5 [4.2; 24.2]+	10.0 [5.2; 17.0]
	*07	71.0 [57.1; 79.0]+	36.8 [27.5; 47.0]+	27.5 [20.2; 41.0]+	1.3 [0.7; 2.1]+	13.4 [6.6; 23.3]+	9.6 [5.5; 15.4]+
	*08	69.7 [56.0; 79.6]	38.0 [28.0; 48.1]+	27.0 [17.0; 37.9]+	1.3 [0.8; 2.3]+	13.5 [3.4; 25.9]	10.0 [4.8; 13.0]+
	*09	67.3 [49.0; 76.7]+	35.0 [21.4; 48.0]+	27.4 [16.6; 37.0]+	1.2 [0.6; 2.2]+	15.2 [6.5; 29.8]+	9.9 [5.7; 15.2]+
	*10	71.2 [58.0; 78.0]+	38.2 [26.4; 50.0]+	26.5 [19.9; 42.0]+	1.4 [0.7; 2.4]+	12.5 [4.6; 24.7]+	10.2 [5.3; 16.7]
	*11	69.0 [52.0; 78.0]+	36.0 [26.4; 47.0]+	26.0 [16.7; 39.0]+	1.3 [0.7; 2.4]+	13.1 [6.6; 25.1]	11.0 [6.5; 18.1]+
	*12	67.7 [54.8; 77.7]+	36.7 [24.7; 46.0]+	25.2 [18.5; 36.9]+	1.4 [0.8; 2.1]+	13.9 [8.2; 25.5]+	10.6 [5.2; 19.1]
	*13	67.2 [52.4; 79.0]+	35.0 [23.8; 46.2]+	26.6 [18.7; 39.0]+	1.3 [0.6; 2.1]+	15.2 [6.7; 28.5]+	9.4 [5.3; 14.6]+
	*14	66.1 [50.7; 80.0]+	35.2 [24.0; 47.0]+	26.0 [19.2; 38.5]+	1.3 [0.7; 2.0]+	13.9 [4.6; 27.2]+	9.4 [5.9; 19.2]
	*15	69.0 [52.0; 77.0]+	34.7 [23.0; 45.5]+	29.3 [16.8; 39.4]+	1.2 [0.7; 2.3]+	14.2 [6.5; 25.7]+	10.1 [6.6; 16.4]+
	*16	74.2 [69.4; 77.7]+	43.6 [36.3; 50.4]+	24.4 [18.8; 27.6]+	1.7 [1.4; 2.7]+	12.5 [7.4; 17.1]+	10.5 [7.7; 15.4]+
DQA1	*01:01	68.0 [51.8; 79.0]+	36.2 [25.0; 47.2]+	25.8 [18.0; 38.2]+	1.3 [0.7; 2.2]+	13.7 [4.5; 29.4]+	9.5 [5.1; 18.1]
	*01:02	70.0 [56.2; 78.6]+	35.4 [24.0; 46.1]+	29.3 [20.0; 39.0]+	1.2 [0.7; 2.1]+	13.7 [6.5; 25.1]+	9.8 [6.3; 15.8]
	*01:03	65.2 [52.0; 78.7]+	33.9 [23.8; 45.8]+	25.6 [16.7; 39.7]	1.4 [0.6; 2.3]	15.7 [7.0; 29.3]+	9.8 [5.3; 14.6]+
	*02:01	71.0 [56.3; 79.0]+	37.0 [27.1; 47.0] +	27.1 [20.0; 40.0]	1.3 [0.7; 2.2]	14.1 [6.5; 22.8]+	9.7 [5.7; 15.7]
	*03:01	70.0 [53.9; 78.7]+	36.8 [25.0; 48.0]+	27.4 [18.7; 37.0]	1.3 [0.7; 2.2]	14.0 [5.3; 26.0]+	10.0 [5.2; 16.2]
	*04:01	71.5 [58.4; 80.7]+	39.0 [26.6; 48.1]+	27.0 [14.0; 40.0]	1.3 [0.6; 3.3]	13.4 [7.5; 21.0]+	10.1 [5.4; 15.0]
	*05:01	69.0 [54.0; 79.0]+	36.0 [26.0; 47.0]+	27.4 [19.0; 39.0]	1.3 [0.7; 2.3]	13.5 [5.6; 25.8]+	10.4 [5.9; 17.5]+
	*06:01	66.0 [36.9; 74.1]+	31.0 [17.5; 44.3]+	25.0 [14.8; 39.0]	1.2 [0.7; 1.8]	14.3 [7.7; 46.2]+	10.5 [5.6; 14.1]
DQB1	*02	70.7 [57.5; 78.7]+	36.0 [26.2; 47.0]+	29.7 [21.0; 41.0]+	1.2 [0.6; 2.1]+	13.2 [5.7; 23.9]+	10.2 [5.2; 16.1]+
	*03:01	69.0 [53.1; 78.1]+	36.1 [25.7; 47.0]+	26.0 [18.0; 38.0]+	1.3 [0.7; 2.2]+	13.8 [5.5; 26.1]+	10.3 [5.9; 17.5]+
	*03:02	71.0 [59.4; 79.4]+	38.2 [29.6; 48.2]+	28.2 [20.0; 35.2]+	1.3 [0.8; 2.1]+	10.6 [3.0; 23.5]+	10.0 [5.2; 15.2]+
	*03:03	67.3 [51.9; 76.3]+	35.2 [21.9; 48.0]+	27.1 [16.5; 36.0]+	1.2 [0.7; 2.4]+	15.5 [7.2; 29.8]+	9.8 [5.8; 15.0]+
	*03:05	74.6 [58.4; 82.2]+	33.7 [25.3; 43.2]+	37.9 [23.4; 44.2]+	0.9 [0.6; 1.6]+	8.2 [6.6; 12.1]+	12.7 [7.7; 14.2]+
	*04:01/*04:02	71.3 [56.0; 81.6]+	40.4 [25.9; 51.5]+	25.3 [15.3; 39.7]+	1.5 [0.7; 2.5]+	13.1 [6.3; 21.0]+	10.5 [5.4; 17.0]+
	*05:01	69.0 [52.0; 79.4]+	37.8 [27.0; 48.1]+	25.1 [18.0; 37.2]+	1.4 [0.8; 2.5]+	13.6 [4.9; 29.7]+	9.3 [5.1; 15.7]+
	*05:02/*05:04	70.2 [51.5; 79.5]+	35.9 [22.5; 45.8]+	25.0 [18.8; 40.9]+	1.3 [0.6; 2.0]+	13.9 [6.1; 23.5]+	9.3 [5.2; 17.0]+
	*05:03	64.8 [49.2; 76.8]+	35.5 [23.1; 47.0]+	25.1 [14.1; 36.8]+	1.5 [0.8; 2.2]+	15.1 [2.3; 32.5]+	12.0 [6.3; 20.6]+
	*06:01	64.5 [51.9; 76.7]+	33.0 [21.5; 43.0]+	26.8 [12.1; 39.8]+	1.2 [0.6; 2.3]+	15.8 [8.3; 26.3]+	9.8 [6.5; 16.3]
	*06:02-8	68.4 [53.6; 78.5]+	35.0 [23.9; 45.8]+	28.0 [19.9; 39.0]+	1.2 [0.7; 2.1]+	14.5 [6.5; 26.4]+	9.6 [5.6; 15.1]+

Note. ⁺p <  0.05 compared to the values for the other allelic groups

 

For most allelic variants at the DRB1 locus (*01, *03, *04, *07, *09, *10, *11, *12, *13, *14, *15), moderate yet statistically significant changes were observed, consisting of an increase in the proportion of CD3⁺ cells and a shift in the CD4⁺/CD8⁺ ratio toward a moderate predominance of the cytotoxic component, which was reflected in a trend toward a decrease in the IRI value to 1.1–1.4. The DRB1*16 allele should be considered significant, as carriers of this allele had the highest proportion of T-helper cells — 43.6 [36.3; 50.4]% — while simultaneously exhibiting a lower proportion of cytotoxic T lymphocytes — 24.4 [18.8; 27.6]% — and the highest IRI value among all alleles — 1.7 [1.4; 2.7].

For the DQA1 locus, the most pronounced changes were observed in carriers of the *06:01 allele, characterized by reduced levels of both CD3⁺ cells and their CD3⁺CD4⁺ subpopulation, which was accompanied by a decrease in the IRI to 1.2. In contrast, volunteers with the DQA1*04:01 allele showed an increased proportion of T-helper cells, whereas for the other variants (*01:01–*05:01), an increase in the percentage of NK cells was observed, remaining within the physiological range.

In individuals with the *03:05 allele combination at the DQB1 locus, the median percentage of CD3⁺CD8⁺ lymphocytes was found to reach 37.9%, exceeding the upper limit of the reference range, while the IRI decreased to 0.9 [0.6; 1.6], indicating a cytotoxic type of immune response. The *04:01/*04:02 allelic pair, in contrast, caused a helper shift (CD3⁺CD4⁺ cells — 40.4% and IRI — 1.5). For *05:03 and *06:01, a moderate reduction in the total T-cell pool was observed (CD3⁺ cell content approximately 65%) with a simultaneous increase in NK cells to 15.1–15.8%.

Discussion

We have previously demonstrated an association between HLA-II gene alleles and the levels of IL-4 and IFN-γ secretion in the blood of high-risk individuals vaccinated/revaccinated with live plague vaccine, living in the natural foci of the disease — the Gorno-Altai highlands and the Tuva mountains [18]. Statistically significant differences in IL-4 production were found in individuals with allele groups *08, *09, *10, *12, *15, and *16 at the HLA-DRB1 locus, and *01:01, *01:02, *03:01, *04:01, *05:01, and *06:01 at the HLA-DQA1 locus, and *02, *03:01, *03:02, *03:05, *05:01, and *06:02–8 at the HLA-DQB1 locus, which may indicate a significant influence of HLA-II gene polymorphism on the body’s immunological reactivity in the context of specific plague prevention. With regard to IFN-γ secretion, allelic groups *04, *08, *14, and *15 were identified at the HLA-DRB1 locus, *02:01 and *03:01 at the HLA-DQA1 locus, and *02, *03:01, *03:02, and *06:02-8 at the HLA-DQB1 locus. Furthermore, O.M. Kudryavtseva et al. identified correlations between the HLA-DRB1*01, *03, *07, and *11 with the production of the anti-inflammatory cytokine IL-10, as well as *04 and *12 with the production of the pro-inflammatory tumor necrosis factor-α [15]. J.A. Musson et al., following immunization with the F1 capsid antigen, identified a number of HLA epitopes restricted by the DR1 molecule, with the T-cell immune response biased toward a single immunodominant epitope near the C-terminus of the Caf1 protein exhibiting non-specific binding to HLA-II [17].

Our results indicate a statistically significant contribution of HLA-II allelic polymorphism to the variability and individuality of humoral immune responses in populations living in the natural foci of Siberian plague. The highest frequency of established significant effects on IgE may reflect the role of HLA-associated mechanisms of allergen and antigen presentation (as in the case of DQB1*03:05), whereas the association of DRB1*09, *13, *14, and *15 with elevated titers of specific IgG points to their potential significance in the formation of post-vaccination immunity. This partially echoes our study [18], which established a correlation between these allelic groups and the production of IL-4 and IFN-γ.

Among the HLA-DRB1 allele groups, *07, *08, *09, *10, *12, *13, *14, and *15 stand out strikingly; for HLA-DQA1, *01:03, *04:01, and *06:01; and for HLA-DQB1, *02, *03:01, *03:02, *03:05, *04:01/*04:02, *06:01, and *06:02-8, in whose samples the levels of total and specific immunoglobulins in serum differ significantly from those of other groups, which is also consistent with previously obtained data on IL-4 levels [18].

Statistically significant differences in blood cell subpopulation composition were observed in virtually every allele group. However, among DRB1 alleles in all samples except *08, as well as for DQA1 alleles *04:01 and *06:01, and among DQB1 alleles *03:05, *04:01/*04:02, *05:03, and *06:01, were significantly distinguished by the total content of T lymphocytes, T helper cells, cytotoxic T lymphocytes, the IRI value, and NK cells.

It has been shown that HLA-II gene polymorphism determines the quantitative and qualitative characteristics of the T-cell and NK-cell components of immunity in individuals vaccinated/revaccinated with live plague vaccine. Carriers of the DRB1*16 allele exhibit a profile characteristic of a Th-oriented response, which is potentially favorable for the development of humoral immunity. In turn, the DQB1*03:05 allele is associated with a predominance of cytotoxic T cells and reduced IRI, which may provide increased efficacy against intracellular pathogens, including Y. pestis, and may also be associated with a risk of more pronounced inflammatory reactions.

Conclusion

Alleles *09, *10, *12, *13, *14, and *15 (DRB1), *04:01 (DQA1), and *03:05 (DQB1) are significant genetic factors involved in the regulatory mechanisms of both cellular and humoral immune responses during the development of vaccine-induced immunity against plague.

Alleles of the DRB1*16 and DQB1*03:05 genes cause significant deviations of key immunological parameters beyond the reference ranges, which allows them to be considered as potential genetic markers of the direction of the cellular immune response during vaccination with live plague vaccine.

The identified probable associations of DRB1*09, *13, *14, and *15 with elevated titers of specific IgG antibodies indicate their significance in the development of vaccine-induced humoral immunity.

Most of the differences we identified in humoral and cellular immunity parameters among the compared allelic groups were detected at the DRB1 locus, which is consistent with the results of other researchers [17].

Nevertheless, it is important to note that the small sample sizes for certain HLA alleles (DQA1*06:01, DQB1*03:04, etc.) may be considered limiting factors in this study, as they could contribute to statistical errors and distort the results. Therefore, to clarify the role of both individual alleles and entire HLA-II haplotypes in the formation of vaccine-induced anti-plague immunity, further research is needed to refine and deepen our understanding of the mechanisms underlying the variability of immune responses to specific plague prophylaxis.

About the authors

Konstantin M. Korytov

Irkutsk Anti-Plague Research Institute

Author for correspondence.
Email: konstmikhkor@yandex.ru
ORCID iD: 0000-0003-1137-6049

senior researcher, Pathophysiological laboratory

Russian Federation, Irkutsk

Valentina I. Dubrovina

Irkutsk Anti-Plague Research Institute

Email: dubrovina-valya@mail.ru
ORCID iD: 0000-0001-8561-6207

Dr. Sci. (Biol.), Head, Pathophysiological laboratory

Russian Federation, Irkutsk

Anna B. Pyatidesyatnikova

Irkutsk Anti-Plague Research Institute

Email: 50anechka@mail.ru
ORCID iD: 0000-0002-6381-4517

researcher, Pathophysiological laboratory

Russian Federation, Irkutsk

Sergey V. Balakhonov

Irkutsk Anti-Plague Research Institute

Email: balakhonov.irk@mail.ru
ORCID iD: 0000-0003-4201-5828

Dr. Sc. (Med.), Professor, Director

Russian Federation, Irkutsk

References

Supplementary files