Distribution of HLA‐DRB1 Alleles and Genotypes With Respect to Plasma Anti‐SARS‐CoV‐2 IgG Titres Among COVID‐19‐Vaccinated Bangladeshi Adults

ABSTRACT

Background

Interindividual variations in vaccine‐induced immunological responses are well‐observed across different populations. This may underlie the highly polymorphic HLA molecules directly involved in antigen presentation to CD4⁺ T cells, leading to the generation of adaptive immunity. With the highest diversity among HLA molecules, HLA‐DRB1 alleles and/or genotypes are often hypothesised to be associated with humoral and cellular immune components, as observed in other antiviral vaccines.

Objective

In this study, we aim to seek an association between HLA‐DRB1 alleles and/or genotypes and plasma anti‐SARS‐CoV‐2 (RBD) IgG levels.

Methods

Here, 98 participants who received two doses of COVID‐19 vaccines were subjected to molecular typing of the HLA‐DRB1 gene in association with the plasma anti‐SARS‐CoV‐2 (RBD) IgG titre.

Results

The seropositive vaccinees had a mean (±SD) anti‐SARS‐CoV‐2 IgG level of 302.89 (±105.64) IU/mL. The most commonly found allele was HLA‐DRB1*15 (28.57%), while other alleles with frequencies above 5% were HLA‐DRB1*07, HLA‐DRB1*12, HLA‐DRB1*10, HLA‐DRB1*13, HLA‐DRB1*04, and HLA‐DRB1*03. The most frequent genotype was HLA‐DRB1*07/15 (16.32%) among these participants. Other genotypes with frequencies above 5% were HLA‐DRB1*10/15, HLA‐DRB1*12/15, HLA‐DRB1*07/12, HLA‐DRB1*13/15, HLA‐DRB1*03/15, HLA‐DRB1*04/10, HLA‐DRB1*07/10, and HLA‐DRB1*07/13. The HLA‐DRB1*15 allele was associated with anti‐SARS‐CoV‐2 IgG levels among the high‐antibody response (HAR) subset for whom more than 180 days (6 months) elapsed post‐vaccination. However, no other alleles or genotypes were found to be directly associated with serological status or plasma anti‐SARS‐CoV‐2 IgG levels among the study participants.

Conclusion

While this study provides insights into the genotypic and allelic distribution of the HLA‐DRB1 gene among the Bangladeshi population, further investigation into allelic haplotypes, including other HLA (Class I and Class II) genes, is warranted to determine inter‐ and intra‐population variability in vaccine‐mediated protection against SARS‐CoV‐2.

1. Introduction

The COVID‐19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) had an unprecedented impact on our lives since its emergence. To curb the pandemic, a widespread vaccination campaign was initiated worldwide by the end of 2020 (Marian 2021). In Bangladesh, the mass vaccination program commenced on 7 February 2021, aiming to develop herd immunity against SARS‐CoV‐2 and curtail its transmission. According to the Directorate General of Health Services (DGHS), Bangladesh, different types of vaccines, including adenoviral vector vaccines (ChAdOx1‐S/Covishield, Ad26.COV2.S, and Sputnik V), mRNA vaccines (BNT162b2 and mRNA‐1273), and inactivated whole virus vaccines (BBIBP‐CorV and CoronaVac), have been administered to individuals. These vaccines develop antibodies against the spike protein of SARS‐CoV‐2 as a signature of humoral immunity (Walls et al. 2020). Precisely, a previous study by our group revealed above 90% seroprevalence in the Bangladeshi population (Saba et al. 2023).

Inter‐individual variation in vaccine‐induced immunological responses against SARS‐CoV‐2 depends on immune‐related genes (Larché 2008). The major histocompatibility complex (MHC), denoted as human leukocyte antigen (HLA), is a cluster of more than 200 genes, known to be the most polymorphic regions of the human genome. HLA molecules are directly involved in the presentation of an extremely diverse repertoire of foreign antigens, modulating the initiation and regulation of immune responses (Blum et al. 2013). T‐cell activation occurs upon encountering processed antigens complexed with HLA molecules. Upon activation, T cells proliferate rapidly and subsequently release cytokines to induce an immune response that recognizes and eliminates cells with this same foreign antigen/HLA complex during subsequent encounters. HLA class II molecules are exclusively expressed on the surface of antigen‐presenting cells (APCs) and are involved in the humoral wing of the adaptive immune response (Mosaad 2015; Poland et al. 2007).

HLA Class II molecules are extremely polymorphic, and over a thousand different HLA Class II alleles are known. Population coverage in relation to MHC variation is further complicated, as different HLA types are expressed at different frequencies in different ethnicities (Quon et al. 2023). Therefore, HLA Class II molecules can be associated with the variation in vaccine responses due to MHC restriction of T‐cell responses (Delshad et al. 2022). Furthermore, among the genes in HLA Class II, DPB1, DQB1, and DRB1 are the most frequently variable (Charonis et al. 2022). According to the IPD‐IMGT/HLA database (www.ebi.ac.uk/ipd/imgt/hla/about/statistics/), the HLA‐DRB1 gene possesses the highest number of alleles among HLA Class II genes (Barker et al. 2023). Therefore, it may be useful to investigate allele‐specific associations of HLA‐DRB1 with the effectiveness of anti‐SARS‐CoV‐2 vaccines.

The vaccine rollout in Bangladesh has been enormously successful, achieving WHO's COVID‐19 immunization coverage of 70% of its population by 2022. Since then, several studies have assessed vaccine‐induced antibody responses among the population. However, the association between HLA alleles and immune responses remains largely unexplored. With an exceptionally high number of alleles and a co‐dominant mode of expression, HLA‐DRB1 presents an important target for studying inter‐individual variation in vaccine‐induced immune responses. Hence, we aimed to investigate the diversity of the HLA‐DRB1 gene and explore its association with COVID‐19 post‐vaccination serology at the population level in Bangladesh.

2. Materials and Methods

2.1. Ethics Statement

This study was approved by the ethical review committee of the Faculty of Biological Sciences, University of Dhaka (Ref#151/Biol. Sci.). Informed consent was obtained from each study participant before blood sample collection.

2.2. Study Design

A total of 98 healthy individuals aged 18 years or above who received at least two doses of any of the following WHO‐approved COVID‐19 vaccines were enrolled in the study: AstraZeneca (adenoviral vector encoding S‐glycoprotein, ChAdOx1‐S), Sinopharm (inactivated whole virus, BBIBP‐CorV), or Moderna (mRNA‐1273) at least a week before sample collection (World Health Organization n.d.). Peripheral blood samples were drawn at a single point in time from each participant. Those reporting flu‐like symptoms 4 weeks before sample collection or a prior positive RT‐PCR test for SARS‐CoV‐2 were excluded. Individuals with diagnoses of acute and chronic infection, for example, cancer, diseases of the immune system, and those undergoing steroid treatment were also excluded.

2.3. Specimen Collection and Processing

Five millilitres of peripheral whole blood were collected from each participant in EDTA vacutainer tubes. Plasma samples were separated immediately by centrifuging at 5000 rpm for 5 min and stored at −80°C until further analysis.

2.4. Quantitative Detection of Anti‐SARS‐CoV‐2 IgG Levels

Levels of anti‐SARS‐CoV‐2 IgG were determined from plasma samples using a DRG SARS‐CoV‐2 (RBD) IgG ELISA (EIA‐6150) kit according to the manufacturer's instructions. This kit detects IgG antibodies against the S1 (RBD) subunit of the spike protein of SARS‐CoV‐2. Test results less than 4.86 DU/mL depicted negative results, while those greater than 5.94 DU/mL were considered positive. Samples resulting between 4.86 and 5.94 DU/mL fell in the grey zone and were also considered seronegative in the present study. IgG levels were expressed as IU/mL by multiplying values in DU/mL by 5.15 (1 DU/mL = 5.15 IU/mL).

2.5. DNA Extraction

Genomic DNA was extracted using the organic solvent‐based DNA extraction method as described previously (Afruza et al. 2013). Extracted DNA was resuspended in TE buffer (1 M Tris, 0.5 M EDTA) and stored at −20°C. The ratio of absorbance at 260 and 280 nm (A _260/280) was used for evaluating the purity of extracted DNA, where ratios of ∼1.8 were considered pure DNA.

2.6. RT‐PCR‐Based Molecular Typing of HLA DRB1

HLA‐DRB1 genotyping was performed using rapid fluorotyping and melt‐curve analysis as described previously (Liu et al. 2007). In this method, specific alleles of the HLA DRB1 gene are determined by a dye‐based RT‐PCR method using allele‐specific primers. Overall, 32 primers, including 10 forward (F) and 20 reverse (R) primers in 21 combinations, were used along with a pair of primers targeting the ERCC5 gene as an internal control. Specific amplifications were ensured using an additional melt‐curve analysis.

2.7. Statistical Analyses

Categorical variables were reported as frequencies (%) and numbers (n). Quantitative continuous variables were expressed as the mean (±SD). Chi‐square tests and Fisher exact tests were performed to assess the statistical association between categorical variables. Associations between categorical and numerical variables were analysed by the Mann‒Whitney U test. Kruskal–Wallis test was conducted to determine the significance of differences between more than two groups. Dunn test was performed post hoc on Kruskal‒Wallis. The Bonferroni method was employed to adjust the p value to correct for multiple testing. Spearman's correlation coefficient was calculated to evaluate the correlation between continuous variables.

The seropositive participants were divided into two age groups as follows: (i) less than or equal to 40 years and (ii) more than 40 years. The seropositive participants were stratified according to the plasma anti‐SARS‐CoV‐2 IgG titre quartiles into the following subsets: (i) low‐antibody response (LAR; IgG < Q1), (ii) medium‐antibody response (MAR; IgG < Q2), (iii) upper antibody response (UAR; IgG < Q3), and (iv) high‐antibody response (HAR; IgG > Q3).

Genotype and allele frequencies of the HLA‐DRB1 gene were calculated as follows:

$$\mathrm{Genotype}\mathrm{frequency}=\frac{\mathit{x}}{\mathit{n}}\times 100$$ (1)

$$\mathrm{Allele}\mathrm{frequency}=\frac{\mathit{x}}{2\mathit{n}}\times 100$$ (2)

where x is the number of individuals having the genotype (1) or allele (2) and n is the total number of participants.

All statistical tests were performed using the R packages tidyverse and rstatix. The propCI function was used to calculate the 95% CI. Statistical significance was set at p < 0.05.

3. Results

3.1. Demographic Characteristics of Study Participants

Among the 98 study participants, 47 were male and 51 were female, with mean (±SD) ages of 43.32 (±10.81) and 40.98 (±11.43) years, respectively (Table 1).

TABLE 1.

Demographic characteristics of the seronegative and seropositive study participants.

Attributes			Serological status
			Seronegative	Seropositive	p value
Study participants		n (%)	31 (31.63)	67 (68.37)
Sex	Male	n (%)	16 (34.04)	31 (65.96)	0.668
	Female	n (%)	15 (29.41)	36 (70.59)
Age		Mean (±SD)	40.548 (±10.25)	42.82 (±11.53)	0.609
Days post‐vaccination		Mean (±SD)	103.16 (±88.81)	141.39 (±79.72)	0.023

Participants who were either seronegative or seropositive for anti‐SARS‐CoV‐2 (RBD) IgG had no history of SARS‐CoV‐2 infection.

3.2. Plasma Anti‐SARS‐CoV‐2 IgG Levels Among Study Participants

Of the 98 study participants, 67 were seropositive (IgG ≥ 30.5 IU/mL), with a mean (±SD) plasma anti‐SARS‐CoV‐2 IgG of 302.89 (±105.64) IU/mL, whereas 31 participants were seronegative (anti‐SARS‐CoV‐2 IgG < 30.5 IU/mL) (Table 2).

TABLE 2.

Plasma anti‐SARS‐CoV‐2 IgG levels among the seropositive study participants classified into groups according to sex, age and the number of days post‐vaccination.

Classification		Mean (±SD) IgG (IU/mL)	p	Adj p value
Sex	Male	299.98 (±105.94)	0.836	0.689
	Female	305.41 (±106.82)
Age group	≤40 years	286.83 (±119.93)	0.441	0.965
	>40 years	315.92 (±92.12)
Days post‐vaccination	<60	265.29 (±104.85)
	60–180	313.03 (±95.17)	0.0798	0.509
	>180	320.16 (±108.48)	0.2304	0.679

Plasma anti‐SARS‐CoV‐2 IgG levels for low (LAR), medium (MAR), upper (UAR), and higher (HAR) antibody response subsets fell within the ranges of 68.02–259.98 IU/mL (Q₁), 259.98–327.46 IU/mL (Q₂), 327.46–380.94 IU/mL (Q₃), and 380.93–468.92 IU/mL (Q₄), respectively.

Among the cohort, 31 male and 36 female participants were seropositive, with mean (±SD) plasma anti‐SARS‐CoV‐2 IgG levels of 299.98 (±105.94) and 305.40 (±106.82) IU/mL, respectively, not varying significantly between the two groups (p = 0.837). Participants' age did not differ significantly between the serogroups (p = 0.609). There was no statistically significant correlation between plasma anti‐SARS‐CoV‐2 IgG levels and seropositive participants' age (r _s = 0.175, p = 0.158). When divided by age groups, plasma anti‐SARS‐CoV‐2 IgG did not vary significantly between the participants aged less than or equal to and more than 40 years (p = 0.441).

The number of days elapsed after administration of the first vaccine dose was significantly higher in the seropositive participants, with a mean (±SD) number of days post‐vaccination of 141.39 (±79.72), compared with a mean (±SD) of 103.16 (±88.81) days in the seronegative group (p = 0.023). However, there was no statistically significant correlation between plasma anti‐SARS‐CoV‐2 IgG levels and the number of days post‐vaccination among the seropositive participants (r _s = 0.211, p = 0.087). The mean (±SD) plasma anti‐SARS‐CoV‐2 IgG levels were significantly higher among participants for whom more than 180 days (6 months) had elapsed post‐vaccination, with a mean (±SD) of 320.15 (±108.48) IU/mL, compared with those for whom less than 60 days (2 months) had elapsed, with a mean (±SD) of 265.29 (±104.85) IU/mL (p = 0.045).

3.3. Allele and Genotype Frequency Distributions of HLA‐DRB1

A total of 12 HLA‐DRB1 alleles and 27 genotypes were identified among the study participants (Table 3).

TABLE 3.

Frequency distribution of HLA‐DRB1 alleles among seronegative and seropositive study participants.

HLA‐DRB1 alleles
Allele	Total n (%)	Seronegative n (%)	Seropositive n (%)	p value	Adj p value
HLA‐DRB1*01	2 (1.02)	1 (1.61)	1 (0.75)	0.21	1
HLA‐DRB1*03	10 (5.1)	4 (6.45)	6 (4.48)
HLA‐DRB1*04	11 (5.61)	2 (3.23)	9 (6.72)
HLA‐DRB1*07	46 (23.47)	11 (17.74)	35 (26.12)
HLA‐DRB1*08	1 (0.51)	0 (0.0)	1 (0.75)
HLA‐DRB1*10	21 (10.71)	8 (12.9)	13 (9.7)
HLA‐DRB1*11	3 (1.53)	3 (4.84)	0 (0.0)
HLA‐DRB1*12	25 (12.76)	7 (11.29)	18 (13.43)
HLA‐DRB1*13	12 (6.12)	4 (6.45)	8 (5.97)
HLA‐DRB1*14	8 (4.08)	5 (8.06)	3 (2.24)
HLA‐DRB1*15	56 (28.57)	17 (27.42)	39 (29.1)
HLA‐DRB1*16	1 (0.51)	0 (0.0)	1 (0.75)

Among the 12 alleles, the HLA‐DRB1*15 allele was present in the highest frequency at 28.57%. Alleles with a frequency above 5% were HLA‐DRB1*07, HLA‐DRB1*12, HLA‐DRB1*10, HLA‐DRB1*13, HLA‐DRB1*04, and HLA‐DRB1*03, in descending order. In contrast, HLA‐DRB1*01, HLA‐DRB1*08, and HLA‐DRB1*16 alleles were among the rare alleles with a frequency below 5%, whereas HLA‐DRB1*09 was absent among the study participants (Figure 1).

FIGURE 1.

Frequency distribution of the alleles of the HLA‐DRB1 gene among the seropositive (yellow) and seronegative (violet) vaccinees. HLA‐DRB1*04, HLA‐DRB1*07, HLA‐DRB1*12, and HLA‐DRB1*15 alleles were comparatively higher in frequency among the seropositive group, and HLA‐DRB1*01, HLA‐DRB1*03, HLA‐DRB1*10, HLA‐DRB1*11, and HLA‐DRB1*14 alleles were comparatively higher in the seronegative group.

On the account of genotype, HLA‐DRB1*07/15 was the most common genotype, with a frequency of 16.32%. Genotypes with frequencies above 5% were HLA‐DRB1*10/15, HLA‐DRB1*12/15, HLA‐DRB1*07/12, HLA‐DRB1*13/15, HLA‐DRB1*03/15, HLA‐DRB1*04/10, HLA‐DRB1*07/10, and HLA‐DRB1*07/13. The rest of the genotypes were comparatively rare, with frequencies below 5% (Figure 2).

FIGURE 2.

Frequency distribution of the genotypes of the HLA‐DRB1 gene among the seropositive (yellow) and seronegative (violet) vaccinees. HLA‐DRB1*07/15 was the most frequent genotype among the study participants.

3.4. Association Between Participants' Serology and HLA‐DRB1 Alleles

The HLA‐DRB1*15 allele occurred most frequently among the seropositive individuals, with a frequency of 29.1%. Apart from the HLA‐DRB1*15 allele, HLA‐DRB1*07 (25.37%), HLA‐DRB1*12 (13.43%), HLA‐DRB1*10 (9.7%), HLA‐DRB1*04 (6.72%), HLA‐DRB1*13 (5.97%), and HLA‐DRB1*03 (5.22%) occurred in frequencies above 5%. Among the minor alleles, HLA‐DRB1*11 was not found among the seropositive individuals. The HLA‐DRB1*15 allele was also the most common among the seronegative individuals (27.42%). With a frequency above 5%, the following alleles were found among the seronegative participants: HLA‐DRB1*07 (17.74%), HLA‐DRB1*10 (12.9%), HLA‐DRB1*12 (11.29%), HLA‐DRB1*14 (8.06%), HLA‐DRB1*13 (6.45%), and HLA‐DRB1*03 (6.45%). Despite a frequency below 5%, HLA‐DRB1*11 (4.84%) was present among the seronegative group, whereas HLA‐DRB1*08 and HLA‐DRB1*16 were absent. However, no HLA‐DRB1 allele was significantly associated with the participants' serological status for plasma anti‐SARS‐CoV‐2 IgG (p = 0.239).

When stratified by the number of days post‐vaccination, among the study participants for whom more than 180 days (6 months) had elapsed after administration of the first vaccine dose, the HLA‐DRB1*07 (p = 0.011), HLA‐DRB1*04 (p = 0.018), HLA‐DRB1*15 (p = 0.026), HLA‐DRB1*03 (p = 0.029), and HLA‐DRB1*13 (p = 0.048) alleles were significantly associated with participants' serological status with respect to the HLA‐DRB1*14 allele. However, upon adjusting for multiple testing, none of the alleles retained a significant association (p = 1.000).

The seropositive participants of the higher antibody response (HAR) subset had a significantly higher frequency of the HLA‐DRB1*15 allele than participants of the LAR subset (p = 0.022).

3.5. Association Between Plasma Anti‐SARS‐CoV‐2 IgG and HLA‐DRB1 Alleles

The plasma anti‐SARS‐CoV‐2 levels varied with the HLA‐DRB1 alleles among the seropositive participants (Table 4).

TABLE 4.

Plasma anti‐SARS‐CoV‐2 IgG levels across HLA‐DRB1 alleles among the seropositive study participants.

HLA‐DRB1 alleles
Allele	Mean (±SD) IgG (IU/mL)	p	Adj p
HLA‐DRB1*01	334.25	0.909	1
HLA‐DRB1*03	305.85 (±87.66)
HLA‐DRB1*04	320.35 (±89.0)
HLA‐DRB1*07	312.27 (±84.18)
HLA‐DRB1*08	282.94
HLA‐DRB1*10	296.09 (±133.81)
HLA‐DRB1*11	—
HLA‐DRB1*12	322.11 (±87.57)
HLA‐DRB1*13	255.77 (±135.89)
HLA‐DRB1*14	366.43 (±53.12)
HLA‐DRB1*15	289.25 (±125.22)
HLA‐DRB1*16	249.86

The seropositive participants with the HLA‐DRB1*12 allele had among the highest mean (±SD) anti‐SARS‐CoV‐2 IgG titre of 322.11 (±87.57) IU/mL. In contrast, seropositive participants with the HLA‐DRB1*13 allele had among the lowest mean (±SD) anti‐SARS‐CoV‐2 IgG titre of 255.77 (±135.89) IU/mL (Figure 3). However, there was no statistically significant association between a particular allele and plasma anti‐SARS‐CoV‐2 IgG levels (p = 0.91).

FIGURE 3.

HLA‐DRB1 alleles with respect to plasma anti‐SARS‐CoV‐2 (RBD) IgG levels among the seropositive participants.

The seropositive participants of the HAR subset with the HLA‐DRB1*15 allele had a significantly higher mean (±SD) anti‐SARS‐CoV‐2 IgG titre of 408.26 (±27.72) IU/mL compared with the LAR subset, where no participant had an HLA‐DRB1*15 allele (p = 0.031). The association remained significant after adjusting for age, sex, and the number of days post‐vaccination (p < 0.001).

3.6. Association of HLA‐DRB1 Genotypes With Participants' Serology and Plasma Anti‐SARS‐CoV‐2 IgG

HLA‐DRB1*10/15 was the most common of the 19 genotypes, with a frequency of 12.9%, found among the seronegative participants. In contrast, HLA‐DRB1*07/15 had the highest frequency (20.9%) among the seropositive participants, followed by HLA‐DRB1*12/15 (11.94%), HLA‐DRB1*07/15 (10.45%), and HLA‐DRB1*10/15 (8.96%). However, there was no statistically significant association between HLA‐DRB1 genotypes and participants' serological status for plasma anti‐SARS‐CoV‐2 IgG (p > 0.05). The frequency distributions of HLA‐DRB1 genotypes are presented in Table 5.

TABLE 5.

Frequency distribution of HLA‐DRB1 genotypes among seronegative and seropositive study participants.

HLA‐DRB1 genotypes
Genotype	Total n (%)	Seronegative n (%)	Seropositive n (%)	p value	Adj p value
HLA‐DRB1*01/10	1 (1.02)	1 (3.23)	0 (0.0)	0.03	1
HLA‐DRB1*01/07	1 (1.02)	0 (0.0)	1 (1.49)
HLA‐DRB1*10/12	3 (3.06)	1 (3.23)	2 (2.99)
HLA‐DRB1*10/15	10 (10.2)	4 (12.9)	6 (8.96)
HLA‐DRB1*11/12	1 (1.02)	1 (3.23)	0 (0.0)
HLA‐DRB1*11/15	1 (1.02)	1 (3.23)	0 (0.0)
HLA‐DRB1*12/13	1 (1.02)	0 (0.0)	1 (1.49)
HLA‐DRB1*12/14	2 (2.04)	2 (6.45)	0 (0.0)
HLA‐DRB1*12/15	9 (9.18)	1 (3.23)	8 (11.94)
HLA‐DRB1*13/15	5 (5.1)	2 (6.45)	3 (4.48)
HLA‐DRB1*14/15	4 (4.08)	2 (6.45)	2 (2.99)
HLA‐DRB1*03/10	1 (1.02)	1 (3.23)	0 (0.0)
HLA‐DRB1*03/14	1 (1.02)	0 (0.0)	1 (1.49)
HLA‐DRB1*03/15	5 (5.1)	3 (9.68)	2 (2.99)
HLA‐DRB1*03/07	3 (3.06)	0 (0.0)	3 (4.48)
HLA‐DRB1*04/10	1 (1.02)	0 (0.0)	1 (1.49)
HLA‐DRB1*04/13	1 (1.02)	0 (0.0)	1 (1.49)
HLA‐DRB1*04/15	5 (5.1)	1 (3.23)	4 (5.97)
HLA‐DRB1*04/07	4 (4.08)	1 (3.23)	3 (4.48)
HLA‐DRB1*07/10	4 (4.08)	1 (3.23)	3 (4.48)
HLA‐DRB1*07/11	1 (1.02)	1 (3.23)	0 (0.0)
HLA‐DRB1*07/12	9 (9.18)	2 (6.45)	7 (10.45)
HLA‐DRB1*07/13	5 (5.1)	2 (6.45)	3 (4.48)
HLA‐DRB1*07/14	1 (1.02)	1 (3.23)	0 (0.0)
HLA‐DRB1*07/15	17 (17.35)	3 (9.68)	14 (20.9)
HLA‐DRB1*07/16	1 (1.02)	0 (0.0)	1 (1.49)
HLA‐DRB1*08/15	1 (1.02)	0 (0.0)	1 (1.49)

Among the seropositive participants, plasma anti‐SARS‐CoV‐2 IgG levels varied across different genotypes, as presented in Table 6.

TABLE 6.

Plasma anti‐SARS‐CoV‐2 IgG levels across HLA‐DRB1 genotypes among the seropositive study participants.

HLA‐DRB1 genotypes
Genotype	Mean (±SD) IgG (IU/mL)	p value	Adj p value
HLA‐DRB1*01/10	334.25	0.799	1
HLA‐DRB1*01/07	—
HLA‐DRB1*10/12	298.76 (±101.95)
HLA‐DRB1*10/15	246.42 (±181.03)
HLA‐DRB1*11/12	—
HLA‐DRB1*11/15	—
HLA‐DRB1*12/13	68.58
HLA‐DRB1*12/14	—
HLA‐DRB1*12/15	344.64 (±70.11)
HLA‐DRB1*13/15	241.44 (±194.87)
HLA‐DRB1*14/15	376.82 (±70.68)
HLA‐DRB1*03/10	—
HLA‐DRB1*03/14	345.66
HLA‐DRB1*03/15	207.04 (±85.48)
HLA‐DRB1*03/07	358.46 (±29.13)
HLA‐DRB1*04/10	415.1
HLA‐DRB1*04/13	293.47
HLA‐DRB1*04/15	308.38 (±131.63)
HLA‐DRB1*04/07	313.7 (±22.17)
HLA‐DRB1*07/10	347.6 (±40.64)
HLA‐DRB1*07/11	—
HLA‐DRB1*07/12	339.25 (±48.43)
HLA‐DRB1*07/13	319.92 (±44.06)
HLA‐DRB1*07/14	—
HLA‐DRB1*07/15	282.25 (±119.17)
HLA‐DRB1*07/16	249.86
HLA‐DRB1*08/15	282.94

The highest mean anti‐SARS‐CoV‐2 IgG titre of 415.1 IU/mL was observed for seropositive participants with the HLA‐DRB1*04/10 genotype, and the lowest mean IgG titre of 68.58 IU/mL was observed in a seropositive participant with the HLA‐DRB1*12/13 genotype (Figure 4).

FIGURE 4.

HLA‐DRB1 genotypes with respect to plasma anti‐SARS‐CoV‐2 (RBD) IgG levels among the seropositive participants.

Nevertheless, no significant association was found between HLA‐DRB1 genotypes and plasma anti‐SARS‐CoV‐2 IgG levels (p = 0.799).

4. Discussion

The allelic distribution of HLA genes is highly variable across human populations. Insights about HLA allele frequencies are associated with different public health interests, such as disease association studies, genetic epidemiology, forensics, and pharmacogenetics. Despite being an important factor in the assessment of vaccine efficacy, the association between HLA alleles and antibody response among the COVID‐19‐vaccinated Bangladeshi population remains unexplored. In this study, we determined allele frequencies of HLA‐DRB1, which is the first report of this sort to our knowledge among the population.

Moreover, our results will add to the limited reports of allelic and genotypic frequencies of HLA genes among the Bangladeshi population. Consistent with the findings of a previous study (Ali et al. 2008), this study revealed the highest frequency for HLA‐DRB1*15 and the second highest frequency for HLA‐DRB*07 and HLA‐DRB1*07/15 to be the most frequent genotype in the Bangladeshi population. In addition, HLA‐DRB1*09 was absent in our study participants and was found to be the rarest among the Bangladeshi population (Ali et al. 2008).

Although no association was found between genotype and serological status of the vaccinees, the HLA‐DRB1*07/15 genotype was more frequent among the seropositive participants, while HLA‐DRB1*10/15 was more frequent among the seronegative participants. A study conducted among healthcare workers in Italy revealed no association between participants' seronegativity and HLA‐DRB1 gene alleles alone. In fact, the HLA‐A*24:02∼C*07:01∼B*18:01∼DRB1*11:04 haplotype was found to be associated with a weaker antibody response among vaccinees (Crocchiolo et al. 2022). A similar study also found no association between HLA‐DRB1 alleles and antibody response after two doses of vaccines among hospital workers in Japan (Khor et al. 2022).

A study found HLA‐DRB1*07 to be associated with seropositivity among vaccinees who had received their second dose of vaccine at least 30 days before sample collection (Gutiérrez‐Bautista et al. 2022). In this study, HLA‐DRB1*07 appeared to be significantly associated with seropositivity among participants for whom more than 180 days (6 months) had elapsed after administration of the first vaccine dose. However, in post hoc analysis, the association was not statistically significant. Interestingly, participants among the HAR subset with the HLA‐DRB1*15 allele had significantly higher anti‐SARS‐CoV‐2 IgG levels than those among the LAR subset, entirely lacking the allele. In fact, studies have reported an association of HLA‐DRB1*15 alleles with cellular responses against SARS‐CoV‐2 (Astbury et al. 2022; Augusto et al. 2023; Bertinetto et al. 2023) as well as with anti‐spike antibody levels (Mentzer et al. 2022). Hence, the HLA‐DRB1*15 allele may be associated with anti‐SARS‐CoV‐2 immunity as an allele and/or as part of a haplotype.

Several methodological limitations have been considered when interpreting the findings. In the subgroup analysis, as statistical power was reduced for evaluating the effects of rare low‑frequency HLA alleles and genotypes, some true associations may remain undetected. The cohort was heterogeneous in terms of vaccine platforms, which, despite adjustment through regression analysis, may dilute allele‑specific effects on antibody responses. Similarly, as a history of SARS‑CoV‑2 infection could not be definitively established for all participants, allele‐specific effects may not be represented by the observed antibody levels.

This study emphasises the importance of studying immunogenetic associations with vaccine response among ethnically diverse populations. While the observations are similar to those among other populations studied in the context of anti‐SARS‐CoV‐2 immunity, this encourage further experiments, including other HLA genes and seeking the association of HLA haplotypes with seroprevalence among the Bangladeshi population.

5. Conclusions

Adding to the existing report on the population's HLA‐DRB1 allelic distribution, this study is the first report among the Bangladeshi population exploring immunogenetic associations of vaccine‐induced anti‐SARS‐CoV‐2 IgG response. Our findings indicate that HLA‐DRB1*15 may have an association with SARS‐CoV‐2 seroprevalence, reiterating the significance of evaluating associations of HLA molecules with vaccine‐induced immune responses.

Author Contributions

P.B. and A.T. performed the laboratory experiments, data curation, formal analysis, and writing of the first and final draft of the manuscript. A.A.S. and M.A. conducted specimen collection, processing, experimental validation and data visualisation. T.Y. contributed to resource arrangements, funding acquisition and review of the manuscript drafts. A.E. acquired funding and provided the resources. A.H.M.N.N. conceived the idea, acquired funding, provided the resources, supervised the research work, and reviewed the first and final draft of the manuscript. All authors carefully reviewed and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.