https://orcid.org/0000-0001-9508-7863
ABSTRACT
To investigate whether the TNF-α, IL-2, IL-4 and IL-10 genes contribute to variations in vaccine-induced immune responses after immunization with the inactivated Japanese encephalitis vaccine (IJEV), a total of 369 individuals who received the IJEV were enrolled. Based on Japanese encephalitis virus (JEV) neutralization antibodies (NAbs), the individuals were divided into seropositive (SP) and seronegative (SN) groups. Then, 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes were genotyped using the TaqMan method. Although there was no association of the TNF-α, IL-2, IL-4 and IL-10 genes with JEV seropositivity triggered by JEV vaccination when all the individuals in the SP and SN groups were compared, differences were observed in a subgroup analysis. In the male group, rs2243291 in the IL-4 gene showed a difference between the JEV SP and SN groups with the overdominant model (P = .045), and the C/G genotypes conferred more JEV seropositivity (OR = 1.87; 95% CI: 1.01–3.49); the CT genotype of rs3093726 in the TNF-α gene showed higher JEV NAbs geometric mean titer (GMT) than the TT genotype (P = .018, CT: 1.677 ± 0.144 vs TT: 1.271 ± 0.039). Furthermore, the rs1800629 genotype in the TNF-α gene and the rs1800896 genotype in the IL-10 gene exhibited a trend of association with JEV seropositivity in the female group, but the difference was not significant. The present study suggested that the polymorphisms in the cytokine genes could be associated with sex-specific JEV NAbs seroconversion. However, more samples should be studied, and further functional verification should be performed.
Introduction
Vaccination is an efficient method for controlling infectious diseases. However, there is interindividual variation in immune responses to vaccines. For example, the seroconversion rate and hepatitis B and measles neutralization antibody levels were different after vaccination with the hepatitis B vaccine (HBV) and the measles vaccine,Citation1,Citation2 which indicated that host genetic polymorphisms may play an important role in the efficacy of vaccines.
Japanese encephalitis (JE) is one of the most serious mosquito-borne infectious diseases.Citation3 To date, four different types of JE vaccines (inactivated mouse brain-derived, live attenuated cell culture-derived, inactivated cell culture-derived, and genetically engineered live attenuated chimeric vaccine) are available in different countries. After immunization, the serum neutralizing antibody positive conversion rates ranged from 64.4% to 93.3% for the inactivated or live attenuated vaccines.Citation4,Citation5 The variations in the positive serum conversion rates indicate that host genetic polymorphisms could play a key role in the efficacy of JE vaccines.
Recently, human leukocyte antigen (HLA) alleles and several single-nucleotide polymorphisms (SNPs) in cytokine genes, such as the pro-inflammatory cytokine TNF-α gene, subsets of the Th1-promoting cytokine IL-2 gene, and the Th2 cytokines IL-4 and IL-10 genes, were investigated to be associated with hypo- or nonresponsiveness and the variable antibody levels in immune responses to different vaccines. The variation of IL-2 gene has been investigated in association with measles vaccine and hepatitis B vaccine (HBV) induced antibody response,Citation6–Citation9 the variation of IL-4 genes were in association with HBV, diphtheria, tetanus, and combined pneumococcal conjugate and polysaccharide vaccines,Citation8,Citation10-Citation12 and the variation of IL-10 genes were in association with diphtheria, tetanus, and measles vaccine.Citation6,Citation7,Citation11 In 2009, Yucesoy et al. investigated the association between cytokine or cytokine receptor gene polymorphisms and the immune response to childhood vaccines (HBV, 7-valent pneumococcal conjugate, and diphtheria, tetanus, acellular (DTaP) pertussis vaccines) and found that SNPs in the TNF-α, IL-12B, IL-4Rα, and IL-10 genes were associated with vaccine-specific immune responses (P < .05).Citation13 Moreover, SNPs in the IL-1β, TNF-α, IL-2, IL-4, IL-10, IL-4Rα, and IL-12B genes were associated with serum immunoglobulin (IgG, IgA, and IgM) levels (P < .05).Citation13 All studies suggested that genetic variations in cytokine genes can influence vaccine-induced immune responses, which in turn may influence vaccine efficacy. As Japanese encephalitis vaccine be considered, our previous study investigated the association of HLA-DRB1, HLA-DPB1, and HLA-DQB1 with the humoral immune response elicited by the inactivated Japanese encephalitis vaccine (IJEV) and showed that HLA-DQB1∗02:01 was significantly associated with JEV seropositivity (P < .05), while HLA-DQB1∗02:02 was significantly associated with JEV seronegativity (P < .05).Citation14 In addition, we found that certain HLA-DRB1 and HLA-DPB1 alleles were associated with higher geometric mean titers (GMTs) than others.Citation14 The association study of cytokine gene variations with vaccine antibody response are particularly important in developing countries where JE is still a major health issue, because it may provide a clue for vaccine efficacy evaluation and new vaccine development.
In the current study, we evaluated the association between polymorphisms of the cytokine genes (TNF-α, IL-2, IL-4, and IL-10) and vaccine-induced antibody responses following immunization with the IJEV in a Mongolian Chinese population. Our results suggested that the genetic variation in cytokine genes may play a role in the immune response to the JE vaccine. However, the SNP association needs to be verified by function studies in the future.
Materials and methods
Ethics statement
All the procedures were in accordance with the ethical standards of the responsible committee on human experimentation (the Institutional Review Board of the Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College) and with the Helsinki Declaration of 1975, which was revised in 2008. Informed consent was obtained from all subjects included in the study.
Subjects
Our previous study reported that a randomized, double-blinded, positive-control, noninferiority IJEV trial was implemented in the Inner Mongolia autonomous region of China from August 2012 to September 2013.Citation14 A total of 1,200 individuals, aged 8 months to 12 years, were enrolled to receive two doses of IJEV on a 0- and 7-day schedule. Considering the blood sample value limitation and the consistence of the test, only 369 individuals of 3 to 12 years old who were negative for JEV-neutralizing antibodies (NAbs) before vaccination were selected for cytokine SNP genotyping in the current study. The positive serum conversion rate and GMTs were used as alternative markers of efficacy of JE vaccines, and a NAb titer of at least 10 has been established to correlate with protection against the JEV.Citation15,Citation16 Based on the NAb titer, the enrolled individuals were divided into seropositive (SP) and seronegative (SN) groups.
Japanese encephalitis vaccine neutralization antibody detection
JEV-specific NAbs were determined by the National Institute for Food and Drug Control using the 50% plaque-reduction neutralization test according to the requirement of the Pharmacopoeia of the People’s Republic of ChinaCitation17 as previously reported.Citation14 As the NAbs at 50% plaque-reduction neutralization titer (PRNT50) of at least 10 have been established as a correlate of protection against development of JE disease in humans,Citation3 PRNT50 <10 or a PRNT50 increased less than fourfold after vaccination indicated negative seroconversion, while PRNT50 > 10 or a PRNT50 with at least a fourfold increase after vaccination indicated positive seroconversion.
Genotyping of the TNF-α, Il-2, IL-4 and IL-10 genes
Genomic DNA was extracted from peripheral lymphocytes using the QIAamp Blood Mini Kit (Qiagen, Hilden, German). Genotyping of three SNPs (rs1800629, rs3093668, and rs3093726) in the TNF-α gene, four SNPs (rs11932411, rs11575812, rs2069762, and rs4833248) in the IL-2 gene, seven SNPs (rs2243247, rs2243248, rs2243250, rs2070874, rs2227284, rs2243291, and rs2243292) in the IL-4 gene and three SNPs (rs1800872, rs1800871, and rs1800896) in the IL-10 gene was performed using a TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA). To determine the accuracy of SNP genotyping by the TaqMan assay, some samples were randomly selected for sequencing to confirm the TaqMan genotyping results.
Statistical analysis
The age and sex of the subjects were compared using Student’s t and chi-square test, respectively. Hardy–Weinberg equilibrium (HWE) was assessed using the Guo and Thompson method. Linkage disequilibrium analysis (LD) was calculated, and a D’ value greater than 0.80 was considered to be in linkage disequilibrium. The haplotypes were constructed based on the genotyping results by the expectation-maximization algorithm.Citation18,Citation19 The χ2 test was used to determine differences in the allele, genotype, and haplotype frequencies between the responder and nonresponder groups, and the Bonferroni correction was used for multiple comparisons. The association between each genotype and the immune response was assessed using the inheritance model analysis in the SNPstats software.Citation20 The Akaike information criterion (AIC) and Bayesian information criterion (BIC) were calculated to determine the best fit inheritance model, which possesses the smallest AIC and BIC values. The association between the SNPs in the TNF-α, IL-4 and IL-10 genes and the antibody levels was analyzed through one-way ANOVA, and Tukey’s correction was used for multiple comparisons. P values < .05 were considered statistically significant. The genotype and allele for each SNP and haplotype-specific risk analysis were calculated, and the odds ratios (OR) and the associated 95% confidence intervals (CIs) were also calculated for allele-specific or haplotype-specific risks.
Results
Subject characteristics
lists the characteristics of the enrolled subjects. All NAbs were negative before vaccination. After vaccination, 160 individuals with PRNT50 > 10 were included in the SP group, while 209 individuals with PRNT50 < 10 were included in the SN group. There was no significant difference in sex or age between the SP and SN groups (P = .066 and P = .346, respectively) (). There was no significant difference in age between the NAbs seropositive and seronegative responses in the male group and female group (P = .336 and P = .128, respectively). In the NAbs seropositive responders, the distribution of antibody levels showed no differences between males (GMT: 1.293 ± 0.338) and females (GMT: 1.238 ± 0.330) (P = .301).
Table 1. Demographic characteristics of the IJEV NAbs SP and SN group
Association of the 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes with the JEV NAbs response triggered by IJEV vaccination
The allele and genotype frequencies of 17 SNPs in the TNF-α, IL-2, IL-4, and IL-10 genes are presented in . The genotype frequencies for the SNPs were in HWE (P > .05). The allele and genotype distributions of the SNPs showed no association with JEV seropositivity triggered by JEV vaccination (P > .05). For the subgroup analysis, when sex was compared, the GG genotype of rs1800629 in the TNF-α gene showed a higher trend in the SP group than in the SN group among females (0.872 VS 0.775, OR = 0.504, 95% CI: 0.230 ~ 1.104); however, the difference was not significant (P = .083). Similarly, the C allele of rs1800896 in the IL-10 gene showed a higher trend in the SP group than in the SN group (0.157 VS 0.098, OR = 1.713, 95% CI: 0.924 ~ 3.178) with no significant difference (P = .085) in the female group ().
Table 2. Allelic and genotypic distribution of 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes in the IJEV NAbs SP and SN group
Table 3. Allelic and genotypic distribution of the rs1800896 in IL-10 and rs1800629 in TNF-α gene in the IJEV NAbs SP and SN groups in females
The other alleles and genotypes of the SNPs were not associated with JEV seropositivity in the female group (P > .05) (data not shown). In the male group, no SNPs associated with JEV seropositivity were observed (P > .05) (data not shown).
Model of inheritance analysis of the 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes with the JEV NAbs response triggered by JEV vaccination
In the current study, no significant differences in the SNPs were found between the SP and SN groups in the model of inheritance analysis (P > .05) (data not shown). For the subgroup analysis, rs2243291 in the IL-4 gene showed a significant difference between the JEV SP and SN groups with the overdominant model (P = .045), and the C/G genotype conferred more JEV seropositivity (OR = 1.87; 95% CI: 1.01–3.49) in the male group (). In addition, rs1800896 in the IL-10 gene showed a trend of a difference between the SP and SN groups with the log-dominant model in the female group; however, the difference was not significant (P = .086) (). No significant differences in the other SNPs were found between the SP and SN groups in the model of inheritance analysis in either the male or female groups (P > .05) (data not shown).
Table 4. Inheritance model analysis of the rs2243291 in IL-4 gene in the IJEV NAbs SP and SN group in males
Table 5. Inheritance model analysis of the rs1800896 in IL-10 gene in the IJEV NAbs SP and SN group in females
Association of the haplotypes in the TNF-α, IL-2, IL-4 and IL-10 genes with the JEV NAbs response triggered by JEV vaccination
The SNPs were considered to construct haplotypes when D’≥0.800 in the LD analysis. After the haplotypes were constructed, there were no differences between the SP and SN groups after Bonferroni correction (P > .05) (data not shown). For the subgroup analysis, the haplotypes of the SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes were not associated with JEV seropositivity in the male and female groups after Bonferroni correction (P > .05) (data not shown).
Association between JEV NAbs GMTs and 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes
A total of 160 individuals in the SP group were included in the analysis of the association between the SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes and the JEV NAbs GMTs. However, there was no significant difference in the GMTs among the different SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes (P > .05) (data not shown). For subgroup analysis, rs3093726 in the TNF-α gene showed significant GMTs among the TT and CT genotypes (P = .018), and the CT genotype showed higher JEV neutralization antibody GMTs than the TT genotype (CT: 1.677 ± 0.144 vs TT: 1.271 ± 0.039) in the male group. The other SNPs were not associated with the JEV neutralization antibody GMTs in the male group (P > .05) (data not shown). In addition, no SNPs were associated with the JEV Nab GMTs in the female group (P > .05) (data not shown).
Discussion
Cytokines play central roles in the regulation of the Th1/Th2 balance in response to vaccine antigens.Citation21–Citation23 Genetic polymorphisms in the genes encoding cytokines representing both Th1 (IL-2) and Th2 (IL-4 and IL-10) subsets showed that the genetic polymorphisms of cytokines were associated with hypo- or nonresponsiveness and variations in antibody levels in the immune responses to different vaccines.Citation6–Citation13 In the present study, we did not identify an association of SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes with JEV seropositivity triggered by JEV vaccination when all individuals in the SP and SN groups were compared, but we investigated sex-specific association in the subgroup analysis.
TNF-α is a pro-inflammatory cytokine that is associated with the regulation of cellular immune responses.Citation24 Several SNPs in the TNF-α promoter regions have been shown to directly affect gene transcription.Citation24,Citation25 In 2012, Ovsyannikova et al. performed a study of the association between cytokines and smallpox vaccination and found that Caucasian individuals demonstrated significant associations between rs3093726 and rs30936687 in the TNF-α gene and secreted IL-1β.Citation26 In the current study, rs3093726 showed significant GMTs between the TT and CT genotypes, and the CT genotype showed higher JEV neutralization antibody GMTs than the TT genotype (CT: 1.677 ± 0.144 vs TT: 1.271 ± 0.039) in the male group. For another SNP, rs1800629, the GG genotype was significantly associated with high levels of hepatitis B neutralizing antibodies induced by HBV vaccination.Citation13 In 2009, Yucesoy et al. reported that rs1800629 may be associated with changes in the amount and function of the gene products, which in turn affects the processing and presentation of antigens by antigen-presenting cells. However, Macedo et al. found that there was no correlation between the rs1800629 polymorphism and the hepatitis B vaccine response in children approximately 1-year-old.Citation27 In the present study, the genotype of rs1800629GG in TNF-α genes showed a trend of association with a higher positive seroconversion rate after the IJEV in the female group, although the difference was not significant. Thus, rs1800629, which is located in the 5ʹ-flanking region of the TNF-α gene, influenced the expression of the TNF-α gene, potentially affecting the immune response to vaccines.
A previous study suggested that transcription of the IL-4 gene is positively regulated by the coordination of multiple promoter and enhancer elements.Citation28 In 2011, Haralambieva et al. reported that rs2243248 was associated with measles virus-specific neutralizing antibody responses, and the AA genotype showed a high virus-specific neutralizing antibody titer.Citation6 In 2017, Youn Roh et al. reported that rs2227284G in the IL-4 gene was significantly more frequent in nonresponder and lower-titer individuals than in high-titer responder individuals among 6-month-old subjects during the response to the HBV vaccine, and the rs2227284G frequencies were significantly different among the three subgroups.Citation29 In the current study, we found that rs2243291 in the IL-4 gene showed a significant difference between the JEV SP and SN groups with the overdominant model in the female group. However, we did not observe the association of the rs2227284 allele and genotypes with the positive seroconversion rate after the IJEV.
As a Th2 type cytokine, IL-10 is a key immune modulating factor with anti-inflammatory activities that inhibits the activation of antigen-presenting cells, thereby influencing T cell responses.Citation30 The polymorphisms located in the 5ʹ-flanking region of the IL-10 gene influence the expression of the IL-10 gene, which is associated with the immune reaction. In 2005, Hohler et al. found that the rs1800896GG genotype was associated with strong anti-HBs responses after vaccination with HBsAg and Hepatitis A.Citation9 In 2008, Smith et al. reported that the rs1800896G and rs1800871T alleles were associated with low IL-10 production,Citation31 and rs1800871 was related to increased cellular and humoral immunity to measles vaccination.Citation7 Then, Yucesoy et al. reported that rs1800896GG was associated with higher antibody responses to HBV and DTaP vaccinations, and rs1800871CC was associated with higher antibody responses to PnPS serotypes.Citation13 In the current study, we found rs1800896GG showed a trend of association with a higher positive seroconversion rate after the IJEV in the female group, but the difference was not significant.
The above results suggested that sex may play a role in the antibody response after the IJEV. The different immune responses between males and females induced by vaccines have been investigated for several immunizations, including the hepatitis B, diphtheria, pertussis, pneumococcus, rabies, measles, malaria and human papillomavirus, influenza, herpes virus, smallpox and Td/T dap vaccines, for which high antibody responses against vaccine antigens were significant greater in females than in males.Citation32–Citation34 These stronger adaptive humoral responses are characterized by higher basal and postvaccination IgG levels and increased B-cell numbers.Citation35 Further studies indicated that sex-based differences in the HLA genes and the IL-4 and IL-10 genes are associated with antibody responses against measles, mumps, tetanus, diphtheria, and hepatitis A vaccines.Citation36 Moreover, in mice, the cytokine response of CD4 + T cells differed between male and female mice, and females exhibited higher Th1 (i.e. IFN-r), Th2 (i.e. IL-4), and regulatory T cell (i.e. IL-10) responses than males;Citation37,Citation38 in humans, peripheral monocytes isolated from males produced higher amounts of TNF-α, IL-1b, and IL-6 but lower amounts of IL-10 compared to cells from females.Citation39,Citation40 Thus, we deduced that the variation in the SNPs in the TNF-α, IL-4, and IL-10 genes may influence the expression of these cytokines, resulting in different JEV antibody responses between male and females.
In the present study, we did not identify an association of the SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes with JEV seropositivity triggered by JEV vaccination when all individuals in the SP and SN groups were compared. However, in the subgroup analysis, rs2243291 in the IL-4 gene showed a significant difference between the JEV SP and SN groups with the overdominant model, and the C/G genotypes conferred more JEV seropositivity; rs3093726 in the TNF-α gene showed significant GMTs between the TT and CT genotypes, and the CT genotype showed higher JEV neutralization antibody GMTs than the TT genotype in the male group.
One limitation of the present study is that the relationship between these SNPs and the cytokine levels, or between the cytokine levels and the immune response triggered by JEV vaccination, has not been investigated. Moreover, the limitation of a smaller group of subjects may restrict the analytical power of the present study. Thus, more samples and functional studies are needed to better clarify and examine the association between these SNPs and antibody responses triggered by JEV vaccination.
Conclusions
In the current study, we evaluated the associations between 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes and the immune responses triggered by JEV vaccination in a Chinese Mongolian population. The results suggested that the polymorphisms in the cytokine genes could be associated with the JEV seroconversion rate after vaccination in the Chinese Mongolian population in a sex-specific manner. The present studies may provide some clue for future vaccine efficacy evaluation and new vaccine development. In the future, larger-scale studies are needed to better clarify and examine the association between cytokines and immune responses triggered by JEV vaccination.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Additional information
Funding
References
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