Associations between Single Nucleotide Polymorphisms and Haplotypes in Cytokine and Cytokine Receptor Genes and Immunity to Measles Vaccination

doi:10.1016/j.vaccine.2011.08.083

Vaccine. Author manuscript; available in PMC 2012 October 19.

Published in final edited form as:

Vaccine. 2011 October 19; 29(45): 7883–7895.

Published online 2011 August 27. doi: 10.1016/j.vaccine.2011.08.083

PMCID: PMC3191314

NIHMSID: NIHMS320308

Associations between Single Nucleotide Polymorphisms and Haplotypes in Cytokine and Cytokine Receptor Genes and Immunity to Measles Vaccination

Iana H. Haralambieva,^a^b Inna G. Ovsyannikova,^a^b Richard B. Kennedy,^a^b Robert A. Vierkant,^c V. Shane Pankratz,^c Robert M. Jacobson,^a^b^d and Gregory A. Poland^a^b^d^*

^aMayo Clinic Vaccine Research Group, Mayo Clinic, Rochester, MN 55905 USA

^bProgram in Translational Immunovirology and Biodefense, Mayo Clinic, Rochester, MN 55905 USA

^cDivision of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN 55905 USA

^dDepartment of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905 USA

^*Address correspondence to: Gregory A. Poland, M.D., Director, Mayo Vaccine Research Group, Mayo Clinic, Guggenheim 611C, 200 First Street SW, Rochester, MN 55905, Phone: (507) 284-4968, Fax: (507) 266-4716, Email: Poland.gregory/at/mayo.edu

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Abstract

Identification of host genetic determinants of measles vaccine-induced immunity can be used to design better vaccines and ultimately predict immune responses to vaccination. We performed a comprehensive candidate gene association study across 801 genetic markers in 56 cytokine/cytokine receptor genes, in a racially diverse cohort of 745 schoolchildren after two doses of MMR vaccine. Using linear regression methodologies we examined associations between SNPs/haplotypes and measles virus-specific immunity.

Forty-eight significant SNP associations with variations in neutralizing antibodies and measles-specific IFNγ Elispot responses were identified (p<0.05). Our study replicated an important previously found association of a functional IL12B genetic variant rs3212227 with variations in measles-specific humoral immunity (p=0.037). Similarly, two previously reported promoter IL10 and IL2 polymorphisms (rs1800890 and rs2069762) demonstrated associations with measles-specific cellular immunity in Caucasians (p≤0.034). Multiple IL7R polymorphisms, including a non-synonymous functional SNP (rs6897932/Thr244Ile), were associated with humoral (p≤0.024) and/or cellular (IFNγ Elispot, p≤0.023) measles-specific immune responses in Caucasians, but not African-Americans. Haplotype level analysis confirmed the association of IL7R genetic variants with measles vaccine-induced immunity in the Caucasian group (global p-value=0.003). Our results validate previous findings and identify new plausible genetic determinants, including IL7R polymorphisms, regulating measles vaccine-induced immunity in a race-specific manner.

Keywords: Measles vaccine, Immunity, Single Nucleotide Polymorphisms, Haplotypes, Cytokine, Cytokine receptor

1. Introduction

Recent advances in the field of immunogenetics have partly revealed the genetic basis of differential susceptibility to infectious diseases and inter-individual differences in vaccine response [1]. Studies (including ours) consistently implicate genetic polymorphisms in key immune response genes, such as cytokine and cytokine receptor genes as important determinants of adverse events and observed variations in immune responses to vaccines such as measles-mumps-rubella (MMR), influenza, smallpox, hepatitis A and B vaccines, pneumococcal conjugate vaccine and diphtheria-tetanus-pertussis vaccine [2-12]. Furthermore, racial, ethnic and gender differences have been associated with differences in immune responses to immunization and infection [2,3,13-18]. Previous studies have also demonstrated race-specific distributions of allelic variants in cytokine genes across populations [19,20]. Although much information has been accumulated in the field of measles immunity, the discovery and validation of genetic drivers of measles-specific immune responses still remains a challenge.

In the current study we performed a high-density, comprehensive, candidate gene genetic study to identify, replicate, and validate SNP associations across 56 key cytokine and cytokine receptor genes with measures of both humoral and cellular immunity to measles vaccine in a large racially diverse cohort of healthy schoolchildren and young adults after two doses of MMR vaccine.

2. Materials and Methods

2.1. Study subjects

The present study was based on a combined sample of 764 subjects from 2 independent age-stratified cohorts of randomly selected healthy schoolchildren and young adults from Olmsted County, MN: cohort 1 consisted of 388 subjects eligible for the current study (age 11-19 years, enrolled in 2006-2007 year) as previously reported [4], and cohort 2 consisted of 376 eligible subjects (age 11-22 years, enrolled in 2008-2009 year). Inclusion criteria required each participant to have documentation of receipt of two doses of measles-mumps-rubella vaccine (MMR). The race/ethnicity reported in the study were self-reported. The Institutional Review Board of the Mayo Clinic approved the study, and written, informed consent was obtained from subjects’ parents/guardians as well as written assent from age-appropriate subjects at the time of enrollment.

2.2. Immune measures

Cytokine assays

The frequencies of measles-specific IFNγ-producing cells were quantified using commercially available IFNγ Elispot kits (R&D Systems, Minneapolis, MN) as previously described [21]. Seven secreted Th1, Th2, and innate/inflammatory cytokines were measured with commercial ELISA kits in PBMC cultures following in vitro virus stimulation using a response surface methodology approach to pre-optimize conditions for MOI and incubation time for each specific cytokine, as previously described [4]. We stimulated cell cultures with live measles virus using the following pre-optimized conditions for MOIs and incubation times: IFNα and TNFα MOI=1.0, 24 hours; IL-2 and IL-10 MOI=0.5, 48 hours; IL-6, IFNγ and IFNλ1 MOI=1.0, 72 hours.

Plaque Reduction Microneutralization Assay (PRMN)

Measles-specific neutralizing antibodies were quantified using a high throughput fluorescence-based PRMN, as previously described [22,23] with the following modifications in the readout. The GFP-positive syncytia were scanned and counted on an automated Olympus IX71 Fluorescent microscope using the Image-Pro Plus Software Version 6.3 (MediaCybernetics). Neutralizing dose (ND₅₀) values, calculated using Karber’s formula, were transformed into mIU/mL values using the 3^rd WHO international anti-measles antibody standard as previously described [22,23].

2.3. SNP selection and genotyping

We selected tagSNPs within and 5kb upstream and downstream of 56 candidate genes, based on the approach of Carlson [4,24] on data from the source with the greatest number of SNPs from among the Hapmap Phase II (http://www.hapmap.org), Seattle SNPs (http://pga.mbt.washington.edu/), NIEHS SNPs (http://egp.gs.washington.edu/), and NCBI (http://www.ncbi.nlm.nih.gov/projects/SNP/), as previously described [25], with SNP minor allele frequencies (MAF) ≥0.05, and a linkage disequilibrium (LD) threshold of r² ≥0.90. A total of 994 SNPs were selected based on this approach. The selected SNPs were genotyped as part of our population-based measles vaccine study using two custom Illumina GoldenGate SNP panels (Illumina Inc., San Diego, CA) for 1,536 and 768 SNPs, following the manufacturer’s protocol, and as previously described [25].

2.6. Statistical methods

The statistical methods described herein are similar to those published for our previous genetic association studies [4,5,25]. Data were descriptively summarized using frequencies and percentages for categorical variables, and medians and inter-quartile ranges (IQR) for continuous variables. Assessments of cytokine secretion and IFNγ Elispot responses resulted in multiple recorded values per outcome both prior to and after stimulation with measles virus. For descriptive purposes, a single response measurement per individual was obtained by subtracting the median of the unstimulated values from the median of the stimulated values. Assessments of antibody levels resulted in only one recorded value per individual. Observed genotypes were used to estimate allele frequencies for each SNP and departures from Hardy-Weinberg equilibrium (HWE) were assessed using either a Pearson goodness-of-fit test or, for SNPs with a minor allele frequency (MAF) of less than 5%, a Fisher exact test [26]. Estimates of pair-wise linkage disequilibrium (LD) based on the r-squared statistic and D’ statistic were obtained using Haploview software, version 3.32 [27].

SNP associations with immune response outcomes were evaluated using regression models. Simple linear regression was used for measles antibody levels. Repeated measures approaches were implemented for the cytokine secretion and IFNγ Elispot variables, simultaneously modeling the multiple observed measurements and using an unstructured within-person variance-covariance matrix to account for within-subject correlations. This was achieved by including the genotype variable in the regression model, together with a variable representing stimulation status. The resulting genotype-by-stimulation status interaction was then tested for statistical significance. Tests of association assumed an ordinal (log-additive) SNP effect using simple tests for trend.

To further explore genomic regions containing statistically significant single-SNP effects, we performed post-hoc haplotype analyses. Posterior probabilities of all possible haplotypes for an individual, conditional on the observed genotypes, were estimated using an expectation-maximization (EM) algorithm [28]. This information was used to define haplotype design variables that estimated the number of each of the haplotypes carried by an individual. Analyses were performed on all resulting common haplotypes (those with an estimated frequency of greater than 1%) using simple least squares linear regression approach for antibody levels and repeated measures approaches for the cellular immune response (CMI) variables. Differences in immune response among common haplotypes were first assessed globally and simultaneously tested for statistical significance using a multiple degree-of-freedom test. Following these global tests, we examined individual haplotype effects. Each haplotype was included in a separate regression analysis, effectively comparing immune response levels for the haplotype of interest against all others combined. Due to phase ambiguity, haplotype-specific medians and inter-quartile ranges could not be calculated. Thus, descriptive summaries were represented using the corresponding t-statistics.

All of the association analyses adjusted for age at enrollment, race (in the combined analysis), gender, age at first and second measles vaccination, and cohort status (cohort 1 versus cohort 2). We used an inverse normal transformation for all cytokine secretion and CMI outcome variables, and a log transformation for the antibody response measure, in all formal tests to account for the skewed nature of the data. All statistical tests were two-sided and, unless otherwise indicated, all analyses were carried out using the SAS software system (SAS Institute, Inc., Cary, NC).

3. Results

3.1 Subjects demographics and immune responses

All demographic and clinical variables of the study cohort, and the immune response measures, are summarized in Table 1. Briefly, our study population had a median age of 15 years at the time of sampling with a median time from second MMR immunization to enrollment of 7.4 years. The cohort consisted of 417 males (55.97%), 598 Caucasians (80.27%), but included also 89 African-Americans (11.95%) and a limited number of other races/ethnicities.

Table 1

Demographic and immunological characteristics of the study cohort

3.2 Genotyping of SNPs in cytokine and cytokine receptor genes

Genotyping was performed on 764 subjects for 994 known SNPs in the cytokine: IL2, IFNG, IL12A, IL12B, IL4, IL5, IL10, IL1A, IL1B, IL6, IL8, IL18, TNFA, CSF2, IFNB1, IL28A/IFNλ2, IL28B, IL29/IFNλ1, and 13 IFNA, and cytokine receptor genes: IL2RA, IL2RB, IL2RG, IFNGR1, IFNGR2, IL12RB1, IL12RB2, IL4R, IL10RA, IL10RB, IL6R, IL6ST, IL7R, IL8RA, IL18R1, IL1R1, IL1R2, IL1RN, CSF2RA, CSF2RB, IFNAR1, IFNAR2, IL28RA, TNFRSF1A, TNFRSF1B. Our overall genotyping sample success rate was 98.75% (including replicate samples), the locus success rate was 94.55%, and reproducibility was 100%. Nineteen DNA samples failed because of low call rates (<95%), leaving 745 subjects for final analysis. In addition, fifty-five SNPs failed the genotyping, one hundred ten SNPs were excluded based on MAF<1%, one SNP was excluded based on having a low call rate (<0.95%), and twenty-seven SNPs were excluded based on being monomorphic. A total of 801 SNPs were used for the final analysis in a set of 745 subjects (including 598 Caucasians and 89 African-Americans).

3.3 Genetic associations

Associations between SNPs in cytokine and cytokine receptor genes and humoral immune responses after measles vaccination

Overall we found 19 significant associations between SNPs within the coding or regulatory gene regions and variations (29 to 72% increase/decrease) in measles-specific neutralizing antibody levels (p<0.05) (Table 2). Of note, in this study we were able to replicate the association of SNP rs3212227 (in LD with rs6859018, D’statistic, a pairwise measure of LD=0.98) located in the 3′UTR region of the IL12B gene with variations in measles-specific antibody levels (p=0.037) [5]. Other interesting findings include three IL4 SNPs (including two promoter SNPs rs2243248 and rs2243247) associated with significant immune outcome variations (53 to 72% increase/decrease in antibody responses, 0.0064≤p≤0.0358) (Table 2). The minor alleles of one coding SNP (rs6897932/Thr244Ile) and one promoter SNP (rs6890853) within the IL7R gene (in LD, D’=0.99), were associated with up to a 34% decrease in measles-specific antibody levels in an allele dose-related manner.

Table 2

SNPs in coding/regulatory regions of cytokine and cytokine receptor genes associated with measles virus-specific neutralizing antibody responses

Race-specific analysis in the Caucasian subjects (n=598) revealed that many of the identified associations with measles-specific antibody levels still remained valid (p<0.05, Table 3), however SNPs rs3212227/IL12B displayed only a suggestive association with immune outcome (p=0.08, data not shown). In addition to the two already reported IL7R SNPs (rs6897932/Thr244Ile and rs6890853, D’=0.99), our analysis demonstrated four additional IL7R SNP associations (including one additional coding SNP rs3194051/Ile356Val; SNPs in LD, D’≥0.99) demonstrating a considerable (approximately 2-fold for rs3194051) allele dose-related increase in antibody responses (p≤0.024, Table 3).

Table 3

Race-specific analyses for associations of SNPs in coding/regulatory regions of cytokine and cytokine receptor genes and measles virus-specific neutralizing antibody responses

We also performed a race-specific analysis for the African-American subjects (n=89). Only two SNP associations (rs2228046/IL6ST and rs2243292/IL4) from the combined analysis were significant in this racial group, and those were different from the SNPs that demonstrated significance in the Caucasian group (Table 3).

Associations between SNPs in cytokine and cytokine receptor genes and cellular immune responses after measles vaccination

Overall, we found 29 significant associations between genetic variations in the coding/regulatory regions of cytokine/cytokine receptor genes and measles virus-specific IFNγ Elispot responses after measles immunization (p<0.05)(Table 4). We found six IL7R genetic variants (all of which were also associated with antibody responses) associated with measles-specific cellular immunity (Table 4). The minor alleles of one coding SNP (rs6897932/Thr244Ile) and one promoter IL7R SNP (rs6890853), in LD (D’=0.99), were associated with up to a 21% increase in measles-specific IFNγ Elispot responses. The minor alleles of four other IL7R SNPs (one additional coding SNP rs3194051/Ile356Val and three SNPs in the 3′intregenic region, in LD, D’≥0.93) demonstrated up to a 40% allele dose-related decrease in measles-specific cellular responses (p≤0.04, Table 4). Two coding synonymous IL10RA SNPs (rs2229115/Thr324Thr and rs4252249/Ala40Ala) were also associated with allele dose-related variations in the immune outcome (p≤0.011). In addition, the minor alleles of two IL4R coding SNPs (rs1805011/Glu400Ala and rs2234900/Leu433Leu) and one LTA coding SNP (rs2229092/His51Pro) demonstrated up to a 72% (for rs2229092) decrease/increase in IFNγ Elispot responses (p≤0.039). Two IL2 promoter SNPs (the previously reported rs2069762 [5] and rs4833248, in LD, D’=1) also demonstrated a significant increase in IFNγ Elispot responses with the representation of the minor allele genotype (p≤0.037).

Table 4

SNPs in coding/regulatory regions of cytokine and cytokine receptor genes associated with measles virus-specific IFNγ Elispot responses

A race-specific analysis in the Caucasian group confirmed many of the above associations found in the combined analysis (Table 5). In particular, the two IL7R SNPs (rs6897932/Thr244Ile and the promoter SNP rs6890853), the two coding IL4R SNPs (rs1805011/Glu400Ala and rs2234900/Leu433Leu) and the LTA coding SNP (rs2229092/His51Pro) reproduced their association, with up to an 87% variation in IFNγ Elispot responses between genotypes in the Caucasian group (p≤0.037). The two IL2 promoter SNPs (rs2069762 and rs4833248) referred to above, also reproduced their association with measles-specific cellular immune responses (p≤0.042). In addition, one previously reported IL10 promoter SNP rs1800890 [5] was associated with variations in IFNγ Elispot responses (p=0.025).

Table 5

Race-specific analyses for associations of SNPs in coding/regulatory regions of cytokine and cytokine receptor genes and measles virus-specific IFNγ Elispot responses

Analysis of the African-American group confirmed the associations (from the combined analysis) for the two coding IL10RA SNPs (rs2229115/Thr324Thr and rs4252249/Ala40Ala), which were associated with significant variations in the immune outcome in this racial group (p≤0.011). One IL4 SNP rs2243291 also reproduced its association in the African-American sample (Table 5). Two additional genetic variants in the promoter region of IL4 (rs2243248 and rs2243250) demonstrated associations with substantial variations (3-fold decrease and 7-fold increase, respectively) in IFNγ Elispot responses (Table 5). One non-synonymous IL4R SNP (rs3024638/Thr256Thr) was associated with an allele dose-related increase in IFNγ Elispot response (p=0.045).

Associations between IL7R haplotypes and measles virus-specific humoral and cellular immunity

The IL7R haplotype level analysis revealed significant associations with measles-specific humoral immunity in Caucasians (global p-value=0.003, Table 6), but not in African-Americans (global p-value=0.259, data not shown). Particularly, the most common IL7R haplotype AAGCGAGAGAAAAAAAGGA was associated with decrease in neutralizing antibodies (allele p-value=0.011), and also with increase in IFNγ Elispot responses (allele p-value=0.019). Another common haplotype GAGAGGGAGCGAGAGGCGA demonstrated significant association with increased antibody levels (p=0.007).

Table 6

IL7R haplotype associations with measles-specific humoral and cellular immune responses in Caucasians

Associations between SNPs in cytokine and cytokine receptor genes and measles-specific secreted cytokines

We found 121 significant associations (p-value<0.01) between SNPs and measles virus-specific cytokine secretion (Supplementary Table 1). Of note, a non-synonymous CSF2RB polymorphism rs1801114/Val652Met demonstrated highly significant associations with the secretion of IL-2 (p=0.00007), IL-6 (p=0.00003) and IFNγ (p=0.001) in the African-American group. We also identified two highly significant intronic SNP associations, IFNGR2 SNP rs9976572 (p=0.000005) and IL1R2 SNP rs13408303 (p=0.00001) associated with measles-specific IL-6 and TNFα secretion (respectively) in the African-American group. (Supplementary Table 1).

4. Discussion

Cytokine and cytokine receptors are intrinsically linked to the generation and regulation of adaptive immune responses, and therefore are reasonable targets for candidate gene-based genetic studies [6,29-32]. We have previously sampled a smaller number of candidate cytokine and cytokine receptor gene polymorphisms/SNPs (n=58) in Caucasians (n=118) and African-Americans (n=85) and reported initial moderate associations between SNPs in IL2, IL10, IL12B, IL4RA, IL12RB, IL6 and TNFA genes and measles vaccine-induced immunity [3,5]. The current comprehensive candidate gene association study aimed to replicate these initial findings in a racially diverse cohort of vaccinated individuals and to identify new plausible genetic variants/genes regulating measles vaccine-induced immunity.

Importantly, our current study confirmed and replicated a significant association of genetic variant rs3212227 located within the 3′UTR region of the IL12B gene with variations in measles-specific humoral immunity in our combined analysis (p=0.037) [5]. However, while discovered in a study of Caucasian subjects [5], this SNP demonstrated a suggestive association (p=0.08) in Caucasians in our current study. There are differences between the discovery and replication studies, which might explain the slightly different levels of significance in the Caucasian subjects, regardless, both findings were in the same direction. An important difference between the two studies is the measurement of the humoral immune outcome used in the analysis. The discovery study measured total measles-specific IgG using EIA, while the current study measured the clinically relevant measles-specific neutralizing antibody levels (anti-H and anti-F antibodies) using PRMN assay. In the discovery study the associations between SNPs and measles-specific humoral and cellular immune responses were assessed using χ2 tests of significance that compared the distribution of SNPs across four quadrants, defined by the extremes of humoral (antibody) and cellular (lymphoproliferative) immune responses [5], while the present study evaluated associations between SNPs and quantitative measures of humoral/cellular immune outcomes in an entire cohort using linear regression methodologies. While we speculate that these and/or other differences in the study design might account for the slightly different results found in Caucasian subjects between the two studies, we have planned subsequent validation of results in a new independent cohort.

Interleukin-12 (IL-12p70), a pivotal cytokine in the induction of Th1 immune responses is a dimer composed of two subunits p40 and p35, encoded by the IL12B and IL12A genes, respectively. Interestingly, the IL12B gene has a relatively high level of conservation among humans [33]. The replicated genetic variant rs321227 (in our combined analysis) that affects the TaqI restriction site within the 3′UTR gene region (TaqI polymorphism) is a known regulatory/functional polymorphism with confirmed effects on the expression of IL12B and the secretion of IL-12, and has been associated with a number of autoimmune diseases, immune/inflammatory disorders and infectious diseases [33-40]. More importantly, the TaqI polymorphism has been implicated in regulation of the antibody response to HBsAg after immunization [6]. The replication of this functional polymorphism in two independent measles vaccine studies with differences in the study design, cohort characteristics, analysis and in the humoral immune outcome (EIA vs. neutralizing antibodies), is highly suggestive of IL12B involvement in the immune response variations observed after measles vaccination. Similarly, one IL2 promoter polymorphism (rs2069762) and one IL10 promoter polymorphism (rs1800890), previously associated with measles-specific antibody and lymphoproliferative responses [5], demonstrated significant associations with IFNγ Elispot responses (as a measure of measles-specific cellular immunity) in the Caucasian group in this study. Particularly, IL2 rs2069762 was also found to be associated with IL-2 secretion in our study (p=0.006, Supplementary Table 1), and is a plausible functional SNP with effects on gene regulation and IL-2 expression, previously known to regulate the response to HBV vaccination [12,41].

Other interesting findings include the multiple IL7R genetic variants that were associated, at the genotype and haplotype level, with both humoral and cellular measles-specific immune responses in the Caucasian sample, but not the African-American sample. The IL7R gene encodes one of the receptor subunits for the IL-7 receptor, IL7Rα, which forms a receptor complex with IL-2RG. The ligand-receptor interaction and signaling induces somatic recombination of the immunoglobulin (Ig) and the T-cell-receptor (TCR) genes, promoting the survival and proliferation of B and T lymphocytes, as demonstrated in animal and human studies, where genetic aberrations lead to severe immune deficiencies [42,43]. Furthermore, as noted by Gregory et al., the presence of membrane-bound IL7Rα is the factor that limits IL7 receptor formation with significant effects on IL-7 signaling, T cell regulation and maintenance [44]. One of the IL7R allelic associations identified in our study pertains to a known causal non-synonymous SNP rs6897932, found in the alternatively spliced exon 6 of IL7R (Chr5p13), with confirmed effects the expression of the gene and the amounts of membrane-bound and soluble protein isoforms [44]. This genetic variant and other polymorphisms in the IL7R locus have been implicated in the susceptibility of immune, autoimmune, inflammatory and degenerative disorders [43-47]. Interestingly, in our study, the minor alleles of the coding causal IL7R polymorphism rs6897932 and a promoter IL7R polymorphism rs6890853 were consistently associated with a substantial decrease (more than 30%) in measles-specific neutralizing antibody response and a modest increase (cross-regulation) in measles-specific cellular IFNγ Elispot response in our Caucasian group, while the other four identified coding/regulatory genetic variants in the same genetic locus were associated with increases and decreases in measles-specific humoral and cellular immunity, respectively. It is plausible that a gene with a critical role in virus-induced immunity (such as IL7R), will likely demonstrate associations with both increases and decreases in adaptive immune response measures for polymorphisms affecting gene regulation/activation, transcriptional activity/gene expression, and/or translation and receptor binding, depending on the SNP specific location and function. Thus our findings, in concert with the observed haplotype effects, strongly suggest the involvement of IL7Rα-related immune response mechanisms in the immune response heterogeneity observed after measles vaccination.

Among other findings, the results of the present study also highlight genetic variants within the IL4 and IL4R genes that are plausible determinants of vaccine-induced immune responses as recognized by other studies [4-7]. Particularly interesting are our findings for a genetic variant in the promoter region of IL4 (rs2243248), which has been associated with multiple measles-specific immune outcomes including neutralizing antibody levels in Caucasians (p=0.003). This same genetic variant has been associated with Herpesvirus-8 lytic antibody titers in HHV-8-positive adults [48]. Another IL4 promoter polymorphism (rs2243250) with likely functional consequences (with effects on IL-4 activity by creating a transcription factor binding site for NFAT-1 [49]), and known to confer susceptibility to subacute sclerosing panencephalitis [50], demonstrated a 7-fold increase in measles-specific cellular immunity/IFNγ Elispot with the representation of the minor allele genotype in our African-American group (p=0.026). Two coding IL4R SNPs (rs2234900 and rs1805011), associated with measles-specific IL-4 secretion in our initial study [5], demonstrated associations with IFNγ responses in Caucasians in our current study, thus further confirming their importance for measles vaccine-induced immunity.

Genetic studies often report inconsistent findings on associations between interrogated genes/polymorphisms and complex traits such as vaccine-induced immunity. The replication data (analysis of previously reported significant SNP associations in independent cohorts) and discovery data (data from interrogation of new genes/SNPs, not studied in our previous studies) presented here are in accordance with the polygenic nature of vaccine response, where multiple genes and interactions are likely to contribute to phenotype. Our findings are more consistent with race-specific, rather than common genetic variants/genes associated with measles-specific immune outcomes in the studied racial groups. A major strength of our study is the ability to validate previous pilot findings on genetic determinants of measles immunity by replication in a relatively large racially-diverse cohort of healthy vaccinees after two doses of MMR vaccine sampled from a region with high vaccine coverage and no circulating wild type measles virus (hence immune measures reflect vaccination). The heterogeneity of the study cohort and the smaller sample size of the different racial groups (particularly the African-American racial group), limited our power, therefore we chose to use the less stringent cutoff value 0.05 in the belief that the risk of false negatives outweighs that of false positives. In addition we reported race-specific associations for the two predominant racial groups. The resultant findings were mostly restricted to the specific groups being analyzed and the results cannot be extrapolated to other races and ethnicities, due to differences in the allele frequencies across populations.

This study, to our knowledge, represents the largest genetic association effort ever performed in measles immunity on non-HLA candidate genes. This enabled us to replicate previous findings, as well as to probe a large number of additional genetic variants and genes. We acknowledge the multiple testing issues and the possibility of false-positives. We would expect approximately 80 associations with antibody and IFNγ Elispot response outcomes by chance alone (assuming independent tests of association at the p=0.05), while we observed 98 associations (48 coding/regulatory and 50 intronic SNPs), which increase our confidence that some of the observed effects are genuine. The replication of previous findings, along with the observed allele dose-response relationships and haplotype effects, cross-regulation of cellular and humoral immunity, the biologically plausible functionality and substantial immune outcome variations for some of the genetic variants further strengthen the significance of our results. Replication of all newly identified associations in an independent cohort, followed by fine mapping and functional studies of the implicated genetic determinants are planned to further extend our findings.

In conclusion, the present study highlights the importance of replicated and newly recognized non-HLA-related genetic variants and genes with likely functional consequences for interindividual variations in immune response to measles vaccine.

*Highlights

We studied 801 SNPs in 56 genes, in a cohort of 745 MMR-vaccinated schoolchildren
We found 48 significant associations with variations in vaccine immune responses
We replicated an association of IL12B rs3212227 with variations in humoral immunity
We replicated two IL10 and IL2 SNP associations with cellular immunity variations
Single SNP and haplotype analysis revealed IL7R associations with vaccine immunity

Supplementary Material

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Acknowledgments

We thank the parents and children who participated in our study and the Mayo Vaccine Research Group nurses for subject recruitment. We thank the Mayo Vaccine Research Group laboratory personnel for technical help with the assays and Matthew J. Phan for assistance in preparing the manuscript. We thank Megan O’Byrne for her contribution to statistical analyses. We thank David Rider and Ying Li for developing the SNP selection algorithm, and Julie M. Cunningham and the Mayo Advanced Genomic Technology Center for assistance with genotyping. The project described was supported by Award Numbers AI33144 and AI48793 (which recently received a MERIT Award) from the National Institute Of Allergy And Infectious Diseases, and 5UL1RR024150-03 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health, and the NIH Roadmap for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute Of Allergy And Infectious Diseases or the National Institutes of Health.

Footnotes

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Disclosures

Dr. Poland is the chair of a DMSB for novel vaccines undergoing clinical study by Merck Research Laboratories. Dr. Jacobson recently served on a Safety Review Committee for a post-licensure study conducted by Kaiser-Permanente concerning Gardasil HPV vaccine funded by Merk & Co. Other authors declare that they have no conflict of interest.

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