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
]. 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
genes and measles vaccine-induced immunity [3
]. 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
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 Taq
I restriction site within the 3′UTR gene region (Taq
I 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
]. 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
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
]. 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
]. 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
genes that are plausible determinants of vaccine-induced immune responses as recognized by other studies [4
]. 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.