Establishing genetic effects for complex traits, such as vaccine-induced immunity, is dependent on the replication of genetic findings in more than one population [31
]. For this reason, we assessed common and potentially important genetic markers for smallpox vaccine–induced humoral immunity across 2 different racial groups. The latter were defined as genetically clustered race and self-declared race (with no disagreement) and were augmented with a collection of subjects who had not self-declared for race but who clustered with one of the racial groups. Because these analyses were performed in subjects with a diverse genetic background, it was important to account for these genetic differences by using genetically defined racial groups and adjusting for additional genetic heterogeneity, even within racial groups.
Seven SNPs, including likely functional promoter and coding SNPs, belonging to the IL18R1 and IL18 genes were the only genetic variants with associations that were consistent between the 2 most prevalent racial groups at the genotype and haplotype level, which suggests the important role of these genes/genetic variants in the modulation of smallpox vaccine–induced humoral immunity.
Interleukin (IL)–18 is a unique and potent proinflammatory cytokine that is known to enhance innate immunity and both Th1- and Th2-driven immune responses, depending on its cytokine milieu [32
]. Upon binding to its receptor IL18R1, IL-18 triggers the recruitment of IL18RAP, which initiates signaling. Of note, IL-18 has been demonstrated to play an important antiviral role in orchestrating the cell-mediated immune response to viruses, including vaccinia virus, which encodes a soluble IL-18–binding protein that promotes virulence by countering IL-18 [35
]. The importance of IL-18 in poxvirus immunity is also suggested by earlier in vivo studies in humans that have demonstrated quick down-modulation of IL18R1
gene expression after smallpox vaccination [39
Our data provide evidence for IL18R1
SNP and haplotype associations with neutralizing antibody levels that were consistent across 2 different racial groups with a high level of significance. Of note, the only coding IL18R1
SNP, the synonymous G/A polymorphism rs1035130 (Phe251Phe), is located in the third immunoglobulin-like domain of the extracellular portion of IL18R1, critical for IL-18 binding and formation of the ternary complex [40
], and is predicted to have a functional effect in splicing regulation. This genetic variant (in addition to other SNPs in the Chr2q12 locus) was associated with bronchial hyperresponsiveness and asthma in European populations [41
]. Promoter variants rs6710885 and rs2287037 occur in the regulatory region of IL18R1
and are predicted to change transcription factor binding sites for GATA family transcription factors and C/EBPb (CCAAT/enhancer binding protein beta), respectively, with a possible effect on gene transcription and immune/inflammatory responses (FastSNP). In a family-based association analysis for asthma, the IL18R1
promoter variant rs2287037 was shown to have significant replicated association (in haplotype analysis) with bronchial hyperreactivity in 2 distinct European populations [43
]. In a study of inflammatory bowel disease, 2 of the referred IL18R1
SNPs (rs1035130/Phe251Phe and rs2287037/promoter) were significantly associated as part of a haplotype block with bowel inflammation and disease susceptibility [31
The only other locus that consistently demonstrated associations with vaccinia-specific neutralizing antibody levels across the 2 racial groups is on 11q22, within the IL18
gene, and includes the intronic SNPs rs2043055 and rs5744280 (in strong LD; P
≤ .023). Genetic variants in IL18
have been linked to a number of inflammatory conditions and immunological and treatment outcomes, including inflammatory bowel disease, cardiovascular disease, bronchial asthma, adverse events following smallpox immunization, viral clearance and treatment response in hepatitis C patients [17
]. Particularly interesting is the reported haplotype-level association of the intronic IL18
SNP rs2043055 identified in our study (in LD with rs5744280) with the risk for development of fever after smallpox vaccine [17
]. Findings of the same variant or variants in 2 independent clinical vaccine trials is highly suggestive of IL18
involvement in the mechanisms underlying biological response heterogeneity to smallpox vaccine. Furthermore, a genome-wide association study in Caucasians provided evidence for the potential functional importance of the other IL18
SNP rs5744280, which has been found to be associated with plasma IL-18 levels [46
], suggesting an effect on protein expression.
A large-scale smallpox vaccination study (US Department of Defense study) evaluated the local and systemic adverse effects after smallpox vaccination, such as the development of myopericarditis (inflammatory heart disease), which was reported to occur predominantly in males (98%) of Caucasian race (86.4%) [50
]. Although these observations might indicate racial genetic differences in the risk of adverse inflammatory responses to smallpox vaccine, a large prospective genetic association study (with larger representation of minorities) is still warranted to elucidate the genetic basis of these dissimilarities.
Interestingly, our genetic association study assessing humoral immune response to smallpox vaccination demonstrated overall gene-level IL18 and IL18R1 associations with weaker effect in the Caucasian group (which might suggest a stronger relative influence of other genes and genetic variants), compared with the African American group. However, the combined analysis resulted in stronger SNP associations, and some of the observed single SNP associations, as well as the global haplotype-level IL18R1 associations, demonstrated a higher level of significance in the Caucasian group, which implies (along with the consistency of significant IL18 and IL18R1 associations across our racial groups) the importance of these genes and polymorphisms for regulating poxvirus humoral immunity in both racial groups.
A major strength of our study is the large, well-characterized, and racially diverse cohort of healthy vaccinia virus–naive adult volunteers, who received a single smallpox vaccine dose, which allowed us to measure immune variables as a true response to vaccination and presented the opportunity to study 2 different racial groups. Another benefit is the measurement of functional neutralizing antibody levels as a better correlate of protection and a clinically relevant immune outcome.
Several limitations of the study have to be addressed. The results cannot be extrapolated to other racial or ethnic groups. The race-specific analyses for Caucasians and African Americans are based on relatively smaller numbers of subjects and therefore have limited power. In addition, owing to the multiple comparisons performed, the possibility of false-positive associations cannot be excluded. However, after correction for FDR, 7 IL18R1 and 2 IL18 SNPs in the combined analysis (including all SNPs that were consistent across races) still remained significant. Although the effect of most single-nucleotide polymorphisms on complex trait outcomes (such as immunity) is more likely to be small with multiple genes or polymorphisms (including human leukocyte antigen) contributing to outcome variation, we were able to detect substantial variation in antibody levels for some of the genotypes. This, along with the biologically plausible connection of the IL-18 pathway with adaptive poxvirus immunity, the replication of pathway-related genes and genetic variants across races, and the probable functionality of certain genetic variants argues against chance findings and increases our confidence that the observed associations highlight important immune regulators in poxvirus immunity.
In conclusion, our study provides evidence for IL18 and IL18R1 genes as plausible candidate genes regulating the humoral immune response to smallpox vaccine in Caucasians and African Americans. Follow-up replication studies, locus-specific fine mapping, and functional studies are planned to determine the causative genes and genetic variants and examine their biological relevance and mechanism of action. Ultimately, understanding the genetic factors that influence poxvirus immunity across races may assist in the development of better vaccines and better approaches for vaccine management.