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Background.Identifying genetic factors that influence poxvirus immunity across races may assist in the development of better vaccines and approaches for vaccine development.
Methods.We performed an extensive candidate-gene genetic screen (across 32 cytokine and cytokine receptor genes) in a racially diverse cohort of 1056 healthy adults after a single dose of smallpox vaccine. Associations between single-nucleotide polymorphisms (SNPs)/haplotypes and vaccinia virus–specific neutralizing antibodies were assessed using linear regression methodologies.
Results.The combined analysis identified 63 associations between candidate SNPs and antibody levels after smallpox vaccination with P < .05. Thirty-one of these were within the IL18R1 and IL18 genes. Five IL18R1 SNPs, including a coding synonymous polymorphism rs1035130 (Phe251Phe) and 2 promoter SNPs (rs6710885, rs2287037), all in linkage disequilibrium, were associated with significant variations in antibody levels in both Caucasians (P ≤ .016) and African Americans (P ≤ .025). Similarly, associations with 2 intronic IL18 SNPs (rs2043055 and rs5744280) were consistent in the Caucasian (P ≤ .023) and African American samples (P ≤ .014). Haplotype analysis revealed highly significant associations between IL18R1 haplotypes and vaccinia virus–specific antibody levels (P < .001, by combined analysis) that were consistent across races.
Conclusions.Our study provides evidence for IL18 and IL18R1 genes as plausible genes regulating the humoral immune response to smallpox vaccine in both Caucasians and African Americans.
Host genetic factors play an important role in determining differential susceptibility to infectious diseases in humans and in explaining the heterogeneity of immune response to infection and vaccination . Cytokines are important regulators of the immune system that maintain a balanced cytokine environment and shape innate and adaptive immune responses. Genetic variations, such as single-nucleotide polymorphisms (SNPs) in cytokine and cytokine receptor genes, can affect gene transcription and regulation and the protein/functional activity of these molecules, thereby altering the resultant immune responses. Our previous research has highlighted the importance of polymorphisms in these genes for variations in humoral and cellular immune responses to multiple vaccines [2–6].
Recently, there has been renewed scientific and public interest in smallpox vaccines and in evaluating the immunologic response to smallpox vaccination, as well as in understanding the underlying mechanisms of immunogenetic variation, because of concerns about bioterrorism and documented outbreaks of monkeypox in Africa and the United States [7, 8]. Neutralizing antibodies induced by infection or immunization play a central role in protection against poxviruses.
Sex, ethnic, and racial differences have been associated with differences in immune responses to both infection and vaccination [2, 3, 9–14]. Although racial differences in inherited SNPs in immune response genes have been identified, most genetic association studies of vaccine-induced immunity have been performed in Caucasian populations, and little is known about other racial groups [3, 15]. A modicum of data are available regarding associations between SNPs in immune response genes and adverse reactions or immunity to smallpox vaccination [16–19]. Therefore, in this study, we examined and sought to identify common SNPs in cytokine and cytokine receptor genes and their associations with variations in vaccinia-specific neutralizing antibody levels in Caucasian and African American populations after receipt of a single dose of Dryvax vaccine.
Our study cohort comprised a sample of 1076 healthy individuals (age, 18–40 y) who participated in the Department of Health and Human Services civilian health care worker smallpox immunization program at the Mayo Clinic in Rochester, Minnesota, and armed forces personnel at the Naval Health Research Center in San Diego, California, as previously described . All study subjects had been vaccinated with a single dose of live-virus Dryvax vaccine (Wyeth Laboratories) within 4 years prior to recruitment and had a documented vaccine vesicular “take,” or development of a pustule at the vaccination site. Institutional Review Board approval was granted for the study, and written informed consent was obtained from each participant.
To measure vaccinia-specific neutralizing antibody levels, we used a standardized, rapid, and sensitive high-throughput neutralization assay (based on a recombinant, β-galactosidase–expressing vaccinia virus) developed at the US Food and Drug Administration and optimized for our use as previously described [11, 20]. Results were defined as the serum dilution that inhibits 50% of virus activity (ID50), estimated using the M estimation approach introduced by Huber . Each serum sample was tested at least 3 times. The high reproducibility of this assay and its ability to provide reliable outcome estimates are reflected in the relatively low mean coefficient of variation (CV, 6.9%), based on multiple measurements [11, 20].
We selected tag SNPs within and 10 kb upstream and downstream of 32 candidate cytokine and cytokine receptor genes using the approach of Carlson et al  on data from the source with the greatest number of SNPs from among the Hapmap Phase II Seattle SNPs, National Institute of Environmental Health Sciences (NIEHS) SNPs, and National Center for Biotechnology Information (NCBI). Candidate SNPs had minor allele frequencies (MAFs) ≥0.05 and a linkage disequilibrium (LD) threshold of r2 ≥ 0.90. Validated SNPs identified by Illumina as having a high likelihood of successful genotyping received priority, as did SNPs with a greater likelihood of having an impact on function (coding nonsynonymous, synonymous, 5′ or 3′ untranslated regions, intronic).
The selected SNPs were genotyped using 2 custom-designed 384-plex Illumina GoldenGate assays (Illumina) following the manufacturer’s instructions and as previously described . SNPs that failed Illumina genotyping were alternatively genotyped using polymerase chain reaction (PCR)–based TaqMan custom designed assays (Applied Biosystems) or via pyrosequencing.
Statistical analyses assessed associations between smallpox-specific neutralizing antibody levels and tagSNPs selected from candidate genes. We used genotype data from Illumina 550 and 650 SNP arrays (obtained from these subjects for other purposes) to classify individuals into Caucasian and African American subgroups. The average genetic similarity between subjects included none of the SNPs that were specifically highlighted in this report. In brief, 22863 SNPs in low LD (r2 < 0.10) from the SNP arrays were selected. A principal components analysis was performed using EIGENSTRAT , and the top ten eigenvectors were extracted. Subjects were clustered into genetically similar groups. The clusters that were predominantly self-identified Caucasians or African Americans were labeled accordingly. Of the 1056 subjects with data in this report, 214 were classified as African American, and 569 were classified as Caucasian. No subjects were misclassified relative to their self-declared race using this approach, but 36 subjects who had not provided a self-declaration were classified as African American in subsequent analyses. The references to Caucasians and/or African Americans through the manuscript, therefore, refer to the genetically clustered groups and not the self-reported race, although the 2 groups are nearly interchangeable. There were no substantial differences between the results obtained using the 2 different definitions of race.
The median of the repeated measurements of neutralizing antibody levels was extracted from each individual and used in descriptive summaries. These values, as well as those from other demographic and clinical variables, were summarized across individuals.
SNP allele frequencies were estimated within racial groups, and departures from Hardy–Weinberg equilibrium (HWE) were assessed within racial groups using a Pearson goodness-of-fit test or a Fisher’s exact test when SNPs with a MAF of <5%. No SNPs were excluded from analysis due to violation of HWE, but significant SNPs with deviation from HWE (HWE P < .001) were highlighted as such in the reported data (1 SNP rs8178561 in Supplementary Table 1). Pair-wise LD estimates were obtained using Haploview, version 3.32 . SNP associations with antibody levels were assessed using all available replicates within each subject, with antibody levels represented on the logarithmic scale, using linear regression models that accounted for the repeated assessments per subject using an unstructured covariance matrix. Primary tests of association assumed an ordinal SNP effect. Analyses using dominant and recessive SNP effects were also performed, although these additional analyses did not differ substantially from the original ordinal tests. Overall tests of significance were performed while adjusting for population stratification using the eigenvectors from EIGENSTRAT. Separate race-specific analyses were also performed. These analyses adjusted for residual population stratification using the top 4 eigenvectors from EIGENSTRAT using genotypes within each racial group. We estimated false discovery rates for each SNP by computing q values as per Storey and Tibshirani [26, 27].
We also examined gene-level associations with our measure of humoral immunity, because the SNPs selection was gene-centric, using the principal components approach of Wang and Abbott . For each gene, we selected enough principal components to explain ≥90% of the variability in the ordinal SNP variables. This group of principal components was simultaneously tested for significance using the regression approach described for single SNPs.
We also performed post hoc haplotype analyses within racial groups for haplotypes with frequencies >1% in the combined data set. Expected design matrices were defined that reflected the number of each candidate haplotype that was expected to be carried by each subject, similar to methods outlined by Schaid et al [29, 30]. The haplotype design variables were analyzed using the regression approach described for single SNPs. Tests of significance and summaries of haplotype effects were extracted.
All of the association analyses were adjusted for age at enrollment, eigenvectors from EIGENSTRAT, sex, and time between smallpox vaccination and blood draw. Combined analyses also adjusted for genetically derived racial groups. All statistical tests were 2-sided, and unless otherwise indicated, all analyses were performed using the SAS software system (SAS Institute).
The demographic characteristics and antibody levels of the study cohort of 1076 healthy adults have been previously described . Briefly, our cohort (n = 1056) consisted predominantly of Caucasians (n = 569; 53.9%) and African Americans (n = 214; 20.3%), primarily men (n = 778; 73.7%) . The median age at enrollment was 24 years (interquartile range [IQR], 18–40 y), and the median time from vaccination to blood draw was 1.3 years (IQR, 0.1–4.1 y). The median for neutralizing antibody ID50 values for all subjects was 132.2 (IQR, 78.8–205.6) .
We genotyped 1076 subjects for 785 known SNPs in the Th1 (IL2, IFNG, IL12A, IL12B), Th2 (IL4, IL10), and innate/inflammatory (IL6, IL18, IL1B, TNFA, IFNA, IFNB1) cytokine genes and the corresponding Th1 (IL2RA, IL2RB, IL2RG, IFNGR1, IFNGR2, IL12RB1, IL12RB2), Th2 (IL4R, IL10RA, IL10RB), and innate/inflammatory (IL6R, IL6ST, IL18R1, IL1R1, IL1R2, IL1RN, IFNAR1, IFNAR2, TNFRSF1A, TNFRSF1B) cytokine receptor genes. Our overall genotyping success rate was 89.3%, and the study success rate was 98.14%. Subject exclusions were made on the basis of inadequate/poor quality DNA (n = 5) or call rates <0.95 (n = 15). SNPs were excluded on the basis of genotyping failure, low call rates (n = 25), or MAF < 0.05 (n = 50). A total of 701 SNPs met all genotyping quality assessments and were used for analysis in the final set of 1056 subjects (including 569 Caucasians and 214 African Americans).
The combined analysis of 1056 subjects at the gene level revealed significant associations between IL18R1 (P = .003) and IL18 genes (P = .02) and humoral immune response to smallpox vaccine. Similarly, we found significant associations between IL18R1 (P = .043) and IL18 (P = .029) genes and antibody levels in our African American subjects, whereas IL18R1 and IL18 gene-level associations in Caucasians were only suggestive (P = .129 and P = .198, respectively).
Overall, we found 31 significant associations (49.2% of all significant associations), at the level P < .05, between SNPs located in the IL18R1 and IL18 genes and vaccinia-specific antibody levels (Table 1). The presence of a homozygous minor allele genotype or heterozygous genotype for 6 IL18R1 SNPs (rs2080289, rs2241116, rs1035130, rs4851570, rs6710885, and rs2287037), in LD (Figure 1), was associated with an increase in vaccinia virus–specific antibody response, and these associations were highly significant (P ≤ .001; Table 1). Two of these SNPs (rs2080289 and rs2241116) demonstrated clear allele dose-response relationships with immune outcome. Two of the genetic variants in this LD block (rs6710885 and rs2287037; r2 = 1) were within the promoter region of the IL18R1 gene, and 1 genetic variant (rs1035130) was a coding synonymous SNP, Phe251Phe. Furthermore, the minor alleles of 2 additional IL18R1 promoter SNPs (rs3755276 and rs10204837; r2 = 1; in LD with 15 intronic SNPs, r2 ≥ 0.63) were associated with a decrease in vaccinia virus–specific antibody levels (P ≤ .031) in an allele dose-dependent manner (Table 1). Six SNPs within the IL18 gene also demonstrated associations with humoral immune responses. The minor alleles of 2 intronic IL18 SNPs (rs2043055 and rs5744280) in LD (r2 = 0.78) were associated with an allele dose-related decrease in antibody levels (P < .001) (Table 1). After correcting for false discovery rate (FDR), 9 SNPs remained significant at the q value = 0.1 (Table 1). Other significant associations for SNPs belonging to cytokine/cytokine receptor genes other than IL18R1 and IL18 are shown in Supplementary Table 1.
Race-specific analysis in the Caucasian subjects (n = 569) revealed that the 6 already cited IL18R1 SNPs (rs2080289, rs2241116, rs1035130/Phe251Phe, rs4851570, rs6710885/promoter, and rs2287037/promoter), in LD (Figure 1), were associated with an increase in humoral immune response in an allele dose-dependent manner (P ≤ .016; Table 2). The observed increase in antibody levels varied between 17.6% and 61% (calculated for a specific SNP as the ratio, minus 1, of the immune outcome observed for the homozygous minor allele genotype to the immune outcome observed for the homozygous major allele genotype, multiplied by 100%). Similarly, in the Caucasian population, the minor alleles of the 2 already cited intronic IL18 SNPs (rs2043055 and rs5744280; r2 = 0.82), were associated with an allele dose-related decrease (18%) in antibody levels (P ≤ .023).
In the African American sample, 5 of the 6 reported IL18R1 SNPs (in Caucasians) in LD (Figure 1; rs2080289, rs1035130/Phe251Phe, rs4851570, rs6710885/promoter, and rs2287037/promoter) had consistent associations with vaccinia virus–specific humoral immune response (P ≤ .025) (Table 3). The 2 most significant genetic variants in the promoter region of IL18R1 (rs6710885 and rs2287037; r2 = 1; P ≤ .002) demonstrated an allele dose-related increase in antibody levels of 78%. In addition, in the African American sample, we observed associations that were similar to those identified in the Caucasian subjects for the intronic SNPs in the IL18 gene (rs2043055 and rs5744280; r2 =0.65), and these minor alleles were associated with an allele dose-related decrease (35% and 21% for rs2043055 and rs5744280, respectively) in antibody levels (P ≤ .014).
The Haploview output for the IL18R1 SNPs consistent across races, which were used in the haplotype analysis, is shown in Figure 1. Among the 6 haplotypes with frequencies ≥1% in our combined study group, the global tests demonstrated highly significant associations between IL18R1 haplotypes and vaccinia virus–specific neutralizing antibody levels both in the combined analysis (global P < .001; Table 4) and in the race-specific analyses (P = .001 for Caucasians and P = .034 for African Americans). Both combined and race-specific analyses demonstrated that the most common major allele IL18R1 haplotype AGGGCA (major alleles of all genetic variants) was associated with decreased antibody levels (P ≤ .027), whereas the rarer minor allele IL18R1 haplotype GAAAAG was associated with increased antibody levels (P ≤ .031).
Establishing genetic effects for complex traits, such as vaccine-induced immunity, is dependent on the replication of genetic findings in more than one population . 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 [32–34]. 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 [35–38]. 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, IL18, and IL18RAP gene expression after smallpox vaccination .
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 , 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, 42]. 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 . 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 .
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, 44–49]. 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 . 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 , 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%) . 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.
Supplementary data are available at The Journal of Infectious Diseases online.
National Institutes of Health (NIH) grant AI 40065 and 1 UL1 RR024150-01 from the National Center for Research Resources, a component of the NIH.
We thank the volunteers who participated in the study; the Mayo Clinic Vaccine Research Group nurses; Drs Margaret Ryan and Kevin L. Russell; the Naval Health Research Center (NHRC) team, for subject recruitment; Jenna E. Ryan and the Mayo Clinic Vaccine Research Group staff, for technical help; Julie M. Cunningham and the Mayo Advanced Genomic Technology Center, for assistance with genotyping; and Robert A. Vierkant and David A. Watson, for their contribution to statistical analyses.