Abstract
Background. Genetic association studies demonstrated a role for cytokine proteins and cytokine or cytokine receptor gene polymorphisms in smallpox vaccine–induced adaptive immunity.
Methods. We examined the association of genetic polymorphisms with cellular (interferon [IFN] γ enzyme-linked immunospot assay [ELISPOT]) immune response to smallpox vaccine in 1076 immunized individuals.
Results. The majority of significant associations were discovered between single-nucleotide polymorphisms/haplotypes in IL18R1 and IL18 genes, in which we previously reported an association with vaccinia virus–induced neutralizing antibody titers in this study cohort. A functional coding IL18R1 polymorphism (rs1035130/Phe251Phe; P = .01) was significantly associated with an allele dose-related increase in IFN-γ production and was also associated with vaccinia-specific neutralizing antibody titers. Significant associations were also found between IL18R1 haplotypes and variations in IFN-γ ELISPOT responses (global P < .0001).
Conclusions. Our data suggest the importance of variants in the IL18R1 and IL18 genetic loci for broad-based smallpox vaccine–induced adaptive immunity.
Keywords: Polymorphism, single nucleotide, smallpox vaccine, vaccinia virus, interleukin-18, interleukin-18 receptor alpha subunit, haplotypes, genetic predisposition to disease, interferon-gamma, viral vaccines, genetic variation, enzyme-linked immunospot assay, european continental ancestry group, African-Americans
Live vaccinia virus–based smallpox vaccine (Dryvax; Acam) is an effective vaccine that induces a robust and long-lasting humoral and cell-mediated immune response [1, 2]. This adaptive immune response is characterized by a broad interindividual spectrum of vaccinia virus–specific antibody titers, secreted cytokine levels and frequencies of interferon (IFN) γ–producing T cells [3–6]. With regard to smallpox vaccine–induced immunity, a number of important factors, including environmental factors, can determine the response to vaccinia vaccination. Among these are genetically determined host factors (gene polymorphisms), which may determine whether or not a person will adequately respond to vaccination and/or develop experience adverse events (AEs).
Our recent study on genetic associations with humoral immune response to smallpox vaccine suggested an important effect of IL18 and IL18R1 genetic variants (single-nucleotide polymorphism [SNPs]) and haplotypes on the vaccinia-induced circulating humoral antibody production [7]. Polymorphism in the IL18R1 gene has also been linked to smallpox vaccine–induced interleukin 1β, tumor necrosis factor α, and interleukin (IL)-2 secretion [8]. In a recent study of 1076 healthy armed forces members, vaccinia-induced IFN-γ and IL-2 were significantly correlated with neutralizing antibody titers after smallpox vaccination [9]. In another study, smallpox vaccine–induced AEs were found to be correlated with increased circulating concentrations of IFN-γ, tumor necrosis factor α, IL-2, IL-5, and IL-10 [4]. In addition to the results associated with cytokine production, it has been shown that quantification of vaccinia-specific T-cell responses (from IFN-γ–producing cells) in immunized persons is important for the characterization of smallpox vaccine–induced cellular immunity [3, 6, 10]. Furthermore, a candidate gene association study identified specific IL1 and IL18 gene haplotypes associated with the development of fever after receipt of smallpox vaccine [11]. These vaccinia immunogenetic studies suggest that cytokine proteins and cytokine gene polymorphisms play a role in smallpox vaccine–induced adaptive immunity and in the development of AEs after smallpox vaccination.
We hypothesized that variations in the adaptive cellular IFN-γ responses after smallpox vaccination are associated with specific genetic markers in host cytokine and cytokine receptor genes. The purpose of this study was to examine genetic associations between individual SNPs and SNP haplotypes in the cytokine and cytokine receptor genes and vaccinia-specific T-cell response by IFN-γ enzyme-linked immunospot assay (ELISPOT).
MATERIALS AND METHODS
Study Subjects
Details of this study's recruitment and study subjects have been provided elsewhere [5–8, 10, 12]. Briefly, we enrolled 1076 healthy subjects (aged 18–40 years). All study subjects had been immunized with a single dose of Dryvax vaccine (Wyeth Laboratories) between 2002 and 2006. A total of 1056 subjects, 580 white and 217 African American, participated in this study. All subjects had a documented vaccine “take” at the vaccination site after immunization. The institutional review boards of both the Mayo Clinic (Rochester, Minnesota) and Naval Health Research Center (San Diego, California) granted permission for the study, and written informed consent was obtained from each subject.
IFN-γ ELISPOT
Our description of the ELISPOT that measures vaccinia-specific IFN-γ in vitro production by CD4+ and CD8+ T cells (ELISPOT kits; R&D Systems) is similar to those we published elsewhere [6, 10, 13]. Briefly, peripheral blood mononuclear cells were stimulated with inactivated vaccinia virus (NYCBOH strain) at a multiplicity of infection of 5 for 24 hours. Plate scanning and spot counting were performed on an ImmunoSpot S4 Pro-Analyzer using ImmunoSpot software, version 4.0 (Cellular Technology). Outcomes are expressed as spot-forming cells (SFCs) per 200 000 peripheral blood mononuclear cells. ELISPOT counts were successfully obtained for all subjects in all replicate assessments, except for 6. In these subjects, all 3 counts were available from the stimulated wells, but only 2 were successfully measured from the unstimulated wells.
SNP Genotyping
The genotyping methods we used for this study were identical to those published elsewhere [7, 8]. We selected tag SNPs within the 32 candidate cytokine and cytokine receptor genes, and 10 kb upstream and downstream of them, using the linkage disequilibrium (LD) tag SNP selection approach from 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/) databases. The selected 785 candidate SNPs were genotyped using 2 custom-designed 384-plex Illumina GoldenGate assays (Illumina), TaqMan custom assays (Applied Biosystems), and pyrosequencing. After removal of failed SNPs and DNA samples, 701 SNPs were used for analysis. We genotyped 1076 subjects for 785 SNPs in candidate cytokine genes (IL2, IFNG, IL12A, IL12B, IL4, IL6, IL10, IL18, IL1B, TNFA, IFNA, and IFNB) and their corresponding receptor genes. A total of 701 SNPs met all genotyping quality assessments and were used for analysis in 1056 subjects. The Illumina genotyping success rate was 89% (701 SNPs yielded genotyping data for analysis of 785 SNPs), and the study sample success rate was 98% (1056 samples yielded genotyping data out of 1076) [7, 8]. Subjects were excluded on the basis of inadequate or poor DNA quality (n = 5) or low call rates (<0.95; n = 15) [7, 8].
Statistical Methods
Medians were used to summarize the ELISPOT data across groups of study subjects. As repeated measurements were obtained for each subject, the subject-specific median was used as a single per-individual response measure in these summaries. Associations between IFN-γ ELISPOT responses and each of the genotyped SNPs in the candidate genes were assessed in all recruited subjects and also within white and African American subgroups, using linear mixed models regression to account for the repeated assessments of ELISPOT counts per subject. An inverse-normal transformation was applied to the data to ensure that modeling assumptions were met. In these analyses, we modeled each SNP genotype using a covariate representing the number of copies of the minor allele carried by each individual, and we adjusted for covariates that could have an impact on observed ELISPOT counts (see footnote to Table 1). We computed Q values, which estimate the probability that an observed P value is a false-positive [14, 15], and we identified SNP associations as meriting future consideration if their Q values were <0.5.
Table 1.
SNPs Associated With Vaccinia-Specific IFN-γ ELISPOT Responses in the Study Cohorta
| Gene | SNP ID | Location | Genotypeb | Subjects With Genotype, No. | IFN-γ, SFCs/2 × 105 Cells, Median (IQR)c | P Valued |
|---|---|---|---|---|---|---|
| IL6 | rs2069827 | Promoter (−1427) | CC | 923 | 51 (23–87) | .0004 |
| CA | 117 | 63 (33–97) | ||||
| AA | 3 | 122 (48–150) | ||||
| IL18R1 | rs6749014 | Intron (boundary) | GG | 307 | 48 (22–92) | .005 |
| GA | 497 | 55 (28–89) | ||||
| AA | 238 | 48 (22–84) | ||||
| IL18R1 | rs4851004 | Intron | GG | 307 | 48 (22–92) | .005 |
| GA | 495 | 55 (28–90) | ||||
| AA | 241 | 48 (22–84) | ||||
| IL18R1 | rs6706002 | Intron | AA | 307 | 48 (22–92) | .005 |
| AG | 496 | 55 (28–89) | ||||
| GG | 239 | 48 (22–84) | ||||
| IL18R1 | rs1420096 | Intron (boundary) | GG | 307 | 48 (22–91) | .005 |
| GA | 491 | 55 (28–90) | ||||
| AA | 239 | 48 (22–84) | ||||
| IL18R1 | rs7584093 | Intron | GG | 309 | 49 (24–91) | .005 |
| GA | 490 | 54 (27–89) | ||||
| AA | 242 | 48 (22–84) | ||||
| IL18R1 | rs1420094 | 3′ Intergenic | GG | 307 | 48 (22–92) | .005 |
| GA | 497 | 55 (28–89) | ||||
| AA | 240 | 48 (22–84) | ||||
| IL2RB | rs2235330 | Intron (boundary) | AA | 671 | 52 (22–85) | .005 |
| AG | 311 | 54 (27–93) | ||||
| GG | 57 | 51 (32–91) | ||||
| IL18R1 | rs6758936 | Intron | GG | 308 | 48 (23–91) | .005 |
| GA | 493 | 55 (27–89) | ||||
| AA | 241 | 49 (22–84) | ||||
| IL18R1 | rs3213732 | Intron | AA | 308 | 49 (24–91) | .006 |
| AG | 493 | 55 (27–89) | ||||
| GG | 242 | 48 (22–84) | ||||
| IL18R1 | rs7556917 | G/GC/T/TT (insertion) | CC | 311 | 49 (22–92) | .006 |
| CA | 476 | 54 (28–89) | ||||
| AA | 235 | 48 (22–83) | ||||
| IL18R1 | rs10204757 | Intron | AA | 307 | 49 (24–91) | .006 |
| AC | 493 | 55 (27–89) | ||||
| CC | 242 | 48 (22–84) | ||||
| IL18R1 | rs2287033 | Intron | AA | 307 | 48 (22–92) | .006 |
| AG | 495 | 55 (27–90) | ||||
| GG | 239 | 48 (22–84) | ||||
| IL18R1 | rs2041739 | Intron | GG | 310 | 48 (22–91) | .007 |
| GA | 493 | 55 (28–89) | ||||
| AA | 239 | 49 (22–85) | ||||
| IL18R1 | rs11465641 | Intron | AA | 309 | 49 (24–91) | .007 |
| AC | 493 | 55 (27–89) | ||||
| CC | 240 | 48 (22–84) | ||||
| IL10 | rs6697497 | 3′ UTR | GG | 1013 | 52 (24–88) | .008 |
| GA | 27 | 60 (32–111) | ||||
| AA | 2 | 91 (60–123) | ||||
| IL18R1 | rs4851570e | Intron | AA | 594 | 51 (24–82) | .011 |
| AG | 374 | 53 (25–95) | ||||
| GG | 76 | 53 (25–98) | ||||
| TNFRS1A | rs505844 | Intron | AA | 647 | 48 (22–83) | .012 |
| AG | 336 | 58 (31–95) | ||||
| GG | 54 | 66 (32–105) | ||||
| IL18R1 | rs1035130e | Synonymous (F251F) | GG | 594 | 51 (24–82) | .013 |
| GA | 372 | 53 (25–95) | ||||
| AA | 78 | 57 (25–98) | ||||
| IL18R1 | rs3771172 | Intron | GG | 620 | 51 (24–82) | .014 |
| GA | 352 | 52 (24–93) | ||||
| AA | 72 | 61 (30–99) |
Abbreviations: A, adenine; C, cytosine; ELISPOT, enzyme-linked immunospot assay; G, guanine; IFN-γ, interferon γ; IQR, interquartile range; SFCs, spot-forming cells; SNP, single-nucleotide polymorphism.
a Analyses were adjusted for sex and race of study participants, age at blood collection (quartiles), time from smallpox immunization to blood collection (quartiles), time from blood collection to assay (quartiles), shipping temperature of the sample (frozen or ambient), and time of year when the sample was shipped (warm weather months [April–September] vs cold weather months [October–March]).
b Values are presented as homozygous major allele/heterozygous/homozygous minor allele.
c Values represent simple medians (IQRs) of the raw data, not the modeled estimates. We show these values because of our use of an inverse-normal data transformation. Because of this transformation and our need to adjust for a number of covariates, and because of the high variability of the ELISPOT counts, the medians may not match the modeled ordinal genotypic trend, even though the model fit was adequate.
d Repeated-measures linear regression modeling approach.
e The SNPs rs4851570 and rs1035130 are in linkage disequilibrium (r2 = 1).
Associations between haplotypes in the IL18R1 gene and IFN-γ ELISPOT responses were tested by extracting covariates representing the expected haplotype dosage for additive haplotype effects in each individual using the haplo.design function implemented in the Haplo.Stats R package (http://cran.r-project.org/web/packages/haplo.stats/index.html), and using these expected dosage variables [16] in linear mixed models that paralleled those used for the single-SNP tests. A global test of significance was obtained for all modeled haplotypes, followed by individual per-haplotype tests. Analyses were conducted in the SAS, version 9.2, and R, version 2.14, (http://www.r-project.org/) statistical software packages.
RESULTS
Associations Between SNPs in Candidate Genes and IFN-γ ELISPOT Responses
The median for vaccinia-specific IFN-γ ELISPOT counts for all subjects was 52 SFCs/200 000 cells (IQR, 24–88). In the combined cohort (n = 1056), we estimated Q values and found 20 SNPs with a <50% chance of false-positive associations with IFN-γ ELISPOT responses (P < .015), as shown in Table 1. Of these, 16 SNPs (80%) were located in the gene encoding IL18R1. One genetic variant was a coding synonymous IL18R1 SNP rs1035130 (Phe251Phe; P = .01; in LD with intronic rs4851570; r2 = 1) associated with an increase in IFN-γ ELISPOT response.
Similarly, in the white cohort (n = 580), we found 20 significant associations (at P < .015) between genetic variants in cytokine or cytokine receptor genes and variations in IFN-γ ELISPOT responses (Table 2). Of these, 13 associations (65%) were identified with SNPs in the IL18R1 gene and these associations were consistent with the overall cohort findings. In addition, an IL6 promoter polymorphism (rs2069827, 51 vs 122 SFCs, P = .0004 for the overall cohort and 56 vs 122 SFCs, P = .001 for white) was significantly associated with a 2-fold allele dose-related increase in IFN-γ ELISPOT counts.
Table 2.
SNPs Associated With Vaccinia-Specific IFN-γ ELISPOT Responses in White Subjectsa
| Gene | SNP ID | Location | Genotypeb | Subjects With Genotype, No. | IFN-γ, SFCs/2 × 105 Cells, Median (IQR)c | P Valued |
|---|---|---|---|---|---|---|
| IL6 | rs2069827 | Promoter (−1427) | CC | 472 | 56 (28–93) | .001 |
| CA | 87 | 68 (36–120) | ||||
| AA | 3 | 122 (48–150) | ||||
| TNF | rs1800610 | Intron | GG | 459 | 58 (30–98) | .002 |
| GA | 99 | 51 (25–83) | ||||
| AA | 4 | 48 (21–85) | ||||
| IL2RG | rs11574625 | Intron (boundary) | AA | 477 | 60 (30–100) | .004 |
| AG | 35 | 31 (15–61) | ||||
| GG | 50 | 55 (27–82) | ||||
| IL2RB | rs12160547 | Intron | AA | 561 | 58 (29–95) | .006 |
| AG | 1 | 145 (145–145) | ||||
| GG | 0 | …e | ||||
| IL2RB | rs3218333 | 3’ UTR | AA | 559 | 58 (29–95) | .006 |
| AG | 1 | 145 (145–145) | ||||
| GG | 0 | …e | ||||
| IL18R1 | rs6749014 | Intron (boundary) | GG | 159 | 63 (35–102) | .008 |
| GA | 295 | 59 (28–96) | ||||
| AA | 108 | 49 (25–81) | ||||
| IL18R1 | rs6758936 | Intron | GG | 159 | 63 (35–102) | .009 |
| GA | 295 | 59 (28–96) | ||||
| AA | 108 | 50 (25–84) | ||||
| IL18R1 | rs3213732 | Intron | AA | 15929 | 63 (35–102) | .0099 |
| AG | 5 | 59 (28–96) | ||||
| GG | 109 | 49 (25–82) | ||||
| IL18R1 | rs10204757 | Intron | AA | 159 | 63 (35–102) | .0099 |
| AC | 259 | 59 (28–96) | ||||
| CC | 109 | 49 (25–82) | ||||
| IL18R1 | rs6706002 | Intron | AA | 159 | 63 (35–102) | .0099 |
| AG | 295 | 59 (28–96) | ||||
| GG | 109 | 49 (25–82) | ||||
| IL18R1 | rs4851004 | Intron | GG | 159 | 63 (35–102) | .0099 |
| GA | 295 | 59 (28–96) | ||||
| AA | 109 | 49 (25–82) | ||||
| IL18R1 | rs1420094 | 3′ Intergenic | GG | 159 | 63 (35–102) | .0099 |
| GA | 295 | 59 (28–96) | ||||
| AA | 109 | 49 (25–82) | ||||
| IL18R1 | rs7584093 | Intron | GG | 159 | 63 (35–102) | .010 |
| GA | 293 | 58 (28–95) | ||||
| AA | 109 | 49 (25–82) | ||||
| IL18R1 | rs3771172 | Intron | GG | 286 | 55 (29–87) | .011 |
| GA | 229 | 64 (28–108) | ||||
| AA | 48 | 54 (35–101) | ||||
| IL18R1 | rs7556917 | G/GC/T/TT (Insertion) | CC | 161 | 63 (35–101) | .012 |
| CA | 284 | 58 (28–95) | ||||
| AA | 105 | 52 (25–82) | ||||
| IL18R1 | rs11465641 | Intron | AA | 159 | 63 (35–102) | .012 |
| AC | 295 | 59 (28–96) | ||||
| CC | 108 | 49 (25–84) | ||||
| IL18R1 | rs2287033 | Intron | AA | 159 | 63 (35–102) | .012 |
| AG | 294 | 59 (28–96) | ||||
| GG | 108 | 49 (25–84) | ||||
| IL18R1 | rs1420096 | Intron (boundary) | GG | 158 | 63 (35–100) | .013 |
| GA | 293 | 60 (29–96) | ||||
| AA | 109 | 49 (25–82) | ||||
| TNF | rs1799724 | Promoter (−1038) | CC | 457 | 58 (30–98) | .013 |
| CT | 100 | 51 (26–84) | ||||
| TT | 5 | 70 (26–87) | ||||
| IL18R1 | rs2080289 | Intron | GG | 286 | 55 (29–89) | .015 |
| GA | 252 | 63 (28–101) | ||||
| AA | 22 | 74 (42–145) |
Abbreviations: A, adenine; C, cytosine; ELISPOT, enzyme-linked immunospot assay; G, guanine; IFN-γ, interferon γ; IQR, interquartile range; SFCs, spot-forming cells; SNP, single-nucleotide polymorphism; T, thymine.
a Analyses were adjusted for subject sex, subject age at blood collection (quartiles); time from smallpox immunization to blood collection (quartiles), time from blood collection to assay (quartiles), shipping temperature of the sample (frozen or ambient), and time of year when the sample was shipped (warm weather months [April–September] vs cold weather months [October–March]).
b Values are presented as homozygous major allele/heterozygous/homozygous minor allele.
c Values reflect simple medians (IQRs) of the raw data, not the modeled estimates. We show these values because of our use of an inverse-normal data transformation. Because of this transformation and our need to adjust for a number of covariates, and because of the high variability of the ELISPOT counts, the medians may not match the modeled ordinal genotypic trend, even though the model fit was adequate.
d Repeated-measures linear regression modeling approach.
e No subjects with genotype.
In the African American cohort (n = 217), 3 genetic variants in the promoter and intronic regions of the IL10 (rs3024503; P = .007) and IL18 (rs5744280 [P = .014] and rs3882891 [P = .015; r2 ≥ 0.38]) genes, respectively, exhibited an allele dose-response relationship with IFN-γ response. No association was found between IL18R1gene polymorphisms and IFN-γ ELISPOT responses among African Americans (Supplementary Table 1).
Associations Between IL18R1 Haplotypes and IFN-γ ELISPOT Responses
As shown in Tables 3 and 4, we identified 5 haplotypes (2 in the combined cohort and 3 in white) in the IL18R1 gene that were significantly associated with variations in IFN-γ ELISPOT responses (global P < .0001). Two specific haplotypes based on the 16 and 14 IL18R1 SNPs (GAAAAGCCGGAAAAGA; t statistic, −5.39; P < .0001) and (GAGAAGCCGAAAGA; t statistic, −5.04; P < .0001) were associated with reduced vaccinia-specific IFN-γ responses in the combined cohort and in whites, respectively. Likewise, IL18R1 haplotypes AGGCGAAAAAGGGGAG (overall cohort; t statistic, 4.63; P < .0001) and AGACGAAAAGGGAG (whites; t statistic, 4.20; P < .0001) demonstrated a significant haplotype-level association for high IFN-γ ELISPOT counts.
Table 3.
IL18R1 Gene Haplotype Associations With Vaccinia-Specific IFN-γ ELISPOT Responses in the Study Cohort
| Allelea | Frequency | Test Statistic (Haplotype t Statistic)b | Allele P Value | Global P Value |
|---|---|---|---|---|
| GGGCGAAAGAAGGGAG | 0.280370341 | 0.62 | .5331 | |
| GGGCGAAAAAGGGGAG | 0.018751575 | 1.08 | .2801 | <.0001 |
| GAAAAGCCGGAAAAGA | 0.459881582 | −5.39 | <.0001 | |
| AGGCGAAAAAGGGGAG | 0.231306574 | 4.63 | <.0001 |
Abbreviations: A, adenine; C, cytosine; ELISPOT, enzyme-linked immunospot assay; G, guanine; IFN-γ, interferon γ; T, thymine.
a IL18R1 genetic variants from left to right: rs3771172, rs6758936, rs2041739, rs7556917, rs7584093, rs3213732, rs10204757, rs11465641, rs1035130, rs6706002, rs4851570, rs6749014, rs4851004, rs1420096, rs2287033, and rs1420094.
b Haplotype effects were calculated using the haplotype t statistic, which shows the direction and magnitude of the estimated haplotypic effect on the IFN-γ ELISPOT measure. Allele P values reflect comparison of individual haplotypes with all other haplotypes combined. Statistical significance was defined as P < .05.
Table 4.
IL18R1 Gene Haplotype Associations With Vaccinia-Specific IFN-γ ELISPOT Responses in White Subjects
| Allelea | Frequency | Test Statistic (Haplotype t Statistic) | Allele P Value | Global P Value |
|---|---|---|---|---|
| GGGCGAAAAGGGAG | 0.254657349 | 0.35 | .7298 | |
| GGACGAAAAGGGAG | 0.004138499 | 0.82 | .4133 | |
| GAGAAGCCGAAAGA | 0.45163355 | −5.04 | <.0001 | <.0001 |
| AGGCGAAAAGGGAG | 0.026272206 | 2.00 | .0452 | |
| AGACGAAAAGGGAG | 0.258648173 | 4.20 | <.0001 |
Abbreviations: A, adenine; C, cytosine; ELISPOT, enzyme-linked immunospot assay; G, guanine; IFN-γ, interferon γ; T, thymine.
a IL18R1 genetic variants from left to right: rs3771172, rs6758936, rs2080289, rs7556917, rs7584093, rs3213732, rs10204757, rs11465641, rs6706002, rs6749014, rs4851004, rs1420096, rs2287033, and rs1420094.
b Haplotype effects were calculated using the haplotype t statistic, which shows the direction and magnitude of the estimated haplotypic effect on the IFN-γ ELISPOT measure. Allele P values reflect comparison of individual haplotypes with all other haplotypes combined. Statistical significance was defined as P < .05.
DISCUSSION
By analyzing 701 candidate gene SNPs in 1056 subjects, which included 580 whites and 217 African Americans, we found 23 significant associations (20 and 3 associations for whites and African Americans, respectively) between SNPs and haplotypes and IFN-γ ELISPOT responses after smallpox vaccination. In our study, the majority of these significant associations were observed between SNPs in the coding, regulatory, and intronic regions of the interleukin 18 receptor alpha chain, IL18R1, and IL18 genes and variations in vaccinia-specific T-cell response by IFN-γ ELISPOT. We identified a functional coding synonymous IL18R1 polymorphism (rs1035130/Phe251Phe; in LD with 5 intronic SNPs; r2 ≥ 0.92) that was significantly associated with an allele dose-related increase (12%) in IFN-γ ELISPOT responses in the combined cohort of subjects. This SNP (Phe251Phe) is located in the third immunoglobulinlike domain in transmembrane IL18R1, which is critical for interleukin 18 (IL-18) binding to its receptor (ie, IL18R1) and IL-18–induced IFN-γ activity [16]. Notably, this coding rs1035130 and several promoter and intronic SNPs (n = 15) in the IL18R1 gene that were previously associated with vaccinia-specific neutralizing antibody titers in the combined cohort (P < .001), whites (P = .005), and African Americans (P = .02) [7] were also associated with IFN-γ ELISPOT responses (P = .01) in the combined cohort of subjects in this study. The association of rs1035130 with IFN-γ ELISPOT and antibody responses can also be explained by LD with other SNPs. In whites, we also identified a specific IL18R1 haplotype (AGACGAAAAGGGAG) that includes rs2080289 (in LD with the coding rs1035130; r2 = 1), in which a G→A substitution was associated with both higher IFN-γ ELISPOT and antibody responses to smallpox vaccine, indicating the potential functional significance of these SNPs (rs2080289 and rs1035130) [7]. One possible mechanism for IL18R1 SNP associations with both IFN-γ ELISPOT and antibody responses may be the influence of IL18/IL18R1–modulated IFN-γ and interleukin 4/interleukin 13 secretion by T-helper (Th) 1 and 2 cells, natural killer cells, monocytes, and macrophages and concomitant regulation of IFN-γ ELISPOT and antibody production after vaccinia virus stimulation [17]. This is consistent with previous findings that control of vaccine-induced immunity is most likely mediated by multigenic interactions, pathways, and networks (ie, “immune response network”) [18].
Key proinflammatory cytokines IL-18 (similarly known as IFN-γ–inducing factor) and IFN-γ, along with the IL18R1 and IL18RAP receptors, play a significant part in the control of innate and adaptive immunity. Receptor IL18R1 belongs to the interleukin 1 receptor family, is expressed in Th1 cells, binds IL-18, and is important for IL18–mediated signal transduction [19]. IL-18 is secreted by antigen-presenting cells, stimulates IFN-γ synthesis and is involved in the synergistic activation of interleukin 12 to promote a Th1-mediated immune response [19]. Type II IFN, IFN-γ, plays a central role in host protection against viral infections, including poxviruses, and mediates antiviral activity via the phosphorylation of STAT1 in the T and natural killer cells and macrophages [20]. Other evidence demonstrates that human poxviruses and poxvirus-encoded proteins, such as soluble IL-18–binding protein, can suppress IL-18–induced IFN-γ secretion [20]. Though IL18R1/IL18RAP and IL18 genetic variants have recently been found to be linked to smallpox vaccine humoral immune response [7] and several diseases, such as inflammatory bowel disease [21], herpes virus seropositivity, and extrinsic asthma [22, 23], little is known about their impact on vaccine-induced cellular IFN-γ ELISPOT responses.
Even though the strongest association was found for IL6 promoter rs2069827 (both in the overall cohort and among whites) and IL10 intronic rs3024503 (in African Americans), the majority of associations were discovered in the IL18R1 and IL18 genes. We found that the occurrence of specific genetic polymorphisms in the IL18R1 genes were consistently associated (13 SNPs) with antigen-specific IFN-γ ELISPOT responses among both the overall cohort and white subjects. These also include the intronic IL18R1 rs6758936 and tagged SNPs rs6749014, rs3213732, rs10204757, rs6706002, rs4851004, rs11465641, rs7584093, and rs1420096 (in strong LD; r2 ≥ 0.99) in the IL18R1 gene cluster region that have been associated elsewhere with vaccinia virus–induced neutralizing antibody titers in the overall cohort and among whites [7]. Again, because IL18R1/IL18 is known to induce synthesis and release of IFN-γ, interleukin 4, and interleukin 13, and to regulate the function of antigen-presenting cells, it is possible that variation in the IL18R1/IL18 genes may play a role in the induction of both vaccine-induced humoral and cellular adaptive immunity.
An interesting observation in our study is the association of the intronic IL18 SNP rs5744280 (in LD with rs3882891; r2 ≥ 0.38) with an allele dose-related decrease in IFN-γ ELISPOT response (38% decrease; P = .01) in the African American cohort. This specific IL18 polymorphism rs5744280 has also been associated elsewhere with an allele dose-related reduction in vaccinia neutralizing antibody levels in whites (18% decrease; P = .02) and among African Americans (21% decrease; P = .01) [7], and it demonstrated a genome-wide association (P = 3.9 × 10−7) with variations in the IL-18 plasma levels among whites in the study by He et al [24]. We speculate that IL18 polymorphism rs5744280 may be of functional relevance or in LD with a functional polymorphism that may modulate expression and production of IL-18 protein. Likewise, Stanley et al [11] observed an association between 3 IL18 haplotypes, which include rs3882891 polymorphism (also associated with an allele dose-related 22% reduction in IFN-γ ELISPOT response in our study), that were associated with a reduced risk for fever (GGAATACAGGTGA haplotype) and also with an augmented susceptibility to fever (GGAATACAGATGA and AGCATACTGATGA haplotypes) after smallpox vaccination. These results suggest the significance of the IL18 gene polymorphism in smallpox vaccine–induced immunity and susceptibility to AEs. Our recent genome-wide association study analysis with vaccinia-specific cell-mediated immunity (CD8+ IFN-γ ELISPOT) demonstrated that 3 of these cytokine receptor genetic variants (rs4851004, rs1420094, and rs2287033) were replicated, with genome-wide significance levels of P = .0119, P = .0125, and P = .0125, respectively, in whites [10].
To our knowledge, this is the first report of that associations between IL18R1 and IL18 gene polymorphisms and smallpox vaccine–induced IFN-γ ELISPOT responses. Our findings indicate that IL18R1 and IL18 gene SNPs, and IL18R1 haplotypes may be important regulators of smallpox vaccine–induced IFN-γ immune responses. Further work is necessary to determine whether our results pertain to other racial or ethnic groups. Because we cannot exclude the probability of false-positive associations, replication and functional studies of causal polymorphisms in large independent cohorts are necessary to confirm these findings. Our data also suggest the possibility that common IL18R1, IL18, and other genetic polymorphisms may be linked to the control of vaccine-induced adaptive immunity (humoral and cell-mediated immunity) and inflammation (the development of AEs) in individuals after smallpox vaccination. Understanding molecular mechanisms behind these associations may help improve smallpox vaccines and will add to our knowledge of vaccine-induced immune responses.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Acknowledgments. We thank the Mayo Clinic Vaccine Research Group in Rochester, Minnesota, the Naval Health Research Center in San Diego, California, and the subjects who participated in our studies. We thank Robert A. Vierkant for his assistance in statistical analysis and Caroline L. Vitse for her editorial assistance.
Financial support. This work was funded by federal funds from the National Institute of Allergies and Infectious Diseases, National Institutes of Health, and Department of Health and Human Services (under contract HHSN266200400065C).
Potential conflicts of interest. G. A. P. is the chair of a safety evaluation committee for nonsmallpox investigational vaccine trials being conducted by Merck Research Laboratories and offers consultative advice on vaccine development to Merck, CSL Biotherapies, Avianax, Sanofi Pasteur, Dynavax, Novartis Vaccines and Therapeutics, PAXVAX, and Emergent Biosolutions. These activities have been reviewed by the Mayo Clinic Conflict of Interest Review Board and are conducted in compliance with Mayo Clinic conflict of interest policies. This research has also been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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