We found that the variations in cell-mediated (cytokine) immune responses to smallpox vaccine are significantly influenced by genetic variants in cytokine and cytokine receptor genes. In total, we discovered 277 significant associations (p<0.05) between polymorphisms in cytokine/cytokine receptor genes (n=32) and variations in vaccinia-specific IFN-α, IL-12p40, IL-1β, IL-2, and TNF-α secretion in Caucasians (Supplemental Tables 1–5
). Multiple non-coding and coding polymorphisms in these genes were putatively associated with variations in cytokine responses, with many of these SNPs associated in an allele dose-related manner. A number of SNP associations (n=13) belonging to the IL1RN, IL2RB, IL4R, IL6, IL10RB, IL12A
, and IL12RB2
genes demonstrated concordance between Caucasians and African-Americans. Furthermore, SNP associations in the IL4R, IL6, IL12A
, and IL12RB2
genes demonstrated allele dose-related responses that were consistent in the two racial groups. As an example, we identified a SNP in the IL1RN
(rs452204) gene whose minor allele is associated with a considerable decrease (1.5-fold for Caucasians and three-fold for African-Americans) in vaccinia-specific IL-2 secretion levels in a dose-related manner. Similarly, the intronic IL12RB2
polymorphism (rs3790567) previously reported to be associated with giant cell-arteritis (Rodriguez-Rodriguez et al., 2011
) demonstrated significant associations with decreased IL-1β secretion in a dose-related manner (1.5-fold for Caucasians and 1.7-fold for African-Americans) after vaccinia virus stimulation. It is important to define the functional consequences of these replicated genetic variants that are associated with inter-individual variations in cytokine responses to smallpox vaccine, and such studies are ongoing in our laboratory.
Polymorphisms in coding and non-coding domains of genes coding for cytokine/cytokine receptors can influence various effects of cytokine biology. Among these effects are altered gene and/or protein expression and function, alternate splice variant usage, altered transcriptional factor activity, methylation and miRNA function (van Deventer, 2000
; Haukim et al., 2002
; Bidwell et al., 2001
; Bidwell et al., 1999
). Cytokine receptor polymorphisms can alter gene expression and affect receptor protein function (van Deventer, 2000
;Hackstein et al., 2001
;van de Vosse et al., 2003
). Functional genetic variants can be found for all cytokines, as well as for their respective receptors. For example, a specific haplotype in the IL4
gene has been demonstrated to be associated with reduced predisposition to the development of fever in smallpox vaccine-naïve individuals (Stanley, Jr. et al., 2007
). One potential mechanism behind the association between the IL4
gene haplotype, which includes the known functional SNP rs2243250, and a decreased risk for fever might be the influence of a Th2 type-modulated increase in IL-4 production and concomitant suppression of IFN-γ production and Th1 responses (Stanley, Jr. et al., 2007
; Nelms et al., 1999
Race-specific analyses demonstrated that a specific polymorphism in the IL2
gene (coding synonymous rs2069763, Leu38Leu) is associated with vaccinia-specific IL-2 production in an allele dose-dependent manner in both racial groups (Caucasians and African-Americans). This specific polymorphism may have a functionally significant effect on the gene product (i.e., IL-2 protein secretion level). The Th1 type cytokine, IL-2, is a critical immunoregulatory cytokine required for T cell proliferation and plays an important role in adaptive immunity (Malek, 2002
). Of interest, a positive correlation (p=0.0009) between IL-2 secretion and vaccinia-specific neutralizing antibody titer was found within this cohort of study subjects (Ryan et al., 2009
; Umlauf et al., 2011
). Also, previously cited IL18R1
SNPs rs2080289 and rs2241116 were associated with both an increased neutralizing antibody titer (p=0.001) and IL-2 (p=0.029), and TNF-α (p=0.023) secretion level, respectively, in an allele dose–dependent manner in Caucasians (Supplemental Tables 4 and 5
) (Haralambieva et al., 2011a
). Notably, the IL2
genetic polymorphism rs2069763 has also been associated with high measles vaccine-induced antibody and high lymphoproliferative responses (Dhiman et al., 2007
). This SNP has also been linked to human papillomavirus (HPV)-related cervical and vulvar cancers (Hussain et al., 2008
). These observations suggest an important role for this genetic variant in the modulation of immune responses to multiple viral pathogens.
Significant variation in immune responses to smallpox vaccine was observed in our study subjects. We found evidence for vigorous production of innate/proinflammatory and Th1 cytokines (IFN-α, IL-12p40, IL-1β, and TNF-α) that indicates the importance of the early innate and inflammatory immune pathways after smallpox vaccination in the generation of protective immunity. These inter-individual variations in cytokine immune responses after smallpox immunization are likely to result in part from genetic variability between individuals. Studies have demonstrated that IL-1β, IL-18, TNF-α, and the interferons are inhibited by specific vaccinia strain proteins (Dunlop et al., 2003
; Moss and Shisler, 2001
), and removal of these inhibitory proteins can reduce the virulence of the resulting vaccinia strains (Reading and Smith, 2003
; Mossman et al., 1996
). These data indicate that alterations in individual cytokine levels, or in the overall balance of cytokines, significantly influence immune response to smallpox vaccine and disease susceptibility.
We found evidence for increased production of IL-1β and TNF-α, which are well recognized proinflammatory cytokines that cause fever in humans (Dinarello, 2004
). This increased cytokine production may be due to actions modulated by a transcription factor-kB (NF-kB) that is required for transcription of several cytokines, including IL-1 and TNF-α (Jin and Wang, 2003
). NF-kB plays a role in regulating cellular responses to viral antigens and infection. Conversely, the binding of NF-kB and other transcription factors (Jak/STAT/IRF) to regulatory regions can be interrupted by promoter polymorphism(s) (Jin and Wang, 2003
). A recent study examining associations between AEs following smallpox vaccination and SNPs in 19 candidate genes found that at the single-gene level, variations within the IL1
genes were associated with adverse responses, specifically fever or vaccine-induced febrile responses after smallpox vaccination (Stanley, Jr. et al., 2007
). Our analysis in Caucasians demonstrated a number of significant associations, at the level p<0.05, between SNPs in the IL1R
(n=15 associations), IL18
(n=6 associations), and IL18R1
(n=28 associations) genes and vaccinia-specific IL-1β secretion levels (Supplemental Table 3
). It is important to replicate these candidate gene SNPs discovered in our work and conduct follow-up studies of genetic variants to gain insights into the functions of replicated polymorphisms and the mechanisms by which they may contribute to smallpox vaccine immunity. Even if these individual SNPs do not provide causative insights into smallpox vaccine immune response, they may lead to further studies providing mechanistic insights (Debouck, 2009
The strengths of our population-based study include a large sample of healthy Caucasian and African-American individuals with well-documented demographic and immunization data. Genotyping across 32 cytokine and cytokine receptor genes allowed us to examine associations of both relatively common and uncommon alleles with vaccinia-specific cytokine responses and with demographic variables of interest.
The limitations of our study include the genotyping of candidate cytokine and cytokine receptor gene families, and no other candidate immune response genes. Therefore, these individual cytokine/cytokine receptor SNPs may influence only a small proportion of the heritability of the phenotype expression (i.e., cellular immunity). Further, our analysis in African-Americans was conducted using a smaller group of subjects and thus has restricted power. Statistical analysis only included Caucasians and African-Americans, narrowing the conclusions regarding genetic associations in other racial or ethnic groups.
Our analyses were conducted in two independent study groups that had different racial backgrounds: African- and Caucasian-American. Because of the genetic differences between these two groups, we must acknowledge that it is possible that genetic control of immune responses may differ between them. However, if there are indeed functional variants that have a direct effect on the development of immune responses to smallpox vaccine, and these functional variants are present in both genetic populations, the presence of a consistent genetic association within each of the two populations provides much stronger evidence in favor of that association.
Additionally, we acknowledge the importance of multiple testing issues in this study. We examined 701 SNPs across five different cytokine secretion measures in the Caucasian cohort, resulting in 3,505 tests of association. Thus, some of the associations reported herein could be false positive associations. Under the complete null hypothesis of no SNP associations and under the assumption of independent tests of hypothesis (an assumption not completely met due to linkage disequilibrium present in some SNP clusters), we would expect approximately 175 statistically significant tests by chance alone. It is noteworthy that we identified 277 SNP associations with p<0.05, suggesting that a large number of our SNP associations are likely to be true positive results. Similarly, of the 277 SNP associations with p<0.05 in the Caucasian cohort, we would expect approximately seven to successfully replicate in the African-American cohort (assuming two-sided p<0.05 and association in the same direction as Caucasian estimate, effectively resulting in a one-sided test with p<0.025). We successfully replicated 13 SNP associations in the African-American cohort, higher than that expected by chance alone.
In conclusion, our results suggest that genetic diversity at cytokine and cytokine receptor loci may contribute to variations in cytokine responses following smallpox vaccination. Vaccine-induced adaptive immune responses in our study subjects were characterized by high levels of innate/proinflammatory and Th1 TNF-α, IL-1β, IL-12p40, and IFN-α cytokines. It is plausible that high production of these cytokines originates from genetic polymorphisms in the cytokine and cytokine receptors and/or activation of downstream signaling molecules and pathways. Several SNP associations in Caucasian subjects with variations in vaccinia-induced secreted IFN-α, IL-12p40, IL-1β, IL-2, and TNF-α cytokines were replicated in African-Americans. These replicated associations were found within the key cytokine and cytokine receptor IL1RN, IL2RB, IL4R, IL6, IL10RB, IL12A, and IL12RB2 genes, indicating these genetic factors have an important role in cellular immunity. This study enhances our understanding of genetic determinants involved in inter-individual variation of cellular immune responses following smallpox vaccination, and may be applicable to other viral diseases as well. Our findings will inform further studies intended to confirm the results reported herein, and may provide novel insights into functional mechanisms behind these genetic associations.