Abstract
Genetic polymorphisms are known to affect responses to both viral infection and vaccination. Our previous work has described genetic polymorphisms significantly associated with variations in immune response to rubella vaccine from multiple gene families with known immune function, including HLA, cytokine and cytokine receptor genes, and in genes controlling innate and adaptive immunity. In this study, we assessed cellular immune responses (IFNγ and IL-6) in a cohort of healthy younger individuals and performed genome-wide SNP analysis on these same individuals. Here, we report the first genome-wide association study focused on immune responses following rubella vaccination. Our results indicate that rs16928280 in protein tyrosine phosphatase delta (PTPRD) and a collection of SNPs in ACO1 (encoding an iron regulatory protein) are associated with interindividual variations in IFNγ response to rubella virus stimulation. In contrast, we did not identify any significant genetic associations with rubella-specific IL-6 response. These genetic regions may influence rubella vaccine-induced IFNγ responses and warrant further studies in additional cohorts in order to confirm these findings.
Introduction
Rubella vaccines were first developed in the late 1960s and early 1970s. The vaccines contain live, attenuated rubella strains (RA27/3 worldwide; TO-336 in China; Takahashi and Matsuura in Japan), and in the United States, are given as components of multivalent vaccine formulations (MMRII or MMRV) (Meyer et al. 1969; Plotkin et al. 1969). Since that time, their use has dramatically decreased the number of rubella infections and cases of congenital rubella syndrome (Reef and Plotkin 2013). The vaccines are remarkably effective; vaccine efficacy and protection rates are estimated to be >94 % (Beasley et al. 1969; Chang et al. 1970; Grayston et al. 1969; Landrigan et al. 1974), which coincide with the 2–5 % of vaccinated individuals who do not seroconvert. Among those who do respond to the vaccine, there is a large degree of variation in immune response, and we have shown that this variability is heritable, with nearly 50 % of the variance in rubella-specific antibodies following immunization being explained by genetic factors (Tan et al. 2001). Additional doses typically boost antibody titers and increase seroconversion rates (>99 %).
Both host and virus genetics have been associated with rubella virus growth in joint tissue and corresponding joint symptoms in vivo (Lund and Chantler 2000; Mitchell et al. 1998). Similarly, rubella vaccine response variability is due, in part, to differences in host genetics (Jacobson et al. 2009; Tan et al. 2001). Our lab has identified a number of genes where genetic polymorphisms are associated with vaccine response including HLA, cytokines and cytokine receptor genes, pattern recognition receptor and antiviral genes, as well as in vitamin receptor and other genes (Dhiman et al. 2007, 2010a; Haralambieva et al. 2010; Kennedy et al. 2010; Ovsyannikova et al. 2004, 2009a, b, 2010a, b, c, 2012a). These findings were the result of focused candidate gene associations studies, and indicated that genetic control of immune responses to rubella-containing vaccines are multigenic in nature (Pankratz et al. 2010), and likely involves a much larger collection of genes than originally believed.
Here, we report the first genome-wide association study conducted in a cohort of school-aged children receiving rubella-containing vaccine. While we did not identify any significant genetic associations with IL-6 response, our data indicate that rs16928280 in PTPRD and a collection of SNPs in ACO1 are associated with variations in IFNγ response to rubella virus stimulation. These genetic regions may influence rubella vaccine-induced cytokine responses and warrant further testing in additional cohorts in order to replicate our findings.
Methods
Subject recruitment and demographics
The study cohort was a large population-based sample of 1,145 healthy children and older adolescents, and healthy adults (age 11 to 22 years), recruited from Olmsted County, MN. This study cohort was enrolled through three separate recruiting phases, recruited at the following various times: (1) 346 children ages 12–18, recruited in 2001–2002 (Ovsyannikova et al. 2004, 2005); (2) 440 children ages 11–18, recruited in 2006–2007 (Haralambieva et al. 2010; Ovsyannikova et al. 2010a); and (3) 388 children ages 11–22, recruited in 2008–2009 (Ovsyannikova et al. 2011, 2012c). The parents of each participant provided parental consent and medical records for 1,101 of the subjects indicated receipt of two doses of measles-mumps-rubella (MMR, Merck) vaccine. The methods described herein are similar or identical to those published for our previous studies (Dhiman et al. 2010a; Haralambieva et al. 2010; Kennedy et al. 2010; Ovsyannikova et al. 2004, 2005, 2009a, b, 2010a, b, c). The Institutional Review Boards of both the Mayo Clinic and the NHRC approved the study, which was performed in accordance with the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from each adult subject, and from the parents of all children who participated in the study.
Rubella-specific cytokine secretion
Cytokine responses to rubella virus stimulation were measured as previously described (Dhiman et al. 2010b; Ovsyannikova et al. 2009b). Briefly, 2 × 106/ml PBMCs were stimulated with the W-Therien strain of rubella virus (a gift from Dr. Teryl Frey, Georgia State University, Atlanta, GA) with optimized multiplicity of infection (MOI: IL-2, IL-6, and IFN-γ: MOI of 5. TNF-α: MOI of 0.05) and incubation times (IL-6, 24 h. IFN-γ, 48 h. IL-2 and TNF-α, 8 days). Cytokine-containing culture supernatants were stored at −80 °C until quantified using BD OptEIA™ Human ELISA kits. Absorbance levels were measured using a Molecular Devices SpectraMax 340PC.
Genome-wide SNP typing and QC
The genome-wide SNP typing protocol used for this study is essentially identical to that used in previously published reports (Kennedy et al. 2012a, b; Ovsyannikova et al. 2012b). Briefly, DNA was extracted from each subject’s blood specimen using the Gentra Puregene Blood kit (Gentra Systems Inc., Minneapolis, MN) and quantified by Picogreen (Molecular Probes, Carlsbad, CA). The genome-wide SNP typing for the cohort (n = 1,052) was performed using the Infinium Omni 1 M-Quad SNP array (Illumina, San Diego, CA). DNA samples underwent amplification, fragmentation, and hybridization onto each BeadChip, which were imaged on an Illumina BeadArray reader. Genotype calls based on clustering of the raw intensity data were made using BeadStudio 2 software. The resulting genotype data on SNPs were exported into SAS for analysis. Quality control checks included genotyping reproducibility, gender checks, removal of SNPs where typing failed in >1 % of subjects, removal of subjects where >1 % of SNPs failed, elimination of monomorphic SNPs, removal of duplicate samples, and a Hardy-Weinberg Equilibrium (HWE) check (SNPs with p < 1e-7 were flagged as having poor genotyping quality). The genotyping success was high, with the average per-SNP call rate being 99.07 % and the average per-subject call rate being 99.07 % for this Omni1M-Quad array.
Population substructure was evaluated with EIGENSTRAT, a principal components-based assessment of genetic similarity (Price et al. 2006). We retained those subjects whose genetic background was no further than 15 % away from the predominant Caucasian cluster toward the Asian or African clusters, as identified by the two most predominant axes of genetic admixture.
Statistical analyses
Demographic and vaccination history features were summarized for the study participants with counts and percentages for nominal features and medians and interquartile ranges for numerical variables, including the outcomes of IFNγ and IL6 immune responses (summarized as the difference between the median simulated and median unstimulated observations). Linear regression models, with generalized estimating equations to account for the multiple measures obtained per subject, were used to test the associations between individual SNPs and the two cell-mediated immune response measures. In these analyses, the primary test of significance assessed the ordinal association between the genotypes of each SNP and inverse normal-transformed immune response measures while adjusting for sex, age, and time between immunization and blood draw. We evaluated whether significance levels were inflated due to unmeasured confounding (Devlin and Roeder 1999), and adjusted the levels of significance with the estimated inflation factor. We summarized the genotype counts and the per-genotype medians and interquartile ranges of the two immune response measures for all SNPs showing suggestive associations with p values less than 1 × 10−6.
Results
Of the original cohort, 897 subjects had usable genotyping and immune outcome data. This cohort was predominantly Caucasian (98.4 %) and, due to the lack of sufficient numbers of subjects of other race/ethnicities, we restricted our analysis to Caucasian individuals. This subset of individuals was 54.9 % male and ranged in age from 11 to 19 years old, with a median age of 15. Most of the subjects received their most recent vaccination at approximately 1 year of age (median age = 10 months; interquartile range, IQR = 5.0–12.0). Furthermore, the median time between second immunization with a rubella-containing vaccine and enrollment was 6.4 years (IQR = 4.6–8.5 years).
Subjects had a median IFNγ response of 6.4 (IQR 1.7–19.7) pg/ml; this is indicative of a relatively weak Th1 response, which confirms an earlier report (Dhiman et al. 2010b). Despite the low levels of IFNγ produced by our subjects’ PBMC samples in response to rubella virus stimulation, we identified a number of SNPs displaying significant (p < 5 × 10−8) associations with IFNγ secretion (Fig. 1 and Table 1).
In contrast to the relatively weak IFNγ response, this cohort had a robust, inflammatory IL-6 response, with a median production of 3,629.3 (IQR 3,095.4–4,003.8) pg/ml. Notwithstanding this robust response, we did not identify any SNPs associated with IL-6 secretion at the genome-wide level of significance set at p < 5 × 10−8 (Fig. 2). Table 2 lists all SNPs associated with IL-6 secretion with p values < 1 × 10−6.
Discussion
We had previously conducted a series of immunogenetic association studies with this cohort; however, those studies involved limited sets of candidate immune-related genes (HLA, cytokines and receptors, pathogen recognition receptors, antiviral, and other genes) (Dhiman et al. 2008, 2010a; Haralambieva et al. 2010; Ovsyannikova et al. 2004, 2005, 2010a, b). In this report, we conducted genome-wide SNP typing in order to perform the corresponding genetic association analyses in a manner that enabled the discovery of potentially novel associations throughout the genome.
Although our cohort displayed robust IL-6 production in response to in vitro rubella virus stimulation, we did not identify any SNPs associated with this response at the pre-set genome-wide level of significance. Two SNPs, rs7218761 in DNAH9 (encoding for a dynein heavy chain subunit) and rs4140752 located near the SLC8A1 gene (encoding a sodium/calcium ion exchanger) had p values suggestive of an effect.
In contrast to the IL-6 findings, our GWAS did reveal a number of SNPs that were significantly associated with IFNγ response to rubella virus stimulation. The most significant SNP, rs16928280, is located in PTPRD. PTPRD codes for protein tyrosine phosphatase delta, which regulates cell signaling processes involved in cell growth, division, and differentiation. PTPRD has been shown to have very low levels of expression in immune cells (Arimura and Yagi 2010) and has yet to be linked to any immune function. We also identified six SNPs on chromosome 9 near the ACO1 gene that were significantly associated with IFNγ response. ACO1 encodes for aconitase 1, an iron-binding protein involved in the conversion of citrate to isocitrate. Aconitase also binds to, and suppresses, translation of ferritin mRNA. Although regulation of iron (primarily sequestration) has been identified as an innate immune defense mechanism as far back as the 1940s (Cartwright et al. 1946), the role of iron regulation in adaptive immune function has not been studied extensively. Our results indicate that the genetic region encompassing ACO1 may be associated with cytokine responses to rubella vaccination and further investigation of this locus is warranted.
Our study is the first reported GWAS with adaptive immune responses to rubella vaccine. As such, it expands the available data regarding genetic control of rubella vaccine-induced immunity. We have identified several genetic regions for further analysis and investigation. As with many studies, ours has several limitations including the small sample size (<1,000); the fact that our observed phenotypes represent subtle variations in immune responses rather than clear cut, dichotomous outcomes; and the multigenic nature of complex biological processes, such as immunity, where each SNP likely has small effects on the phenotype in question. Our intent is to conduct replication studies in additional cohorts of rubella-vaccinated subjects, followed by fine mapping through any genetic regions where we identify replicated associations. This approach allows us to overcome many of the limitations of the current study. We set a genome-wide level of significance at p < 5 × 10−8; however, evidence suggests that genetic associations not meeting this level of significance may also be real and worth pursuing (Panagiotou and Ioannidis 2012). With this in mind, additional loci will be carried forward into the replication studies in order to definitively assess whether or not they are truly associated with immune responses to rubella.
References
Arimura Y, Yagi J (2010) Comprehensive expression profiles of genes for protein tyrosine phosphatases in immune cells. Sci Signal 3:rs1
Beasley RP, Detels R, Kim KS, Gale JL, Lin TL, Grayston JT (1969) Prevention of rubella during an epidemic on Taiwan. HPV-77 and RA 27-3 rubella vaccines administered subcutaneously and intranasally HPV-77 vaccine mixed with mumps and-or measles vaccines. Am J Dis Child 118:301–306
Cartwright GE, Lauritsen MA, Humphreys S, Jones PJ, Merrill IM, Wintrobe MM (1946) The anemia associated with chronic infection. Science 103:72–73
Chang TW, DesRosiers S, Weinstein L (1970) Clinical and serologic studies of an outbreak of rubella in a vaccinated population. N Engl J Med 283:246–248
Devlin B, Roeder K (1999) Genomic control for association studies. Biometrics 55:997–1004
Dhiman N, Ovsyannikova IG, Cunningham JM, Vierkant RA, Kennedy RB, Pankratz VS, Poland GA, Jacobson RM (2007) Associations between measles vaccine immunity and single-nucleotide polymorphisms in cytokine and cytokine receptor genes. J Infect Dis 195:21–29
Dhiman N, Ovsyannikova IG, Vierkant RA, Pankratz VS, Jacobson RM, Poland GA (2008) Associations between cytokine/cytokine receptor single nucleotide polymorphisms and humoral immunity to measles, mumps and rubella in a Somali population. Tissue Antigens 72:211–220
Dhiman N, Haralambieva IH, Kennedy RB, Vierkant RA, O’Byrne MM, Ovsyannikova IG, Jacobson RM, Poland GA (2010a) SNP/haplotype associations in cytokine and cytokine receptor genes and immunity to rubella vaccine. Immunogenetics 62:197–210
Dhiman N, Haralambieva IH, Vierkant RA, Pankratz VS, Ryan JE, Jacobson RM, Ovsyannikova IG, Poland GA (2010b) Predominant inflammatory cytokine secretion pattern in response to two doses of live rubella vaccine in healthy vaccinees. Cytokine 50:24–29
Grayston JT, Detels R, Chen KP, Gutman L, Kim KS, Gale JL, Beasley RP (1969) Field trial of live attenuated rubella virus vaccine during an epidemic on Taiwan. Preliminary report of efficacy of three HPV-77 strain vaccines in the prevention of clinical rubella. JAMA 207:1107–1110
Haralambieva IH, Dhiman N, Ovsyannikova IG, Vierkant RA, Pankratz VS, Jacobson RM, Poland GA (2010) 2’-5’-Oligoadenylate synthetase single-nucleotide polymorphisms and haplotypes are associated with variations in immune responses to rubella vaccine. Hum Immunol 71:383–391
Jacobson RM, Ovsyannikova IG, Poland GA (2009) Genetic basis for variation of vaccine response: our studies with rubella vaccine. Paediatr Child Health 19:S156–S159
Kennedy RB, Ovsyannikova IG, Vierkant RA, Jacobson RM, Poland GA (2010) Effect of human leukocyte antigen homozygosity on rubella vaccine-induced humoral and cell-mediated immune responses. Hum Immunol 71:128–135
Kennedy RB, Ovsyannikova IG, Pankratz VS, Haralambieva IH, Vierkant RA, Jacobson RM, Poland GA (2012a) Genome-wide genetic associations with IFNgamma response to smallpox vaccine. Hum Genet 131:1433–1451
Kennedy RB, Ovsyannikova IG, Pankratz VS, Haralambieva IH, Vierkant RA, Poland GA (2012b) Genome-wide analysis of polymorphisms associated with cytokine responses in smallpox vaccine recipients. Hum Genet 131:1403–1421
Landrigan PJ, Stoffels MA, Anderson E, Witte JJ (1974) Epidemic rubella in adolescent boys. Clinical features and results of vaccination. JAMA 227:1283–1287
Lund KD, Chantler JK (2000) Mapping of genetic determinants of rubella virus associated with growth in joint tissue. J Virol 74:796–804
Meyer HM Jr, Parkman PD, Hobbins TE, Larson HE, Davis WJ, Simsarian JP, Hopps HE (1969) Attenuated rubella viruses. Laboratory and clinical characteristics. Am J Dis Child 118:155–165
Mitchell LA, Tingle AJ, MacWilliam L, Horne C, Keown P, Gaur LK, Nepom GT (1998) HLA-DR class II associations with rubella vaccine-induced joint manifestations. J Infect Dis 177:5–12
Ovsyannikova IG, Jacobson RM, Vierkant RA, Jacobsen SJ, Pankratz VS, Poland GA (2004) The contribution of HLA class I antigens in immune status following two doses of rubella vaccination. Hum Immunol 65:1506–1515
Ovsyannikova IG, Jacobson RM, Vierkant RA, Jacobsen SJ, Pankratz VS, Poland GA (2005) Human leukocyte antigen class II alleles and rubella-specific humoral and cell-mediated immunity following measles-mumps-rubella-II vaccination. J Infect Dis 191:515–519
Ovsyannikova IG, Jacobson RM, Vierkant RA, O’Byrne MM, Poland GA (2009a) Replication of rubella vaccine population genetic studies: validation of HLA genotype and humoral response associations. Vaccine 27:6926–6931
Ovsyannikova IG, Ryan JE, Vierkant RA, O’Byrne MM, Pankratz VS, Jacobson RM, Poland GA (2009b) Influence of host genetic variation on rubella-specific T cell cytokine responses following rubella vaccination. Vaccine 27:3359–3366
Ovsyannikova IG, Dhiman N, Haralambieva IH, Vierkant RA, O’Byrne MM, Jacobson RM, Poland GA (2010a) Rubella vaccine-induced cellular immunity: evidence of associations with polymorphisms in the Toll-like, vitamin A and D receptors, and innate immune response genes. Hum Genet 127:207–221
Ovsyannikova IG, Haralambieva IH, Dhiman N, O’Byrne MM, Pankratz VS, Jacobson RM, Poland GA (2010b) Polymorphisms in the vitamin A receptor and innate immunity genes influence the antibody response to rubella vaccination. J Infect Dis 201:207–213
Ovsyannikova IG, Vierkant RA, Pankratz VS, Jacobson RM, Poland GA (2010c) Extended LTA, TNF, LST1 and HLA gene haplotypes and their association with rubella vaccine-induced immunity. PLoS One 5:e11806
Ovsyannikova IG, Haralambieva IH, Vierkant RA, O’Byrne MM, Jacobson RM, Poland GA (2011) The association of CD46, SLAM and CD209 cellular receptor gene SNPs with variations in measles vaccine-induced immune responses: a replication study and examination of novel polymorphisms. Hum Hered 72:206–223
Ovsyannikova IG, Haralambieva IH, Kennedy RB, Pankratz VS, Vierkant RA, Jacobson RM, Poland GA (2012a) Impact of cytokine and cytokine receptor gene polymorphisms on cellular immunity after smallpox vaccination. Gene 510:59–65
Ovsyannikova IG, Kennedy RB, O’Byrne M, Jacobson RM, Pankratz VS, Poland GA (2012b) Genome-wide association study of antibody response to smallpox vaccine. Vaccine 30:4182–4189
Ovsyannikova IG, Pankratz VS, Vierkant RA, Jacobson RM, Poland GA (2012c) Consistency of HLA associations between two independent measles vaccine cohorts: a replication study. Vaccine 30:2146–2152
Panagiotou OA, Ioannidis JP (2012) What should the genome-wide significance threshold be? Empirical replication of borderline genetic associations. Int J Epidemiol 41:273–286
Pankratz VS, Vierkant RA, O’Byrne MM, Ovsyannikova IG, Poland GA (2010) Associations between SNPs in candidate immune-relevant genes and rubella antibody levels: a multigenic assessment. BMC Immunol 11:48
Plotkin SA, Farquhar JD, Katz M, Buser F (1969) Attenuation of RA 27-3 rubella virus in WI-38 human diploid cells. Am J Dis Child 118:178–185
Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38:904–909
Reef SE, Plotkin SA (2013) Rubella vaccine. In: Plotkin SA, Orenstein WA, Offit PA (eds) Vaccines, 6th edn. Elsevier/Saunders, Edinburgh
Tan PL, Jacobson RM, Poland GA, Jacobsen SJ, Pankratz VS (2001) Twin studies of immunogenicity–determining the genetic contribution to vaccine failure. Vaccine 19:2434–2439
Acknowledgments
We appreciate the study subjects and their families’ willingness to participate in our study. We thank the Mayo Clinic nurses and study coordinators for their efforts in subject recruitment, Dr. Julie Cunningham and the Mayo Medical Genome Facility Genotyping Core for their assistance with genotyping efforts. We also thank Megan O’Byrne, Beth Larrabee, and Nathaniel Warner for their contributions to the statistical analyses. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers R01AI33144 and AI048793 (which recently received an NIH MERIT award). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Competing interests
Dr. Poland is the chair of a Safety Evaluation Committee for novel investigational vaccine trials being conducted by Merck Research Laboratories. Dr. Poland offers consultative advice on vaccine development to Merck & Co. Inc., CSL Biotherapies, Avianax, Sanofi Pasteur, Dynavax, Novartis Vaccines and Therapeutics, PAXVAX Inc, and Emergent Biosolutions. Drs. Poland and Ovsyannikova hold patents related to vaccinia and measles peptide research. These activities have been reviewed by the Mayo Clinic Conflict of Interest Review Board and are conducted in compliance with the Mayo Clinic Conflict of Interest policies. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with the Mayo Clinic Conflict of Interest policies. The other authors do not have any conflicts of interest.
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Kennedy, R.B., Ovsyannikova, I.G., Haralambieva, I.H. et al. Genome-wide SNP associations with rubella-specific cytokine responses in measles-mumps-rubella vaccine recipients. Immunogenetics 66, 493–499 (2014). https://doi.org/10.1007/s00251-014-0776-3
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DOI: https://doi.org/10.1007/s00251-014-0776-3