Associations Between SNPs in Toll-like Receptors and Related Intracellular Signaling Molecules and Immune Responses to Measles Vaccine: Preliminary Results

Abstract

Toll-like receptors (TLRs) represent the critical “bridge” between innate and adaptive immunity to viral pathogens. We hypothesized that single nucleotide polymorphisms (SNPs) that potentially influence the expression/function of TLRs and their associated intracellular signaling molecules contribute to variations in humoral and cellular immunity to measles vaccine. We genotyped 190 randomly selected subjects (12–18 years old), previously vaccinated with two doses of measles, for known SNPs in TLR 2, 3, 4, 5, 6, 7, 8 and 9, and their associated intracellular signaling genes. Specific SNPs in the TLR 2, 3, 4, 5, 6, MyD88 and MD2 genes were associated with measles-specific humoral and cellular immunity. Heterozygous variants for rs3775291 (Phe412Leu) and rs5743305 (−926bp in promoter region) of the TLR3 gene were associated with low antibody and lymphoproliferative responses (p ≤ 0.02) to measles vaccination. Heterozygous variants for rs4986790 (Gly299Asp) and rs4986791 (Ile399Thr) in the TLR4 gene demonstrated higher levels of (p ≤ 0.02) IL-4 secretion. Heterozygous variants for SNPs in TLR5 (rs5744174) and TLR6 (rs5743818) were associated with higher levels of (p ≤ 0.02) IFN-γ secretion. In addition, SNPs in MyD88 and MD2, intracellular molecules that associate with TLRs, also demonstrated associations with variations in antibody and IL-10 production (p ≤ 0.03). Thus, we identified specific SNP associations between TLRs and their associated signaling molecules that have a known role in viral immunity and variations in both humoral and cellular immunity following measles vaccination. These data contribute to understanding the immunogenetic mechanisms underlying variations in the immune response to measles vaccine.

Introduction

Toll-like receptors (TLRs) have emerged as important sensors for viral recognition that act as “bridging molecules” between innate and adaptive immunity [1–4]. TLRs are able to discriminate between different viruses and bacteria by pathogen-associated molecular patterns (PAMPs) and thereby direct a pathogen specific immune response [2, 5]. Upon stimulation, TLRs trigger a cascade of adaptor and intracellular signaling molecules that lead to induction of innate and adaptive immunity [3, 6].

In recent years, it has been suggested that signaling through TLRs may play a role in measles virus pathogenicity and immunogenicity. Wild-type, but not vaccine strains of measles virus, can activate TLR2 expressing human T cells, which leads to subsequent secretion of proinflammatory cytokines, such as IL-6, and up-regulated surface expression of signaling lymphocyte activation molecule (SLAM) [7]. Wild-type measles virus is also known to suppress TLR4 mediated IL-12 induction in dendritic cells [8]. In contrast, the attenuated strains of measles virus are known to induce the expression of TLR3 via an interferon-dependent mechanism triggered as a part of the host response [9]. Measles virus recognition and specificity by TLRs can be influenced by genetic variations in the interaction domains between virus and host receptors. For example, a single amino acid mutation of asparagine at position 481 to tyrosine in the measles virus hemagglutinin (H) protein abolishes the ability of wild-type measles virus to activate via TLR2 [7]. We hypothesize that genetic variations in the TLRs and their associated signaling molecules, that play an important role in measles virus recognition, could result in variable immune responses to measles vaccination.

Materials and Methods

Study Cohort and Immune Characterization

Our study subjects (n = 190) were randomly sampled from a previously well-characterized study cohort [10, 11]. Measles virus-specific IgG levels were measured in sera using commercially available Enzygnost anti-masern-virus/IgG enzyme immunoassay (Dade Behring Marburg, Germany) following the manufacturer’s instructions [12]. IFN-γ, IL-2, IL-4, IL-10 and IL-12p40 secretion in response to measles virus stimulation was determined in PBMC cultures by ELISA as previously described [13, 14]. Measles virus-specific lymphoproliferative responses were measured using a 3[H]-thymidine incorporation assay as described previously [12]. Briefly, all assays were performed in triplicates at a pre-optimized time point (72 hrs) and stimulation dose (75 plaque forming units/well). Results were expressed as antigen-specific stimulation indices (SI), defined as the ratio between the median counts per minute (cpm) of measles vaccine-stimulated wells and the median cpm of control wells. Stimulation indices of three or higher were considered to be an indicator of positive lymphoproliferative response. PHA (5μg/ml) was used as a positive control.

SNP Selection and Genotyping

A panel of 96 known SNPs (located in the coding, intronic and regulatory regions 10kb upstream and downstream for each gene) from TLRs (TLR 2–9) and their associated intracellular signaling molecules (NF-κB, MyD88, IRAK, TRAF6, IkK, CD14, MAD3 and MD2) were selected from a literature review and public databases. We tested only those SNPs that had a reported minor allele frequency >5% in the Caucasian population. The nomenclature used for the description of the variants follows that described by den Dunnen and Antonarakis [15].

Genotyping Methods

Genomic DNA was extracted from frozen clotted blood using the Puregene® extraction kit (Qiagen Inc., Valencia, CA). Multiplex PCR and SNP analyses were carried out using the GenomeLab SNPstream platform (Beckman Coulter Inc., Fullerton, CA) as described previously [16].

Statistical Methods

The statistical methods used to determine associations between SNPs in TLR and associated intracellular signaling molecules and immune measures after measles vaccination have been previously described in detail [17]. Briefly, all data were descriptively summarized using frequencies and percentages for all categorical variables, and medians and inter-quartile ranges for all continuous variables. TLRs and associated signaling molecule SNPs were examined on a genotypic level, creating three levels for each SNP: homozygous major, heterozygous and homozygous minor alleles. A cut-off at p-value ≤0.03 for associations between SNPs and immune responses to measles was selected prior to examining data results. We tested for SNP-specific deviation from Hardy-Weinberg Equilibrium (HWE) using chi-square goodness-of-fit tests and excluded all SNPs out of HWE (p-value <0.001) [18]. Associations of SNPs in TLR and associated intracellular signaling molecules with measles virus-induced immune responses were carried out using analysis of covariance (ANCOVA) methods. SNPs were modeled assuming a general (co-dominant) genotypic effect. To better assess the effects of multiple testing, we supplemented the p-values from the ANCOVA models with their associated q-values [19, 20]. Briefly, the q-values are based on the concept of false discovery rates and can be interpreted as the expected proportion of false positive results among all features at least as extreme as the observed result. All analyses were adjusted for the potential confounding effects of age, gender, race, age at first MMR vaccination and age at 2^nd measles-mumps-rubella (MMR) vaccination. Due to data skewness, p-values were calculated based on log-transformed values for IgG and lymphoproliferative measures, and rank-transformed values for cytokine secretion measures. All statistical tests were two-sided, and all analyses were carried out using the SAS system (SAS Institute Inc., Cary, NC).

Results

Demographic and Immunological Variables of Study Cohort

The study cohort was primarily Caucasian (94.2%) with a median age of 15 years and had near even gender representation (males = 54%). Median ages at first and second MMR were 15.6 months and 12 years, respectively. The median (interquartile range, IQR) measles-specific IgG response was 1430 (646, 2482) IU/L and for proliferation response as measured in stimulation indices (SI) was 3.5 (2.0, 6.1). The median (IQR) for secreted levels of signature Th1 cytokines (IL-12p40 and IFN-γ) was 7.3 (2.7, 20.0) pg/mL and 66.5 (13.1, 198.1) pg/mL, respectively. The median (IQR) for secreted levels of signature Th2 cytokines (IL-4 and IL-10) was 11.5 (4.5, 24.3) pg/mL and 30 (11.5, 75.5) pg/mL, respectively.

SNP Associations between TLRs/Associated Intracellular Molecules and Immune Responses following Measles Vaccination

A total of 29 significant SNP associations (p ≤ 0.03) were identified between TLRs and their associated intracellular signaling molecules and measures of humoral and cellular immunity following measles vaccination (Table 1 and 2). Out of these, 11 SNPs were located in the coding and the regulatory regions of the genes corresponding to TLRs and their associated intracellular signaling molecules (Table 1). Minor SNP allele variant GG for rs6853 located in the 3′ UTR in MyD88 was associated with a lower antibody level (272 IU/L vs. 1430 IU/L; p = 0.001; q = 0.33) as compared to the major allele variant (Table 1). The heterozygous variants for two SNPs (rs3775291; non-synonymous, Phe412Leu and rs5743305;-976bp in promoter region) in the TLR3 gene resulted in significantly lower (p ≤ 0.02; q ≤ 0.46) measles-specific antibodies as compared to the homozygous variants (Table 1).

Table 1.

Associations between SNPs located in coding and regulatory regions in TLRs and associated intracellular signaling molecules and measles-specific immune responses

Gene	SNP ID	Location	Function	Genotype	N*	Median response	P- value#	Q- value†
						Ab (IU/L)
MyD88	Rs6853	3′UTR	-	AA/AG/GG	149/38/3	1430/1583/272	0.001	0.33
TLR3	Rs3775291	Ex4 + 601C > T	Phe412Leu	GG/GA/AA	106/67/17	1602/1025/2133	0.02	0.46
TLR3	Rs5743305	−976T > A	-	AA/AT/TT	36/93/59	1412/1063/1670	0.004	0.35
						IL-4 (pg/mL)
TLR4	Rs4986790	Ex4 + 636A > G	Gly299Asp	AA/AG/GG	169/21/0	11/23/-	0.02	0.44
TLR4	Rs4986791	Ex4 + 936C > T	Ile399Thr	CC/CT/TT	169/21/0	10/23/-	0.009	0.35
						IL-10 (pg/mL)
MD2	Rs11466004	Ex5 + 85C > T	Ser157Pro	CC/CT/TT	177/6/0	31/4/-	0.03	0.46
						IL-12p40 (pg/mL)
TLR2	Rs3804100	Ex3 + 1366T > C	Ser450Ser	TT/TC/CC	159/29/2	8/5/133	0.01	0.35
						IFN-γ (pg/mL)
TLR3	Rs3775296	5′UTR	-	GG/GT/TT	134/50/6	44/112/101	0.03	0.46
TLR6	Rs5743818	Ex1 + 756G > T	Ala644Ala	TT/TG/GG	96/76/18	51/78/38	0.02	0.46
TLR5	Rs5744174	Ex4 + 936C > T	Leu616Phe	CC/CT/TT	42/87/61	72/82/37	0.002	0.33
						LPA (SI)
TLR3	Rs5743305	−976T > A	-	AA/AT/TT	36/93/59	3.7/3.1/4.4	0.003	0.35

Phe-Phenylalanine, Leu- Leucine, Gly-Glycine, Asp-Aspartic acid, Ile-Isoleucine, Thr-Threonine, Ser-Serine, Pro-Proline, Ala-Alanine, Ex-Exon, A-Adenine, C-Cytosine, G-Guanine, T-Thymine, LPA-lymphoproliferative assay; SI- stimulation index

P-values ≤0.03 are shown

^*Values presented as homozygous major allele/heterozygous/homozygous minor allele.

^#Two degree-of-freedom p-value from analysis of covariance adjusting for age, gender, race, age at first MMR and age at second MMR

^†Corresponding q-value, based on the concept of false discovery rates. Values can be interpreted as the expected proportion of false positive results among all features at least as extreme as the observed result.

Table 2.

Associations between SNPs located in the intronic regions in TLRs and associated intracellular signaling molecules and measles-specific immune responses

Gene	SNP ID	Genotype	N*	Median response	P-value#	Q-value†
				IL-4 (pg/mL)
IkK	Rs5029748	CC/CA/AA	95/77/17	10/18/6	0.02	0.44
TLR4	Rs7864330	TT/TG/GG	165/20/0	11/23/-	0.02	0.46
				IL-10 (pg/mL)
IkK	Rs3747811	TT/TA/AA	47/105/37	15/39/30	0.005	0.35
TLR3	Rs1879026	CC/CA/AA	144/36/4	32/16/79	0.01	0.35
				IL-12p40 (pg/mL)
NFkB	Rs1801	CC/CG/GG	27/90/65	5/7/11	0.03	0.48
TLR5	Rs1773727	AA/CA/CC	66/83/42	5/9/11	0.004	0.35
				IFN-γ (pg/mL)
TLR5	Rs1773726	GG/GA/AA	57/84/34	37/85/70	0.005	0.35
TLR5	Rs1773729	TT/TG/GG	56/84/44	38/81/72	0.01	0.35
TLR5	Rs1291584	TT/TG/GG	61/87/42	37/82/72	0.002	0.33
TLR5	Rs5744159	GG/CG/CC	67/81/40	37/82/72	0.007	0.35
TLR5	Rs851138	TT/TG/GG	54/87/42	22/82/72	0.001	0.33
TLR5	Rs851139	GG/GA/AA	67/86/37	37/77/73	0.009	0.35
TLR5	Rs851186	GG/TG/TT	60/88/42	37/81/72	0.005	0.35
NFkB	Rs230494	AA/GA/GG	61/94/35	66/94/21	0.01	0.35
NFkB	Rs230544	CC/TC/TT	56/96/37	66/73/38	0.02	0.46
NFkB	Rs230547	CC/TC/TT	170/19/1	58/116/18	0.007	0.35
NFkB	Rs1585213	GG/GA/AA	61/96/29	64/74/16	0.01	0.36
TRAF6	Rs5030419	CC/CG/GG	135/46/5	72/22/94	0.02	0.46

Phe-Phenylalanine, Leu- Leucine, Gly-Glycine, Asp-Aspartic acid, Ile-Isoleucine, Thr-Threonine, Ser-Serine, Pro-Proline, Ala-Alanine, Ex-Exon, A-Adenine, C-Cytosine, G-Guanine, T-Thymine

Values ≤0.03 are shown

^*P-values presented as homozygous major allele/heterozygous/homozygous minor allele

^#Two degree-of-freedom p-value from analysis of covariance adjusting for age, gender, race, age at first MMR and age at second MMR

^†Corresponding q-value, based on the concept of false discovery rates. Values can be interpreted as the expected proportion of false positive results among all features at least as extreme as the observed result

The heterozygous variants for two non-synonymous SNPs (rs4986790; Gly299Asp and rs4986791; Ile399Thr) in the TLR4 gene were associated with significantly higher (p ≤ 0.02; q ≤ 0.44) secretion levels of the prototypic Th2 cytokine, IL-4, as compared to the corresponding major allele variants (Table 1). However, no allele dose-related response could be seen for this SNP, as we did not have any minor allele variant in our study population. The heterozygous genotype TC for a non-synonymous SNP (rs11466004; Ser157Pro) in the MD2 intracellular signaling molecule gene was associated with lower levels of IL-10 secretion (4 pg/mL vs. 31pg/mL; p = 0.03; q = 0.46) as compared to the major SNP allele variant CC. Again, due to a lack of minor SNP allele variant TT, no allele dose-related response could be determined (Table 1). Genotype TT for a synonymous SNP rs3804100 (Ser450Ser) in the TLR2 gene resulted in decreased levels of measles-specific IL-12p40 secretion as compared to the minor SNP allele variant CC (8 pg/mL vs. 133pg/mL; p = 0.01; q = 0.35).

For the prototypic Th1 cytokine, IFN-γ, heterozygous SNP variant GT for a SNP located in the 5′UTR (rs3775296) of the TLR3 gene was associated with significantly higher levels of (p = 0.03;q = 0.46) IFN-γ secretion as compared to homozygous alleles (Table 1). Similarly, heterozygous variants for a synonymous SNP (rs5743818; Ala644Ala) in the TLR6 gene and a non-synonymous SNP (rs5744174; Leu616Phe) in the TLR5 gene were associated with significantly higher (p ≤ 0.02; q ≤ 0.46) levels of secreted IFN-γ as compared to the homozygous variants.

The heterozygous variant for rs5743305 located in the 5′ region of the TLR3 gene was associated with a significantly lower (p = 0.003; q = 0.35) lymphoproliferative response as compared to the homozygous variants (Table 1). We also identified significant associations (p ≤ 0.03) between 18 intronic SNPs in the TLR (3, 4 and 5) and associated signaling genes (IkK, NFκB and TRAF6) and variations in cellular immune response to measles vaccination (Table 2).

Discussion

The genetic mechanisms underlying measles virus modulation of host immunity in response to vaccination are poorly defined and are under active research. In the present study, we identified significant associations between SNPs in TLRs 3, 4, 5 and 6 and the downstream intracellular signaling molecules, MyD88 and MD2, with variations in both antibody and cellular responses following measles vaccination. The associations between TLR3 and measles vaccine immunity are particularly intriguing as TLR3 has been previously identified as a prime target for laboratory adapted, but not wild-type measles virus strains in the generation of host immunity [9]. Tanabe et al [9] reported that laboratory adapted and vaccine strains of measles virus, including Edmonston, up-regulate the expression of TLR3 in human dendritic cells via enhanced IFN-β secretion. The 500bp region upstream of exon 1 is characterized as a measles virus-responsive segment in the TLR3 gene. This region contains the NF-κB and STAT (family of eukaryotic transcription factors that mediate the response to a large number of cytokines and growth factors) binding sites, and an interferon-stimulated response element (ISRE) located 30bp upstream of exon 1 in the promoter region of the TLR3 gene, which has also been identified as an IFN-β induction site [9]. We identified a SNP in the 3′ UTR of TLR3 (rs5743305 at −976bp of TLR promoter) that demonstrated an association between heterozygous variant AT and low antibody and low lymphoproliferative responses. Specific deletion mutations in the TLR3 promoter region, specifically in the ISRE and STAT binding sites, are known to dramatically decrease promoter activity and hence TLR expression [9]. Since rs5743305 is located in the promoter region within 1 kb upstream of exon 1 in the TLR3 gene in close proximity to the regulatory regions, it can be postulated that it might modulate or influence the promoter activity of TLR3. The GA variant of a non-synonymous SNP also in the TLR3 gene was associated with lower antibody production. Non-synonymous SNPs can directly alter protein expression by aberrant trafficking to cell surface [21], and function by changing protein structure and conformation.

TLR2 and TLR4 are known to stimulate innate and adaptive immune responses to wild-type measles viruses [7, 22] [8]. Wild-type measles virus strains activate cells specifically via TLR2, induce SLAM expression and activate a TLR-responsive cytokine profile such as IL-6, IL-12p40 and IL-1α/β [7]. Vaccine strains of measles virus lack the ability to activate TLR2 due to a single amino acid mutation in the hemaglutinin protein of measles virus [7]. In addition, wild-type measles virus infected dendritic cells are rendered defective in IL-12 production in response to TLR4 stimulation [8]. We found heterozygous variants for two non-synonymous SNPs (Gly299Asp and Ile399Thr) in the TLR4 gene to be associated with higher IL-4 secretion to the measles vaccine strain of measles virus. These two SNPs have been studied extensively in association with septic shock after infection with gram negative bacteria, premature birth, myocardial infarction and allograft rejection [23]. Since only wild-type and not vaccine strains of measles viruses and bacteria are known to signal through TLR4, the biological relevance of the associations between SNPs in TLR4 and measles vaccine-induced immunity needs further validation. It is also possible that the associations observed between TLR4 SNPs and measles vaccine immunity may be spurious and not in agreement with the current literature for two reasons. First, very low level IL-4 responses were detected in response to measles stimulation using a modified IL-4 receptor blocking assay [14] and the differences in the magnitude of IL-4 secretion, though statistically significant, were small across genotypes. Second, there were no subjects carrying a homozygous minor allele to confirm the additive effect of the minor allele on IL-4 secretion. Similarly, we had a very small representation of a minor allele variant for a synonymous SNP in TLR2 that may have skewed the IL-12 response. Hence, these associations need verification and validation in a larger cohort to understand their biological significance.

We also found associations between SNPs in TLR5 and TLR6 genes and variations in IFN-γ secretion in response to measles virus stimulation. TLR5 and TLR6 (a co-receptor for TLR2) are known receptors for bacterial derivatives [5, 6, 24]. Hence, the significance of the associations between TLR5 and TLR6 and measles immunity is currently unknown.

The effects of TLRs are mediated by a complex network of intracellular molecules that trigger the signal from the cell surface to the nucleus for activation of antiviral response genes. Therefore, we also looked at associations between SNPs in genes of intracellular signaling molecules associated with TLRs and the immune response to measles. A minor allele variant for a SNP in the 3′ UTR of MyD88, an intracellular adaptor molecule that signals for most of the TLRs, was found to be associated with a lower antibody response to measles. The role of MyD88 signaling has been implicated in the generation of long-term antibody response to viral infection using a MyD88 knockout mice model [25]. MD2, another molecule that associates intra-cellularly with TLR4, demonstrated decreased IL-10 secretion in the presence of the heterozygous variant. Again, due to limited sample size, we did not observe any minor allele variant for this SNP. We also found several intronic SNPs in TLR and their associated intracellular molecule genes to be significantly associated with variations in cellular immune responses to measles vaccine. No direct relevance or mechanism of action of intronic SNPs on gene function has been reported. However, multiple indirect mechanisms have been proposed by which intronic SNPs can influence gene function such as: i) intronic SNPs can prevent the correct splicing of introns and hence affect the intron splicing efficiency. ii) intronic SNPs may alter splicing and result in premature stop codons or exon deletion and hence generate aberrant or instable mRNA. iii) SNPs within introns may create cryptic splice sites that can affect the production of functional mRNA [26–29].

Overall, consistent with the limited literature on the role of TLRs in measles vaccine-induced immunity, we found specific SNPs in the coding and regulatory regions of TLRs, specifically TLR3, that were significantly associated with variations in antibody and cellular immune responses to measles vaccination. Ours is the first report suggestive of the possible influence of SNPs in TLR and their associated intracellular signaling genes in modulating the immune response to measles vaccine. These findings are consistent with the fact that multiple genetic variations in candidate immune response genes contribute to the complex architecture of the immune response, and are important in understanding the immunogenetics of vaccine response. We assessed associations between a large number of SNPs and several different measures of immune response and, therefore, multiple testing issues are a legitimate concern. However, we found a higher number of significant associations than we would expect by chance alone (29 vs. 20 significant results assuming independent hypothesis tests and a type I error rate of 0.03) indicating a possible genetic component to measles immune response. Furthermore, the q-values for a few of the more significant SNPs indicate the relatively low probability of a false positive result.[20] Note that the calculation of the q-values assumes independent tests of hypothesis, which we may not have due to linkage disequilibrium across SNPs. Thus, these results should be interpreted with a certain level of caution. Other limitations of our study are the small sample size and modest P-values due to the hypothesis-generating nature of this study. New guidelines for SNP association studies for candidate genes in humans suggest the use of lower p-values [30], hence the associations we identified need further validation in a larger cohort.