Vitamin D, leptin and impact on immune response to seasonal influenza A/H1N1 vaccine in older persons

ABSTRACT

Background: Influenza-related complications are highest in the elderly. Vaccine efficacy is lower due to immunosenescence. Vitamin D's immunomodulatory role was studied in the context of vaccine response.

Methods: We evaluated the effect of baseline 25-(OH) D on vaccine-induced immunological response in a cohort of 159 healthy subjects ages 50–74 in Rochester, MN, who received one dose of seasonal trivalent 2010–2011 influenza vaccine, containing A/California/H1N1- like virus. We examined correlations between 25-(OH) D, leptin, and leptin-related gene SNPs to understand the role of leptin and vitamin D's effects.

Results: The median (IQR) baseline for total 25-(OH) D was 44.4 ng/mL (36.6–52.2 ng/mL). No correlation was observed with age. No correlation between 25-(OH) D levels and humoral immune outcomes existed at any timepoint. There was a weak positive correlation between 25-(OH) D levels and change (Day 75-Day 0) in influenza-specific granzyme-B response (r=0.16, p=0.04). We found significant associations between 3 SNPs in the PPARG gene and 25-(OH) D levels (rs1151996, p=0.01; rs1175540, p= 0.02; rs1175544, p=0.03).

Conclusion: Several SNPs in the PPARG gene were significantly associated with baseline 25-(OH) D levels. Understanding the functional and mechanistic relationships between vitamin D and influenza vaccine-induced immunity could assist in directing new influenza vaccine design.

KEYWORDS:

• H1N1 subtype
• influenza A virus
• influenza vaccines
• leptin
• receptors
• vitamin D
• vitamin D deficiency

Introduction

The burden of influenza in terms of influenza-related hospitalization and mortality among adults is consistently higher in the elderly, the obese and those with medical co-morbidities.¹ The inactivated trivalent influenza vaccine (TIV) offers an efficacy against influenza of between 0–53% among adults above age 65 compared to 70–90% of younger adults depending on the virus strain.^2,3 This difference has been linked to immunosenescence, lower protective antibody titer, and sub-optimal T cell responses in the elderly. Cell-mediated immunity (CMI) may be a better correlate of vaccine effectiveness in the elderly, but gold-standard assays are not available.⁴ Obesity is an independent risk factor for influenza-related complications, and higher body mass index (BMI) has been associated with diminished humoral and CMI response to influenza.⁵

Vitamin D has emerged as a potent immunomodulator, and there is tremendous interest in the effect of vitamin D status and vitamin D receptor gene polymorphisms on the clinical outcome and susceptibility to certain infectious diseases.^6-8 There are limitations involving studies that have examined the relationship of vitamin D status and influenza vaccination-induced immune response. Numerous knowledge gaps remain, as the studies to date have only assessed humoral immunity, and many studies have been conducted in very selected patient populations. Few studies have included subjects already on vitamin D supplementation.^9-13

Epidemiological studies have demonstrated a higher incidence of vitamin D deficiency in elderly and obese populations.^14,15 Recent findings from a longitudinal study of older adults suggest leptin may mediate obesity-related vitamin D deficiency.¹⁶ Leptin, a hormone produced in adipose tissue, has notable immunomodulatory effects.¹⁷ We have previously demonstrated our findings on the association of leptin and leptin-related gene polymorphisms on the immune response to influenza A/H1N1 vaccine in older persons.¹⁸

In this study, we examined the effect of baseline serum 25-(OH) D status on both humoral and CMI responses following a single vaccination with the licensed seasonal trivalent 2010–2011 influenza vaccine (containing influenza A/California/H1N1 like virus) in a healthy older cohort. Our primary aim was to correlate baseline 25-(OH) D levels with humoral and CMI response to influenza A/H1N1 vaccine. We also examined associations between baseline 25-(OH) D with baseline leptin levels and leptin-related single nucleotide polymorphisms (SNPs), as leptin may act as a mediator of vitamin D's actions. This study aimed to be hypothesis-generating and may help inform future studies.

Results

Study subjects

The demographics and clinical variables of the study subjects are illustrated in Table 1. The median age was 59.5 years, and most subjects were Caucasian. The median baseline 25-(OH) D level was 44.4 ng/mL (IQR 36.6–52.2) ng/mL. Eight subjects had baseline 25-(OH) D levels below 25 ng/mL. Women (n = 97) had significantly higher baseline total 25-(OH) D levels (median 47.2 ng/ml, IQR 38.1–53.0) ng/ml, compared to men (median 41.0 ng/ml, IQR 35.6–48.3) ng/ml, p = 0.029 (Fig. 1a). Baseline 25-(OH) D level did not correlate with age.

Table 1. Demographics and descriptive baseline variables.

Variable	N	Median (IQR) or N (%)
Age (years)	159	59.5 (55.3, 66.3)
Gender	159
Female		98 (61.6%)
Male		61 (38.4%)
Race	159
Caucasians		157 (98.7%)
Others		2 (1.3%)
BMI (kg/m^2)	147	28.2 (24.5; 33.1)
Baseline (Day 0) Leptin (pg/mL)	147	8,100 (4,325; 15,860)
Baseline (Day 0) 25-OH D level (ng/mL)	158	44.4 (36.6; 52.2)

IQR, interquartile range; BMI, body mass index.

The median BMI was 28.2 kg/m² (IQR 24.5, 33.1); 52 subjects were obese (BMI ≥ 30 kg/m²). The baseline leptin level was measured in 147 subjects, with median level of 8,100 pg/mL (IQR 4,325–15,860) pg/mL. Baseline 25-(OH) D levels did not correlate with BMI (Fig. 1b), but there was suggestion of a weak positive correlation between baseline 25-(OH) D and baseline leptin levels (Spearman's rank correlation coefficient 0.15, p=0.08), as shown in Figure 1c.

Summary of immune outcome measures to influenza A/ H1N1 vaccine

The key immune outcome measures to influenza A/H1N1 vaccine in this study cohort across all timepoints are illustrated in Table 2. The median baseline (Day 0) influenza A/H1N1 HAI titer was 1:80 (IQR 1:40, 1:320), which means 75% of the cohort was already immune to influenza A/H1N1 based on a cut-off HAI titer of 1:40. ¹⁸ H1N1 VNA titers were similar at baseline compared to H1N1 HAI titer. There was a significant increase in humoral immune outcome measures following influenza vaccination, specifically HAI titer, VNA titer, and B-cell ELISPOT responses from baseline to Day 28. ¹⁹

Table 2. Distribution of humoral and cellular immune response variables across the time-points in study cohort.

Immune response variable	No. of subjects	Day 0	Day 28	Day 75	P value^a	P value^b
Influenza A/H1N1 HAI titer, median (IQR)	159	1:80 (1:40, 1:320)	1:320 (1:160, 1:640)	1:320 (1:80, 1:640)	<0.0001	<0.0001
Influenza A/H1N1 VNA titer, median (IQR)	159	1:80 (1:40, 1:320)	1:320 (1:160, 1:640)	1:320 (1:160, 1:640)	<0.0001	<0.0001
B-cell ELISPOT (SFC^c, median, IQR)	159	9.0 (3.0, 20.0)	35.0 (15.3, 57.5)	21.5 (9.3, 36.3)	<0.0001	<0.0001
Granzyme B ELISPOT (SFC^c, median, IQR)	159	4.0 (1.0,12.5)	5.0 (1.0, 13.0)	7.0 (2.0, 14.0)	0.393	0.340

^a p value; day 28 vs day 0,

^b p value; day 75 vs day 0,

^c SFC: spot forming counts/ 2 × 10⁵ PBMCs (peripheral blood mononuclear cells)

The distribution of specific subpopulations of T regulatory cells, CD4+ T cells, CD8+ T cells, and B cells, as measured by flow cytometry, are shown in Table S1. There was no significant change in secreted cytokine/chemokine levels across the timepoints, and the level of secreted cytokines/chemokines at baseline was low, as illustrated in Table S1.

Correlation between baseline 25-OH D level and immune measures to influenza A/H1N1 vaccine

The baseline 25-(OH) D level did not correlate with HAI A/H1N1 at Day 28, change in HAI at Day 28 compared to Day 0, or with HAI at Day 75. Figure 1d shows correlation between baseline 25-(OH) D levels with Day 28 HAI. There was no correlation between baseline 25-(OH) D level and VNA, as well as B-cell ELISPOT response across the timepoints.

We found a weak positive correlation between baseline 25-(OH) D levels and change in influenza-specific granzyme-B response at Day 75 compared to Day 0 (Spearman's rank correlation coefficient 0.16, p = 0.04) (Fig. 1e).

There was no correlation between baseline 25-(OH) D level and changes in subpopulations of immune cells (specifically dendritic cells, regulatory T cells, CD4+ and CD8+ T cells), and cytokine response (specifically IL-10, RANTES, MIP-1b, MCP-1) at any timepoint. There was a weak negative correlation observed between baseline 25-(OH) D and change in HLA expression on classical monocytes at Day 28 compared to Day 0, and B cell frequencies (percent of live cells) (see Fig. S1).

Association between SNPs in leptin and leptin-related genes and baseline 25-(OH) D level

The linear regression model was adjusted for gender and leptin levels. Results of these analyses are shown in Table 3, along with SNPs with p ≤ 0.10. Three SNPs in the PPARG gene were potentially associated with baseline 25-(OH) D levels in adjusted analyses (p ≤ 0.05). We found the following significant associations: homozygous genotype AA for an intronic rs1151996 in the PPARG gene; homozygous genotype CC for an intronic rs1175540 in the PPARG gene; and homozygous genotype GG for an intronic rs1175544 in the PPARG gene were associated with higher median baseline 25-(OH) D level compared to the heterozygous genotypes.

Table 3. Associations between SNPs in leptin-related genes and baseline 25-(OH) D levels in study cohort.

Outcome measure	Gene	SNP ID	Location	Genotype^a	N	Median (IQR)	p value^b	p value^c
Baseline 25- (OH) D level (ng/mL)	PPARG	rs1151996	intron	AA	41	47.8 (40.7, 55.4)	0.0288	0.0103
				AC	73	42.9 (35.9, 50.8)
				CC	26	43.2 (34.9, 58.8)
	PPARG	rs1175540	intron	CC	49	47.8 (37.2, 55.4)	0.0435	0.0167
				CA	66	43.0 (37.0, 49.5)
				AA	25	42.4 (34.7, 58.5)
	PPARG	rs1175544	intron	GG	54	47.4 (37.8, 54.1)	0.0553	0.0258
				GA	62	42.3 (36.5, 49.5)
				AA	24	43.2 (35.4, 58.6)
	ADIPOR1	rs1342387	intron	GG	43	41.1 (33.8, 48.7)	0.0506	0.0538
				GA	70	45.8 (37.6, 53.0)
				AA	27	47.2 (40.8, 52.4)
	PPARG	rs17793951	intron	AA	54	46.9 (40.4, 53.0)	0.0975	0.0584
				AG	65	41.5 (35.8, 51.3)
				GG	21	45.5 (37.8, 58.5)

^a Values are presented as homozygous major allele/heterozygous/homozygous minor allele

^b Unadjusted p value (linear regression model using SNPs classified as no minor allele vs at least one minor allele)

^c Adjusted p value from linear regression model, when adjusted for gender and baseline leptin level.

Discussion

Older persons have impaired immune responses to influenza vaccination secondary to immunosenescene, inflammaging, and impaired T cell responses to vaccination. ^20,21 Vitamin D levels are typically lower in the elderly due to factors involving vitamin D metabolism, such as less efficient synthesis of vitamin D in the skin, and decreased renal production of active 1,25-(OH)₂D.¹⁵

There has been significant and growing interest in the non-skeletal actions of vitamin D.²² Vitamin D has a robust action on the innate immune response, for example producing an antimicrobial peptide (cathelicidin), and triggering a shift to a Th2-type cytokine response compared to Th1 (characterized by increased levels of IL-4, IL-5, IL-10 and reduced levels of IL-2, IFN-γ, and TNF-α). The action of vitamin D on B cell response is less clear.²³

Although serum 25-(OH) D levels are a good predictor of vitamin D status (with a half-life of 2–3 weeks), the active metabolite of vitamin D, 1,25-(OH)₂ D, is produced via renal 1-alpha-hydroxylation. Of significance, 1,25-(OH)₂D is also produced locally in immune cells via CYP27B1 (cytochrome P450, family 27, subfamily B, polypeptide 1) conversion of systemically delivered 25-(OH) D, which performs intracrine actions mediated by binding to the vitamin D receptor (VDR). VDR is widely expressed in many tissues, including immune cells.^24,25 Importantly, the vitamin D level threshold that is relevant to its numerous non-skeletal actions has not been defined.

The primary objective of this study was to evaluate the effect of baseline serum 25-(OH) D level on adaptive humoral and cellular immune response outcomes to influenza vaccination in older adults. We did not find a significant correlation for baseline 25-(OH) D levels with humoral immune outcomes such as HAI (which is in agreement with other studies),^9,12,26 VNA or B-cell ELISPOT. Previous studies have not evaluated CMI outcomes.^9,12,26 We found a weak positive correlation between baseline 25-(OH) D levels and change in influenza-specific granzyme B cellular response at Day 75 compared to Day 0 (Spearman's rank correlation coefficient 0.16, p = 0.04). Granzyme B is a serine protease produced by NK cells, dendritic cells and cytotoxic T cells. It is known to induce cytotoxic T cell-mediated apoptosis of virus infected host cells.²⁷ Granzyme B has been evaluated as a promising marker of CMI response to influenza vaccine, and influenza-specific granzyme B responses have been shown to negatively correlate with increasing age.^27,28

Our secondary objective was to evaluate associations between baseline 25-(OH) D with baseline leptin levels and leptin-related gene SNPs. Leptin is produced by adipocytes and, along with its well established roles in metabolism, it has also been shown to have immunomodulatory effects.¹⁷ Leptin levels are known to be higher with increasing adiposity. Elevated leptin levels in the obese state have been observed to result in attenuated leptin signaling (leptin resistance) and impairment in both innate and adaptive influenza-specific immunity.^29,30 Obese patients have a higher prevalence of vitamin D deficiency, as demonstrated in epidemiological studies.³¹ The exact underlying mechanisms for this are not entirely clear.^30,32,33 Vitamin D is stored in both fat and muscle, and adipose tissue cells express VDR, CYP27B1 and 24-hydroxylase.³² We observed a slight non-significant negative correlation of 25-(OH) D with BMI.

Data from studies investigating the relationship between vitamin D and leptin levels, as well as vitamin D and leptin signaling pathways, suggest the 2 are interrelated.^16,34,35 Findings from clinical studies suggest leptin may mediate obesity-related vitamin D deficiency, and that vitamin D and leptin are inversely correlated.³⁴ Leptin receptors and VDR are widely distributed in various tissues. There is supportive evidence that leptin and vitamin D signaling and metabolism pathways interact and influence each other; in one study leptin reduced gene expression of 1-alpha-hydroxylase and production of 1,25-(OH)₂ D.³⁵ However, hyperleptinemia in the context of leptin resistance ^17,36 may modify the relationship between serum 25-(OH) D and leptin. We found suggestion of a positive correlation between baseline 25-(OH) D levels and baseline leptin levels (r = 0.15, p = 0.08). Only 33% of our subjects had BMI > 30 kg/m²; however, our cohort was reasonably representative of Minnesota's population based on Centers for Disease Control (CDC)-published data for state obesity prevalence estimates.³⁷ We did not have access to other measures of obesity e.g., waist-to-hip circumference ratio, body fat percentage. An examination of the relationship between vitamin D and leptin categorizing by BMI did not demonstrate a significant trend in a linear regression model (data not shown). Few studies have shown that statin use may increase 25-(OH) D levels, hence information on statin use (and other medications) would be important variables while evaluating relationship between obesity, leptin and 25-(OH) D.^38-41 The relationship between 25-(OH) D, adiposity and leptin is likely more complex with multiple areas of regulation and feedback loops.

We also evaluated the effect of leptin-related gene SNPs on baseline 25-(OH) D levels in this cohort. Peroxisome proliferator-activator receptors are nuclear hormone receptors and transcriptional co-activators, with 3 known distinct isoforms: PPAR-α, PPAR-δ; and PPAR-γ.⁴² PPAR isoforms play key roles in cellular and bone metabolism, cellular differentiation and development, inflammation and immune response (PPAR-γ modulates macrophage and dendritic cell function), as well as adipocyte differentiation and adipogenesis.^42,43 VDR and PPAR isoforms are nuclear hormone receptors and use RXR as a heterodimer.⁴² Several in vitro studies have recently demonstrated that VDR and PPAR-γ signaling pathways interact and there is evidence of “crosstalk,” although the detailed mechanisms have not been elucidated.^42,44,45 In one study, PPAR-γ was found to monopolize RXR-α available for VDR binding and there was an inverse relationship between VDR and PPAR-γ basal expression in these cell lines.⁴⁴ The promotor region of certain human PPAR genes contains a potent VDRE (vitamin D response element).⁴⁶ In our regression models, we found significant associations with homozygous genotypes in 3 intronic SNPs in the PPARG gene with baseline 25-(OH) D levels, and another intronic SNP in the PPARG gene was marginally significant (p = 0.058). Although these were all intronic SNPs, it is possible they are in LD (linkage disequilibrium) with a functional SNP. We have previously examined associations between leptin-related gene SNPs and influenza-vaccine induced immune outcomes. The SNP rs1175540 was associated with allele-dependent B-cell ELISPOT response at Day 28, and rs17793951 was associated with allele-dependent increase in HAI titer at Day 28 in this study cohort.¹⁸ Based on the aforementioned interaction between VDR and PPAR genes, there could be an interaction influencing systemic vitamin D levels, but our results are not able to define a causal relationship.

We also found an intronic SNP in the adiponectin receptor 1 (ADIPOR1) gene associated with 25-(OH) D levels (p=0.054)—a finding that has not been reported in the literature. The ADIPOR1 gene encodes a protein that acts as a receptor for adiponectin. Adiponectin is secreted by adipocytes, and regulates glucose levels and fatty acid catabolism.⁴⁷ Future replication studies should also involve measuring adiponectin levels.

The strengths of this study include the more extensive evaluation of immune-related outcomes compared to other studies evaluating the effect of 25-(OH) D on influenza vaccine-induced immune response that have only evaluated HAI titers. The association of leptin and leptin-related gene SNPs with baseline 25-(OH) D is also novel in the context of vaccine-induced immune response.

This was a retrospective convenience cohort study, which has certain inherent limitations. Vitamin D supplementation information was not available for this cohort, and while this would not affect correlations studied for our primary objective, it may affect correlations between vitamin D and other variables (e.g., BMI, leptin, age). Although our cohort had a range of 25-(OH) D levels, most of the subjects were “vitamin D sufficient” using the conventional definition of vitamin D deficiency based on bone health. A recently published population-based retrospective cohort study over the time period of 2002–2011, conducted using the Rochester Epidemiology Project, demonstrated a significant rise of age- and sex-adjusted incidence of 25-(OH) D > 50 ng/mL from 9 to 233 per 100,000 person-years through the course of the study. This increase was noted to be highest in women and in persons above 65 years of age and was postulated to be due to increasing use of vitamin D supplementation during the study period. ⁴⁸ Our observation of 25-(OH) D levels appears to be reflective of this trend in the population where our subjects were recruited (also in the context of months of study recruitment); therefore we did not observe any inverse correlation of 25-(OH) D with age. Our study aimed to investigate the effect of baseline 25-(OH) D on immune outcomes, and the “threshold” that is relevant to vitamin D's immunomodulatory actions is not known. A larger sample size with a larger range of 25-(OH) D levels, specifically enough proportion with lower levels will be better powered to find an effect if one exists.⁴⁹ Our cohort was predominantly Caucasian; a similar study will need to be repeated in populations with different races and ethnicities.

The finding of no significant correlation of 25-(OH) D level with humoral outcomes following influenza vaccination, including HAI, is consistent with other studies.^9,10,12 The immune responses studied were for the influenza A/H1N1 strain (majority of subjects had baseline H1N1 HAI titer ≥ 1:40); it will be important to evaluate responses to A/H3N2 and B strains as well. This is the first study, to our knowledge, to assess the effect of 25-(OH) D levels on CMI outcome to influenza vaccination. Granzyme B is a surrogate marker for cytotoxic T cell response, and we found a weak positive correlation with change in granzyme B from Day 75 to Day 0, and 25-(OH) D. Vitamin D has been shown to encourage a Th2 skewed phenotype compared to Th1.^23,24 Replicating our finding involving granzyme B and studying vaccine-induced Th1- and Th2-like responses with relation to vitamin D is likely to be informative.

We have evaluated 25-(OH) D and leptin, leptin-related gene SNPs in conjunction with various aspects of adaptive and humoral and CMI response to influenza A/H1N1 vaccine in older persons. An adequately powered study that includes other measures of obesity, and information on any recent change in BMI or body weight, will be better positioned to further investigate the relationship between vitamin D and leptin. Our findings on SNP associations will need to be replicated and functional studies will need to be performed. There are still numerous unanswered questions. Studying the impact of vitamin D gene-related polymorphisms will provide further insight on the complex interactions that likely exist in vitamin D signaling and phenotype in the context of vaccine-induced immune response. A number of vitamin D gene-related polymorphisms are functional polymorphisms (such as FokI in VDR), although the functional consequence of all polymorphisms is not completely known at this time.^8,50 Vitamin D gene-related polymorphisms have been studied in vaccine-induced immune outcomes for other vaccines.^51,52

Finally, the strategy of vitamin D supplementation or use as a vaccine adjuvant needs to be explored further, instead of merely optimizing “homeostatic” 25-(OH) D status.^{10-13,26,53,54} Several studies involving mature adult mice that were immunized subcutaneously or intramuscularly with inactivated vaccine (inactivated polio vaccine,⁵³ Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine,⁵⁴ and hepatitis B surface antigen (HBsAg)⁵⁵), co-administered with 1,25-(OH)₂D3 demonstrated production of antigen-specific mucosal immunity (IgA and IgG antibodies), as well as enhanced systemic immune responses. It is likely that vitamin D is not an independent predictor of a binary outcome, but may be influential through its complex immune physiological interactions. Understanding the functional and mechanistic relationships between vitamin D and influenza vaccine-induced immunity could assist in directing new influenza vaccine design, such as using vitamin D or a vitamin D-like synthetic analog as an adjuvant in influenza vaccination.

Methods

Subjects

The study recruitment occurred between August and October 2010, and the process has been described in further detail elsewhere.¹⁸ A total of 159 subjects (50–74 years of age) were enrolled for a larger NIH-funded influenza vaccine study in Rochester, MN. All subjects were healthy, without any known immunocompromising conditions, and received one dose of 2010–2011 trivalent inactivated influenza vaccine (TIV, Fluarix), containing influenza A/H1N1/California/2009-like virus. Ninety-five of the 159 subjects (59.7%) had received the 2009 A/H1N1 monovalent vaccine. Of the 159 subjects, 134 (84.3%) received the 2009 seasonal influenza vaccine. Subjects with influenza symptoms at the time of enrollment or during the course of the study were excluded. All study subjects provided venous blood samples at Day 0 (prior to vaccination), Day 28 and Day 75 following vaccination to assess immunological response to the vaccine. The Day 28 timepoint represents peak adaptive response, and the Day 75 timepoint represents a “return to homeostasis” response. Height and weight were obtained on 147 subjects to assess body mass index (BMI). The Mayo Clinic Institutional Review Board approved this study. All subjects had provided informed, written consent.

25-(OH) vitamin D assay

Baseline (Day 0) serum 25-(OH) D levels were measured in stored sera samples (n=158) using liquid chromatography-tandem mass spectrometry at the Mayo Clinic immunochemical core laboratory. The analytic measurement range for total 25-(OH) D (D2+D3) was 6–400 ng/mL.

Leptin assay

Baseline (Day 0) leptin (non-fasting) serum concentrations were measured using the Human Leptin ELISA (R&D Systems, Minneapolis, MN). The level of sensitivity for the leptin assay was 7.8 pg/ml, which has been previously reported.¹⁸ The intra-class correlation coefficient (ICC) for this assay was 0.97.

Influenza hemagglutination inhibition (HAI) and viral neutralization (VNA) assays

Details of the HAI and VNA assays have been previously described.^56,57 HAI assays were performed on Day 0, Day 28 and Day 75 samples using a standard WHO protocol with 0.6% solution of turkey red blood cells by measuring HAI titers against the influenza vaccine strain A/California/7/2009/H1N1. The ICC was 0.91 for the HAI assay (Day 0) and 0.83 for the VNA assay (Day 0).

All serum samples were tested for antibody titer against influenza A/H1N1 strain only.

B cell ELISPOT assay

Influenza virus-specific IgG secreting B cells at Days 0, 28 and 75 were quantified using the Human IgG ELISpot^PLUS kit (Mabtech) as previously described.²¹ The ELISPOT plates were coated with influenza A/California/7/2009/H1N1 virus (50,000 TCID₅₀ per well). ImmunoSpot® S4 Pro Analyzer and ImmunoSpot® version 4.0 software (Cellular Technology Ltd.; Cleveland, OH) was used to analyze the plates.⁵⁸ The ICC for the baseline samples stimulated with influenza virus for this assay was 0.88.

Granzyme B assay

Influenza-specific granzyme B-positive cells in peripheral blood mononuclear cell (PMBC) cultures on Days 0, 28 and 75 were quantified using the BD™ Human Granzyme B ELISpot kit (BD Biosciences; San Jose, CA) as previously described.²² PBMCs were either left unstimulated or stimulated with influenza A/California/7/2009/H1N1 virus at a multiplicity of infection (MOI) of 0.5 for 24 hours.²⁸ The ICC for the baseline samples stimulated with influenza virus for this assay was 0.62.

Flow cytometry (innate, B cells, regulatory T cells, and activated T cells)

PBMCs from each subject at Days 0, 28 and 75 were subjected to the following 3 conditions: influenza A/H1N1-stimulated; phorbol 12-myristate 13-acetate (PMA)-stimulated; and unstimulated. For specific markers used for T cell activation, T regulatory cell (T regs), B cell and innate panels see supplementary data for the methods.

Multiplex cytokine/chemokine assay

We assessed a panel of cytokines/chemokines in the sera of all subjects at Days 0, 28, and 75 with details on methods available in supplementary data. The cytokines/chemokines that had delectable levels, with some detectable changes across all timepoints (IL-10, RANTES, MIP-1b, MCP-1) were used for association analyses with 25-(OH) D.

SNP selection and genotyping

The SNP selection and genotyping has been described elsewhere.¹⁸ Briefly, 96 tagSNPs from 12 candidate leptin signaling related genes were selected using source databases, such as International Hapmap Phase II, NIEHS SNPs, Seattle SNPs, and the 1000 Genomes Project. These genes included leptin (LEP), leptin receptor (LEPR), adiponectin (ADIPOQ), adiponectin receptor 1 (ADIPOPOR1), adiponectin receptor 2 (ADIPOR2), Janus kinase (JAK2), signal trasuducer and activator of transcription (STAT3), suppressor of cytokine signaling 3 (SOCS3), protein tyrosine phosphatase N1 (PTPN1), C-reactive protein (CRP), peroxisome-proliferator-activated receptor γ (PPARG), and ghrelin/obestatin (GHRL) as has been previously described.¹⁸ Illumina BeadXpress Reader with CeraCode digital microbead technology (Illumina Inc.; San Diego, CA) was used for detection of multiple SNPs in a given DNA sample. Three SNPs were excluded from analysis due to failing QC.

Statistical methods

Demographic results and clinical characteristics of the cohort are presented as medians and interquartile ranges (IQR) for continuous variables, and counts and proportions for categorical variables. Baseline 25-(OH) D level was analyzed as a continuous variable for all analyses. Spearman's rank correlation was used to test the associations between baseline 25-(OH) D level with influenza-vaccine specific adaptive immune response at Day 0, Day 28, Day 75, and the changes from Day 0 to Day 28, and Day 75. Spearman's correlation was also used to determine if factors such as age, obesity, and baseline leptin levels correlate with baseline 25-(OH) D levels. The Wilcoxon rank sum test was used to test for differences in baseline 25-(OH) D levels between genders. Finally, linear regression was used to evaluate associations between leptin-related gene SNPs and baseline 25-(OH) D levels, with SNPs as the independent variable (SNPs were grouped based on whether a minor allele is present). Gender and baseline leptin levels were included as covariates in the model instead of applying multiple correction methods to the SNP analyses; results are presented with p-values. Statistical analyses were not adjusted based on subjects' prior vaccination history information.

Disclosure of potential conflicts of interest

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, Dynavax, Novartis Vaccines and Therapeutics, Emergent Biosolutions, Adjuvance, and Microdermis. Drs. Poland and Ovsyannikova hold 2 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 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 Mayo Clinic Conflict of Interest policies.

Acknowledgment

The authors wish to thank Caroline L. Vitse for her editorial assistance.

Funding

Research reported in this abstract was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under award number U01AI089859, and by the Division of Infectious Diseases, Mayo Clinic, Rochester, MN. This publication was made possible by CTSA Grant Number UL1 TR000135 from the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.