ABSTRACT
Background: Vitamin D is an immunomodulating hormone, which has been associated with susceptibility to infectious diseases.
Methods: Serum vitamin D levels in 135 children ages 3–17 y were measured at baseline and hemagglutinin influenza antibody titers were measured pre- and 21 d post influenza vaccination with live attenuated influenza vaccine (LAIV) or inactivated influenza vaccine (IIV). Height and weight were derived from the electronic medical record and were used to calculate body mass index (BMI).
Results: Thirty-nine percent of children were ages 3–8 years; 75% were black, 34% were obese (BMI ≥ 95th percentile); vitamin D levels were >20 ng/ml in 55%. In linear regression analyses, post vaccination antibody titers for LAIV B lineages (B Brisbane and B Massachusetts) were significantly higher among those with lower vitamin D levels and among younger participants (P < 0.05). No associations between vitamin D levels and responses to LAIV A strains (A/H1N1 and A/H3N2) or to any IIV strains or lineages were found.
Conclusion: Low vitamin D levels were associated with higher response to LAIV B lineages in the 2014–2015 LAIV, but not related to LAIV A or any IIV strains.
Introduction
Immune response to infectious agents or the vaccines designed to prevent them is complex and influenced by an increasing number of physiologic and metabolic factors. Among those factors receiving attention are serum vitamin D levels and elevated body weight. Vitamin D is thought to play a role in both innate and adaptive immune responses to infection.Citation1 Previous studies have demonstrated that increased incidence of certain infections including influenza are associated with lower levels of vitamin D.Citation2 Correspondingly, a lower relative risk (RR) of influenza (0.58 (95% CI: 0.34–0.99; P = 0.04)) was reported among children with vitamin D supplementation compared with children taking placebo.Citation3 The mechanism for these findings may be related to the effect of vitamin D levels on influenza vaccine immunological response, yet inconsistent associations between vitamin D levels and serological response to influenza vaccine have been reported.Citation4,5
Vitamin D insufficiency (< 30 ng/ml) and deficiency (< 20 ng/ml) have been found to be associated with obesity in males and females, for both children and adults.Citation6-9 Furthermore, obesity has been associated with dysregulated cytokine production, reduced natural killer cell activity, altered CD4:CD8T cell balance, and decreased response to antigen stimulation.Citation10 Although Sheridan et al.Citation11 reported significantly higher antibody fold increases among adults at one month following influenza vaccination associated with increasing body mass index (BMI), at 12 months post vaccination, approximately 50% of these obese individuals showed ≥ 4-fold decrease in their antibody titer. Talbot et al.Citation12 reported positive associations between BMI and serologic response to influenza only for A(H3N2) virus among older adults.
The purpose of this study was to examine serological response to influenza vaccine in children 3 -17 y of age in relation to serum vitamin D levels measured immediately before vaccination and to BMI.
Results
Of the 173 children enrolled in the study, 23 children did not complete both blood draws, 10 did not have height and weight data available and 5 did not have vitamin D levels measured, leaving 135 children for analysis. Demographic characteristics of the participants, overall and by the type of vaccine received are shown in . Thirty-nine percent (53/135) were ages 3–8 years, one half (54%) was female, and the majority (75%) self-identified their race as Black. Nearly one half of participants (45.2%) had deficient vitamin D levels at baseline with baseline levels ranging from 4 to 40 ng/ml. Over one-third (34%) of the children were considered to be obese (BMI ≥ 95th percentile). All but 9 of the children had received at least one previous influenza vaccine. Demographic characteristics of participants receiving the 2 types of influenza vaccine did not differ except that significantly more males than females received LAIV (, P < 0.05).
Table 1. Demographics and variables of interest by vaccine type.
Age group, vitamin D level, obesity and Day 0 antibody titers were included a priori in linear regression analyses (). For LAIV, neither A virus subtype was related to any variable other than its baseline antibody titer; whereas, for both B virus lineages, Day 21 antibody titers were significantly lower among older children, those with higher vitamin D levels (≥ 20 ng/ml) and lower Day 0 antibody titers. For IIV, Day 21 antibody titers were only significantly associated with Day 0 antibody levels for both A(H1N1) virus and B(Brisbane) virus lineage whereas, Day 21 A(H3N2) virus titers were significantly associated with Day 0 titers, younger age and lower BMI, and Day 21 B/Massachusetts virus (Yamagata lineage) titers were significantly associated with younger age and Day 0 titers.
Table 2. Factors related to Day 21 Log2 titers by influenza vaccine type (LAIV or IIV) and strain or lineage by linear regression.
Discussion
Nearly half of the children in this study had vitamin D levels considered to be suboptimal for good health; this differs from Europe where approximately 80% of adolescentsCitation16 and the United States where 19% of children 1–11 y have been reported to have deficient vitamin D levels and higher proportions of non-white children had deficient vitamin D.Citation17 In contrast to US obesity prevalence (17% overall and 20–22% for non-white children),Citation18 more children in this study were obese (one third was at or above the 95th percentile for BMI for their age and sex).
The association of vitamin D levels with immune response to influenza vaccine has not been extensively studied. In this study, lower vitamin D levels were associated with higher antibody titers for influenza B vaccine virus lineages in the LAIV vaccines only. In previous research, lower vitamin D levels have been found to be associated with higher antibody response to 4 human papilloma virus (HPV) vaccine strains among young adult men.Citation19 Moreover, there is mounting evidence that low vitamin D level is related to onset of and higher disease activity of autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis and type I diabetes,Citation20,21 as well as risk of childhood asthma.Citation22
Several studies suggest a role of vitamin D in both innate and adaptive immunity. Vitamin D is essential for the regulation of human genesCitation23 and is a known promoter of cathelicidin,Citation24 which increases production of hCAP-18Citation25 and enhances macrophage function and innate immunity. These associations are believed to explain the observed association between vitamin D levels, cathelicidin expression and intracellular killing of Mycobacteria tuberculosis,Citation26 as well as the inverse association between vitamin D levels and risk of upper respiratory tract infections.Citation27,28 Vitamin D may also influence the development of adaptive immunity by inhibiting B cell proliferation, differentiation and immunoglobulin secretionCitation29,30 and by suppressing T cell proliferation,Citation31 resulting in a shift from more pro-inflammatory Th1- to anti-inflammatory Th2 cell responses.Citation32 Furthermore, vitamin D has been associated with the induction of T regulatory cells.Citation33 It is therefore reasonable to assume that vitamin D may have a role in response to influenza vaccines.
Younger age was associated with higher antibody titers for both B lineages in LAIV and to the A(H3N2) virus subtype and B/Massachusetts virus (Yamagata lineage) for IIV. In addition, baseline titer was a significant variable in all regression analyses. Baseline titer levels may reflect past vaccinations, past infections or both; HAI analyses are unable to distinguish between them. Body weight was not significantly related to antibody titer response to influenza vaccine.
A meta-analysis had shown that LAIV was more effective among younger children than older children,Citation34 leading the US Advisory Committee on Immunization Practices to recommend preferential use of LAIV for children 2–8 y old in the 2014–15 influenza season. Subsequent research on influenza vaccine effectiveness found that LAIV was not more effective than IIV in this age group. Specifically, in 2014–2015, LAIV effectiveness against A(H3N2) virus was 11% (95%CI = -1, 21) and against B virus (Yamagata lineage) was 54% (95% CI = 41, 64).Citation35
In our serology data, to the extent that LAIV was eliciting an immune response (i.e., B lineages), it was significantly and inversely associated with vitamin D levels. Small sample size among the IIV recipients may have masked a similar response to this form of the vaccine. Previous research in adults did not find an association of influenza vaccine response with vitamin D levels.Citation5
Strengths and limitations
Although the coefficients for vitamin D were all in the same direction, the modest sample size might have obscured a more consistently significant finding. Because the study was observational, unmeasured biases could exist and causality cannot be determined. The relationship of vitamin D and obesity to the immune system's response to vaccine antigens is complex and should be further investigated in larger groups across different vaccines and across various infectious diseases.
Conclusion
Low serum vitamin D levels were associated with higher response to the 2014–2015 LAIV B virus (Yamagata and Victoria lineages), but not to LAIV A viruses or to IIV, among children and adolescents.
Methods
This study was approved by the University of Pittsburgh Institutional Review Board. Written informed consent was obtained from parents of all study participants.
Subjects, eligibility and enrollment
From September through December 2014, healthy children aged 3 through 17 y were recruited from 3 primary care offices in the Pittsburgh area. Exclusion criteria included body weight <17 kg, having an immunosuppressive disease, taking immunosuppressive medicine or high dose oral steroids, having a severe allergy to the influenza vaccine or its components, and prior vaccination with the current season's (2014–2015) influenza vaccine. Based on funding, likelihood of being able to recruit eligible children with the desired vaccination history and age groups, and the need to make return visits for blood draws, a desired total sample size of 175 was set, with children divided by age group (3–8 and 9–17 years). Recruitment goals were set for each age group and each of 4 combinations of previous season's vaccine (LAIV, IIV or no vaccine) and current season's vaccine (LAIV or IIV). When the goal was reached for a given group, recruitment for that group ceased.
Parents provided demographic data and permission to extract data from the electronic medical record (EMR). Height and weight measured within 6 months of enrollment were derived from the EMR and were used to calculate BMI. Using 2000 Centers for Disease Control and Prevention (CDC) growth chartsCitation13 age- and sex-specific BMI percentiles were calculated. Childhood obesity was defined as being at or above the 95th percentile of BMI. A dichotomous variable was generated for non-obese (< 95th percentile) versus obese (≥ 95th percentile) children.
Specimen collection and processing
On day 0, participants provided blood samples then received the influenza vaccine of their choice (live attenuated influenza vaccine (LAIV) or inactivated influenza vaccine (IIV) per CDC recommendations) as part of their clinical care; they returned on day 21 post vaccination (range 20–35 days) for an additional blood draw. Blood samples were collected using a) serum separator tubes (SST) for hemagglutination inhibition (HI) assays to estimate antibody titers, and b) glass mononuclear cell preparation (CPT) tubes for determination of 25-hydroxy vitamin D levels (day 0 only).
Sample processing and antibody titers
Within 4–6 hours of collection blood samples were spun at 3200 rpm for 10–15 minutes. Serum (SST) and plasma (CPT) were collected and stored at -70°C until analysis. Antibody responses were measured by hemagglutination inhibition (HI) assays using turkey erythrocytes as described previously.Citation14 HI titers were defined as the reciprocal of the highest dilution of virus that caused complete hemagglutination. Testing was performed by Battelle contract laboratory (Aberdeen, MD) after proficiency assessment by CDC.
Vitamin D assays
Vitamin D assays were performed with a Waters ultra-performance liquid chromatography with tandem mass spectrometer that detected molecular weight for 25-hydroxy vitamin D2 and D3. The first quadruple mass analyzer was tuned for the parent ions. The second mass analyzer was tuned for specific daughter ions; these are detected by the photomultiplier system. The instrument's coefficient of variation was 10 for both vitamin D2 and D3.
Statistical analyses
Vitamin D2 (range = 4–12 ng/ml) and D3 (range = 4–60 ng/ml) were combined for a total vitamin D level for analysis. The Institute of Medicine (IOM) recommends concentrations of vitamin D ≥ 20 ng/ml as optimal for skeletal health of US childrenCitation15 thus, normal vitamin D was defined as ≥ 20 ng/ml and deficient vitamin D was defined as <20 ng/ml.
The antibody titers used in the regression models were log2-transformed because they were skewed. The outcome variables were the log2-transformed Day 21 antibody titers each influenza A virus subtype and B virus lineage.
Demographic characteristics at baseline of recipients of each vaccine type were compared using Chi square and Fisher's exact tests. Age groups (3–8 y vs. 9–17 years), vitamin D levels, obesity status (BMI <95th percentile or ≥ 95th percentile), and baseline log2 transformed antibody titers were included in linear regression analyses to determine their relationships to Day 21 antibody titers for each vaccine virus. Statistical significance was set at P < 0.05. SAS 9.4 (SAS Institute, Cary, NC) and SPSS v.24 (IBM Corp., Armonk, NY) were used for analyses.
Disclosure of potential conflicts of interest
Drs. Zimmerman, Lin and Ms. Moehling have research funding from Sanofi Pasteur, Inc. Drs. Zimmerman, Lin, and Nowalk and Ms. Moehling have research funding from Pfizer, Inc. and Merck & Co., Inc.
Acknowledgment
The authors thank the Special Chemistry and Reference Laboratory of UPMC for vitamin D assays.
Funding
This work was supported by the Centers for Disease Control and Prevention (CDC), through a cooperative agreement with the University of Pittsburgh (U01 IP000467) and by the National Institutes of Health (NIH grants UL1 RR024153 and UL1TR000005) to the University of Pittsburgh. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of CDC nor the NIH.
References
- Aranow C. Vitamin D and the immune system. J Investige Med 2011; 59(6):881-886; PMID:21527855; https://doi.org/10.2310/JIM.0b013e31821b8755
- Maguire JL, Russell ML, Smieja M, Walter SD, Loeb M. Low serum 25-hydroxyvitamin D level and risk of upper respiratory tract infection in children and adolescents. Clin Infect Dis 2013: 57(3):392-7; cit289.
- Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, Ida H. Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren. Am J Clin Nutr. 2010; 91(5):1255-60; PMID:20219962; https://doi.org/10.3945/ajcn.2009.29094
- Science M, Maguire JL, Russell ML, Smieja M, Walter SD, Loeb M. Serum 25-hydroxyvitamin D level and influenza vaccine immunogenicity in children and adolescents. PloS one 2014; 9(1):e83553; PMID:24427274; https://doi.org/10.1371/journal.pone.0083553
- Sundaram ME, Talbot HK, Zhu Y, Griffin MR, Spencer S, Shay DK, Coleman LA. Vitamin D is not associated with serologic response to influenza vaccine in adults over 50 years old. Vaccine 2013; 31(16):2057-61; PMID:23453766; https://doi.org/10.1016/j.vaccine.2013.02.028
- Pereira-Santos M, Costa P, Assis A, Santos C, Santos D. Obesity and vitamin D deficiency: a systematic review and meta-analysis. Obes Rev 2015; 16(4):341-349; PMID:25688659; https://doi.org/10.1111/obr.12239
- Brock K, Huang W-Y, Fraser D, Ke L, Tseng M, Stolzenberg-Solomon R, Peters U, Ahn J, Purdue M, Mason RS, et al. Low vitamin D status is associated with physical inactivity, obesity and low vitamin D intake in a large US sample of healthy middle-aged men and women. J Steroid Biochem Mol Biol 2010; 121(1):462-466; PMID:20399270; https://doi.org/10.1016/j.jsbmb.2010.03.091
- Ganji V, Zhang X, Shaikh N, Tangpricha V. Serum 25-hydroxyvitamin D concentrations are associated with prevalence of metabolic syndrome and various cardiometabolic risk factors in US children and adolescents based on assay-adjusted serum 25-hydroxyvitamin D data from NHANES 2001–2006. Am J Clin Nutr 2011; 94(1):225-233; PMID:21613551; https://doi.org/10.3945/ajcn.111.013516
- Gilbert-Diamond D, Baylin A, Mora-Plazas M, Marin C, Arsenault JE, Hughes MD, Willett WC, Villamor E. Vitamin D deficiency and anthropometric indicators of adiposity in school-age children: a prospective study. Am J Clin Nutr 2010; 92(6):1446-1451; PMID:20926524; https://doi.org/10.3945/ajcn.2010.29746
- Karlsson EA, Beck MA. The burden of obesity on infectious disease. Exp Biol Med 2010;235(12):1412-1424; PMID:21127339; https://doi.org/10.1258/ebm.2010.010227
- Sheridan PA, Paich HA, Handy J, Karlsson EA, Hudgens MG, Sammon AB, Holland LA, Weir S, Noah TL, Beck MA. Obesity is associated with impaired immune response to influenza vaccination in humans. Int J Obes 2012; 36(8):1072-1077; PMID:22024641; https://doi.org/10.1038/ijo.2011.208
- Talbot H, Coleman L, Crimin K, Zhu Y, Rock MT, Meece J, Shay DK, Belongia EA, Griffin MR. Association between obesity and vulnerability and serologic response to influenza vaccination in older adults. Vaccine 2012; 30(26):3937-3943; PMID:22484350; https://doi.org/10.1016/j.vaccine.2012.03.071
- Prevention Centers for Disease Control and Prevention. A SAS Program for the 2000 CDC Growth Charts (ages 0 to <20 years). 2016; http://www.cdc.gov/nccdphp/dnpao/growthcharts/resources/sas.htm. Accessed October 14, 2016.
- Network WHOGIS. Serological diagnosis of influenza by haemagglutination inhibition testing. In: Manual for the laboratory diagnosis and virological surveillance of influenza. Geneva, Switzerland: WHO; 2011.
- Ross AC, Manson JE, Abrams SA, Aloia JF, Brannon PM, Clinton SK, Durazo-Arvizu RA, Gallagher JC, Gallo RL, Jones G, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: What clinicians need to know. J Clin Endocrinol Metab 2011; 96(1):53-58; https://doi.org/10.1210/jc.2010-2704
- Moreno LA, Valtueña J, Pérez-López F, González-Gross M. Health effects related to low vitamin D concentrations: beyond bone metabolism. Ann Nutr Metab 2011; 59(1):22-27; PMID:22123633; https://doi.org/10.1159/000332070
- Mansbach JM, Ginde AA, Camargo CA. Serum 25-hydroxyvitamin D levels among US children aged 1 to 11 years: do children need more vitamin D? Pediatrics 2009; 124(5):1404-1410; PMID:19951983; https://doi.org/10.1542/peds.2008-2041
- Prevention Centers for Disease Control and Prevention. Childhood Obesity Facts. 2015; https://www.cdc.gov/obesity/data/childhood.html. Accessed October 14, 2016.
- Zimmerman RK, Lin CJ, Raviotta JM, Nowalk MP. Do Vitamin D levels affect antibody titers produced in response to HPV vaccine? Hum Vaccin Immunother 2015; 11(10):2345-9
- Makariou S, Liberopoulos EN, Elisaf M, Challa A. Novel roles of vitamin D in disease: what is new in 2011? Eur J Inter Med 2011; 22(4):355-362; PMID:21767752; https://doi.org/10.1016/j.ejim.2011.04.012
- Holick MF. Vitamin D deficiency. N Eng J Med 2007; 357(3):266-281; PMID:17634462; https://doi.org/10.1056/NEJMra070553
- Man L, Zhang Z, Zhang M, Zhang Y. Association between vitamin D deficiency and insufficiency and the risk of childhood asthma: evidence from a meta-analysis. Int J Clin Exp Med 2015; 8(4):5699; PMID:26131154
- Tavera-Mendoza LE, White JH. Cell defenses and the sunshine vitamin. Sci Am 2007; 297(5):62-72; https://doi.org/10.1038/scientificamerican1107-62
- Wang T-T, Nestel FP, Bourdeau V, Nagai Y, Wang Q, Liao J, Tavera-Mendoza L, Lin R, Hanrahan JW, Mader S, et al. Cutting edge: 1, 25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immunol 2004; 173(5):2909-2912; PMID:15322146; https://doi.org/10.4049/jimmunol.173.5.2909
- Yim S, Dhawan P, Ragunath C, Christakos S, Diamond G. Induction of cathelicidin in normal and CF bronchial epithelial cells by 1, 25-dihydroxyvitamin D 3. J Cyst Fibros 2007; 6(6):403-410; PMID:17467345; https://doi.org/10.1016/j.jcf.2007.03.003
- Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006; 311(5768):1770-1773; PMID:16497887; https://doi.org/10.1126/science.1123933
- Science M, Maguire JL, Russell ML, Smieja M, Walter SD, Loeb M. Low serum 25-hydroxyvitamin D level and risk of upper respiratory tract infection in children and adolescents. Clin Infect Dis 2013; 57(3):392-397; PMID:23677871; https://doi.org/10.1093/cid/cit289
- Ginde AA, Mansbach JM, Camargo CA. Association between serum 25-hydroxyvitamin D level and upper respiratory tract infection in the Third National Health and Nutrition Examination Survey. Arch Intern Med 2009; 169(4):384-390; PMID:19237723; https://doi.org/10.1001/archinternmed.2008.560
- Chen S, Sims GP, Chen XX, Gu YY, Chen S, Lipsky PE. Modulatory effects of 1, 25-dihydroxyvitamin D3 on human B cell differentiation. J Immunol 2007; 179(3):1634-1647; PMID:17641030; https://doi.org/10.4049/jimmunol.179.3.1634
- Lemire JM, Adams J, Sakai R, Jordan S. 1 alpha, 25-dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells. J Clin Invest 1984; 74(2):657-661; PMID:6611355; https://doi.org/10.1172/JCI111465
- Bhalla AK, Amento E, Serog B, Glimcher L. 1, 25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation. J Immunol 1984; 133(4):1748-1754; PMID:6206136
- Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O'Garra A. 1α, 25-Dihydroxyvitamin D3 has a direct effect on naive CD4+ T cells to enhance the development of Th2 cells. J Immunol 2001; 167(9):4974-4980; PMID:11673504; https://doi.org/10.4049/jimmunol.167.9.4974
- Gregori S, Casorati M, Amuchastegui S, Smiroldo S, Davalli AM, Adorini L. Regulatory T cells induced by 1α, 25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance. J Immunol 2001; 167(4):1945-1953; PMID:11489974; https://doi.org/10.4049/jimmunol.167.4.1945
- Osterholm MT, Kelley NS, Sommer A, Belongia EA. Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. Lancet Infect Dis 2012; 12(1):36-44; PMID:22032844; https://doi.org/10.1016/S1473-3099(11)70295-X
- Zimmerman RK, Nowalk MP, Chung J, Jackson ML, Jackson LA, Petrie JG, Monto AS, McLean HQ, Belongia EA, Gaglani M, et al. 2014–2015 Influenza vaccine effectiveness in the United States by vaccine type. Clin Infect Dis 2016; 63(12):1564-1573.