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Article

Baseline Serum Vitamin A and D Levels Determine Benefit of Oral Vitamin A&D Supplements to Humoral Immune Responses Following Pediatric Influenza Vaccination

by
Nehali Patel
1,
Rhiannon R. Penkert
1,
Bart G. Jones
1,
Robert E. Sealy
1,
Sherri L. Surman
1,
Yilun Sun
2,
Li Tang
2,
Jennifer DeBeauchamp
1,
Ashley Webb
1,
Julie Richardson
3,
Ryan Heine
1,
Ronald H. Dallas
1,
A. Catharine Ross
4,
Richard Webby
1,5 and
Julia L. Hurwitz
1,5,*
1
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
2
Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
3
Department of Pharmaceuticals, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
4
Department of Nutritional Sciences, Pennsylvania State University, University Park, PA 16802, USA
5
Department of Microbiology, Immunology and Biochemistry University of Tennessee Health Science Center, Memphis, TN 38163, USA
*
Author to whom correspondence should be addressed.
Viruses 2019, 11(10), 907; https://doi.org/10.3390/v11100907
Received: 29 June 2019 / Revised: 17 September 2019 / Accepted: 25 September 2019 / Published: 30 September 2019
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
Browse Figures
Versions Notes

Abstract

:
Maximizing vaccine efficacy is critical, but previous research has failed to provide a one-size-fits-all solution. Although vitamin A and vitamin D supplementation studies have been designed to improve vaccine efficacy, experimental results have been inconclusive. Information is urgently needed to explain study discrepancies and to provide guidance for the future use of vitamin supplements at the time of vaccination. We conducted a randomized, blinded, placebo-controlled study of influenza virus vaccination and vitamin supplementation among 2 to 8 (inclusive) year old children over three seasons, including 2015–2016 (n = 9), 2016–2017 (n = 44), and 2017–2018 (n = 26). Baseline measurements of vitamins A and D were obtained from all participants. Measurements were of serum retinol, retinol-binding protein (RBP, a surrogate for retinol), and 25-hydroxyvitamin D (25(OH)D). Participants were stratified into two groups based on high and low incoming levels of RBP. Children received two doses of the seasonal influenza virus vaccine on days 0 and 28, either with an oral vitamin supplement (termed A&D; 20,000 IU retinyl palmitate and 2000 IU cholecalciferol) or a matched placebo. Hemagglutination inhibition (HAI) antibody responses were evaluated toward all four components of the influenza virus vaccines on days 0, 28, and 56. Our primary data were from season 2016–2017, as enrollment was highest in this season and all children exhibited homogeneous and negative HAI responses toward the Phuket vaccine at study entry. Responses among children who entered the study with insufficient or deficient levels of RBP and 25(OH)D benefited from the A&D supplement (p < 0.001 for the day 28 Phuket response), whereas responses among children with replete levels of RBP and 25(OH)D at baseline were unaffected or weakened (p = 0.02 for the day 28 Phuket response). High baseline RBP levels associated with high HAI titers, particularly for children in the placebo group (baseline RBP correlated positively with Phuket HAI titers on day 28, r = 0.6, p = 0.003). In contrast, high baseline 25(OH)D levels associated with weak HAI titers, particularly for children in the A&D group (baseline 25(OH)D correlated negatively with Phuket HAI titers on day 28, r = −0.5, p = 0.02). Overall, our study demonstrates that vitamin A&D supplementation can improve immune responses to vaccines when children are vitamin A and D-insufficient at baseline. Results provide guidance for the appropriate use of vitamins A and D in future clinical vaccine studies.

1. Introduction

Vitamin A and D metabolites function as nuclear hormones and have profound influences on innate and adaptive immune activities [1,2,3,4,5,6]. These micronutrients have each been shown to correlate positively with immune responses in a portion of small animal and clinical studies [1,2,3,7,8,9,10,11,12,13]. Vitamin A, for example, was described by Rahman et al. to improve immune responses toward a diptheria vaccine [14]. Chadha et al. showed that vitamin D correlated positively with immune responses toward influenza virus in a set of cancer patients [15] and others reported that vitamin D could control a variety of infectious pathogens including influenza viruses in humans [9,16,17,18]. Vitamins A and D function in part by binding nuclear hormone receptors. Receptors include the retinoic acid receptor [RAR], the peroxisome proliferator-activated receptor β/δ [PPAR β/δ], and the vitamin D receptor [VDR], each complexed with retinoid X receptor (RXR) as a heterodimer [19,20]. Receptors regulate gene expression by binding nuclear hormone response elements (NHRE) in mammalian DNA [8,21,22,23,24,25,26,27]. Recently, we discovered NHRE within key promoter, enhancer, and switch sites in immunoglobulin gene loci. We found that nuclear hormone receptors could bind these sites, indicating a direct influence of nuclear hormones on antibody expression [28,29,30,31].
Despite the positive influences described above, clinical studies have yielded disparate results [2,3,4,6,7,8,14,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]. When Hanekom et al. tested vitamin A therapy in HIV-infected individuals, they identified no improvements in responses toward influenza virus vaccines [43] and when Lee et al. tested correlations between vitamin D levels and antibody titers after influenza virus vaccinations, significant correlations were not found [49]. An influenza virus vaccine study by Sundaram et al. revealed no consistent association between vitamin D levels and the vaccine-induced antibody response in older adults, although there was a greater frequency of post-vaccination ‘sero-protection’ against H1N1 among vitamin D deficient individuals in the first year of the study [50].
A lack of clarity as to when and how vitamins influence immune responses toward vaccines is concerning because vitamin A and D deficiencies and insufficiencies are prevalent worldwide, affecting both developed and developing countries [11]. Low vitamin levels render individuals vulnerable to infectious diseases [9,13]. Although vitamin supplementation programs are common in developing countries where deficiencies are a known public health concern, they cannot be implemented worldwide without a comprehensive understanding of benefits and risks.
Surprisingly, although there is considerable interest in the independent effects of vitamins A and D on the immune response, the two vitamins are rarely examined together. This is despite the knowledge that vitamins A and D influence numerous cell functions, are closely related, and can be cross-regulated [8,20,24,26,27,51,52,53,54,55,56,57,58]. Unfortunately, when clinical studies are designed to evaluate the influences of vitamin supplements on the immune response, the baseline vitamin A and/or D levels of study participants are often unknown, and the potential consequences of vitamin cross-regulation are rarely considered.
To address knowledge gaps, we designed a pediatric randomized controlled study of influenza virus vaccination that measured baseline vitamin A (using retinol-binding protein [RBP] as a surrogate [59]) and 25(OH)D levels among healthy children. Participants were stratified into two groups based on high and low baseline RBP levels and randomized to receive a vitamin A and D supplement (A&D) or a placebo control immediately prior to influenza virus vaccination. The hemagglutination inhibition (HAI) assay, a standard in the field for the assessment of influenza virus vaccines, was used to measure immune responses [60]. We discovered that baseline vitamin levels were critical parameters that determined not only antibody responses toward the influenza virus vaccine but the influence of vitamin supplementation on the immune response.

2. Materials and Methods

2.1. Clinical Protocol

2.1.1. Enrollment

A randomized, placebo-controlled clinical study was conducted at St. Jude Children’s Research Hospital (St. Jude) in Memphis, TN to examine the influences of baseline vitamin levels and vitamin supplementation on immune responses to the influenza virus vaccine (https://clinicaltrials.gov/ct2/show/NCT02649192). The study was conducted in accordance with the Declaration of Helsinki, and the protocol (FluVIT, Pro00006109, 5 December 2015) was approved by the Institutional Review Board of St. Jude. Healthy children between 2 to 8 (inclusive) years of age were enrolled over three influenza virus seasons including season 2015–2016 (n = 9), season 2016–2017 (n = 44), and season 2017–2018 (n = 26). Informed consent was given by parents or guardians and assent was given by minors when age-appropriate. Comprehensive histories of previous vaccinations and influenza virus exposures were not available. Participants were excluded from study entry if they had any chronic illness, developmental delay, or neurological disorder. They were also excluded if they were known to have received an influenza vaccine for the current season or were routinely taking a daily vitamin supplement.

2.1.2. Randomization and Masking

During the screening visit, sera were collected for measurements of baseline serum RBP (usually present in blood at a 1:1 molar ratio with retinol [59]) for stratification of participants into “high” and “low” groups. Stratification to the “high” (vitamin A sufficient) group was based on a measurement of ≥ 22,000 ng/mL RBP (approximating ≥ 1.05 µM retinol) [59,61,62,63]. Each group was then randomized to receive A&D as an oral gummy vitamin supplement (20,000 IU and 2000 IU per gummy for vitamins A and D, respectively) or a matched gummy that lacked vitamins (placebo). Stratification was performed to prevent an imbalance between placebo and A&D study groups. Gummies were formulated by Regel PharmaLab (Memphis, TN, USA). The components were vitamin A palmitate liquid (#30-3124, PCCA USA, Houston, TX, USA), vitamin D3 liquid (#30-1033, PCCA USA), gelatin base (#30-1520, 830 mg/dose, PCCA USA), tangerine oil flavor (#30-2155, 1µL/dose, PCCA USA), steviol glycosides 95% (8.7 mg/dose, PCCA USA), citric acid USP anhydrous fine granular (12.5 mg/dose, Letco Medical LLC, Decatur, AL, USA), polysorbate 20 NF liquid (2 µL/dose, PCCA USA), and silica gel micronized powder (4 mg/dose, Letco). Gummies were administered to participants on day 0 and day 28 prior to influenza virus vaccination. Participants and healthcare providers were blinded throughout the vaccination and collection procedures.

2.1.3. Vaccine Components

In season 2015–2016, the vaccine was FluMist® (AstraZeneca, Cambridge, UK), whereas in seasons 2016–2017 and 2017–2018, the vaccines were Fluzone® Quadrivalent (Sanofi Pasteur, Lyon, France) for children < 3 years of age and Fluarix® Quadrivalent (GlaxoSmithKline, Brentford, UK) for children of ≥ 3 years of age. These changes were due to revised recommendations by the Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control and Prevention (CDC). Vaccine components changed in each influenza virus season. In 2015–2016, components were A/CA/7/09 H1N1, A/Switzerland/9715293/13 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008. In 2016–2017 components were A/CA/7/09 H1N1, A/Hong Kong/4801/14 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008, and in 2017–2018, components were A/Michigan/45/2015 H1N1, A/Hong Kong/4801/14 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008.

2.1.4. Blood Sample Collection

Blood samples were collected at screening (≤10 days from day 0) and on days 0, 28, and 56. Participants who completed the day 28 visit were considered evaluable.

2.2. RBP, Retinol, and 25(OH)D Measurements

Blood samples were preserved in the cold and dark according to standard guidelines. Levels of RBP were measured with an enzyme-linked immunosorbent assay (ELISA, R&D Systems [Minneapolis, MN, USA] human RBP4 Quantikine kits).
Retinol was measured by extraction from test samples under conditions of UV-blocked lighting with samples of SRM-968f human reference sera (NIST, https://www-s.nist.gov/srmors/orderingSRMs.cfm) used as controls. Samples were diluted with HPLC-grade water and then absolute ethanol and HPLC grade hexane. Tubes were vortexed, incubated for 30 min in the dark, and centrifuged. Hexane layers were removed. A second hexane extraction followed, which was combined with the first, and evaporated to dryness under nitrogen. Methanol:water:acetonitrile (10:20:70 by volume) was added followed by vortexing. Samples were transferred to UPLC vials, briefly centrifuged, and loaded into a Waters Acquity UPLC tray for injection onto a Waters Acquity BEH C-18 reverse phase column using methanol:water:acetonitrile (10:20:70) as the mobile phase. Standards of purified all-trans-retinol (0 to 0.8 pmol/10 µL) provided a standard curve (R2 > 0.99).
25(OH)D was tested in the Pathology Department at St. Jude using the Roche Elecsys Vitamin D ELISA (Roche, Basel, Switzerland) that measures 25(OH)D metabolites of cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). Vitamin D sufficiency was defined as ≥ 30 ng/mL 25(OH)D.

2.3. Hemagglutination Inhibition (HAI) Assay

The HAI assay was conducted to evaluate antibody responses to each of the 4 components in the seasonal influenza virus vaccine. Briefly, antigen (~4 agglutination doses representing each antigen) was added to wells of a 96-well plate containing serial dilutions of test sera (initiated with a 1:10 serum dilution followed by serial 1:2 dilutions, each tested in duplicate). After 30 min incubation at room temperature, 50 microliters of 0.5% vol/vol turkey red blood cells were added to each well and plates were incubated at room temperature for an additional 30 min. Titers were recorded as the highest dilution that inhibited hemagglutination. A few participants did not return for the day 56 visit. A score of 5 (1/2 the lowest dilution tested) was given if no HAI activity was detected. If two different values were observed for duplicate samples, the geometric mean value was used for graphing and calculation purposes.

2.4. Statistical Analyses

Results for each vaccine season were tested independently due to differences in vaccine composition each year. Spearman’s rank correlation was applied to evaluate relationships involving ordinal variables. A generalized estimating equation (GEE) model was used to assess the effect of influenza vaccine plus A&D over time on HAI responses to each antigen among participants with different baseline vitamin levels. Specifically, the GEE model was constructed with log2-transformed HAI titers as the response, age, race, and a three-way interaction among time, baseline vitamin levels, and study groups as covariates, and the first order autoregressive (AR1) as the working correlation structure. Data analyses were performed using SAS 9.4 (SAS Institute, Inc, Cary, NC, USA) and GraphPad Prism software (San Diego, CA, USA).

3. Results

3.1. Participant Characteristics

Age, sex, race, and baseline vitamin levels are shown for all three seasons and all participants in Table 1. As shown, a different vaccine was administered to study participants each year, and FluMist® was only used in season 2015–2016 due to revised recommendations by the CDC. As expected, baseline RBP and retinol levels were positively correlated (Spearman’s correlation r = 0.7, p < 0.0001), emphasizing that RBP can serve as a surrogate for retinol. An additional test of RBP was conducted on day 56 for placebo and A&D groups, but day 56 values were not significantly different from day 0 values in either group. Baseline HAI means and ranges are shown for each season (Table 1). For HAI, data were not combined among years because vaccines and consequent immune responses differed. Full details for all participants and all years are provided in Table A1, Table A2, Table A3, Table A4, Table A5 and Table A6.
Table 2 shows the frequencies of ≥ 4-fold increases in HAI titers by year and influenza virus antigen on days 28 and 56. As shown, the largest participant number was in season 2016–2017 (n = 44). Accordingly, results from this season were used for the primary analyses. For other seasons, descriptive analyses were performed due to limited available data.
For several vaccine components, baseline HAI titers were highly variable among study participants, a situation that may confound comparisons between participants and study groups. Fortuitously, for the B/Phuket/3073/13 vaccine in the 2016–2017 season, when enrollment was highest, baseline titers were all negative (highlighted in Table 1).

3.2. Vitamin Supplements were Beneficial to Children with Insufficient Vitamin A and D Levels at Baseline

To determine how baseline vitamin levels affected immune responses and benefits of vitamin supplementation, we characterized each participant from season 2016–2017 as being sufficient or insufficient/deficient for vitamins A or D at baseline, and accordingly assigned participants to one of four groups (e.g., the “Low A/Low D” group included individuals who were insufficient or deficient for both vitamins A and D). Cut-offs for sufficiency (termed “high”) were ≥ 22,000 ng/mL for RBP and ≥ 30 ng/mL for 25(OH)D. The fold-change of HAI titers on days 28 and 56 compared to baseline were examined for each group (Table 2 and Figure 1.) The percentages of participants with a ≥ 4-fold rise in HAI activity on day 28 or day 56 compared to baseline are shown in Table 2. Of particular interest, we found that children who were insufficient or deficient for both vitamins A and D (Low A/Low D) benefitted significantly from the A&D supplement (Table 2 and Figure 1.).

3.3. Vitamin Supplements were Ineffective or Inhibitory of Vaccine Responses among Children with Sufficient Levels of Vitamins A and/or D

The benefit of A&D supplementation differed for children with sufficient levels of vitamins A and/or D at baseline compared to children who were insufficient or deficient. As shown in Figure 1, A&D benefits were not observed when children exhibited sufficient vitamin levels at baseline. In fact, for some responses, the A&D supplement conferred an inhibitory effect on HAI responses. Results help explain discrepancies in previous clinical studies by showing that the benefit of a vitamin supplement can be determined by the study participant’s baseline vitamin levels.

3.4. Baseline RBP Correlates Positively, while Baseline 25(OH)D Correlates Negatively, with Immune Responses Toward Influenza Vaccine Components.

We next examined how baseline serum vitamin A and D levels correlated with HAI toward the four vaccine components in season 2016–2017 (A/CA/7/09 H1N1, A/Hong Kong/4801/14 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008). Correlations were evaluated between baseline RBP levels and HAI titers (including absolute HAI titers on day 28 and day 56, peak HAI titers, or changes in HAI titers between days 0 and 28 or days 0 and 56). As shown in Figure 2A and 2B, baseline RBP was positively correlated with HAI.
This result was more pronounced in the placebo group (Figure 2A) compared to the A&D group. Surprisingly, the same result was not observed for 25(OH)D. In this case, there were often negative correlations between 25(OH)D and the HAI immune response (i.e., a high 25(OH)D level at study entry associated with a low HAI response).
The negative correlation was more pronounced in the A&D group (Figure 2D) compared to the placebo group. Individual patient data for two of these correlations are shown in Figure 2E–F. As shown, the baseline RBP value was positively and significantly correlated with the day 28 HAI response toward B/Phuket/3073/13 in the placebo group (r = 0.6, p = 0.003, Figure 2E), and the baseline 25(OH)D value was negatively and significantly correlated with the day 28 HAI response toward B/Phuket/3073/13 in the A&D group (r = −0.5, p = 0.02, Figure 2F).

4. Discussion

Our clinical study was designed to investigate how baseline vitamins A and D influence the immune response to influenza virus vaccines. We found that baseline vitamin A levels (scored by RBP) correlated positively with immune responses, particularly toward the B/Phuket/3073/13 vaccine component in the placebo group in season 2016–2017. However, negative correlations were observed between baseline 25(OH)D and the immune response, particularly in the group that received vitamin supplementation. The vitamin A&D supplement significantly improved immune responses toward the B/Phuket/3073/13 vaccine in season 2016–2017 but only when baseline vitamin A and D levels were insufficient at baseline. The supplement had no significant effect or weakened the HAI response when baseline RBP and 25(OH)D levels were sufficient.
Our results were somewhat surprising given that vitamins are often viewed as positively associated with immune responses. Our observation that 25(OH)D correlated negatively with vaccine-induced HAI appeared contrary to the report by Chadha et al. who observed a positive influence of vitamin D on the immune response to influenza virus in cancer patients [15]. Of note, not all researchers have agreed that vitamins are beneficial. Some authors have argued that “too much” vitamin can have a negative influence on the immune response to vaccines [5,34,40,44,45]. Lee et al. (described above), Principi et al. [41], and Kriesel et al. [64] each failed to identify an influence of vitamin D on influenza virus-specific responses. In the latter two studies vitamin D supplements were administered to influenza virus vaccine participants but supplements did not improve vaccine-induced immune responses. A study by Lin et al. [45] showed a negative association between vitamin D levels and responses to type B influenza viruses. Our data help to explain the differing results and interpretations of previous literature by showing that baseline vitamin A and baseline vitamin D levels each affect outcome; we found that the benefit of a vitamin A&D supplement was only evident when children were insufficient or deficient in both RBP and 25(OH)D at study entry.
To explain why 25(OH)D could have a negative influence on immune responses to influenza virus vaccines, we consider the vitamin’s capacity to clear pathogens. Vitamin D upregulates cathelicidin, an anti-microbial peptide [65,66] that can denature and clear both bacteria and viruses, including influenza virus. Perhaps children who were 25(OH)D replete (due to diet and/or the A&D supplement) rapidly cleared vaccine antigens, thus removing the trigger for an antibody response. The influenza virus vaccine is not adjuvanted and may have been particularly vulnerable to cathelicidin-induced damage. A suggestion of rapid vaccine clearance in the presence of high vitamin levels was similarly proposed by Semba et al. when their vitamin A supplementation of 6 month old infants was found to inhibit antibody responses to the measles vaccine [44]. Another explanation for vitamin D inhibition of HAI may relate to the vitamin’s capacity to alter innate and adaptive immune cell trafficking in vivo, and to inhibit B cell proliferation and survival in vitro [1,67]. One final consideration concerns the contrasting influences of vitamins A and D; vitamin D has been positively correlated with serum IgM and IgG3, whereas vitamin A has been positively correlated with other isotypes that might better support HAI [11,68,69,70]. If vitamin D and its receptor blocked the binding of RAR to DNA response elements, vitamin D may have inhibited positive influences of vitamin A [27].
Our study had limitations because more than one factor could have influenced HAI responses and data interpretation. The phenotypes and functions of individual T cell populations, B cell populations and innate immune cells were not analyzed in our study. T cell and antigen presenting cell (APC) populations are particularly important as drivers of B cell activation, proliferation, and antibody expression. Each can be influenced by vitamin levels. In vitamin A deficient mice, for example, CD103 is significantly upregulated on dendritic cell (DC) and T cell membranes [1,12]. CD103 is the αE component of the αEβ7 integrin, which binds the epithelial cell marker E-cadherin. Changes in homing receptor expression affect DC and T cell trafficking/residence and thereby alter antigen presentation and the “help” T cells provide to virus-specific B cells [1,12,71]. The plethora of indirect and direct influences of nuclear hormones on the B cell response (including nuclear hormone receptor binding to enhancers and switch regions of the immunoglobulin heavy chain locus [28,29,58]) may explain some of the complex outcomes of clinical studies. Our primary data were from season 2016–2017 with the highest enrollment; smaller datasets from other seasons were sufficient only for observation and not for confirmatory analyses. We note differences between HAI responses to the four components in each vaccine. For example, among individuals with low baseline RBP and low baseline 25(OH)D levels in the 2016–2017 season, the most significant improvements in vaccine-induced responses in the A&D group compared to the placebo group were toward the B/Phuket/3073/13 vaccine component. In this case, our analyses of vaccine-induced immune responses benefitted from the harmonious and negative HAI responses toward B/Phuket/3073/13 among study participants at baseline; in contrast, study participants exhibited variable and confounding baseline responses toward the other three vaccine components (Table 1). Another limitation of our study was that subclinical or clinical exposures to cross-reactive viral antigens by study participants before or during vaccination may have contributed to outcomes [72,73]. One individual in the placebo group of the 2016–2017 season (ID #58, Table A2) had a confirmed influenza virus infection and one individual in the placebo group of season 2017–2018 (ID #66, Table A3) reported an exposure to family members with confirmed influenza virus infections.
Despite the limitations noted above, our data provide an explanation for conflicting results in previous vitamin supplementation studies. As in our study, responses to vaccine antigens and vitamin supplements in other studies were likely dependent on baseline vitamin levels [40]. Unfortunately, in past studies, baseline levels of vitamin A, vitamin D, or both, were often untested and/or unreported. Our study provides guidance for the suitability of adding vitamin A and D measurements to future clinical vaccine protocols to improve data interpretation and thereby improve vaccine efficacy and infection control in children. Finally, our results support a recommendation to use vitamin A and D supplements with influenza vaccines in areas where children are frequently vitamin deficient or insufficient but not in areas where children are frequently replete for vitamins A and D.

Author Contributions

Conceptualization, J.L.H.; data curation, N.P., R.R.P., B.G.J., R.E.S., S.L.S., J.D., A.W., R.H., and J.L.H.; formal analysis, Y.S. and L.T.; investigation, N.P., R.R.P., B.G.J., R.E.S., S.L.S., Y.S., L.T., J.D., A.W., J.R., R.H., R.H.D., A.C.R., R.W., and J.L.H.; methodology, N.P., R.R.P., B.G.J., R.E.S., S.L.S., Y.S., L.T., J.D., A.W., J.R., R.H., R.H.D., A.C.R., R.W., and J.L.H.; project administration, N.P. and J.L.H.; resources, N.P., R.R.P., B.G.J., R.E.S., L.T., J.D., A.W., J.R., R.H.D., A.C.R., R.W., and J.L.H.; supervision, N.P. and J.L.H.; validation, Y.S. and L.T.; writing—original draft, J.L.H.; writing—review and editing, N.P., R.R.P., B.G.J., R.E.S., S.L.S., Y.S., L.T., J.D., A.W., J.R., R.H., R.H.D., A.C.R., R.W., and J.L.H.

Funding

The study was supported in part by NIH DK41479 (to AC Ross), NIH NCI P30CA21765, and ALSAC. AstraZeneca supported the study by providing FluMist vaccine in 2015.

Acknowledgments

We thank AstraZeneca for provision of the FluMist vaccine in 2015. We thank C-H. (Gina) Wei of Pennsylvania State University for assistance with the retinol measurements. We thank D. Jay of St. Jude’s Department of Pathology for 25(OH)D measurements. We thank S-G. Clyburn of St. Jude’s Department of Infectious Diseases for assistance with regulatory affairs. We thank P. Flynn and A. Gaur of St. Jude’s Department of Infectious Diseases for support in the initiation and implementation of the clinical study.

Conflicts of Interest

J.L.H. reports authorship on a patent for Sendai virus-based vaccine development.

Appendix A

Table
Table A1. Patient Characteristics 2015–2016.
Table A1. Patient Characteristics 2015–2016.
IDSexAgeRaceDateRBP (ng/mL)Retinol (µg/dL)Vitamin D (ng/mL)Assn.
1Female3Black2/11/201627,12326.5841.66A
2Male7Black2/15/201625,60731.3621.14B
3Female2Black2/15/201625,05623.0623.99A
4Female8Black2/16/201620,52330.9814.39B
5Male4Black2/24/201612,97012.1517.76A
6Female6Black2/24/201616,24822.1024.3B
7Female8Black2/24/201624,31437.7717.16B
8Male4Black2/29/201621,66033.5921.52B
9Female5Black2/29/201619,77321.4922.29A
Assignments (Assn.) were A = Placebo, B = Vit A&D.
Table
Table A2. Patient Characteristics 2016–2017.
Table A2. Patient Characteristics 2016–2017.
IDSexAgeRaceDateRBP (ng/mL)Retinol (µg/dL)Vitamin D (ng/mL)Assn.
13Female8White9/26/201626,20831.4840.32B
14Male7Black10/6/201619,96740.6925.67A
15Male2Black10/6/201618,62825.4240.83B
16Male5Black10/14/201629,27040.7420.46B
17Male7Black10/14/201622,03942.7217.83A
19Male3Black10/18/201614,71717.4817.52A
20Female5Black10/21/201628,40833.5519.94B
21Female2Black10/21/201622,69133.4124.42A
22Male2White10/31/201622,77332.6332.1A
23Male4Black11/1/201625,28438.8738.07B
24Female4Black11/1/201621,51134.7532.94A
25Female8Black11/4/201628,50941.7326.36A
26Female8Black11/4/201627,40737.2831.06A
27Male3Black11/2/201621,07734.7243.38B
28Female8White11/10/201631,60245.5633.12B
29Female5White11/10/201628,46236.7135.72B
30Female8White11/18/201616,26237.4035.09B
31Female6White11/18/201618,03334.6933.81B
32Female7White11/18/201616,51327.4730.6B
33Male2White11/18/201620,78932.8133.9A
34Female3White11/22/201619,76330.2547.91A
35Male7Black11/29/201621,60437.0123.56B
36Female4Black12/5/201622,32233.5518.34A
38Female2Black12/13/20169,56318.0614.29B
39Male3Black12/13/201618,42337.1038.66A
40Female5Black12/13/201620,55443.1341.98A
41Female2Black12/29/201615,62029.5218.44A
42Female8Black12/29/201623,45331.0621.21A
45Female3Black1/5/201728,08854.8727.05B
46Female4Black1/5/201718,41630.0023.72A
47Female3Black1/13/201723,04434.9230.79B
48Female8Black1/19/201727,11339.9419.17B
49Female2Black1/20/201718,94626.1520.18B
50Male6Black1/20/201721,19634.1616.4B
51Male4Black1/24/201723,40236.7420.44A
53Female5Black1/26/201724,51835.9221.74A
54Female6Black1/26/201722,32732.2827.14A
55Female6Black1/27/201714,55119.5723.7B
57Male7White2/9/201732,56355.8131.88A
58Female8White2/20/201719,93429.5631.52A
60Female6Black3/3/201721,40833.0115.95B
61Female4White3/7/201722,00731.9035.13A
62Male5White3/7/201724,65030.9532.71B
64Male2Black3/31/201728,60236.9042.95B
Assignments (Assn.) were A = Placebo, B = Vit A&D.
Table
Table A3. Patient Characteristics 2017–2018.
Table A3. Patient Characteristics 2017–2018.
IDSexAgeRaceDateRBP (ng/mL)Retinol (µg/dL)Vitamin D (ng/mL)Assn.
66Male3White9/18/201726,71038.1133.6B
67Female8Black9/20/201730,55543.6531.28B
68Female8White10/13/201725,71137.7043.25A
70Male2White10/20/201722,60034.3039.52B
71Male8White10/20/201721,55643.4130.74A
72Male2White10/20/201730,10936.3834.36A
73Male2White10/20/201729,91741.6353.05A
74Female2White10/30/201717,97326.5334.74B
75Female2Black11/2/201722,49933.3417.04B
78Female2Black11/15/201720,17828.5356.76A
79Male6Black11/16/201720,11334.3326.05A
80Female8Black11/17/201716,13725.5113.68B
81Female2Black11/17/201720,11229.3823.76A
83Male2Black12/5/201735,50147.4431.49B
84Female8Black12/8/201715,69221.5323.13A
85Male3Black12/12/201718,80926.2728.4B
86Male8Black12/12/201731,92044.3818.75A
87Male8Black1/9/201837,53951.8835.06B
88Male7Black1/9/201823,07430.4937.53A
89Female7White2/19/201819,05527.3025.16B
90Female7Black2/20/201826,64338.9326.8A
92Female8Black3/13/201830,63636.4210.06B
94Male2Black3/28/201813,34818.4920.32B
95Female4Black3/26/201826,15729.9412.97A
96Male2Black4/5/201820,86628.0253.52A
97Male2White4/23/201825,63836.5239.14A
Assignments (Assn.) were A = Placebo, B = Vit A&D.
Table
Table A4. HAI results 2015–2016.
Table A4. HAI results 2015–2016.
A.Day 0Day 28Day 56
IDA/CA/7/09 H1N1A/Switz/9715293/13 H3N2B/Phuket/3073/13B/Brisbane/60/2008A/CA/7/09 H1N1A/Switz/9715293/13 H3N2B/Phuket/3073/13B/Brisbane/60/2008A/CA/7/09 H1N1A/Switz/9715293/13 H3N2B/Phuket/3073/13B/Brisbane/60/2008
1<10<10<10<10<10320/640<10<10<10160<10<10
2<1064040<10<10320160<10<1032080<10
3<10<10<10<10<10160<10<10<101280<10<10
4160<10<10<10160<10<10<10320<10<10<10
520160<10<10<10320<10<10<10320<10<10
6<10<10<10<10<101280<10<10<101280<10<10
740160/80<10<10<10<10<10<10<1080<10<10
8<10<10<10<10<10640<10<10<10320<10<10
940320/640<10<1040320<10<10<10640<10<10
Table
Table A5. HAI results Season 2016–2017.
Table A5. HAI results Season 2016–2017.
IDDay 0Day 28Day 56
A/CA/7/09 H1N1A/Hong Kong/4801/14 H3N2B/Phuket/3073/13B/Brisbane/60/2008A/CA/7/09 H1N1A/Hong Kong/4801/14 H3N2B/Phuket/3073/13B/Brisbane/60/2008A/CA/7/09 H1N1A/Hong Kong/4801/14 H3N2B/Phuket/3073/13B/Brisbane/60/2008
13<10640<10<10640204801064064020480<10160
1440<10<10<10320640<10<10640640<10<10
15<10<10<10<10<10<10<10<108040<10<10
16320/640320<10<10640512080801280256080<10
1732040<10<10640640808012803204020
19<10320<10<10<102560/5120<10320N/SN/SN/SN/S
204080<10<106405120408064051204040
2110<10<10<101601280404032012804040
2210<10<10<1012801280/25608032025602560160320
232080<10<10320320403201603204080/160
24<10<10<10<101280320403206401601080/160
2510<10<10<1016016080160320160160160
26<10320<10<103201024040203205120<10<10
27320/640<10<10<10640160<1020N/SN/SN/SN/S
28<10160<10<10640320<10<10160320<10<10
29<10<10<10<1012801280<10<106401280<10<10
30<102560<10<10<10512080<10<102560<10<10
31<10650<10<10<10640<10<10<102560/1280<10<10
32<10<10<10<10320640<10<10640640<10<10
33<10<10<10<10320640/12802040640640<10<10
34<10<10<10<10<10<10<10<10160<10<10<10
35160160<10<10640256080<1012802560<10<10
36<10320<10<10<102560<10801602560/1280<10<10
38<10<10<10<10640640/1280808012801280160<10
39160160<10<106402560801606401280/256080160
4040160<10<103201601604064016080<10
41320<10<10<102560<10<10<102560160<10<10
42<10160<10<10256012803206402560640/1280320320
456402560<10160640160/2560<1016012801280/256080160
466401280<10<1012802560<103206402560<10160
47<10640<10<10<1010240<10<108010240<10<10
48160320<10<101280256016012801280512080640
49320<10<10<1064064080<10320640<10<10
503201280<10<10128025608080/1606401280<1080
51320160<10<106402560<1080128025608080/160
5380<10<10801280160160<10N/SN/SN/SN/S
5432080<10<10128012801603202560128080160
55<10<10<10<103201280<10<106402560<10<10
57<10160<10<1012801280/256032080640128016040
58<10160<10<10320320<10320/64032064080320
60320640<10<1025605120<102560/512025602560<102560
61<101280<10<101601280<10<101601280<10<10
62<10320<10<101280320<1020640160<10<10
64<10<10<10<1016080<10<10160160<10<10
N/S = no sample.
Table
Table A6. HAI results 2017–2018.
Table A6. HAI results 2017–2018.
IDDay 0Day 28Day 56
A/Michigan/45/2015 H1N1A/Hong Kong/4801/14 H3N2B/Phuket/3073/13B/Brisbane/60/2008A/Michigan/45/2015 H1N1A/Hong Kong/4801/14 H3N2B/Phuket/3073/13B/Brisbane/60/2008A/Michigan/45/2015 H1N1A/Hong Kong/4801/14 H3N2B/Phuket/3073/13B/Brisbane/60/2008
66640160<10<102560160<10<105120320<10<10
67<10320<1016025601280160<1025602560<10320/640
68<10<10<10<10402560<1032025601280<10640
70<1080<10<101280640<10<1040640<10<10
71160320<10<10160640<10801280640<10<10
72<10<10<10<1016010<10<10320<10<10<10
73<10<10<10<101010<10<108040<10<10
74<10<10<10<1032080<10<106408080<10
75<10<10<10<1016080<10320320160<10320
78<10320<10<101605120<10<1012805120<10<10
79320320<10<102560512080<102560256080<10
801280640<1016025601280<10160/320640128080160/320
81320<10<10<10640320<10<10128064080/16080
833201601040640102401603201280256080/160320
8412801280801605120512080/16080/16025601280160320
8532080<10<10128051204080640640<10<10
8612801280<10<1051205120<10<10102402560/5120<1080
871601280<10<1012805120<10<10640/1280256080<10
881601280160806406403201606401280160160
895120<10<10<102560320<10<102560/5120160<10<10
9080<10<10<105120320<101605120320160160
92160640<10<1051202560<101605120256040/8080
94320<10<10<10640160<10<10N/SN/SN/SN/S
951280320<10<10640640<10<106403208080
96320/640320<10<10512010240<10<1025605120<10<10
97<10<1080<1016016080<10160<10<10<10
N/S = no sample.

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Figure 1. Estimated fold change in hemagglutination inhibition (HAI) responses compared between placebo (black) and vitamin A and D supplement (A&D, red) study groups at different time points of the study in season 2016–2017. Participants were placed into one of four groups based on vitamin levels. Cut-offs for sufficiency (termed “high”) were ≥ 22,000 ng/mL for retinol-binding protein (RBP) and ≥ 30 ng/mL for 25(OH)D. Groups were “Low A/Low D”, n = 10; “High A/Low D”, n = 12; “Low A/High D,” n = 11; “High A/High D,” n = 11 (see Table 2). For each of the four viruses, the GEE model was constructed with log2-transformed HAI titers as the response, age, race, and a three-way interaction among time, incoming vitamin levels, and study groups as covariates, and the first order autoregressive (AR1) as the working correlation structure. Estimated mean fold changes are plotted with 90% confidence intervals. p-values were obtained from post hoc comparisons from GEE models (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 1. Estimated fold change in hemagglutination inhibition (HAI) responses compared between placebo (black) and vitamin A and D supplement (A&D, red) study groups at different time points of the study in season 2016–2017. Participants were placed into one of four groups based on vitamin levels. Cut-offs for sufficiency (termed “high”) were ≥ 22,000 ng/mL for retinol-binding protein (RBP) and ≥ 30 ng/mL for 25(OH)D. Groups were “Low A/Low D”, n = 10; “High A/Low D”, n = 12; “Low A/High D,” n = 11; “High A/High D,” n = 11 (see Table 2). For each of the four viruses, the GEE model was constructed with log2-transformed HAI titers as the response, age, race, and a three-way interaction among time, incoming vitamin levels, and study groups as covariates, and the first order autoregressive (AR1) as the working correlation structure. Estimated mean fold changes are plotted with 90% confidence intervals. p-values were obtained from post hoc comparisons from GEE models (* p < 0.05, ** p < 0.01, *** p < 0.001).
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Figure 2. Relationships between baseline vitamin levels and HAI responses in season 2016–2017. Spearman correlation coefficients are plotted for comparisons between baseline vitamin levels and HAI titers among participants enrolled during the 2016–2017 season. R values are plotted on the Y axis in A–D. Positive correlations are indicated in green and negative correlations are indicated in red. The top row shows RBP correlations with HAI titers in placebo (A) and A&D (B) groups. Specifically, correlations are shown between baseline RBP and day 0 HAI, baseline RBP and day 28 HAI, baseline RBP and day 56 HAI, baseline RBP and peak HAI, baseline RBP and changes in HAI between days 0 and 28, and baseline RBP and changes in HAI between days 0 and 56. * p < 0.05; ** p < 0.01; *** p < 0.001. The middle row substitutes 25(OH)D for RBP in placebo (C) and A&D (D) groups. In the bottom row, detailed correlative data are shown for RBP versus B/Phuket/3073/13 HAI titers on day 28 in the placebo group (n = 22, some values overlap) (E), and 25(OH)D versus B/Phuket/3073/13 HAI titers on day 28 in the A&D group (n = 22, some values overlap) (F).
Figure 2. Relationships between baseline vitamin levels and HAI responses in season 2016–2017. Spearman correlation coefficients are plotted for comparisons between baseline vitamin levels and HAI titers among participants enrolled during the 2016–2017 season. R values are plotted on the Y axis in A–D. Positive correlations are indicated in green and negative correlations are indicated in red. The top row shows RBP correlations with HAI titers in placebo (A) and A&D (B) groups. Specifically, correlations are shown between baseline RBP and day 0 HAI, baseline RBP and day 28 HAI, baseline RBP and day 56 HAI, baseline RBP and peak HAI, baseline RBP and changes in HAI between days 0 and 28, and baseline RBP and changes in HAI between days 0 and 56. * p < 0.05; ** p < 0.01; *** p < 0.001. The middle row substitutes 25(OH)D for RBP in placebo (C) and A&D (D) groups. In the bottom row, detailed correlative data are shown for RBP versus B/Phuket/3073/13 HAI titers on day 28 in the placebo group (n = 22, some values overlap) (E), and 25(OH)D versus B/Phuket/3073/13 HAI titers on day 28 in the A&D group (n = 22, some values overlap) (F).
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Table
Table 1. Baseline patient characteristics.
Table 1. Baseline patient characteristics.
CharacteristicsAll Seasons
FluMist, Fluzone, or Fluarix		2015–2016 Season
FluMist		2016–2017 Season
Fluzone or Fluarix		2017–2018 Season
Fluzone or Fluarix
Placebo (n = 40)Vitamin A&D (n = 39)Placebo
(n = 4)		Vitamin A&D (n = 5)Placebo
(n = 22)		Vitamin A&D (n = 22)Placebo
(n = 14)		Vitamin A&D (n = 12)
Age groups, n (%)
2–4 years22 (55 %)16 (41%)3 (75%)1 (20%)12 (54.5%)8 (36%)7 (50%)7 (58%)
5–8 years18 (45 %)23 (59%)1 (25%)4 (80%)10 (45.5%)14 (64%)7 (50%)5 (42%)
Sex, n (%)
Female23 (57.5%)23 (59%)3 (75%)3 (60%)14 (64%)14 (64%)6 (43%)6 (50%)
Male17 (42.5%)16 (41%)1 (25%)2 (40%)8 (36%)8 (36%)8 (57%)6 (50%)
Race, n (%)
White11 (27.5%)11 (28%)0 (0%)0 (0%)6 (27%)7 (32%)5 (36%)4 (33%)
Black29 (72.5%)28 (72%)4 (100%)5 (100%)16 (73%)15 (68%)9 (64%)8 (67%)
RBP, n (%)
<22,000 ng/mL18 (45%)19 (49%)2 (50%)3 (60%)10 (45.5%)11 (50%)6 (43%)5 (42%)
≥22,000 ng/mL22 (55%)20 (51%)2 (50%)2 (40%)12 (54.5%)11 (50%)8 (57%)7 (58%)
Vitamin D, n (%)
<30 ng/mL21 (52.5%)21 (54%)3 (75%)5 (100%)12 (54.5%)10 (45.5%)6 (43%)6 (50%)
≥30 ng/mL19 (47.5%)18 (46%)1 (25%)0 (0%)10 (45.5%)12 (54.5%)8 (57%)6 (50%)
Retinol, n (%)
<20 µg/dL2 (5%)3 (7.7%)1 (25%)0 (0%)1 (4.6%)2 (9%)0 (0%)1 (8.3%)
20–30 µg/dL11 (27.5%)8 (20.5%)3 (75%)1 (20%)3 (13.6%)3 (14%)5 (36%)4 (33.3%)
>30 µg/dL27 (67.5%)28 (71.8%)0 (0%)4 (80%)18 (81.8%)17 (77%)9 (64%)7 (58.3%)
Baseline HAI titer (log2), median (min, max)
B/Phuket---#---2.32 (2.32, 2.32)2.32 (2.32, 5.32)2.32 (2.32, 2.32)2.32 (2.32, 2.32)2.32 (2.32, 7.32)2.32 (2.32, 3.32)
B/Brisbane------2.32 (2.32, 2.32)2.32 (2.32, 2.32)2.32 (2.32, 6.32)2.32 (2.32, 7.32)2.32 (2.32, 7.32)2.32 (2.32, 7.32)
H1N1 *------3.32 (2.32, 5.32)2.32 (2.32, 7.32)3.32 (2.32, 9.32)2.32 (2.32, 9.32)7.32 (2.32, 10.32)7.82 (2.32, 12.32)
H3N2 *------4.82 (2.32, 8.82)2.32 (2.32, 9.32)6.82 (2.32, 10.32)7.32 (2.32, 11.32)8.32 (2.32, 10.32)6.82 (2.32, 10.32)
The numbers and characteristics of study participants in all seasons and in each of the three seasons are shown, with levels for RBP, vitamin D (25(OH)D), and retinol. HAI titers are shown for each season. #HAI titers were not combined among seasons because the vaccine changed each year. In 2015–2016, vaccine components were A/CA/7/09 H1N1, A/Switzerland/9715293/13 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008. In 2016–2017 components were A/CA/7/09 H1N1, A/Hong Kong/4801/14 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008, and in 2017–2018 components were A/Michigan/45/2015 H1N1, A/Hong Kong/4801/14 H3N2, B/Phuket/3073/13, and B/Brisbane/60/2008. When an HAI value was below detection, the sample was given a value of 5 (log2 2.32). Tests were conducted in duplicate and geometric means were determined. The negative baseline responses toward B/Phuket/3073/13 among all participants in season 2016–2017 are highlighted. For conversion, 30 µg/dL retinol = 1.05 µmol/L; 20 ng/mL 25(OH)D = 50 nmol/L. * H1N1 and H3N2 components differed between seasons.
Table
Table 2. HAI response (≥4 fold change) after the first or second vaccine doses.
Table 2. HAI response (≥4 fold change) after the first or second vaccine doses.
B/Phuket/3073/13B/Brisbane/60/2008H1N1*H3N2*
PlaceboA&DPlaceboA&DPlaceboA&DPlaceboA&D
HAI Response(≥4 Fold Change) after the 1st Dose (Day 28 HAI Titer Compared to Day 0)
2015–2016 (n = 9)0/4 (0%)1/5 (20%)0/4 (0%)0/5 (0%)0/4 (0%)0/5 (0%)2/4 (50%)2/5 (40%)
2016–2017 (n = 44)13/22 (59%)9/22 (41%)17/22 (77%)10/22 (45%)16/22 (73%)14/22 (64%)16/22 (73%)15/22 (68%)
2017–2018 (n = 26)1/14 (7%)3/12 (25%)3/14 (21%)4/12 (33%)10/14 (71%)8/12 (67%)9/14 (64%)10/12 (83%)
2016–2017 (n = 44)
Low A and Low D (n = 10)0/4 (0%)4/6 (67%)2/4 (50%)3/6 (50%)2/4 (50%)5/6 (83%)2/4 (50%)5/6 (83%)
High A and Low D (n = 12)6/8 (75%)3/4 (75%)7/8 (88%)3/4 (75%)5/8 (63%)2/4 (50%)8/8 (100%)3/4 (75%)
Low A and High D (n = 11)4/6 (67%)1/5 (20%)5/6 (83%)1/5 (20%)5/6 (83%)1/5 (20%)3/6 (50%)2/5 (40%)
High A and High D (n = 11)3/4 (75%)1/7 (14%)3/4 (75%)3/7 (43%)4/4 (100%)6/7 (86%)3/4 (75%)5/7 (71%)
HAI Response (≥4 Fold Change) after the 2nd Dose (Day 56 HAI Titer Compared to Day 0)
2015–2016 (n = 9)0/4 (0%)0/5 (0%)0/4 (0%)0/5 (0%)0/4 (0%)0/5 (0%)2/4 (50%)2/5 (40%)
2016–2017 (n = 41)11/20 (55%)6/21 (29%)12/20 (60%)6/21 (29%)19/20 (95%)15/21 (71%)16/20 (80%)15/21 (71%)
2017–2018 (n = 25)4/14 (29%)5/11 (45%)5/14 (36%)3/11 (27%)12/14 (86%)8/11 (73%)7/14 (50%)8/11 (73%)
2016–2017 (n = 41)
Low A and Low D (n = 9)0/3 (0%)1/6 (17%)1/3 (33%)2/6 (33%)2/3 (67%)4/6 (67%)2/3 (67%)5/6 (83%)
High A and Low D (n = 11)6/7 (86%)4/4 (100%)6/7 (86%)2/4 (50%)7/7 (100%)2/4 (50%)7/7 (100%)3/4 (75%)
Low A and High D (n = 10)3/6 (50%)0/4 (0%)3/6 (50%)0/4 (0%)6/6 (100%)2/4 (50%)4/6 (67%)2/4 (50%)
High A and High D (n = 11)2/4 (50%)1/7 (14%)2/4 (50%)2/7 (29%)4/4 (100%)7/7 (100%)3/4 (75%)5/7 (71%)
The vitamin group with the greater frequency of responses toward each vaccine component in season 2016–2017 is highlighted. *H1N1 and H3N2 changed between years. A few participants did not return for the day 56 visit.

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Patel, N.; Penkert, R.R.; Jones, B.G.; Sealy, R.E.; Surman, S.L.; Sun, Y.; Tang, L.; DeBeauchamp, J.; Webb, A.; Richardson, J.; Heine, R.; Dallas, R.H.; Ross, A.C.; Webby, R.; Hurwitz, J.L. Baseline Serum Vitamin A and D Levels Determine Benefit of Oral Vitamin A&D Supplements to Humoral Immune Responses Following Pediatric Influenza Vaccination. Viruses 2019, 11, 907. https://doi.org/10.3390/v11100907

AMA Style

Patel N, Penkert RR, Jones BG, Sealy RE, Surman SL, Sun Y, Tang L, DeBeauchamp J, Webb A, Richardson J, Heine R, Dallas RH, Ross AC, Webby R, Hurwitz JL. Baseline Serum Vitamin A and D Levels Determine Benefit of Oral Vitamin A&D Supplements to Humoral Immune Responses Following Pediatric Influenza Vaccination. Viruses. 2019; 11(10):907. https://doi.org/10.3390/v11100907

Chicago/Turabian Style

Patel, Nehali, Rhiannon R. Penkert, Bart G. Jones, Robert E. Sealy, Sherri L. Surman, Yilun Sun, Li Tang, Jennifer DeBeauchamp, Ashley Webb, Julie Richardson, Ryan Heine, Ronald H. Dallas, A. Catharine Ross, Richard Webby, and Julia L. Hurwitz. 2019. "Baseline Serum Vitamin A and D Levels Determine Benefit of Oral Vitamin A&D Supplements to Humoral Immune Responses Following Pediatric Influenza Vaccination" Viruses 11, no. 10: 907. https://doi.org/10.3390/v11100907

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