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Does Vitamin D Deficiency Affect the Immunogenic Responses to Influenza Vaccination? A Systematic Review and Meta-Analysis

Influenza virus infection is a major global public health problem, and the efficacy of influenza vaccination is not satisfactory. Vitamin D is involved in many immune-mediated inflammatory processes. The impact of vitamin D levels on the immunogenic response to influenza vaccination is not clear. We performed a comprehensive literature search and systematic review of studies that investigated vitamin D and influenza vaccination. Data pertaining to study population, vaccine components, vitamin D levels, and immunogenic response were analyzed. Nine studies, with a combined study population of 2367 patients, were included in the systematic review. Four studies were included in the meta-analysis to investigate the influence of vitamin D deficiency (VDD) on the seroprotection (SP) rates and seroconversion (SC) rates following influenza vaccination. We found no significant association between vitamin D level and the immunogenic response to influenza vaccination. However, strain-specific differences may exist. We observed lower SP rates of influenza A virus subtype H3N2 (A/H3N2) and B strain in VDD patients than patients with normal vitamin D levels (A/H3N2: 71.8% vs. 80.1%, odds ratio (OR): 0.63, 95% confidence interval (CI): 0.43–0.91, p = 0.01; B strain: 69.6% vs. 76.4%, OR: 0.68, 95% CI: 0.5–0.93, p = 0.01). However, the SP rates of A/H1N1 and SC rates of all three strains were not significantly different in VDD and control groups. In conclusion, no association was observed between VDD and immunogenic response to influenza vaccination.


Introduction
Influenza is an important infectious disease and a major contributor to the global disease burden. It is estimated to be responsible for 250,000-500,000 deaths annually, especially among the December 2017 were eligible for inclusion. The Cochrane Collaboration Central Register of Controlled Clinical Trials, Cochrane Systematic Reviews, and ClinicalTrials.gov databases were searched manually for additional references. Two authors (M.-D.L. and C.-Y.L.) conducted the search independently, while disagreements were resolved through discussion with the third author (C.-H.L.).
Two independent reviewers (M.-D.L. and C.-Y.L.) assessed the eligibility of each paper after the initial search was conducted. The inclusion criteria were (1) inclusion of a controlled group in the study design; (2) administration of influenza vaccination; (3) measurement or supplementation of vitamin D; (4) reporting of at least one immunological response to influenza vaccination. The following criteria were excluded: (1) articles irrelevant to the topic; (2) duplicate publications; (3) trials with a crossover study design; (4) animal studies; and (5) case reports and studies which excluded a controlled group.

Data Extraction and Quality Assessment
Two authors (M.-D.L. and C.-Y.L.) used the Cochrane Review risk of bias assessment tool independently to evaluate the quality of all eligible articles. Each publication was assessed with its adequacy of randomization, allocation concealment, blinding methods, implementation of the intent-to-treat analysis, dropout rate, complete outcomes data, selective data reporting, and other potential biases.
The articles were scrutinized, and the data pertaining to the following variables were extracted: study population; influenza vaccine components; vitamin D levels; supplementation of vitamin D; details of vaccine immune responses; and adverse effects. Discrepancies between the two independent evaluations for potential articles were determined through discussion and consensus. The primary outcome was seroprotection (SP) of influenza vaccination, which was defined as antibody titer after vaccination ≥40 as measured by micro-neutralization. The secondary outcome was seroconversion (SC), which was defined as a fourfold increase in antibody titer after influenza vaccination. The SP and SC were assessed based on the hemagglutination inhibition (HI) antibody titers. The HI antibody titer that equals the maximum dilution capable of inhibiting the agglutination of guinea pig red blood cells was with the influenza viruses under standardized conditions [29]. Other comparative variables contained the components of the vaccine, the prevalence of VDD, vitamin D supplementation, and severe adverse effects.

Data Synthesis and Analysis
In order to determine differences in the efficacy of influenza vaccination in VDD groups and control groups, the authors extracted, analyzed and compared the immunogenicity data from all the studies. A random effects model was employed due to the significant (and expected) heterogeneity among the studies [30]. The outcomes are presented as point estimates with 95% confidence intervals (CIs). I-square and Cochran's Q tests were used to test the heterogeneity across studies. A p-value < 0.10 for chi-squared test of the Q statistic or an I-square > 50% was considered indicative of statistically significant heterogeneity [31]. To observe the effect on the overall results, a sensitivity analysis was performed by repeating the analysis after sequential exclusion of one study at a time. Potential publication bias was assessed through the observation of the symmetry of funnel plots and the use of Egger's test [32]. Review Manager (version 5.3.5, Cochrane Community: Lisbon, Portugal, available online: http://community.cochrane.org/tools/review-production-tools/revman-5/revman-5-download) was used for our analyses.

Description of Studies and Quality Assessment
The flowchart schematic illustration of the literature search and study-selection criteria is presented in Figure 1. Nine publications were included in our qualitative synthesis and critical review after the examination. (Table 1) [33][34][35][36][37][38][39][40][41]. Two trials conducted during different influenza seasons with different patient numbers, vaccine components, and years of study were published in the same article and regarded as two independent studies [37]. Consequently, nine publications compromising ten studies were enrolled in our systematic review. Most studies (7/10) were conducted in the USA, among which three studies were performed in children. Two studies involved (human immunodeficiency virus) HIV-infected individuals, and one study investigated influenza vaccination in patients with prostate cancer [35,39,40]. Trivalent inactivated influenza vaccine (TIV) was administered in most studies (9/10). In total, 2367 patients were enrolled in these studies; the female-to-male ratio was 1:1.14. Vitamin D levels were measured in eight studies, and the percentage of patients with VDD varied widely between different studies (0.9-45%). The cutoff values used to determine VDD were 20 ng/mL in three studies and 25 ng/mL in the other three studies. Vitamin D supplementation was performed in four studies [38,39,41].
The Cochrane assessment tool shows that most of the included studies had a low potential for bias. The detailed quality assessment of each included study is presented in Table S2.

Data Synthesis and Meta-Analysis
Based on the basis of currently available evidence, no association was found between VDD and the immunogenic response to influenza vaccination (Table 1). Positive association was found in two studies, and negative association was found in two studies. VDD was not associated with the immunogenic response in the other five studies. Data pertaining to immunogenic response including SP and SC following influenza vaccination were extracted for further meta-analysis. Ultimately, four studies with a combined study population of 1517 patients were included in our meta-analysis [35][36][37][38]. Data pertaining to vitamin D levels were unavailable for these studies, and therefore these studies were not eligible for the meta-analysis [34,[39][40][41]. High baseline SP rate (75%) of enrolled individuals was reported in one study [34]. One study was excluded in the meta-analysis because of different outcome parameters [33]. In Crum-Cianflone's study, the study population was classified into two groups (HIV-infected and HIV-uninfected), and the groups were compared separately [35]. Sundaram's study was conducted in two different seasons with different influenza vaccines and was regarded as different studies [37]. Finally, four publications with six studies were enrolled in the final meta-analysis.
No serious adverse effects were reported. The funnel plots were also assessed.

Discussion
Our systematic review found no association between VDD and the immunogenic response to influenza vaccination based on the currently available evidence. However, strain-specific differences may exist, and decreased SP rates of A/H3N2 and B strain were observed in patients with VDD (6.8-8.3% difference in SP rates with low heterogeneity). VDD may decrease the immunogenic response to influenza vaccination, and correction of VDD may potentially improve the protective efficacy of influenza vaccination. Further studies are warranted to elucidate the influence of VDD on individual vaccine strains and the impact of vitamin D supplementation.
Influenza is a highly contagious disease characterized by widespread antigenic variations. Although the influenza vaccination is widely used nowadays, it remains an important health threat worldwide. The effectiveness of current influenza vaccination is not adequate, and protection rates in elderly people are probably as low as 30% [2,42,43]. Multiple factors are responsible for the low protective efficacy; these include antigen drift, seasonal mismatch, and manufacture technique limitations [2,44,45]. Immunosenescence, gradual deterioration of the immune system brought on by natural aging, poor nutritional status, and higher rates of comorbid diseases also play important roles in the hyporesponsiveness of influenza vaccination, especially in the elderly [46]. Efforts have been made to improve the immune response to influenza vaccination. One commonly used strategy involves the use of adjuvants [47]. Additionally, TIV with high doses (four times the standard dose) and intradermal injection were shown to induce significantly higher antibody response; however, these strategies are not widely used [3]. Nutritional interventions (e.g., use of probiotics) may provide a simple, convenient and practical solution [48,49]. VDD may affect the immune response to influenza vaccination, and correction of VDD may help enhance the humoral response. Our results suggest that vitamin D levels do not affect the immunogenicity of influenza vaccination and routine vitamin D supplementation is not recommended. However, VDD may attenuate the immunogenic response to specific strains, such as strain A/H3N2 and strain B. Compared with strains A/H1N1 and A/H3N2, the immunogenic response to strain B was shown to be poorer in previous reports [5,6]. The immunogenic responses were inconsistent between different vaccine strains and variations of pre-vaccinated antibody levels may contribute to the observed differences. Sometimes, one of the vaccine strains may be included in the following years, therefore booster effects may interfere the immunogenic responses to the same strain. For example, A/California/7/2009 (H1N1) has been included in the suggested vaccine component every year between 2010-2017 but the accompanying strains A/H3N2 and B have changed every year. Differences in immunogenic response to vaccination with different component strains of influenza were found; however, the role of strain difference remains unclear. Our systematic review showed that strain A/H3N2 had the highest SP and SC rates ( Table 2). Further studies are warranted to clarify the influence of vitamin D on the individual strain components.
In addition to its important role in bone metabolism, the anti-inflammatory properties of vitamin D have been investigated in the last decade [7,8]. Vitamin D receptors are present in many systems, and vitamin D was found to play important roles in various diseases. Vitamin D is not only a steroid hormone but also an important immune modulator. Patients with VDD were shown to have a higher risk of several cancers [17,19]. VDD is also prevalent in patients with critical illnesses and may contribute to death because of cardiovascular disease, cancer and other causes [9,12,16,50]. Supplementation of vitamin D may help prevent these diseases or improve treatment outcomes. Increased risk of infection with influenza virus in patients with VDD has been observed as well [20]. However, the underlying mechanisms are not well characterized. Both VDD and influenza infection are more common in cold seasons. Lower solar radiation may contribute to VDD and affect the susceptibility to influenza infection. Vitamin D may also alter the immune response to influenza infection. Additionally, VDD may affect the effectiveness of influenza vaccination and increase the risk of influenza infection. Our systematic review found no obvious association between VDD and immunogenicity of influenza vaccine. Concomitant supplementation of vitamin D during influenza vaccination is not needed from this viewpoint.
Although the importance of vitamin D has been recognized in recent years, there is no clear consensus on the definition of VDD [51][52][53]. Generally, vitamin D supplementation is recommended for pregnant women and newborns [54]. The cutoff values of vitamin D levels used to define VDD are not standardized, and different definitions of VDD are recommended by different societies. On the basis of the concentration of 25(OH)D, a serum concentration of 50 nmol/L (20 ng/mL) is sufficient for 97% of the population according to the recommendations of the Institute of Medicine published in 2010 [52,54]. The cutoff level recommended by the Endocrine Society is 30 ng/mL [53]. Similarly, a cutoff level of 20 ng/mL was adopted in half of the enrolled studies, and 25 ng/mL was used in the other studies ( Table 1). Use of different definitions may result in different outcomes and interfere with the comparison of response rates. Furthermore, the prevalence of VDD tends to vary in different countries [25][26][27]. A similar phenomenon was also observed in our systematic review. The reported prevalence of VDD varied widely in the different studies (range, 0.9-45%).
Moreover, vitamin D supplement was administered in three studies. The recommended daily dosage in children is 400-1000 IU [52][53][54]. However, single-day high-dose vitamin D therapy (stoss therapy) has also been investigated. Further studies are required to investigate the optimal dosage and protocol for vitamin D supplementation to enhance the immunogenic response to influenza vaccination.
Our study had some limitations. First, much heterogeneity was observed with respect to the study design, study participants, and the study period. The cutoff values of VDD were not the same among the studies, and data pertaining to vitamin D levels were not available for all studies. Therefore, not all the enrolled studies were eligible for meta-analysis, and further large-scale studies are warranted to confirm our findings. Second, the prevalence of VDD varied widely among the studies, and the causation of VDD is multifactorial. Presence of risk factors for VDD may interfere with the immunogenic response to influenza vaccination as well. Furthermore, although SP rates and SC rates are regarded as reliable parameters for evaluation of immunogenicity of influenza vaccination, the pre-vaccination baseline antibody titers were not similar in the included studies. Vitamin D supplementation was not administered in all studies. Moreover, the components of the influenza vaccine and the prevalent influenza strains were different each year. Ideally, it is more valuable to compare the immunogenic response in a similar study population with different vitamin D levels, but with similar pre-vaccination baseline antibody titer, the same vitamin D supplementation, and the same influenza vaccine.

Conclusions
Our study suggests that VDD is not associated with the immunogenic response to influenza vaccination based on the currently available data. Routine correction of VDD to enhance the immunogenicity of influenza vaccination is not recommended. However, strain-specific differences may exist, and VDD may attenuate the immune response to strain A/H3N2 and strain B following influenza vaccination. Adjuvant vitamin D supplementation may improve the immunogenic response to specific strains. Further studies are warranted to elucidate the influence of VDD on individual vaccine strains and the impact of vitamin D supplementation.
Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/10/4/409/s1, Table S1: Detailed search strategy of systematic review, Table S2: Risk of bias assessment of each included study.