Evaluation of BCG Vaccine Immunogenicity in Relation to Vitamin D Status in a Group of Egyptian Children

Background: Vitamin D has an immuomodulatory role in vaccine response and this may be mediated via its action on antigen presenting cells with the most potent effects on dendritic cells. Aim of work: was to determine if there is an association between circulating 25(OH)D concentrations and BCG vaccine cytokine response (interferon gamma) level. Methods: A cross sectional study included 80 infants aged 4 - 9 months from the pediatric outpatient clinic of Al-Zahraa university hospital (40 with normal weight and 40 with underweight). Participants were subjected to history taking, clinical examination, and laboratory investigations, particularly 25 hydroxy vitamin D [25(OH)D] and BCG vaccine-induced interferon gamma (IFN-γ). Results: The study showed that the underweight group had a lower socioeconomic level and higher percentage of breastfeeding compared with the normal weight group. Most infants in our study received vitamin D supplementation (80% and 62.5% in normal and underweight groups, respectively). The average serum level of 25(OH)D was relatively high (>100 ng/ml) in both normal and underweight groups. The mean levels of IFN-γ in response to BCG vaccine were not significantly different between the normal and underweight groups of infants in our study. Also, there was a statistically-significant moderate correlation between serum levels of 25(OH)D and BCG vaccine-induced IFN-γ in both normal (r = 0.58) and underweight (r = 0.64) groups. Conclusion: A good vitamin D status was observed in all studied infants irrespective of their nutritional status and significant correlation with BCG vaccine induced IFN-γ.


Introduction
Vitamin D was first characterized like a vitamin in the 20th century and now it is recognized as a prohormone. Two major forms of vitamin D are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is synthesized in the skin of humans and is consumed in the diet via the intake of animal-based foods, mainly fish oils, whereas vitamin D2 is derived from plant sources, is not largely human-made, and added to foods. Vitamins D2 and D3 forms differ only in their side chain structure. The differences do not affect metabolism (i.e., activation), and both forms have the prohormone function.
Vitamin D obtained from sun exposure, food, and supplements is biologically inactive (either the vitamin D2 or D3) and must undergo activation through 2 consecutive enzymatic hydroxylation reactions occurring in the liver and kidney.
Dietary vitamin D (either vitamin D2 or D3) is usually absorbed at the small intestine with other dietary fats. The presence of fats in the lumen triggers bile acids release, which initiate emulsification and support the formation of lipid-containing micelles, which diffuse into enterocytes. Once absorbed, exogenous vitamin D is packaged into chylomicrons, and thus is transported to the liver.
In recent years, researches have illuminated many functions of vitamin D other than its traditionally known roles in calcium homeostasis [1]. The important extra-skeletal action of vitamin D has now attracted tremendous interest [2]. Many cells from the innate and adaptive immune system express VDR. In addition, antigen presenting cells (macrophages and dendritic cells) and T and B lymphocytes express CYP27B1 (1-a -hydroxylase), which leads to local production of the active 1, 25-(OH)2 D3 from serum 25-(OH) D, that lead to tissue intracrine and paracrine action. These immune cells express CYP27B1only when activated by invading organism. This facilitates enhanced generation of antimicrobial peptides cathelicidin and beta-definsin 2 [3]. Vitamin D has an immuomodulatory role in vaccine response and this may be mediated via its action on antigen presenting cells with the most potent effects on dendritic cells [4]. The aim of this study was to determine if there is an association between circulating 25(OH)D concentrations and BCG vaccine cytokine response (interferon gamma) level and to correlate between cytokine response to BCG vaccine, vitamin D concentration and nutrition status of those infants.

Patients and Methods
A cross sectional study that was carried on 80 infants aged 4 -9 months, either nutritionally adequate or malnourished (simple underweight according to  Ethical consideration: An informed consent was obtained from parents of all patients before getting them involved in the study and confidentiality of all data were ensured any of patients has the right to withdraw from the study at any time without giving any reason.

Statistical Analysis
Data were collected, coded, revised and entered to the statistical package for Social Science (IBM SPSS) version 20. The data were presented as number and percentages for h qualitative data, mean, standard deviation and ranges for the quantitative data with parametric distribution and median with inter quartile range (IQR) for the quantitative data with non parametric distribution.
Chi-square test was used in the comparison between two groups with qualitative data and Fisher exact test was used instead of the Chi-square test when the expected count in any cell found less than 5. Independent t-test was used in the comparison between two groups with quantitative data and parametric distribution and Mann-Whitney test was used in the comparison between two groups with quantitative data and non parametric distribution. Spearman correlation coefficients were used to assess the significant relation between two quantitative parameters in the same group.

Results
In this study mean of age z score were 0 with range from −1.58 to 1.29 and mean of weight z score were 0 with range from −1.79 to 2.08. There was statistically significant increase low socioeconomic in underweight group and statistically significant increase in normal weight group in comparison to under weight group with weight, length, weight z score and length z score.
This study showed statistically significant increase breast feeding in underweight group and increase formula feeding in normal weight group. Also, there was statistically significant increase with Hb, urea and creat in normal weight group in comparison to under weight group.
In this study, there was no statistically significant difference between vit D and  This table showed that there was statistically significant increase low socioeconomic in underweight group and there was statistically significant increase weight, length, weight z score and length z score in normal weight group in comparison to underweight group. This table showed that there was statistically significant increase in infants receiving breast feeding and more prevalent in under weight group while formula feeding are more prevalent in normal weight group. Open Journal of Pediatrics

Discussion
Effective vaccination requires a good immune response to produce protective immune cells and/or immunoglobulins against future infections. However, several factors may affect immune status and response to vaccination, including nutritional status. Moreover, vitamin D has been found to have immunomodulatory effects and may affect the immune response to infections and vaccination [6]. In this cross-sectional study, we aimed at investigating the association among vitamin D status, cytokine response to BCG vaccination, and nutritional status in infants. We included 80 infants aged 4 -9 months from the pediatric Regarding epidemiological data, we found that infants in the underweight group had a lower socioeconomic level compared with that of the normal weight group. Egypt is one of the developing countries with a high rate of childhood malnutrition. As of 2014 data, the rates of underweight, wasting, and stunting in Egyptian children under five years were 6%, 8%, and 21%, respectively. Low socioeconomic status is associated with poor food security, poor environmental conditions, higher rate of infections, and lower educational levels of parents. All these factors contribute to malnutrition in infants and children [7] [8].
Lack of breastfeeding is an important cause of malnutrition in infants [9].
However, the percentage of breastfeeding in the underweight group (85%) is higher than that in the normal weight group (65%). Other factors, like the low socioeconomic status as discussed above, may have a more pronounced effect to the extent outweighing the benefits of breastfeeding; prolonged breastfeeding without proper weaning may also contribute [8] [9]. The lower levels of hemoglobin in infants with underweight could be attributed to the associated deficiencies of vitamins and minerals, particularly iron. [12].

Most infants in our study received vitamin D supplementation (80% and
On the other hand, both groups of infants have normal serum levels of urea and creatinine, but the normal weight group has a statistically significant higher level. Serum urea and creatinine are affected by several factors, such as age, gender, dietary intake, and body mass; the latter may obviously explain the difference between groups. Indeed, serum creatinine may be a poor indicator for impaired kidney function in patients with underweight/low body mass [13]. The average serum level of 25(OH)D was relatively high (>100 ng/ml) in both normal and underweight groups. Serum 25(OH)D remains the optimal indicator for vitamin D status thanks to its longer half-life. It may be difficult to specify the exact "normal" level of serum 25(OH)D, but there is a consensus on defining levels below 12.5 ng/ml (30 nmol/l) as vitamin D deficiency and between 12.5 -20 ng/ml (30 -50 nmol/ml) as vitamin D insufficiency [11] [14] [15]. Accordingly, none of infants in our study has vitamin D deficiency or insufficiency.
This result disagrees with prior studies.
Two studies included Egyptian mothers and their infants. In one study, the average levels of 25(OH)D were 16.8 ng/ml and 3.2 ng/ml in mothers and infants, respectively [16]. In the other study, the mean level of 25(OH)D was 16.7 ng/ml in infants. It is interesting that 40% of mothers in the last study had a 25(OH)D level of <20 ng/mL, although their mean level was higher at 32.6 ng/mL because some cases had very high levels [17].
The higher mean level of vitamin D in our study could be attributed to vitamin D supplementation (more than 70% of cases) and regular sun exposure (more than 40% of cases). The recent guidelines emphasizing daily vitamin D supplementation since birth and increased awareness of the community may contribute to improved vitamin D status in infants and children compared with earlier studies [11]. However, we acknowledge that our results may not be generalizable, given the limited number of cases, and should be confirmed by prospective large-scale studies.
The mean levels of IFN-γ in response to BCG vaccine were not significantly different between the normal and underweight groups of infants in our study.

A. A. Abdelgawad et al. Open Journal of Pediatrics
Results of IFN-γ release assay in response to BCG vaccination are affected by several factors, such as ethnicity, genetic factors, and time-lapse since vaccination [18]. Tuberculosis is a major health concern all over the world. BCG vaccination has been proven to offer variable protection against tuberculous infection (27%), active diseases (71%), and progression from infection to active disease (58%) [19]. BCG is included in the mandatory immunization program in Egypt [20].
Our study shows a statistically-significant moderate correlation between serum levels of 25(OH)D and BCG vaccine-induced IFN-γ in both normal (r = 0.58) and underweight (r = 0.64) groups. This underscores the immunomodulatory role of vitamin D and its possible important effect on the immune response to vaccine [2] [21].
Vitamin D may affect the immune response to vaccination through activation of dendritic cells (via toll-like receptors by vaccine antigen ± adjuvant) and intracellular upregulation of CYP27B1 with stimulation of cytokine production and T-cell proliferation. This results in suppression of Th1 response-like cytokines (IFN-γ, IL-2) and the increase of both Th2 (IL4, IL5, IL10, IL13) and Th17 (IL-17) response-like cytokines. Vitamin D may also affect the proliferation and antibody-production of B-cells. Despite 1α,25(OH)2D may blunt B-cell functions, it may paradoxically stimulate a vaccine-induced immune response by its effect on innate immunity [2] [22].
There are several observational and clinical studies on the association between vitamin D status and vaccine effectiveness. However, the results are conflicting and reveal some methodologic weaknesses as well as limiting and confounding factors [21].
For example, in a retrospective study on 200 patients with chronic kidney disease, vitamin D deficiency was associated with poor antibody response to Hepatitis B vaccination [23]. However, in a randomized controlled trial on 31 hemodialysis patients with hepatitis B booster vaccine, the intravenous 4 µg of paricalcitol (an active vitamin D analog) during hemodialysis three times weekly was compared with placebo. There was no difference in the in vivo memory response or in vitro proliferation and release of cytokines (IL-2, IL-6, TNF-α, and INF-γ) [24].
Lalor et al. [22] compared the serum levels of 25(OH)D and IFN-γ in BCG vaccinated and unvaccinated groups of infants at two points (3 and 12 months of age). There was a moderate inverse correlation between vitamin D status and IFN-γ response to BCG vaccine at infants aged three months; infants with higher vitamin D levels have a lower IFN-γ response. These results disagree with the positive correlation found in our study. Of note, the levels of 25(OH)D in this study were markedly low compared with that of our study. In addition, the correlation was weaker when assessed in infants at 12 months of age compared with that at 3 months of age [22].
Another interesting point in Lalor et al. [22] study is that BCG vaccinated infants had a six-time higher vitamin D level than that of unvaccinated infants.

A. A. Abdelgawad et al. Open Journal of Pediatrics
They proposed that mycobacterial antigens in BCG may increase the production of the 25 hydroxylase enzymes, resulting in increased production of 25(OH)D [22]. We did not include control (unvaccinated) cases in our study to investigate this point.
In a randomized control study in China, 597 infants with poor BCG vaccine response, as evidenced by Tuberculin Skin Test (TST), were revaccinated with BCG vaccine. Infants were divided into two groups, one of which (intervention group) received supplementation with combined vitamins D and A. TST was repeated after three months. Results show that supplementation with combined vitamins D and A improved immune response to BCG vaccination compared to control. However, levels of vitamin D or cytokines were not evaluated in this study [25].
Our study has some limitations. First, we assessed the immune response to BCG vaccination by only IFN-γ release assay. Second, we assessed IFN-γ level in infants aged 4 -9 months. This might result in the variability of IFN-γ levels, which are affected by time-lapse after BCG vaccination. Third, it would be meaningful to have a control group of unvaccinated infants. However, this was not applicable because BCG is a part of the Egyptian obligatory immunization schedule for all infants. Fourth, our study has a small number of cases. Results of our study need to be confirmed by future large-scale studies.

Conclusion
Our study showed a relatively good vitamin D status in included infants irrespective of their body weight; this may be attributed to vitamin D supplementation and regular exposure to sun. We found a statistically significant moderate correlation between vitamin D status and immune response to BCG vaccine (as assessed by IFN-γ release assay). These results are important particularly for developing countries, where children are at high risk of both vitamin D deficiency and infections.