Are plasma mineral levels related to antibody response to influenza vaccination in older adults?

abstract

Introduction: An effective immune response to vaccination may be related to nutritional status. This study examined the association of plasma mineral levels with hemagglutination inhibition (HI) titers produced in response to influenza vaccine in older adults. Methods: Prior to (Day 0) and 21 (range = 19–28) days after receiving the 2013–14 influenza vaccine, 109 adults ages 51–81 years, provided blood samples. Serum samples were tested for HI activity against the A/H1N1 and A/H3N2 2013–2014 vaccine virus strains. Plasma minerals were collected in zinc-free tubes and assayed by inductively coupled plasma mass spectrometry. HI titers were reported as seroprotection (≥1:40) and seroconversion (≥ 4-fold rise from Day 0 (minimum HI = 1:10) to Day 21). Both HI titers and mineral values were skewed and thus log₂ transformed. Magnesium (Mg), phosphorus (P), zinc (Zn), copper (Cu), iron (Fe), potassium (K) and the Cu to Zn ratio were tested. Logistic regression analyses were used to determine the associations between mineral levels and seroconversion and seroprotection of HI titers for each influenza A strain. Results: Participants were 61% white, 28% male, 39% diabetic, and 81% overweight/obese with a mean age of 62.6 y. In logistic regression, Day 21 A/H1N1 seroprotection was associated with P and Zn at Day 21(P < 0.05). Seroconversion of A/H1N1 was associated with Day 21 Cu, P, and Mg (P < 0.03). Day 21 A/H3N2 seroprotection and seroconversion were associated with Day 21 P (P < 0.05). Conclusions: Phosphorus was associated with seroprotection and seroconversion to influenza A after vaccination; these associations warrant additional studies with larger, more diverse population groups.

Keywords:

• immunogenicity
• influenza vaccine
• plasma minerals

Introduction

Adults over age 65 y comprise the largest number of deaths from influenza and other acute respiratory infections,¹ largely attributable to older adults' inability to mount a sufficient immune response to infecting antigens, as well as their diminished immune response to vaccines.² The mechanisms for this change in immune function have not been clearly elucidated. Some research indicates that poor nutritional status from inadequate or imbalanced intake, drug-nutrient interactions, or chronic disease states, can result in inadequate levels of protein, vitamins and minerals.³ In general, lower serum levels of minerals such as selenium (Se), zinc (Zn) and copper (Cu) are associated with lower immune response.^4,5 Supplementation to normal serum levels of minerals is generally believed to improve immune response,^4,6 although some data suggest otherwise.⁷

Most research on trace minerals and immune function has focused on Zn, Cu, Se and iron (Fe) and has been performed among the elderly, because insufficient blood levels of these minerals have been observed in older adults and may play a role in immune function and response.^4,5,8,9,10 Moreover, phosphorus and potassium have been implicated in immune cell function.^11,12 Few studies of the relationship between trace mineral levels and immune function have been specific to influenza vaccination.⁶ The purpose of this study was to examine the relationships between detectable serum mineral levels tested using a set of standards developed for broad applications including healthcare, and immune response, specifically hemagluttination inhibition (HI) titers to influenza A strains, among ambulatory, middle-aged to older adults.

Results

Demographic characteristics of participants are shown in Table 1. Participants' mean age was 62.6 y and mean BMI was 32; they were 61% white, 28% male, 39% diabetic, and 81% were overweight or obese (BMI ≥ 25). Of the 6 minerals tested, 4 were outside clinically normal ranges at Day 0: Fe and P were high and K and Mg were low. At Day 21, Fe and P were higher than the normal range and Mg was lower. There were no relationships identified between mineral levels and diabetes status, except for Mg, but some mineral levels varied by BMI and race. Not surprisingly, BMI differed significantly between those with and without diabetes (35.8 vs. 29.8, respectively).

Table 1. Baseline Demographic Characteristics and Mineral Levels at Day 0 and Day 21.

Variable		n (%)		n (%)
Race	Non-white	48 (44)	White	61 (56)
Sex	Male	31 (28)	Female	78 (72)
Diabetes	Yes	39 (36)	No	70 (64)
Overweight (BMI ≥25)	Yes	88 (81)	No	21 (19)
Age, years, mean (SD)		62.6 (7.2)
BMI, mean (SD)		32.0 (7.5)
Mineral level	Day 0 Mean (SD)		Day 21 Mean (SD)		Normal values
Copper	21 (4) µmol/L		21 (6) µmol/L		12.5–21.0 µmol/L
Iron	2.14 (0.90) mg/L^*		2.24 (3.36) mg/L^*		0.5–1.7 mg/L
Potassium	3.17 (0.83) mEq/L		4.15 (1.88) mEq/L		3.5–5.4 mEq/L
Magnesium	1.4 (0.2) mEq/L^**		1.40 (0.19) mEq/L^**		1.5–2.4 mEq/L
Phosphorus	5.71 (0.88) mEq/L^*		6.0 (1.2) mEq/L^*		2.5–4.5 mg/dL
Zinc	12 (6) µmol/L		16 (15) µmol/L		9–16 µmol/L
Cu/Zn ratio	1.87 (0.75) µmol/L		1.67 (0.68) µmol/L		—

Notes. BMI=weight/height² (kg/m²).

* Above normal range.

** Below normal range.

In logistic regression, no Day 0 mineral levels were significantly associated with seroprotection (≥40) against either A/H1N1 or A/H3N2 at Day 0 (Appendix Table A1). Table 2 shows the relationship of Day 0 mineral levels to Day 21 seroprotection and seroconversion for A/H1N1 (top) and A/H3N2 (bottom). Immune response to A/H1N1, measured as either seroprotection or seroconversion, was not related to the circulating level of any Day 0 trace mineral level. Immune response to A/H3N2 measured by Day 21 seroprotection was not related to any Day 0 mineral level (Table 2 bottom). However, seroconversion of A/H3N2 antibody titers was significantly less likely when iron was high (P < .04)

Table 2. Relationship of Mineral Levels at Day 0 to Immune Response to Influenza Vaccine, Adjusted for Race.

	Antibody Titers for H1N1
	Seroprotection (≥40) at Day 21			Seroconversion (4-fold rise) at Day 21
Mineral level (log2) at Day 0	Odds ratio	95% CI	P value	Odds ratio	95% CI	P value
Zinc	1.87	0.53–6.58	0.33	0.64	0.15–2.65	0.54
Copper	1.07	0.24–4.68	0.93	2.19	0.39–12.24	0.37
Phosphorus	1.37	0.24–7.75	0.72	2.35	0.31–17.7	0.41
Magnesium	1.41	0.18–11.08	0.75	3.36	0.29–38.82	0.33
Potassium	1.15	0.38–3.47	0.80	0.58	0.17–1.96	0.38
Iron	1.48	0.70–3.10	0.30	1.02	0.45–2.34	0.96
Copper/Zinc ratio	0.80	0.43–1.49	0.47	1.65	0.83–3.30	0.15
Multivariate model including obesity
Copper	—	—	—	8.41	1.21–58.5	0.03
Phosphorus	—	—	—	7.58	1.38–41.7	0.02
Magnesium	—	—	—	15.00	1.13–198.8	0.04
	Antibody Titers for H3N2
	Seroprotection (≥40) at Day 21			Seroconversion (4-fold rise) at Day 21
Mineral level (log2) at Day 0	Odds ratio	95% CI	P value	Odds ratio	95% CI	P value
Zinc	1.49	0.40–5.46	0.55	1.50	0.41–5.54	0.55
Copper	0.81	0.17–3.75	0.78	1.64	0.34–7.87	0.54
Phosphorus	1.42	0.24–8.50	0.70	3.95	0.61–25.80	0.15
Magnesium	1.60	0.19–13.54	0.67	7.56	0.76–75.5	0.09
Potassium	2.35	0.76–7.29	0.14	1.57	0.47–5.20	0.46
Iron	0.52	0.24–1.12	0.09	0.41	0.18–0.95	0.04
Copper/Zinc ratio	0.73	0.38–1.40	0.35	1.05	0.55–2.02	0.88

Table 3 shows the likelihood of seroprotection and seroconversion at Day 21, given Day 21 mineral levels. This table contains, in addition to the minerals shown in Table 2, odds ratios and 95% confidence intervals for 3 minerals with significantly different mean values between Day 0 and Day 21. It also contains results from multivariate regression analyses for the uncorrelated mineral levels. The likelihood of seroprotection against A/H1N1 was significantly related to higher levels of zinc and phosphorus, to larger differences between Day 0 and Day 21 phosphorus levels and to phosphorus levels when accounting for magnesium, phosphorus, copper and copper to zinc ratio. Whereas, seroconversion against A/H1N1 was significantly more likely with higher levels of Cu, P, Mg with and without controlling for the other minerals.

Table 3. Relationship of Day 21 Mineral Levels to Immune Response to Influenza Vaccine, Adjusted for Race.

	Antibody Titers for H1N1
	Seroprotection (≥40) at Day 21			Seroconversion (4-fold rise) at Day 21
Mineral level (log2) at Day 21	Odds ratio	95% CI	P value	Odds ratio	95% CI	P value
Zinc	4.07	1.02–16.20	0.05	1.52	0.34–6.71	0.58
Copper	2.16	0.48–9.79	0.32	10.09	1.50–68.07	0.02
Phosphorus	6.15	1.56–24.23	0.01	7.28	1.40–37.90	0.02
Magnesium	1.04	0.15–7.43	0.97	14.22	1.26–160.44	0.03
Potassium	1.45	0.72–2.89	0.30	1.95	0.91–4.22	0.09
Iron	1.37	0.75–2.48	0.30	1.07	0.54–2.10	0.86
Copper/Zinc ratio	0.79	0.39–1.60	0.51	1.25	0.57–2.73	0.58
Potassium, difference Day 0 to Day 21	1.21	0.70–2.11	0.50	1.71	0.94–3.11	0.08
Phosphorus, difference Day 0 to Day 21	7.10	1.59–31.8	0.01	5.65	0.99–32.17	0.051
Iron difference, Day 0 to Day 21	1.04	0.60–1.81	0.89	1.05	0.56–1.99	0.87
Multivariate models including Magnesium, Phosphorus, Copper:
Phosphorus	5.64	1.46–21.86	0.01	6.63	1.25–35.10	0.03
Copper	—	—	—	7.68	1.24–47.42	0.03
Multivariate models including Magnesium, Phosphorus, Copper/Zinc: ratio
Phosphorus	6.69	1.61–27.86	0.01	—	—	—
	Antibody Titers for H3N2
	Seroprotection (≥40) at Day 21			Seroconversion (4-fold rise) at Day 21
Mineral level (log2) at Day 21	Odds ratio	95% CI	P value	Odds ratio	95% CI	P value
Zinc	2.04	0.51–8.20	0.32	0.63	0.16–2.48	0.51
Copper	2.45	0.51–11.78	0.26	3.99	0.77–20.68	0.10
Phosphorus	4.16	1.06–16.32	0.04	7.27	1.61–32.92	0.01
Magnesium	0.75	0.10–5.80	0.79	3.94	0.47–32.78	0.21
Potassium	0.94	0.47–1.89	0.86	0.88	0.43–1.81	0.73
Iron	1.49	0.80–2.78	0.21	1.00	0.54–1.85	1.00
Copper/Zinc ratio	0.82	0.39–1.70	0.59	1.38	0.67–2.86	0.38
Potassium, difference Day 0 to Day 21	0.80	0.46–1.39	0.43	0.86	0.48–1.53	0.60
Phosphorus, difference Day 0 to Day 21	4.38	0.99–19.49	0.52	3.90	0.83–18.34	0.08
Iron, difference Day 0 to Day 21	2.19	1.16–4.15	0.16	1.66	0.90–3.07	0.11
Multivariate models including Magnesium, Phosphorus, Copper:
Phosphorus	3.86	1.00–14.86	0.05	7.47	1.66–33.58	0.01
Multivariate models including Magnesium, Phosphorus, Copper/Zinc ratio:
Phosphorus	—	—	—	6.13	1.35–27.80	0.02

Only Day 21 phosphorus level was significantly associated with both Day 21 seroprotection and seroconversion against A/H3N2 (Table 3 bottom; P = 0.04 and 0.01, respectively).

When uncorrelated minerals (Mg, P, Cu and Mg, P, Cu/Zn ratio) were included in multivariate regression, P and Mg were significantly related to Day 21 seroprotection and seroconversion of H1N1 (P < 0.05) (Table 3). P was related to Day 21 seroprotection and seroconversion of H3N2 when Cu was included in the model and to Day 21 seroconversion when Cu/Zn ratio was included (P < 0.05).

These regression analyses were repeated while controlling for BMI. There was no change in the direction or significance of the relationships shown in the models without BMI.

Discussion

In this exploratory study of ambulatory, middle age and older adults, plasma mineral levels were highly skewed and correlated. Relationships of plasma minerals with antibody response to influenza vaccination were mixed.

For the primary analyses, no Day 0 mineral levels were significantly associated with seroprotection or seroconversion of A/H1N1 or A/H3N2 at Day 21, except that a lower Day 0 iron level was related to seroconversion of A/H3N2 this may be related to the fact that average iron levels were above normal values. Further research should look at different populations including those with differing baseline nutrient levels.

Higher mineral levels at 21 d post influenza vaccination were associated with seroprotection against influenza A/H1N1 (iron and phosphorus) and seroconversion of A/H1N1 (copper, magnesium and phosphorus). Higher phosphorus level 21 d post vaccination was also associated with seroprotection against and seroconversion of influenza A/H3N2. When combined in regression, Mg and P continued to affect immune responses to the influenza vaccine A strains with P related to seroprotection against A/H1N1 and seroprotection and seroconversion against A/H3N2 influenza strains. This study did not find positive relationships between immune response to influenza vaccine and zinc, a mineral commonly found to be related to immune function.^3–5 A randomized controlled trial of elderly adults supplemented with vitamins, minerals, vitamins and minerals, or neither found that the minerals only- and vitamins plus minerals-supplemented groups had higher antibody response to influenza vaccination, although there was no difference among groups on number of individuals with respiratory infections.⁶ Whereas, another randomized controlled trial that supplemented elderly participants with zinc reported no change in immune response to influenza vaccine in spite of restoration of circulating zinc levels.⁷ While copper is known to have a positive effect on immune response,⁴ this study found a positive relationship between immune response to influenza vaccine and copper level only when being overweight was included in the model. Day 0 magnesium and phosphorus levels were also significantly related to immune response to A/H1N1 levels when being overweight was included in the model. Other research has reported no relationship of obesity to immune response to influenza vaccine,¹³ but that study did not include mineral levels. No research was found linking magnesium or phosphorus with antibody response to influenza vaccine in either normal or overweight persons.

Differences between response to A/H1N1 and A/H3N2 are not unexpected because the A/H1N1 vaccine component (A/California/7/2009) has existed since 2009 and participants have been vaccinated repeatedly with it, whereas the A/H3N2 vaccine component (A/Texas/50/2012) was new in 2013–14.

The major finding of the current study is the strong association between Day 21 phosphorus levels and both Day 21 seroprotection and seroconversion for both influenza A strains. The literature suggests a prominent role for phosphorus for lymphocyte and monocyte proliferation and activation.^14,15 Phosphate (PO₄) the major biological ion of phosphorus has been shown to modulate CCR2 chemokine receptor in monocytes and CCR2 is essential for the maturation of dendritic cells and lymphocytes.^16-19 We speculate that phosphorus-replete status is beneficial in mounting appropriate antibody response to influenza vaccine.

The strengths of this study include the inclusion of a younger, healthier group of adults than has typically been studied, and the separate examination of the 2 influenza A strains. The study also had limitations including the fact that a majority of the participants were overweight who may differ in trace mineral metabolism, and the analyses were limited to immune responses and did not include clinical outcomes. The Day 0 mineral levels may also be predictors of immune response to influenza vaccination, but the skewed nature of the data may have masked associations. Due to its exploratory nature and modest sample size, future studies might focus on larger sample sizes, other population groups and clinical outcomes. Correlations among some of the mineral levels limited the models that contained multiple minerals simultaneously in adjusted logistic regression modeling.

Conclusion

The phosphorus level at Day 21 was associated with seroprotection and seroconversion to influenza A after vaccination. These preliminary results indicate the complexity of the relationships among micronutrients and immune function. Further studies with larger sample sizes and other more diverse, demographic groups are warranted.

Materials and methods

Subjects

Eligible patients aged 51 y and older, who had not yet received the 2013–2014 trivalent inactivated influenza vaccine (TIV) were enrolled from 9/30/2013 to 11/25/2014; influenza virus did not circulate widely in the community during that time period, thus the participants were not monitored for influenza. Exclusion criteria included documented contraindications to TIV,²⁰ Guillain-Barré syndrome, dementia or Alzheimer's disease, estimated life expectancy <2 years, medical treatment requiring immunosuppressant therapy or diagnosis of an immunocompromising condition, or concurrent participation in another influenza vaccine research study. Consented participants had blood drawn before receiving the 2013–2014 standard-dose, unadjuvanted intramuscular TIV (Day 0) and, because trace minerals have been shown to vary during the course of an infection or inflammatory state,^21,22 at approximately Day 21 (range, 19–28 d post vaccination), when antibody peaks. BMI was calculated from baseline self-reported height and weight. The primary aim was to determine the effect of Day 0 and Day 21 mineral levels on Day 21 antibody outcomes, specifically hemagluttination inhibition (HI) titers to influenza A strains, among ambulatory, middle aged to older adults.

Serum samples were aliquotted and frozen at −80°C until assayed. Study procedures, informed consent and data collection documents were reviewed and approved by the University of Pittsburgh Institutional Review Board (IRB approval #PRO11060173).

Hemagglutination inhibition assay

The HI assay protocol was adapted from the CDC laboratory-based influenza surveillance manual as previously described²³ and assessed functional antibodies to the hemagglutinin able to inhibit agglutination of turkey erythrocytes. To inactivate non-specific inhibitors, sera were treated with receptor destroying enzyme prior to being tested.^24–28 The HI titers were determined in assays by the reciprocal dilution of the last well that contained non-agglutinated red blood cells and were conducted in duplicate. Positive and negative serum controls were included for each plate.

Each serum sample was tested in HI assays against the A strains in the 2013–2014 TIV (A/California/7/2009 [H1N1]; A/Texas/50/2012 [H3N2]) viruses. Reference sera from ferrets infected with each of the viruses that seroconverted to the infection were used as positive controls. Seroprotection was defined as a HI titer ≥1:40 and seroconversion was: 1) ≥4-fold rise in HI titer from Day 0 (>1:10) to Day 21; or 2) a Day 21 titer of 1:40, if the Day 0 titer were <1:10.

Measurement of mineral concentrations

Whole blood was collected into heparinized, zinc-free tubes and plasma was separated at 4°C at 10,000 rpm for 5 min. After centrifugation, a subaliquot of the supernatant was diluted 100-fold with 2% nitric acid (sub-boil distilled Trace Metal^TM Grade nitric acid, Thermo Fisher Scientific, Waltham, MA) to ensure a consistent matrix. Dilutions were measured by mass. Internal standard spikes of beryllium, germanium and thallium were added to the diluted solution to assess instrument drift during analysis. Mineral concentrations in these diluted solutions were measured on a PerkinElmer NexION 300X (Waltham, MA) inductively coupled plasma mass spectrometer (ICP-MS). The instrument was calibrated before each run utilizing a 5-point concentration curve. Drift was assessed and blanks were measured every 10 samples. Diluted solution concentrations were transformed to actual concentrations using the mass measurements recorded during dilution. Detection limits were down to 1 ng/L); values ≤1 ng/L were not included in analysis. Analytical precision was assessed with sample duplicates. This study included levels of magnesium (Mg), phosphorus (P), zinc (Zn), copper (Cu), iron (Fe), and potassium (K), which were at detectable levels.

Statistical analyses

Mineral distributions were examined visually and appreciable outliers were removed from analysis. Due to skewed distributions, the remainder was log₂ transformed. Differences between Day 0 and Day 21 mineral levels were calculated and compared using nonparametric tests. Associations between BMI and log₂ transformed mineral levels were tested using Pearson correlations and associations between log₂ transformed mineral levels and presence of diabetes and race were tested using one-way ANOVA. Correlation analyses were also conducted to determine relationships among minerals. Because many of the mineral levels were significantly correlated, each mineral was included separately in a logistic regression that controlled for race, for its relationship to immune response to both A influenza virus strains using seroprotection (yes/no) and seroconversion (yes/no) as the binary outcome variables. These measures were chosen as they are the commonly used measures of immunologic response after influenza vaccination. Day 0 mineral levels were tested against seroprotection at Day 0 and Day 21, and seroconversion at Day 21. Day 21 mineral levels were tested against seroprotection at Day 21 and seroconversion at Day 21. Multivariate regressions using the backward stepwise procedure were conducted for the 3 uncorrelated minerals, Mg, Cu and P. Minerals with significantly different levels between Day 0 and Day 21 (K, Mg, P, Cu/Zn ratio) were included in logistic regressions with Day 21 seroprotection and seroconversion as the outcome variables. Regression analyses were repeated, adding BMI as a covariate. Significance was set at alpha ≤0.05.

Disclosure of potential conflicts of interest

Drs. Zimmerman and Lin have research funding from Merck & Co., Sanofi Pasteur and Pfizer Inc. Dr. Nowalk has research funding from Pfizer, Inc. and Merck & Co. Dr. Ross has research funding from Sanofi Pasteur.

Funding

This investigation was supported in part by an investigator initiated grant #003978 from Sanofi Pasteur, Inc. The findings and conclusions in this report are those of those authors and do not necessarily represent the views of Sanofi Pasteur, Inc. Dr. Kaynar (alone) received financial support from the NIH via grant R01 HL126711.

Appendix

Table A1. Relationship of Day 0 Mineral Levels to Influenza Vaccine Antibody Titers at Day 0 (baseline).

	Seroprotection (≥40) at Day 0 for A/H1N1			Seroprotection (≥40) at Day 0 for A/H3N2
Mineral level (log2) at Day 0	Odds ratio	95% CI	P value	Odds ratio	95% CI	P value
Zinc	1.31	0.35–4.89	0.69	1.46	0.36–5.97	0.60
Copper	0.35	0.07–1.68	0.19	0.95	0.19–4.81	0.95
Phosphorus	0.99	0.16–6.02	0.99	0.15	0.02–1.11	0.06
Magnesium	1.12	0.13–9.78	0.92	0.62	0.06–6.04	0.68
Potassium	1.09	0.35–3.41	0.89	2.34	0.65–8.45	0.20
Iron	1.59	0.75–3.40	0.23	0.66	0.29–1.48	0.31
Copper/Zinc ratio	0.71	0.36–1.42	0.34	0.81	0.40–1.64	0.55