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*
ICDDR,B: Centre for Health and Population Research, Dhaka 1000, Bangladesh;
Society for Applied Research, Calcutta, India;
**
University of Alabama at Birmingham, Birmingham, AL;
Massachusettes Public Health Biologic Laboratories, Boston, MA; and
John Hopkins University, Baltimore, MD
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • infants • vitamin A • diphtheria • pertussis • tetanus • antibody • immunization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Barclay et al. 1987
, Barreto et al. 1994
,
Ghana VAST Study Team 1993
, Glaziou and Mackerras 1993
, Hussey and Klein 1990
). The exact
mechanism by which vitamin A reduces morbidity and mortality is
unknown. However, vitamin A improves the immune responses to a variety
of antigens (Dennert 1984
, Nauss 1986
,
Olson 1994
, Semba 1994
). Because of the
potential beneficial effect of vitamin A on child survival, routine
administration of vitamin A every 6 mo to children >6 mo of age has
been incorporated into child survival programs. Recently, vitamin A
administration in infants <6 mo of age using the routine Expanded
Program on Immunization
(EPI)3
contact has also been suggested (WHO 1982
). However, the
effect of simultaneous administration of large-dose vitamin A
administration and diphtheria, pertussis and tetanus (DPT) vaccination
in infants <6 mo of age on the seroconversion to these vaccines is
unknown. In this study, we examined the effect of simultaneous vitamin
A administration and DPT vaccination on the antibody response to these
vaccines. |
SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was conducted in the EPI clinic of the International Center
for Diarrhoeal Disease Research, Dhaka, Bangladesh (ICDDR,B: Center for
Health and Population Research). ICDDR,B treats ~120,000 patients
with diarrhea each year; most of these are children. All children <2 y
of age are routinely immunized at discharge (Islam et al. 1992
) on the basis of the missed opportunity concept
(WHO 1989
). Infants aged 6–17 wk who were scheduled to
receive their first dose of DPT and oral polio (OPV) vaccines were
enrolled after written informed consent was received from their
parents. Infants with diarrhea, respiratory tract infections or other
infections were not included. The study was approved by the Ethical
Committee of the ICDDR,B.
Study design.
This study was part of a double-blind, randomized placebo-controlled trial. Infants (n = 200) were randomly assigned to receive either 15 mg (50,000 IU) of oral vitamin A or placebo. The infants were fed retinyl palmitate or placebo and then immunized with DPT and OPV vaccines. The second and third doses of supplement were given along with the vaccine after 4 and 8 wk, respectively. The manufacturer of the DPT vaccine was Pasteur Marieux (Paris, France). We used two lots of vaccine produced on March 8, 1993 and April 5, 1993, respectively. A vaccine cold chain monitor (MonitorMark Indicator, Berlinger Gantersxhwil, Switzerland) was attached continuously to monitor the vaccine cold chain. The vaccine used in the EPI clinic of ICDDR,B hospital was transported directly from the EPI Headquarters in Dhaka to our clinic, which took only 10–15 min. The vaccines were used much earlier than the expiration date. The immunization shot was given by one of two nurses who were stationed at the EPI clinic. Once a vial was opened, the left-over portion was discarded. Each day, the clinic requires several vials of vaccine because a large number of patients attend the clinic for immunizations. Our subjects were recruited during the morning hours; thus they obviously received a freshly opened vaccine. Venous blood (1 mL) for serum retinol and antibody titers assay was obtained on enrollment and again 1 mo after the third dose.
This study reported here was part of the larger trial described above
(n = 200) in which the effects of large doses of
vitamin A on acute toxicity and morbidity were evaluated. Blood was
preserved from those infants whose mothers consented to blood drawing.
Because all of the available paired (pre- vs. post-treatment) samples
were analyzed, and the overall trial was randomized and double blind,
selection bias was unlikely. We had paired samples from 56 infants, a
number adequate to detect a difference in postsupplementation antibody
concentrations between the supplemented and placebo groups at the 5%
probability level with 80% power. This sample estimation was
calculated on the basis of a previous study in which vitamin A
supplementation was shown to increase the tetanus immunoglobulin (Ig) G
antibody (Semba et al. 1992
). The baseline
characteristics of the 56 infants did not differ from those of the
other infants in the original cohort studied (n = 200). The effects of vitamin A supplementation on acute toxicity
(Mahalanabis et al. 1997
), morbidity (Rahman et al. 1996
), cell-mediated immunity (Rahman et al. 1997
) and seroconversion to oral polio vaccine (Rahman et al. 1998
) in the same cohort of 200 children were reported
earlier.
Laboratory methods.
Serum IgG antibodies to diphtheria, tetanus and pertussis were assayed
by ELISA as previously described (Englund et al. 1997
,
Siber et al. 1991
). Briefly, ELISA plates were coated
with a 1 mg/L solution of diphtheria toxoid, tetanus toxoid or
pertussis toxin (Massachusetts Public Health Biologic Laboratories,
Boston, MA) in PBS. Assays utilized a standard of pooled adult serum
calibrated by the method of Zollinger and Boslego (1981)
. Results are reported as mg/L. During analysis, the
passively acquired maternal antibody titer (baseline value) was
subtracted (half-life was taken as 1 mo) to calculate the antibody
titer measured at 3 mo (Robertson 1993
).
Serum retinol was analyzed using HPLC (Waters, Millipore, Bedford, MA)
(Catignani and Bieri 1983
).
Statistical analysis.
Analyses were done using the statistical package for Social Science
(SPSS/PC+, Chicago, IL). Categorical analysis was done using the
-square test. Comparison of continuous variables was done with
Student’s t test for normally distributed data and the
Mann-Whitney test for skewed data. Because antibody levels were
highly skewed, a t test was done after log
transformation, and the antibody levels are presented as geometric
means. A paired t test was done to compare the
postsupplementation serum retinol value with the baseline value.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mean age of these infants was 2.5 mo and 93% were
breast-fed. The serum retinol concentrations at entry did not
differ between the two groups; 89% had serum retinol < 0.70
µmol/L and 38% had serum retinol < 0.53
µmol/L (Table 2
). Among the infants with low serum retinol, 86% had normal
C-reactive protein, and the rest had a slightly elevated serum
C-reactive protein. The serum concentrations had increased in both
groups after 3 mo when they were ~5.5 mo old (P < 0.001; paired t test). The postsupplementation mean serum
retinol concentration tended to be greater in the vitamin A group
(P = 0.22). Baseline antibodies to diphtheria,
pertussis and tetanus did not differ between the vitamin A and placebo
groups (Table 3
). The antibody concentration to diphtheria after vitamin A
supplementation was significantly greater in the vitamin A group than
in the placebo group (P = 0.029). The postdose
diphtheria antibody concentration was 17 times the initial value in the
vitamin A group and 6 times that in the placebo group. The
post-vitamin A supplementation IgG antibodies to tetanus and
pertussis were not different between the two groups (Fig. 1
). After adjusting for age, sex, nutritional status, baseline
retinol and C-reactive protein, the greater response to diphtheria
antibody in vitamin A–supplemented infants remained significant
(P = 0.019). Age of the child was also associated
with a greater antibody response (P = 0.05).
Association of other variables with postvaccination antibody levels was
not significant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Ross 1992
,
Semba 1994
). Physiologic concentrations of retinol
augment IgM synthesis by cord-blood mononuclear cells and IgG
synthesis by mononuclear cells in the peripheral blood of adults
(Israel et al. 1991
, Wang et al. 1993
).
The increased production of immunoglobulin is mediated by increased
T-helper cell numbers and enhanced production of cytokines, which
in turn induce B cells to differentiate into greater numbers of
immunoglobulin-secreting cells (Wang and Ballow 1993
, Wang et al. 1993
). However, not all
responses to T-cell dependent antigens are increased by vitamin A,
nor is the production of all cytokines increased, e.g., 
-interferon
is decreased. This study showed enhanced antibody response to
diphtheria after simultaneous vitamin A administration and DPT
immunization. Vitamin A deficiency is associated with a reduction of
both humoral (Carman et al. 1989
, Pasatiempo et al. 1989
, Smith et al. 1987
, Smith and Hayes 1987
) and cell-mediated immunity (Chandra and Au 1981
, Mark et al. 1983
, Nauss et al. 1979
and 1985
), which may explain in large part the increased
mortality in vitamin A–deficient children (Sommer et al.1983
and 1984
). The beneficial effect of vitamin A supplementation
on mortality reduction is well established. In our earlier report from
the same cohort, we demonstrated that infants who received vitamin A
experienced respiratory tract infections for significantly fewer days
compared with the placebo group, although the number of episodes was
similar (Rahman et al. 1996
). This suggests that the
severity of infections was less in vitamin A–supplemented infants,
which may be attributed to enhanced immune function.
In this study there was no effect of simultaneous vitamin A
administration on antibody responses to pertussis and tetanus vaccines.
In an earlier study in Bangladesh, Brown et al. (1980)
found no effect of vitamin A on the antibody response to tetanus
toxoid. In contrast, Semba et al. (1992)
reported that
vitamin A supplementation increased the response to tetanus vaccine in
children aged 3–6 y. One explanation for the different findings
between these two studies is that Brown et al. gave intramuscular
injections of vitamin A simultaneously with intramuscular tetanus
immunizations to vitamin A–deficient children. Vitamin A acts in a
hormone-responsive manner by upregulating the immune system
(Blomhoff 1992
, Semba 1994
). The immune
system of a vitamin A–deficient child might require more time to
respond after vitamin A supplementation. Therefore, it is possible that
there was not adequate time to observe an immune-enhancing effect
in that study. The other difference was that the children in the study
of Semba et al. were much older. In this study, we administered three
large doses of vitamin A at 1-mo intervals, and antibody was measured 1
mo after the last dose. Hence, there was adequate time for vitamin A to
exert an immune-enhancing effect. However, antibody responses to
tetanus and pertussis were not observed in our study. The mean age of
our study infants was only 2.5 mo at the time of enrollment, making
them much younger than the children of previous studies. In very young
infants, antibody response to tetanus may be affected by passive
immunity, especially if their mothers were immunized with two doses of
tetanus toxoid during pregnancy to prevent neonatal tetanus.
One of the important reasons why Brown et al. (1980)
did
not find a significant effect of vitamin A supplementation on immune
response to tetanus toxoid could be that their method of assay was the
mouse protection assay. In this assay, the patient’s serum or plasma
is mixed with a known quantity of tetanus toxoid and then injected into
a mouse to see if the mouse dies. This is a relatively crude assay, and
the mouse protection assay did not detect even a primary antibody
response to tetanus toxoid in that study. An ELISA detects (with great accuracy) a primary IgG response to tetanus toxoid.
Why tetanus and pertussis antibody responses were not enhanced, despite
an immune-enhancing effect on diphtheria, is not clear. The
diphtheria and tetanus vaccines are toxoid, whereas the pertussis
vaccine is a whole-cell derivative. This difference in vaccine
preparation could be one factor. The immune response to vaccines can
differ on the basis of the type of antigen because this can affect the
type of antigen-presenting cells (e.g., dentritic or macrophase)
that will be involved. These antigen-presenting cells can produce
different cytokine patterns [e.g., macrophaes will produce interleukin
(IL)-12; IL-12 production is impaired by vitamin A deficiency). Thus,
the difference in responses among the vaccines may be related to an
underlying specific effect of vitamin A on, for example, IL-12
production. If IL-12 is not needed for the response to pertussis and
tetanus, this may explain the lack of response to vitamin A. Another
explanation could be a difference in vaccine potency. One of the
limitations of our study is that we did not measure the vaccine
potency. However, the rise of IgG antibody in both the vitamin
A–supplemented and placebo groups suggests that the vaccine potency
was good. Also, the enhanced immune response to diphtheria indicates
that the vaccine potency was well maintained. In an earlier report on
the same cohort of children, we found that the combined
cell-mediated immune responses to diphtheria, tetanus and
tuberculin were greater in vitamin A–replete infants who received
vitamin A supplementation than in infants who received placebo
(Rahman et al. 1997
). Small sample size is also a
possible reason for not observing a significant difference in vaccine
responses. However, in this study, there was not even a trend for
differences between the two groups.
Our data demonstrate that vitamin A supplementation at routine
immunization enhances the antibody response to diphtheria. Although the
antibody responses to tetanus and pertussis were not affected in terms
of increased antibody levels, a previous report based on cutaneous
responses suggests a beneficial effect of giving vitamin A with these
antigens (Rahman et al. 1997
). Because vitamin A
deficiency occurs early in life in developing countries, vitamin A
administration at EPI contact might be beneficial in improving both
humoral and cell-mediated immunity. Administration of vitamin A as
part of routine EPI visits has the potential to enhance immune
responses while reducing the program cost of administering vitamin A at
a separate time.
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FOOTNOTES |
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3 Abbreviations used: DPT, diphtheria, pertussis and tetanus; EPI, Expanded Program on Immunization; ICDDR,
International Centre for Diarrhoeal Disease Research; Ig, immunoglobulin; IL, interleukin; OPV, oral polio vaccine.
Manuscript received February 16, 1999. Initial review completed April 12, 1999. Revision accepted July 29, 1999.
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