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,2
*
ICMR Advanced Center for Diarrheal Disease Research, Department of Pediatrics and
Center for Community Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India
2To whom correspondence should be address.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin A • measles immunization • infants • immune response
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Fawzi et al. 1993
, Glasziou and Mackerras 1993
). Measles immunization given to infants at 9 mo
of age provides the first realistic opportunity for supplementation.
Semba et al. (1995
) reported that 30 mg (100,000
IU) of vitamin A coadministered with a measles immunization
at 6 mo of age reduced seroconversion compared to a placebo. This
effect was primarily observed in infants who had detectable maternal
antibody at baseline. It was hypothesized that immune enhancement by
vitamin A may limit the ability of a live-virus vaccine to
establish infection, a view supported by the decreased incidence of a
vaccine-induced skin rash in the vitamin A group. These
observations by Semba et al. (1995
) caused serious
concerns, in many developing countries, regarding the safety of the
coadministration of vitamin A and measles vaccine to infants 9 mo of
age, and some critics suggested abandoning this practice in ongoing
programs (Ross 1995
). In most developing countries,
coverage under vitamin A prophylaxis programs is low after the measles
immunization contact, this contact is therefore an important
opportunity for supplementation. To determine the impact on the antibody response to the vaccine 12 wk after immunizing infants, at 9 mo of age, with 30 mg of vitamin A coadministered with a standard titer Edmonston-Zagreb measles vaccine, we conducted an individually randomized, double-blind, placebo-controlled trial in a Delhi slum.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted in an urban slum of Delhi. In 1–5-y old
children seeking treatment at a health facility in a neighboring slum,
the prevalence of clinical vitamin A deficiency was ~3.5% and that
of subclinical deficiency (serum vitamin A levels of 
0.7 µmol/L)
was 37% (Bhandari et al. 1997
).
In a multi-center study conducted in an adjoining area at the same
time as the present study, vitamin A status of 9-mo-old infants was
assessed by serum retinol and the Modified Relative Dose Response
(MRDR)3
assay (WHO/CHD 1998
). The mean serum retinol was 0.89
± 0.36 µmol/L, 29.6% infants had concentrations 0.7
µmol/L, and 57.5% had inadequate vitamin A stores (MRDR > 0.06).
Sample size.
The a priori hypothesis was that 30 mg of vitamin A coadministered with
a measles vaccine to infants at 9 mo of age would, 12 wk after
immunization, result in a 10% reduction in seroconversion to measles.
Assuming that 92% of controls would seroconvert after the measles
immunization (Job et al. 1991
), the required sample size
was calculated to be 238 infants per cell at 95% confidence level and
90% power (Smith and Morrow 1991
). To allow for the
possibility of the observed values for the control group being
different from those assumed, and for 10% attrition, 309 infants per
cell were enrolled.
Enrollment, randomization and study intervention.
A household survey was conducted, and ~700 infants between 6 and 9 mo of age were identified. If informed parental consent was available, infants were enrolled when they became 9 mo old. Infants were not included in the study if they had a previous history of measles, contact with a case of measles or measles immunization, or if they had received a dose of vitamin A in the previous 4 mo. This information was ascertained from the caregiver. Infants who had any serious illness requiring hospitalization or had signs of vitamin A deficiency (Bitot's spots, corneal xerosis or keratomalacia) at enrollment were also excluded.
Infants were randomly assigned to receive vitamin A or a placebo by using a simple randomization scheme with random permuted blocks of size eight, i.e., four infants each out of every eight infants enrolled were randomized to receive vitamin A or a placebo. Vitamin A and the placebo were packed in individual dose capsules (courtesy of World Health Organization); the two types of capsules (placebo, vitamin A) were identical in color, shape and size. The active ingredient in the vitamin A capsules was 30 mg of retinol palmitate, whereas the placebo capsules contained soybean oil. Backup doses were provided for each infant in case the infant spat out the allocated supplement.
Infants received two capsules during the study period, one at 9 mo with the measles vaccine and the other at 12 mo of age. Those assigned to receive the placebo at 9 mo of age were given 30 mg vitamin A at 12 mo of age. Infants given the vitamin A supplement at 9 mo of age received the placebo at 12 mo. This scheme ensured that all infants received 30 mg vitamin A by 12 mo of age without interfering with the double-blind design of the study.
The study protocol was reviewed and approved by the All India Institute of Medical Sciences ethics committee.
Immunization and follow up.
Subjects were vaccinated with a single dose, standard titer Edmonston-Zagreb measles vaccine (same manufacturing lot, obtained from Swiss Serum Institute, Berne, Switzerland), within 20 min of capsule administration, at 9 mo of age, in the study clinic.
All infants were visited at home by a field worker on post vaccination days 1, 2, 7, 14 and 21 to monitor adverse effects of the vitamin A and measles vaccine. On these visits, the mother was queried about any illness symptoms in the infant since the last visit; the infant was also examined physically, particularly for a measles-like skin rash.
Laboratory tests.
About 3 mL venous blood was obtained from all infants prior to measles vaccination and 12 wk thereafter. Serum was separated by centrifugation at 1500 rpm for 10 min within 30–45 min and stored at -20°C.
ELISA was performed to estimate antibody titers by using the modified
method of Voller and Bidwell (1976)
.
The antigen for ELISA was prepared from the same Edmonston-Zagreb vaccine strain of measles vaccine that was used for immunization. Different aliquots were pooled to obtain ~2 L of supernatant, and the virus was partially purified by ultracentrifugation. ELISA was standardized by using the International Standard Anti-Measles Serum (66/202) obtained from the National Institute for Biological Standards and Controls, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK. The pre- and post-vaccination samples from one study subject were always tested in the same plate; the samples were coded to ensure that the laboratory staff could not identify pre-immunization from post-immunization samples. All test serums were tested in duplicate with and without the antigen-coated wells. The values of IgG antibodies against measles virus in the test samples was calculated in reference to the standard curve for the same plate.
Analysis.
Seroconversion was defined as a four-fold rise in antibody titer
over the expected antibody titer caused by the presence of baseline
antibody. This titer at the time of obtaining the post immunization
sample was calculated assuming a 6-wk half-life of the maternally
acquired antibody (Markowitz et al. 1990
).
Eighteen (11 in the vitamin A and 7 in the placebo group) infants had evidence of past measles infection at enrollment (pre-immunization antibody titer >2500) although they had no history suggestive of measles infection. Because the objective of the study was to evaluate the impact of vitamin A on the response to primary measles vaccination (previous history of measles was one of the exclusion criteria), it was an a priori decision to exclude infants who had a very high antibody titer at baseline. These 18 infants were therefore excluded from the analysis.
The analysis was performed using EPI INFO version 6 and SPSS for Windows software. Categorical variables were compared using the chi-square test and quantitative outcomes by Student's t-test. Differences were considered significant at P < 0.05. A multiple linear regression analysis restricted to malnourished infants was performed. In this regression analysis, the outcome variable was the post-immunization measles antibody titer on the logarithmic scale, while vitamin A or placebo treatment, sex, breast feeding, immunization status and presence of baseline measles antibody were explanatory variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The pre-immunization blood specimen was available from all the
618 enrolled infants, but the post-immunization specimen could not
be obtained in 83 of these infants (vitamin A, 39; placebo, 44).
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).
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) suggested a differential
impact of vitamin A on the geometric mean antibody titer in infants
with weight for age Z-score -2 (P < 0.01). A
subgroup analysis was, therefore, performed with infants stratified by
weight for age. The geometric mean antibody titer in the subgroup of
infants with weight for age Z-score -2 was significantly
greater in the vitamin A group compared to that in the placebo group,
but there were no differences in seroconversion rates. Seroconversion
rates and geometric mean titers did not differ between the two study
groups among the well-nourished children (Table 3
).
|
). Vitamin A treatment was found to significantly improve the antibody
titer in the subgroup of malnourished infants, after adjustment for
potential confounding factors, including sex, breast feeding,
immunization status and presence of baseline measles antibody
(P = 0.009).
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38°C, did not differ between the two
intervention groups (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
In the study by Semba et al. (1995)
, the adverse impact
of vitamin A on measles vaccine immunogenicity at 6 mo of age occurred
largely in the presence of significant levels of maternally acquired
antibody. We did not, however, find an interaction of vitamin A impact
with the presence of baseline antibody in the present study. Unlike the
findings of the Indonesian study (Semba et al. 1995
), we
observed no differences in the incidence of a measles-like rash
after vaccination.
The improved antibody response to measles vaccine in malnourished
infants observed in this study is biologically plausible. Malnourished
infants are more likely to have subclinical vitamin A deficiency.
Several human and animal studies have reported increased T-cell
help, down-regulation of interferon-gamma (IFN)-
and
isotype switching to IgA and IgG1 after vitamin A supplementation,
resulting in an improved humoral response (Friedman et al. 1991
, Cantorna et al. 1995
, Semba et al. 1993
, Tokuyama and Tokuyama 1996
).
Administration of vitamin A with tetanus toxoid in young children in
Bangladesh did not result in an increased antibody response to the
vaccine (Brown et al. 1980
). Another study in Indonesian
children reported an improved antibody response in the vitamin
A-treated children compared to those given a placebo, when
Diphtheria-Pertussis-Tetanus (DPT) vaccine was given 2 wk after
supplementation (Semba et al. 1992
). Animal studies have
clearly demonstrated that vitamin A repletion of depleted animals
causes significantly improved antibody response to protein (T
cell-dependent) and polysaccharide (T cell-independent type 2)
antigens (Ross 1991
).
In the multiple linear regression analysis restricted to malnourished infants, the factors associated with a higher post-immunization antibody titer were vitamin A administration, being not previously immunized with DPT or Oral Polio vaccines, and the presence of baseline measles antibody. There is a plausible explanation for the paradoxical finding of higher antibody titer at the end of the study in those who had baseline antibody. As expected, both seroconversion rate (75.7% vs. 90.6%, P < 0.001) and geometric mean difference between the 12-mo and baseline antibody titers (70.3 vs. 112.3, P = 0.06) were lower in the infants who had baseline antibody compared to those who had no antibody at baseline. Therefore, we attribute the higher post-immunization antibody titer in these infants to the remaining baseline antibody after decay in 12 wk, despite a poorer response to the measles vaccine.
The observed improvement in vaccine immunogenicity in malnourished infants that were administered vitamin A in this study should be interpreted with caution; this finding is based on a subgroup analysis. Because randomization was not performed at the subgroup level, baseline characteristics of the two intervention groups may have differed. This seems unlikely in the present situation because, in a multiple linear regression model restricted to malnourished infants, the impact of vitamin A on post-immunization antibody titer remained significant even after the adjustment for potentially confounding factors.
Plaque reduction neutralization (PRN), hemagglutination inhibition and
ELISA may all be used for measuring the antibody response to measles
vaccine. The most meaningful test for the serologic evaluation of
immunity status to viral infections including measles is the
neutralization test. Several earlier studies have compared the results
of PRN with those of ELISA and have shown that ELISA is a
sensitive and reliable assay and could represent an alternative to the
PRN (Cremer et al. 1985
, De Souza et al. 1991
, Neumann et al. 1985
). Also, the present
study was designed to compare the antibody response between the two
intervention groups and not for evaluating the absolute protection
against measles. Therefore, ELISA was chosen as the more convenient and
less expensive assay.
The major implication of these findings is that they set to rest the controversy about the current recommendation of combining vitamin A supplementation with measles immunization of infants at 9 mo of age in public health programs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: DPT,
diphtheria-pertussis-tetanus; IFN-, interferon-gamma; MRDR,
modified relative dose response; PRN, plaque reduction neutralization.
Manuscript received August 27, 1998. Initial review completed February 16, 1999. Revision accepted May 10, 1999.
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REFERENCES |
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