A Randomized, Controlled Trial of the Impact of Alternative Dosing Schedules on the Immune Response to Human Rotavirus Vaccine in Rural Ghanaian Infants
A Randomized, Controlled Trial of the Impact of Alternative Dosing Schedules on the Immune Response to Human Rotavirus Vaccine in Rural Ghanaian Infants
George Armah 2
Kristen D. C. Lewis 5
Margaret M. Cortese 4
Umesh D. Parashar 4
Akosua Ansah 1
Lauren Gazley 5
John C. Victor 5
Monica M. McNeal 3
Fred Binka 0
A. Duncan Steele 5
0 University of Health and Allied Health Services , Ho , Ghana
1 Navrongo Health Research Centre
2 Noguchi Memorial Institute for Medical Research, University of Ghana , Legon
3 Department of Pediatrics, Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center , Ohio , USA
4 Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention , Atlanta , Georgia
5 PATH, Seattle , Washington , USA
(See the editorial commentary by Cunliffe and Kang on pages 1673-5, and major article by Zaman et al on pages 1686-93.) Background. The recommended schedule for receipt of 2-dose human rotavirus vaccine (HRV) coincides with receipt of the first and second doses of diphtheria, pertussis, and tetanus vaccine (ie, 6 and 10 weeks of age, respectively). Alternative schedules and additional doses of HRV have been proposed and may improve vaccine performance in low-income countries. Methods. In this randomized trial in rural Ghana, HRV was administered at ages 6 and 10 weeks (group 1), 10 and 14 weeks (group 2), or 6, 10, and 14 weeks (group 3). We compared serum antirotavirus immunoglobulin A (IgA) seroconversion (≥20 U/mL) and geometric mean concentrations (GMCs) between group 1 and groups 2 and 3. Results. Ninety-three percent of participants (424 of 456) completed the study per protocol. In groups 1, 2, and 3, the IgA seroconversion frequencies among participants with IgA levels of <20 U/mL at baseline were 28.9%, 37.4%, and 43.4%, respectively (group 1 vs group 3, P = .014; group 1 vs group 2, P = .163). Postvaccination IgA GMCs were 22.1 U/mL, 26.5 U/mL, and 32.6 U/mL in groups 1, 2, and 3, respectively (group 1 vs group 3, P = .038; group 1 vs group 2, P = .304). Conclusions. A third dose of HRV resulted in increased seroconversion frequencies and GMCs, compared with 2 doses administered at 6 and 10 weeks of age. Since there is no correlate of protection, a postmarketing effectiveness study is required to determine whether the improvement in immune response translates into a public health benefit in low-income countries. Clinical Trials Registration. NCT015751.
Rotavirus is a leading cause of diarrhea-associated mortality
among children <5 years of age in low-income countries
], with countries in sub-Saharan Africa exhibiting
the highest rates of rotavirus-associated mortality . In
Ghana, rotavirus is a common cause of diarrhea, circulating
from approximately September/October to February/March
]. Prior to widespread introduction of rotavirus vaccine,
rotavirus was responsible for approximately 50% of diarrheal
hospitalizations among children aged <5 years .
Two orally administered human rotavirus vaccines (HRVs),
Rotarix (GSK Biologicals, Rixensart, Belgium) and RotaTeq
(Merck, West Point, Pennsylvania), are recommended by the
World Health Organization (WHO) for inclusion in the infant
immunization series. While antirotavirus immunoglobulin A
(IgA) seroconversion frequencies with these vaccines range
between approximately 87% and 95% in high-income countries
], rates in Africa and Asia have been consistently
lower, between 36% and 78% [
]. Similarly, IgA
geometric mean concentrations (GMCs) in LICs are approximately
5–10 times lower than those in HICs [
], and, while vaccine
efficacy against severe rotavirus diarrhea in HICs is high [
there is moderate efficacy among children in LICs [
The reasons postulated for the observed reduced response to
rotavirus vaccines in LICs include the presence of high maternal
antirotavirus antibody titers, interference by the first dose of
coadministered oral polio vaccine (OPV), altered gut microbiota,
enteropathy, and malnutrition [
]. In LICs, HRV is
administered in a 2-dose schedule at 6 and 10 weeks of age (WHO
Expanded Programme on Immunization [EPI] visits 1 and 2).
Pragmatic ways to overcome the effect of some of these factors
might be to administer a third dose at 14 weeks of age (EPI visit 3)
or, although not considered practical in maximizing EPI
coverage, delay administration of the standard 2-dose schedule to a
slightly older age, when maternal antibody levels have declined
and when there is lower risk of interference from concomitant
administration with the first OPV dose.
Previous studies assessed whether alternate schedules
improve vaccine responses in infants from LICs. One study
evaluated the immunogenicity and efficacy of HRV through 2 years
of age in South Africa and Malawi, finding consistent trends
indicating that a 3-dose HRV schedule (6, 10, and 14 weeks of
age) may be optimal as compared to a 2-dose schedule
administered at an older age (10 and 14 weeks of age) [
]. In an
earlier South African immunogenicity study, infants receiving 2
doses of a lower-dose HRV at 6 and 10 weeks of age had
lower immune responses as compared to infants receiving 2
doses at 10 and 14 weeks of age [
]. Thus, the increases in
immunogenicity and efficacy of a 3-dose schedule identified in the
South Africa and Malawi study may be even more pronounced
when compared to a 2-dose schedule given at an earlier
recommended schedule (6 and 10 weeks of age).
To investigate whether a 3-dose schedule of HRV
administered at 6, 10, and 14 weeks of age is superior to a 2-dose
schedule in which vaccine is given at the recommended earlier ages
(6 and 10 weeks of age), we conducted a phase 4, single-center,
individually randomized, open-label trial in rural Ghana.
Secondary objectives included assessing whether 2 doses of HRV
administered at a delayed schedule of 10 and 14 weeks of
age were superior to 2 doses administered at 6 and 10 weeks
of age, comparing the effect of high versus low levels of
maternally derived antirotavirus immunoglobulin G (IgG) on the
immune response to vaccination, and describing vaccine-type
Study Site and Subjects
This study was conducted in rural Navrongo (Upper East
Region, Ghana). HRV (Rotarix) was introduced into the Ghana
National EPI in May 2012 as a 2-dose schedule, given at 6 and
10 weeks of age. When infants were between 42 and 55 days old,
parents were invited to learn more about the study and provide
written informed consent. Infants were subsequently enrolled
and vaccinated between September and December 2012. Infants
eligible for study vaccination were between 6 and 7 weeks of age
at the time of enrollment, generally healthy, free of
contraindications to receipt of rotavirus vaccine, born at full term
(≥36 weeks of gestation), not underweight (ie, <2000 g), the
only child <16 weeks of age living in the compound, and had
not previously received rotavirus vaccine. The trial was
conducted according to the principles of the Declaration of
Helsinki (2008), in compliance with good clinical practice
guidelines, and approved by the following ethics review
committees: the Ghana Health Service Ethics Review Board, the
Navrongo Health Research Center Institutional Review Board
(IRB), the Noguchi Memorial Institute for Medical Research
(NMIMR) IRB, the Centers for Disease Control and Prevention
IRB, and the Western IRB (Olympia, Washington).
Randomization and Procedures
All infants eligible for vaccination were randomly assigned to
receive liquid HRV at 6 and 10 weeks of age (group 1), 10 and 14
weeks of age (group 2), or 6, 10, and 14 weeks of age (group 3).
Randomization was conducted using a computer-generated
algorithm, with infants assigned to study groups in blocks of 3 in a
1:1:1 ratio. A baseline blood specimen was collected prior to
receipt of the first dose of HRV. A stool specimen was also collected
before each HRV dose. All study groups received their rotavirus
vaccinations concomitantly with routine EPI vaccines (OPV,
pneumococcal conjugate vaccine, and pentavalent diphtheria,
pertussis, tetanus, Haemophilus influenzae type b, and hepatitis
B vaccine). Breastfeeding was not restricted.
Trained field workers visited participants at their homes to
collect stool specimens 4 and 7 days (±1 day) after each HRV
dose. All participants were asked to report to the study clinic
4 weeks (+2 weeks) following the last dose of HRV, to provide
a postvaccination blood specimen (specimens were obtained
from group 1 at 14 weeks and from groups 2 and 3 at 18
weeks); group 1 participants also had a blood specimen
collected during their last visit, at 18 weeks of age. The last follow-up
visit occurred in February 2013. Participants were followed
throughout the study for serious adverse events (SAEs). All
SAEs were reviewed and vetted by an independent safety
monitor. Masked serum and stool specimens were shipped on dry
ice and stored frozen (temperature, −20°C) at the Regional
Rotavirus Reference Laboratory, NMIMR (University of Ghana,
Legon) until testing (for stool specimens) or further shipment
(for serum specimens).
At the NMIMR laboratory, rotavirus antigen in stool was
detected by an enzyme immunoassay (EIA; ProSpecT, Oxoid, United
Kingdom). All stool samples testing positive for rotavirus by
EIA were characterized by reverse transcription–polymerase
chain reaction to identify the vaccine-like genotype G1P[
]. Sera were analyzed at Cincinnati Children’s Hospital Medical
Center Laboratory for Specialized Clinical Studies (Cincinnati,
Ohio) by an enzyme-linked immunoassay, as previously
described, to detect and quantify serum antirotavirus IgA or
IgG antibody concentrations [
]. Seroconversion was
defined as the detection of an antirotavirus IgA concentration of
≥20 U/mL in infants with an IgA concentration of <20 U/mL
at the time of receipt of their first rotavirus vaccine (6 weeks of
age for groups 1 and 3 and 10 weeks of age for group 2).
follow-up period during the rotavirus season, the primary
measure of seroconversion and GMCs in group 1 was based on the
higher of the 2 postvaccination antirotavirus IgA concentrations
measured in serum specimens obtained at 14 weeks or 18 weeks.
GMCs were defined as the exponential of the mean logarithmic
transformation of the antirotavirus IgA and IgG antibody
responses. For quantitative analyses, serum antirotavirus IgA
values of <20 U/mL were converted to 10 U/mL to produce results
comparable to those of other studies, even though the limit of
quantification of the assay was 7 U/mL. The GMCs and 95%
CIs of the IgG antibody responses before vaccination and IgA
antibody responses after vaccination were compared between
groups, using the t test. The effect of maternally derived
serum IgG on HRV IgA seroconversion was also evaluated,
using (1) the Fisher exact test to compare IgA seroconversion
frequencies within each group, stratified by IgG level before
vaccination (ie, low [≤25th percentile] vs high [≥75th percentile]), with
cut points determined by the distribution within each arm; and
(2) a fixed-effects logistic regression model, with rotavirus IgA
seroconversion after vaccination as the dependent variable and the
level of rotavirus maternally derived IgG antibody before
vaccination as an independent variable. In these analyses of effects of
maternally derived antibody, all valid serum IgA and IgG values
were used. SAEs were summarized by group and by relationship
to HRV in terms of counts and percentages.
Enrollment of 152 participants per study group was estimated to
provide 90% power to detect an increase in seroconversion from
35% in group 1 to 55% in group 3 [
], using a 2-sided χ2 test
(α = 0.05) for proportions and accounting for a 15% attrition rate.
Of 456 participants screened and randomly assigned to the 3
study arms, 455 (99.8%) received HRV (Figure 1); 152, 152,
and 151 participants were in groups 1, 2, and 3, respectively.
The majority (93.2%) of randomized participants met the
study criteria for inclusion in the PPE serologic analysis; only
2 participants were seropositive at baseline (Figure 1). Baseline
characteristics were similar across groups and a very high
proportion completed each follow-up visit within protocol-defined
windows (Table 1). Per WHO recommendations, the majority
of participants included in the PPE (>97% in each group)
received a birth dose of OPV at ≤4 weeks of age. All participants
received concomitant OPV.
Serum Antirotavirus IgA Responses
In the PPE population, 28.9% (95% CI, 22.1%–36.8%), 37.4%
(95% CI, 29.8%–45.7%), and 43.4% (95% CI, 35.5%–51.6%)
of participants in groups 1, 2, and 3, respectively, seroconverted,
based on the primary measure (Table 2). Participants receiving
3 doses of HRV had a significantly higher seroconversion
frequency (absolute rate difference, 14.5%; 95% CI, 3.3%–25.1%;
P = .014) than those receiving 2 HRV doses at 6 and 10 weeks
of age (group 1), but those receiving 2 HRV doses on a delayed
schedule (group 2) did not have a significantly higher
seroconversion frequency (absolute rate difference, 8.5%; 95% CI,
−2.5% to 19.3%; P = .163) than those in group 1. When
measuring seroconversion frequencies at 4 weeks following the final
dose of HRV (14 weeks for group 1), there were larger increases
in group 3 (43.4%) and group 2 (37.4%) as compared to group 1
(20.4%). While not as large, this also held true when
seroconversion in group 1 was measured 8 weeks after dose 2.
In the PPE population, using the primary measure, GMCs were
22.1 U/mL (95% CI, 17.4–28.2 U/mL), 26.5 U/mL (95% CI, 20.7–
34.0 U/mL), and 32.6 U/mL (95% CI, 24.7–43.2 U/mL) for
participants in groups 1, 2, and 3, respectively (Table 3). Participants
receiving 3 doses of HRV had significantly higher GMCs than
those receiving 2 doses at 6 and 10 weeks of age (GMC ratio,
1.47; 95% CI, 1.02–2.13; P = .038), but participants receiving 2
doses on a delayed schedule did not have significantly higher
GMCs than those receiving doses at 6 and 10 weeks of age
(GMC ratio, 1.20; 95% CI, .85–1.69; P = .304). Based on
comparisons between groups of GMCs at only the 14-week visit (GMC,
16.8 U/mL; 95% CI, 13.8–20.4 U/mL) or 18-week visit (GMC,
19.1 U/mL; 95% CI, 15.4–23.8 U/mL) for group 1, the differences
between study groups increased, resulting in a significant
difference between groups 1 and 2 when the 14-week measurement
was used (GMC ratio, 1.58; 95% CI, 1.15–2.16; P = .005).
Among only participants who seroconverted based on the
primary outcome measure, postvaccination GMCs were 156.7
U/mL (95% CI, 101.3–242.5 U/mL), 135.5 U/mL (95% CI,
95.0–193.3 U/mL), and 153.1 U/mL (95% CI, 103.6–226.2 U/
mL) for groups 1, 2, and 3, respectively (Table 2). There were
no significant differences between groups 1 and 3 (GMC
ratio, 0.98; 95% CI, .54–1.76; P = .938) or groups 1 and 2
(GMC ratio, 0.86; 95% CI, .50–1.50; P = .600). These results
were similar regardless of whether the final specimen used for
comparison between study groups was based on only the week
14 or week 18 specimen for group 1.
Maternal Antibody Evaluations
The GMC of maternally derived rotavirus measured at 6 weeks
of age was similar in both group 1 (279.9 U/mL; 95% CI,
237.2–330.4 U/mL) and group 3 (269.7 U/mL; 95% CI,
232.6–312.8 U/mL; P = .743; Table 4), while the IgG GMC
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measured at 10 weeks in group 2 was statistically significantly
lower, at 193.4 U/mL, than that in group 1(P = .002) and
group 3 (P = .003). Within each arm, when using the findings
for serum specimens obtained 4 weeks after vaccination, the
seroconversion frequency was higher among infants with low
maternally derived IgG levels (≤25th percentile), compared with
infants with high maternally derived IgG levels (≥75th
percentile; group 1, 38.9% vs 5.6% [P = .001]; group 2, 60.0% vs 28.6%
[P = .015]; and group 3, 52.8% vs 36.1% [P = .236]; Table 4).
After controlling for maternally derived IgG level, infants
vaccinated with 3 doses of HRV had a significantly increased
likelihood of seroconversion as compared to those who received 2
doses at 6 and 10 weeks of age (odds ratio, 1.38; 95% CI, 1.07–
1.76; P = .012), while infants who received 2 doses at 10 and 14
weeks of age did not have a significantly increased likelihood of
seroconversion as compared to those vaccinated with 2 doses at
6 and 10 weeks of age (OR, 1.12; 95% CI, .86–1.45; P = .400).
Postvaccination Shedding of Vaccine-Type Virus
Only 5% of participants (17 of 338) were found to be shedding
vaccine-type rotavirus in stool, based on analysis of specimens
collected 4 or 7 days after receipt of any vaccine dose (group 1, 6
of 121; group 2, 5 of 99; group 3, 6 of 118). All cases of shedding
occurred after the first dose with the exception of one occurring
after the second dose.
SAEs occurred in 8 participants; 1 (0.7%) in group 1 (a case of
gastroenteritis), 4 (2.6%) in group 2 (1 case of abscess of the left
jaw, 2 cases of sepsis, and 1 death from congenital hydrocephalus),
and 3 (2.0%) in group 3 (2 cases of acute respiratory infection and
1 case of gastroenteritis). All SAEs were classified by the
independent safety monitor as unrelated to HRV or study procedures.
Earlier studies have elicited mixed results regarding the
immunological benefit of adding a third dose of HRV to the infant
schedule so that the vaccine is provided at 6, 10, and 14
weeks of age [
10, 12, 19
]. In this study in rural Ghana, 3 doses
of HRV resulted in significantly improved antirotavirus IgA
seroconversion frequencies and GMCs as compared 2 doses
given at the WHO-recommended ages of 6 and 10 weeks. A
higher likelihood of seroconversion following 3 HRV doses
was also found after controlling for maternally derived IgG
concentration. In contrast, 2 doses of HRV given on a delayed
schedule at 10 and 14 weeks of age increased the seroconversion
frequency when compared to the 6- and 10-week arm, but this
difference was not significant. As expected, the conservative
approach of using the highest IgA titer in 6- and 10-week blood
specimens collected 4 and 8 weeks following the last dose of
HRV shifted the differences between study arms toward the
null. Although there was significant improvement in the
immune response (an additional 14.5% of infants seroconverted)
when a third dose of HRV was added to the recommended
2-dose regimen in these Ghanaian children, the seroconversion
frequency in this arm is still far lower than that among children
receiving 2 doses in HICs [
While the results from this study in Ghana differ from a
similar study conducted in Pakistan, where no significant
differences in seroconversion frequencies or GMCs were observed
between the standard schedule and a delayed 2-dose schedule
or a 3-dose schedule [
], they are consistent with those from
prior studies of HRV in Africa investigating the effects of a
delayed 2-dose schedule (at ages 10 and 14 weeks) as compared to
(1) 2 doses administered at younger ages (6 and 10 weeks) and
(2) 3 doses (6, 10, and 14 weeks of age) [
]. Although not
powered to differentiate between the 6- and 10-week arm and
the 10- and 14-week arm, results from a South African
immunogenicity study demonstrated an even larger, though not
statistically significant, increase in seroconversion frequencies and
GMCs among participants receiving a lower-dose HRV at the
older age as compared to the current study . In an HRV
efficacy and immunogenicity subset study conducted in South
Africa and Malawi [
], 3 doses of HRV, administered at 6, 10,
and 14 weeks of age, resulted in modestly increased antirotavirus
IgA seroconversion frequencies (South Africa, 66.7%; Malawi,
57.1%) as compared to 2 doses given at 10 and 14 weeks of age
(South Africa, 57.1%; Malawi, 47.2%). Although not powered to
differentiate between the 2 HRV arms, the efficacy of the vaccine
to prevent severe rotavirus gastroenteritis through 2 rotavirus
seasons was also higher among 3-dose recipients (South Africa,
85.0%, Malawi, 42.3%), compared with 2-dose recipients
(South Africa, 32.0%, Malawi, 34.0%) [
], suggesting that a
third dose of HRV, even when compared to 2 doses provided at
an older age, provides a boost or provides another opportunity to
better protect children through their second year of life.
In addition to concomitant administration of OPV with the
first dose of rotavirus vaccine at 6 weeks of age, maternal antibody
has also been cited as one of the probable causes of the low
immune response to rotavirus vaccines in children in LICs,
including in Africa [
]. The baseline IgG GMC was significantly lower
in group 2, in which serum specimens were collected at 10 weeks
of age, as compared to groups 1 and 3, in which serum specimens
were collected at 6 weeks of age. The findings from this study, in
which a third dose of HRV reduced the impact of maternally
derived IgG antibodies on IgA seroconversion, are similar to those
of studies in Pakistan and Vietnam, which found that the
frequency of IgA seroconversion was reduced among participants
with higher levels of maternally derived IgG before vaccination
]. In addition, IgA GMCs were not significantly different
between the 3-dose and 2-dose arms among infants who
seroconverted in the current study, indicating that infants receiving a
third dose of HRV have an additional chance to respond to the
vaccine at an older age, after maternal IgG antibody levels have
waned and the first dose of OPV is administered.
Vaccine-like virus shedding in this study was similar to
results from trials in other developing countries in which OPV
was concomitantly administered. Like other trials, shedding
primarily occurred following the first vaccination [
Bangladesh and South Africa, where HRV doses were provided at 12
and 16 weeks of age and at 6, 10, and 14 weeks of age,
respectively, approximately 5% and 13% of participants, respectively,
shed rotavirus following the first dose [
There were a few limitations to this study. First, we attempted
to balance the person-time exposure to wild-type infection
across study groups by using the highest titer of either the
14week or 18-week visit for group 1, although the group 1 results
from the 4-week postvaccination visit may be a more valid
comparison than this more conservative measure. Second, a baseline
blood sample was only collected immediately prior to the
10week HRV dose in group 2, while it was collected prior to the
6-week HRV dose for groups 1 and 3, allowing participants in
these groups an additional 4 weeks to be exposed to wild-type
infection, which would have been falsely counted as an immune
response to the vaccine. However, rotavirus season began later
than usual in 2012, with the first rotavirus cases detected via
routine surveillance in December and only 2 participants,
both in group 1, seropositive prior to receipt of HRV. Finally,
while serum IgA is not a mechanistic correlate of protection
], it has been used in the development of all live attenuated
rotavirus vaccines to demonstrate vaccine take and is the best
surrogate marker available [
]. Thus, the improved immune
responses observed in this and other African studies could
indicate that there would be improved clinical protection, even if
modest, in African populations.
Although there were only modest increases in immune
responses in this study and mixed results in other trials, it is
possible that administration of 2 doses on a delayed schedule or a
third dose in LIC populations may result in a large clinical
benefit. As administration of 2 doses of HRV on a delayed schedule
(1) misses the opportunity to vaccinate children at the first EPI
visit and prevent disease early in life and (2) was deemed
operationally suboptimal by the WHO [
], inclusion of a third dose
of HRV may be the most feasible option for improving clinical
benefit. Since inclusion of a third dose of HRV in the EPI
schedule would increase both cold chain capacity and vaccine costs, a
postmarketing study designed to investigate the effectiveness
and operational considerations of a third HRV dose could
determine whether this schedule results in public health benefit
and provide compelling evidence for policy change in LICs.
Acknowledgments. We thank all families who participated in this trial;
the research team in Ghana, Ms Margaret Atta-Poku, Mr Edward Sobe, Mr
Clement Narh, and the entire rotavirus study group at the Noguchi
Memorial Institute for Medical Research and the Navrongo Health Research
Centre, for their contributions to the study; and Dr Eileen Klein, from the
University of Washington and Seattle Children’s Hospital, for her role as
the independent medical monitor for this study.
Disclaimer. The findings and conclusions in this report are those of the
authors and do not necessarily represent the official position of the Gates
Foundation, PATH, or the Centers for Disease Control and Prevention.
Financial support. This work was supported by PATH through
funding from the Bill & Melinda Gates Foundation (grant OPP1017334).
Potential conflicts of interest. M. C. M. has a laboratory service
agreement with GSK and Merck. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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