Am. J. Trop. Med. Hyg., 72(2), 2005, pp.

189197
Copyright 2005 by The American Society of Tropical Medicine and Hygiene

RANDOMIZED, DOUBLE-BLIND, PHASE III, PIVOTAL FIELD TRIAL OF THE

COMPARATIVE IMMUNOGENICITY, SAFETY, AND TOLERABILITY OF TWO
YELLOW FEVER 17D VACCINES (ARILVAX AND YF-VAX) IN HEALTHY
INFANTS AND CHILDREN IN PERU
VIVIAN E. BELMUSTO-WORN, JOSE L. SANCHEZ, KAREN MCCARTHY, RICHARD NICHOLS,
CHRISTIAN T. BAUTISTA, ALAN J. MAGILL, GIOVANNA PASTOR-CAUNA, CARLOS ECHEVARRIA,
VICTOR A. LAGUNA-TORRES, BILLEY K. SAMAME, MARIA E. BALDEON, JAMES P. BURANS, JAMES G. OLSON,
PHILIP BEDFORD, SCOTT KITCHENER, AND THOMAS P. MONATH
Sutter Institute for Medical Research, Sutter Health, Sacramento, California: Divisions of Retrovirology and Communicable Diseases
and Immunology, Walter Reed Army Institute of Research, Washington, District of Columbia; U.S. Naval Medical Research
Detachment, Lima, Peru; Acambis Research Limited, Cambridge, United Kingdom; Acambis Inc., Cambridge, Massachusetts;
Ministry of Health, Sullana, Peru

Abstract. We conducted a randomized, double-blind, phase III yellow fever (YF) vaccine trial among 1,107 healthy
children in Sullana in northern Peru. The safety and efficacy (by measurement of geometric mean neutralizing antibody
titer responses) were determined for two YF vaccines, ARILVAX (n 738) and YF-VAX (n 369). Seroconversion rates were higher (94.9%) in ARILVAX than in YF-VAX (90.6%) recipients. The two-sided 95% confidence
interval (YF-VAXARILVAX) was (12.8% to 2.5%), indicating that the higher seroconversion rate for Arilvax
was significant. Post-vaccination (30-day) mean log10 neutralization indices were found to be similar for both products:
1.32 for ARILVAX and 1.26 for YF-VAX (P 0.1404, by analysis of variance). A similar number of subjects in each
group reported at least one adverse event (AE); 441 (59.8%) for ARILVAX versus 211 (59.9%) for YF-VAX. Most
(591; 96.7%) of these were of a mild nature and resolved without treatment. There were no treatment-related serious
AEs. This is the first randomized, double-blind comparison of two YF vaccines in a pediatric population; both vaccines
were shown to be highly immunogenic and well-tolerated.
Adverse reactions to ARILVAX and YF-VAX have
generally been mild and of low frequency.1 In published studies, self-limited mild and local reactions (erythema, swelling,
and pain at the injection site) and systemic reactions (feeling
ill, headache, myalgia, headache, and fever with or without
symptoms) have occurred in a minority of adult subjects 57
days after immunization.68
In Peru, YF vaccination has been included in the Expanded
Program of Immunization (EPI) of the Ministry of Health
(MoH) since 2000 and is recommended for all children beginning at nine months of age.9 Implementation of this vaccination policy has been highly variable, however, due in part
to shortages of YF vaccine. Moreover, vaccine use varies
across regions according to the risk of YF transmission. Continued occurrence of isolated and clusters of cases of YF
among Peruvians has continued to occur during the past 25
years. Indeed, Peru has had the highest incidence of YF in
South America during this interval.1 The YF virus is maintained in enzootic/epizootic sylvatic cycles involving nonhuman primates and forest tree-top dwelling mosquitoes. Humans generally are infected when they intrude into forested
areas (so-called jungle YF).10
Many cities in coastal areas of northern Peru are infested
with Ae. aegypti and thus are receptive to virus introduction
and resulting urban epidemics, in which humans rather than
monkeys serve as the principal host. Cases of YF have also
been increasing in Peru in the last few years in association
with specific hydrographic river basins,10,11 with some recent
cases reported in regions of greater geographic proximity
(within 100 miles) of the city of Sullana. Moreover, a pilot
review of immunization records conducted in mid 2001 in five
MoH immunization clinics in this Ae. aegypti-infested city
indicated that less than 3% of age-eligible children had received YF vaccine (Sanchez JL and others, unpublished data).
Since Sullana lies outside the YF enzootic area and YF vac-

INTRODUCTION
Yellow fever (YF) is a severe mosquito-borne hemorrhagic
disease that is endemic and epidemic in tropical South
America and Africa. It has the potential for introduction in
other areas, including the United States, where the Aedes
aegypti mosquito vector is present. The etiologic agent is the
prototype of the family Flaviviridae of single-stranded RNA
viruses that includes dengue and West Nile viruses.1 The clinical spectrum of YF infections can vary from subclinical to
severe systemic disease characterized by hepatitis, renal failure, bleeding diatheses, and cardiovascular collapse, and the
case fatality rate can be as high as 2050%.1,2 The World
Health Organization (WHO) estimates that 200,000 cases of
YF occur in endemic areas each year.3 In addition, there have
been several reports in recent years of deaths among unvaccinated tourists traveling to endemic areas.1,4
An attenuated YF 17D vaccine strain was developed in
1936 by serial passage of the wild-type parental Asibi virus in
mouse and chick embryo cells and is manufactured in a number of countries worldwide.1 More than 400 million persons
have been immunized with YF 17D vaccines since 1937. However, no controlled clinical studies of these vaccines have
been conducted in infants and children, who represent the
principal population in which vaccination is indicated in endemic areas. The origin, derivation, production, and genomic
sequences of these vaccines have been previously described.1,5 The vaccines, which are manufactured according to
standards developed by WHO must be administered at approved vaccination centers. Despite increasing demands for
the vaccine, the number of manufacturers has decreased from
13 in 1980 to 8 in 2000.1 YF-VAX (Aventis-Pasteur, Swiftwater, PA) is the only vaccine currently approved by the
Food and Drug Administration (FDA) and is manufactured
in the United States.

189

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BELMUSTO-WORN AND OTHERS

cine shortages have continued to exist, YF vaccination has not

been a priority in coastal regions of Peru. Therefore, Sullana
provided an ideal location for a clinical trial of YF vaccines
because children who might travel to nearby enzootic regions
of Peru or be exposed to urban outbreaks in the future would
benefit from vaccination.
We present the results of a randomized, double-blind,
phase III vaccine trial conducted among 1,107 healthy children in Sullana in northern Peru. The objectives of the study
were to determine the safety, tolerability, and efficacy (by
measurement of neutralizing antibody responses) of two YF
vaccines, ARILVAX and YF-VAX and to assess the consistency in production of three different lots of ARILVAX.
This research was carried out under an Investigational New
Drug Application approved by FDA in accordance with
Good Clinical Practice regulations and clinical research
guidelines established by the basic principles defined in the
U.S. 21 Code of Federal Regulations Part 312 and the Declaration of Helsinki. Prior to study start, the protocol (and
modifications) and the informed consent and assent forms
were reviewed and approved by a properly constituted Institutional Review Board (IRB) as recognized by the FDA.
MATERIALS AND METHODS
Vaccines. The YF vaccines compared were ARILVAX,
produced by Evans Vaccines Limited (formerly Evans Medical Limited; now part of Chiron Vaccines, Speke, Liverpool,
United Kingdom) and YF-VAX, manufactured by AventisPasteur Laboratories Inc., Swiftwater, PA). Both vaccines are
manufactured in embryonated chicken eggs and both vaccines
meet the international standards and requirements for YF
vaccines specified by WHO.12 There are minor differences in
passage histories and final formulations of ARILVAX and
YF-VAX; however, the two vaccines are estimated to be
greater than 99% homologous at the nucleotide sequence
level.13,14 The indication for use of these vaccines is the prevention of YF in persons traveling to, or living in endemic
areas of, South America and Africa. Vaccine is also indicated for laboratory personnel who might be exposed to virulent YF virus. As for all live vaccines, neither ARILVAX
nor YF-VAX vaccine contains a bacteriostatic agent.
ARILVAX is one of three YF 17D vaccines currently approved by WHO for supply to United Nations agencies.
Manufacture of YF 17D vaccine in the United Kingdom,
performed according to WHO specifications, commenced in
1945 at the Wellcome Foundation Limited (Wellcome, London, United Kingdom). In 1964, a leukosis-free vaccine was
introduced to the United Kingdom market and in 1976, the
vaccine was improved with the addition of stabilizers. The
vaccine was also approved for marketing in six other European Community Member States (Austria, Belgium, Finland,
Ireland, The Netherlands, and Sweden) and in eight other
countries (Hong Kong, Israel, Malaysia, Norway, Singapore,
South Africa, Switzerland, and Thailand). In 19951996,
manufacture of ARILVAX was initiated at Evans Medical
Limited (Leatherhead, United Kingdom) by inoculation of
chicken embryos with seed virus acquired from Wellcome.
The only significant difference between the Wellcome and
Evans Medical manufacturing processes, apart from the site
of manufacture, is that the current vaccine does not contain
antibiotics (polymyxin B sulfate and neomycin sulfate).

The three lots of ARILVAX used in this study were

manufactured by Evans Vaccines Limited from seed virus
acquired from Wellcome. ARILVAX is supplied as a
freeze-dried powder in a single dose vial containing not less
than 4.4 log10 plaque-forming units (PFU)/0.5-mL dose of YF
17D virus at release and which will contain not less than 3.7
PFU/0.5 mL at the end of shelf life. The virus content meets
or exceeds the WHO specification for potency.12 The vaccine
also contains orthophosphate, potassium chloride, potassium
dihydrogen orthophosphate, sorbitol, and porcine gelatin.
The vaccine is reconstituted in 0.75 mL of diluent (water for
injections) and 0.5 mL is injected by the subcutaneous route.
After reconstitution, the vaccine appears opalescent and is
pink-brown in color.
YF-VAX is licensed by the FDA in the United States. It
is also supplied as a freeze-dried powder in single dose or
five-dose vials containing not less than 5.04 log10 PFU/0.5-mL
dose of the attenuated 17D virus. The virus content meets or
exceeds the WHO specification for potency.12 The vaccine
contains sorbitol and porcine gelatin without added salts. It is
reconstituted in 0.6 mL of preservative-free sodium chloride
United States Pharmacopeia, and 0.5 mL is injected by the
subcutaneous route. After reconstitution, the vaccine appears
slightly opalescent and is light orange in color.
Trial design and study population. This study was designed
as a randomized, double-blind vaccine trial that was conducted in healthy children 9 months to 10 years of age (inclusive) in Sullana, a city in the northern coastal region of
Peru. The study sponsor was Acambis Inc. The study was
conducted by the U.S. Naval Medical Research Detachment
(NMRCD) in Lima, Peru. The protocol was reviewed by the
Ministry of Health of Peru and by the Scientific Review Committee of the Walter Reed Army Institute of Research. The
protocol and supporting documents were reviewed and approved by three IRBs, including the Universidad Peruana
Cayetano Heredia, the U.S. Naval Medical Research Center,
and the Surgeon Generals Human Subjects Research Review
Board (HSRRB). Clinical monitoring of the study was conducted by staff of PRA International (Lenexa KS), a contract
research organization. At monitoring visits, the study binder
was reviewed and updated, verification of informed consent
was performed, and a 100% source document verification of
data in the case report form (CRF) was performed to ensure
accuracy and reporting of adverse events (AEs). Drug accountability was also performed. A Peruvian physician served
as Medical Monitor and was able to independently report
AEs to the Peruvian IRB and the HSRRB. A Data and
Safety Monitoring Board (DSMB) was established consisting
of two physicians in Peru and one in the United States who
reviewed all serious AEs. Data entry was the responsibility of
Acambis and Syne qua non Limited (Norfolk, United Kingdom). Statistical analyses were provided by Syne qua non
Limited and ViruStat Limited (North Wales, PA).
Five recruitment centers (health posts and clinics) within
the city were selected based on staff availability and logistical
considerations. The initial objective was to be able to vaccinate and complete follow-up of 1,050 subjects assuming a loss
to follow-up of no greater than 510%. Planned recruitment
was for 750 children in the nine month to five years of age
group (cut-off at the fifth birthday) and for an additional
300 children in the 510-year-old age group (cut-off at the
10th birthday). Within each of the two groups, a 2:1 ratio of

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PEDIATRIC PHASE III TRIAL OF TWO YF VACCINES

ARILVAX to YF-VAX was followed. Vaccination was

conducted between the months of May and November 2002,
inclusive.
Randomization. A stratified randomization procedure was
followed to ensure equal distribution of vaccination by age
group and sex. Additionally, for the ARILVAX vaccine
product only, randomization was also stratified according to
production lot (that is, to achieve enrollment and follow-up of
at least 167 subjects for each of the three conformance lots).
All study staff who dealt with subjects in the study, as well as
co-investigators and laboratory personnel who performed
laboratory testing at NMRCD-Lima and Acambis Inc., remained blinded to the vaccine type and lot assignments.
Study enrollment criteria and follow-up procedures. Participation in the study was limited to infants and children
meeting the inclusion and exclusion criteria. Exclusion criteria included children falling outside of the age parameters,
those who were unwell or demonstrated poor growth, those
who had received another vaccine in the previous 30 days or
who had documented YF vaccination in their personal or
clinic immunization records, those who had immunosuppressive illness or were taking immunosuppressive medications,
and those who gave reliable histories of egg allergies. Children who were due for routine childhood vaccinations would
receive them according to Peruvian MoH guidelines and were
re-scheduled for participation in the study once the 30-day
period elapsed and if the parents still wished to have their
child participate.
The study procedures are outlined in Table 1. The vaccination center was equipped with a full-time study pediatrician
and nurse as well as with the necessary emergency facilities
for maintenance care in response to a serious allergic reaction. Children were vaccinated with one of the two vaccines
administered subcutaneouly into the deltoid region of the upper arm. They were kept at the center for a minimum of 30
minutes to monitor for any systemic allergic reactions. Children failing to present for scheduled clinic appointments were
visited in their homes.
Safety was assessed by recording all AEs that occurred

after vaccination. On all clinic visits (Table 1), a structured

interview was conducted for AEs and the diary cards were
reviewed. All AES were recorded in the case report forms,
including severity, the investigators assessment of causality
(relationship to study vaccine), start date and end date, and
whether treatment was required. Children failing to present
for scheduled appointments were visited in their homes. Parents/guardians were instructed to return to the clinic if the
child developed a fever (oral temperature 38C/100.4F) or
if they were in any way concerned with the health of their
child during the follow-up period (days 131). Subjects who
developed a generalized febrile illness within the first 10 days
post-vaccination were carefully evaluated. The studys on-site
investigator (VEB-W) determined if there was a plausible
explanation for the illness, such as a respiratory infection. If
there was no plausible explanation, a blood sample was taken
to help determine the nature of illness (by liver function tests
[LFTs] and viremia).
Demographic and clinical information was recorded on
standardized data forms (source documents) and transcribed
into CRFs, which were then sent in hard copy for data entry
and statistical analysis with copies retained at the Sullana
study site under lock and key until study termination.
All subjects completing the 31-day follow-up received confirmation of immunization against YF, which was deemed
valid by Peruvian MoH authorities.
Antibody tests. Neutralizing antibody is the principal mediator of immunity elicited by YF 17D vaccines and has been
proven to be correlated with protection from disease in nonhuman primates.6,15 Sera collected on days 1 and 31 were
tested for neutralizing antibodies to YF and serum collected
on day 1 was tested for evidence of pre-existing immunity to
dengue virus serotypes 1, 2, 3, and 4 by an enzyme-linked
immunosorbent assay and a plaque-reduction neutralization
test (PRNT) at NMRCD-Lima. Neutralizing antibody titers
to YF were measured at Acambis Inc. using a constant serum
varying virus assay performed in cell culture previously standardized by Bureau of Biologics (FDA) (Rockville, MD).15,16
Acambis Inc. has used this method to establish a validated

TABLE 1
Study procedures and schedule
On study period day (range)
Study day

Clinic visit
Informed consent
Inclusion/exclusion criteria
Medical history
Physical examination
Vital signs
Blood collections
Serum for yellow fever neutralizing antibody
Serum for dengue neutralizing antibody
Baseline AST, ALT (retention sample)
AST, ALT, quantitative viremia
Randomization
Vaccination
Diary card
Subject interviews for adverse events

X
X
X
X
X
X

X*

X*

X*

X
X
X
X
X
X

11 (1114)

31 (2438)

X
At any time days 210
Days 110

* Interim physical examinations are performed at other visits at the discretion of the investigator, if necessary, to investigate adverse events or clinical laboratory abnormalities.
Serum for liver function (aspartate aminotransferase [AST], alanine aminotransferase [ALT]) frozen for subsequent analysis as required to assist in the interpretation of adverse events during
days 210.
Blood tests (AST, ALT, and viremia) to investigate unexplained febrile illness occurring between days 2 and 11.
AST and ALT repeated on day 31 if abnormal results were obtained on samples tested between days 2 and 11 and repeated at day 31 to determine resolution.

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BELMUSTO-WORN AND OTHERS

neutralization test in Vero cells. This test measures the neutralizing capacity of serum.
Seroconversion to YF virus was defined as a log10 neutralization index (LNI) 0.7. The LNI is calculated as the log10
difference in virus titer of a mixture of serum and virus between baseline (pre-immunization) and post-immunization
samples. The post-vaccination serum from a subject who did
not seroconvert had to exhibit an LNI 0.7 compared with
the pre-vaccination serum. The cut-off value for a positive
LNI 0.7 was established by protection studies in nonhuman primates and represents the antibody titer required to
protect against lethal challenge.15 In addition, an LNI cut-off
0.7 was used as the endpoint for seroconversion in a previous clinical trial comparing ARILVAX with YF-VAX
conducted in the United States by Acambis Inc.6
Statistical methods. Sample size. We estimated that a
sample size of 144 subjects per treatment group (all age
ranges) would be required to show a study power of 0.80 in a
one-sided test for non-inferiority at a significance level (alpha
error) of 0.05 and with a 5% exclusionary percentage when
the underlying seroconversion rates are equal and 97% in
each group. All 700 subjects in the ARILVAX treatment
group and 350 in the YF-VAX treatment group (total
1,050) were to be tested for a YF serologic response. The
sample size for efficacy exceeded that required based on statistical assumptions because it could not be predicted in advance what proportion of subjects would not be evaluable for
efficacy (seronegative to YF at baseline). Moreover, prior
dengue immunity may interfere with the immune response to
YF vaccine, reducing the seroconversion rate and increasing
the sample size for demonstrating non-inferiority.
The sample size estimations also established an upper
bound of 0.004 for the 95% confidence interval (CI) for the
incidence of a severe AE (SAE) in the case that such an event
was not observed among the 700 subjects in the ARILVAX
treatment group and an upper bound of 0.009 for the 95% CI
for the 350 subjects in the YF-VAX treatment group.
For the objective of demonstrating clinical consistency of
three ARILVAX conformance lots considered in a pairwise analysis, a sample size for conformance of 62 subjects per
group was estimated to provide a study power of 0.90 in a
one-sided test of non-inferiority at an alpha significance level
of 0.025 (or an overall 0.025 level alpha test of equivalence)
with a 10% exclusionary percentage when the underlying seroconversion rates are equal and 97% in each group.
Primary analysis. Since the primary goal of this study
was to demonstrate non-inferiority in immunogenicity of
ARILVAX compared with YF-VAX, the primary efficacy analysis consisted in comparing the proportion of subjects who seroconverted to YF virus 30 days post-vaccination
in the two vaccine groups. Initially, non-inferiority was defined as the seroconversion rate following ARILVAX
not being lower than the seroconversion rate following
YF-VAX by more than a clinically acceptable difference of
5%. Following subsequent discussions with the FDA, a narrower limit of confidence for non-inferiority was applied. The
non-inferiority test was repeated using a one-sided test at a
significance level of 0.025. Moreover, the upper limit for inferiority was calculated as the one-sided 97.5% confidence
limit. Finally, the two-sided 95% CI for the difference of proportions between treatment groups was also calculated. These
tests were performed to evaluate the proportion of subjects

seroconverting to YF following vaccination with either

ARILVAX or YF-VAX up to the day 31 visit from the
combined age groups, 9 months to 10 years inclusive. These
analyses were repeated for the following age groups: 918
months, > 1836 months, > 3660 months, and 60 months plus
one day to 10 years. Only study subjects without prior YF
immunity at baseline, and who actually received one of the
two study vaccines and were tested for YF neutralizing antibody on day 31, were included in the per-protocol population.
Secondary analysis. The secondary goal of this study was
the comparison of geometric mean titers (GMTs) between
the two vaccine groups. Analyses were performed to show
that the 95% CI on the ratio of the two treatment group
geometric mean YF neutralizing antibody titers (expressed as
mean LNI), rules out a half-fold decrease and a two-fold
increase (ARILVAX to YF-VAX). Analyses were repeated for the following age groups: 918 months, > 1836
months, > 3660 months, and > 60 months to 10 years.
Clinical consistency. A pairwise comparison of the geometric mean YF neutralization titer (expressed as mean LNI)
was used to test the three ARILVAX clinical consistency
lots for homogeneity. Use of this analysis shows that the 95%
CI on the ratio of means rules out both a half-fold decrease
and a two-fold increase in geometric mean YF neutralizing
antibody titers from the combined age groups (9 months to 10
years inclusive) between each ARILVAX lot on day 31. A
secondary analysis of ARILVAX lot homogeneity was carried out using equivalence tests using the 95% CI for the
difference of proportions of subjects who seroconverted.
Where pairwise CIs all fall between 10% and +10%, the
limits identified in the study protocol, the three lots were to
be considered equivalent.
Supplemental analyses. Supplemental analyses were performed to address the role of host factors (sex, age, and sex by
age) and consistency of YF antibody response between the
three ARILVAX lots. Other analyses included a comparison of the mean LNI on day 31 between age groups with and
without adjustment for pre-existing dengue immunity
(present or absent), and ARILVAX conformance lot effects.
Safety analysis. Safety and tolerability were assessed by
comparison of the incidence (expressed in percent) of AEs
across the two treatment groups by a chi-square test (Fishers
exact test as required). Separate analyses were performed to
determine whether subjects with and without antibodies to
YF or dengue at baseline differed with respect to the incidence of AEs in the two vaccine groups.
RESULTS
Demographic and baseline characteristics of participants. A total of 1,107 children were eligible for participation
and were randomly assigned to a study group at the time of
vaccination. Of these, 738 received ARILVAX and 369
received YF-VAX (an exact 2:1 ratio of vaccine group randomization was achieved). There were no age and sex differences in terms of the population receiving vaccination at baseline (Table 2). At baseline, 36 (26 receiving ARILVAX
and 10 receiving YF-VAX) children were found to be YF
immune; 82 (54 and 28 in the two treatment groups, respectively) did not have two samples taken for serologic testing,

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PEDIATRIC PHASE III TRIAL OF TWO YF VACCINES

TABLE 2
Description of the study population (for all vaccinees)
Statistic

ARILVAX
(n 738)

YF-VAX
(n 369)

Caucasian
Black
Mixed
Mean
Standard deviation
9 months to 18 months
18 months + 1 day to 36 months
36 months + 1 day to 60 months
60 months + 1 day to 10 years
Male
Female

8 (1.1)
1 (0.1)
729 (98.8)
48.6
30.44
144 (19.5)
131 (17.8)
243 (32.9)
220 (29.8)
357 (48.4)
381 (51.6)

4 (1.1)
0 (0)
365 (98.9)
50.1
30.67
59 (16.0)
77 (20.9)
123 (33.3)
110 (29.8)
178 (48.2)
191 (51.8)

Demographic feature

Ethnicity
no. (%)
Age (months)
Age group
no. (%)
Sex
no. (%)

P*

1.0000
0.4421
0.3999

1.0000

* T-tests used for continuous variables; chi-square tests for categorical variables. YF yellow fever.
Age derived from date of day 1 visit and date of birth. Years/months have been rounded down to the lowest whole number, where applicable.

one child had a baseline titer that could not be determined,

and seven children otherwise did not complete the protocol.
This left a final (per protocol population) sample of 981
(nARILVAX 652; nYF-VAX 329) children for endpoint
analyses.
There were also no statistically significant differences between treatment (vaccine) groups in terms of the proportion
of children randomized to each vaccine by study site, mean
age, sex, weight (in kg), height (in cm), body mass index
(kg/m2), pulse (beats per minute), allergy history, anaphylactic reaction history, or significant pre-existing medical conditions. No differences were found for the pre-existing (baseline) prevalences of antibody to YF (4.1% versus 3.0%) or
antibody to dengue by PRNT50 (14.3% versus 15.0%) between the ARILVAX and YF-VAX treatment groups,
respectively.
Efficacy. Overall, 619 (94.9%) of the 652 ARILVAX
and 298 (90.6%) of the 329 YF-VAX recipients in the per
protocol final sample seroconverted to YF virus. The seroconversion rate for ARILVAX was statistically noninferior to that for YF-VAX (P < 0.0001, by one-sided test of
non-inferiority). Moreover, the upper limit of the 97.5% CI
for YF-VAX minus ARILVAX was 0.025%, indicating
that ARILVAX actually produced a significantly higher
response rate than YF-VAX. The two-sided 95% CI for
YF-VAX minus ARILVAX ranged from 12.8% to
2.5%, which also indicates that ARILVAX produced a
significantly higher seroconversion rate than YF-VAX (P <
0.05). Analysis for each of the four age groups indicated in all
cases a significantly higher seroconversion rate, as well as
non-inferiority between treatment groups (Table 3). The difference between seroconversion rates for the two vaccines

was most striking in the lowest age groups. In addition, no

differences in seroconversion rates were detected between
the male and female participants (P 0.82).
Notably, the seroconversion rates did not vary according to
baseline level of dengue immunity or vaccine administered
(Table 4). Among dengue immune and non-immune participants, 93.6% and 94.4% in the ARILVAX group versus
82.7% and 92.0% in the YF-VAX group, respectively, seroconverted. Baseline immunity to specific dengue serotypes
was not found to affect the YF seroconversion rates following
vaccination.
Yellow fever antibody titer response. The absolute level
(GMT) of the YF vaccine-induced antibody response (expressed as the mean YF LNI) was not found to differ in dengue
immune versus non-immune individuals (Table 4). Overall, the
mean ( SD) LNI was 1.32 ( 0.56) for ARILVAX compared
with 1.26 ( 0.65) for YF-VAX (Table 5). Using analysis
of variance to calculate the 95% CI for the difference in mean
LNI, we found that there was no difference between the
ARILVAX and YF-VAX treatment groups at day 31
(P 0.1404). More importantly, the 95% CI on the ratio of
geometric mean YF neutralizing antibody titers was 0.9563
1.3706, which falls within the interval 0.52.0, thus establishing
equivalence of ARILVAX and YF-VAX in terms of neutralizing antibody titer levels. Additional sub-analysis for each of
the four age groups showed no differences between the two
vaccine groups (Table 5).
Tests for ARIVAX lot consistency. Six hundred fiftytwo doses of ARILVAX were administered from three
conformance lots as follows: lot 760467, n 216; lot 760468,
n 217; and, lot 761294, n 219. The 95% CI on the ratio
of means ruled out both a half-fold decrease and a two-fold

TABLE 3
Yellow fever (YF) seroconversion rates at day 31 (per protocol population) by age groups

Feature

Total evaluated
Age group
918 months
> 1836 months
> 35 years
> 510 years

ARILVAX
% (no./No.)

YF-VAX
% (no./No.)

Noninferiority
P*

One-sided
97.5% for
inferiority*

Two-sided 95%
confidence
interval

94.9 (619/652)

90.6 (298/329)

< 0.0001

0.025

(0.128, 0.025)

95.8 (115/120)
94.6 (106/112)
93.1 (202/217)
96.6 (196/203)

88.5 (46/52)
86.2 (50/58)
92.3 (108/117)
92.2 (94/102)

0.0016
0.0018
0.0231
0.0003

0.010
0.006
0.049
0.009

(0.191, 0.010)
(0.201, 0.006)
(0.076, 0.049)
(0.114, 0.009)

* One-sided test of non-inferiority at the 2.5% level of significance with 5% non-inferiority bound based on Chans test.

194

BELMUSTO-WORN AND OTHERS

TABLE 4
Comparison of yellow fever (YF) immune response 30 days after
vaccination for subjects with and without baseline dengue (any
serotype) immunity with paired samples (by vaccine study group)*
ARILVAX group

YF-VAX group

Baseline
dengue
immunity

Seroconversion
rate

Mean
YF LNI

Seroconversion
rate

Mean
YF LNI

Present
Absent

88/94 (93.6%)
32/573 (94.4%)

1.449
1.336

43/52 (82.7%)
23/288 (92.0%)

1.332
1.254

* No statistically significant difference for comparison of seroconversion rates (P

0.0980) or mean YF log10 neutralization index (LNI) (P 0.1659) between vaccine study
groups within a linear regression model accounting for dengue immunity as a host factor.

increase in geometric mean YF neutralizing antibody titers

from the combined age groups (9 months to 10 years inclusive) between each ARILVAX lot on day 31, establishing
equivalence of the three lots. Moreover, the proportion of
children who seroconverted to each lot was very similar ranging from 94.0% to 96.3%.
Effect of host (sex and age) factors. In a model of host
factors including age and sex analyzed using unconditional
logistic regression, the overall YF seroconversion rate for
subjects vaccinated with ARILVAX continued to be significantly higher compared with that after vaccination with
YF-VAX (P 0.01). Moreover, when stratifying for dengue
immunity using the model applied to the entire per protocol
population (n 981), we found that the difference in seroconversion rates between ARILVAX and YF-VAX was
retained (P 0.03). Within the model, none of the host
factors had a statistically significant impact on the YF antibody seroconversion rate. The significance (P) values for the
host factors that included sex, age, and sex by age were 0.82,
0.49, and 0.46, respectively. Similar regression analysis based
on log mean YF neutralizing index (LNI) antibody titers at
day 31 also indicated that the role of host factors did not
influence final YF antibody response (P 0.50).
Safety assessments. No SAEs were experienced by children receiving ARILVAX; however, two children had
three unrelated, though serious, AEs after YF-VAX. One
child had an episode of bronchial pneumonia and another a
urinary tract infection as well as a documented diarrheal episode attributed to an enteropathogenic Escherichia coli infection.
The first subject was a six-year-old girl who developed fever 13 days after vaccine administration and was subsequently
hospitalized. The workup showed a urine culture positive for
Klebsiella, as well as a stool culture positive for enteropathogenic E. coli. Results of liver function studies were normal.

She was treated with appropriate antibiotics and recovered

uneventfully.
The second subject was a 10-month-old boy who was hospitalized the day after vaccination with a bronchial obstruction, which was diagnosed as focal pneumonia by chest radiograph. Further inquiry showed that the subject had developed
a cough two days before vaccination, suggesting that the process was occurring prior to vaccine administration. The subject was treated with antibiotics and also recovered uneventfully. No subjects had a febrile syndrome clinically suspicious
of YF vaccineassociated viscerotropic disease (YEL-AVD)
requiring liver function or viremia investigations.
A third subject who did not meet the criteria for an SAE
presented on day 31 follow-up with scleral icterus. Preliminary LFTs showed mildly elevated levels of liver enzymes and
subsequent blood work showed IgM for hepatitis A. Hepatitis
A is endemic throughout this population and given the time
frame of the event (approximately 29 days after vaccine administration), it is highly unlikely that the jaundice was vaccine related. This child was followed until resolution of symptoms was documented.
The incidences of reporting one or more AEs were almost
identical (Table 6) between the two vaccine study groups. Of
these, investigators blinded to the vaccine identity determined approximately half the subjects were reporting AEs
related to vaccination. The majority of related AEs were mild
in nature and resolved within 2448 hours post-vaccination.
The profile of commonly reported (> 5% of subjects) AEs
following each vaccine was similar (Table 7). Given the fact
that these reports include events that may or may not be
related to vaccination, the profile includes common pediatric
conditions. In addition to these common AEs, it should be
noted that only 28 (3.8%) reports of injection site pain were
received following ARILVAX vaccination and 5 (1.4%)
following YF-VAX administration, although it is accepted
that because of the age of the subjects in this study, underreporting of such symptoms most probably occurred.
DISCUSSION
For the purposes of international travel certification, YF
revaccination is required every 10 years.1,17 However, immunity has been documented to last more than 35 years18 and is
probably life long. Thus, durability of immunity could not be
tested in a clinical trial setting. In our study, we demonstrated
that children receiving ARILVAX met the primary endpoint showing statistical non-inferiority in seroconversion
rates compared with the active control, YF-VAX. Indeed,

TABLE 5
Geometric mean yellow fever (YF) neutralization antibody titer on day 31 (per protocol population) for age groups*
Feature

Total evaluated
Age group
918 months
> 1836 months
> 35 years
> 510 years

ARILVAX
Mean LNI (SD)

YF-VAX
Mean LNI (SD)

1.32 (0.56)

1.26 (0.65)

0.1404

0.059

1.31 (0.51)
1.36 (0.58)
1.27 (0.56)
1.33 (0.56)

1.16 (0.61)
1.24 (0.62)
1.23 (0.51)
1.35 (0.80)

0.0850
0.2086
0.4549
0.8398

0.157
0.122
0.047
0.016

* LNI log10 neutralization index; CI confidence interval.

By analysis of variance (analysis one-way for difference between treatments).

Mean difference

95% CI

0.0194, 0.1369
0.022, 0.337
0.069, 0.313
0.076, 0.169
0.171, 0.139

PEDIATRIC PHASE III TRIAL OF TWO YF VACCINES

TABLE 6
Summary of adverse events (AEs) by age group and severity
Parameter

ARILVAX
n 738 (%)

YF-VAX
n 369 (%)

Number of reported AEs

Subjects with 1 AE
918 months old reporting AE
1836 months old reporting AE
3660 months old reporting AE
60120 months old reporting AE
Subjects reporting related AE
Mild related AE
Moderate related AE
Severe related AE

1,365
441 (59.8)
98 (68.1)
90 (68.7)
156 (64.2)
97 (44.1)
216 (29.3)
396 (53.7)
12 (1.6)
0

612
221 (59.9)
49 (83.1)
54 (70.1)
62 (50.4)
56 (50.9)
111 (30.1)
195 (52.8)
8 (2.2)
0

the upper limit of the 97.5% CI for YF-VAX minus ARILVAX was 2.5%, indicating that ARILVAX actually
produced a significantly higher response rate than YF-VAX.
The two vaccines elicited equivalent neutralizing antibody titers.
Interestingly, for both ARILVAX and YF-VAX vaccines, seroconversion rates in Peruvian children were lower
than those recently reported in adults in the United States
(99%).6 The overall seroconversion rate in children who received ARILVAX (94.9%) was higher than that in children
who received YF-VAX (90.6%), and the difference in seroconversion rate was most pronounced in the two youngest age
groups (95.8% versus 88.5% in children 918 months old and
94.6% versus 86.2% in children 1836 months old). In Africa,
young children represent the age group at highest risk, and
this would also be true in South America if YF virus were
introduced into coastal regions and transmitted by the urban
vector Ae. aegypti. The reason for the higher seroconversion
rates in adults6 than in children is unclear. It is possible that
both the ethnic background of the population and age may
have played a role. In the adult study reported by Monath and
others,6 Hispanics had slightly lower antibody titers than Caucasians. One previously published report from Latin America
(Brazil) noted that young children had lower immune responses than older persons to YF vaccine.19
Factors that can potentially affect the immune response to
YF 17D vaccine include 1) pre-existing immunity to antigenically related, cross-protective flaviviruses, such as dengue viruses; 2) immunosuppression due to underlying diseases or
drug treatment; 3) severe malnutrition; and 4) pregnancy. The

TABLE 7
Common adverse events reported (incidence 5%)*
MedDRA (diagnostic)
terminology

ARILVAX
n 738 (%)

YF-VAX
n 369 (%)

Pyrexia
Pharyngitis
Diarrhea NOS
Nasopharyngitis
Appetite decreased NOS
Malaise
Cough
Pharyngotonsillitis
Headache
Vomiting NOS

197 (26.7)
129 (17.5)
91 (12.3)
64 (8.7)
62 (8.4)
57 (7.7)
55 (7.5)
45 (6.1)
44 (6.0)
43 (5.8)

98 (26.6)
54 (14.6)
40 (10.8)
32 (8.7)
34 (9.2)
26 (7.0)
20 (5.4)
21 (5.7)
26 (7.0)
11 (3.0)

* MedDRA Medical Dictionary for Regulatory Activities; NOS not otherwise specified.

195

incidence of illness following infection with wild-type YF virus is higher in males, but it is uncertain whether this is due to
epidemiologic or host susceptibility factors.1 In the phase III
trial conducted among adults in the United States, males had
higher mean neutralizing antibody titers than females.6 However, in this study among young children in Peru, no such
effect was observed.
Many areas of YF endemicity overlap with those of dengue
transmission, particularly in South America. Therefore, dengue immunity was investigated to determine whether this
modulates the immune response to YF vaccine. Previous wild
dengue infection with residual immunity has been shown to
reduce the immune response to YF vaccine,20 and dengue
immunity was shown to partially cross-protect against YF in a
monkey model.21 There is no evidence of this effect in the
data collected here, and quite the contrary, it would appear
that the mean YF antibody (LNI) response for dengueimmune children after vaccination is higher than that among
children which are not dengue-immune, suggesting flavivirus
antigenic cross-reactivity. Dengue infection, particularly with
serotypes 1 and 2, is evidently common among young children
of the Sullana city area. While the response to ARILVAX
is comparable to that observed among children not immune
to dengue, the proportions of children responding to YFVAX is less than 90%. The trend to lesser seroconversion
rates for YF-VAX in dengue-immune individuals compared
with ARILVAX is not statistically significant. This may be
due to the small sample sizes or reflects the overall lesser
response to YF-VAX regardless of dengue immunity.
The underlying reasons for dengue modulation of YF infection or vaccination deserves further study, and is particularly relevant in Peru and other South American countries
where since the early 1990s, dengue has invaded the YFendemic region. As early as 1923, dengue immunity was suggested as the basis for resistance to YF in long-term residents
of endemic areas, and was later proposed as a barrier to introduction of YF into Asia.22 However, the evidence for interference with 17D vaccine in humans is conflicting and discrepancies across studies may be due to the number of prior
Flavivirus infections, the breadth of heterotypic response, or
the identity of the viruses responsible for prior infection.
In a phase III clinical trial of ARILVAX and YF-VAX
conducted in the United States in 1999,6 significantly more
subjects in the YF-VAX group (71.9%) experienced one or
more drug-related AEs when compared with subjects in the
ARILVAX group (65.3%; P 0.008). The difference between subjects receiving these treatments was due to a higher
rate of local reactions in the YF-VAX group. Interestingly,
we did not find such AE rates to be any different among
young Peruvian children.
In the United Kingdom and the United States, approximately 6,0009,000 infants and 3,0004,000 children between
9 months and 10 years of age annually receive ARILVAX
or YF-VAX, respectively. There is no evidence that the
safety profile of these vaccines differs in children who are
9 months old from that of adult subjects. Yellow fever vaccineassociated neurotropic adverse events (YEL-AND, formerly called post-vaccinal encephalitis) are a rare, age-related
complications of YF vaccination. Encephalitis has been reported in the published literature in 25 cases since standardization of YF vaccine manufacture in 1945, of which 16 cases
were in infants 7 months old, prior to establishment of a

196

BELMUSTO-WORN AND OTHERS

minimum age for vaccination.1 The incidence of encephalitis

following YF vaccination in children 9 months of age is not
known with precision, but is believed to be very low. Full
recovery from encephalitis is the rule, but one fatal case occurred in the United States in a three-year-old child.23
Hypersensitivity reactions to egg proteins, gelatin, or other
allergens contained in YF 17D vaccine are rare. The incidence of such reactions has historically been estimated to be
approximately 1 per million,17 but recent data suggest that
this may be an underestimation. Post-marketing reports of
generalized allergic reactions to YF-VAX between 1991 and
1997 indicate an incidence of 1 in 131,000.1,24 An analysis of
Vaccine Adverse Event Reporting System data for YFVAX was conducted by the Centers for Disease Control and
Prevention. During the interval 19901997, there were an estimated 1.3 million doses administered to civilians and 31
SAEs defined as neurologic or systemic reactions persisting
greater than 48 hours, giving a rate of 2.3 per 100,000 vaccinees. The incidence of SAEs was significantly higher in elderly subjects.25
During a mass immunization campaign in 19992000 in
Brazil, deaths associated with YF 17D vaccination occurred in
a five-year-old child and a 22-year-old adult.22 The clinical
presentation and pathologic evidence suggested that the vaccine caused hepatitis and a syndrome similar to wild-type YF.
These reactions appeared to be host-related rather than due
to mutation(s) in the vaccine virus. Serious AEs of this kind
appear to be extremely rare.27,28 Unfortunately, the sample
size in this study was too small to identify rare serious AEs
such as systemic allergic reactions, YEL-AND, or YELAVD.
Finally, the lot-to-lot consistency of production of
ARILVAX was demonstrated in this study by comparing
geometric mean (LNI) antibody titers and seroconversion
rates to the three lots. Each lot of ARILVAX produced
seroconversion rates in excess of 94%, all greater than levels
achieved by YF-VAX. Therefore, no lot of ARILVAX
was considered inferior to YF-VAX and the ARILVAX
lots were assessed to be equivalent.
No controlled clinical trials of ARILVAX and YFVAX (or any of the other YF 17D vaccines) had been previously conducted among infants and children. This is the
largest such study ever conducted and as recently shown in
adults,6 confirms that these two vaccines are well tolerated
and highly immunogenic. It is noteworthy that YF 17D vaccines, including ARILVAX and YF-VAX, have been
used for decades in travelers 9 months of age without recognized safety problems, and YF 17D vaccines produced by
major manufacturers (Biomanguinhos, Rio de Janeiro, Brazil;
Institute Pasteur, Dakar, Senegal; and Aventis-Pasteur, Lyon,
France) are used in infants starting at nine months of age
undergoing routine immunization in the EPI in South
America and Africa. There have been recent increases in
demand for YF vaccines associated with a concomitant expansion of YF virus circulation in endemic countries, as well
as an increase in travel by non-immune persons from developed countries. ARILVAX represents an additional, welltolerated and immunogenic vaccine, which can now be added
to the armamentarium of YF vaccines available to travelers
and other populations at risk of exposure. This study was one
of several trials that were conducted for the purposes of obtaining approval from the U.S. FDA for use in the United

States. The clinical data on safety and immunogenicity in children contained in this report will be summarized on the product label. If approval is granted, the vaccine will be sold and
distributed in the United States for protection of travelers,
military, and laboratory personnel.
Received June 9, 2004. Accepted for publication August 17, 2004.
Acknowledgments: We thank Dr. Nancy Rivera (PRA International)
for her continued and superb monitoring of this study. We express
our sincere appreciation to Eppie Chang (Sutter Institute for Medical
Research, Sutter Health) for her generous support of Vivian E. Belmusto-Worn in this study. We also express our sincere appreciation to
the innumerable staff at the U.S. Naval Medical Research Detachment-Lima laboratory for their excellent support in all aspects of this
study. Lastly, we are indebted to all study participants and field workers in the city of Sullana, Peru, who with their commitment and hard
work made this study possible.
Financial support: This study was supported by a Cooperative Research and Development Agreement between Acambis Research
Limited (Cambridge, United Kingdom) and the U.S. Naval Medical
Research Center (Silver Spring, MD), NCRADA-NMR-01-1233
(September 2001).
Disclaimer: The opinions and assertions made by the authors do not
reflect the official position or opinion of the U.S. Department of the
Navy or Army or the Peruvian Ministry of Health.
Disclosure: Some of the authors of this paper wish to disclose that
they are Acambis employees and may hold stock in Acambis. This
statement is made in the interest of full disclosure and not because
the authors consider this to be a conflict of interest.
Authors addresses: Vivian E. Belmusto-Worn, U.S. Naval Medical
Research Center Detachment, Lima, Peru, American Embassy-Lima,
Unit 3800, APO AA 34031-3800, Telephone: 51-1-561-2882/3848,
Fax: 51-1-561-3042, E-mail: [email protected]. Jose L.
Sanchez, Department of Epidemiology and Threat Assessment, U.S.
Military HIV Research Program, Division of Retrovirology, Walter
Reed Army Institute of Research, 13 Taft Court, Suite 200, Rockville,
MD 20850, Telephone: 301-251-5000, Fax: 301-762-4177, E-mail:
[email protected]. Karen McCarthy, Clinical Operations,
Acambis Research Limited, Peterhouse Technology Park, 100 Fulbourn Road, Cambridge CB1 9PT, United Kingdom, Telephone: 441223-275-300, Fax: 44-1223-416-300, E-mail: karen.mccarthy@
acambis.com. Richard Nichols, Acambis Inc., 38 Sidney Street,
Cambridge, MA 02139, Telephone: 617-761-4200, Fax: 617-494-1741,
E-mail: [email protected]. Christian Bautista, Data Center,
U.S. Naval Medical Research Center Detachment, Lima, Peru,
American Embassy-Lima, Unit 3800, APO AA 34031-3800, Telephone: 51-1-561-2882/3848, Fax: 51-1-561-3042, E-mail:
[email protected]. Alan J. Magill, Walter Reed Army
Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD
20910, Telephone: 301-319-9959, Fax: 301-319-9227, E-mail:
[email protected]. Giovanna Pastor-Cauna, PRA International, Jr. Incahuasi 539, Urbanizacion Mangomarca, Lima,
Peru, Telephone/Fax: 51-1-459-2943, E-mail: [email protected].
Carlos Echevarria, PRA International, Pasaje San Jose 120,
Magdalena, Lima, Peru, Telephone: 51-1- 263-7231, Cell Phone: 511-9992-4422, E-mail: [email protected]. Victor A. Laguna-Torres,
HIV Program, U.S. Naval Medical Research Center Detachment,
Lima, Peru, American Embassy-Lima, Unit 3800, APO AA 340313800, Telephone: 51-1-561-2882/3848, Fax: 51-1-561-3042, E-mail:
[email protected]. Billey K. Samame, Sullana, Peru,
Telephone: 51-74-93-5324, E-mail: [email protected]. Maria E.
Baldeon, Ministry of Health, Direccion de Salud Lima-Este, El Agustino, Lima, Peru, Telephone: 51-1-363-0909 (Annex 200), Fax: 51-1362-7056, E-mail: [email protected]. James P. Burans, National
Biodefense Analysis and Countermeasures Center, U.S. Army Medical Research and Materiel Command, Attn: MCMR/ZT, 504 Scott
Street, Building 810, Suite 204, Fort Detrick, Frederick, MD 217025012, Telephone: 301-619-7363, E-mail: james.burans@det.
amedd.army.mil. James G. Olson, Virology Department, U.S. Naval
Medical Research Center Detachment, Lima, Peru, American Embassy-Lima, Unit 3800, APO AA 34031-3800, Telephone: 51-1-561-

PEDIATRIC PHASE III TRIAL OF TWO YF VACCINES

2882/3848, Fax: 51-1-561-3042, E-mail: [email protected].

Philip Bedford, Clinical Operations and Regulatory Affairs, Acambis
Research Limited, Peterhouse Technology Park, 100 Fulbourn Road,
Cambridge CB1 9PT, United Kingdom, Telephone: 44-1223-275-300,
Fax: 44-1223-416-300, E-mail: [email protected]. Scott
Kitchener, Centre for Military and Veterans Health, Mayne Medical
School, Herston, Brisbane, Queensland 4006, Australia, Telephone:
61-407-366733, E-mail: [email protected]. Thomas P.
Monath, Acambis Inc., 38 Sidney Street, Cambridge, MA 02139,
Telephone: 617-761-4200, Fax: 617- 494-1741, E-mail: thomas.
[email protected].
Reprint requests: Administrative Assistant, Research Support Section, U.S. Naval Medical Research Center Detachment, Lima, Peru,
American Embassy-Lima, Unit 3800, APO AA 34031-3800, Telephone: 51-1-561-2882/3848, Fax: 51-1-561-3042, E-mail: rlescano@
nmrcd.med.navy.mil.

13.

14.

15.
16.
17.

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