Full text of DOI:10.1086/315859

1331
Evaluation of a Live, Cold-Passaged, Temperature-Sensitive, Respiratory
Syncytial Virus Vaccine Candidate in Infancy
Peter F. Wright,1 Ruth A. Karron,3 Robert B. Belshe,4
Juliette Thompson,1 James E. Crowe, Jr.,1
Thomas G. Boyce,1 Lisa L. Halburnt,1
George W. Reed,2,a Stephen S. Whitehead,5
Edwin L. Anderson,4 Alec E. Wittek,6 Roberta Casey,2
Maryna Eichelberger,2 Bhagvanji Thumar,2
Valerie B. Randolph,6 Stephen A. Udem,6
Robert M. Chanock,5 and Brian R. Murphy5
1Vanderbilt Vaccine Center, Department of Pediatrics,
2
and Department of Preventive Medicine, Vanderbilt University School
3
of Medicine, Nashville, Tennessee; Center for Immunization Research,
Department of International Health, School of Hygiene and Public
4
Health, Johns Hopkins University, Baltimore, Maryland; Departments
of Medicine and Pediatrics, Division of Infectious Diseases, St. Louis
5
University Health Sciences Center, St. Louis, Missouri; Laboratory
of Infectious Diseases, National Institute of Allergy and Infectious
6
Diseases, National Institutes of Health, Bethesda, Maryland; Wyeth
Lederle Vaccines and Pediatrics, Pearl River, New York
A live-attenuated, intranasal respiratory syncytial virus (RSV) candidate vaccine, cpts-248/
404, was tested in phase 1 trials in 114 children, including 37 1–2-month-old infants—a target
age for RSV vaccines. The cpts-248/404 vaccine was infectious at 104 and 105 plaque-forming
units in RSV-naive children and was broadly immunogenic in children 16 months old. Serum
and nasal antibody responses in 1–2 month olds were restricted to IgA, had a dominant
response to RSV G protein, and had no increase in neutralizing activity. Nevertheless, there
was restricted virus shedding on challenge with a second vaccine dose and preliminary evidence
for protection from symptomatic disease on natural reexposure. The cpts-248/404 vaccine
candidate did not cause fever or lower respiratory tract illness. In the youngest infants, how-
ever, cpts-248/404 was unacceptable because of upper respiratory tract congestion associated
with peak virus recovery. A live attenuated RSV vaccine for the youngest infant will use cpts-
248/404 modified by additional attenuating mutations.
Respiratory syncytial virus (RSV) is the leading cause of
severe viral respiratory disease in infants and children [1]. It is
also an important cause of severe respiratory disease in the
elderly [2], immunocompromised patients of all ages [3, 4], and
infants with underlying cardiopulmonary disease [5]. It is con-
sidered a major infectious trigger for reactive airway disease
[6]. RSV infections are estimated to account for ∼90,000 pe-
diatric hospitalizations and 4500 deaths yearly in the United
States [7]. RSV causes a yearly epidemic during the winter
months, with a high penetrance in the first years of life. Of the
2 serologically distinct subgroups, RSV A and B, RSV A viruses
appear to be slightly more virulent and are more commonly
isolated [8]. To be effective, a vaccine must protect against RSV-
associated lower respiratory tract disease in very young infants,
because the peak age of hospitalization is in the second and
third months of life [9].
To introduce an RSV vaccine into the pediatric immunization
schedule, the following properties of the vaccine must be assessed:
(1) its safety in infancy, (2) the effect of maternal antibody on
its infectivity, (3) the effect of immunological immaturity or trans-
placental maternal antibody on its immunogenicity [10, 11], and
(4) its efficacy against natural RSV infection. By analogy with
vaccines given in infancy against other pathogens, multiple doses
are expected to be required.
Live, attenuated, intranasally administered RSV vaccines
have been under development since the late 1960s. At that time,
the parent strain of the lineage under current investigation
(cold-passaged [cp] RSV) was evaluated in seronegative children
as young as 2 years old [12]. In seronegative children, cp RSV
caused mild respiratory illness that was temporally associated
with virus shedding [13].
In parallel, there was an effort to develop a formalin-inacti-
vated vaccine. This inactivated vaccine failed to protect vaccine
recipients and led to enhanced illness on natural exposure to
virus [14]. That enhanced illness has profoundly influenced the
Received 7 March 2000; revised 12 July 2000; electronically published 22
September 2000.
Presented in part: American Pediatric Society/Society for Pediatric Re-
search meeting, Washington, DC, May 1998, and Keystone Symposium on
Molecular Approaches to Human Viral Vaccines, Snowbird, Utah, April
1999.
Informed consent was obtained from parents or guardians of volunteers,
and the human experimentation guidelines of the US Department of Health
and Human Services and of the relevant institutions were followed in the
conduct of the clinical research.
Financial support: National Institutes of Heath (NIH; AI-15095 and
GCRC RR0095) and Wyeth Lederle Vaccines and Pediatrics. This work is
part of a continuing program of research and development between NIH
and Wyeth Lederle Vaccines and Pediatrics through CRADA numbers AI-
000030 and AI-000087.
a
Present affiliation: University of Massachusetts, Worchester.
Reprints or correspondence: Dr. Peter F. Wright, D7219 MCN, Division
of Infectious Disease, Dept. of Pediatrics, Vanderbilt University School of
Medicine, Nashville, TN 37232 (peter.wright@mcmail.vanderbilt.edu).
The Journal of Infectious Diseases 2000;182:1331–42
᭧ 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18205-0007$02.00

1332
Wright et al.
JID 2000;182 (November)
development of an RSV vaccine by delaying the evaluation of
other inactivated or subunit vaccines that might follow similar
antigen processing and presentation pathways and have atten-
dant safety concerns [15]. Consequently, live vaccines that mimic
natural infection have been pursued as the safest strategy for
immunizing young children. Wild-type (wt) RSV infection does
not exhibit enhanced disease during reinfection. Intranasal in-
fection should induce mucosal immunity, which contributes to
protection against RSV [16, 17] and should lead to a balance
between Th1 and Th2 immunological responses without the risk
of enhanced illness [18].
Recently, a series of further attenuated candidate vaccines
[19–21] were derived by chemical mutagenesis of cp RSV. cp RSV
was subjected to 2 rounds of chemical mutagenesis, and tem-
perature-sensitive (ts) mutant derivatives were generated. The
non–ts-attenuating mutations of the cp RSV parent virus were
anticipated to work in concert with the subsequently introduced
ts mutations, to yield further attenuated, genetically stable vi-
ruses. The ts phenotype of the cpts vaccine is more stable than
that of a prototype ts virus, RSV ts-1, which also was evaluated
in children [22, 23]. The mutations in the cpts vaccines are distinct
from mutations in the overly attenuated RSV ts-2 that block
replication in the human respiratory tract [24]. The first 2 vaccines
evaluated in the cpts lineage, cpts-248/955 and cpts-530/1009,
were either insufficiently attenuated or were transmitted among
seronegative children 6–36 months old [25].
of maternal antibodies, immunogenicity in the presence of ma-
ternal antibodies, ability of the first dose of vaccine to restrict
replication of a second dose, transmissibility, phenotypic sta-
bility of the cpts-248/404 vaccine, the level of attenuation of
RSV vaccines that is needed for the very young infant, and
preliminary evidence of protection from symptomatic illness
during natural reexposure to wt RSV.
Materials and Methods
Viruses. Isolation and characterization of cpts-248/404 (shut-
off temperature for plaque formation of 35ЊC–36ЊC) has been de-
scribed elsewhere [19, 20]. A viral suspension for clinical trials was
produced in Vero cells and found to be free of adventious agents
by Dr. Louis Potash (lot RSV A-25; Nova Vax, Bethesda, MD).
The titer of cpts-248/404 (lot RSV A25) prepared for clinical eval-
uation was 105.3 pfu/mL. When necessary to achieve the planned
titer for inoculation, this virus suspension was diluted in L-15 me-
dium (BioWhittaker, Walkersville, MD). Nineteen vaccine and 11
placebo recipients in the 6–24-month-old group received cpts-248/
404 vaccine (lot LRSV-404-301) prepared by Wyeth-Lederle Vac-
cines and Pediatrics (Pearl River, NY) that had a titer of 108 pfu/
mL. To achieve the planned titer for inoculation, this virus sus-
pension was diluted in phosphate buffered saline with sucrose-
phosphate-glutamate. The results with both vaccine lots are similar,
and the results are combined.
Clinical studies in children. Because the parental strains from
which the further attenuated cpts-248/404 was derived had very
limited infectivity in adults [12, 25], adult studies were not per-
formed, and initial studies were done in seropositive children 15–59
months old. Children were enrolled in this randomized, double-
blind, placebo-controlled phase I study at 3 study sites, with a 2:
1 ratio of vaccine to placebo recipients. Children were eligible to
participate if they were healthy and if all other family members
and day care contacts were у6 months old. Before enrollment,
children were screened for the presence of RSV serum-neutralizing
antibodies by a complement-enhanced, 60% plaque reduction neu-
tralization test [26]. Those with titers 11:40 were considered RSV
seropositive. Each study subject received 0.5 mL of vaccine or
placebo as intranasal drops. Children were examined daily for 10
days after vaccine administration. Each study group of 4–6 children
was unblinded after clinical observations were completed, so that
continuous monitoring of safety could be maintained among sites.
Because of the high level of attenuation of a dose of 105 pfu in
17 seropositive children, studies were then performed in 74 sero-
negative children 6–24 months old at a dose of 105 or 104 pfu. The
first 31 children immunized with 105 pfu of RSV cpts-248/404 vac-
cine were examined on days 0–10 and day 14. Subsequently, the
remaining children immunized with 105 pfu and all children im-
munized with 104 pfu were examined and had samples taken on
days 0, 4 or 5, 7 or 8, and 10 or 11. Using the latter schedule,
studies then were done in infants 3–5 months old at a dose of 105
pfu and then in 1–2-month old infants at a dose of 105 pfu or 104
pfu. Although some infants had maternally derived antibodies in
their serum, these study subjects had not been previously infected
with RSV, because they were born after the previous annual RSV
epidemic. One month after the first dose of cpts-248/404, all avail-
The present candidate, cpts-248/404, is among the most atten-
uated of the current series of cpts vaccines on the basis of in vitro
and in vivo analysis. Because its shut-off temperature for plaque
formation is between 35ЊC and 36ЊC, it is unlikely to replicate
efficiently at human core body temperature, 37ЊC. The cpts-248/
404 vaccine is highly restricted in its replication in chimpanzees.
Only 101.3 pfu/mL was recovered from the upper respiratory tract
(levels 11000-fold lower than that of wt RSV), and virus was not
recovered from the lungs, despite the fact that infection was in-
itiated by direct intratracheal instillation. When passive RSV
antibodies were given before immunization of 2 chimpanzees to
mimic transplacental maternal antibodies, an inapparent infec-
tion with cpts-248/404 occurred without documented virus shed-
ding and a limited antibody response that, nevertheless, protected
these animals against challenge with wt RSV 28 days later. There
was an enhanced neutralizing antibody response after challenge
with wt RSV [19].
In the present study, cpts-248/404 underwent testing in pro-
gressively younger children, which resulted in the first admin-
istration of a live, attenuated RSV vaccine to the target age
group for vaccination of infants—namely, those !2 months old.
In the youngest age group, cpts-248/404 vaccine caused mild-
to-moderate upper respiratory congestion, precluding it from
being a candidate for efficacy trials in early infancy. Neverthe-
less, valuable information was derived from this study, includ-
ing the quantity of vaccine virus required to infect a 1–2-month
old infant, the level of vaccine virus replication in the presence

JID 2000;182 (November)
Live RSV Vaccine in Infancy
1333
able 1–2-month-old vaccine and placebo recipients were given a
second dose of the vaccine or placebo to which they were originally
randomized.
purified F or G protein in carbonate buffer and were blocked with
0.5% gelatin in phosphate buffered saline with 0.05% Tween
(PBST). Sera were diluted in PBST with 0.5% gelatin and 2% fetal
calf serum on antigen-coated and noncoated wells. After 1 h of
incubation, the plates were washed and were incubated with goat
anti–human IgG or IgA alkaline phosphate conjugate (Jackson
ImmunoResearch, West Grove, PA) for 1 h. Color development
Children were observed for 1–2 h on each study day in an out-
patient setting. Febrile illness was defined as a rectal temperature
у38.1ЊC. Respiratory illness was categorized as upper respiratory
tract infection (URI), defined as rhinorrhea or pharyngitis of у2
consecutive days duration, or lower respiratory tract infection
(LRI), defined as persistent rhonchi, rales, or wheezing. Cough for
у2 consecutive days was recorded without assignment as to the
site of involvement of the respiratory tract [23].
used 1 mg/mL of
D-nitrophenylphosphate (Sigma, St. Louis) in
diethanolamine buffer. Optical densities (OD) were read at 405 nm
wavelength, the OD was subtracted for the corresponding blank
well, and the end-point dilution at 0.2 OD was calculated. The
results were expressed as log2 of end point, with a positive response
defined as a у4-fold increase in antibody titer.
Isolation, quantitation, and identification of virus. Nasal wash
specimens for virus isolation were obtained on each day of obser-
vation from all subjects and from those with illness reported in the
3 weeks after immunization, as described elsewhere [25]. Fresh or
snap-frozen samples were inoculated into 2 sets of tissue culture
tubes that contained either Vero or HEp-2 tissue culture cell mono-
layers and were incubated at 32ЊC. Viral isolates from these cultures
were identified as RSV by use of an indirect immunofluorescence
assay (IFA; Bartels Microscan; Baxter Healthcare, Bellevue, WA).
RSV in fresh or snap-frozen specimens was titered by plaque assay
on HEp-2 cell monolayer cultures maintained under a semisolid
overlay at 32ЊC, as described elsewhere [24], and results were ex-
pressed as log10 pfu/mL. For purposes of calculation, samples in
which virus was not detected were assigned a titer of 100.6 pfu/mL.
Phenotypic characterization of viral isolates. The level of tem-
perature sensitivity of virus present in snap-frozen nasal wash spec-
imens was determined by plaque titration in HEp-2 cell monolayers
at 32ЊC, 36ЊC, 37ЊC, 38ЊC, and 39ЊC, as described elsewhere [25].
Virus isolates from nasal wash samples that showed a significantly
altered ts phenotype (an increase in shut-off temperature for plaque
formation у2ЊC) were prepared for analysis by either 2 passages
on HEp-2 monolayers or by 1 round of plaque purification on
HEp-2 cell monolayers. The level of attenuation of isolates with
altered ts phenotype was assessed by examining their level of rep-
lication in BALB/c mice, as described elsewhere [27].
Nasal wash samples were tested for the presence of IgA anti-
bodies to purified RSV F and G proteins by a kinetic ELISA
(kELISA) originally developed for influenza [28]. The increase in
absorbance in milliOD/min of each nasal wash IgA value was ex-
pressed as a dilution of a standard serum curve run in the assay
that gave the same increase in absorbance. kELISA values !5
milliOD/min were defined as negative. For pre- and postsamples
with RSV-specific antibody, the RSV-specific results were adjusted
for their total IgA concentration, as measured by a radial immu-
nodiffusion assay using secretory IgA standards (Binding Site, San
Diego, CA). Replicate experiments determined that a у4-fold dif-
ference in the standardized, adjusted result was at the 95% confi-
dence interval (CI) for the test. Therefore, pre- to postimmunization
changes of this magnitude or paired samples that went from neg-
ative to positive were considered to demonstrate a mucosal anti-
body response.
Surveillance. By use of methods described elsewhere [25], RSV
vaccine recipients, placebo recipients, and age-matched controlsub-
jects were monitored during the subsequent RSV season for the
occurrence of wt RSV–associated illness, to determine whether im-
munization with live attenuated vaccine prevented RSV disease or
modified the clinical response to subsequent infection with RSV.
Parents were contacted on a weekly basis throughout the time when
wt RSV was identified in the 3 groups, in which trials were con-
ducted. If a child developed a respiratory illness that met one of
the definitions used during the initial vaccine evaluation, the child
had a clinical assessment and viral culture. Before and after the
RSV epidemic season, children had serum drawn to measure the
incidence of RSV infection in the study population, as judged by
increases in serum neutralizing antibody titer. The surveillance was
not blinded; participant families and investigators knew the child’s
vaccine status.
Genetic characterization of viral isolates. Viral isolates that
showed an altered ts phenotype were further characterized by se-
quence analysis. Reverse transcription–polymerase chain reaction
(RT-PCR) amplification of viral RNA was performed for regions
of cpts-248/404 known to contain determinants of the ts and at-
tenuation phenotypes, as described elsewhere [25]. The nucleotide
sequence of mutations specific to cp RSV and cpts-248/404 was
determined by use of Sequenase 2.0 (USB, Cleveland, OH), as
described by Whitehead et al. [27].
Immunological assays. Sera and nasal wash specimens were
obtained before and either 4 or 8 weeks after initial immunization
for measurement of RSV-specific antibodies. A third serum and
nasal wash specimen was obtained 1 month after the second dose
from study subjects who received either vaccine or placebo at 1–2
months old. For comparison, additional sera and nasal wash sam-
ples were available from 18 children 1–2 months old who were not
enrolled in these trials but were hospitalized for illness caused by
wt RSV infection. Sera were tested for antibodies to RSV by a
plaque neutralization assay [26] and for IgG and IgA antibodies
to RSV fusion (F) and attachment (G) proteins by an end-point
titration in a modification of an ELISA described elsewhere [25].
In brief, Nunc polysorb plates were coated with 20 ng/well of either
Data analysis. Infection with RSV cpts-248/404 vaccine was
defined as the isolation of cpts RSV, a у4-fold increase in serum
RSV neutralizing antibody titer, and/or a у4-fold increase in у2
of the ELISA-based assays. In infants with residual maternal an-
tibodies at the time of immunization, an increase was calculated
as being 4-fold above the anticipated 28 day half-life (t1/2) of passive
antibodies. All comparisons of antibody titer by age were made
after the first dose of vaccine. Titers were expressed as reciprocal
mean log2. The Mann-Whitney U test (2-tailed) was used to com-
pare the mean titers among the groups. Comparison of tabulated
data (e.g., comparison of frequency of illness among vaccine and
placebo recipients and comparison of immunological responses)
was made using 2-tailed Fisher’s exact tests. The k statistics were

1334
Wright et al.
JID 2000;182 (November)
Vaccine recipients given the 105 dose of vaccine shed virus
with high frequency (79%) and at a moderately high level. The
mean peak titer was 104.2 log10 pfu/mL of nasal wash. The lower
mean peak titer of vaccine virus (102.4 log10 pfu/mL) shed by
recipients of the 104 dose may be methodological, because the
samples were collected by nasal swabs rather than by nasal
washes. Thirty-four of the 45 6–24-month-old vaccine recipients
tested who had been given 104 or 105 pfu developed a serum-
neutralizing antibody response, with an increase in mean neu-
tralizing titer to ∼1:300. The dose of vaccine did not influence
the frequency or magnitude of the antibody response (table 2).
Serum IgG ELISA responses to F and G proteins were con-
cordant with neutralizing antibody responses (k values of .76
and .74, respectively).
Response of 3–5-month-old infants to RSV cpts-248/404. In
the next study, 16 infants (10 vaccine and 6 placebo recipients)
3–5 months old were given 105 pfu of cpts-248/404. One vaccine
recipient developed a fever of 38.2ЊC, but LRI was not seen.
There was a suggestion that URI was more frequent in vaccine
recipients (7 of 10) versus placebo recipients (2 of 6; P p .30;
table 1). Vaccine virus was recovered from 3–5-month old vac-
cine recipients, including 5 with residual maternal neutralizing
antibodies, at levels comparable with older RSV-naive infants
(figures 1 and 2). This finding suggests that maternally acquired
serum antibody did not restrict nasal replication of the atten-
uated cpts vaccine, which is an observation confirmed in the
youngest age group (figure 2). Systemic and mucosal immune
responses were lower in frequency and in magnitude than those
observed for vaccine recipients 16 months old; however, the
computed to measure concordance of antibody responses. The as-
sociations among antibody response, age, and prevaccine antibody
were estimated using logistic regression analysis.
Results
Response of seropositive 15–59-month-old children to cpts-248/
404. In this age group, the frequency of illness was compa-
rable between the 11 vaccine and 6 placebo recipients (table 1).
Virus was not recovered from seropositive vaccine recipients
given 105 pfu, and the only antibody response was seen in a
single study subject who had an increase in mucosal IgA an-
tibody to the RSV G protein (table 2). Therefore, RSV cpts-
248/404 is the most restricted in the cpts series evaluated, to
date, in regard to infectivity for seropositive children.
Response of 6–24-month-old seronegative infants and children
to RSV cpts-248/404. The cpts-248/404 vaccine was highly
infectious and immunogenic in 6–24-month-old seronegative
vaccine recipients. After a single administration of either 104
or 105 pfu, ∼90% of vaccine recipients exhibited evidence of
infection (tables 1 and 2). There was no difference in the fre-
quency of illness seen in the vaccine and placebo recipients,
although the ability to detect differences was limited by the
high frequency of minor illness seen in children of this age group
over a 10-day period of close observation. Notably, 1 vaccine
recipient had mild wheezing associated with shedding of cpts
RSV, and 2 vaccine recipients had rhonchi (1 with evidence of
RSV infection). One placebo recipient developed mild wheezing
temporally associated with an adenovirus infection.
Table 1. Clinical and virological responses of infants and children to respiratory syncytial virus (RSV) cpts-248/404 or placebo.
Virus isolation (nasal wash)
Percentage with indicated illness
Mean peak titer,
Otitis
media
Study subjects (age), dose in pfu
Virus given
n
Shedding, %
log10 pfu/mLa
Fever
URI
LRI
Cough
Seropositive children (15–59 months)
105
cpts-248/404
Placebo
11
6
0
0
0.6b
0.6b
18
33
36
33
0
17
0
17
9
0
Seronegative children (6–24 months)
105
cpts-248/404
Placebo
cpts-248/404
Placebo
38
20
11
5
79
0
55
0
4.2
29
40
18
20
68
70
100
60
8
5
0
0
24
30
36
20
11
30
9
0.6b
2.4
104
0.6b
20
Infants (3–5 months)
105
cpts-248/404
Placebo
10
6
70
0
4.2
10
0
70
33
0
0
10
0
0
17
0.6b
Infants (1–2 months)
Dose 1
105
cpts-248/404
Placebo
cpts-248/404
Placebo
17
8
7
76
0
100
0
4.0
0
0
14
0
65
0
86
33
0
0
0
0
18
0
43
33
6
0
0
0
0.6b
4.9
104
3
0.6b
Dose 2
105
cpts-248/404
Placebo
cpts-248-404
Placebo
15
7
7
27
0
0
3.8
7
14
0
40
0
33
33
0
0
0
0
7
0
0
0
0
0
0
0
0.6b
0.6b
0.6b
104
3
0
0
NOTE. LRI, lower respiratory tract infection; URI, upper respiratory tract infection.
a
Calculated for infected volunteers only.
Lowest limit of detection of the assay was 0.7 log10 pfu/mL. For samples without detectable plaques, a titer of 0.6 was assigned.
b

1336
Wright et al.
JID 2000;182 (November)
virus shedding in each infant !6 months old with his/her age
(figure 2A) or level of maternal derived antibody (figure 2B)
demonstrates that virus replication in the nasopharynx was
independent of these variables.
Even when the expected decay in maternal antibody was
considered in the calculations, 1–2-month-old vaccine recipients
receiving either dose of vaccine rarely developed an increase in
serum neutralizing antibody or an IgG ELISA response to F
or G protein (table 2). In 18 infants of the same age hospitalized
with wt RSV, infection serum neutralizing responses were also
seen infrequently; only 30% had a у4-fold increase. In contrast,
29 of 35 6–24-month-old vaccine recipients who shed virus de-
veloped a neutralizing antibody response, which is significant
when compared with the 1–2-month-old vaccines (P ! .001).
In contrast to their failure to develop a serum neutralizing
and IgG-based ELISA antibody responses, the 1–2-month-old
vaccine recipients frequently developed a serum IgA response
to RSV G and F glycoproteins (table 2 and figure 3). The
frequency and magnitude of the IgA antibody response to the
G glycoprotein of the 1–2-month-old vaccine recipients did not
differ from that of 6–24-month-old vaccine recipients. In con-
trast, the frequency of the IgA response to the F glycoprotein,
combining the results of 104 and 105 doses by the 1–2-month-
old vaccine recipients (41%), was less than that of the 6–24-
month-old vaccine recipients (67%) and also less than that to
the G glycoprotein (82%) in the 1–2-month-old vaccine recip-
ients. Therefore, these findings indicate that 1–2-month-oldvac-
Figure 1. Recovery of respiratory syncytial virus (RSV) cpts-248/
404 from the upper respiratory tract of RSV-naive infants and young
children on selected days after vaccine administration. Mean titer of
virus shed by children 1–3 months old (ࡗ), 3–5 months old (Ⅵ), and
6–24 months old (᭡). SD is shown by upward deflection lines.
small number of 3–5-month-old children did not allow for sta-
tistical comparisons.
Response of 1–2-month-old infants to initial immunization.
In a 2-dose regimen, 105 and 104 doses of vaccine were given
to infants 1–2 months old, which is the target age group for
RSV vaccination. Twenty-five children were studied; 17 received
105 pfu of vaccine, 7 received 104, and 11 received placebo (table
1). Seventeen of the vaccine recipients developed a clinical syn-
drome characterized by nasal congestion that occurred most
typically between days 8 and 12. There were no signs of LRI
on repeated examinations, although 6 mothers of vaccine re-
cipients reported that their infants had a mild cough. Symptoms
of congestion were linked temporally with the peak of virus
shedding. Fifteen of 18 infants who shed 1103 pfu of RSV per
milliliter of nasal wash experienced congestion, fussiness while
trying to sleep, and mild difficulty with feeding, which lasted
∼24 h. Virus shedding was not accompanied by the profuse
rhinorrhea typically seen with wt RSV infection, nor did any
of the infants have LRI, otitis media, or fever. One of 11 placebo
recipients and 1 of 4 vaccine recipients who did not shed virus
had mild congestion on days 2 and 3 after receiving vaccine B,
a significant difference in the rate of illness, compared with that
of vaccine recipients from whom cpts-248/404 was recovered
(P p .0002). The same pattern and frequency of congestion was
seen in 105 and 104 vaccine recipients.
Thirteen of 17 vaccine recipients shed virus after the first 105
dose. All 7 vaccine recipients receiving 104 pfu shed virus in an
amount equivalent to that observed for vaccine recipients re-
ceiving 105 pfu. In figure 1, the magnitude of virus shedding is
compared among RSV-naive infants 6–24 months, 3–5 months,
and 1–2 months old on days 4–6, 7–9, and 10–12 (constant
days of sampling throughout the study). Peak virus shedding
was similar for all ages. The lack of a correlation of the peak
Figure 2. Peak recovery of respiratory syncytial virus (RSV) cpts-248/
404 from the upper respiratory tract is independent of age (A) or level
of maternally acquired serum antibody (B). ࡗ, Individual volunteers.

JID 2000;182 (November)
Live RSV Vaccine in Infancy
1337
Figure 3. Maturation of the systemic and mucosal immune responses to respiratory syncytial virus cpts-248/404 vaccine with age. Percentage
of children with a systemic or mucosal antibody response to the indicated immunoglobulin isotype is shown graphically by age. F, fusion protein;
G, attachment protein.
cine recipients preferentially respond to the RSV G protein with
an IgA response and that this response was similar to that of
older vaccine recipients and was not influenced by titer of ma-
ternally derived antibodies.
high level of resistance to replication of the second dose. There
was an absolute correlation of protection from reinfection with
detection of serum IgA antibody to the G protein (P ! .0001),
although, whenever an immune response to the first dose was
documented by any assay in either serum or nasal wash, re-
infection did not occur (table 3). Antibody increases after the
second dose of vaccine were largely restricted to those vaccine
recipients not infected with the first dose (table 2).
Mucosal IgA responses were also seen in the 1–2-month-old
infants (table 2 and figure 3). The mucosal response to G protein
was also more frequent than that to F protein. Study of 18
infants of the same age hospitalized with wt RSV detected IgA
mucosal responses in 61% to the F protein and 50% to the G
protein, which are frequencies slightly higher than that observed
for 1–2-month-old vaccine recipients. Neutralizing activity to
RSV could be not detected in 10 available postvaccination nasal
washes from 1–2-month-old vaccine recipients when tested at
a dilution of 1:4.
Factors influencing the immune responses of infants !24
months old to cpts-248/404 vaccine. Multivariate analysis of
Response of 1–2-month-old infants to second dose of vaccine.
The relatively mild nature of the symptoms associated with the
first dose allowed us to give the planned second dose of vaccine
or placebo 4–6 weeks later to 22 available vaccine recipients
(15 who received 105 and 7 who received the 104 dose) and to
10 placebo recipients (table 1). Virus was recovered from only
2 of 19 vaccine recipients who had shed virus after the first
dose. Two of 3 who had not shed virus after the first dose of
vaccine were infected, leaving 1 child who did not shed virus
after either dose. Nasal congestion was observed in each of the
vaccine recipients who shed RSV, but only in 2 of 18 vaccine
recipients from whom virus was not recovered and in 1 of 10
placebo recipients (P p .002). The inverse relationship between
peak virus shedding with the first and second dose of vaccine
is shown in figure 4. Infection with the first dose provided a
Figure 4. Influence of peak virus shedding on virus recovery, with
the initial dose of respiratory syncytial virus cpts-248/404 given at 1–2
months old and a second dose of vaccine given 1 month later.

1338
Wright et al.
JID 2000;182 (November)
Table 3. Correlates of protection against a second dose of respiratory
syncytial virus (RSV) cpts-248/404 vaccine when the initial dose was
given at 1–2 months old.
these 3 nasal wash samples was amplified at permissive temper-
ature (32ЊC), and isolates then were amplified further at both
32ЊC and 37ЊC. The higher shut-off temperature of these am-
plified virus suspensions was confirmed, and the recovered virus
was shown to be of vaccine origin by nucleotide sequence anal-
ysis. The nucleotide sequences of the genomic regions of these
recovered viruses that contain the attenuating mutations present
in cpts-248/404—namely, the set of 5 cp mutations [29], the 248
mutation [30], and the 404 mutation in the M2 gene start se-
quence [31]—were determined. The recovered virus was found
to have sustained a single CrA nucleotide substitution at nu-
cleotide position 9 of the M2 gene start sequence. The mutation
in the recovered vaccine occurred at the same position that spec-
ifies the 404-M2 attenuating mutation (table 4). It is interesting
to note this substitution is not a reversion to the wt RSV T
nucleotide at this position.
To assess the effect of this nucleotide substitution on the
attenuation phenotype of the resulting virus, virus isolated from
the vaccine recipient on day 15 was studied in mice and com-
pared with wt RSV A2, cpts-248 (the parent of cpts-248/404),
and cpts-248/404 viruses that were evaluated at the same time
in mice. The viruses from the vaccine recipient replicated to the
same level in the upper and lower respiratory tract of mice as
cpts-248, which indicates that the loss of the distinguishing M2-
404 mutation returned the virus to the same level of attenuation
as cpts-248 (table 4). In a previous study, a recombinant virus
counterpart of cpts-248/404, constructed by site-directed mu-
tagenesis to lack the M2-404 mutation, was shown to have the
same temperature sensitivity and attenuation phenotype as cpts-
248 virus [27].
Surveillance. To examine whether infection with live at-
tenuated cpts-248/404 RSV vaccine modified the frequency or
severity of wt RSV–associated illness, young infants and se-
ronegative children (i.e., RSV-naive individuals) immunized in
these trials and age-matched control subjects in the same clinical
care setting were closely followed through the subsequent RSV
epidemic season. As in previous studies with live attenuated
RSV vaccines, enhanced illness did not occur during reinfection
of vaccine recipients with wt RSV [22, 25].
Indeed, the surveillance provided preliminary evidence that
the live attenuated RSV vaccine induced a measure of protec-
tion (table 5). The rate of subsequent wt RSV infection, as
judged by a serological increase, was very high and did not
differ between 6–24-month-old vaccine recipients and age-
matched control subjects, with 37% overall becoming infected.
Indeed, in those !6 months old, there was a significantly higher
seroconversion rate in the vaccine recipients than in the age-
matched control subjects, which we suggest might result from
the vaccine priming the immune system to mount a booster
response. There was a marginally significant lower rate of chil-
dren with subsequent symptomatic wt RSV infection in 6–24-
month-old vaccine recipients (4%) than in age-matched control
Shed
virus
No
response
Shed
virus
Specimen tested, assay used
Response
Serum
RSV neutralizing antibody
IgA ELISA for RSV F protein
IgA ELISA for RSV G protein
IgG ELISA for RSV F protein
IgG ELISA for RSV G protein
Nasal wash
0
9
18
0
0
0
0
0
0
22
13
4
20
19
4
4
4
4
4
1
RSV neutralizing antibody
IgA ELISA for RSV F protein
IgA ELISA for RSV G protein
0
3
8
0
0
0
9
13
7
1
1
1
NOTE. Data are no. of vaccine recipients with indicated antibody response
to first dose who shed virus after the second dose.
the correlation of increase in antibody titer at 4–8 weeks after
immunization with dose of vaccine and level of preexisting ma-
ternal antibodies in the infants !24 months old showed that
preexisting neutralizing antibodies inhibited or masked an in-
crease in neutralizing antibodies (odds ratio [OR], 4.0; 95% CI,
1.7–9.5; P p .035). IgG antibody responses to the F protein
(P ! .001) and the G protein (P ! .001) were significantly de-
creased or masked in a direct relationship to the level of pre-
existing homologous antibodies but were not influenced by age.
The frequency (OR, 0.85; 95% CI, 0.72–0.99; P p .03) and
magnitude (P p .02) of IgA responses to F, but not to G, were
significantly depressed by maternally acquired IgG antibody
level to the homologous protein but not by age.
Evidence for lack of transmission of cpts-248/404. Vaccine
and placebo recipients often came into close contact with one
another for brief periods during follow-up examinations. How-
ever, virus was not recovered from placebo recipients, and only
1 of 52 seronegative control subjects developed a serological re-
sponse. Transmission had been shown under similar circum-
stances with earlier vaccines in this lineage [25]. A more formal
demonstration of the level of transmission will be performed with
this and future candidates in a day care setting, as described
elsewhere [23].
Effect of breast-feeding on magnitude of virus shedding.
Seventeen of 18 children who were exclusively breast-fed shed
virus with a mean peak titer of 104.5, whereas 7 of 12 exclusively
bottlefed shed virus with a lower mean peak titer of 103.5.
Stability of ts and attenuation phenotype of the vaccine. Of
the 176 specimens tested, 173 had a level of temperature sensi-
tivity of plaque formation within 1ЊC of that of the cpts-248/404
vaccine virus, which indicates the high level of phenotypic sta-
bility of this virus after replication in susceptible infants and
children. Specimens obtained from a single 11-week-old vaccine
recipient on days 15, 16, and 17, however, contained an RSV
isolate that produced plaques at 38ЊC, which is 2ЊC higher than
the highest temperature at which cpts-248/404 produces plaques.
This vaccine recipient experienced a pattern of congestion similar
to that seen in other infants receiving this vaccine. Virus from

JID 2000;182 (November)
Live RSV Vaccine in Infancy
1339
Table 4. Respiratory syncytial virus (RSV) shed by a vaccine recipient on days 15, 16, and 17.
Nucleotideb
Mean titer
Virus titer at indicated
temperature, log10 pfu/mL
in mice,a
log10 pfu/g tissue
Mutation
cp
248
404-M2c 404-L
Nasal
Variable
cp
248 404-M2 32ЊC 36ЊC 37ЊC 38ЊC 39ЊC turbinate Lung 1938 6313 7228 9453 13565 10989
7605
12046
Compared virus
wt RSV A2
cpts-248
cpts-248/404
Day RSV recovered
Day 15
Ϫ
ϩ
ϩ
Ϫ
ϩ
ϩ
Ϫ
Ϫ
ϩ
5.2
6.4
4.9
5.0
6.4
5.1
6.1d !0.7
!0.7
5.1
5.2
!0.7
!0.7
4.5
3.4
2.0
4.3
3.1
2.4
G
A
A
A
C
C
C
T
T
G
A
A
C
T
T
A
T
T
T
T
C
T
T
A
3.6d !0.7
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
Ϫ
6.5
6.4
6.5
6.3
6.2
6.3
6.1
6.1
6.0
6.0d !0.7
6.0d !0.7
6.0d !0.7
3.6
ND
ND
3.3
ND
ND
A
A
A
C
C
C
ND
ND
ND
A
A
A
ND
ND
ND
ND
ND
ND
A
A
A
ND
ND
ND
Day 16
Day 17
Passage temperaturee
32ЊC
ϩ
ϩ
ϩ
ϩ
Ϫ
Ϫ
6.3
5.5
6.1
4.9
6.1
5.0
6.0d !0.7
4.8d !0.7
3.6
3.4
3.5
3.3
A
A
C
C
T
T
A
A
T
T
T
T
A
A
A
A
37ЊC
NOTE. RSV was recovered from a single vaccine recipient on days 15, 16, and 17 and was passaged twice on HEp-2 cells at 32ЊC. Virus has a nucleotide
substitution at the 404 mutation site in the M2 gene start signal, which reduced its temperature sensitivity and attenuation in mice to a level similar to that of RSV
cpts-248. Shut-off temperature is defined as the lowest restrictive temperature at which a у100-fold reduction of plaque titer is observed and is underlined. cp, cold-
passaged; ND, no data; wt, wild type; ϩ, presence of mutation; Ϫ, absence of mutation.
a
Groups of 5 mice under light anesthesia were given 106 pfu of the indicated virus in a 0.1-mL inoculum. After 4 days, virus titer was determined in the nasal
turbinate and lung tissues.
b
Numbered from 30 end of negative-sense (viral) RNA. Nucleotide assignments are given in the positive sense.
M2 gene start signal (nucleotide 7605 is underlined): A2 wt, GGGGCAAATA; cpts-248/404, GGGGCAAACA; and isolate, GGGGCAAAAA.
Pin-point plaque size.
c
d
e
Virus present in the day 15 nasal wash sample was grown at either 32ЊC or 37ЊC on HEp-2 cells. Five plaques were picked at each temperature, and passage was
continued at either 32ЊC or 37ЊC. Results are shown for only plaque no. 4 at each temperature, although remaining plaques were tested and had the same level of
temperature sensitivity and the same nucleotide sequence as those shown here.
subjects (20%; table 5). There were too few LRIs to reach any
conclusions of vaccine efficacy.
panzee studies [19, 20]. Therefore, the young infant is a more
permissive host for RSV than the chimpanzee. The absence of
lower respiratory tract illness in the vaccine recipients isconsistent
with the failure to recover the vaccine candidate from the chim-
panzee’s trachea and the shut-off temperature of cpts248/404,
which is less than that of human core body temperature, 37ЊC.
The level of passively acquired maternal antibodies and the
infant’s age did not have an effect on replication of this atten-
uated RSV strain in the nasopharynx. This observation is similar
to data with bovine parainfluenza [34] and rotavirus vaccines
given at the same age [35] and indicates that the mucosal route
of administration of a live attenuated vaccine virus permits rep-
lication in the upper respiratory tract or gastrointestinal tract,
despite the presence of a moderately high titer of serum anti-
bodies. Very high levels of infused RSV serum antibodies can
limit upper respiratory tract replication in experimental animals,
but this is not an efficient process. Also, it should be noted that
the effectiveness of high-titered RSV antibody in prophylaxis
against human disease is presumed to be primarily the result of
passive transfer of antibody across the mucosal epithelium into
the lumen of the lower respiratory tract [36]. Our study also
suggests that breast-feeding does not interfere with RSV repli-
cation, although breast milk may have antibodies to RSV [37].
In contrast, maternal antibodies and/or age had an inhibitory
effect on neutralizing antibody responses and inhibited ormasked
RSV IgG-binding antibody responses. The vaccine recipients in
the youngest age group rarely developed serum antibody re-
Discussion
The strategy of passaging virus at low temperature to generate
vaccine candidates has been successfully used to derive topically
administered vaccines for influenza [32] and parainfluenza type
3 [33] that are appropriately attenuated, immunogenic, geneti-
cally stable, and, as shown in the case of influenza, highly effi-
cacious on exposure to wt influenza virus [32]. Similar cp deri-
vation of a vaccine for RSV yielded a mutant that was only
partially attenuated [13]. The current systematic approach to ex-
ploring the correlates of virulence and using animal models to
determine attenuation and infectivity has been very predictive of
behavior of candidate vaccine strains in humans and in estab-
lishing a rank-order attenuation of such vaccine mutants for the
young seronegative child.
This is the first time that a live attenuated RSV vaccine can-
didate has been evaluated in 1–2-month-old infants, the target
age for vaccination, which allows us to examine factors that
influence safety, infectivity, and immunogenicity of an RSV vac-
cine in early infancy. The demonstration that cpts-248/404 re-
tained sufficient residual virulence to cause mild symptomatic
URI in 1–2-month-old infants and to readily infect the respi-
ratory tract and to attain a titer у104 pfu of virus per milliliter
of nasal wash would not have been predicted from the chim-

1340
Wright et al.
JID 2000;182 (November)
Table 5. Surveillance of cpts-248/404 vaccine recipients and unvaccinated control subjects
during the subsequent respiratory syncytical virus (RSV) epidemic.
No. (%) who developed
RSV-associated illnessa
Serological
response
Otitis
RSV
Age at immunization, subject
n
Any
URI
media
LRI
Total
positive
!6 Months
Vaccine recipientsb
28
51
6 (21)
14 (28)
5 (18)
13 (26)
2 (7)
5 (10)
0 (0)
2 (4)
25
37
12c
4
Unimmunized age-matched controlsd
6–24 Months
Vaccine recipientsb
28
70
1 (4)e
14 (20)
0 (0)f
9 (13)
1 (4)
6 (9)
1 (4)
1 (1)
24
52
7
21
Unimmunized age-matched controlsd
NOTE. LRI, lower respiratory tract infection; URI, upper respiratory tract infection.
a
Culture positive at time of illness. Serological response is у4-fold increase in neutralizing antibody.
One vaccine recipient 6–24 months and 4 vaccine recipients !6 months were not included in surveillance
b
because they had no evidence of vaccine infection.
c
P ! .01 when comparing serological responses in vaccine recipients and control subjects.
Age-matched participants were !9 months old at the beginning of the winter surveillance (i.e., were RSV
d
naive).
e
P p .059 when comparing total illness in vaccine recipients and control subjects.
P p .056 when comparing URI in vaccine recipients and control subjects.
f
sponses that could be detected by a sensitive plaque-reduction
neutralization assay or an IgG ELISA assay to purified F and
G protein. This lack of a detectable serum response is not unique
to vaccine-induced immunity. As we report in this article and as
has been documented elsewhere [38], !50% of young infants hos-
pitalized with culture-documented wt RSV infection demonstrate
a neutralizing response. It is also possible that a low-level IgG
response is masked by residual maternal antibody, since the level
of maternal IgG antibodies to RSV F and G proteins in the 1–2-
month-old infants is comparable with that seen in the 6–24-
month-old children after vaccination, but vaccination did not
decrease the expected decay of maternally acquired RSV IgG
antibodies, and neutralizing antibody titers were low enough that
an increase might have been detected. Passive transfer of RSV
antibodies in animals suppress the immune response to RSV F
and G proteins expressed by vaccinia virus, despite the fact that
the virus vector is not inhibited in its growth. These antibody-
suppressed animals are susceptible to RSV reinfection [39]. Pre-
vious studies in human infants infected with wt RSV virus have
suggested that both age, acting primarily on F protein responses,
and the level of maternal antibodies, acting primarily on G pro-
tein responses, influence the response to infection with wt RSV
[11]. Multivariate analysis suggested that, in this study, the sup-
pression of the all IgG responses and the IgA antibody response
to F from the vaccine virus were a function of the level of ma-
ternal acquired antibodies and not of age.
IgA in the respiratory tract is inherently more difficult to mea-
sure, because of collection methodology and rapid clearance of
IgA in secretions. The IgA responses were not accompanied by
a measurable increase in nasal wash or serum neutralizing anti-
body in the 1–2-month-old vaccine recipients.
The unexpected focus of the infant’s immune system on the
RSV G protein strongly suggests that a live attenuated RSV
vaccine will need to be a bivalent vaccine that contains RSV
subgroups A and B, because the G proteins of the RSV sub-
groups are only ∼50% related by amino acid sequence and only
5% related antigenically [40].
The cpts-248/404–infected vaccine recipients were highly re-
sistant to infection with the second dose of vaccine. Vaccine
virus was not recovered from any vaccine recipient who de-
veloped any immunological response to the first dose of vaccine.
The second dose of vaccine induced a slight boost in immunity
in vaccine recipients infected with the first dose but did not
lead to the enhanced neutralizing antibody response seen in the
chimpanzee model [19]. Because the second dose of vaccine did
infect 2 of 3 children not infected by the first dose, an argument
can be made for 2 doses of vaccine as a minimum schedule for
immunization.
Each of the vaccine recipients protected against a second dose
of vaccine developed a serum IgA response to the G protein
after the initial dose, whereas all those infected with the second
dose failed to mount such a response to the initial dose. Im-
munity induced by vaccinia virus that expresses RSV G has
been demonstrated, although the degree of protection was less
than that seen with a vaccinia virus F recombinant [41]. It is
possible that the protection afforded in the present study by
IgA antibodies to the RSV G protein were mediated by neu-
tralizing IgA antibodies below the level of our detection, but
it is also possible that mucosal IgA antibodies are mediating
protection by a mechanism other than, or in addition to, viral
Serum IgA responses to the RSV G protein proved to be the
most consistent response in the youngest infants. It is reason-
able to suggest that the IgA serum antibody is a direct result
of replication of vaccine virus in the mucosa of the respiratory
tract and reflects a response that originated in the mucosal arm
of the immune system. Mucosal IgA responses to RSV F and
G protein were also seen, although with a lower frequency than
that seen in serum, again more commonly to the G protein.

JID 2000;182 (November)
Live RSV Vaccine in Infancy
1341
neutralization. Proposed mechanisms of antiviral action for IgA
beyond neutralization include antibody-dependent cellular cy-
totoxicity [42] and intracellular interruption of virus replication
during trancytosis of IgA across the epithelial mucosa [43]. The
critical response may be mucosal IgA memory on secondary
challenge, as reported for RSV in calves whose primary re-
sponses are inhibited by colostrums [44]. Others have reported
cell-free and cell-bound IgA antibody in the nasopharynx in
the course of primary RSV infection in children and its lack
of correlation with neutralizing activity [45]. Work with other
viral model systems has suggested that protection may be seen
in the absence of a neutralizing antibody response that is me-
diated by components of the cellular immune response mea-
sured by lymphoproliferation, as shown in monkeys infected
with measles [46]. An advantage of using a live attenuated vac-
cine is that it can theoretically stimulate a balanced immune
response, including antibodies, MHC class I restricted CD8ϩ
cytotoxic T cell response, and a CD4ϩ T cell memory.
ronegative children 16 months old to prevent the substantial
burden of RSV-associated URI, otitis media, and milder LRI
seen at that age. For the 1–2-month-old infant, у1 additional
attenuating mutations need to be introduced in cpts-248/404 to
generate a vaccine that is slightly more attenuated. This can be
readily achieved using reverse genetics [47, 48]. Equally im-
portant to the optimization of an RSV vaccine will be an un-
derstanding of the immune mechanisms by which resistance to
reinfection with vaccine virus is conferred in the young child.
Future studies will investigate whether protection against a sec-
ond dose of vaccine continues to translate into protection
against reinfection with wt virus and/or the amelioration of the
severity of illness because of RSV in the young child, as seen
in this study.
References
1. Glezen WP, Denny FW. Epidemiology of acute lower respiratory disease in
children. N Engl J Med 1973;288:498–505.
Our inability to infect seropositive children with the atten-
uated cpts-248/404 is another indication of RSV immunity. The
marked inhibition of vaccine virus replication by prior natural
infection is similar to that seen with influenza and parainfluenza
type 3 viruses, and, collectively, they are a strong indicator that
effective immunity to respiratory viruses can be induced. It is,
however, important to emphasize that the resistance observed
to vaccine challenge was to an attenuated virus. Finally, the
vaccine recipients 6–24 months old had decreased RSV-asso-
ciated illness on subsequent exposure to wt RSV. The observed
decrease in subsequent wt RSV illnesses by vaccine recipients
is an encouraging signal that a similarly derived RSV vaccine
may prove to be efficacious when tested for protection against
LRI. It is clear from serological studies that wt RSV reinfection
occurs frequently in vaccine recipients, as it does after natural
infection, and thus cannot serve as a marker for vaccine efficacy.
Despite the high level of replication in fully susceptible young
vaccine recipients, cpts-248/404 maintained the full ts pheno-
type in 173 of 176 isolates; however, 3 isolates from 1 vaccine
recipient exhibited a decrease in their level of temperature sen-
sitivity and in their attenuation in rodents. The 3 virus isolates
retained the set of 5 cp mutations and the 248 mutation in the
L protein of RSV and exhibited a level of temperature sensi-
tivity and attenuation for rodents that were characteristic of
viruses that contain these mutations. The alteration in the level
of temperature sensitivity and partial loss of attenuation for
rodents resulted from a single nucleotide substitution in the M2
gene start sequence at the same position as the original atten-
uating mutation. As indicated below, a more attenuated RSV
vaccine than cpts-248/404 is being sought. It is anticipated that
the introduction of an additional attenuating mutation will re-
sult in a concomitant increase in the stability of the attenuation
phenotype.
2. Falsey AR, Cunningham CK, Barker WH, et al. Respiratory syncytial virus
and influenza A infections in hospitalized elderly. J Infect Dis 1995;172:
389–94.
3. Harrington RD, Hooton RD, Hackman RC, et al. An outbreak of respiratory
syncytial virus in a bone marrow transplant center. J Infect Dis 1992;165:
987–93.
4. Hall CB, Powell KR, MacDonald NE, et al. Respiratory syncytial viral
infection in children with compromised immune function. N Engl J Med
1986;315:77–81.
5. MacDonald NE, Hall CB, Suffin SC, Alexson C, Harris PJ, Manning JA.
Respiratory syncytial virus infection in infants with congenital heart dis-
ease. N Engl J Med 1982;307:397–400.
6. Sigurs N, Bjarnason R. Sigurbergsson F, Kjellman B, Bjorksten B. Asthma
and immunoglobulin E antibodies after respiratory syncytial virus bron-
chiolitis: a prospective cohort study with matched controls. Pediatrics
1995;95:500–5.
7. Anderson LJ, Parker RA, Strikas RL. Association between respiratory syn-
cytial virus outbreaks and lower respiratory tract deaths of infants and
young children. J Infect Dis 1990;161:640–6.
8. Walsh EE, McConnochie KM, Long CE, Hall CB. Severity of respiratory
syncytial virus infection is related to virus strain. J Infect Dis 1997;175:
814–20.
9. Kim HW, Arrobio JO, Brandt CD, et al. Epidemiology of respiratory syn-
cytial virus infection in Washington, DC. I. Importance of the virus in
different respiratory disease syndromes and temporal distribution of in-
fection Am J Epidemiol 1973;98:216–25.
10. Kovarik J, Siegrist C-A. Immunity in early life. Immunol Today 1998;19:
150–2.
11. Murphy BR, Alling DW, Snyder MH, et al. Effect of age and preexisting
antibody on serum antibody response of infants and children to the F
and G glycoproteins during respiratory syncytial virus infection. J Clin
Microbiol 1986;24:894–8.
12. Friedewald WT, Forsyth BR, Smith CB, Gharpure MA, Chanock RM. Low-
temperature–grown RS virus in adult volunteers. JAMA 1968;203:690–4.
13. Kim HW, Arrobio JO, Pyles G, et al. Clinical and immunological response
of infants and children to administration of low-temperature adapted
respiratory syncytial virus. Pediatrics 1971;48:745–55.
14. Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K.
Respiratory syncytial virus disease in infants despite prior administration
of antigenic inactivated vaccine. Am J Epidemiol 1969;89:422–34.
15. Crowe JE Jr. Immune responses of infants to infection with respiratoryviruses
The cpts-248/404 vaccine is not an acceptable RSV vaccine
candidate for the youngest infant. It might have a role in se-

1342
Wright et al.
JID 2000;182 (November)
and live attenuated respiratory virus vaccine candidate vaccines. Vaccine
32. Belshe RB, Mendelman PM, Treanor J, et al. The efficacy of live attenuated,
cold-adapted, trivalent, intranasal influenza virus vaccine in children. N
Engl J Med 1998;338:1405–12.
1998;16:1423–32.
16. Mills J 5th, VanKirk JE, Wright PF, Chanock RM. Experimental respiratory
syncytial virus infection of adults. J Immunol 1971;107:123–30.
17. Watt PJ, Robinson BS, Pringle CR, Tyrell DAJ. Determinants of suscepti-
bility to challenge and the antibody response of adult volunteers given
experimental respiratory syncytial virus vaccines. Vaccine 1990;8:231–6.
18. Graham BS, Bunton L, Wright PF, Karzon DT. The role of T cell subsets
in the pathogenesis of primary infection and reinfection with respiratory
syncytial virus in mice. J Clin Invest 1991;88:1026–33.
19. Crowe JE Jr, Bui PT, Siber GR, Elkins WR, Chanock RM, Murphy BR.
Cold-passaged, temperature-sensitive mutants of human respiratory syn-
cytial virus (RSV) are highly attenuated, immunogenic, and protective in
seronegative chimpanzees, even when RSV antibodies are infused shortly
before immunization. Vaccine 1995;13:847–55.
33. Karron RA, Wright PF, Newman FK, et al. A live human parainfluenza type
3 vaccine is attenuated and immunogenic in healthy infants and children.
J Infect Dis 1995;172:1445–50.
34. Karron RA, Makhene M, Gay K, Wilson MH, Clements ML, Murphy BR.
Evaluation of a live attenuated bovine parainfluenza type 3 vaccine in
two- to six-month-old infants. Pediatr Infect Dis J 1996;15:650–4.
35. Kobayashi M, Thompson J, Tollefson SJ, Reed GW, Wright PF. Tetravalent
rhesus rotavirus vaccine in young infants. J Infect Dis 1994;170:1260–3.
36. Groothuis JR, Simoes EA, Levin MJ, et al. Prophylactic administration of
respiratory syncytial immune globulin to high-risk infants and young chil-
dren. Respiratory Syncytial Virus Immune Globulin Study Group. N Eng
J Med 1993;329:1524–30.
20. Crowe JE Jr, Bui PT, Davis AR, Chanock RM, Murphy BR. A further
attenuated derivative of a cold-passaged temperature sensitive mutant of
human respiratory syncytial virus retains immunogenicity and protective
efficacy against wild-type challenge in seronegative chimpanzees. Vaccine
1994;12:783–91.
21. Crowe JE Jr, Bui PT, Davis AR, Hung PP, Chanock RM, Murphy BR.
Satisfactorily attenuated and protective mutants derived from a partially
attenuated cold-passaged respiratory syncytial virus mutant by introduc-
tion of additional attenuating mutations during chemical mutagenesis.
Vaccine 1995;13:847–56.
22. Kim HW, Arrobio JO, Brandt CD, et al. Safety and antigenicity of temper-
ature sensitive (ts) mutant respiratory syncytial virus (RSV) in infants and
children. Pediatrics 1973;52:56–63.
23. Wright PF, Shinozaki T, Fleet W, Sell SHW, Thompson J, Karzon DT. Eval-
uation of a live, attenuated respiratory syncytial virus vaccine in infants.
J Pediatr 1976;88:931–9.
24. Wright PF, Belshe RB, Kim HW, Van Voris LP, Chanock RM. Administration
of a highly, attenuated, live respiratory syncytial virus vaccine to adults
and children. Infect Immun 1982;37:397–400.
25. Karron, RA, Wright PF, Crowe JE Jr, et al. Evaluation of two live, cold-
passaged, temperature-sensitive respiratory syncytial virus (RSV) vaccines
in chimpanzees, adults, infants and children. J Infect Dis 1997;176:1428–36.
26. Coates HV, Alling DW, Chanock RM. An antigenic analysis of respiratory
syncytial virus isolates by a plaque reduction neutralization test. Am J
Epidemiol 1966;83:299–313.
27. Whitehead SS, Firestone CY, Collins PL, Murphy BR. A single nucleotide
substitution in the transcription start signal of the M2 gene of respiratory
syncytial virus vaccine candidate cpts248/404 is the major determinant of
the temperature-sensitive and attenuation phenotypes. Virology 1998;247:
232–9.
37. Fishaut M, Murphy D, Neifert M, Mcintosh K, Ogra P. Bronchomammary
axis in the immune response to respiratory syncytial virus. J Pediatr 1981;
99:186–91.
38. Brandenburg AH, Groen J, van Steensel-Moll HA, et al. Respiratory syn-
cytial virus specific serum antibodies in infants under six months of age:
limited serological response upon infection. J Med Virol 1997;52:97–104.
39. Murphy BR, Olmsted RA, Collins PL, Chanock RM, Prince GA. Passive
transfer of respiratory syncytial virus (RSV) antiserum suppresses the
immune response to the RSV fusion (F) and large (G) glycoproteins ex-
pressed by recombinant vaccinia viruses. J Virol 1988;62:3907–10.
40. Johnson PR, Olmsted RA, Prince GA, et al. Antigenic relatedness between
glycoproteins of human respiratory syncytial virus subgroups A and B:
evaluation of the contributions of F and G proteins to immunity. J Virol
1987;61:3163–6.
41. Olmsted RA, Elango N, Prince GA, et al. Expression of the F glycoprotein
of respiratory syncytial virus by a recombinant vaccinia virus: comparison
of the individual contributions of the F and G proteins to host immunity.
Proc Natl Acad Sci USA 1986;83:7462–6.
42. Cranage MP, Gardner PS, McKintosh K. In vitro cell-dependent lysis of
respiratory syncytial virus–infected cells mediated by antibody from local
respiratory secretions. Clin Exp Immunol 1981;43:28–35.
43. Mazanec MB, Nedrud JG, Kaetzel CS, Lamm ME. A three-tiered view of
the role of IgA in mucosal defense. Immunol Today 1993;14:430–5.
44. Kimman TG, Westenbrink F, Schreuder BEC, Straver PJ. Local and systemic
antibody response to bovine respiratory syncytial virus infection and re-
infection in calves with and without maternal antibodies. J Clin Microbiol
1987;25:1097–115.
45. McIntosh K, McQuillin J, Gardner PS. Cell-free and cell-bound antibody in
nasal secretions from infants with respiratory syncytial virus infection.
Infect Immun 1979;23:276–81.
28. Snyder MH, Banks S, Murphy BR. Determination of antibody response to
influenza virus surface glycoproteins by kinetic enzyme-linked immuno-
sorbent assay J Clin Microbiol 1988;26:2034–40.
46. Binnendijk RS, Poelen MCM, van Amerogngen G, de Vries P, Osterhaus
ADME. Protective immunity in macaques vaccinated with live attenuated,
recombinant, and subunit measles vaccines in the presence of passively
acquired antibodies. J Infect Dis 1997;175:524–32.
29. Connors M, Crowe JE Jr, Firestone CY, Murphy BR, Collins PL. A cold-
passaged, attenuated strain of human respiratory syncytial virus contains
mutations in the F and L genes. Virology 1995;208:478–84.
30. Crowe JE Jr, Firestone C-Y, Whitehead SS, Collins PL, Murphy BR. Ac-
quisition of the ts phenotype by a chemically mutagenized cold-passaged
human respiratory syncytial virus vaccine candidate results from the ac-
quisition of a single mutation in the polymerase (L) gene. Virus Genes
1996;13:269–73.
47. Collins PL, Hill MG, Camargo E, Grosfeld H, Chanock RM, Murphy BR.
Production of infectious human respiratory syncytial virus from cloned
cDNA confirms an essential role for the transcription elongation factor
from the 5 proximal open reading frame of the M2 mRNA in gene ex-
pression and provides a capability for vaccine development. Proc Natl
Acad Sci USA 1995;92:11563–7.
48. Whitehead SS, Firestone C-Y, Karron RA, et al. Addition of a missense
mutation present in the L gene of respiratory syncytial virus (RSV)
cpts530/1030 to RSV vaccine candidate cpts248/404 increases its attenu-
ation and temperature sensitivity. J Virol 1999;73:871–7.
31. Firestone CY, Whitehead SS, Collins PL, Murphy BR, Crowe JE Jr. Nucleo-
tide sequence analysis of the respiratory syncytial virus subgroup A cold
passaged (cp) temperature sensitive (ts) cpts-248/404 live attenuated virus
vaccine candidate. Virology 1996;225:419–22.