829
Live Subgroup B Respiratory Syncytial Virus Vaccines that Are Attenuated,
Genetically Stable, and Immunogenic in Rodents and Nonhuman Primates
James E. Crowe, Jr.,* Phuong T. Bui, Cai-Yen Firestone,
Mark Connors,* William R. Elkins, Robert M. Chanock,
and Brian R. Murphy
Respiratory Viruses Section, Laboratory of Infectious Diseases, National
Institute ofAllergy and Infectious Diseases, National Institutes of
Health, Bethesda, Maryland
Optimal immunization of neonates against disease caused by respiratory syncytial virus (RSV)
probably will require multiple doses of a vaccine containing viruses of both subgroups A and
B. Live subgroup B RSV mutants were generated containing multiple attenuating mutations, ts
(temperature-sensitive) and non-ts (host range), that were introduced by prolonged passage in cell
culture or by chemical mutagenesis. The cold-passaged (cp)-52 mutant was restricted in replication
compared to wild type virus in rodents and nonhuman primates. In addition, the attenuation
phenotype of cp-52 was stable after prolonged replication in immunosuppressed rodents. One or
two ts mutations were then introduced into the cp-52 mutant to generate additional candidate
vaccine strains that were more attenuated in vivo than the cp-52 parental virus. Tests in humans
are being done to determine if one or more of the RSV B-1 mutants exhibit a satisfactory balance
between attenuation and immunogenicity.
Respiratory syncytial virus (RSV) is the most common cause
of serious viral lower respiratory tract disease in infants and
children worldwide [1]. Two subgroups of RSV, designated A
and B, have been distinguished on the basis of antigenic relatedness and nucleotide sequence information. The RSV subgroups are '""25% related by reciprocal cross-neutralization assay
[1]. Viruses of each subgroup cause severe lower respiratory
tract disease that often requires hospital admission. Therefore,
prevention of such disease likely will require a high level of
protection against lower respiratory tract infection by virus of
either subgroup. Studies of the prevalence of the subgroup A
and B viruses in sequential epidemics in Finland and of children
undergoing sequential first and second infection with RSV suggest that naturally acquired infection imparts a relatively higher
protection against disease caused by the homologous subgroup
virus [2, 3]. However, it is clear from these and other studies
that the immunity induced by wild type (wt) RSV infection is
transient after first infection, allowing subsequent reinfection
with virus of either subgroup. To achieve optimal immunization
of the very young infant for protection against RSV-associated
bronchiolitis and pneumonia, it is likely that a bivalent vaccine
containing subgroups A and B components given in repeated
Received 18 April 1995; revised 5 December 1995.
Presented in part: American Society for Virology, Madison, Wisconsin, 913 July 1994 (abstract W9-9).
Financial support: Cooperative Research and Development Agreement with
Wyeth Ayerst Laboratories, Radnor, PA.
Reprints or correspondence: Dr. James E. Crowe, Jr., D-7235 MCN, VUMC,
Nashville, TN 37232-2581.
Present affiliation: Department of Pediatrics, Division ofInfectious Diseases,
Vanderbilt University Medical Center, Nashville, Tennessee (J.E.C.); Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland (M.C.).
The Journal of Infectious Diseases
This article is in the public domain.
1996; 173:829-39
doses will be required. Our laboratory is pursuing the development of a live attenuated RSV vaccine for two reasons: Live
virus infection induces a balanced immune response that includes
both serum and secretory neutralizing antibodies and cytotoxic
T cells, none of which are induced efficiently by inactivated or
subunit vaccines, and infection of seronegative infants with wt
RSV [1, 4, 5] or its attenuated mutants [6-8] has not been
associated with potentiation of disease on subsequent natural
infection such as was seen with formalin-inactivated RSV [9,
10]. The pursuit of live attenuated mutants as vaccine candidates
in the 1970s was abandoned because of the difficulty in identifying mutants that were both genetically stable and satisfactorily
attenuated [6-8]. More recent studies confirmed that subgroup
A RSV mutants that possessed only one or two attenuating
mutations exhibited genetic instability during replication in a
fully susceptible host [11 -14]. In contrast, we observed that
subgroup A RSV mutants that possessed at least three attenuating
mutations exhibited a very high level of stability of the temperature-sensitive phenotype during prolonged replication in nude
mice or chimpanzees [12-14]. The most genetically stable subgroup A mutants tested contain both host range and ts attenuating
mutations that appear to act in concert to achieve a genetically
stable virus. Some of these subgroup A mutants are promising
vaccine candidates, yet alone they are unlikely to induce a reasonably durable cross-protection against illness caused by infection with heterologous subgroup B viruses.
In the present study, we generated attenuated RSV subgroup
B mutants that possess multiple attenuating mutations selected
by multiple passages in cell culture at low temperature or induced by chemical mutagenesis. We investigated the level of
replication, immunogenicity, and genetic stability of these candidate vaccines in rodents and primates.
Materials and Methods
Cell culture. Primary African Green monkey kidney (AGMK)
cells were used for initial isolation of the RSV B-1 wt virus. WHO
830
Crowe et al.
Vero cells, from a well-characterized stock of this continuous line
of African Green monkey kidney cells, were obtained from the
World Health Organization via the American Type Culture Collection (ATCC, Rockville, MD) at passage 134. Cells banked at passage 138 were recertified to be free of adventitious agents and
were maintained as described [12]. Vero cells were not used beyond passage 150. MRC-5 cells, a human diploid fibroblast cell
line frozen by Flow Laboratories (Rockville, MD) at passage 14,
were certified for use in human vaccines and maintained as described [11]. MRC-5 cells were not used beyond passage 30. HEp2 cells, a human epithelial transformed cell line obtained from
ATCC at passage 364, were maintained as described [12]. HEp-2
cells were not used beyond passage 400. Vero or HEp-2 cell monolayer cultures grown on 24-well tissue culture plates were used
for all virus assays as described [15].
Viruses. The passage history of the RSV B-1 wt strain and its
mutant derivatives is summarized in figure 1. The B-1 wt virus
was derived from a recent field isolate as follows. The original
isolate (provided by R. Belshe, St. Louis University Medical Center), designated RSV B/WV/14617/'85, was recovered during the
winter of 1985 from the endotracheal tube saline wash of a 7week-old infant undergoing mechanical ventilation for nosocomially acquired RSV bronchiolitis/pneumonia. The virus was isolated
after direct passage of the saline wash in primary AGMK cells,
then passaged twice in AGMK cells and 6 times in MRC-5 cells,
biologically cloned by 3 consecutive plaque-to-plaque passages in
MRC-5 cells, then passaged once in MRC-5 cells and 4 times in
WHO Vero cells. The virus suspension obtained from the supernatant of the fourth passage in Vero cells was designated lot B-1
and found to be free of adventitious agents by L. Potash (PR!
DynCorp, Rockville, MD).
This virus then was passaged 18 times at progressively lower
temperatures (lowest temperature, 21°C) in MRC-5 cell monolayer
cultures using L-15 medium containing 2% fetal bovine serum,
gentamicin, amphotericin, and glutamine in closed flasks maintained inside metal coffins immersed in temperature-controlled water baths. This cold-passaged virus, cp-18, was cloned biologically
by 3 consecutive plaque-to-plaque passages in Vero cells at 32°C
and passaged twice in Vero cells to yield the suspension designated
RSV B-1 cp-23. The cp-23 virus then was passaged 29 times
further at suboptimal temperatures (lowest temperature was 22°C).
Three different virus clones were obtained in Vero cell monolayer
cultures at 32°C by consecutive plaque-to-plaque passages of 3
separate cp-52 plaque progeny (designated cp-52/2B5, cp-52/3C1,
and cpsp-52/1Al [a small-plaque variant]). Subsequently, a biologically cloned suspension of the cp-12 virus was obtained from the
frozen suspension of the 12th cold passage material by 3 consecutive plaque-to-plaque passages in Vero cell monolayer cultures at
32°C, as shown in figure 1. Temperature-sensitive (ts) mutants of
cp-52/2B5 were derived by chemical mutagenesis using 5-fluorouracil (Sigma, St. Louis) during replication of virus in Vero cell
monolayer cultures as described [12, 13]; 1440 plaque progeny
were screened, and 8 of these exhibited a decrease in plaque number or size at 38°C in HEp-2 cell monolayer cultures. These 8
mutants that exhibited the temperature-sensitive phenotype were
biologically cloned by 3 plaque-to-plaque passages in Vero cells
and subsequently amplified in Vero cells. One of these mutants,
designated the cpts-176 mutant, was used to initiate a second round
of chemical mutagenesis with 5-fluorouracil. The suspension of
JID 1996; 173 (April)
cpts-176 used for mutagenesis contained plaque-purified virus that
was subsequently amplified twice in Vero cells. Two.· of 1152
plaques derived from the mutagenesis of cpts-176 appeared to
have acquired a second ts mutation because these clones exhibited
restriction of plaque formation at 36°C, a temperature at which
the parent virus cpts-176 was not restricted. These two mutants
were cloned biologically by 3 serial plaque passages and designated cpts-176/427 or cpts-176/1072.
The isolation and characterization of the RSV A2 wt virus and
its attenuated derivatives, cpts-248, cpts-248/804, cpts-248/404,
cpts-530, and cpts-530/1009, were described previously [12-14,
16]. These viruses were evaluated for comparative purposes in
these studies. The parainfluenza virus type 3 wt strain JS used for
infection of control animals in cross-protection studies has been
described [17]. The subgroup B wt RSV WV/401R/'90 virus was
a field isolate from Huntington, West Virginia (provided by M.
Mufson, Marshall University School of Medicine, Huntington,
WV) and the subgroup B Wash/18537/'62 virus was described
previously [18]. These subgroup B wt viruses were selected for
challenge studies because they differed from each other in ELISA
binding pattern to a commercially available panel of RSV-specific
murine monoclonal antibodies (Chemicon, Temecula, CA). The
subgroup A RSV wt viruses RSB89-6190 and RSB89-6256 were
1989 field isolates from Birmingham, United Kingdom, previously
described [19,20] and provided by C. Pringle (University of Warwick, Coventry, UK). These isolates represent a range of genetically diverse strains within circulating subgroup A isolates, based
on deduced amino acid sequence of the G glycoprotein. The relatedness of G glycoproteins from subgroup A viruses was as follows:
strain A2 versus strain 89-6190, 87%; strain A2 versus strain 896256, 88%; strain 89-6190 versus strain 89-6256, 90% [12, 20].
Virus characterization. The efficiency of plaque formation of
virus present in tissue culture harvests, lung, or nasal turbinate
(NT) homogenates of cotton rats or nasopharyngeal (NP) or tracheal lavage fluids collected from experimentally infected monkeys or chimpanzees was determined on HEp-2 or Vero cell monolayer cultures maintained under a semisolid overlay at various
temperatures as described [11]. Detection ofplaques was facilitated
by staining monolayers by an immunoperoxidase procedure as
described [21]. When determining the titer of subgroup A or B
virus in experiments involving dual infection of cotton rats, the
staining procedure was modified to use subgroup A- or B-specific
murine monoclonal antibodies (92-11 C or 102-1 OB, respectively;
provided by L. Anderson, CDC, Atlanta). Preliminary experiments
using virus stocks of known titer demonstrated that as little as 1.0
log,o pfulmL virus of one subgroup could be detected in the presence of up to 6.0 10gIO pfu/mL virus of the heterologous subgroup
(data not shown). Ciprofloxacin (25 J.lg/mL) was added to the
overlay of cultures used to quantitate virus recovered from monkeys or chimpanzees.
Immunologic studies. Serum RSV neutralizing antibodies
were quantitated by a complement-enhanced 60% plaque reduction
neutralization assay [18] using RSV A2 in HEp-2 monolayer cultures or RSV B-1 in Vero cell monolayer cultures stained by the
immunoperoxidase procedure [21]. Serum IgG antibodies binding
to RSV F or G glycoprotein were quantitated in an ELISA using
F or G glycoprotein. that had been immunoaffinity-purified from
RSV subgroup A (A/Long/'56)- or subgroup B (B/Wash/18537/
'62)-infected celllysates as described [22, 23].
RSV BIWV/146171'85
(originally isolated in 10 AGMK) *
!
16 passages in AGMK, MRC-5, and Vero cell
monolayer cultures (including biological cloning
comprising three plaque-to-plaque passages)
RSV B-1 wild-typet
I
MRC-5 cells at progressively
+112lowerpassages
temperature (32° to 21°C)
in
I
plaque-purified x 3
oneIoned cp- 12 ---.. in Vero cells at 320C ---.. RSV B-1 cp-12
I
!
6 passages in MRC-5 cells at progressively lower temperature followed
by plaque-purification x 3 in Vero cells at 32°C (these plaque passages
were designated cp-19, -20, -21), and 2 passages in Vero cells
IRSV B-1 cp-23/1Al!
I
29 passages in Vero cells at progressively
+lower temperature (26°C to 22°C)
oncloned cp-52
3 clones were derived from the cp-52 suspension
by plaque-purification x 3 in Vero cells at 32°C
I RSV B-1 cpsp-52/1Al
I
I
RSV B-1 cp-52/3Cl
I RSV B-1 cp-52/2B5 I
I
t
chemical mutagenesis of 2B5 clone and selection of'ts mntants
mutants were then plaque purified x 3
RSV B-1 cpts-176 I and others
chemical mutagenesis
selection of ts mutants
mutants plaque purified x 3
RSV B-1 cpts-176/427
RSV B-1 cpts-176/1072
Figure 1. Passage history of RSV B-1 cold-passaged (ep) temperature-sensitive (ts) vaccine candidates. * Original isolate, RSV BIWV/
14617/'85, was from tracheal sample from infant being mechanically ventilated for RSV bronchiolitis. t After passage and triple plaque
purification in cell culture as indicated, the fifth virus passage designated RSV B-1 was used for derivation of cold-passaged strains. Cell
culture was used to isolate or cold-passage RSV B-1 viruses (AGMK, primary African Green monkey cells; MRC-5, human diploid lung cell
line; Vero, well-characterized World Health Organization line of African Green monkey kidney cells [continuous cell lineD. sp, small plaque.
832
Crowe et al.
Animals. Respiratory pathogen-free cotton rats (inbred Sigmodon hispidus) were obtained from the National Cancer Institute
(Frederick, MD) and used at 4-12 weeks of age. Cotton rats were
maintained in microisolator cages throughout the study. Animals
were anesthetized with methoxyflurane inhalation at the time of
virus inoculation. Animals in groups of 6 were inoculated intranasally on day 0 with 105.5 pfu of mutant or wt RSV in a 0.2-mL
inoculum. Four days after inoculation, cotton rats were sacrificed
by CO2 asphyxiation, and NT and lung tissues were obtained separately for quantitation of virus and characterization of recovered
virus as described [15, 21].
To test the genetic stability of cp-52/2B5 in vivo, cotton rats
were infected intranasally with 105.5 pfu in a 0.2-mL inoculum,
then immunosuppressed by intraperitoneal injection of 75 mg of
cyc1ophosphamidelkg of body weight (Sigma) on postinfection
days 3, 6, 9, and 12. This regimen was determined in preliminary
dosage and interval studies to allow prolonged replication of RSV
cp-52 to occur in the respiratory tract without causing excessive
mortality due to immunosuppression (data not shown). In two
preliminary studies, virus could not be isolated from lung homogenates of cp-52-infected immunosuppressed cotton rats 14 days
after infection; therefore, this tissue was not tested subsequently.
However, viral replication in nasal turbinates was active at that
time, at a mean titer of 3.2 loglo pfulg (range, 2.3-3.9). Animals
were sacrificed on day 14. NT homogenates obtained for virus
quantitation as described [15, 21] were inoculated without freezing
onto Vero cell monolayers that were incubated in liquid culture
medium at 32°C. Supernatants from the first tissue culture passage
of NT homogenates ("isolates") were harvested when extensive
cytopathologic effect was observed (average of 7 days), frozen in
triplicate, stored at - 70°C, and then quantitated by plaque assay
at 32°C in Vero cells. To determine ifthe day 14 isolates retained
the attenuation phenotype of the input cp-52 virus (i.e., significant
restriction of replication compared with the B-1 wt virus), the 7
isolates with the highest titers in both original NT homogenates
and isolates, the cp-52 input virus, and the B-1 wt virus were each
used at a dose of 105.5 pfu to inoculate a group of 8 immunocompetent cotton rats. The mean titer of the original NT homogenates
used to generate these 7 isolates was 3.6 10glO pfulg (range, 3.53.9). NT and lung homogenates from animals infected with isolates
were obtained for virus titration on day 4 after infection.
Twenty-two African Green monkeys (Cercopithecus aethiops)
from a Caribbean colony were housed in modified Horsfall isolator
units. The animals, obtained from Spriggs Scientific (Perkasie,
PA), had a body weight ranging from 3.2 to 5.6 kg. Twelve young
male or female chimpanzees (Pan troglodytes) weighing 6.8-20
kg (age range, 1-4 years) were pair-housed in large glass isolator
suites and maintained as described [24]. The chimpanzees were
on loan from the Southwest Foundation for Biomedical Research
(San Antonio, TX), the M. D. Anderson Cancer Center (Bastrop,
TX), or the Coulston Foundation (White Sands, NM). These monkeys and chimpanzees lacked detectable serum neutralizing antibodies for RSV B-1 or RSV A2 (titer, < 1: 10). Groups of 4 or 8
monkeys were inoculated with either A2 wt, B-1 wt, B-1 cp-23,
or B-1 cp-52/2B5 virus by both the intranasal and intratracheal
routes with a dose of 105.5 pfu in a 1.0-mL inoculum at each site.
One month later, the monkeys were challenged with wt RSV A2
or wt RSV B-1 in a similar fashion. Groups of 2 or 4 seronegative
chimpanzees were infected with RSV B-1 or B-1 cp-5212B5 by
lID 1996; 173 (April)
both the intranasal and intratracheal routes with a dose of 104 or
105 pfu in a 1.0-mL inoculum at each site. After inoculation of
virus, NP swab specimens were collected from each monkey or
chimpanzee under ketamine anesthesia for quantitation of the
amount of virus shed on days 1-10, 13, 16, and 20, and tracheal
lavage specimens were collected on days 2, 4, 6, 8, 10, 13, 16,
and 20 as described [11]. The amount of rhinorrhea was estimated
daily and assigned a score of 0-4 by an experienced observer (0
= none, 1 = trace, 2 = mild, 3 = moderate, 4 = severe). After
wt virus challenge of previously immunized monkeys, NP swab
specimens were collected every day, and tracheal lavage specimens
were collected every other day for 10 days, to estimate quantity
of virus shed.
Results
In vitro characterization of cp mutants. Biologically
cloned viruses were derived from each of 3 cold-passaged virus
suspensions after 12, 23, or 52 passages of the RSV B-1 strain
in cell culture at low temperature (32°-21°C). The plaque phenotype and efficiency of plaque formation of these viruses were
determined in Vero or HEp-2 cell monolayer cultures at 32,
39, or 40°C (data not shown). The RSV B-1 wt virus formed
plaques of equal size and with equal efficiency in both .cell
types at all three temperatures. The plaque size of the cp-23 and
cp-52 mutants was decreased ~50% at 39 or 40°C compared to
the permissive temperature of 32°C. In most experiments (5 of
7 tests) the number of plaques produced by cp-52 at 39° or
40°C was not reduced significantly compared to plaque titer at
32°C (i.e., a ~ 100-fold reduction at the higher temperatures
was not observed). Despite multiple passages in cell culture at
temperatures as low as 21°C, the B-1 cp mutants did not exhibit
a cold-adapted phenotype when evaluated by plaque formation
or growth yield in cultures incubated at 20, 23, 24, or 25°C
(data not shown).
Level of replication of cp mutants in cotton rats. Preliminary analysis of the level of replication of the RSV B-1 wt
virus in rodent species, including miCe (strains BALB/c, DBAI
2NCR, C57L/J, NIH Swiss, and PINCR), golden Syrian hamsters, and cotton rats, indicated that the virus replicated to a
high level in both the nasal turbinates and lungs only in cotton
rats (data not shown). This host range restriction for the B-1
virus was in contrast to the RSV subgroup A strain A2 that
replicated to high level in all rodent species tested. A high
level of replication of the B-1 virus in cotton rats (~1 as pfu/
g of tissue) was observed on day 3, 4, or 5 after intranasal
inoculation of 105 or 106 pfu, with the peak titer occurring on
day 4 (data not shown). The level of replication of the B-1 wt
virus, cp-12, cp-23, or cp-52 viruses was compared in the NT
and lungs of cotton rats after intranasal inoculation of 10 5.5 pfu
(table 1). Because derivatives of the subgroup A RSV A2 wt
virus had been characterized previously in rodents, chimpanzees, and humans, we included the A2 wt virus and a promising
subgroup A vaccine candidate, A2 cpts-530/l009, for comparative purposes in this study [12]. The B-1 cp-12 mutant exhibited
RSV Subgroup B Vaccine Candidates
JID 1996; 173 (April)
Table 1.
833
Cold-passaged derivatives of RSV B-1 wild type virus are attenuated in cotton rats.
Virus titer (mean loglO pfulg of tissue ± SE)
Nasal turbinates
Experiment 1
Virus
RSV
RSV
RSV
RSV
RSV
RSV
RSV
RSV
B-1 wild type
B-1 ep-12/BIA
B-1 ep-23/lAl
B-1 epsp-52/lAI
B-1 ep-52/2B5
B-1 ep-52/3CI
A2 wild type
A2 epts-530/1009
± 0.14
ND
ND
1.7 ± 0.11
1.8 ± 0.25
1.8 ± 0.14
5.9 ± 0.09
3.2 ± 0.11
4.7
Lungs
Experiment 2
5.1
3.3
2.4
2.1
2.2
± 0.10
± 0.15
± 0.36
± 0.27
± 0.30
ND
5.4 ± 0.07
2.1 ± 0.22
Experiment 1
5.4 ± 0.15
ND
ND
3.0 ± 0.13
1.8 ± 0.11
1.8 ± 0.14
6.6 ± 0.06
2.1 ± 0.19
Experiment 2
5.8 ±
4.4 ±
3.2 ±
2.3 ±
<1.5
ND
6.1 ±
1.7 ±
0.08
0.10
0.31
0.07
0.06
0.12
NOTE. Groups of 6 cotton rats were infected intranasally under light methoxyflurane anesthesia with 105.5 pfu of indicated virus on day 0; tissue homogenates
were obtained on day 4, and virus titer was determined by titration of homogenate suspensions on Vero cell monolayer cultures incubated at 32°C for 10 days
in experiment 1, 7 days in experiment 2. ND = not done. Wild type RSV A2 (subgroup A) and its attenuated epts mutant 530/1009 were studied for purpose
of comparison. ep, cold-passaged; ts, temperature-sensitive; sp, small plaque.
a 30- or lOO-fold reduction in replication in the lungs or NT,
respectively, compared with the B-1 wt virus, while the cp-23
mutant was 400- or 500-fold restricted in those sites. The cp52/2B5 mutant was the most restricted mutant, exhibiting a
~20,000- or lOOO-fold reduction in the lungs or NT, respectively. These cold-passaged viruses are considered to have host
range mutations because they do not exhibit a significant defect
in replicative capacity in vitro but are restricted in growth in
a semipermissive animal host. The stepwise increase in restriction of replication with increase in number of cold passages
exhibited by the cp-12, cp-23, and cp-52 viruses suggests that
the cp-52/2B5 mutant had acquired at least three distinct attenuating mutations. This interpretation is made with the caveat that
the cloned cp-12/B1A mutant tested was derived by plaque
purification from the uncloned cp-12 virus that was the actual
progenitor of the subsequent cp-23 and cp-52 mutants (see
figure 1). Thus, it is possible, though unlikely, that the genetic
determinants of the attenuation phenotype of the cp-12/B 1A
virus are not present in the cp-23 and cp-52 mutants derived
from the uncloned cp-12 suspension.
Genetic stability of cp-52 after replication in immunosuppressed cotton rats. It was important to determine if the attenuation phenotype of the cp-52 virus (restriction of replication
in cotton rats compared to the B-1 wt virus) was retained after
multiple cycles of replication in immunosuppressed animals.
Virus isolated after 14 days of replication of cp-52 in immunosuppressed cotton rats subsequently was administered to immunocompetent cotton rats to determine if it retained the attenuation phenotype (table 2). Each of the isolates recovered after
14 days of replication in the immunosuppressed animals was
as restricted in replication as the input virus (table 2). These
findings indicate that the constellation of the three putative
attenuating mutations in cp-52 confers a high level of stability
of the attenuation phenotype, even after prolonged replication
in immunosuppressed animals.
Level ofattenuation, immunogenicity, andprotective efficacy
against wt viral challenge in African Green monkeys. It was
reported recently that RSV can replicate to reasonably high
levels in African Green monkeys [25]. We therefore evaluated
the level of replication of the RSV B-1 viruses in this animal
(table 3). The B-1 wt virus replicated to a low level in both
the NP and trachea of these animals (10-fold lower in the NP
than the subgroup A wt strain RSV A2). The B-1 cp-23 virus
was 3-fold or ;::dO-fold restricted compared with the B-1 wt
virus in the NP or trachea, respectively, while the cp-52 mutant
was lO-fold or ~ lO-fold restricted in those sites, respectively.
The B-1 cp-23 and B-1 cp-52 mutants each induced a high
level of serum neutralizing antibodies; the 10-fold-Iower level
of serum antibodies induced by cp-52 is consistent with its
greater restriction of replication. Both cp-23 and cp-52 induced
resistance against homologous wt, virus challenge, resulting in
~30-fold or ;:=dO-fold reduction in wt virus shedding in the
NP or trachea compared with the level of shedding observed
in naive animals infected with B-1. Infection with RSV A2 or
B-1 wt viruses induced complete protection against challenge
with the heterologous subgroup strain in this species (data not
shown).
Level of attenuation and immunogenicity of cp-52 in seronegative chimpanzees. Chimpanzees have proved to be an
extremely useful animal model for the evaluation of candidate
RSV subgroup A live virus vaccines [11-13]. Therefore, we
compared the level of replication of the cp-52 mutant with that
of the wt strain B-1 in seronegative chimpanzees after intranasal and intratracheal administration of 104 or 105 pfu (table 4).
The cp-52 mutant was ~300-fold restricted in replication in
the NP compared with wt virus and completely restricted in the
lower respiratory tract, even though the mutant was inoculated
directly into the trachea. Both the subgroup B RSV wt strain
B-1 and the subgroup A wt strain A2 replicate to high level
and cause upper respiratory tract disease in one-third of experi-
834
Crowe et al.
JID 1996; 173 (April)
Table 2. RSV B-1 cp-52/2B5 (cold-passaged) mutant retains its attenuation phenotype after prolonged
replication in immunosuppressed cotton rats.
Virus titer (mean loglo pfu/g of tissue ± SE)
Virus
Nasal turbinates
Lungs
RSV B-1 wild type
RSV cp-52/2B5 (input virus)
cp-52/2B5 isolates from
immunosuppressed cotton rats
Isolate I
Isolate 2
Isolate 3
Isolate 4
Isolate 5
Isolate 6
Isolate 7
3.9 ± 0.03 (6/6)
2.0 ± 0.07 (8/8)
4.8 ± 0.12 (6/6)
<1.5 (0/8)
1.5 ± 0.13
1.5 ± 0.13
1.5 ± 0.16
1.3 ± 0.09
1.2 ± 0.00
1.5
<1.5
<1.5
<1.5
< 1.5
< 1.5
<1.5
(5/8)
(6/8)
(3/8)
(4/8)
(2/8)
1.2 ± 0.00 (3/8)
1.3 ± 0.06 (3/8)
± 0.04 (1/8)
(0/8)
(0/8)
(0/8)
(0/8)
(0/8)
(0/8)
NOTE. Groups of 6 or 8 cotton rats were infected intranasally under light methoxyflurane anesthesia with 10 5.5
pfu of indicated virus in O.l-mL inoculum on day O. Animals were sacrificed by CO 2 inhalation on day 4, and tissue
homogenates were obtained for quantitation of virus titer by plaque titration on Vero cell monolayer cultures. Isolates
1-7 were recovered in previous experiment from nasal turbinate homogenates of 7 different immunosuppressed
cotton rats 14 days after infection with cp-52/2B5. Inoculum used here was obtained after single passage in Vero
cell culture of virus present in nasal turbinate homogenate suspension of previous experiment. Data in parentheses
indicate proportion of animals from which virus was detected at titer of ~ 1.2 loglo pfu/g (nasal turbinates) or ~ 1.5
loglo pfu/g (lungs).
mentally infected adult human volunteers (Clements ML, unpublished data). However in the present study, the B-1 wt virus
replicated to a level 200-fold or 3000-fold lower in the NP or
trachea, respectively, in chimpanzees than did the wt strain A2
in our previous studies [11], indicating that chimpanzees are
less useful for evaluation of B-1 mutants than for A2 mutants.
The B-1 wt virus caused mild upper respiratory symptoms in
chimpanzees, while the cp-52 mutant did not. These findings
indicate that the cp-52 virus was attenuated for the upper and
lower respiratory tract of chimpanzees and cotton rats. Infection
with cp-52 induced serum RSV ELISA antibodies to the F
glycoprotein and neutralizing antibodies detectable 1 month
after immunization, although at a lower level than that observed
for wt virus infection. The 10 5 -pfu dose of cp-52 induced a
significantly higher level of postinfection RSV serum antibodies than the 104 -pfu dose, even though viral shedding was not
detected in either group.
Does interference ofviral replication occur during coinfection of cotton rats with the B-1 cp-52 and A2 cpts-530/1009
viruses? Because our goal is to develop a bivalent vaccine
containing attenuated mutants of both subgroup A and B RSV,
we addressed the issue of whether interference in viral replication occurs during simultanebus infection with the B-1 cp-52
mutant and the subgroup A vaccine candidate A2 cpts-5301
Table 3. Evaluation of level of attenuation, immunogenicity, and efficacy against challenge of RSV B-1 candidate live virus vaccines in
African Green monkeys.
Immunization
Challenge with homotypic wild type RSV
Peak titer
Virus
A2 wild type
B-1 wild type
B-1 cp-23
B-1 cp-52/2B5
Nasopharynx
Trachea
Mean prechallenge reciprocal
serum neutralizing antibody
titer to homotypic virus
3.2
2.2
1.7
1.2
1.2
1.6
<0.7
<0.7
53,232
2048
4705
239
Peak titer
Wild type
challenge virus
Nasopharynx
Trachea
A2
B-1
B-1
B-1
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
NOTE. Animals were infected intranasally and intratracheally with 1055 pfu of indicated virus at each site in 1.0-mL inoculum on day 0, using groups of 4
animals for each virus. Titers are expressed as mean 10glO pfu/mL of sample, as determined by plaque assay on HEp-2 cell monolayer cultures for RSV A2 wild
type and Vero cell monolayer cultures for RSV B-1 wild type and its derivatives. Serum neutralizing antibody titers were determined by complement-enhanced
60% plaque reduction in HEp-2 (RSV A2 wild type) or Vero (RSV B-1 wild type) cell monolayer culture. Animals were challenged by inoculation intranasally
and intratracheally with 1055 pfu of indicated virus at each site in 1.0-mL inoculum on day 28 after initial immunization.
JID 1996; 173 (April)
Table 4.
RSV Subgroup B Vaccine Candidates
835
RSV B-1 cp-52/2B5 mutant is attenuated and immunogenic in seronegative chimpanzees.
NOTE. Animals were infected with indicated dose in both nasopharynx and trachea using 1.0-mL inoculum for each site. Mean rhinorrhea scores for individual
animals represent sum of daily scores for 8 days surrounding peak day of viral shedding, or day of highest rhinorrhea score if virus was not detected, divided
by 8. 4 is highest score; 0 is lowest score (represents complete absence of detectable rhinorrhea). Neutralizing antibody titers were determined by complementenhanced 60% plaque reduction in Vero cell monolayer cultures. Individual values are average of 2 tests.
1009 that is currently in phase I trials in humans. Experiments
in vitro indicated that the level of replication of these viruses
was not aff:::\.c;·~d by the replication of the heterologous virus
during simu1 caneous multicycle infection of Vero cell monolayers after inoculation at an MOl of 0.1 (data not shown).
Evidence for interference in vivo also was sought. Three groups
of 6 cotton rats were inoculated with 105 pfu of the wt strain
B-1 alone, wt RSV A2 alone, or B-1 and A2. Similarly, another
3 groups of 6 animals were inoculated with 105 pfu of B-1 cp52 alone, A2 cpts-530/l009 alone, or both cp-52 and cpts-530/
1009. Significant interference was not observed in the level of
replication of these pairs of wt or mutant viruses during dual
infection of the NT or lungs of cotton rats, except for a slight
reduction of wt B-1 (from 4.6 to 3.6 loglo pfu/g) in the NT
(but not in the lungs) during simultaneous infection with wt
A2 (table 5).
Protection by infection with cp-52 against subsequent challenge with representative wt strains of subgroup A or B in
cotton rats. Previous studies have demonstrated that infection
with subgroup A or B wt viruses induces a high level of resistance against replication of homologous or heterologous subgroup RSV in cotton rats [26]. An experiment was done to
determine if infection produced by the subgroup B attenuated
mutants induced a similar level of protection against challenge
with representative wt viruses ofboth subgroups. Immunization
with B-1 cp-52 (or A2 cpts-530/l 009 studied for comparison)
induced a ~ 10,000-fold restriction of replication of virus after
homologous or heterologous wt virus challenge in the cotton
rat (table 6). In addition, the candidate subgroup A or B vaccines were, as expected, broadly protective against diverse
members of the homologous subgroup (i.e., B-1 cp-52 protected against BfWV/401R/'90 and BfWash/18537/'62, and A2
cpts-530/l009 protected against A/RSB89-6l90 and A/RSB6156; data not shown).
Isolation and characterization of ts mutants derived from
cp-52. Although the B-1 cp-52 mutant was highly restricted
during experimental infection of all laboratory animals tested,
it might not be sufficiently attenuated for use in the target
population of fully susceptible human infants. In addition, we
previously observed that the most attenuated and genetically
stable live attenuated subgroup A RSV mutants were those that
possess both host range and temperature sensitivity mutations
[13, 14]. Therefore, we sought to introduce ts mutations into
the host range-restricted cp-52 virus. The cp-52 mutant was
mutagenized with 5-fluorouracil, and 1440 plaques were characterized. Eight plaque progeny were temperature-sensitive,
and in addition, 1 of these designated cptssp-14l5 exhibited
both the small plaque phenotype « 50% wt plaque size at
32°C) and a new host range restr:iction phenotype (failure to
form plaques in HEp-2 cell monolayers) (table 7). The efficiency of plaque formation at multiple temperatures of the ts
mutants of cp-52 was examined because previous studies have
demonstrated a correlation between the level of attenuation of
a ts mutant and its degree of temperature sensitivity in vitro
[12-14,27]. The plaque titer of the wt RSV strain B-1 or cp52 was not reduced significantly in HEp-2 cells at 39°C, while
cp-52 was temperature-sensitive at 40°C in Vero cells in these
experiments. The shutoff temperature of the ts mutants, that
is, the lowest temperature at which plaque titer was reduced
at least 100-fold relative to the titer at permissive temperature
(32°C), ranged from 35 to 37°C in HEp-2 cells and from 37
to 40°C in Vero cells (table 7). Thus, the mutants derived from
cp-52 exhibited a wide spectrum of temperature sensitivity.
The level of replication of these mutants in cotton rats was
evaluated, although the high level of restriction of replication
of the cp-52 parent virus in vivo precluded the demonstration
of large differences in level of replication between the parent
virus and its ts derivatives (table 7). Nonetheless, the ts mutants
lID 1996; 173 (April)
Crowe et al.
836
Table 5. Subgroup B RSV does not interfere with replication of subgroup A virus during coinfection
of cotton rats.
Mean virus recovery (loglO pfulg) : :!: : SE
Nasal turbinates
Virus
RSV A titer
A2 wild type (wt)
B-1 wt
A2 wt + B-1 wt
A2cpts-530/l 009
B-1 cp-52
A2 cpts~530/1009 + B-1 cp-52
5.4 : :!: : 0.08
5.2 : :!: : 0.11
3.2 : :!: : 0.09
2.8 : :!: : 0.13
RSV B titer
Lungs
RSV A titer
RSV B titer
5.8 : :!: : 0.07
4.6 : :!: : 0.03
3.6 : :!: : 0.07
2.4 : :!: : 0.08
2.0 : :!: : 0.14
5.7 : :!: : 0.08
1.9 : :!: : 0.15
1.8 : :!: : 0.08
5.4 : :!: : 0.12
5.0 : :!: : 0.05
<1.5
<1.5
NOTE. Groups of 6 animals were infected with 105 pfu of single wt virus or attenuated mutant or mixture of 105
pfu each of2 viruses intranasally under light methoxyflurane anesthesia on day O. All inoculations were administered in
O.I-mL volume. Subgroup A- or B-specific titers were determined by separately staining duplicate methanol-fixed
cell monolayer culture plaque titers of tissue homogenates in irnmunoperoxidase procedure using subgroup A-or
B-specific monoclonal antibody.
exhibited a greater restriction of replication in cotton rats compared to the ep-52 parent, as indicated by a lower proportion
of inoculated animals from which virus was isolated. All but
one ts mutant (epts-1313) exhibited a decrease in immunogenicity in cotton rats compared to the ep-52 parent (table 7).
Therefore, the epts mutants could be distinguished from their
ep-52 parental virus by their temperature-sensitive phenotype
in two cell types and their greater restriction of replication and
diminished immunogenicity in cotton rats.
In the present study, we sought to generate mutants with the
widest possible range of attenuation for subsequent evaluation
in humans. We derived two mutants from the B-1 epts-176
virus that each exhibited a lower shutoff temperature in both
Vero and HEp-2 cells. The epts-176/1072 mutant had a 36 or
38°C shutoff temperature in HEp-2 or Vero cells, respectively,
while the epts-176/427 mutant had a 35 or 37°C shutofftemperature in those cells. Because the epts-176 parent virus is highly
restricted in replication in cotton rats, the ts derivatives epts-
Table 6. RSV A2 cpts-53 0/1 009 and RSV B-1 cp-52 vaccine candidates are protective in cotton rats against challenge with wild type
(wt) virus of either subgroup A or B.
Lung titer of challenge virus
(mean loglO pfulg of tissue:::!::: SE)
Immunizing virus
RSV A2 wt
RSV B-1 wt
RSV A2 cpts-530/1009
RSV B-1 cp-52
Control (PIV-3 wt)
:s;;1.2
1.9 : :!: : 0.15
6.2 : :!: : 0.13
:s;;1.2
:s;;1.2
5.0 : :!: : 0.09
NOTE. Groups of 6 cotton rats were immunized intranasally under light
methoxyflurane anesthesia with 105.5 pfu of virus in 200-J.tL volume on day
0, then challenged with 105 .5 pfu of indicated virus 28 days later. Lungs were
harvested 4 days after challenge. PIY-3, parainfluenza virus type 3.
176/427 and epts-176/1072 were not tested in vivo. However,
on the basis of previous experience with other RSV mutants
that possess a similar degree of temperature sensitivity, the
acquisition of mutations specifying a 35 or 36°C shutoff temperature should render the epts-176/1 072 and epts-176/427 mutants more restricted in replication in humans than their epts176 parent virus.
Discussion
Previous efforts in our laboratory to develop safe and effective live attenuated RSV mutants did not succeed because of
genetic instability of the candidate vaccine strains and failure
to achieve a satisfactory level of attenuation. Other laboratories
have used similar classical biologic procedures to derive live
attenuated mutant viruses of subgroup A or B, yet genetically
stable, satisfactorily attenuated vaccine candidates have not
been identified [28, 29]. Our strategy to overcome both obstacles was to generate a large number of mutants possessing a
wide range of level of attenuation specified by three or more
attenuating mutations. Preferably, the mutations would belong
to different classes such as temperature sensitivity, host range,
or small plaque. Since RNA viral polymerases have a relatively
high error rate and lack a proofreading function, genetic
changes are inevitable for live attenuated RNA viruses during
replication in susceptible hosts. When viruses containing a single attenuating mutation, such as a missense mutation specifying the temperature-sensitive phenotype, replicate in a relatively nonpermissive site (e.g., the 37°C lower respiratory
tract), a strong selective pressure exists for the amplification
of virus with altered phenotype. Viruses that possess multiple
attenuating mutations, both ts and non-ts, are less likely to
undergo simultaneous reversion or suppression of these multiple attenuating mutations. The high level of stability of the
JID 1996; 173 (April)
RSV Subgroup B Vaccine Candidates
837
Table 7. Efficiency of plaque formation, in vitro host range restriction, in vivo replication, and immunogenicity of temperature-sensitive
mutants derived from cp-52 or cpts-176.
Efficiency of plaque formation (lOglO pfulmL) in Vero or HEp-2 cell
monolayer cultures at temperature
caC)
Vero
Experiment,
virus
Experiment 1
B-1 wild
type*
cp-52*
cpts-1313
cpts-452
cpts-I091
cpts-784
cptssp-1415§
cpts-176
cpts-1229
cpts-1324
Experiment 2
B-1 wild
type*
cp-52*
cpts-176
cpts-176/
1072
cpts-176/427
HEp-2
32
37
38
39
40
32
35
36
37
38
39
6.0
6.1
6.2
6.2
5.6
5.6
6.1 t
6.0
5.8
5.8
6.0
6.1
5.8
5.8
5.0
5.2
5.3 t
5.7
4.6
2.8 t
6.0
5.9
5.2
5.1
4.2
4.6
4.5 t
4.8
3.8
<0.7
5.9
5.4
4.7
3.2
3.2
3.2
2.2 t
<0.7
2.6
<0.7
5.8
3.1
2.8
1.8
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
5.8
5.4
3.9 t
5.6
5.1 t
4.3 t
<0.7
5.4 t
5.1
5.1
5.7
5.2
3.0
5.2
4.7 t
4.0 t
<0.7
4.8 t
4.9
5.0t
5.6
5.1
<0.7
5.2
5.2 t
4.1 t
<0.7
5.0t
5.1
5.0
5.6
5.0
<0.7
3.3 t
<0.7
<0.7
<0.7
<0.7
4.4 t
<0.7
5.7
5.0
<0.7
3.1 t
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
5.5
4.7 t
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
7.2
6.6
6.5
7.0
6.2
6.1
7.1
6.2
4.9
7.1
5.6
2.9 t
7.0
5.2 t
1.2 t
6.9
6.1
5.7
6.7
5.8
5.1 t
6.7
5.8
2.9 t
6.5
5.7
1.9 t
6.6
5.7
1.0t
6.0
6.1
4.9
4.0 t
2.2
<0.7
1.0t
<0.7
5.2
5.5
3.5 t
2.2 t
1.0t
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
<0.7
Virus titer in nasal
turbinates of cotton
rats (mean 10glO
pfulg of tissue of 6
animals ± SE)
4.3 ± 0.05
1.7 ± 0.11
ND
1.3 ± 0.06
1.5 ± 0.11
1.1 ± 0.14
1.4 ± 0.10
1.0 ± 0.21
<0.7 (0/6)
0.8 ± 0.11
(6/6)
(6/6)
(3/6)
(4/6)
(1/6)
(3/6)
(3/6)
(1/6)
Postinfection serum
neutralizing antibody
titer induced vs.
RSV B-1 (reciprocal
mean log2 ± SE)
15.3
12.8
14.8
9.8
4.8
4.2
8.6
8.6
10.1
7.3
±
±
±
±
±
±
±
±
±
±
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.25
0.69
0.42
0.93
0.18
0.45
0.63
0.62
0.32
0.79
NOTE. Cotton rats were inoculated intranasally with 5.0 10glO pfu under light methoxyflurane anesthesia on day 0, and sacrificed by CO2 inhalation on day
4. Wild type RSV B-1 replicated to titer of 4.4 ± 0.25 (6/6 animals) in lungs; virus was not detected from lungs of animals infected with any mutants (lower
limit of detection = 1.5 10glO pfulmL); therefore, lung data are not shown. Nos. in parentheses indicate proportion of animals from which virus was recovered.
ND, not done. Underlines indicate in vitro shutoff temperature (lowest restrictive temperature at which ~ IOO-fold reduction of plaque titer was observed).
* Different lots were used in experiments 1 and 2.
t Small plaque phenotype «50% wild type plaque size).
t Pinpoint plaque phenotype « 10% wild type plaque size).
§ Plaques were not observed in HEp-2 cells at any temperature; therefore, shutoff temperature was not assigned for HEp-2 cells.
attenuation phenotype of many live attenuated RNA virus vaccines and vaccine candidates may be associated with the possession of multiple mutations of different types, for example
subgroup A RSV mutants [13, 14], the cp-45 parainfluenza
type 3 vaccine candidate [17], the cold-adapted influenza N
Ann Arbor/6/60 vaccine [30], and the Sabin 3 polio strain [31].
We derived mutants from the RSV B-1 wt strain by passaging the virus multiple times in cell culture at low temperature.
Viruses isolated from passage 12,23, or 52 exhibited increasing
levels of restriction of replication in cotton rats, which suggests
the sequential acquisition of at least three distinct attenuating
mutations in the cp-52 virus. While three mutations most likely
account for the different levels of growth restriction, other
explanations are possible. For instance, a single missense mutation in the cp-12 virus could have been altered to a different
amino acid substitution at the same site associated with greater
growth restriction in subsequent passages; alternatively, a single deletion in the cp-12 virus could have been extended on
further passage. However, the high level of genetic stability of
the attenuation phenotype of cp-52 demonstrated after pro-
longed replication in vivo argues for the presence of multiple
independent mutations that contribute to the attenuation phenotype. The complete nucleotide sequence of several of these
viruses is being determined, and this information should reveal
the type and number of mutations that could be involved in
attenuation. Since the cp-52 virus replicated efficiently in vitro
and did not acquire a cold-adapted or temperature-sensitive
phenotype, its attenuating mutations are considered to be of
the host range restriction type. We chose this mutant for further
manipulation because it likely contained three or more attenuating mutations and because it was attenuated, immunogenic,
protective against wt challenge, and genetically stable in vivo.
The exact level of attenuation of live RSV subgroup B mutants required for safe use in human infants is not known. In
other studies, a significant correlation was observed for subgroup A RSV cpts mutants between level of replication in
rodents and chimpanzees [13, 14]. There is a discordance between the level of replication of the RSV B-1 wt virus in
chimpanzees and the level in humans because of host range
differences. The RSV B-1 wt virus replicated to a high level
838
Crowe et al.
and caused upper respiratory disease in a significant proportion
of seropositive adult volunteers (Clements ML, unpublished
data). However, this virus replicated 200 or 3000 times less
well in the NP or trachea, respectively, of seronegative chimpanzees. Thus, the chimpanzee appears to be a less permissive
host for the B-1 wt virus than is an adult human. The reason
for such host range differences is not clear. In addition, we
have observed that while the B-1 wt virus and the subgroup A
wt strain A2 each replicate to high level and cause upper respiratory disease in a similar proportion of adult human volunteers
(Clements ML, unpublished data), the B-1 virus is significantly
more restricted in replication than A2 in four species of animals
(mice, hamsters, Green monkeys, and chimpanzees). Again,
the mechanisms underlying these host range differences remain
undefined. Therefore, one must interpret with caution the absolute level of replication of subgroup B mutants in chimpanzees.
Studies with other live attenuated respiratory virus vaccine
candidates have shown that a relative restriction of replication
in a fully susceptible host of between 1000- and 1O,000-fold
compared to wt virus is desirable. Replication of the cp-52
virus was not detected by recovery of virus after inoculation
of seronegative chimpanzees with 104 or 105 pfu, indicating at
least a 300-fold restriction of replication compared with the wt
virus.
The cp-52 virus, while highly attenuated in semipermissive
animal hosts, might not be sufficiently attenuated for the fully
susceptible human infant. Therefore, we sought to derive ts
mutants from cp-52 to obtain viruses that represent an even
greater degree of attenuation. Several ts mutants with unusual
properties were derived from cp-52. The cpts-1324 mutant was
the most temperature-sensitive (shutoff temperature, 37°C) and
the most restricted in vivo. The cptssp-1415 mutant acquired
three additional in vitro phenotypes: small plaque, temperature
sensitivity (shutoff temperature, 39°C in Vero cells), and host
range restriction (failure to form plaques in HEp-2 cell monolayers). The cpts-1313 mutant exhibited a host range-dependent temperature-sensitive phenotype because it was temperature-sensitive in HEp-2 cells but not in Vero cells. The
cpts-176 virus was chosen for further mutagenesis because it
represented a virus possessing at least four putative attenuating
mutations and an intermediate level of temperature sensitivity
(39°C shutoff in Vero cells, and 37°C shutoff in HEp-2 cells).
Two ts mutants, designated cpts-176/427 and cpts-176/1072,
were derived that exhibited a lower shutoff temperature than
their cpts-l 76 parent. These two cpts viruses likely possess at
least five attenuating mutations. These mutants would be expected to be more restricted in replication in vivo in a susceptible host than the cpts-176 parent virus, although we could not
test this directly because of the near complete restriction of
replication of cpts-176 in cotton rats. Ultimately, the level of
attenuation of these viruses for the target population of human
infants must be determined during clinical trials. We plan to
initiate clinical trials with the cpts-176 virus, because it is a
vaccine candidate likely to be highly restricted in replication
JID 1996; 173 (April)
in humans. If this mutant proves to be over- or underattenuated
for seronegative infants, further testing will proceed .with viruses exhibiting a different level of attenuation above or below
that of cpts-176.
In summary, we have isolated and characterized a panel of
live attenuated subgroup B mutants that hold promise as vaccine candidates because they possess multiple (up to five) attenuating mutations of different types, a high level of genetic
stability, and a wide range of level of attenuation. Our goal is
to determine through clinical trials which, if any, of the subgroup B mutants possess the appropriate level of attenuation
for seronegative infants, then to develop a bivalent formulation
of RSV mutants containing both subgroup A and B mutants.
Acknowledgments
We thank Jin Lin Du, Ernest Banks, and Heather Henderson
for excellent technical support and Todd Heishman for editorial
assistance.
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