Growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract.

Abstract

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in infants and children. To study RSV replication, we have developed an in vitro model of human nasopharyngeal mucosa, human airway epithelium (HAE). RSV grows to moderate titers in HAE, though they are significantly lower than those in a continuous epithelial cell line, HEp-2. In HAE, RSV spreads over time to form focal collections of infected cells causing minimal cytopathic effect. Unlike HEp-2 cells, in which wild-type and live-attenuated vaccine candidate viruses grow equally well, the vaccine candidates exhibit growth in HAE that parallels their level of attenuation in children.

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JOURNAL OF VIROLOGY, July 2005, p. 8651–8654
0022-538X/05/$08.00?0
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 13
doi:10.1128/JVI.79.13.8651–8654.2005
Growth of Respiratory Syncytial Virus in Primary Epithelial Cells
from the Human Respiratory Tract
Peter F. Wright,1,2,4* Mine R. Ikizler,1Ricardo A. Gonzales,1Kecia N. Carroll,1
Joyce E. Johnson,4and Jay A. Werkhaven3
Departments of Pediatrics,1Microbiology and Immunology,2Otolaryngology,3and Pathology,4
Vanderbilt University Medical Center, Nashville, Tennessee
Received 17 November 2004/Accepted 1 March 2005
Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in infants
and children. To study RSV replication, we have developed an in vitro model of human nasopharyngeal
mucosa, human airway epithelium (HAE). RSV grows to moderate titers in HAE, though they are significantly
lower than those in a continuous epithelial cell line, HEp-2. In HAE, RSV spreads over time to form focal
collections of infected cells causing minimal cytopathic effect. Unlike HEp-2 cells, in which wild-type and
live-attenuated vaccine candidate viruses grow equally well, the vaccine candidates exhibit growth in HAE that
parallels their level of attenuation in children.
Respiratory syncytial virus (RSV) causes severe respiratory
illness in infancy (10) and recurrent upper respiratory tract
infections in older children and adults (11) and is an important
cause of morbidity in the elderly (7). In pathological specimens
from human cases, RSV grows exclusively in epithelial cells
lining the respiratory tract (17). The clinical impact of RSV
must be explained in the context of growth in epithelial cells
and in the innate and adaptive immune response that RSV
infection initiates in the associated lymphoid tissues lining the
respiratory tract.
RSV growth was examined in primary adenoid epithelial
cells, human airway epithelium (HAE), derived from tissue
removed at adenoidectomy. We have previously described our
HAE model and its support of the growth of influenza virus (6,
18). Surgical removal of adenoids is most often performed for
hypertrophy and partial airway or eustachian tube obstruction
(2). Although chronic inflammatory changes are seen occasion-
ally, the microscopic adenoid anatomy is usually normal. The
adenoids have a transitional epithelium with representation of
ciliated cells, Clara cells, and mucin-producing goblet cells (6).
We hypothesized that examination of RSV replication in HAE
would give important clues to key events in the virus replica-
tion cycle and host response to infection.
Exploration of the growth of RSV in primary respiratory
cells has been largely confined to studies of tracheal organ
cultures in animal species. In a bovine model, with a bovine
RSV strain, growth was seen without alteration in ciliary func-
tion and with virus primarily in the subepithelium (15); never-
theless, RSV is a significant respiratory pathogen in young
cattle (1). In a ferret tracheal ring RSV growth was maximal
between 5 and 7 days (12). As in the bovine model, no histo-
logic changes or diminution of ciliary activity was seen and
RSV was detected on the lamina propria and serosal surface of
ferret tracheal ring. In a piglet tracheal ring, destruction of
ciliated cells and growth of virus over a 17- to 19-day period
were observed. By fluorescence, brightly staining cells scat-
tered in the epithelial layer were seen with no virus in the
subepithelium (8). The animal models of RSV in differentiated
epithelial cells and in organ cultures emphasize the need to
explore the human model, as the studies show sharp dichoto-
mies in the site of replication of RSV.
In a human tracheal ring organ culture, there were multinu-
cleated cells with cytoplasmic inclusions and diminished ciliary
activity (12). By fluorescence, RSV was confined to the super-
ficial epithelial layer with selective infection of a single cell
surrounded by uninfected cells (12). Some tracheal rings were
from a fetus as young as 18 weeks, in which there should be
limited development of the immune system, suggesting that the
restriction in numbers and types of cells infected is not immu-
nologically mediated. It appeared on electron microscopy that
* Corresponding author. Mailing address: Vanderbilt University
Medical Center, Department of Pediatrics, D-7235 MCN, 1215 21st
Ave., Nashville, TN 37232. Phone: (615) 322-2250. Fax: (615) 343-
9723. E-mail: peter.wright@vanderbilt.edu.
FIG. 1. Growth of RSV in respiratory epithelial tissue culture sys-
tems. Titers of RSV strains were determined by plaque assay on HEp-2
cell monolayer cultures maintained under semisolid overlay at 37°C for
wild-type RSV and at 32°C for live-attenuated mutants as previously
described (13). Virus recovery was significantly greater from HEp-2
cells than from primary human epithelial cells (HAE) (P ? 0.01 at 48
and 72 h).
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ciliary cells were infected (12). Studies of RSV histopathology
in the human lung have shown only isolated cells in the bron-
chioles and alveoli that are infected (17). In contradistinction,
extensive staining of exfoliated cells in intraluminal airway
debris is seen, suggesting that infected cells may be rapidly
shed into the airway (J. E. Johnson, unpublished data). A
recent paper has demonstrated more uniform superficial epi-
thelial cell localization of RSV in ciliated human airway epi-
thelial cells using RSV expressing a green fluorescent protein
(GFP) (20).
Our current studies have focused on RSV in adenoid epi-
thelial cells and have involved quantitation of virus growth and
extent and pattern of RSV infection by immunofluorescence
and immunohistochemistry and correlation of level of growth
in HAE of a series of live-attenuated RSV vaccine candidates
with their levels of attenuation in animal models (3, 4) and
adults and children (13, 19).
With the approval of the Vanderbilt Institutional Review
Board, adenoids were obtained from surgeries performed for
independently defined clinical indications (2). The isolation
and growth of primary epithelial cells from adenoidal tissue
were previously described by our group (6, 18). Cells were
grown on the collagen matrix of Vitrogen 100 (Cohesion, Palo
Alto, CA) at 37°C under 5% CO2as a “submerged culture” or
in Transwell collagen-coated inserts (Corning Inc., Corning,
NY). The experiments were performed as soon as the cells
were confluent, 8 to 14 days, at which time the ciliary cell
population was approximately 10% and the cells were one to
three layers thick. These cells are different in site of origin and
length of time in culture (2 weeks versus 5 to 6 weeks) from the
uniformly ciliated, multilayered population of cells described
by Zhang et al. (20). They were used at a point when resistance
was greater than 300 ? · cm2as measured by an epithelial volt
ohmmeter (World Precision Instruments, Sarasota, FL).
Respiratory syncytial virus in primary epithelial cells. RSV
grew to a significantly lower titer in HAE than in HEp-2 cells
in parallel experiments done with the same low multiplicity of
input (MOI) of 0.01 PFU per cell (Fig. 1). Pairwise compari-
sons of growth in the two cell lines were done by both t test and
Mann-Whitney test. A low MOI limited residual virus from the
FIG. 2. Demonstration of the limited growth and focal nature of the infection of RSV in HAE. Shown are expression of GFP by rgRSV in
(A) HEp-2 cells and (B) HAE at 72 h after initiation of infection and results of immunoperoxidase staining with a goat biotin-conjugated antibody
to RSV (Biodesign International, Saco, ME) in (C) HAE and (D) an adenoid organ culture.
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inoculum and thus increased the capacity to demonstrate rep-
lication. The lower initial growth in HAE, the target cells for
RSV infection, was somewhat surprising; nevertheless, the in-
creasing titer of virus in HAE indicated that RSV underwent
productive infection (one-way analysis of variance was used to
test trend over time). The titer of virus recovered from epithe-
lial cells reached a plateau between 48 and 72 h. However,
virus could be recovered at sustained high titers for at least 30
days with no visible alteration in microscopic appearance of the
cells and with continued ciliary activity.
Several aspects of RSV growth in HAE were explored to
help identify causes of limited initial viral replication. Recovery
of virus from HAE at 24 and 72 h was directly influenced by the
initial MOI of virus. The titer at 72 h was 103.65with an MOI
of 0.01 and 105.5with an MOI of 1. This indicates limitation in
the spread of newly formed virus in HAE. The growth of RSV
A2, an extensively laboratory-adapted strain, and that of a
recently isolated RSV A strain were comparable. Fetal bovine
serum concentrations between 0 and 10% had no effect on
virus growth in both HEp-2 cells and HAE. Titers of RSV in
supernatants and cell lysates were the same in both cell sys-
tems. Using a Transwell insert, RSV growth was seen only with
apical application of virus, and release of newly produced virus
was also from the apical surface.
In HAE RSV infection remains focal whereas all cells in
HEp-2 monolayers become infected. The pattern of fluores-
cence with the rgRSV A2 strain with the GFP insert demon-
strates this, as does immunohistochemistry of both HAE and
an infected adenoid organ culture (Fig. 2). This pattern is also
seen in the human lung of infants dying of RSV infection (17).
Possible explanations for the more limited growth in HAE
appear to be that, even with a high multiplicity, RSV is selec-
tive in the cells in which it will replicate, is influenced in
replication by innate immunity, or has a limitation in cell-to-
cell spread.
Attenuated respiratory syncytial virus strains in HAE. The
isolation and characterization of wild-type RSV strain A2,
cold-passaged RSV A2 strain 3131, strain cp, further-attenuated
cpA2 derivatives (rA2cp530/1009, rA2cp248/955, rA2cp248/404,
and rA2cp248/404/1030?SH), and vaccines with deletions of
the NS2 gene have been described previously (5). All have
been evaluated in an ongoing clinical vaccine development
program (13, 19) in a slow, iterative process of evaluation of
strains in adults and progressively younger children. Although
animal models have given important clues to the level of at-
tenuation of strains (5), a tissue culture system in which atten-
uation could be predicted would be helpful. Towards that end
RSV vaccines that had been evaluated in humans and/or chim-
panzees were compared to wild-type virus in HAE cells at
three temperatures, 32°C, 35°C, and 37°C. Each vaccine was
from an experimental vaccine lot with titers of between 105and
106PFU/ml, and each experiment was repeated two to four
times. In HAE the progressively more attenuated derivatives
of cpA2 at permissive temperatures mirrored their growth in
young children and infants (Table 1). Two candidates,
rA2cp248/404 and rA2cp248/404/1030?SH, were considered
sufficiently attenuated to give to infants, although the former
caused mild congestion in infants (19). rA2cp248/404/
1030?SH has an excellent safety profile with limited but con-
sistent replication even in the fully susceptible child (R. Kar-
ron, unpublished data). The introduction of the NS2 deletion
(14) into three prototype vaccine candidates caused a 3- to
40-fold decrease in growth in HAE (Table 1) but not in HEp-2
cells. These findings parallel the growth of these strains in
children, results in which rA2cpNS2 grew to a limited extent in
seropositive children, indicating partial attenuation, and the
rA2cp248/404?NS2and rA2cp530/1009NS2
highly attenuated with limited growth in seronegative children.
Evaluation in chimpanzees also suggests attenuation associ-
ated with deletion of NS2 (Table 1) (16). Growth in HEp-2
cells of the attenuated mutants was generally equivalent to that
of wild type at 32°C, though influenced by temperature sensi-
tivity, so that most of the attenuated growth was limited at 37°C
or even 35°C. There was a significant correlation between
strainswere
TABLE 1. Correspondence of RSV growth in HAE cells and chimpanzee models with that in seronegative children
Clinical assessmentVaccine strain
Recovery of RSV in indicated system (PFU/ml)
HEp-2a
HAECa
Chimpanzeesb
Seronegative childrenb
(% of children infected)
32°C35°C37°C
Virulent virus
Insufficiently attenuated for
seronegative children
A2wt
Cp
530/1009
?NS2
248/955
ts1
248/404
6.5
4.5
4.8
5.3
5.2
5.0
5.2
3.7
3.1
4.0
2.5
4.0
3.2
4.0
4.3
3.7
3.4
3.2
4.2
3.2
3.8
4.8
3.9
3.6
2.1
3.0
2.2
2.6
5.4
5.1
3.6
3.8
4.6
4.2
2.5
Not tested
Not specified (100)
4.5 (100)
Not specified (100)
4.4 (88)
3.7 (96)
4.2 (79)Appropriately attenuated
for seronegative children;
insufficiently attenuated
for infants
Appropriately attenuated
for seronegative children
and infants
Overly attenuated for
seronegative children;
not tested in infants
248/404?SH
248/404/1030?SH
5.9
5.9
3.7
2.6
2.6
2.4
Not tested
1.7
2.14.3 (100)
2.5 (100)Not tested
ts26.01.3
?1
?1.02.2Not specified (14)
248/404?NS2Not tested3.0 2.2 2.2Not tested2.8 (40)
aMean titer of two to four experiments at 72 h with the input virus titer between 105and 106PFU/ml for each vaccine candidate.
bMean peak titer over course of infection.
VOL. 79, 2005NOTES8653

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growth in the chimpanzee and that in HAE in ranking of titer
(Spearman’s rho correlation using rank P ? 0.03) and in ab-
solute titer (Pearson correlation [two-tailed], P ? 0.03). The
growth in HAE approached significance in comparison with
growth in seronegative children, 0.07, by rank and by titer. The
chimpanzee data were not a significant predictor of growth of
RSV attenuated strains in seronegative children. Limited
growth in HAE, below 3.0 logs, was predictive of attenuation in
the most susceptible seronegative children. This was true for
the vaccines derived by sequential mutation from the original
cold-adapted vaccine (9) and those with attenuation conveyed
by deletion of the NS2 gene (16). Although clinical trials in
humans are essential, it appears that HAE represents a very
effective screening step for comparing attenuation of vaccine
candidates. This concept is strengthened by the demonstration
that effective attenuating mutations in three other viruses, in-
fluenza A, influenza B, and parainfluenza type 3 virus, all limit
growth in HAE compared to that of their parent strain (18).
This work was supported by NO1-AI-65298, Respiratory Mucosal
Pathogens Unit.
The contributions of Sandra Yoder and Sarah E. Winbourn to the
work in this paper are gratefully acknowledged. Live-attenuated RSV
vaccines were provided by Brian Murphy, Laboratory of Infectious
Disease, NIAID, NIH, Bethesda, MD, or by Wyeth Lederle Vaccines,
Pearl River, NY (Valerie Randolph). The wild-type RSV A2 recom-
binant green fluorescent protein-expressing construct, rgRSV, was
used in several experiments (kindly supplied by Mark Peeples, Rush-
Presbyterian-St. Luke’ s Medical Center, Chicago, IL).
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