Division of Viral and Rickettsial Diseases,
National Center of Infectious Diseases, Centers for Disease Control
and Prevention, Atlanta, Georgia 30333
A better understanding of the immune response to live and
formalin-inactivated respiratory syncytial virus (RSV) is important for
developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in
bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c
mice intranasally infected with live RSV or intramuscularly inoculated
with formalin-inactivated RSV (FI-RSV). After RSV infection, both class
I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV
infection were less than class I-restricted CTLp frequencies through
day 14 p.i., during which class I-restricted CTLp frequencies
remained elevated, but then declined by 48 days p.i. The frequencies of
class II-restricted CTLps in the BAL were 2- to 10-fold less than those
of class I-restricted CTLps. For spleen cells, frequencies of both MHC
class I- and II-restricted CTLps to live RSV were similar. In contrast,
class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with
FI-RSV immunization and help define possible mechanisms of enhanced
disease after FI-RSV immunization. In addition, these studies provide a
quantitative means to address potential vaccine candidates by examining
both MHC class I- and II-restricted CTLp frequencies.
 |
INTRODUCTION |
Respiratory syncytial virus (RSV)
infection in infants and young children often results in lower
respiratory tract disease and is a high priority for vaccine
development (1, 2). Attempts to develop an effective live,
inactivated, or subunit vaccine have been unsuccessful (24, 25,
28). Early efforts at vaccinating young children with a
formalin-inactivated RSV (FI-RSV) vaccine failed to protect the
children from naturally acquired infection and actually enhanced lower
respiratory tract disease upon later virus infection (2, 15, 24,
25). This enhanced disease has created concern about the safety
of any nonlive RSV vaccine and, consequently, understanding the
pathogenesis of FI-RSV-induced enhanced disease is critically important
to vaccine development. Studies with BALB/c mice suggest that induction
of memory T cells producing Th2-like cytokines, as a result of FI-RSV
vaccination, may be key to the pathogenesis of enhanced disease
(6, 16, 28, 32, 40). Th2-like cytokine mRNA has been
demonstrated in cells from lung tissue or bronchoalveolar lavage (BAL)
specimens after RSV challenge of FI-RSV-immunized mice (17, 32,
40). In addition, in vivo studies using antibody (Ab) blockade
showed that the enhanced histopathology in FI-RSV-immune mice
challenged with live virus could be eliminated by using
anti-interleukin-4 (IL-4) and anti-IL-10 Abs but not anti-IL-12 Abs
(6). Recent evidence suggests that CD8+ T
lymphocytes may be important in directing the type of inflammatory response to RSV in challenge of G glycoprotein-sensitized mice (21, 31).
One aspect of the FI-RSV immune response that has not been well
characterized is the cytotoxic T-lymphocyte (CTL) response. There is
limited information on major histocompatibility complex (MHC) class
I-restricted CTLs after FI-RSV immunization (29), while the
information about the CTL response after live-RSV infection has been
well documented. Several studies have shown class I-restricted CTLs to
kill predominantly target cells expressing the M, N, or F RSV protein
(5, 7, 9, 26, 29, 41). The role of CTLs in the immune
response to RSV is well illustrated by in vivo depletion studies with
BALB/c mice (8, 18, 30). These studies suggest that both
CD4+ (class II) and CD8+ lymphocytes are
important for clearing RSV and that both contribute to the inflammatory
response associated with infection. A vaccinia virus construct
expressing RSV membrane-associated, nonglycosylated protein M2
has been affiliated with short-term protection in the BALB/c
mouse (7). This protein does not induce neutralizing Abs,
and therefore, protection likely is mediated by CTLs. Passive transfer
of CD8+ T lymphocytes has been associated with both
clearance of the virus and enhanced histopathology (1).
In this report, we describe studies of CTL precursor (CTLp) frequencies
in both live-RSV-infected and FI-RSV-immunized mice for MHC class I-
and class II-restricted target cells. These studies demonstrate clear
differences in the CTLp response between RSV and FI-RSV immunizations
and provide additional approaches to identifying potential
FI-RSV-induced enhanced disease mechanisms.
| |
MATERIALS AND METHODS |
Animals.
Four- to 6-week-old, specific-pathogen-free female
BALB/c mice were purchased from Harlan Sprague-Dawley Laboratories
(Indianapolis, Ind.). The mice were housed in microisolator cages and
fed sterilized water and food ad libitum.
Viruses and FI-RSV vaccine.
The A2 (Long) strain of RSV and
human parainfluenza virus type 3 (HPIV3) were grown in Vero cells in
tissue culture medium (TCM) consisting of RPMI 1640 medium (GIBCO
Laboratories, Grand Island, N.Y.) supplemented with 2%
heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Salt
Lake City, Utah), 1% L-glutamine, and 1%
antibiotic-antimycotic (all from GIBCO). Upon the appearance of
cytopathic effects, the medium was decanted and replaced with a minimal
volume of Dulbecco's modified phosphate-buffered saline and frozen at
70°C. The flask was thawed, and any remaining adherent cells were
scraped off with a cell scraper (Costar, Cambridge, Mass.) and
collected. The cells and supernatant were centrifuged at 2,000 × g for 15 min at 4°C. The resulting supernatant was collected, divided into aliquots, and stored at 70°C. A portion of
the virus stock was purified by using a discontinuous sucrose gradient
as previously described (27). The titer was determined by
methylcellulose plaque assay on Vero and HEp-2 cells.
FI-RSV and FI-HPIV3 vaccine was prepared as described previously
(40). Briefly, 1 part formalin (Sigma, St. Louis, Mo.) was
incubated with 4,000 parts clarified virus lysate for 3 days at 37°C
and pelleted by centrifugation for 1 h at 50,000 × g. The volume of virus was adjusted to a 1:25 dilution of
the original volume in minimum essential medium (MEM; GIBCO) and
subsequently precipitated with aluminum hydroxide (4 mg/ml; Sigma),
resuspended in 1/100 of the original volume in serum-free MEM, and
stored at 4°C.
Infection, immunization, challenge, and sampling.
Mice were
anesthetized by intraperitoneal administration of 2,2,2-tribromoethanol
(Avertin) and then infected intranasally (i.n.) with 106
PFU of RSV or HPIV3 or intramuscularly immunized with a 106
PFU equivalent of FI-RSV or FI-HPIV3 in the superficial gluteal muscle.
A portion of the mice immunized with FI-RSV or FI-HPIV3 was i.n.
challenged 3 weeks after immunization with 106 PFU of live
RSV or HPIV3. Prior to removal of BAL or spleens on days 3, 4, 5, 7, 10, 14, and 48 postinfection (p.i.), mice were anesthetized by
intraperitoneal administration of 2,2,2-tribromoethanol and
exsanguinated by severing the right caudal artery. All organs were
collected on ice in Hanks balanced salt solution.
MHC class I- and II-restricted CTLp assays.
The class
I-restricted target cells used were the mouse mastocytoma line P815
(ATCC TIB 64), and the class II-restricted target cells were a subclone
of the B-cell lymphoma line A20 (ATCC TIB 208). Both cell lines were
maintained in RPMI 1640 (GIBCO) containing 10% FBS (Hyclone) plus 1%
antibiotic-antimycotic (GIBCO). The respective target cells were
prepared by suspending 106 cells in 1.0 ml of serum-free
MEM (GIBCO) containing 107-PFU/ml RSV (or a multiplicity of
infection of 10 PFU/cell) in cell lysate (or a comparable dilution of
uninfected cell control lysate) for 18 h at 37°C, followed by
addition of 1.0 ml of MEM containing 10% FBS and 200 µCi of
51Cr (Na2CrO4; Amersham, Arlington
Heights, Ill.), and incubating them for an additional 2 h at
37°C. The cells were then washed and resuspended to an appropriate
concentration in TCM comprised of suspension-MEM (GIBCO) containing
10% FBS (Hyclone), 1% essential amino acids, 2% nonessential amino
acids, 2% sodium pyruvate, 2% L-glutamine, 1%
antibiotic-antimycotic (all from GIBCO), and 50 µM 2-mercaptoethanol
(Sigma).
Virus-specific CTLp prevalence was determined by using a modification
of a well-established limiting-dilution assay (20, 30, 33,
35-37). In brief, different dilutions of effector cells in 0.1 ml of TCM were added to wells (24 wells/dilution) of round-bottom, 96-well microtiter plates (Costar) with 0.1 ml of antigen
(Ag)-presenting cells (APCs). The APCs were syngeneic splenocytes that
had been incubated in a serum-free MEM (GIBCO) containing 1,000-PFU/ml RSV for 2 to 3 h at 37°C and resuspended at 107
cells/ml in TCM containing 20% EL4.IL-2 supernatant (the lymphoma cell
line EL4.IL-2 [ATCC TIB 181] endogenously secretes IL-2). The
effector cells and APCs were incubated at 37°C for 7 days in a
humidified atmosphere. The contents of individual wells were then
divided in two, placed into replica plates, and incubated for 6 h
with 104 51Cr-labeled, RSV-infected or
mock-infected target cells. The virus-specific CTLp frequency was
estimated by using linear regression and 95% confidence intervals
about the slope of the regression line and plotting the number of cells
versus the number of nonresponding cultures. A responding well was
defined as one in which the mean 51Cr release from
RSV-infected targets plus effector cells was 
3 standard deviations
from the mean 51Cr release from control wells containing
uninfected target cells plus effector cells. The virus-specific CTLp
frequency was estimated according to the Poisson equation at the 37%
nonresponding culture point (F0) along the slope
of the linear regression line. The 95% confidence intervals were used
to determine significance, which is indicated by P < 0.05.
Confirmation of MHC class II-specific cytolysis.
To confirm
class II-specific cytolysis, purified
anti-I-Ad/I-Ed monoclonal Ab 2G9 (PharMingen)
was incubated with effector cells and either P815 or A20 target cells.
The purified anti-I-Ad/I-Ed monoclonal Ab
blocked the cytolysis of the A20 target cells but not that of P815
target cells, demonstrating that cytolysis of A20 target cells was MHC
class II restricted, as expected (data not shown).
| |
RESULTS |
Primary CTLp frequencies after live-RSV infection.
Limiting-dilution analysis was performed during the acute immune
response to i.n. RSV infection (Tables 1
and 2). Significant numbers of both MHC
class I- and II-restricted, virus-specific CTLps could be detected in
the BAL (Table 1) and spleen (Table 2) as early as days 4 (spleen) and
5 (BAL) p.i. The class I-restricted CTLp frequencies at day 5 p.i. in the BAL were significantly (P < 0.05) higher
(Table 1; 1:46,500 to 1:50,200) than the class II-restricted
CTLp frequencies (Table 1; 1:145,000 to 1:162,100). The
frequencies of both class I-restricted CTLps remained relatively invariant through day 12 p.i. CTLp frequencies ranged from 1:5,300 at day 7 p.i. to 1:7,500 at day 14 p.i. The frequencies of
class II-restricted CTLps were similarly stable, ranging from
1:22,800 at day 7 p.i. to 1:38,000 at day 14 p.i. However,
the frequencies of both class I-and II-restricted CTLps
examined at 48 days p.i. showed an approximately 10-fold decline (Table
1). In contrast, the class I-and II-restricted CTLp frequencies
in the spleen were similar. The class I-restricted CTLp frequencies
remained stable from day 5 to day 12 p.i. (Table 2; range, 1:2,200
at day 5 to 1:6,400 at day 12) and then decreased about sevenfold by
day 48 p.i. (Table 2; range, 1:26,900 to 1:33,400). The class
II-restricted CTLp frequency reached a maximum on day 10 p.i.
(Table 2; range, 1:2,000 to 1:2,900) and decreased about eightfold by
day 48 p.i. (Table 2; range, 1:14,200 to 1:21,200). Both the
kinetics and frequencies of class I-restricted CTLps are
comparable to those observed with other respiratory virus
infections in mice, including Sendai and influenza virus infections
(10-12). The RSV-specific, class II-restricted CTLp
frequencies have not been previously reported but are similar to those
reported for Sendai virus (20). Heterologous virus control
infection with HPIV3 did not generate a detectable RSV-specific CTLp
frequency (>106) in either the BAL (Table 1) or the
spleen (Table 2).
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TABLE 1.
RSV-specific CTLp frequencies for MHC class I- and
II-restricted lymphocytes isolated from BAL fluid of BALB/c
mice at various time points post-i.n. infection with RSV or an
HPIV3 controla
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TABLE 2.
RSV-specific CTLp frequencies for MHC class I- and
II-restricted lymphocytes isolated from the spleens of BALB/c
mice at various time points post-i.n. infection with RSV or an
HPIV3 controla
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Primary CTLp frequencies after FI-RSV vaccination.
The
CTLp frequencies in the spleen after FI-RSV vaccination were
qualitatively different than those observed after live-RSV challenge
noted above (i.e., higher frequencies of class II-restricted CTLps
and lower frequencies of class I-restricted CTLps; Table 3). There were too few BAL cells after
FI-RSV vaccination to study CTLp frequencies. In the spleen, class
I-restricted CTLp frequencies were the highest at day 10 (range,
1:14,600 to 1:18,700) and rapidly decreased to between 1:38,500 and
1:65,200 by day 48 p.i. (Table 3). Class II-restricted CTLp
frequencies were higher than class I-restricted CTLp frequencies
throughout the study period. The class II-restricted CTLps reached
their highest numbers at day 10 p.i. (Table 3; range, 1:1,700 to
1:3,300) and decreased only about twofold at day 48 p.i. (Table 3;
range, 1:2,700 to 1:3,900). RSV-specific CTLp frequencies could not be detected (>106) following immunization with FI-HPIV3
(Table 3).
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TABLE 3.
RSV-specific CTLp frequencies for MHC class I- and
II-restricted lymphocytes isolated from the spleens of BALB/c
mice at various time points postvaccination with FI-RSV or
an FI-HPIV3 controla
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CTLp frequencies after RSV challenge of FI-RSV-vaccinated
mice.
Upon live-RSV challenge of FI-RSV-vaccinated mice, a
dramatic increase in class I-restricted CTLp frequencies was
observed, as well as an initial decline in the class II-restricted
CTLp frequency (Table 4). The class
II-restricted CTLp frequency ranges decreased from 1:11,900 to
1:12,200 on day 0 to 1:19,700 to 1:24,600 on day 3 p.i. and then
increased to between 1:8,200 and 1:12,700 on day 4 p.i. and to
1:3,100 to 1:8,200 on day 6 p.i. (Table 4). In contrast, the class
I-restricted CTLp frequencies increased approximately 10-fold
following challenge of FI-RSV-vaccinated mice, ranging between
1:111,300 and 1:113,600 at day 0 (3 weeks post-FI-RSV immunization)
and between 1:12,600 and 1:14,400 at day 3 p.i. (Table 4). As
expected, the CTLp frequencies detected were higher earlier in mice
previously immunized (with FI-RSV) than in unimmunized mice (Table
4). HPIV3 infection, FI-HPIV3 immunization, or HPIV3 challenge of
FI-HPIV3-immunized mice did not generate significant RSV-specific
CTLps during the periods examined (Table 4). RSV challenge of
FI-HPIV3-immunized mice resulted in CTLp frequencies comparable to
those observed in mice receiving RSV alone (Table 4). HPIV3 challenge
of HPIV3-immunized mice also did not generate detectable RSV-specific
CTLps (Table 4).
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TABLE 4.
RSV-specific CTLp frequencies for MHC class I- and
II-restricted lymphocytes isolated from the spleens of BALB/c mice
challenged with RSV or HPIV3 3 weeks after intramuscular immunization
with FI-RSV or FI-HPIV3a
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| |
DISCUSSION |
In this study, we examined RSV-specific CTLp frequencies in BAL
samples and spleen cells of BALB/c mice after live-virus infection, compared to FI-RSV immunization. After live-RSV infection, both MHC
class I-and II-restricted CTLps were noted, with class
I-restricted CTLps most common in BAL cells throughout the study,
and in spleen cells early in the acute response phase. By day 10 p.i. in the spleen, class II-restricted CTLps were more frequent
than class I CTLps. In contrast, FI-RSV immunization induced a much
higher frequency of class II-restricted CTLps through day 14 p.i. This difference between the live-RSV and FI-RSV immunizations
demonstrates the value of using CTLp frequencies to quantitate the CTL
response and the importance of examining both class I-and
II-restricted CTLp responses.
The preponderance of class II-restricted CTLps after FI-RSV
vaccination was expected, given the noninfectious nature of FI-RSV. As an extracellular Ag, it must be endocytosed for processing, which is
a route that favors class II Ag presentation but does not necessarily
limit class I presentation. The delay observed in the development of
the class I-restricted response in FI-RSV-immunized mice may
reflect inefficient transport of this extracellular Ag to the
endoplasmic reticulum, the site where class I molecules are loaded with
peptides for presentation to CD8+ T lymphocytes. The
extracellular source of RSV Ag affiliated with FI-RSV immunization
has also been suggested as responsible for the induction of memory T
cells that produce Th2-like rather than Th1-like cytokines (6, 32,
40). Class II-restricted CTL responses have been noted for a
number of other RNA viruses, including parainfluenza and Sendai viruses
(4, 10, 14), measles viruses (38, 39), and
coronavirus (19) in the family Paramyxoviridae
and recently for influenza virus (13, 34) in the family
Orthomyxoviridae. Limiting dilution analysis of parainfluenza virus-specific CD4+ and CD8+ T
lymphocytes from the peripheral blood of adults showed relatively high
frequencies for both CD4+ (1:4,700) and CD8+
(1:2,500) CTLps (10). Measles virus infection in humans has been shown to induce both HLA class I-and class II-restricted CTLs specific to the transmembrane fusion glycoprotein (39). In mice, Sendai virus-specific class II-restricted T-cell responses have been investigated and defined (4, 13). Class
II-restricted, Sendai virus-specific T-cell hybridomas have been
generated, and their reactivity has been characterized to the level of
specific peptide sequences in the matrix protein (4).
Similarly, class II-restricted (CD4+) precursors were
detected in mice during the acute immune response to Sendai virus
(13, 14). Mouse hepatitis virus, a coronavirus, has been
found to induce only class II-restricted CTLs in both BALB/c and
C57BL/6 mice (19). Thus, generation of class
II-restricted cells probably is a common component of the immune
response to RNA viruses.
Although this is the first study to examine MHC class I-and
II-restricted, RSV-specific CTLp frequencies, a number of
investigators have studied RSV-specific CTLs. Most of this work has
focused on class I-restricted CTLs (1, 5, 7, 18, 26,
29), and one study identified class II-restricted CTLs in
cell lines developed from spleen cells from BALB/c mice and peripheral
blood mononuclear cells from humans (29). In this study, no
effector CTLs were detected after FI-RSV immunization. The
inability to detect RSV-specific effector CTLs after FI-RSV
immunization may be due to differences in sensitivity and culture
conditions between bulk-culture CTL assays and limiting-dilution
culture assays.
It is appealing to speculate that the shift in CTLp frequency from a
predominately class II-restricted response following FI-RSV
vaccination to a predominately class I-restricted response following live-virus challenge is related to the shift from Th2-type cytokines toward Th1-type cytokines after RSV infection in
FI-RSV-immunized mice (40). The data in Table 4 shows
that the CTLp response generated by FI-RSV immunization, although
predominantly class II restricted, is also significantly class I
restricted. One possible mechanism for generating class
I-restricted CTLps is by heat shock protein channeling of RSV
peptides to the class I pathway, as has been reported for tumor
peptides (3). Subsequent challenge of FI-RSV-immune mice
with live RSV would then rapidly expand memory CD8+ T
lymphocytes and limit the Th2-type phenotype, as has been previously described (21, 31). This could offer a possible explanation for the lack of enhanced disease in FI-RSV-vaccinated children who
experienced a second RSV infection. Further studies of the frequency and characteristics of RSV-specific CTLps should help to
characterize disease-enhancing components of the immune response to
RSV.
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Abstract
Introduction
