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*
Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, United Kingdom; and
Department of Respiratory Medicine, St. Mary’s Medical School, Paddington, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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enzyme-linked
immunospot and 51Cr release assay in local and systemic
lymph nodes, and their induction was dependent on the use of a mucosal
adjuvant. CTL elicited by peptide immunization afforded protection
against RSV challenge, but also enhanced weight loss. CTL-mediated
viral clearance was not dependent on IFN- since depletion using
specific mAb during RSV challenge did not affect cellular recruitment
or viral clearance. Depletion of IFN- did, however, reduce the
concentration of TNF detected in lung homogenates of challenged mice
and largely prevented the weight loss associated with CTL-mediated
viral clearance. Mice primed with the attachment glycoprotein (G)
develop lung eosinophilia after intranasal RSV challenge. Mucosal
peptide vaccination reduced pulmonary eosinophilia in mice subsequently
immunized with G and challenged with RSV. These studies emphasize that
protective and immunoregulatory CD8+ CTL responses can be
mucosally elicited using enterotoxin-based mucosal adjuvants but that
resistance against viral infection may be accompanied by enhanced
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The exacerbation of disease in humans immunized with
formalin-inactivated RSV has been modeled in murine studies using
recombinant vaccinia virus (rVV) engineered to express the RSV
attachment protein G (rVV-G). BALB/c mice primed with rVV-G and
challenged with RSV are more resistant to infection, but develop
enhanced weight loss and an immunopathology characterized by pulmonary
eosinophilia (4, 5). In mice, CD4+ T
cells secreting type 2 cytokines are necessary for this response since
their depletion eliminates eosinophilia (6). Eosinophilia
in mice can be prevented if RSV-specific CD8+ T
cells are activated before or at the same time as
CD4+ T cells (7). Although BALB/c
mice develop lung eosinophilia after G protein priming, normal C57BL/6
mice do not. Removal of functional CD8+ T cells
using specific Ab or TAP-1, 
2-microglobin, or
CD8 gene knockout mice renders C57BL/6 mice susceptible to eosinophilia
(8). CD8+ T cells are therefore
critical regulators of Th2-driven eosinophilic lung disease and it has
been proposed that vaccines which drive RSV-specific
CD8+ T cells may prevent immunopathology caused
by CD4+ Th2 cells (8).
CD8+ T cells are associated with immunity to RSV. RSV-specific CD8+ T cells can be detected in the blood of previously infected adults in whom CTL responses are associated with reduced clinical symptoms (9). CD8+ T cells also mediate resistance to RSV in murine models. BALB/c mice primed with rVV expressing the RSV second matrix protein (M2) mount a CD8+ T cell response and develop protective immunity (10). In BALB/c mice, the H-2Kd-restricted peptide corresponding to residues 82–90 of the M2 protein, which is conserved in subgroup A and B RSVs, is necessary and sufficient to induce protective CD8+ T cell responses (11, 12). Successful RSV vaccination strategies designed to exploit the protective role of M282–90-specific CD8+ T cells include DNA vaccines (13) and intranasal (i.n.) administration of a chimeric M2 peptide (14). Although strong RSV-specific CTL can mediate resistance to infection, viral clearance can also be associated with acute and sometimes fatal pulmonary disease (15).
Intranasal vaccination against RSV represents an attractive approach
since host immune cells are likely to be primed in relevant lymphoid
tissues. CD8+ T cell responses are, however,
typically difficult to induce by simple introduction of nonliving Ag
into the respiratory tract. We have previously shown that i.n.
coadministration of peptide or protein Ags with bacterial enterotoxins
like Escherichia coli heat-labile (LT) enterotoxin augments
Ag-specific CD8+ CTL responses (16).
Induction of CD8+ CTL responses using LT as an
adjuvant is independent of functional TNF (C.P.S. and G.D.,
unpublished observations), IL-12, IFN-, and
CD4+ T cell help (16). The adjuvant
activity of enterotoxins for CTL induction extends to nontoxic mutant
derivatives of LT containing attenuating single amino acid
substitutions in their enzymatically active A subunits (e.g., LTK63).
LTK63 is molecularly defined (17), has well described
mucosal adjuvant properties (18, 19, 20), and may represent a
safe adjuvant for use in humans.
We have employed a mucosal vaccination strategy that exploits the
adjuvant properties of LT and LTK63 to elicit
CD8+ CTL responses to the dominant CTL epitope in
the M2 protein of RSV. The CTL elicited by immunization-mediated
resistance to RSV challenge via a mechanism independent of IFN-.
Furthermore, mucosally elicited M282–90-specific
CD8+ T cells reduced pulmonary eosinophilia in
mice subsequently primed with the G protein and challenged with RSV.
These are the first studies describing induction and characterization
of protective CTL responses against an RSV Ag by combining mucosal
immunization with defined mucosal adjuvants.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were purchased from Harlan Olac (Bicester, U.K.) and
used when 8 to 10 wk old. RSV and recombinant vaccinia virus expressing
the rVV-G, matrix protein (M2), or control -galactosidase
(rVV--gal) were grown in HEp-2 cells and assayed for infectivity as
previously described (21). All stocks were free of
Mycoplasma infection (determined by DNA hybridization;
Gen-Probe, San Diego, CA).
Ags and Abs
A peptide with sequence SYGSINNI, corresponding to residues
82–90 of the RSV M2 protein, was purchased from Zinsser Analytic
(Maidenhead, Berkshire, U.K.) and was >95% pure. Wild-type porcine LT
and LTK63 were kindly provided by M. Pizza and R. Rappuoli (Chiron
Vaccines, Siena, Italy). The neutralizing anti-IFN- Ab from
clone XMG1.2 was purified from ascites fluid.
Immunizations and challenge
For i.n. immunization, groups of mice were lightly anesthetized
and 30 µl of Ag in PBS was applied to the nasal nares on days 0 and
14. In control groups, mice were scarified once on the rump on day 0
with 3 x 106 PFU rVV-M2 or rVV--gal in a
final volume of 10 µl. Three to four mice from each group were killed
on day 28 when superficial cervical lymph nodes and spleens were
removed for CTL and enzyme-linked immunospot (ELISPOT) assays. The
remaining mice were challenged i.n. with 3 x
106 human RSV (A2 strain, 50 µl). On selected
days postchallenge, four to five mice were killed by injection of 3 mg
pentobarbitone.
Cytokine depletion
Depletion of IFN- was performed by i.p. injection of 0.25 mg
rat anti-mouse IFN- Ab (clone XMG1.2) starting 2 days before RSV
challenge and then every other day for 7 days. Control mice received an
equivalent amount of an irrelevant, isotype-matched mAb on the
same days.
Cell recovery
Bronchoalveolar lavage (BAL) was performed on selected days postchallenge by methods described previously (8). Briefly, the lungs of each mouse were inflated six times with 1 ml of 12 mM lidocaine in DMEM. A total of 100 µl of this BAL fluid from each mouse was retained for cytospin analysis and the rest was immediately diluted into ice-cold RPMI 1640 containing 10% FCS (Sigma, St. Louis, MO), 2 mM/ml L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin (complete RPMI, cRPMI). Cells were harvested by centrifugation and resuspended at 106 cells/ml for flow cytometric analysis. The BAL supernatant was stored at -70°C for subsequent analysis of cytokine content.
Flow cytometric analysis of intracellular and cell surface Ags
Cells were stained with rat anti-mouse fluorochrome-conjugated Abs to CD4 (QR), CD8 (PE), and CD45RB (FITC) (PharMingen, Oxfordshire, U.K.) for 30 min on ice, washed, and then fixed for 20 min at room temperature with 2% formaldehyde. All samples were analyzed on a Coulter Epics Elite flow cytometer (Coulter, Palo Alto, CA) collecting data on at least 40,000 lymphocytes.
Enumeration of eosinophils
Eosinophils were enumerated by microscopic examination of Giemsa-stained cytocentrifuge preparations. At least 300 cells/sample were examined.
Lung virus titer
RSV titers were assessed in lung homogenates on day 2, 4, or 7 after virus challenge as previously described (22). Briefly, lungs were removed from four to five mice per group and homogenized. After centrifugation at 7000 rpm for 1 min, 100 µl of supernatant was removed for cytokine analysis. The remaining supernatant was titrated in doubling dilutions on 60–80% confluent HEp-2 cell monolayers in 96-well flat-bottom plates. Twenty-four hours later, monolayers were washed and incubated with peroxidase-conjugated goat anti-RSV Ab (Biogenesis, Poole, U.K.). Infected cells were detected using 3-amino-9-ethylcarbazole, infectious units being enumerated by light microscopy.
ELISPOT for murine cytokines
The ELISPOT assays were performed as previously described
(23) and all Abs were purchased from PharMingen. Briefly,
nitrocellulose-based 96-well microtiter plates (Multiscreen-HA;
Millipore, Hertfordshire, U.K.) were coated overnight at 4°C with 50
µl/well of either anti-IFN- (4 µg/ml; R46A2) or
anti-IL-5 (4 µg/ml; TRFK5) mAb diluted in carbonate buffer (pH
9.6). After washing three times with filtered PBS, all wells were
blocked with 200 µl of cRPMI for 2–3 h at 37°C. Following removal
of the blocking media, 3-fold serial dilutions of spleen or lymph node
cells (mediastinal or superficial cervical) from individual mice were
added to the wells in duplicate (maximum 5 x
105 cells/well in 200 µl of media) and
incubated for 20 h at 37°C in 5% CO2 in
the presence or absence of 1 µM M282–90
peptide. In some experiments, cells were also incubated in the presence
of 1 µM of the H-2Kd-binding peptide
corresponding to residues 91–99 of listeriolysin O from Listeria
monocytogenes. Similarly, in some experiments, cells were
incubated in the presence of 50 µl of culture supernatant from
hybridoma cultures of either GK1.5 (anti-mouse CD4) or YTS169
(anti-mouse CD8). Cells were removed by washing three times with
PBS, followed by an additional three times with PBS/Tween 20 (0.05%
v/v), then 50 µl of the biotinylated anti-IFN- (XMG1.2) or
anti-IL-5 (TRFK4) Abs (1 µg/ml in filtered PBS/Tween 20) was
added to each well for 2 h. After washing plates five times with
filtered PBS/Tween 20, a 1/1000 dilution of extravadin-alkaline
phosphatase (Sigma) was added to all wells for 1–2 h at room
temperature. Finally, after washing three times with PBS/Tween 20 and
once with PBS alone, a solution of 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium (fast 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium; Sigma) was added as substrate. Spots
representing single IFN-- or IL-5-producing cells were counted using
a dissecting microscope. The number of peptide-specific spot-forming
cells (SFCs)was determined by subtracting the number of spots obtained
with cells stimulated with media from those stimulated with
peptide.
Cytokine ELISA
Capture ELISAs for the detection of TNF were performed using Maxisorp 96-well plates (Nunc, Roskilde, Denmark). An OptEIA sandwich ELISA (PharMingen) was used to detect TNF according to the manufacturer’s instructions. Ab pairs for TNF were G281-2626 (capture) and MP6-XT3 (detection). Bound Abs were detected using streptavidin-HRP followed by tetramethylbenzidine and hydrogen peroxidase. Optical densities were read at 450 nm. The concentration of cytokine in each sample was calculated from a standard curve generated with recombinant cytokine. The detection limit for TNF was 25 pg/ml.
Restimulation and 51Cr release assays
Spleens and superficial cervical lymph nodes were aseptically removed and single-cell suspensions were prepared by passing organs from individual mice through 100-µm nylon sieves (Marathon Laboratories, London, U.K.). After lysis of splenic erythrocytes with Tris-ammonium chloride, a total of 3.5 x 107 leukocytes from the spleen was cultured for 6 days in upright T25 tissue culture flasks in 10 ml of cRPMI in the presence of 3 x 106 RSV-infected P815 cells. P815 cells were infected with RSV A2 at a multiplicity of infection of 10:1 for 2 h at 37°C and then washed three times with cRPMI before addition to splenocytes. Graded numbers of effector cells, consisting of cells that remained viable after the 6-day culture period, were harvested and cocultured in triplicate with 10,000 Na51CrO4-labeled P815 cells or P815 cells pulsed with 1 µM M282–90. In some experiments, effector cells were also cultured with P815 cells pulsed with the H-2Kd-binding peptide residues 91–99 of listeriolysin O from L. monocytogenes. Effector cells were cocultured with target cells for 4–5 h at the indicated E:T ratios in 200 µl of cRPMI. A total of 100 µl of culture supernatant was then removed into 100 µl of OptiPhase Hisafe scintillation mixture (Fisher, Leicestershire, U.K.) and the amount of Na51Cr O4 was measured using a 1450 Microbeta liquid scintillation counter (Wallac, Milton Keynes, U.K.). The percentage of killing was calculated using the following formula: ([experimental release - spontaneous release]/[maximum release - spontaneous release]) x 100, where spontaneous release represents the counts obtained when the target cells were incubated in culture media without effectors and maximum release represents the counts obtained when the target cells were lysed with 1% Triton X-100. Each experiment was repeated at least twice.
Statistical analysis
Mean IFN- ELISPOT numbers were compared using the
nonparametric Mann-Whitney U test. A p value of
<0.05 was regarded as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that enterotoxin-based mucosal adjuvants
can augment CTL responses to i.n. coadministered CTL peptides
(16). To assess the immunogenicity of
M282–90, BALB/c mice were immunized i.n. on day
0 and 14 with 20 µg of M282–90 alone or 20
µg of M282–90 coadministered with 1 µg of LT
or 5 µg of LTK63. In a previous study, coadministration of 5 µg of
LTK63 with CTL peptides was necessary for effective CTL induction
(16). To compare the magnitude of the response elicited by
mucosal immunization with an established potent method of eliciting
M2-specific CTL, separate groups of mice were immunized with 3 x
106 PFU of rVV expressing the M2 protein or an
irrelevant Ag, -gal. Coadministration of
M282–90 in the presence of either LT or LTK63
elicited splenic T cells which produced IFN- in an ELISPOT assay
upon secondary restimulation with the M282–90
peptide (Fig. 1
A). The
responses in immunized mice were specific for the
M282–90 peptide as cells did not respond when
stimulated with a H-2Kd-binding peptide from
L. monocytogenes (data not shown). Addition of an
anti-CD8 mAb, but not an anti-CD4 mAb, to ELISPOT wells largely
abrogated spot formation, indicating that the responding cells were
CD8+ T cells (data not shown). The number of
splenic peptide-specific IFN- SFCs elicited by i.n. coadministration
of peptide with adjuvant was similar regardless of whether LT or LTK63
was used, but was significantly greater than the response measured in
mice immunized with toxin alone or peptide alone. The number of
M282–90-specific IFN- SFCs elicited by
parenteral vaccination with rVV-M2 exceeded the number obtained by
mucosal immunization in the spleen (p = 0.002)
(Fig. 1A), but not significantly in the superficial cervical
lymph node (Fig. 1B). To examine the longevity of the
mucosally induced T cell response, IFN- SFCs were measured 103 days
after vaccination. The M282–90-specific
CD8+ T cell responses elicited by two mucosal
immunizations remained detectable, albeit at a reduced frequency, 103
days after the initial immunization (Fig. 1C).
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). These data show
that M2-specific CD8+ T cells are induced,
cytolytic, and long-lived when LT or LTK63 are used as adjuvants.
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Since LT/LTK63 were able to adjuvant M2-specific CTL, we evaluated
whether these CTL were protective against RSV challenge. Mice immunized
as previously described were i.n. challenged with 2 x
106 PFU of RSV A2 to determine whether mucosally
elicited CD8+ T cells accelerate the natural rate
of viral clearance, which normally occurs by day 7 postchallenge. On
day 2 post-RSV challenge, the viral load in lung tissue was similar
(between 101 and 2 x
102 PFU/lung) in all mice irrespective of the
immunization regimen used. On day 4 however, mice immunized with rVV-M2
had a significantly reduced viral burden compared with mice receiving
rVV expressing -gal (p < 0.001). Mucosally
elicited CTL also accelerated RSV clearance. Mice i.n. immunized with
the M282–90 peptide along with LT or LTK63 had
significantly lower viral loads compared with mice that received
peptide alone or adjuvant alone (Fig. 3A).
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B). The number of activated/memory
CD4+ T cells recovered from BAL was statistically
similar in all groups. Additionally, in some experiments, individual
CTL-primed mice lost weight during the course of viral infection (data
not shown).
Resistance to RSV challenge in peptide-immunized mice is
independent of IFN-
mAb depletion of IFN- was performed to assess the role of this
cytokine in resistance to RSV infection and vaccine-enhanced weight
loss in mice primed mucosally with M282–90. Mice
were i.n. immunized with M282–90 plus LTK63,
M282–90 alone, or LTK63 alone on days 0 and 14
and challenged with RSV A2 on day 28. Just before, and during the
challenge infection, half the mice in each group were administered an
anti-IFN- mAb. The remaining mice received an isotype control
mAb. Mice i.n. immunized with M282–90 plus LTK63
and treated with the control mAb had significantly lower RSV lung
titers on day 4 postchallenge compared with control animals (Fig. 4A). Depletion of IFN- in
similarly immunized mice did not abrogate vaccine-induced resistance to
infection (Fig. 4A). Furthermore, IFN- depletion did not
significantly affect the T cell infiltrate into the pulmonary airways
as measured by flow cytometric analysis of cells recovered from BAL
(data not shown). Although IFN- depletion did not affect viral
clearance, it did abrogate the marked decline in body weight and
ruffled appearance of nondepleted mice mucosally primed with
M282–90 and RSV challenged (Fig. 4B).
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enhances expression of a variety of soluble factors from immune
cells, one of which, TNF, is associated with immunologically driven
weight loss. The amount of TNF in supernatants of lung homogenates was
measured to determine whether IFN- depletion affected TNF levels in
RSV-infected tissue. TNF concentrations were significantly lower in
M282–90-primed mice depleted of IFN- compared
with identically immunized, but nondepleted mice (Fig. 4C).
Levels of IL-4 and IL-5 were below detectable limits, suggesting there
was a not a shift toward an increase in Th2-type cells. Mucosally elicited M282–90-specific CD8+ T cells prevent eosinophilia in G-primed and RSV-challenged mice: evidence for decreased Th2 response
In mice, sensitization to the RSV G protein results in severe
inflammatory lung disease during subsequent infection with RSV
(24, 25). A characteristic feature of this disease is
pulmonary eosinophilia. One potential approach for preventing
pathogenic immune responses in mice, and potentially in humans, is via
the priming of RSV-specific CD8+ T cell responses
before Ag sensitization/vaccination. To this end, the ability of
mucosally and parenterally elicited
M282–90-specific CTL to prevent eosinophilia
in rVV-G primed mice was compared. To mucosally prime
M282–90-specific CTL, BALB/c mice were i.n.
immunized with M282–90 plus LTK63 on days 0 and
14. Parenterally primed mice received 3 x
106 PFU of rVV-M2 intradermally (i.d.) on day 7.
All mice were scarified with rVV-G on day 21, then challenged with RSV
on day 35. Consistent with previous results (Fig. 3B), flow
cytometric analysis of cells recovered from BAL on day 7 postchallenge
demonstrated a lymphocyte infiltrate consisting predominantly of
activated CD8+ T cells in CTL-primed mice (data
not shown). At the same time point, Giemsa staining of cells recovered
from BAL indicated that mice mucosally immunized with
M282–90 alone or LTK63 alone had pulmonary
eosinophilia (Fig. 5). However, five of
six mice mucosally immunized with M282–90 plus
LTK63 had fewer numbers of eosinophils, but this difference did not
reach statistical significance (p = 0.06) for
the whole group (Fig. 5). Vaccination of mice with rVV-M2 2 wk before
sensitization with the RSV G protein completely suppressed the
development of pulmonary eosinophilia, as has been shown previously
(7) (Fig. 5). All mice, regardless of the immunization
regimen used, had undetectable levels of RSV in lung tissue (data not
shown).
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, top). Conversely, there
were significantly more cells making IFN- in
M282–90-primed mice compared with mice with
pulmonary eosinophilia (Fig. 6, bottom). These data show
that a dual vaccination regimen with both a Thl- and Th2-inducing
vaccine can decrease the immunopathological effects of Th2
vaccine-enhanced pulmonary eosinophilia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
CD4+ T cell help (16). The present
study extends these observations by describing the induction of
protective CD8+ CTL responses to a dominant
RSV epitope by mucosal vaccination and the modulation of RSV-induced
immunopathology. The requirements of an RSV vaccine are stringent. An ideal RSV vaccine should reduce the incidence and severity of RSV A- and B-related disease in adult and infant populations, must be efficacious in the presence of maternal Abs, and would be compatible with existing pediatric vaccines. Importantly, any RSV vaccine must not induce enhanced pathology when the host is infected with homologous or heterologous viruses. In murine studies, successful RSV vaccination approaches include DNA vaccines (13, 30), purified protein vaccines (31, 32), peptide vaccines (33, 34), and live attenuated viruses (35). The novelty of the approach used in this study is that it combines reasonably efficient CTL induction with mucosal delivery of nonliving Ag. Furthermore, unlike other studies, this is the first to examine the influence of different immunization regimens on previously well-characterized aspects of RSV immunopathology. Given the history of RSV vaccines (2, 3), this represents an important but neglected aspect of studies of experimental RSV vaccines in mice.
LT and LTK63 adjuvanted CD8+ CTL responses to the M282–90 peptide after two i.n. immunizations. M282–90-specific T cell responses were detected in both systemic (spleen) and local (cervical lymph node) lymphoid compartments and remained detectable for at least 3 mo after vaccination. The M282–90-specific T cells elicited by peptide vaccination proliferated when incubated with RSV-infected targets in secondary CTL assays, suggesting these T cells also recognized naturally processed M2. The magnitude of the M282–90-specific T cell responses elicited using LT or LTK63 was similar to that previously achieved in mice immunized with the dominant H-2Kb-binding epitope from OVA (16). The mechanisms through which bacterial enterotoxins mediate their adjuvant affects for i.n. coadministered Ags remains unclear. Nonetheless, clues are provided in studies defining the APC responsible for presentation of i.n. administered peptides. Porgador et al. (36) showed that after i.n. coimmunization of Ova257–264 with cholera toxin, only ex vivo dendritic cells from the nasal-associated lymphoid tissue could present Ova257–264 to Ova-specific MHC I-restricted T cells in vitro. These results suggest that bacterial enterotoxins directly or indirectly modulate dendritic cells in the nasal-associated lymphoid tissue such that they become competent for productive T cell priming.
Mice with mucosally or parenterally elicited
M282–90-specific T cell responses were more
resistant to RSV challenge. The levels of resistance achieved via
mucosal vaccination in this study is of a similar magnitude to that
achieved in previous studies using either DNA immunization
(13) or i.n. administration of a chimeric peptide
containing the M282–90 sequence
(14). Resistance to infection in immunized mice correlated
with the magnitude of the M282–90-specific T
cell response as measured by IFN- ELISPOT. Mice immunized with
rVV-M2 mounted the strongest M282–90-specific T
cell responses, and, concordantly, had the lowest lung RSV titers.
Immunity to infection in these mice has previously been shown to occur
solely through the actions of M282–90-specific
CD8+ T cell responses (11, 37).
Resistance to RSV infection in mucosally immunized mice was achieved
despite Ab depletion of IFN-. This suggests that mucosally elicited
M282–90-specific T cells mediate their effector
function primarily via killing of infected host cells and/or expression
of cytokines other than IFN-. These results are in contrast to those
of Hsu et al. (13), who showed that IFN- was essential
for resistance to RSV when M282–90-specific T
cells were elicited by DNA vaccination. The contrasting routes of
immunization, the method and duration of cytokine depletion (minimal in
the study by Hsu et al. (13)), the timing of the RSV
challenge, and the magnitude of the elicited
M282–90-specific CD8+ T
cell response may all contribute to the apparent differential
requirement for IFN- in these studies.
CD8+ T cells are central to the two different
immunopathologies described in the murine model of RSV. In persistently
infected -irradiated BALB/c mice, adoptive transfer of high numbers
of a CTL line or clone was accompanied by an acute and sometimes lethal
respiratory disease characterized by lung hemorrhage and frequent
neutrophil efflux (15). A contrasting immunopathology,
characterized by Th2 immune responses and pulmonary eosinophilia,
occurs in mice primed with the RSV G protein and challenged with RSV.
RSV-specific CD8+ T cells producing IFN- have
been proposed as negative regulators of this Th2 response
(8).
In the studies described here, resistance to RSV in CTL-primed mice
occurred at the expense of weight loss, although this was variable
between experiments. Depletion of IFN- during RSV challenge of
M282–90-primed mice successfully prevented
weight loss without adversely affecting viral clearance, suggesting an
immunological basis for enhanced disease. TNF may represent a prime
immunological candidate for this wasting phenomenon since
M282–90-primed mice depleted of IFN-, which
did not suffer weight loss, had significantly reduced levels of TNF in
supernatants of lung homogenates. TNF, whose expression is augmented by
IFN-, is produced by monocytes, macrophages, and T lymphocytes and
plays a critical role in inflammation and infection. However, exuberant
TNF induction during viral infection can have detrimental consequences
which include weight loss and wasting, thymic atrophy, and an increase
in serum glucocorticoids (38). We hypothesize that TNF
expression, partly driven by IFN--producing
M282–90-specific CD8+ T
cells, may play a critical role in the wasting illness that occurs in
M282–90-primed mice challenged with
RSV.
Numerous studies implicate CD4+ T cells
expressing Th2 cytokines as being central to enhanced disease in mice
vaccinated with rVV-G and subsequently infected with RSV
(8). It has been proposed that vaccines designed to
enhance CD8+ T cell recognition of RSV Ags might
avoid disease caused by CD4+ Th2 cells
(8). In this study, the priming of
M282–90-specific CD8+ T
cells before rVV-G vaccination modulated the magnitude of the pulmonary
eosinophilia and Th2 T cell response which occurred during subsequent
RSV challenge. We hypothesize that IFN- produced by RSV-specific
CD8+ T cells during challenge infection
suppresses eosinophilia by limiting the expansion of G-specific Th2
cells and down-regulating expression of the potent eosinophil
chemoattractant eotaxin. IFN- is recognized as a potent inhibitor of
eotaxin synthesis (39). These results are consistent with
previous studies in which the priming of RSV fusion protein-specific
CD8+ T cells before rVV-G vaccination suppressed
the development of Th2 T cells and eosinophilia during challenge
infection (7, 8). However, our results are the first to
show that immune-modulating CD8+ T cells can be
elicited by mucosal peptide vaccination. Furthermore, our results argue
that the anatomical site of CD8+ T cell priming
may be relatively unimportant in the context of limiting the
development of G-specific CD4+ Th2 cells.
The results of this study could help in the design of vaccination strategies which aim to prime protective, multivalent immune responses to RSV Ags while limiting immunopathological disease during natural infection. Conceivably, a vaccination strategy which fulfills these aims would also impact on the frequency of childhood wheezing resulting from RSV lower respiratory tract illness in infancy. Taken together, our results highlight the fine balance which exists between the protective and disease-producing effects of RSV-specific T cells. Indeed, a major hurdle for RSV vaccine development will be to completely dissociate these two elements.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cameron P. Simmons, Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AZ, U.K.
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; i.n., intranasal; i.d., intradermal; LT, E. coli labile-toxin; rVV, recombinant vaccinia; G, attachment protein of RSV; M2, second matrix protein of RSV; -gal, -galactosidase; ELISPOT, enzyme-linked immunospot; BAL, bronchoalveolar lavage; SFC, spot-forming cell. cRPMI, complete RPMI.
Received for publication July 6, 2000. Accepted for publication October 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Int. Immunol. 10:1441. is a potent inhibitor of eotaxin synthesis in vitro. Int. Immunol. 11:1001.This article has been cited by other articles:
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  J. Dietrich, C. Andersen, R. Rappuoli, T. M. Doherty, C. G. Jensen, and P. Andersen Mucosal Administration of Ag85B-ESAT-6 Protects against Infection with Mycobacterium tuberculosis and Boosts Prior Bacillus Calmette-Guerin Immunity J. Immunol., November 1, 2006; 177(9): 6353 - 6360. [Abstract] [Full Text] [PDF]
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 N. W. Lukacs, M. L. Moore, B. D. Rudd, A. A. Berlin, R. D. Collins, S. J. Olson, S. B. Ho, and R. S. Peebles Jr Differential Immune Responses and Pulmonary Pathophysiology Are Induced by Two Different Strains of Respiratory Syncytial Virus Am. J. Pathol., September 1, 2006; 169(3): 977 - 986. [Abstract] [Full Text] [PDF]
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 K. A. Herd, S. Mahalingam, I. M. Mackay, M. Nissen, T. P. Sloots, and R. W. Tindle Cytotoxic T-Lymphocyte Epitope Vaccination Protects against Human Metapneumovirus Infection and Disease in Mice J. Virol., February 15, 2006; 80(4): 2034 - 2044. [Abstract] [Full Text] [PDF]
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E. A. W. Claassen, P. A. A. van der Kant, Z. S. Rychnavska, G. M. van Bleek, A. J. Easton, and R. G. van der Most Activation and Inactivation of Antiviral CD8 T Cell Responses during Murine Pneumovirus Infection J. Immunol., November 15, 2005; 175(10): 6597 - 6604. [Abstract] [Full Text] [PDF]
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 P. J. M. Openshaw Antiviral Immune Responses and Lung Inflammation after Respiratory Syncytial Virus Infection Proceedings of the ATS, August 1, 2005; 2(2): 121 - 125. [Abstract] [Full Text] [PDF]
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 P. J. M. Openshaw and J. S. Tregoning Immune Responses and Disease Enhancement during Respiratory Syncytial Virus Infection Clin. Microbiol. Rev., July 1, 2005; 18(3): 541 - 555. [Abstract] [Full Text] [PDF]
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A. E. Williams, L. Edwards, I. R. Humphreys, R. Snelgrove, A. Rae, R. Rappuoli, and T. Hussell Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease J. Immunol., December 15, 2004; 173(12): 7435 - 7443. [Abstract] [Full Text] [PDF]
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 C. Bartholdy, W. Olszewska, A. Stryhn, A. R. Thomsen, and P. J. M. Openshaw Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis J. Gen. Virol., October 1, 2004; 85(10): 3017 - 3026. [Abstract] [Full Text] [PDF]
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 E. C. Lavelle, A. Jarnicki, E. McNeela, M. E. Armstrong, S. C. Higgins, O. Leavy, and K. H. G. Mills Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent J. Leukoc. Biol., May 1, 2004; 75(5): 756 - 763. [Abstract] [Full Text] [PDF]
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T. J. Harvey, W. J. Liu, X. J. Wang, R. Linedale, M. Jacobs, A. Davidson, T. T. T. Le, I. Anraku, A. Suhrbier, P.-Y. Shi, et al. Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles J. Virol., January 1, 2004; 78(1): 531 - 538. [Abstract] [Full Text] [PDF]
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 K. Schulze, E. Medina, G. S. Chhatwal, and C. A. Guzman Identification of B- and T-Cell Epitopes within the Fibronectin-Binding Domain of the SfbI Protein of Streptococcus pyogenes Infect. Immun., December 1, 2003; 71(12): 7197 - 7201. [Abstract] [Full Text] [PDF]
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G. M. van Bleek, M. C. Poelen, R. van der Most, H. F. Brugghe, H. A. M. Timmermans, C. J. Boog, P. Hoogerhout, H. G. Otten, and C. A. C. M. van Els Identification of Immunodominant Epitopes Derived from the Respiratory Syncytial Virus Fusion Protein That Are Recognized by Human CD4 T Cells J. Virol., December 20, 2002; 77(2): 980 - 988. [Abstract] [Full Text] [PDF]
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 F. J. Culley, J. Pollott, and P. J.M. Openshaw Age at First Viral Infection Determines the Pattern of T Cell-mediated Disease during Reinfection in Adulthood J. Exp. Med., November 18, 2002; 196(10): 1381 - 1386. [Abstract] [Full Text] [PDF]
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L. Stevceva, X. Alvarez, A. A. Lackner, E. Tryniszewska, B. Kelsall, J. Nacsa, J. Tartaglia, W. Strober, and G. Franchini Both Mucosal and Systemic Routes of Immunization with the Live, Attenuated NYVAC/Simian Immunodeficiency Virus SIVgpe Recombinant Vaccine Result in Gag-Specific CD8+ T-Cell Responses in Mucosal Tissues of Macaques J. Virol., October 11, 2002; 76(22): 11659 - 11676. [Abstract] [Full Text] [PDF]
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) and P815 cells
pulsed with 1 µM M282–90 (•).
