Previous Article | Next Article 
J Virol, May 1998, p. 4080-4087, Vol. 72, No. 5
BBSRC, Institute for Animal Health, Compton,
Newbury, Berkshire RG20 7NN, United
Kingdom,1 and
Centro Nacional de
Biologia Celular y Retrovirus, Instituto de Salud Carlos III,
Majadahonda, 28220 Madrid, Spain2
Received 18 December 1997/Accepted 29 January 1998
In order to investigate if immune responses to the fusion (F)
protein of respiratory syncytial virus (RSV) could be influenced by
cytokines, recombinant vaccinia viruses (rVV) carrying both the F gene
of RSV and the gene for murine interleukin-2 (IL-2), IL-4, or gamma
interferon (IFN- Infection of inbred mouse strains
with a number of pathogens has revealed that the selective
differentiation and development of effector T cells have profound
implications for disease resistance or disease susceptibility. Th1-like
immune responses, producing high levels of interleukin-2 (IL-2)
and gamma interferon (IFN- The development of naive Th cells into Th1- or Th2-like cells is
influenced by the cytokine microenvironment upon activation. Thus, IL-4
can direct the development of Th cells into Th2 cells (20, 33,
41) while IL-12 or IFN- The BALB/c mouse model of respiratory syncytial virus (RSV) infection
has revealed that T lymphocytes and the cytokines that they produce
play an important role in determining the outcome of RSV infection
(3, 15, 42, 43). Of particular interest is evidence
that the fusion (F) and the attachment (G) proteins of RSV prime for
different Th-cell responses in BALB/c mice (1). Thus,
recombinant vaccinia viruses (rVV) expressing the F protein of RSV
prime for cytotoxic T lymphocytes (CTL) and a Th1 response, resulting in a characteristic polymorphonuclear (PMN) efflux in the
lungs of mice following RSV challenge. In contrast, rVV expressing the
G protein prime for a Th2 response, which induces large numbers of
eosinophils in pulmonary exudate following RSV challenge (1, 27). The cytokines produced as a result of these different Th responses are therefore reflected by characteristic changes in pulmonary pathology of vaccinated mice following RSV challenge.
Most studies to date have only examined the effects of vaccinia virus
(VV)-expressed cytokines on primary immune responses. In the studies
described here, we investigated the effects of cytokines on the
establishment of memory to the F glycoprotein of RSV expressed in rVV.
The ability of the coexpressed cytokines IL-2, IL-4, and IFN- Viruses.
rVV were constructed according to standard methods
briefly described below. Two parental VVs were used: strain WR and
strain vRB12, a strain WR-derived virus defective for plaque formation (5). The vRB12 virus lacks the gene encoding protein VP37; insertion plasmid pRB21 provides a complete copy of the VP37 gene, allowing rVV to be selected on the basis of plaque formation
(5). Plasmid pSCF and the corresponding rVV VA-F have been
described elsewhere (21) and are referred to here as VSCF.
Plasmid pRBF was obtained by subcloning a fragment of plasmid LF1
containing the F gene of the Long strain of RSV (7) into the
StuI-HindIII-cleaved pRB21 vector and was
used to produce the rVV VRBF. The coding regions for murine cytokine
genes were obtained from plasmids pCD-IL-2 for IL-2 (47),
pCD2A-E3 for IL-4 (19), and pRB322-IFN-
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Recombinant Vaccinia Virus Coexpressing the F Protein of
Respiratory Syncytial Virus (RSV) and Interleukin-4 (IL-4) Does Not
Inhibit the Development of RSV-Specific Memory Cytotoxic T
Lymphocytes, whereas Priming Is Diminished in the Presence of
High Levels of IL-2 or Gamma Interferon
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) were constructed. In vitro characterization of rVV
revealed that insertion of the cytokine gene into the VP37 locus of the
vaccinia virus genome resulted in 100- to 1,000-fold higher expression
than insertion of the same gene into the thymidine kinase (TK) locus.
In comparison, only a two- to fivefold difference in the level
of expression of the F protein was observed when the gene was inserted
into either of these two loci. Mice vaccinated with rVV expressing the
F protein and high levels of IL-2 or IFN- cleared rVV more rapidly
than mice inoculated with a control rVV and developed only low levels
of RSV-specific serum antibody. In addition, these recombinants were
much less effective at priming RSV-specific memory cytotoxic T
lymphocytes (CTL) and IFN- production by spleen cells than rVV
expressing the F protein alone. In contrast, mice vaccinated with rVV
expressing high levels of IL-4 showed signs of delayed rVV clearance.
RSV-specific serum antibody responses were biased in favor of
immunoglobulin G1 (IgG1) in these mice, as there was a significant
reduction in IgG2a antibody responses compared with serum antibody
responses in mice vaccinated with rVV expressing the F protein alone.
However, vaccination with rVV expressing the F protein together with
high levels of IL-4 did not alter the development of RSV-specific
memory CTL or IFN- production by RSV-restimulated splenocytes.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) (25, 26, 28), are protective
against the intracellular pathogens Leishmania major and
Candida albicans (31, 36), whereas hosts that
mount Th2-like responses are susceptible to progressive infection. In
contrast, Th2 cells which secrete IL-4, IL-5, IL-6, and IL-10 (23,
26) are protective against extracellular pathogens such as
Trichuris muris and Borrelia burgdorferi
(22, 36), and the induction of Th1 responses is
nonprotective. Hence, most pathogens are usually preferentially
susceptible to one type of immune response, and the identification of
strategies for the induction of specific types of immunity will aid
vaccine design.
can induce the development of Th cells
into Th1 cells (6, 16, 34). Furthermore, cytokines produced
by one Th subset can block the production or activity of cytokines
produced by the other subset (13, 40). This feedback mechanism allows the possibility of the use of vector vaccines expressing cytokine genes to manipulate the microenvironment to favor
the development of appropriate protective immune responses. Both Th1
and Th2 cells provide B-cell help to antibody-producing B cells, but
the differential secretion of IL-4 and IFN- can regulate the
relative quantities of immunoglobulin G1 (IgG1), IgG2a, and IgE that
are made (10).
to
influence the isotype of RSV-specific antibody, CTL, and Th priming was
assessed. This approach provides a model for studying the role of Th
subsets and T-lymphocyte-virus interactions and may be a practical
approach for the design of effective vaccines.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
for IFN-.
Cytokine genes were subcloned into the SmaI-cleaved pSC11
plasmid or into the StuI-HindIII-cleaved
pRB21 plasmid. rVV were obtained following the transfection of parental
VV-infected CV-1 cells with the corresponding recombinant plasmids. At
48 h postinfection, viral progeny were recovered. WR-derived
single rVV were selected in HuTK
143B cells containing 25 µg of
bromodeoxyuridine ml
1. vRB12-derived single rVV were
selected in CV-1 cells by their ability to plaque. vRB12-derived double
rVV expressing the F protein and a given cytokine were selected in
HuTK 143B cells by their ability to produce blue plaques in the
presence of
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal).
The resulting rVV and site of insertion of the RSV F protein gene or
the murine cytokine gene within the VV genome are indicated in Table
1.
TABLE 1.
Ability of rVV expressing different levels of murine
cytokines to protect mice against RSV challenge 3 weeks after
vaccination
Mice. Six-week-old specific-pathogen-free BALB/c female mice, obtained from Charles River Breeding Laboratories, were inoculated intraperitoneally (i.p.) with 2 × 106 PFU of rVV. Serum samples were obtained on weeks 1 to 3 postinoculation by bleeding live mice from the tail vein. Three weeks postimmunization, mice were challenged intranasally (i.n.) with approximately 105 PFU of the A2 strain of RSV. Five days after challenge, groups of five mice were killed and the titer of RSV in lung homogenates was determined by a plaque assay (45). Other groups of five mice were killed and subjected to one round of bronchoalveolar lavage (BAL) as described previously (8), but with 1 ml of 12 mM lidocaine in phosphate-buffered saline. The numbers of neutrophils and eosinophils in cytocentrifuge preparations of BAL cells stained with May-Grunwald Giemsa stain were counted. Between 300 and 400 cells per mouse were examined on each slide. BAL cells for use in flow cytometric analysis were isolated in the same manner; lungs were subjected to three successive rounds of lavage, and cells from groups of five mice were pooled.
To examine the kinetics of VV clearance, groups of four mice were inoculated i.p. with 2 × 106 PFU of rVV. At intervals after virus inoculation, mice were killed with an overdose of sodium pentobarbital. Samples of liver and spleen were obtained and homogenized to yield 10 or 20% suspensions as described previously (45). Virus infectivity in tissue samples was assayed on HTK cell monolayers incubated for 48 h at 37°C. Titers of virus were expressed as log10 PFU per gram of tissue.Flow cytometry. Two color flow cytometric examinations of isolated BAL cells were done with rat anti-mouse CD4 coupled to fluorescein isothiocyanate (Sigma, Poole, United Kingdom) and biotinylated rat anti-mouse CD8 (Pharmingen, San Diego, Calif.) followed by streptavidin-phycoerythrin (Southern Biotechnology Associates, Birmingham, Ala.). Levels of background staining were assessed with irrelevant isotype-matched monoclonal antibody (MAb) controls (Pharmingen). Staining was analyzed on a FACScan (Becton Dickinson, Mountain View, Calif.).
Antibody assays. The presence of antibodies to RSV was determined by an enzyme-linked immunosorbent assay (ELISA) as described previously (39). Bound antibody was detected by adding goat anti-mouse IgG serum coupled to horseradish peroxidase (Kirkegaard and Perry Laboratories Inc., Gaithersburg, Md.) or horseradish peroxidase-conjugated rabbit anti-mouse IgG1, IgG2a, or IgG2b (ICN Biomedicals Inc., Thame, United Kingdom).
Cytokine assays.
Splenocytes from mice immunized 3 weeks
previously were stimulated with RSV-infected autologous splenocytes as
described elsewhere (14). Supernatants were harvested on
days 1 to 4 and assessed for cytokine production (see below). Cytokines
present in the supernatant or cell-associated material of VV-infected
cells were assessed with a cytokine-specific antigen capture ELISA.
Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight with 50 µl of rat anti-mouse IL-2 MAb (JES6-1A12), rat anti-mouse IL-4 MAb
(BVDV-1D11), or rat anti-mouse IFN- MAb (R4-6A2), (all MAbs from
Pharmingen) at 2 µg/ml in 0.1 M NaHCO3 (pH 8.2). After
being washed with phosphate-buffered saline containing 0.05% Tween 20 (PBSTw) the plates were blocked for 2 h with PBSTw containing 5%
pig serum. Samples or standards (recombinant murine IL-2, IL-4, or
IFN-; Pharmingen) were added and incubated for 20 h at 4°C. The plates were washed and then incubated with 100 µl of biotinylated anti-IL-2 MAb (JES6-5H4), anti-IL-4 MAb (BVDV-24G2), or anti-IFN- MAb (XMG1.2) (all MAbs from Pharmingen) at 1 µg/ml followed by avidin-peroxidase (Sigma) before the addition of
3,3',5,5'-tetramethylbenzidine and hydrogen peroxide in 0.1 M sodium
acetate buffer (pH 6.0) as a substrate. To determine the concentration
of cytokine, mean background levels (±2.5 standard deviations [SD])
were subtracted from the mean values for triplicate samples.
CTL assays. Spleen lymphocytes from mice immunized 4 or 5 weeks earlier with rVV were restimulated in vitro with RSV-infected splenocytes for 5 days as described previously (14) and used as a source of secondary CTL. Target cells for cytotoxicity assays were BCH4 cells, which is a BALB/c fibroblast line (H-2d) persistently infected with the Long strain of RSV (9), BALB/c fibroblasts (44), and the mouse fibroblast line L-929 (H-2k). Cell lines were used uninfected or infected with the A2 strain of RSV and were labelled with 51Cr as described previously (14). Lytic units (LU) were taken as the number of effector cells necessary to cause 33% specific lysis (44).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In vitro expression of the F protein and murine cytokines by
rVV.
The construction of double rVV allowed insertion of the F
glycoprotein or cytokine gene into either of two loci within the VV
genome, namely, the thymidine kinase (TK) or VP37 locus. In order to
determine whether the site of insertion and/or promoter usage
influenced the levels of expression of the F protein or of the
cytokines, supernatants or cell-associated antigens from CV-1 cells
infected with an equivalent multiplicity of infection of different rVV
were compared. Levels of expression of the F protein under the control
of the p7.5 promoter (inserted into the TK locus) were two- to fivefold
lower than those in constructs expressing the F protein under the
control of a synthetic early or late promoter in the VP37 locus
(5). Results of typical endpoint titrations are shown in
Table 1. Expression of the IL-2, IL-4, or IFN- cytokine genes in the
VP37 locus resulted in 100- to 1,000-fold more cytokine in both
supernatants and cell-associated material than did expression in the TK
locus (Table 1). Furthermore, the level of IFN- in both supernatants
and cell-associated material from VRBF.mIFN--infected CV-1 cells was
below the level of detection in the ELISA. rVV that coexpressed the F
protein and high levels of cytokines are referred to hereafter as
VV-F.mIL-2high, VV-F.mIL-4high, and VV-F.mIFN-high. Similarly, rVV
that expressed low levels of cytokines are given the suffix "low".
Coexpression of IL-2 or IFN- reduces antibody responses to
RSV.
The antibody responses induced by the various rVV in mice
were examined to determine whether the coexpression of murine cytokines with the F protein could influence antibody responses to RSV. Groups of
mice inoculated 3 to 4 weeks previously with rVV expressing the F
protein either alone or together with murine cytokines developed RSV-specific antibody, with endpoint titers ranging from
log10 3 to 5 (Table 1). Antibody responses induced with
VV-F.mIL-4high or VV-F.mIL-4low had titers similar to that induced by
VSCF, but significantly lower titers were obtained in mice vaccinated
with rVV expressing the F protein together with IL-2 or IFN-.
or IL-4, whereas titers of IgG2a and IgG2b antibodies
were similar to those detected in mice vaccinated with VRBF. There was
no significant difference in antibody titers by 3 weeks postvaccination
(Fig. 1A).
|
),
VV-F.mIL-2low (
), VVF.mIFN-
), or VV-F.mIL-4low (
) or
with (B) VSCF (high, little or no IgG1, IgG2a, or IgG2b
serum antibody was detected, whereas high levels of all three isotypes
were detected in sera from VSCF-vaccinated mice. Furthermore, titers of
all antibody isotypes were significantly lower in mice given
VV-F.mIL-2high and VV-F.mIFN-high than in those given VSCF 3 weeks
after vaccination. Serum samples were not taken in the first week from
mice immunized with VV-F.mIL-4high. A mortality of 100% was observed
for mice immunized with VRBmIL-4, and up to 20% mortality was observed
for some groups of mice immunized with VV-F.mIL-4high. These mice
appeared ill by 7 days, with a ruffled coat and peritoneal edema, but
the majority made a good recovery and showed no signs of ill health 9 to 11 days after immunization. This outcome contrasted with that for
mice immunized with rVV expressing low levels of IL-4 (VRBF.mIL-4),
which showed no ill effects. There were no significant differences in
IgG1 antibody titers in sera from mice vaccinated with VSCF or
VV-F.mIL-4high 3 weeks after inoculation. However, a significant
reduction in IgG2a antibody titers was detected 2 weeks after
vaccination with VV-F.mIL-4high, and IgG2a antibody titers were still
significantly lower than those in mice vaccinated with VSCF 3 weeks
previously (Fig. 1B).
Although there was evidence that higher levels of the F protein were
expressed from the VP37 locus than from the TK locus (Table 1), there
were no significant differences in IgG1, IgG2a, or IgG2b serum antibody
responses in mice vaccinated with either VSCF or VRBF. These findings
indicate that differences in RSV-specific antibody isotypes were due to
the effects of the different levels of cytokines produced by the rVV
and did not simply reflect differences in the level of expression of
the F protein by the recombinant viruses. We therefore chose to further
characterize the immune responses elicited by rVV that expressed high
levels of cytokines.
High levels of IL-2 or IFN- attenuate rVV replication,
whereas high levels of IL-4 increase virulence in euthymic
mice.
In order to determine the effect of high levels of
cytokines on the replication of rVV, the virulence of
VV-F.mIL-2high, VV-F.mIFN-high, and VV-F.mIL-4high was
compared with that of VSCF after i.p. immunization of mice
with 2 × 106 PFU of the various rVV. Samples of liver
and spleen were removed from groups of four mice on days 1, 2, 3, 4, and 9 after immunization. Virus was recovered from the spleens of
VSCF-immunized mice on days 1 to 4 and from the livers on days 1 and 2 and was cleared from these tissues by day 9 (Table
2). In contrast, virus was recovered from
the spleens of three of four VV-F.mIL-2high-immunized mice on day 2 and
two of four mice on day 3 but not from the livers at any time after
vaccination. Virus was not recovered from the spleens after day 3. Replication of VV-F.mIFN-high appeared to be severely restricted in
vivo, as virus was recovered only from the spleen of one of four mice
inoculated 24 h previously (Table 2). Although the levels of virus
recovered from the spleens of VV-F.mIL-4high-immunized mice were
similar to those recovered from the spleens of VSCF-immunized mice
(Table 2), the levels of virus recovered from the livers of
VV-F.mIL-4high-immunized mice were 10-fold higher. Furthermore,
clearance of VV-F.mIL-4high might have been delayed compared with that
of VSCF, as virus was still detectable in one of four mice 9 days after
inoculation (Table 2). Thus, the resolution of rVV infection was
delayed in mice given virus carrying the gene encoding IL-4, whereas
rVV carrying the gene encoding IL-2 or IFN- was cleared more rapidly from the spleen and liver than was VSCF.
|
Priming of RSV-specific CTL memory is inhibited by rVV expressing
high levels of IL-2 and IFN- but not by rVV expressing high levels
of IL-4.
Studies with rVV expressing individual RSV proteins have
shown that the F protein primes BALB/c mice for RSV-specific CTL (2, 29). In order to investigate whether CTL priming by the F protein could be influenced by the cytokines IL-2, IL-4, and IFN-,
secondary CTL were generated from splenocytes of mice immunized with
rVV expressing F together with high levels of cytokines and stimulated
with RSV in vitro. In order to estimate the relative RSV-specific
CTL activities in spleens from mice vaccinated with the different
recombinants, LU/106 cells were calculated.
high was significantly
reduced (Fig. 2) (0.2 LU/106 cells). Nevertheless, the
level of CTL activity in splenocytes from these mice was higher than
that observed in splenocytes from mice vaccinated with rVV
expressing -galactosidase (VA-gal) (0.0004 LU/106
cells) (Fig. 2).
|
).
Cytokine production is reduced by RSV-specific memory T cells from
mice vaccinated with rVV expressing IL-2 or IFN- but not IL-4.
In order to study the effect of coexpressed cytokines on Th priming,
the levels of cytokines produced by isolated immune splenocytes following in vitro restimulation with RSV were analyzed (Table 3). IL-2 was detectable in all cultures
and peaked at 72 h. The highest levels were produced by
lymphocytes from VSCF-primed mice, although similar levels were also
found in supernatants of restimulated splenocytes from mice primed with
VV-F.mIL-2high, VV-F.mIL-4high, and VV-F.mIFN-high. IFN- was
detectable in supernatants from all restimulated splenocyte cultures
and was still increasing on day 4. The highest levels were found in
culture supernatants from VSCF-primed mice, and the lowest levels were
found in supernatants from mice primed with VV-F.mIL-2high and
VA-gal. VV-F.mIL-2high-primed mice produced almost 8-fold less
IFN- than did VSCF-primed mice, while VV-F.mIFN-high-primed
mice produced only 2.5-fold less. Surprisingly, the level of IFN-
produced by lymphocytes from mice vaccinated with VV-F.mIL-4high was
similar to that produced by lymphocytes from VSCF-primed mice. IL-10
was also found in all culture supernatants, and levels were still
increasing on day 4. The highest levels of IL-10 were produced by mice
vaccinated with VSCF or VV-F.mIL-4high, and the concentrations were
approximately six- to eightfold higher than those produced by mice
primed with VV-F.mIL-2high or VV-F.mIFN-high. Only low levels of
IL-10 and IL-2 were detected in splenocyte supernatants from
VA-gal-vaccinated mice. IL-4 and IL-5 were not detected in
supernatants from any of the lymphocyte cultures.
|
Ability of rVV to protect against RSV challenge.
Protection
against RSV challenge was examined 4 weeks after immunization with the
various rVV (Table 1). All rVV expressing the F protein of RSV induced
protection against a subsequent RSV infection, as virus was not
recovered from the lungs of any of the vaccinated mice 5 days after RSV
challenge (Table 1). In order to determine if the high levels of
cytokines produced by rVV affected RSV titers after challenge, mice
were immunized with rVV that did not express the F protein, namely,
VRBmIL-2 or VRBmIFN-, and challenged. These mice were not protected
against RSV infection, and titers of RSV recovered from the lungs of
these mice were similar to those in the control (VA-gal-treated)
group (Table 1).
Vaccination with rVV expressing F together with high levels of
IL-2, IL-4, or IFN- does not have a major impact on the pulmonary
inflammatory response after RSV challenge.
To determine the
influence of the cytokines IL-2, IL-4, and IFN- on the pulmonary
cell responses of vaccinated mice 5 days after RSV challenge, animals
were subjected to repeated BAL. The numbers of leukocytes recovered
from the lungs of mice immunized with VSCF, VV-F.mIL-2high,
VV-F.mIL-4high, and VV-F.mIFN-high were increased 5 days after RSV
challenge compared with those in mice primed with VA-gal (Table 1).
Analysis of BAL showed that VSCF-vaccinated mice developed neutrophil
(PMN) efflux into the lungs (median value, 39 × 103/ml). PMN efflux also occurred in groups of mice
inoculated with VV-F.mIL-2high (88 × 103/ml),
VV-F.mIL-4high (45 × 103/ml), and VV-F.mIFN-high
(52 × 103/ml). Fewer leukocytes were present in BAL
from mice immunized with VA-gal, and this finding was reflected in
the decreased numbers of neutrophils in the lungs (18 × 103/ml). Few eosinophils (<1%) were present in BAL from
any of the mice.
with the F protein on the recruitment of CD4+ and
CD8+ subsets into the lungs 5 days after RSV challenge was
examined by flow cytometry. Although there was a slight reduction in
the number of CD4+ cells in BAL from mice immunized with
VV-F.mIFN-high or VV-F.mIL-2high 5 days after RSV challenge compared
with the number recovered in BAL from VSCF- or VV-F.mIL-4high-immunized
mice, there was no significant change in the ratio of CD4+
to CD8+ T cells, and CD8+ T-cell numbers always
exceeded CD4+ T-cell numbers (Table
4).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Immunization of mice with rVV that coexpressed the genes for
murine IL-2, IL-4, or IFN- and the F protein of RSV allowed us to
investigate the influence of these factors on the development of
immunity to RSV. The data presented here show that high levels of
cytokine expression by rVV containing the gene for IL-2 or IFN- in
the VP37 locus markedly reduced the replication of the rVV in euthymic
mice. This reduction in virus replication resulted in a reduction in
all RSV-specific antibody isotypes, a marked reduction in CTL priming,
and a reduction in IFN- and IL-10 production. In contrast, although
IL-4 biased the RSV-specific antibody response in favor of IgG1,
priming of RSV-specific CTL and IFN- production were not adversely
affected.
Previous studies demonstrated that an rVV expressing murine IL-2 was
less virulent in immunodeficient mice than a control rVV but did not
show any significant attenuation based on the kinetics of virus
clearance in euthymic mice (30). However, our findings that
IL-2 and IFN- resulted in more rapid clearance of rVV from the
spleen and liver are similar to those reported previously, where IL-2
and IFN- enhanced the clearance of rVV from the ovaries of euthymic
mice (17, 18). In contrast, inclusion of the gene encoding
IL-4 resulted in greater replication and delayed clearance of rVV
from the liver and spleen. This observation is similar to those
reported previously, where clearance of rVV from the ovaries and
clearance of influenza virus from the lungs were delayed by IL-4
(24, 35).
Infection of mice with rVV expressing IL-4 appears to be lethal in
female but not in male CBA/H mice, with a mean time to death of 5 to 7 days (4). This difference in susceptibility to infection has
been attributed to the high titers of rVV recovered from the ovaries of
infected mice (17). However, others have reported that CBA/H
mice are able to clear rVV expressing IL-4 (35). We found
that VRBmIL-4 (high levels of IL-4) was lethal in female BALB/c mice.
This virus is thymidine kinase positive and is therefore likely to grow
to higher titers than VV-F.mIL-4high, which is thymidine kinase
negative but which expresses a level of IL-4 similar to that expressed
by VRBmIL-4 in vitro. The observed peritoneal edema in mice immunized
with an rVV expressing IL-4 (VV-F.mIL-4high) is similar to that
reported elsewhere (4); however, we observed no adverse
effects or signs of peritonitis in mice immunized with VV-F.mIL-4low.
This vascular leak syndrome is similar to that seen following the
systemic administration of recombinant murine IL-2 (32), and
we also saw increased peritoneal swelling in mice immunized with
VV-F.mIL-2high but not in mice immunized with VV-F.mIL-2low. These
observations suggest that the high levels of cytokine production in
VV-F.mIL-2high, VV-F.mIL-4high, and VV-F.mIFN-high compared with
VV-F.mIL-2low, VV-F.mIL-4low, and VV-F.mIFN-low act in a systemic
rather than a localized fashion.
It is well known that lymphokines can control immunoglobulin isotype
selection in vivo. IFN- has been shown to enhance IgG2a and inhibit
IgG1 production (10, 11). In contrast, IL-4 can induce
activated B lymphocytes to secrete IgG1 and IgE (37, 46).
Although immunization with rVV expressing F together with low levels of
IL-2 or IFN- (VRBF.mIL-2 or VRBF.mIFN-) induced significantly
lower titers of RSV-specific antibody than did immunization with VRBF,
we did not find the preferential induction of any particular isotype.
Furthermore, antibody titers and isotypes induced by VV-F.mIL-4low- or
VRBF-immunized mice were similar. These findings are broadly in
agreement with other published data, where the genes encoding IL-2,
IL-4, and IFN- were inserted into the TK locus (18, 35),
but are in contrast to the more dramatic reduction in serum antibody
responses to RSV induced by rVV expressing F together with high levels
of IL-2 or IFN- (VV-F.mIL-2high or VV-F.mIFN- high,
respectively), where the cytokine genes were inserted into the VP37
locus. However, the reduction in antibody responses was not isotype
specific. In contrast, high levels of IL-4 (VV-F.mIL-4high) resulted in
a preferential induction of IgG1 antibody. Thus, although the IgG1
titer was similar to that in sera from VSCF-immunized mice, the IgG2a
titer was significantly reduced in animals vaccinated with
VV-F.mIL-4high. The abilities of IL-4 to induce IgG1 antibody and to
inhibit IgG2a antibody in VV-F.mIL-4high-immunized mice but not in
VV-F.mIL-4low-immunized mice suggest that high levels of IL-4 are
required to exert an effect on B-cell responses during infection with
VV.
Although IL-4 biased the antibody isotype in favor of IgG1, therefore reflecting a Th2-like immune response, few eosinophils were detected in the BAL of VV-F.mIL-4high-vaccinated mice following RSV challenge. Cytokines in culture supernatants from spleen cells of mice scarified with rVV expressing the G protein of RSV are typically Th2-like following restimulation with RSV in vitro, and large numbers of eosinophils are present in the BAL following RSV challenge (1, 27). However, the route of vaccination is critical to inducing a pulmonary eosinophilic response following RSV challenge (4a). Hence, vaccination by the i.p. route with rVV expressing the G protein does not result in an eosinophilic response, whereas scarification with the same rVV does. The abilities of VV-F.mIL-4high to induce a Th2-like immune response to the F protein when mice are vaccinated by dermal scarification and to induce a pulmonary eosinophilic response after RSV challenge are currently under investigation.
Perhaps surprisingly, the priming of RSV-specific CTL was not impaired
in mice vaccinated with VV-F.mIL-4high. It has been reported that the
VV-specific precursor CTL frequency in spleens determined 6 days after
infection of mice with an rVV expressing IL-4 was 12-fold lower than
that in mice infected with a control rVV and remained suppressed
throughout the course of the infection (35). The expression
of IL-2, IL-12, and IFN- mRNAs was also reduced during the acute
phase of rVV infection in these mice (35). This
down-regulation of Th1 cytokines and antiviral CTL responses is a
likely explanation for the delayed rVV clearance and increased virus
titers in mice immunized with VV-F.mIL-4high. However, our findings
suggest that the development of Th1 and CTL memory for a coexpressed F
protein is not impaired by IL-4 at the time of vaccination. Delayed
virus clearance and reduced CTL activity were also observed for
IL-4-treated mice during primary influenza virus infection
(24) and for mice that constitutively overexpressed IL-4
during RSV infection (12). Our experimental design differs
in one significant respect from those of other groups, who assayed CTL
activity while virus-expressed or exogenous IL-4 was still present
during an acute viral infection (24, 35). We assayed CTL
activity from a memory rather than an effector cell population at a
time when virus-expressed IL-4 was no longer present in the system. The
ability of secondary CTL to lyse RSV-infected target cells in vitro may
not necessarily correspond to their ability to clear virus in vivo. CTL
generated from influenza virus-infected mice and restimulated in vitro
in the presence of IL-4 retained their ability to lyse appropriate
targets in vitro but did not clear virus following adoptive transfer to
influenza virus-infected mice (24). The ability of
RSV-specific CTL from VV-F.mIL-4high-primed mice to clear virus as
readily as CTL from VSCF-primed mice following adoptive transfer into
infected recipients is yet to be tested.
Splenocytes from VSCF-immunized mice restimulated with
RSV-infected autologous splenocytes produce an array of
cytokines, including IL-2, IL-3, IL-4, and IL-5, although the
latter two are produced at significantly lower levels than those
produced by splenocytes from mice vaccinated with an rVV expressing the G protein of RSV (1). We found that the predominant
cytokines detectable in supernatants from splenocytes of mice
vaccinated with rVV expressing the F protein and restimulated in vitro
with RSV were IL-2, IL-10, and IFN-. These findings are essentially the same as those reported by Srikiatkhachorn and Braciale
(38). However, those investigators found that the effector
T-lymphocyte population exhibits a cytokine profile different from that
of memory T-lymphocyte precursors (38). We are therefore
currently investigating the cytokine profile of restimulated
splenocytes obtained from VV-F.mIL-4high-immunized mice 5 days after
i.n. challenge with RSV to determine whether the coexpression of IL-4 in rVV can prime for an effector Th2-like response following RSV challenge.
Data obtained from this study indicate that although a Th2 cytokine
(IL-4) present at the time of acute rVV infection resulted in delayed
virus clearance, there was no effect on the development of RSV-specific
memory CTL or IFN- production, and mice were resistant to subsequent
RSV challenge. It therefore appears that the coexpression of IL-4 with
the F protein in a live-virus vector delivery system (rVV) failed to
subvert the Th1 memory response on subsequent exposure to RSV.
Further studies are in progress to examine the ability of
coexpressed cytokines to influence the response to rVV expressing the G
protein of RSV, a protein known to induce a Th2-like immune
response. These studies suggest that the changes observed in the
vaccine-induced immune response are the result of an altered antigen
load due to differences in viral vector replication, rather than of a
direct effect of the cytokines on the developing immune response. In
order to determine if the immune response to the F or G protein of RSV
can be influenced by cytokines, further studies with plasmids
carrying RSV F or G protein and cytokine genes are
currently in progress.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by the European Union (grant PL920489) and the Ministry of Agriculture, Fisheries and Food.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, United Kingdom. Phone: 1635 578411. Fax: 1635 577263. E-mail: Gary.Bembridge{at}BBSRC.AC.UK.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Alwan, W. H., and P. J. Openshaw. 1993. Distinct patterns of T- and B-cell immunity to respiratory syncytial virus induced by individual viral proteins. Vaccine 11:431-437[Medline]. |
| 2. | Alwan, W. H., F. M. Record, and P. J. M. Openshaw. 1993. Phenotypic and functional characterization of T cell lines specific for individual respiratory syncytial virus proteins. J. Immunol. 150:5211-5218[Abstract]. |
| 3. | Alwan, W. H., F. M. Record, and P. J. M. Openshaw. 1992. CD4+ T-cells clear virus but augment disease in mice infected with respiratory syncytial virus. Comparison with the effects of CD8+ T-cells. Clin. Exp. Immunol. 88:527-536[Medline]. |
| 4. | Andrew, M. E., and B. E. H. Coupar. 1992. Biological effects of recombinant vaccinia virus-expressed interleukin 4. Cytokine 4:281-286[Medline]. |
| 4a. | Bembridge, G. P., et al. Unpublished data. |
| 5. | Blasco, R., and B. Moss. 1995. Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158:157-162[Medline]. |
| 6. | Bradley, L. M., D. K. Dalton, and M. Croft. 1996. A direct role for IFN-gamma in regulation of Th1 cell development. J. Immunol. 157:1350-1358[Abstract]. |
| 7. |
Cristina, J.,
J. A. Lopez,
C. Albo,
B. Garcia-Barreno,
J. Garcia,
J. A. Melero, and A. Portela.
1990.
Analysis of genetic-variability in human respiratory syncytial virus by the RNase A mismatch cleavage method subtype divergence and heterogeneity.
Virology
174:126-134[Medline].
|
| 8. |
Denny, F. W.,
D. Taylor-Robinson, and A. C. Allison.
1972.
The role of the thymus-dependent immunity in Mycoplasma pulmonis infections of mice.
J. Med. Microbiol.
5:327-335 |
| 9. | Fernie, B. F., E. C. Ford, and J. L. Gerin. 1981. The development of BALB/c cells persistently infected with respiratory syncytial virus: presence of ribonucleoprotein on the cell surface. Proc. Soc. Exp. Biol. Med. 167:83-86[Medline]. |
| 10. | Finkelman, F. D., and J. Holmes. 1990. Lymphokine control of in vivo immunoglobulin isotype selection. Annu. Rev. Immunol. 8:303-333[Medline]. |
| 11. | Finkelman, F. D., I. M. Katona, T. R. Mosmann, and R. L. Coffman. 1988. IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol. 140:1022-1027[Abstract]. |
| 12. | Fischer, J. E., J. E. Johnson, R. K. Kuli-Zade, T. R. Johnson, S. Aung, R. A. Parker, and B. S. Graham. 1997. Overexpression of interleukin-4 delays virus clearance in mice infected with respiratory syncytial virus. J. Virol. 71:8672-8677[Abstract]. |
| 13. |
Florentino, D. F.,
M. W. Bond, and T. R. Mosmann.
1989.
Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.
J. Exp. Med.
170:2081-2095 |
| 14. |
Gaddum, R. M.,
R. S. Cook,
S. G. Wyld,
J. A. Lopez,
R. Bustos,
J. A. Melero, and G. Taylor.
1996.
Mutant forms of the F protein of human respiratory syncytial (RS) virus induce a cytotoxic T lymphocyte response but not a neutralizing antibody response and only transient resistance to RS virus infection.
J. Gen. Virol.
77:1239-1248 |
| 15. | Graham, B. S., G. S. Henderson, Y. Tang, X. Lu, K. M. Neuzil, and D. G. Colley. 1993. Priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with respiratory syncytial virus. J. Immunol. 151:2032-2040[Abstract]. |
| 16. |
Hsieh, C.-S.,
S. E. Macatonia,
C. S. Tripp,
S. F. Wolf,
A. O'Garra, and K. M. Murphy.
1993.
Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages.
Science
260:547-549 |
| 17. | Karupiah, G., B. Coupar, I. Ramshaw, D. Boyle, R. Blanden, and M. Andrew. 1990. Vaccinia virus-mediated damage of murine ovaries and protection by virus-expressed interleukin-2. Immunol. Cell Biol. 68:325-333. |
| 18. | Kohonen-Corish, M. R. J., N. J. C. King, C. E. Woodhams, and I. A. Ramshaw. 1990. Immunodeficient mice recover from infection with vaccinia virus expressing interferon-gamma. Eur. J. Immunol. 20:157-161[Medline]. |
| 19. |
Lee, F.,
T. Yokota,
T. Otsuka,
P. Meyerson,
D. Villaret,
R. Coffman,
T. Mosmann,
D. Rennick,
N. Roehm,
N. Smith,
A. Zlotnik, and K.-I. Arai.
1986.
Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities.
Proc. Nat. Acad. Sci. USA
83:2061-2065 |
| 20. |
Le Gros, G.,
S. Z. Ben-Sasson,
R. Seder,
F. D. Finkelman, and W. E. Paul.
1990.
Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells.
J. Exp. Med.
172:921-929 |
| 21. |
Lopez, J. A.,
R. Bustos,
A. Portela,
B. Garcia-Barreno, and J. A. Melero.
1996.
A point mutation in the F1 subunit of human respiratory syncytial virus blocks its cell surface transport at an early stage of the exocytic pathway.
J. Gen. Virol.
77:649-660 |
| 22. |
Matyniak, J. E., and S. L. Reiner.
1995.
T helper phenotype and genetic susceptibility in experimental Lyme disease.
J. Exp. Med.
181:1251-1254 |
| 23. | Moore, K. W., A. O'Garra, R. de Waal Maldfyt, P. Vieira, and T. R. Mosmann. 1993. Interleukin 10. Annu. Rev. Immunol. 11:165-190[Medline]. |
| 24. |
Moran, T. M.,
H. Isobe,
A. Fernandez-Sesma, and J. L. Schulman.
1996.
Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.
J. Virol.
70:5230-5235 |
| 25. | Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Gredlin, and R. L. Coffman. 1986. Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348-2357[Abstract]. |
| 26. | Mosmann, T. R., and R. L. Coffman. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145-173[Medline]. |
| 27. |
Openshaw, P. J.,
S. L. Clarke, and F. M. Record.
1992.
Pulmonary eosinophilic response to respiratory syncytial virus infection in mice sensitized to the major surface glycoprotein G.
Int. Immunol.
4:493-500 |
| 28. | Paul, W. E., and R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell 76:241-251[Medline]. |
| 29. |
Pemberton, R. M.,
M. J. Cannon,
P. J. M. Openshaw,
L. A. Ball,
G. W. Wertz, and B. A. Askonas.
1987.
Cytotoxic T-cell specificity for respiratory syncytial virus proteinsfusion protein is an important target antigen.
J. Gen. Virol.
68:2177-2182 |
| 30. | Ramshaw, I. A., M. E. Andrew, S. M. Phillips, D. B. Boyle, and B. E. H. Coupar. 1987. Recovery of immunodeficient mice from a vaccinia virus/IL-2 recombinant infection. Nature 329:545-546[Medline]. |
| 31. | Romani, L., A. Mencacci, E. Cenci, R. Spaccapelo, P. Mosci, P. Puccetti, and F. Bistoni. 1993. CD4+ subset expression in murine candidiasis. J. Immunol. 150:925-931[Abstract]. |
| 32. | Rosenstein, M., S. E. Ettinghausen, and S. A. Rosenberg. 1986. Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2. J. Immunol. 137:1735-1742[Abstract]. |
| 33. | Schmitt, E., R. Van Brandwijk, H.-G. Fischer, and E. Rude. 1990. Establishment of different T cell sublines using either interleukin 2 or interleukin 4 as growth factors. Eur. J. Immunol. 20:1709-1715[Medline]. |
| 34. | Seder, R. A., and W. E. Paul. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12:635-673[Medline]. |
| 35. |
Sharma, D. P.,
A. J. Ramsay,
D. J. Maguire,
M. S. Rolph, and I. A. Ramshaw.
1996.
Interleukin-4 mediates down regulation of antiviral cytokine expression and cytotoxic T-lymphocyte responses and exacerbates vaccinia virus infection in vivo.
J. Virol.
70:7103-7107 |
| 36. | Sher, A., and R. L. Coffman. 1992. Regulation of immunity to parasites by T cell and T-cell derived cytokines. Annu. Rev. Immunol. 10:385-409[Medline]. |
| 37. |
Snapper, C. M.,
F. D. Finkelman, and W. E. Paul.
1988.
Differential regulation of IgG1 and IgE synthesis by interleukin 4.
J. Exp. Med.
167:183-196 |
| 38. | Srikiatkhachorn, A., and T. J. Braciale. 1997. Virus-specific memory and effector T lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection. J. Virol. 71:678-685[Abstract]. |
| 39. |
Stott, E. J.,
G. Taylor,
L. A. Ball,
K. Anderson,
K. K.-Y. Young,
A. M. Q. King, and G. W. Wertz.
1987.
Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.
J. Virol.
61:3855-3861 |
| 40. | Street, N. E., J. H. Schumacher, T. A. Fong, H. Bass, D. F. Florentino, J. A. Leverah, and T. R. Mosmann. 1990. Heterogeneity of mouse helper T cells: evidence from bulk cultures and limiting dilution cloning for precursors of TH1 and TH2 cells. J. Immunol. 144:1629-1639[Abstract]. |
| 41. | Swain, S. L., A. D. Weinberg, M. English, and G. Huston. 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145:3796-3806[Abstract]. |
| 42. | Tang, Y., and B. S. Graham. 1994. Anti-IL-4 treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic T lymphocyte activity in mice challenged with respiratory syncytial virus. J. Clin. Invest. 94:1953-1958. |
| 43. | Tang, Y. W., and B. S. Graham. 1995. Interleukin-12 treatment during immunization elicits a T-helper cell type 1-like immune response in mice challenged with respiratory syncytial virus and improves vaccine immunogenicity. J. Infect. Dis. 172:734-738[Medline]. |
| 44. |
Taylor, G.,
E. J. Stott, and A. J. Hayle.
1985.
Cytotoxic lymphocytes in the lungs of mice infected with respiratory syncytial virus.
J. Gen. Virol.
66:2533-2538 |
| 45. |
Taylor, G.,
E. J. Stott,
M. Hughes, and A. P. Collins.
1984.
Respiratory syncytial virus infection in mice.
Infect. Immun.
43:649-655 |
| 46. | Vitetta, E. S., J. Ohara, C. Myers, J. Layton, P. H. Krammer, and W. E. Paul. 1985. Serological, biochemical, and functional identity of B cell-stimulatory factor 1 and B-cell differentiation factor for IgG1. J. Exp. Med. 161:1726-1731. |
| 47. |
Yokota, T.,
N. Arai,
F. Lee,
D. Rennick,
T. Mosmann, and K.-I. Arai.
1985.
Use of a cDNA expression vector for isolation of mouse interleukin-2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells.
Proc. Natl. Acad. Sci. USA
82:68-72 |
J Virol, May 1998, p. 4080-4087, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Johnstone, C., Guil, S., Rico, M. A., Garcia-Barreno, B., Lopez, D., Melero, J. A., Del Val, M.
(2008). Relevance of viral context and diversity of antigen-processing routes for respiratory syncytial virus cytotoxic T-lymphocyte epitopes. J. Gen. Virol.
89: 2194-2203
[Abstract] [Full Text] -
Riou, C., Dumont, A. R, Yassine-Diab, B., Haddad, E. K, Sekaly, R.-P.
(2006). IL-4 influences the differentiation and the susceptibility to activation-induced cell death of human naive CD8+ T cells. Int Immunol
18: 827-835
[Abstract] [Full Text] -
Bukreyev, A., Belyakov, I. M., Prince, G. A., Yim, K. C., Harris, K. K., Berzofsky, J. A., Collins, P. L.
(2005). Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus. J. Virol.
79: 9515-9526
[Abstract] [Full Text] -
Kienzle, N., Olver, S., Buttigieg, K., Groves, P., Janas, M. L., Baz, A., Kelso, A.
(2005). Progressive Differentiation and Commitment of CD8+ T Cells to a Poorly Cytolytic CD8low Phenotype in the Presence of IL-4. J. Immunol.
174: 2021-2029
[Abstract] [Full Text] -
Johnstone, C., de Leon, P., Medina, F., Melero, J. A., Garcia-Barreno, B., Val, M. D.
(2004). Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus. J. Gen. Virol.
85: 3229-3238
[Abstract] [Full Text] -
Bartlett, N. W., Dumoutier, L., Renauld, J.-C., Kotenko, S. V., McVey, C. E., Lee, H.-J., Smith, G. L.
(2004). A new member of the interleukin 10-related cytokine family encoded by a poxvirus. J. Gen. Virol.
85: 1401-1412
[Abstract] [Full Text] -
Johnson, T. R., Hong, S., Van Kaer, L., Koezuka, Y., Graham, B. S.
(2002). NK T Cells Contribute to Expansion of CD8+ T Cells and Amplification of Antiviral Immune Responses to Respiratory Syncytial Virus. J. Virol.
76: 4294-4303
[Abstract] [Full Text] -
Kienzle, N., Buttigieg, K., Groves, P., Kawula, T., Kelso, A.
(2002). A Clonal Culture System Demonstrates That IL-4 Induces a Subpopulation of Noncytolytic T Cells with Low CD8, Perforin, and Granzyme Expression. J. Immunol.
168: 1672-1681
[Abstract] [Full Text] -
Chang, J., Srikiatkhachorn, A., Braciale, T. J.
(2001). Visualization and Characterization of Respiratory Syncytial Virus F-Specific CD8+ T Cells During Experimental Virus Infection. J. Immunol.
167: 4254-4260
[Abstract] [Full Text] -
Johnson, T. R., Fischer, J. E., Graham, B. S.
(2001). Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine. J. Gen. Virol.
82: 2107-2116
[Abstract] [Full Text] -
Jackson, R. J., Ramsay, A. J., Christensen, C. D., Beaton, S., Hall, D. F., Ramshaw, I. A.
(2001). Expression of Mouse Interleukin-4 by a Recombinant Ectromelia Virus Suppresses Cytolytic Lymphocyte Responses and Overcomes Genetic Resistance to Mousepox. J. Virol.
75: 1205-1210
[Abstract] [Full Text] -
Raggo, C., Habermehl, M., Babiuk, L. A., Griebel, P.
(2000). The in vivo effects of recombinant bovine herpesvirus-1 expressing bovine interferon-{gamma}. J. Gen. Virol.
81: 2665-2673
[Abstract] [Full Text] -
Bembridge, G. P., Rodriguez, N., Garcia-Beato, R., Nicolson, C., Melero, J. A., Taylor, G.
(2000). DNA encoding the attachment (G) or fusion (F) protein of respiratory syncytial virus induces protection in the absence of pulmonary inflammation. J. Gen. Virol.
81: 2519-2523
[Abstract] [Full Text] -
Coupar, B. E. H., Oke, P. G., Andrew, M. E.
(2000). Insertion sites for recombinant vaccinia virus construction: effects on expression of a foreign protein. J. Gen. Virol.
81: 431-439
[Abstract] [Full Text] -
Bembridge, G. P., Lopez, J. A., Bustos, R., Melero, J. A., Cook, R., Mason, H., Taylor, G.
(1999). Priming with a Secreted Form of the Fusion Protein of Respiratory Syncytial Virus (RSV) Promotes Interleukin-4 (IL-4) and IL-5 Production but Not Pulmonary Eosinophilia following RSV Challenge. J. Virol.
73: 10086-10094
[Abstract] [Full Text] -
Aung, S., Tang, Y.-W., Graham, B. S.
(1999). Interleukin-4 Diminishes CD8+ Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo. J. Virol.
73: 8944-8949
[Abstract] [Full Text] -
Bukreyev, A., Whitehead, S. S., Bukreyeva, N., Murphy, B. R., Collins, P. L.
(1999). Interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity. Proc. Natl. Acad. Sci. USA
96: 2367-2372
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»