Download Mutant forms of the F protein of human respiratory syncytial (RS

Journal of General Virology (1996), 77, 1239-1248.
1239
Printed in Great Brita#l
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
Ruth M. Gaddum, 1. Roy S. Cook, 1 Sara G. Wyld, 1 Juan A. L6pez, 2 Regla Bustos, 2
Jos6 A. Melero 2 and Geraldine Taylor 1
Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK and ~Centro Nacional de Biologia
Celular y Retrovirus, Instituto de Salud 'Carlos III', Majahonda, 28220 Madrid, Spain
Vaccinia virus (vv) recombinants expressing either wildtype (VA-F) or mutant forms (VA-FT, VA-FR47, VAFS 1 to VA-FS6) of the fusion (F) protein of respiratory
syncytial (RS) virus were examined for their ability to
elicit antibody, cytotoxic T lymphocytes (CTL) and
protection against RS virus infection in BALB/c mice.
Cells infected with the VA-F and VA-FT recombinants
expressed the F protein on their surface and mice
vaccinated with these recombinants developed RS virus
neutralizing antibodies. The VA-FR47 recombinant
expressed a mutant form of the F protein (with six amino
acid changes from the wild-type) in which both proteolytic processing of the F 0 precursor and its transport
to the cell surface were inhibited. These mutants induced
Introduction
Human respiratory syncytial (RS) virus is a pneumovirus
of the family Paramyxoviridae and is a major cause of
lower respiratory tract infections in infants and young
children (Stott & Taylor, 1985; McIntosh & Chanock,
1990). The pathogenesis of the disease is poorly
understood, reinfection is common and there is currently
no effective vaccine available (Pringle, 1987; Kimman &
Westenbrink, 1990). In an early vaccine trial, children
exposed to natural RS virus infection, following administration of a formalin-inactivated vaccine, exhibited
exacerbated disease (Fulginiti et al., 1969; Kim et al.,
1969). Enhanced pathology has also been observed in
cotton rats and mice inoculated with formalininactivated virus (Prince et al., 1986; Vaux-Peretz &
Meignier, 1990) and in mice vaccinated with vaccinia
virus (vv) recombinants expressing individual RS virus
proteins (Stott et al., 1987; Openshaw et at., 1992). The
* Author for correspondence. Fax +44 1635 577263.
0001-3784 © 1996 SGM
transient protection against RS virus infection although
they did not induce RS virus neutralizing antibodies, or
antibodies detectable by ELISA. All the vv recombinants
were able to induce an RS virus-specific, MHC class I
rest-icted CTL response. Vaccination of mice with a
second set ofvv recombinants expressing mutant forms
of the F protein showed that the replacement Phe to Ser
at amino acid 237 either alone or in combination with
others abolished the neutralizing antibody response but
did not affect priming of CTLs. These results demonstrate that long-term protection against RS virus
infection in mice vaccinated with recombinant vv
expressing the F protein is more dependent upon the
induction of an antibody rather than a CTL response.
enhanced pathology seen in vaccine recipients may
involve Th2 responses as depletion of either CD4 + T cells
or of both interleukin-4 (IL-4) and IL-10 abrogated the
pathology observed in mice vaccinated with formalininactivated virus (Connors et al., 1992b, 1994). Further,
passive transfer of RS virus-specific CD4 ÷ or CD8 + T
cells cleared virus from the lungs of infected mice but
augmented lung pathology (Cannon et al., 1988; Alwan
et al., 1992, 1994). In contrast, passively transferred
antibodies, which reduce virus titres in the lungs, do not
exacerbate pathology in RS virus-infected mice or cotton
rats (Taylor et al., 1984, 1992; Walsh et al., 1984). These
findings suggest that a successful vaccine should stimulate a long-lasting antibody response, but it is not clear
if it is also important to stimulate a cytotoxic T
lymphocyte (CTL) response.
The F glycoprotein of RS virus is the major crossprotective antigen between strains (Stott et al., 1987;
Olmsted et al., 1986) and is expressed on the surface of
infected cells. The F protein is synthesized as a F 0
precursor (69kDa) and is co-translationally Nglycosylated and post-translationally cleaved into two
subunits, F 1 (49 kDa) and F 2 (20 kDa), which are linked
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R. M. Gaddum and others
Table 1. Characterization o f the antibody response induced in mice by recombinant vv expressing mutant forms o f the
F protein of R S virus
Expt no. Virus
A m i n o acid
changes
Intact F
protein on
cell surface
ELISA titre*
VACC
F
Reactivity in
Western blott
Neutralization
titre:~
+
+
+
< 1
6
7
< 1
+
+
+
ND
+
+
ND
ND
ND
ND
ND
ND
ND
ND
VA-5C
VA-F
VA-FT
VA-FR47
262
67, 120, 223, 237,
262, 442
+
+
-
5-0
4.1
4.2
4.3
< 2
4.2
4-1
< 2
VA-F
VA-FS1
VA-FS2
VA-FS3
VA-FS4
VA-FS5
VA-FS6
223
237
223,
223,
237,
223,
+
+
+
-
2.5
2"7
3'0
2'8
3'1
3.0
3.0
2-6
2.6
1
1
2"1
1
1
237
262
262
237, 262
<
<
<
<
* log10 titre of antibody to either extracts of VA-5C infected cells (VACC) or immunoaffinity purified F protein (F).
t Reactivity of mouse sera with the F1 subunit.
:~ log 2 titre that inhibited cytopathic effect in a RS virus microneutralization test.
NO, Not determined.
by disulphide bonds (McIntosh & Chanock, 1990).
Neutralizing and fusion-inhibiting MAbs to the F protein
not only protect against RS virus infection in mice,
cotton rats and calves (see Taylor, 1994; Walsh et al.,
1984) but can clear an established infection (Tempest et
al., 1991 ; see Taylor, 1994). In addition, the F protein is
a target for both human and murine CTL (Pemberton et
al., 1987; Nicholas et al., 1990; Connors et al., 1991;
Cherrie et al., 1992) and Th responses (Openshaw et al.,
1988; Alwan et al., 1993). Vaccination of mice with
recombinant vv expressing the F protein alone induces
neutralizing antibody, a Thl and a CTL response.
Adoptive transfer of F protein-specific T cell lines
reduced the levels of virus in the lungs of RS virusinfected mice (Alwan et al., 1994). However, the role of
antibodies and T cell responses in the protection
mediated by the F protein is not clear. Immunization of
mice with vv recombinants expressing the F protein
protected against RS virus challenge following depletion
o f C D 4 + and CD8 + T cells prior to challenge (Connors et
al., 1992a), suggesting that antibodies to the F protein
were able to protect against RS virus challenge independent of a T cell response. However, resistance in
vaccinated and depleted mice may have been mediated
locally by residual levels of T cells or, since the vv
recombinants were administered both intraperitoneally
(i.p.) and intranasally (i.n.), it is possible that mucosal
IgA antibodies mediated protection in depleted animals
(Connors et al., 1992a). In this study, recombinant vv
expressing F genes that contain mutations which result in
defective maturation of the F 0 precursor, so that the
native protein is not expressed on the surface of the
infected cell (L6pez et al., 1996), have been examined for
their ability to elicit CTLs, induce neutralizing antibodies
and protect against RS virus challenge in BALB/c mice.
The results support the view that long-lasting protection
against RS virus challenge in mice induced by the F
protein is mediated by neutralizing antibodies.
Methods
Viruses. The h u m a n RS viruses of the Long strain, the A2 strain and
the escape m u t a n t R47F/4, selected with M A b 47F (Garcia-Barreno et
al., 1989) were grown in HEp-2 monolayers and purified from culture
supernatants as described previously (Garcia-Barreno et al., 1988).
Recombinant vv expressing native F protein (VA-F) from the Long
strain RS virus and m u t a n t forms of the F protein in which expression
at the surface of infected cells was inhibited, and vv containing plasmid
pSC11 without an insert for use as a recombinant control (VA-5C),
were produced and characterized (L6pez et al., 1996). The amino acid
changes in the F protein of the m u t a n t s and their effect on cell surface
expression are summarized in Table 1. The recombinant w were grown
in HEp-2 cells by inoculation with 5 p.f.u./cell and incubation at 37 °C
until a cytopathic effect was observed in greater than 75 % of the cells
(usually 48 h). The cells were then scraped into the medium, pelleted
and resuspended in 10 mM-Tris-HC1 pH 8.8. The cell suspension was
sonicated for 2 m i n and centrifuged at 1000g for 5 m i n . The
supernatant was removed and the pellet resuspended as before and the
process repeated. The supernatants were pooled and stored at - 70 °C.
Mice. Six-week-old specific-pathogen-free B A L B / c female mice (H2a), obtained from Charles Rivers, were inoculated i.p. with
1 x 106 p.f.u, of recombinant vv. Serum samples were obtained from
the mice immediately before i.n. challenge with approximately 105 p.f.u.
of the A2 strain of RS virus. Five days after challenge, groups of five
mice were killed and the RS virus titre in lung homogenates was
determined by plaque assay on secondary calf kidney cells (Taylor et al.,
1984). Mean titres o f virus from groups were compared by unpaired
Student's t-test. Serum was obtained from further groups of five mice
killed 7 days after challenge; the lungs from these mice were fixed in
formol-sublimate and histological sections were stained with haemotoxylin and eosin as described previously (Taylor et al., 1984). Cells
were recovered from lung washings according to the m e t h o d of D e n n y
et al. (1972), but using 1 ml of PBS.
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Immune responses to R S virus F protein mutants
1241
RSV
50
40
%'%°°%
%'%%°°
'q
.t,/)
_~, 3O
O
%
°°o
%%
%°
%%o°
°r-i................... r-
Q,.
oo 20
10
o ~-.4k_.~_,--,.,~
40:1
2ff::1
E:T
1~1
Fig. 1. Cytotoxic activity of splenocytes from mice inoculated with RS virus, VA-F,
VA-FT or VA-FR47, tested at decreasing effector: target (E:T) ratios on uninfected
BALB/c fibroblasts (O), BCH4 cells ( ~ ) , uninfected L-929 cells ( ~ ) or RS virusinfected L-929 cells (@).
5T1
VA-FT
VA-F
VA-FR47
50
50
50
[0 3.
4O
°%°°%
40
¢/}
~, 30
"g ~0
"%
%%°
".°°
%%
tO
0~
20
"1................... q
"°°°°%°
=o
.=o
g 30
%°
°%
'°%
°%
%
°•
~o
20
"',%
10
40:1
"°%°°°
2b!1
E:T
11~1
5T1
40:1
20:1
Antibody assays. The presence of antibodies to RS virus and vv
recombinants was determined by ELISA, as described previously
(Taylor et al., 1992). Micro-neutralization tests were carried out by
incubating 1-5 x 105 p.f.u, of the Long strain of RS virus with twofold
serial dilutions of sera for 60 min at 37 °C. The virus antibody mixtures
were then used to infect HEp-2 monolayers grown in 96-well tissue
culture plates. After 3 days at 37 °C, monolayers were fixed with 10 %
formaldehyde and stained with crystal violet. Neutralization titres were
estimated from the highest dilution of sera that inhibited the destruction
of cell monolayers, compared with an antiserum control.
Western immunoblotting was carried out using cell extracts that
were separated into individual proteins by SDS PAGE, electrotransferred to nitrocellulose paper (Towbin et al., 1979) and blocked
overnight with 5 % non-fat dried milk in PBS at room temperature. The
paper was cut into strips and incubated for 2 h with 1 : 100 dilutions of
the individual mouse sera diluted in blocking buffer, followed by
incubation with biotinylated anti-mouse Ig, horseradish peroxidasestreptavidin-peroxidase and 4-chloro-l-naphthol as substrate.
Generation o f secondary CTLs in vitro. Spleen lymphocytes from
mice immunized with w recombinants a minimum of 4 weeks earlier
were restimulated in vitro with RS virus-infected splenocytes. Thus,
spleen lymphocytes from uninfected mice were pelleted and incubated
in 1 ml of medium containing 0-4 p.f.u./cell of the A2 strain of the virus
E:T
1~1
°'°-o..°o.
%°°°o.,[~
o~
°%
[3.°°°..,.°....O.O..o
o
'%
"%
'°,
5~'1
10
°o
E:T
1
sT1
for 90 min at 37 °C. Spleen cells from vaccinated mice were purified
using a Histopaque gradient (density 1.083, Sigma), washed and
incubated with 3 x 106 RS virus-infected control splenocytes, at an
effector:stimulator ratio of 5:1, for 5 days in RPMI 1640 containing
10 % fetal calf serum, 2 mM-glutamine, 5 x 10 5 M-fl-mercaptoethanol,
1 0 0 U / m l penicillin and 100 gg/ml streptomycin. After this time
effector cells were harvested, counted and used in chromium release
assays.
Target cells and cvtotoxicity assays. Target cells for cytotoxicity
assays were BCH4 cells, a line of BALB/c embryo fibroblasts
persistently infected with the Long strain of RS virus (Fernie et al.,
1981) or BALB/c fibroblasts (Taylor et al., 1985) infected in suspension
with 2-5 p.f.u./cell of the A2 strain of RS virus for 90 rain at 37 °C
and incubated for a further 21 h to allow viral protein expression before
use in CTL assays. The mouse fibroblast line L-929 (11-2 k) was infected
as described above. YAC-1 cells were included to detect natural killer
(NK) activity and uninfected BALB/c fibroblasts were used as a
control. Suspensions of infected and control target cells were prepared
by trypsinization of monolayers. Cells were labelled as a pellet with
100 taCi Na2~lCrO4 (Dupont) per 106 cells for 90 min at 37 °C, and
washed three times in medium before 5 x 103 targets were plated out
into each well of a V-bottomed 96-well plate. Effector cells were added
to give a final volume of 150 gl per well; microtitre plates were
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1242
R. M. Gaddum and others
centrifuged and incubated for 4 h at 37 °C in 5% COz/air. The
percentage specific release of alCr = [(c.p.m. experimental-c.p.m.
spontaneous)/(c.p.m, m a x i m u m - c . p . m , spontaneous)] x 100. Spontaneous release was the value obtained with medium alone and
maximum release was determined following incubation of target cells
with 1% SDS. Each value was the mean of three replicates.
Table 2. Percentage specific lysis of RS virus-infected
BALB/c fibroblasts by lymphocytes treated with
complement alone or complement and Lyt 2 + antibody,
from RS virus-infected or vaccinated mice, at an
effector : target ratio of 40." 1
Depletion o f C D 8 ÷ cells'. Washed effector cells were incubated in Lyt
2÷-specific M A b (clone YTS 169.4, Seralab) diluted 1:125 in 2 % FCS
in RPMI for 1 h at 4 °C (100 ~1 per 10° cells). The cells were washed
twice in PBS and incubated with rabbit complement (Sigma) diluted
1:15 in 2 % FCS in RPMI (100 gl per 106 cells) for 1 h. The cells were
washed as before and used directly in a chromium release assay.
Virus that
mice were
inoculated
with:
Results
Antibody response in mice vaccinated with vv expressing
mutant F protein
To determine whether the mutations within the VA-FT,
VA-FR47 and VA-FS1 to VA-FS6 recombinants (shown
in Table 1) influenced the immunogenic properties of the
F protein, the antibody responses induced by the
constructs were examined in mice. The vv recombinants
which included a replacement of a Phe to a Ser at amino
acid 237, either alone or in combination with other
mutations, inhibited the transport of the F protein to the
cell surface before F protein reached the medial Golgi
compartment (L6pez et al., 1996). Sera were tested by
ELISA for the presence of antibodies to vv and to
purified RS virus F protein (Table 1). All vaccinated
mice developed antibodies to vv. However, only the mice
vaccinated with VA-F, VA-FT, VA-FS1 and VA-FS4
recombinants, which resulted in cell surface expression
of the F protein, developed antibody which recognized
purified F protein. The amino acid change (Phe to Ser) at
position 237 was common to all of the recombinants
which failed to induce an antibody response to the F
protein in mice. Sera from mice given VA-F and VA-FT
neutralized virus infectivity, whereas sera from mice
given VA-FR47, a recombinant containing a mutation
which causes the F protein to be retained within the cell,
did not contain neutralizing antibody (Table 1).
Further characterization of sera from vaccinated
animals by Western blotting showed that VA-F, VA-FT
and VA-FR47 were all able to elicit antibodies that
recognized the denatured F 1 subunit of either Long or
R47F/4 viruses (Table 1). In addition, sera from mice
inoculated with recombinants VA-FS1 to VA-FS6 all
contained antibodies that reacted by Western blot with
the F~ subunit.
Generation of a CTL response
It has been shown previously that recombinant vv
expressing the F protein of RS virus primes BALB/c
mice for a RS virus specific CTL response (Pemberton et
Specific lysis (%) by lymphocytes* treated with:
Complement alone
Complement + Lyt 2 +
antibody
27
22
22
ND
43
ND
27
27
40
38
0
0
0
ND
13
ND
0
0
15
11
RSV
VA-F
VA-FT
VA-FR47
VA-FS1
VA-FS2
VA-FS3
VA-FS4
VA-FS5
VA-FS6
* Spleen cells obtained from mice inoculated i.n. with RS virus or
i.p. with recombinant vv 4 weeks previously.
~D, Not determined.
al., 1987; Alwan & Openshaw, 1993). Therefore, the
ability of vv recombinants expressing mutant forms of
the F protein to induce CTL was investigated. Secondary
CTLs were generated following in vitro stimulation of
splenocytes from mice infected with either RS virus or
the vv recombinants. Lymphocytes from mice inoculated
with RS virus, VA-F, VA-FT or VA-FR47 specifically
lysed BCH4 cells but not uninfected BALB/c fibroblasts
(Fig. 1). There was less than 6 % lysis of the NK sensitive
YAC-1 cell line (data not shown), suggesting that the
lysis observed was not due to NK cells. Similarly, no
more than 5 % lysis was observed for either uninfected
cells or RS virus-infected MHC rots-matched L-929 (H2 ~) cells. Pre-incubation of the effector cells with Lyt 2+
(CD8-specific) MAb and complement eliminated or
greatly reduced the cytotoxic activity of the lymphocytes
(specific lysis less than 2 %), demonstrating that the CTL
response induced by the recombinant vv was mediated
by CD8 + cells (Table 2).
Further experiments demonstrated that splenocytes
from mice vaccinated with VA-FS1, VA-FS2, VA-FS3,
VA-FS4, VA-FS5 or VA-FS6 were all able to generate
RS virus-specific CTL responses (Fig. 2). Lysis of RS
virus-infected BALB/c fibroblasts and of BCH4 (H-2 a)
cells was considerably greater than that of the uninfected
BALB/c fibroblasts or of virus-infected L-929 (H-2 ~)
cells, indicating that lysis was mainly MHC-restricted.
However, where appreciable lysis of RS virus-infected L929 MHC-mismatched targets was observed, lysis of
YAC-1 cells was also seen indicating that in some of the
experiments shown in Fig. 2 up to 35 % of the total lytic
activity may have been mediated by NK cells. As seen
previously, lysis of infected cells was either abolished or
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1243
Immune responses to R S virus F protein mutants
1
100
80 "..
80
100
VA-FS3
VA-FS2
VA-FS1
100
80
R
.~_
60
•J~ 60
s0
O
t~
O
9
0
Q)
Q.
40
20
""19,.
.............. • ....
40:1
20:1
10:1
5:1
U
"'~.
-to
Q.
03 4O
03 4O
2O
2O
0
40:1
20:1
E:T
10:1
0
40:1
5:1
20:1
VA-FS4
100 ]
80
80[
5:1
VA-FS6
VA-FS5
100
10:1
E:T
E:T
80
6o
Q
2.
6o
0
n
03 40
03 40
40
""lZl..
o~
°"°°o~].°.°
20
20
0
40:1
20:1
10:1
5:1
20
...... _ ............ "41'.......
o
~
40:1
20k:1
......
1~1
5:~
E:T
E:T
00
40:1
9 --........,...~
~''''''~ ..... ""'"-.
20:1
10:1
5:1
E:T
VA-F
100 t
80[
.to
60
°"°""°""rl
.rE_
O
4o
20
0 ~ " " ~
40:1
20:1
o
10:1
E:T
o
5:1
Fig. 2. Cytotoxic activity of splenocytes from mice vaccinated with VA-F, VA-FS1, VA-FS2,
VA-FS3, VA-FS4, VA-FS5 or VA-FS6, tested at decreasing effector:target (E:T) ratios on
uninfected BALB/c fibroblasts (O), RS virus-infected BALB/c fibroblasts (O), BCH4 cells
(r~) or RS virus-infected L929 cells (II,).
greatly reduced if the effector cells were incubated first
with Lyt 2 + MAb and complement, indicating that the
predominant effector cells were CD8 + T cells (Table 2).
Lysis o f RS virus-infected cells by effector cells treated
with antibody to Lyt 2 + and complement may have been
mediated by CD4 + CTL or possibly residual CD8 + CTL.
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R. M. Gaddum and others
Table 3. Abili O, of vv recombinants to protect mice against RS virus infection 4 and 16
weeks after vaccination
Antibody response to RS virus*
Expt
no. Vaccine
Pre-challenge
Post-challenge
VA-F
VA-FT
VA-FR47
None
4.4_+0.4
3.8 ± 0-8
< 1.5
< 1.5
5.2_+0.5
4.7_+ 0.2
2.4_+0.7
< 1.5
VA-F
VA-FS 1
VA-FS2
VA-FS3
VA-FS4
VA-FS5
VA-FS6
None
4.5-+0.5
3.7 _+0.4
< 1.5
1"7+0'5
4'l _+0'3
< 1"5
< 1"5
< 1"5
ND
ND
ND
ND
ND
NO
ND
ND
RS virus titre in
lungs (loga0 p.f.u./g)t
< 1.7;~
< 1.7~
4.0_+0.3
4.6_+0-1
(0/5)§
(0/5)
(5/5)
(5/5)
Lung wash
cell count
(log10 cells/ml)][
5.4¶_+0.2
5.4¶ _+0.1
5.3¶___0-2
4-9_+0-2
< 1"7++ (1/5)
2"0 _+0'9:~ (2/5)
4.0-1-0.5 (5/5)
4'0+ 1"1 (4/4)
2'2_+0"8++ (2/5)
5"0-t-0'4 (5/5)
4"4+0'2 (5/5)
4'3+0'5 (5/5)
ND
ND
ND
ND
ND
ND
ND
ND
* Mean _+SD log10 titre of antibody to RS virus determined by ELISA at 4 weeks (Expt no. 1) or 16 weeks (Expt
no. 2) after vaccination, and 7 days after RS virus challenge.
t Mean_+sD titre of RS virus in lungs 5 days after challenge.
++ Probability of difference in virus titres between vaccinated and control mice < 0-0001, calculated by Student's
t-test.
§ No. of mice infected/total.
II Lung wash cell counts 7 days after RS virus challenge.
¶ Lung wash cell counts in vaccinated mice were significantly different by Student's t-test from those of
unvaccinated controls, P < 0.02.
ND, Not determined.
However, since lysis of YAC-1 cells was observed, it is
likely that the remaining activity was due to NK cells.
There was no significant difference between lysis of RS
virus-infected cells by untreated lymphocytes and those
treated with or without complement alone.
Ability of vv recombinants to protect against RS virus
infection at different times after vaccination
Protection against RS virus infection was examined
following vaccination with vv recombinants expressing
intact or mutant forms of the F protein. Four weeks after
vaccination with VA-F, VA-FT or VA-FR47, mice
vaccinated with recombinant VA-FR47 failed to develop
antibodies to RS virus which could be detected by
ELISA (Table 3). Although VA-FR47 was unable to
induce antibodies detectable by ELISA, there was
evidence that priming had occurred as the antibody titre
increased following RS virus challenge when compared
with unvaccinated controls. However, this priming effect
of the humoral immune response was insufficient to
confer protection and the titres of the virus recovered
from the lungs of these mice were not significantly
different from unvaccinated controls (Table 3).
In a separate experiment, the VA-FS1 to VA-FS6
mutants were also examined for their ability to protect
mice 16 weeks after vaccination. Mice vaccinated with
VA-FS2, VA-FS3, VA-FS5 and VA-FS6 did not develop
an RS virus antibody response that was detectable by
ELISA. Furthermore, these animals were not protected
against RS virus infection (Table 3). Mice given VA-FS1
and VA-FS4, which had either or both of the 223 (Phe to
Leu) or 262 (Asn to Tyr) mutations, were able to
generate RS virus antibodies and were protected against
RS virus infection as shown by the reduction in RS virus
titre in the lungs when compared with unvaccinated
controls (Table 3).
The effect of vaccination on the development of
pneumonic lesions was studied by counting the number
of cells in lung washes obtained 7 days after RS virus
challenge and by microscopic examination of lung
sections. There was a three- to fourfold increase in the
number of cells in the lung washings obtained from all
vaccinated mice, 7 days after RS virus infection,
compared with unvaccinated, RS virus-infected animals
(Table 3). Lung lesions from mice vaccinated 4 weeks
previously were examined 7 days after RS virus challenge
and comprised peribronchiolar and perivascular accumulations of lymphocytes (Fig. 3 C, D). Virus challenge of
mice which had been vaccinated with a recombinant
control (VA-5C) resulted in only a small amount of
cellular infiltrate (Fig. 3B), similar to that seen in
unvaccinated, RS virus-infected mice (unpublished observations), whereas there was no cellular infiltrate in lung
sections from mice inoculated with virus-free cell
supernatant (Fig. 3 A). The cellular infiltrate was much
greater in mice that had been vaccinated with the VA-F
or VA-FR47 recombinant prior to challenge (Fig. 3 C, D
respectively) when compared with RS virus-infected
controls.
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Immune responses to R S virus F protein mutants
1245
).j
~r
,.)
r
,(<
,r
Fig. 3. Sections of lung tissue stained with haemotoxylin and eosin to show cellular infiltrates in mice inoculated i.n. with tissue culture
fluid (A) and in mice vaccinated with VA-5C (B), VA-F (C) or VA-FR47 (D). The bar represents 0-1 mm.
Table 4. Ability of recombinant vv to protect mice
against R S virus infection 6 and 10 days after
vaccination
Expt
no.
1
2
Vaccine
Route
VA-FT
i.n.
VA-FS2
i.n.
VA-fl Gal
i.n.
VA-F
VA-FS2
VA-p Gal
VA-F
VA-FS2
VA-/~ Gal
i.n.
i.n.
i.n.
i.p.
i.p.
i.p.
Day of
challenge
RS virus titre in lungs*
(log10 p.f.u./g)
6
10
6
10
6
10
10
10
10
10
10
10
19+_0.6T
1'9 4-0'6t
2.0 4- 0.9T
2.2 4- 1.2++
4.3 _+0.3
4.14-0.1
< 1.7
2"44- l'0t
4-94-0-4
< 1,7
3"0+_0-5§
4.94-0.3
* Mean s D t i t r e o f R S v i r u s i n lungs 5 days after challenge.
~ P < 0.005 by Student's t-test.
P < 0.05 by Student's t-test.
§ P < 0.0005 by Student's t-test.
Resistance to RS virus infection in mice primed with
recombinant vv expressing the M2 (22K) protein, which is
the major CTL antigen in BALB/c mice (Openshaw et al.,
1990), was short-lived and mediated by primary CTLs,
i.e. those induced by vaccination and not requiring
restimulation by RS virus (Connors et al., 1991). Thus,
protection against RS virus infection observed in the
lungs of mice vaccinated 10 days previously had largely
waned by day 28 post-vaccination. Since recombinant vv
expressing mutant forms of the F protein that remained
intracellular primed CTL but did not protect mice
challenged at 28 days post-vaccination or later, the
ability of these recombinants to protect against infection
when mice were challenged 6-10 days after vaccination
was studied. Mice were, therefore, vaccinated with VAF and VA-FT expressing native F protein or VA-FS2,
which contained only the mutation responsible for the
retention of the F protein within the cytoplasm. A
significant reduction in lung virus titre was observed
following RS virus challenge in mice vaccinated either
i.n. or i.p. with VA-F, VA-FT or VA-FS2 6 or 10 days
previously when compared with the control animals
vaccinated with recombinant vv expressing the flgalactosidase protein (Table 4).
Discussion
Vaccinia virus recombinants expressing mutant forms of
the F protein of RS virus in which both the proteolytic
processing of the F 0 precursor and its transport to the
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1246
R. M . Gaddum and others
cell surface were inhibited did not induce a neutralizing
antibody response but did prime CTLs. These observations allowed the relative contribution of these components of the immune response in resistance against RS
virus infection to be studied. Priming of CD8 + CTL, in
the absence of a neutralizing antibody response, induced
only a transient resistance to RS virus infection in
BALB/c mice and suggests that prolonged resistance to
infection requires the production of a protective antibody
response. Although vv recombinants expressing F genes
containing Phe ~-~7~ Ser induced antibodies that reacted
with denatured F protein in a Western blot, only those
mutants able to induce neutralizing antibodies or
antibodies which recognized native F protein in ELISA
protected against RS virus infection. This indicates that
cleavage of F 0 and correct folding of the F protein are
essential for the induction of a protective antibody
response.
The transient resistance to infection seen in vaccinated
mice was similar to that observed by Connors et al.
(1991) who showed that resistance to RS virus infection
induced by recombinant vv expressing the M2 and N
proteins of RS virus is relatively short-lived. Thus, mice
vaccinated simultaneously by the i.n. and i.p. routes with
these recombinants exhibited significant resistance to RS
virus infection after 9 days, but this resistance, which was
mediated by a primary CTL response (Connors et al.,
1992), had largely waned 28 days after vaccination.
Vaccinia virus recombinants expressing viral proteins
have been shown to induce CTL responses, which vary in
their ability to protect against infection. Complete longlasting protection against murine cytomegalovirus can
be achieved by vaccinating mice with a vv recombinant
expressing the immediate-early protein pp89 (Del Val et
al., 1991). In contrast, although CTLs were induced
following vaccination of mice with a vv recombinant
expressing a minigene of the influenza nucleoprotein, the
CTLs did not accelerate influenza virus clearance and did
not protect (Lawson et al., 1994). The nature of the
expressed protein may dictate whether a protective
response evolves, though the reasons for this are unclear.
Perhaps a protein containing a greater number of CTL
epitopes induces a more protective immune response
because more CTL cells are activated. Alternatively, the
site of virus replication or the target host cell may
influence the capacity of CTLs to mediate protection.
Furthermore, use of vv vectors to introduce RS virus
proteins to the immune system may affect the CTL
response. It is possible that the presence of many vv
peptides capable of binding to the MHC proteins could
alter the immunodominance of peptides normally presented to CTL when only RS virus proteins are available,
and thus modify precursor frequency and the effectiveness of a memory CTL response.
The MHC background of the host animal also has a
direct effect on the CTL response as the amino acids
within the peptide binding groove of each MHC molecule
will determine which viral peptides can be accommodated and thus presented to CTL (for review see
Rammensee et al., 1995). Protection against lymphocytic
choriomeningitis virus (LCMV) is virtually exclusively
mediated by virus-specific CTLs (Zinkernagel & Welsh,
1976), and Hany et al. (1989) demonstrated that different
levels of CTL activity and h7 vivo protection could be
generated depending on the MHC-protein combination.
Splenocytes from mice of different MHC backgrounds
which had been primed with LCMV in vivo were
stimulated in vitro with vv recombinants expressing the
NP or G protein of LCMV. A protein-MHC combination which resulted in high levels of specific lysis was
generally associated with longer-lasting protection than
protein-MHC combinations which resulted in only low
CTL responses.
The transient nature of the resistance to RS virus
induced by VA-FS2 is consistent with the time-course of
a primary CTL response (Bennink & Yewdell, 1990).
These studies suggest that CTLs are more effective at
restricting RS virus replication, at least within the first 5
days of infection, than CTLs generated from a memory
population. Memory T cells are not immediately available to deal with a secondary infection and it takes 3 to
5 days for memory CTL precursors to differentiate to a
stage where they can mediate effector functions (Doherty
& Sarawar, 1994). Therefore, restimulation of memory
CTL, whilst not affecting peak titres of virus, may
accelerate virus clearance from immunized mice. This
response may be of particular importance in children
where virus shedding may occur for up to 3 weeks (Hall
et al., 1976). It remains to be determined whether
accelerated clearance of RS virus from the lungs of
vaccinated mice, when compared with controls, can be
observed at times later than 5 days post-infection.
Studies on the adoptive transfer of RS virus-primed
memory CTLs have shown that virus may be cleared
from the lungs of persistently infected X-irradiated mice,
provided that the CTLs were administered early after
infection (Cannon et al., 1987). Cells given 2 weeks after
virus infection were unable to restrict virus replication. A
later study, involving transfer of primed T cells to
immunocompetent BALB/c mice soon after virus infection, also showed that virus could be cleared by CD8 +
cells but that T cell transfer was associated with enhanced
lung pathology, respiratory distress and high mortality
(Cannon et al., 1988). Perhaps stimulating memory
CTLs in vitro selectively amplifies a CTL population
which has stronger antiviral activities or is more highly
activated than CTLs induced by a single immunization in
vivo. Alternatively, it is possible that the number of
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bntnune responses to R S virus F protein mutants
adoptively transferred RS virus-specific CTLs exceeded
that induced after RS virus challenge of vaccinated mice.
Nevertheless, there was a marked influx of lymphocytes
into the lungs of mice vaccinated with vv recombinants
expressing the RS virus F protein in this study and the
lymphocyte infiltration was greater than that observed in
the lungs of control mice. Following RS virus challenge,
the number of lymphocytes was 25-fold greater in
bronchoalveolar lavage from mice primed with vv
expressing the F protein compared with mice given wildtype vv (Openshaw et al., 1992). In these studies, CD8 +
T cells outnumbered CD4 + T cells by 4: 3. The rate of loss
of CD45RB from the CD8 + T cells was maximal 9 days
after RS virus challenge, indicating that the cells were
activated.
In conclusion, vaccines against RS virus that rely
solely on an MHC class I restricted CTL response may
provide only transient resistance to infection, although
they may reduce the severity of illness by enhancing the
clearance of virus later in infection. It is likely that to
achieve long-term protection against RS virus infection,
vaccines will need to induce a protective humoral
response.
This work was funded by MAFF and the European Union
(BIOTECH PL920489). J.A.M. was also supported by the Comision
Interministerial de Ciencia y Technologia (SAF94-0045) and J.D.L.
was a recipient of a postdoctoral fellowship from Instituto de Salud
' Carlos III'.
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