J. gen. Virol. (1986), 67, 623-629. Printed in Great Britain
623
Key words: RSV/cross-reaction/cytotoxic T cells/antigenic variation
Marine Cytotoxic T Cells Specific to Respiratory Syncytial Virus
Recognize Different Antigenic Subtypes of the Virus
By C H A R L E S R. M. B A N G H A M * I " AND B R I G I T T E A. A S K O N A S
National Institute for Medical Research, The Ridgeway, Mill Hill, London N W 7 1AA, U.K.
(Accepted 17 January 1986)
SUMMARY
The aim of this study was to establish whether cytotoxic T cells (Tc), raised against
respiratory syncytial virus (RSV) in the mouse, are specific to the strain of immunizing
virus, or cross-reactive between virus strains. Several recent studies using monoclonal
antibodies have begun to define the antigenic variation among strains of RSV. It is
likely that a successful RSV vaccine will need to contain antigenic determinants from
more than one subtype, but since only the highest levels of neutralizing antibody are
able to give complete protection against RSV infection, a vaccine may also need to
elicit a cellular immune response. We have recently described H-2-restricted, RSVspecific Tc following RSV infection in the mouse; we present here evidence that
polyclonal RSV-specific Tc in the mouse recognize syngeneic target cells infected with
every human strain of RSV tested, whatever the subtype. The only RSV strain that
appeared not to be recognized was bovine RSV, which seems unable to infect mouse
cells; however, bovine cells, infected with bovine RSV and fixed with glutaraldehyde,
primed mice for Tc which recognized human strains of RSV.
INTRODUCTION
The importance of the antigenic heterogeneity of respiratory syncytial virus (RSV) in natural
infection is little understood. In the 1960s it was shown that the extent of this heterogeneity is
small compared with some viruses, for example influenza virus, but it was then shown by
cross-neutralization assays (Coates & Chanock, 1962; Coates et al., 1963; Doggett & TaylorRobinson, 1965) and reciprocal plaque reduction assays (Coates et al., 1966) that antigenic
differences could be identified between RSV strains.
Recent studies using monoclonal antibodies to human RSV have identified two (Gimenez et
al., 1984; Stott et al., 1984a; Ward et al., 1984; Mufson et al., 1985) or three (Anderson et al.,
1985) distinct antigenic groups. While these respective authors examined many different virus
isolates, the studies concur in grouping the Long and A2 strains (with other early strains)
separately from the 8/60, 18537 and RSN-2 strains, although Anderson et al. (1985) were able to
define further antigenic differences between the A2 (and A1) strains and the Long strain. In this
paper, we shall use the designation of Anderson et al. (see Table 1) of groups (subtypes) 1 to 3.
Although passively transferred antibody gives some protection against RSV infection (Parrott
et al., 1973 ; Downham et al., 1976; Walsh et al., 1984; Taylor et al., 1984), high antibody levels
are required for complete protection (Prince et al., 1985), and immunity is only gradually
acquired in infants after repeated infections with the virus (Glezen et al., 1981). Also, reinfection by the same strain can occur in the face of neutralizing antibody (Coates et al., 1963;
Beem, 1967; Henderson et al., 1979). A successful vaccine may, therefore, need to elicit a cellular
immune response to RSV in addition to antibody; there is evidence in man (Fishaut et al., 1979),
cotton rats (Sun et al., 1983) and mice (M. J. Cannon & E. J. Stott, unpublished observations)
that cell-mediated immunity plays a part in combating RSV infection. We have recently
t Present address: Nuffield Department of Clinical Medicine, Job.n Radcliffe Hospital, Oxford OX3 9DU,
U.K.
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624
C, R. M, BANGHAM AND B. A. ASKONAS
described the generation in mice of RSV-specific, major histocompatibility complex- ( M H C ) restricted cytotoxic T cells (Tc) (Bangham et al., 1985). In view of the antigenic subtypes now
identified among RSV strains, it is important to ascertain whether these Tc are cross-reactive,
i.e. whether Tc raised against one RSV subtype recognize strains of the other subtype(s). W e
show here that there is indeed qualitative cross-reaction, which is apparently complete, between
the three antigenic subtypes of h u m a n RSV identified by Anderson et al. (1985).
METHODS
Mice. Female BALB/c mice (aged 1 to 3 months) were obtained from the specific pathogen-free colony bred at
the National Institute for Medical Research.
Virus; plaque assay. The A2, Randall, 8/60, 18537 and bovine 127 strains of RSV were given by E. J. Stott
(Institute for Research on Animal Diseases, Compton, U.K.). The RSS-2 and RSN-2 strains were given by
Professor C. R. Pringle (University of Warwick, Covent~, U.K.). Each strain was grown in HEp-2 cells in H21
(DMEM) medium with 10% foetal calf serum (FCS), penicillin (60 l.tg/ml), streptomycin (100 p.g/ml) and
glutamine (300 ~tg/ml),harvested at 72 h after infection (120 h in the case of the 18537 strain), and stored without
clarification in 1 ml aliquots in liquid nitrogen. Table 1 shows the origin of these strains, and their classification by
Anderson et al. (1985). The plaque assay was done on monolayers of HEp-2 cells, incubated for 5 days at 33 °C with
an overlay containing 0.25 % agaruse, as described previously (Bangham et al., 1985). Bovine nasal mucosa cells
infected with bovine RSV (Compton 127 strain) and fixed with 0.075% glutaraldehyde (Stott et al., 1984b) were
supplied by E. J. Stott.
Immunization of mice. Mice were given 50 ~tlintranasally (i.n .) of a 1 : 5 dilution of stock virus (A2 or 8/60 strains;
equivalent to 2 x 105 p.f.u.) in phosphate-buffered saline (PBS-A), under pentobarbitone anaesthesia [30 mg/kg
intraperitoneally(i.p.)]. BCH4 cells (see Target cells below), fixed with 0.075 % glutaraldehyde for 5 rain (obtained
from E. J. Stott) were used to immunize mice against the Long strain of RSV: mice were given two doses i.p., each
of 105 cells, separated by 3 weeks, with or without 20 ~tg Quil A (a saponin derivative) as adjuvant. Mice
immunized against the bovine strain were either given stock 127 strain virus i.n. (as above), or fixed infected
bovine nasal mucosa cells using the same regimen as for the fixed BCH4 cells.
Generatmn ofTc. Tc memory cells were derived from the spleens of mice primed 3 to 8 weeks earlier (see above),
and re-stimulated in vitro, as described before (Bangham et al., 1985): briefly, 3 x 106 autologous spleen cells
(stimulators) were incubated for I to 2 h at 37 °C with 300 p.1of stock virus, and then incubated for 5 days at 37 °C
with 1.5 x 107 spleen cells (responders) in 20 ml RPMI 1640 medium with 10% FCS, and antibiotics and
glutamine as above. The virus strain used to re-stimulate the spleen cells was the same as the priming strain, except
that spleen cells from mice primed with fixed BCH4 cells or fixed infected bovine nasal mucosa cells were restimulated with the A2 strain.
Target cells and cytotoxicity assay. A continuous line of BALB/c fibroblasts, and a branch (BCH4) of the same
line persistently infected with the Long strain of RSV (Fernie et al., 1981), were obtained from E. J. Stott. For use
as targets in the cytotoxicity assay, BALB/c fibroblasts were incubated with 300 ~tlof stock virus (a multiplicityof
infection of 0.1 to I p.f.u./cell) for 90 rain at 37 °C, then incubated in a 6 cm diam. Petri dish (Nunc) in RPMI 1640
medium with 10% FCS, antibiotics and glutamine as above, for 2 days, or 6 days in the case of the 18537 strain. Tc
were assayed by a chromium release assay, as previously described (Bangham et al., 1985), using 2 x 104 target
cells/well. The percentage specific lysis (L) was calculated as follows: L = [(sample c.p.m. - background
c.p.m.)/(total c.p.m. - background c.p.m.)] x 100, where total c.p.m, is the radioactivity released by targets
treated with Triton X-100 detergent. Background release of 5~Cr ranged between 10 and 35% of total c.p.m.
RESULTS
R S V cross-reaction o f Tc raised against A 2 or 8/60 strains
The Tc prepared from mice immunized with the 8/60 strain o f RSV recognized BALB/c
fibroblasts infected with every RSV strain tested (Fig. 1), whatever its antigenic subtype (Table
1). The same target cells were lysed by Tc from mice p r i m e d with A2 strain RSV (re-stimulated
with the A2 strain in vitro), with approximately the same rank order of percentage specific lysis
(Fig. 2). The differences in lysis between different virus strains, at a given effector : target (E : T)
ratio, do not necessarily reflect antigenic variation among the target virus strains (see
Discussion), since the strains differed in titre (Table 1), in growth rate (Coates et al., 1966), and
possibly in infectivity.
The 18537 strain, which grew considerably more slowly in culture than any other strain used
here, produced no c.p.e, in the BALB/c fibroblasts at 2 days post-infection, and the fibroblasts
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Cross-reaction of anti-RSV T cells
50
i
i
~
i
i
40
t
30
~
50
Rand l
8/60
20
10
E: T ratio
Fig. 1
i
t
40
~
40
~
=
20
Control
625
_
-"~t [
5
2.5
20
40
20
10
E: T ratio
Fig. 2
5
2.5
Fig. 1. Effectors from BALB/c mice primed i.n. with 8/60 strain RSV, and re-stimulated in vitro with
the 8/60 strain, specifically lysed target BALB/c fibroblasts infected for 48 h with RSV strains from
each antigenic subtype. Control: uninfected fibroblasts.
Fig. 2. Effectors from BALB/c mice primed i.n. with A2 strain RSV, re-stimulated in vitro with the A2
strain, lysed target BALB/c fibroblasts infected for 48 h with strains of RSV from each of the antigenic
subtypes. Control: uninfected fibroblasts.
Table 1. Origin, antigenic subtype and titre of R S V strains examined
Origin
~"
r
Strain
Long
Randall
8/60
18537
RSN-2
Bovine 127'
A2
RSS-2
Year
1956
1958
1960
1962
1972
1972
1961
1980
•
Country
U.S.A.
U.S.A.
Sweden
U.S.A.
U.K.
U.K.
Australia
U.K.
Classification
(Anderson et at., 1985)
Group 1
Group 2
t
Group 3
t
Titre
(logtop.f.u./ml)
:~
5.5
6.9
6.7
6.6
7.3
6.9
7.1
* The bovine 127 strain was assayed on calf kidney cells (Stott et al., 1984b); all other strains were assayed as
described in Methods.
* Not assigned by Anderson et aL (see Results).
:~Long strain used only in fixed infected cells in this study (see Methods).
were not lysed when used in a cytotoxicity assay at this time. These target cells were therefore
incubated for 5 or 6 days before use in the cytotoxicity assay, when the cells showed extensive
c.p.e. Fig. 3 shows that these targets were then recognized and lysed by Tc from mice inoculated
i.n. with the A2 strain; Tc raised against the 8/60 strain lysed these targets with equal efficiency
(not shown).
Priming for memory Tc by glutaraldehyde-fixed, infected cells
That Tc from mice immunized with Long strain RSV recognized the A2 strain is shown in
Fig. 4. These mice were given glutaraldehyde-fixed BCH4 cells i.p. as described under Methods.
Their spleens were removed 3 weeks after the second dose, and the spleen cells re-stimulated in
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626
C. R. M. BANGHAM AND B. A. ASKONAS
40
i
,
i
i
5O
iI
I
I
I
I
I
4O
m
"~
m
sts
"~ 30
"~ 20
"i
o
11
10~
I
32
I
16
I
8
E: T ratio
Fig. 3
I
4
Iq
2
0
I
32
I
16
I
8
E: T ratio
Fig. 4
I
4
-""~
Fig. 3. Tc raised against the A2 strain lysed BALB/c fibroblasts infected with the 18537 strain for 6
days.
Fig. 4. Two doses of glutaraldehyde-fixed, RSV-infected cells primed BALB-c mice for memory Tc
specific to RSV. O, BCH4 target cells (persistently infected with Long strain RSV); O, uninfected
BALB/c fibroblast targets. - - , Effectors from mice given two i.p. doses (see text) of 105 fixed infected
bovine nasal mucosa cells (infected with the bovine 127 strain); - - - , effectors from mice given two
doses of fixed BCH4 cells i.p. In all cases, spleen cells were re-stimulated in vitro with the A2 strain.
vitro with A2 strain RSV; this re-stimulation was, of course, likely to select for Tc cross-reacting
between the Long and A2 strains. Neither a second dose of cells, nor Quil A adjuvant, was
essential to elicit these Tc; this regimen was used because it also induces an effective secondary
antibody response (Stott et al., 1984b). This finding complemented our earlier, converse
observation (Bangham et al., 1985), in which Tc raised against the A2 strain recognized the
Long strain.
Bovine R S V primed for R S V cross-reactive memory To, but failed to form a target for Tc
Fig. 4 also shows that BALB/c mice given two i.p. doses of glutaraldehyde-fixed bovine nasal
mucosa cells, infected before fixation with bovine 127 strain RSV, induced Tc memory cells
which recognized BCH4 target cells (which carry the Long strain of RSV). This induction was
approximately as efficient as induction of Tc by the fixed BCH4 cells. Again, these Tc
precursors were re-stimulated in vitro using the A2 strain: however, 5 days in vitro was too short
for a primary Tc response to RSV to develop, and so this RSV-specific response was evidence of
epitope sharing between human and bovine strains. E. J. Stott (personal communication) groups
the bovine RSV strains with the human 8/60, RSN-2 and 18537 strains, i.e. group 2 of Anderson
et al. (1985). Thus, in these experiments, Tc raised against a group 2 (bovine 127) strain, restimulated by a group 3 strain (A2), recognized cells infected with a group 1 strain (Long).
Despite the cross-reactivity shown above, it was not possible to prime BALB/c mice i.n. with
bovine 127 strain RSV: mice inoculated in this way did not develop any Tc activity against
BCH4 targets (results not illustrated). In addition, BALB/c fibroblasts infected for 2 days in vitro
with the bovine 127 strain, at 33 °C (the permissive temperature for this strain), failed both to
show any c.p.e, and to form a target for Tc raised against the A2 strain (not illustrated).
However, the bovine 127 strain was able to induce Tc which recognized cells infected with
human strains of RSV (Fig. 4). These results therefore demonstrate that, within the limits of this
study, polyclonal anti-RSV Tc do not discriminate between antigenic subtypes of RSV.
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Cross-reaction o f anti-RSV T cells
627
DISCUSSION
The recent studies using monoclonal antibodies to group RSV strains into subtypes (see
Introduction for references) have extended the early observations made with hyperimmune
polyclonal antisera, of antigenic differences between certain strains of RSV. It seems clear that
the main serological differences lie between two groups, group A of Mufson et al. (1985) (groups
1 and 3 of Anderson et al., 1985), and Mufson's group B (group 2 of Anderson et al.). Mufson et
ak used a competition ELISA to identify epitopes on the different virus strains tested, and they
found that the greatest difference between groups A and B was in the G protein, where five of
the six epitopes identified were distinct. They concluded, from the extent of this difference, that
these two antigenic subtypes separated in evolution relatively early, and that G proteins from
representatives of both their groups A and B may be necessary in an effective RSV vaccine.
Anderson et al. (1985) divided the first subtype further, separating the Australian isolates A 1
and A2 on the basis of their reaction with two monoclonal antibodies (one anti-N, one anti-G).
However, they concluded that groups 1 and 3 were antigenically similar, and group 2
antigenically more distinct.
It is important to examine the role of T cell immunity to RSV for two main reasons: first,
because antibody is incompletely protective (see Introduction), and so an effective vaccine may
need to elicit a T cell response; second, because the paradoxically severe RSV infection seen in
some infants who had previously received the formalin-inactivated vaccine (in the 1960s) may
have been due to damage by a cellular effector mechanism (McIntosh & Fishaut, 1980; Karzon,
1983). The characteristics of the T cells elicited by a potential RSV vaccine must therefore be
known.
We have recently shown that RSV-specific Tc cells can be elicited in mice by live or u.v.inactivated RSV (Bangham et al., 1985). Work is in progress to determine whether these Tc cells
limit the replication of RSV in the mouse's lung. In the present paper, we show that mixed
(polyclonal) Tc from mice primed i.n. (and re-stimulated in vitro) with either A2 (group 3) or 8/60
(group 2) strains recognize all of the virus strains tested, including strains from each of the three
antigenic groups. And mice primed with Long strain RSV (in the form of glutaraldehyde-fixed
BCH4 cells) recognized the A2 strain (other viruses not tested here as targets). These
observations parallel those made on (for example) influenza virus, where polyclonal Tc do not
distinguish between the serologically distinct type A strains (Zweerink et al., 1977), and confirm
our previous finding (Bangham et al., 1985) of Tc cross-reaction between different RSV strains,
albeit variable in magnitude (as judged by the extent of lysis in the chromium release assay).
The observed RSV cross-reaction of anti-RSV Tc cells, in the face of considerable antigenic
differences in the G protein (Mufson et al., 1985), suggests that the cross-reactive Tc cells
recognize either a common site on the G protein distinct from these monoclonal antibody
epitopes, or a different, possibly internal viral antigen(s), as appears to be the case in influenza
virus (Townsend & Skehel, 1984), where the nucleoprotein serves as a major cross-reactive
determinant for Tc. Possible target antigens for cross-reactive Tc include the F (fusion) and 22K
proteins of RSV, both of which are associated with the membrane of infected cells. It is probable
that Tc cell clones could be selected which would be specific to certain strains of RSV, but
whether the use of such clones would provide the same subtype classification as that provided by
monoclonal antibodies is a matter for speculation.
Quantitative differences between strains in this recognition (i.e. differences in percentage
specific lysis at a given E : T ratio) do not necessarily imply only partial cross-reaction; since the
virus strains differ in their titre, plaque morphology, c.p.e, and rate of growth (Coates et al.,
1966), the extent of infection or even the pattern of surface antigen expression could vary from
one strain to another. The clearest example of this seen in the present study was the 18537 strain,
which was found to grow slowly both in ferrets (Coates et aL, 1963) and in vitro (Coates et al.,
1966); we observed no c.p.e, until 4 days post-infection, and there was no specific lysis of
BALB/c fibroblast targets infected with this strain for only 48 h, which was sufficient for target
formation for all other human strains examined here.
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628
C. R. M. B A N G H A M A N D B. A. A S K O N A S
There was no simple relation between the titre of virus used to infect targets and the
percentage specific lysis; for example, targets infected with the Randall strain (logio p.f.u, titre
5.5) and those infected with the RSS-2 strain (logto p.f.u, titre 7.1) were lysed to a similar extent
by Tc raised against either the 8/60 strain (Fig. 1) or the A2 strain (Fig. 2). This similarity
suggests a close antigenic relationship between the Randall and RSS-2 strains, but this cannot be
definitely concluded from the present data, for the reasons given above.
The extent of the cross-reaction is emphasized by the experiments (Fig. 4) in which Tc raised
against bovine RSV and re-stimulated with A2 strain (group 3) recognize target cells infected
with the (group 1) Long strain. In this case the bovine RSV, presented on xenogeneic (bovine)
cells, is presumably being processed and re-presented by host antigen-presenting cells, although
attempts to demonstrate such antigen processing in vitro by thioglycollate-induced macrophages
were not successful (results not shown). Stott et al. (1984b) found that these fixed, infected
bovine cells protected calves against RSV infection better than each of two live virus vaccines,
and that protection did not correlate with neutralizing antibody titre. It is possible that more
efficient induction of Tc accounted for the difference in efficacy between these vaccines.
The failure of i.n. inoculation with bovine RSV to induce Tc which recognize targets infected
with human RSV strains, and the reciprocal failure of bovine RSV-treated mouse cells to form
targets for anti-A2 strain Tc, might be due to a difference in virus receptor specificity, which
would preclude infection of mouse cells by the bovine RSV; infection of mouse cells with bovine
strains of RSV has not been demonstrated, either in viva or in vitro.
These results indicate that, as with influenza-specific Tc, polyclonal anti-RSV Tc recognize
different subtypes of the virus.
We should like to thank Drs E. J. Stott and G. Taylor for their continuing generosity and advice. C.R.M.B. is
supported by an MRC Training Fellowship.
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