The Effects of Strain Variation on Respiratory
Syncytial Virus infection and Immunity
13
Sande C1*, Cane P2, Nokes DJ1,3
1.KEMRI/Wellcome
1
KEMRI/W ll
T
Trustt R
Research
hP
Programme, Kilifi K
Kenya 2
2. H
Health
lth P
Protection
t ti
A
Agency, L
London
d
UK 3
3. U
University
i
it off
Warwick Coventry
Warwick,
Coventry,UK
UK
*Corresponding author address: KEMRI/Wellcome Trust Research Programme, P.O. Box 230 – 80108 Kilifi, Kenya.
kemri-wellcome org
Email: csande@kilifi
csande@kilifi.kemri-wellcome.org
Objectives
Introduction
A, A
3 -5
6 - 17
18+
0 -2
3- 5
B, A
6 - 17
log (10)) ND50
0 -2
18+
B, B
6 - 17
18+
Infecting: RSV B(2003
B(2003,2008),
2008) Test: RSV B(2008)
3
2.5
2
1.5
3 -5
6 - 17
18+
0 -2
3- 5
6 - 17
0 -2
18+
3 -5
6 - 17
18+
0 -2
3- 5
6 -17
18+
age (months)
Acute sera
Convalescent sera
Acute sera
Convalescent sera
• Generally,
Generally the neutralising antibody response increases with age
age. Convalescent titres are significantly
higher when the group identity of the test and infecting strain are matched.
Fig 5. Acute/convalescent responses from RSV infections arising in the
2000s. (a) sera from RSV A infections between 2005 and 2006 reacted
against a 2006 RSV A strain (left panel) and an RSV A strain from 1961
(right panel). (b) sera from RSV B infections in 2008 reacted against a
2008 RSV B strain (right panel) as well as an RSV B strain from 1960
(left panel)
•The magnitude of response did not vary despite the
temporal distances between test and infecting strains.
strains
Four fold seroconversion rates in seronegative infants (acute titres < 130)
Discussion
A, B
500 1 ,000 1,50
00
A, A
• We found evidence that RSV
antibodies are strongly group
specific
p
a
and
da
are poo
poorly
y cross
o
reactive.
41
40
33
31
20
18
16
11
9
10
7
6
5
4
2
1
41
40
33
31
20
18
16
11
9
10
8
7
6
5
4
2
1
0
B A
B,
B B
B,
•Supports hypothesis of immune mediated group
replacement
•Suggests future vaccines based on one strain may
alter RSV epidemiology.
epidemiology
41
40
0
33
3
31
20
18
8
16
6
11
7
6
5
4
2
1
41
40
0
33
3
31
20
18
8
16
6
11
10
0
9
8
7
6
5
4
2
1
0
• Despite over 40 years of antigenic
drift, no significant loss of serological
recognition was evident.
age (months)
Notes: ages on X axis represent the mean responses o f all individ uals within that ag e catergor y
panel heading
p
g s represen
p
t the infecting
g group
g p and the test gro
g up
p re spectively
p
y
Acute sera
Convalescent sera
pan els A,A and A,B represent homologous/heterologo us resp onses in the same sampl e set, simil arly for panels B,A and B,B
Fig 2. Seroconversions in seronegative infants/children mainly occurred when the infecting and test groups were matched
Effect of the 20 amino acid duplication on the neutralising response
Neutralisation of a BA strain by sera from infections with RSV Bs with/without duplication
Infecting: RSV B without duplication
Infecting: RSV B with duplication
• The magnitude of the neutralising
35
3.5
response against an RSV B strain
that contained the 20 amino acid
duplication did not differ between
individuals infected with RSV B
strains with or without the
duplication.
3
2.5
2
3 -5
6 -17
18+
0 -2
3-5
6 - 17
Fig 3. Age matched responses against RSV B BA strain (containing the duplication) by
infants/children who were infected by RSV B with/without the duplication
Neutralising antibody responses in the convalescent phase
T t Vi
Test
Virus G
Group
neutrralising titre
e
6
8
10
Infecting group
(conv phase
titres)
(Titre 95% CI)
(Titre,
A
4
B
P value
0
10
20
30
age (months)
40
50
Fig 4.
4 Relationship between age and neutralising titre
A
B
6 93 (6.75
6.93
(6 75-7
7.12)
12)
5 78 (5.57
5.78
(5 57-5
5.99)
99)
6 61(6 0-6
6.61(6.0
6.64)
64)
0.026
• The 20 amino acid duplication did
not result in loss of serological
recognition
g
3852
18+
Relationship between neutralsing antibody titre and age
•Future vaccines may not need periodic updates.
Duration of group/cross specific neutralising
responses in
i primary
i
& secondary
d
infections
i f ti
Acute sera
Convalescent sera
Note: th e test virus in both panel s is an RSV B con taini ng the dup lication
•Suggests
S
antigenic
i
i d
drift
if iis not a mechanism
h i
ffor
immune escape
Ongoing work
•This observation was evident in all
age classes
l
ttested
t d
2
• Neutralising titres were determined using
the Plaque Reduction and Neutralisation Assay
as previously described (Coates,1966).
W adopted
d t d th
d tto countt
• We
the Eli
Elispott reader
plaques which significantly increased
throughput without sacrificing accuracy.
•Neutralising
eut a s g ttitres
t es were
e e calculated
ca cu ated using
us g tthe
e
Spearman-Karber method (Cohen et al,
2007).
2007) Results are expressed as neutralising
dose 50, ND50 (+/- 95% CL)
log (10) N
ND50
Infecting: RSV B (2003
(2003,2008),
2008) Test: RSV B (1960)
Methods
Plaque Reduction &
Microneutralisation
c o eut a sat o Assay
ssay
3- 5
3.5
age (months)
• A nasal specimen and acute blood were
collected from children admitted to KDH
with WHO defined severe or very severe
pneumonia. Samples were screened for
RSV Ag by immunofluorescent antibody
test. Convalescent blood was collected
from RSV positives.
0 -2
Neutralisation of a contemporary RSV B strain (2008) vs RSV B (8/60 - 1960)
Fig 1. Heterologous and homologous neutralising antibody responses across different age classes.
0 -2
Sample set
18+
Acute sera
Convalescent sera
(b)
A, B
Panel headings denote infecting group & test group respectively
At the population level,
immunity to primary
i f ti
infections
will
ill be
b strongly
t
l
group specific.
Responses to contemporary
infections will
ill be poorly
poo l cross
c oss
reactive against
g
older strains.
‰We investigated group & strain
specific antibody responses
following RSV infections in children
aged 0 to 47 months during RSV
epidemics of 2006,
2006 2007
((dominated by
y RSV A)) and
2003,2008 (dominated by RSV B)
in Kilifi Dist
District
ict Hospital (KDH)
(KDH), a
rural hospital on the Kenyan coast.
6 - 17
age (months)
log (10) RSV ND50
‰
3 -5
age (months)
3.5
3
2.5
2
1.5
0 -2
‰ We
W h
hypothesized:
th i d
‰
0 -2
500
0 1,000 1,500
‰
1.5
3.5
3
5
3
2.5
2
1.5
RSV
V ND5 0
‰
2.5
Homologous & heterologous neutralising antibody responses
Antigenic drift within
attachment and Fusion
proteins
There are underlying
mechanisms through which
temporal selection pressures
confer a transmission
advantage to one group
relative to the other at a
particular point in time (White
ett al,
l 05)
05).
Decline & extinction of strains
is driven by immune pressure
A ti
Antigenic
i d
drift
ift might
i ht b
be driven
d i
by
y immunity
y
35
3.5
RSV group specific neutralising antibody response
Cyclical alterations in
dominance between its two
main antigenic groups,
groups A and
B.
Emergence and extinction of
within-group
within
group strains &
periodically global dominance
off recently emergent strains
‰This suggests that :
‰
Infecting: 2006 RSV A strains, Test: A2
Results
(eg a recent 20amino acid duplication in
the G protein of RSV B isolates that has
attained global dominance).
‰
Infecting: 2006 RSV A strains, Tes t: 2006 GA2 strain
2
10
‰
Neutralisation of a contemporary RSV A strain (2006) vs A2 (1961)
3. Determine the serological effect of the recent 20 amino acid
3
duplication
log (1
10) ND50
‰
(a)
3
8
‰ RSV epidemiology
id
i l
iis
characterized by:
Effect of over 40 years of antigenic
drift on the neutralising
g response
p
2.Determine cross reactivity
y between RSV A/B
/ strains from the
2000s with RSV A/B strains from the 1960s.
9
‰ Neutralising antibodies are
directed at two variable surface
expressed
p
g
glycoproteins,
y p
, G and F.
1. Quantify homologous and heterologous group neutralising
antibody responses following primary and subsequent infection
and estimate cross neutralisation titres
8
g
‰ RSV re-infects throughout
life
despite the development of an
effective
ff ti
b
butt ttransient
i t neutralizing
t li i
antibody response.
response
Results
6 28 (6.11
6.28
(6 11-7
7.1)
1)
0.003
Table 1. Age adjusted homologous and heterologous
neutralising antibody titres (titres expressed as natural log)
• Age adjusted homologous antibody titres were significantly higher than the heterologous titres in the
convalescent phase (Table 1). Analysis of children >5 months (to remove influence of maternal
antibody) showed (a) homologous group A titres to be significantly higher than heterologous titres
against
i
RSV A ((p<0.05)
0 05) and
d (b) h
homologous
l
group B titres
i
to b
be higher
hi h than
h
heterologous
h
l
titres
i
((p
value <0.005).
• Measurement of RSV
A and B neutralising
antibodies in a cohort
of infants recruited at
birth and followed up to
2 years of age
•The relative
magnitude of RSV A
and RSV B specific
antibodies at all time
points within during
follow up
p will be tested
•The relative duration
of these responses
following an antigen
confirmed
fi
d iinfection
f ti
primary and
subsequent infections
will be measured
•Some preliminary data
(for RSV A specific
antibodies only) are
shown on the right.
g
RSV A Neut ralising ant ibody t it re
1200
1000
800
600
400
200
0
A
A2 ELISA ant ibody
4.3
3.8
33
3.3
2.8
2.3
1.8
1.3
A
0
3
6
9
12 15 18 21 24 27 30
age (mo nths)
3866
3500
3000
2500
2000
1500
1000
500
0
4
35
3.5
A
3
25
2.5
2
15
1.5
0
3
6
9
12 15 18 21 24 27 30 33
5034
1000
3.3
800
2.8
A
600
2.3
400
1.8
200
0
1.3
0
5
10
15
20
25
Note:
o e red
ed a
arrow
o indicates
d a es a
an a
antigen
ge confirmed
o
ed infection.
e o
ACKNOWLEDGMENTS
RSV research group-kilifi, parents/guardians of infants who
donated samples, KEMRI/Wellcome Trust Research Programme,
The Wellcome Trust for generous support (Grant refs 083085
(PhD Studentship) and 084633 (Programme Grant).
30