RSV is a major cause of respiratory disease in infants and older adults. This study examined B cell correlates of protection against RSV infection in adults experimentally infected with RSV. The study found that: 1) RSV-specific nasal IgA levels correlated strongly with protection from RSV infection, while serum neutralizing antibody levels did not. 2) Antibody-secreting cells induced by RSV infection correlated with increased antibody levels but these levels declined rapidly after infection. 3) Antibody levels against RSV returned to baseline within 6 months, suggesting that strategies to enhance B cell memory against RSV may be needed.

1. Quality of antigen-specific B cell responses as a correlate
of protection against respiratory syncytial virus infection
Laura Medrano González, Maximillian S. Habibi, Agnieszka Jozwik, Spyridon Makris, Allan Paras,
John P. DeVincenzo, Cornelis A.M. de Haan, Peter J. M. Openshaw and Christopher Chiu
Centre for Respiratory Infection, National Heart and Lung Institute, Imperial College London
Respiratory syncytial virus (RSV) is the most important cause of childhood
hospitalisation due to respiratory disease. Individuals are susceptible to re-
infection throughout life and RSV is a frequent cause of bronchiolitis and severe
respiratory tract disease, especially in infants and older adults. Although
potential vaccines against RSV have been studied for the last five decades,
there is still no vaccine available nor any effective specific treatment. B cells are
responsible for humoral immune responses and are the most well-understood
determinants of protective immunity induced by vaccines. However, it remains
unclear why RSV re-infects people throughout life even in the context of a
healthy mature immune system and the contribution of antibody in protection
against infection has still not been precisely defined. Using an experimental
human infection model, in which healthy adult volunteers were inoculated with
live RSV, we examined B cell correlates of protection against RSV infection.
INTRODUCTION
RESULTS
RSV-specific nasal IgA correlates significantly better with protection from infection than
serum neutralisation but virus-specific antibody is remarkably short-lived after human
experimental infection. We hypothesise that RSV vaccines designed to induce local IgA as
well as systemic antibodies will have greater efficacy. However, the short-lived nature of
anti-RSV antibodies even following natural infection implies that strategies to enhance B
cell memory may be required.
CONCLUSIONS
Figure 5. RSV-specific antibodies return to steady-state levels rapidly after convalescence. Serum
neutralizing antibody (a) was determined by plaque reduction neutralization and nasal IgA by ELISA (b). In
the majority of cases, both serum and nasal antibody levels increased immediately following infection but
were not maintained by 6 months post-finection
Viral
inocula+on
n=
61
44%
uninfected
(qPCR
undetectable)
n
=
27
56%
infected
(culture
OR
qPCR
posi+ve
on
>
2
consecu+ve
days)
n
=
34
32%
no
symptoms
68%
common
cold
symptoms
Figure 1. Clinical data. (a) Intranasal inoculation in 61 healthy adult volunteers with RSV A
Memphis 37 resulted in a 56% infection rate as determined by qPCR. RSV detection in nasal
washes was achieved by quantitative PCR and subjects were deemed infected if PCR+ on ≥2
consecutive days. (b) There was no difference in age, sex or ethnicity between outcomes.
Antibody-secreting cells by flow cytometry
0 7 14 21 28
0
2
4
6
8
Day post-inoculation
Antibodysecretingcells
(%ofCD3-CD20-lymphocytes)
Day post-inoculation
ASCfrequency(SFU/106
PBMCs)
0
200
400
600
800
0 3 7 10 14 28
Uninfected
0 3 7 10 14 28 0 3 7 10 14 28
0
200
400
600
800
RSV-IgG
Infected
RSV-IgA RSV-IgM
RSV+
ASC
Ig+
ASC
Day 0 Day 7 Day 10 Day 14 Day 28
Figure 4. Plasmablasts are responsible for the post-infection increments in anti-RSV antibodies. (a &
b) An increased % of plasmablasts were observed by flow cuytometry around day 10 post-inoculation from
infected PBMCs. These are short-lived, decreasing rapidly after day 10 post-inoculation. (c) Ig-producing and
RSV-specific ASCs were observed by to peak at day 10 post-infection. (d) The ASC response is dominated
by IgA- and IgG-producing cells with minimal IgM response, indicative of a recall response. (e) Peak IgA+
ASCs correlate with fold-increase in nasal RSV-specific IgA, suggesting that these cells are the source.
Uninfected
(n = 27)
Infected
(n = 34)
Median (range)
age (years)
23
(18-39)
21
(18-50)
Gender (M:F) 17:10 18:16
Ethnicity
White 21 28
Black 4 1
Asian 0 2
Mixed 2 1
Other 0
KEY POINTS
• Despite moderately high levels of virus-specific antibodies, ~60% of
adults were susceptible to RSV infection on challenge
• Virus-specific serum antibody levels did not correlate with protection
against RSV infection
• In contrast, mucosal IgA in the upper respiratory tract correlated
strongly with protection and could be used to predict likelihood of
infection
• Virus-specific B cells are induced by RSV infection and correlate with
rise in antibody levels
• Antibody levels against RSV fall to baseline levels within 6 months,
predisposing to recurrent infection
ACKNOWLEDGEMENTS
This work was founded by the Medical Research Council and Wellcome Trust
a)
c)
b)
d)
a)
b)
c)
D
d)
e)
a)
b)
b)
a)
Figure 2. Serum antibody correlates poorly with protection from RSV infection. Serum
was taken at baseline (7-14 days pre-inoculation) and 28 days post-inoculation. (a) Serum
neutralising antibody levels were determined by plaque reduction neutralisation in uninfected
(○) vs. infected (●) subjects, 4 infant samples (∆) and 3 reference serum standards (■). (b)
Logistic regression of probability of protection and baseline serum neutralising antibody levels.
(c) Serum RSV IgG and (d) IgA an+body
levels
were
determined
by
ELISA
in
uninfected
(●)
vs.
infected
(○)
subjects.
6 8 10 12 14 16
0.0
0.2
0.4
0.6
0.8
1.0
Nasal anti- RSV- IgA (log2 titre)
Probabilityofprotection
OR = 1.9 (1.2 œ3.4) *
protec%on'to'serum'neutralizing'an%body'
Figure 1: RSV-specific nasal IgA is a superior correlate of protection to serum neutralization titer. (a) Healthy adult
volunteers were inoculated with RSV A M37 via nasal drops and followed up with blood and nasal sampling for 28 days. (b)
RSV detection in nasal washes was achieved by quantitative PCR and subjects were deemed infected if PCR+ on ≥2
consecutive days. Common-cold symptoms were determined by a cumulative modified Jackson score. (c) Serum and
nasal lavage were taken at baseline (7-14 days pre-inoculation) and 28 days post-inoculation. Serum neutralizing antibody
was determined by plaque reduction neutralization and nasal IgA by ELISA. Baseline serum neutralising (top), nasal IgA
against whole RSV (middle), and nasal anti-RSV fusion (F) protein (bottom) antibody levels in uninfected (○) vs. infected
(●) subjects. Serum neutralization includes 4 infant samples (∆) and 3 reference serum standards (■ Wyeth 06937, 06938,
06939, (d) Logistic regression of probability of protection and baseline serum neutralising (top), nasal whole RSV-IgA
(middle), and nasal RSV fusion (F) protein IgA (bottom) antibody levels. Predicted probability (solid line) and pointwise
95% confidence band (shaded) are shown. OR = odds ratio (and 95% confidence interval), P-values for unpaired Mann-
Whitney-Wilcoxon (U) test or Chi-squared test are shown (ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001).
Complete'data'for'61'subjects'(1'subject'is'excluded'
from'nasal'lavage'as'sample'was'too'dilute'to'
measure'anything'(subject'AW'from'2012))'
Figure 1: RSV-specific nasal IgA is a superior correlate of protection to serum neutralization titer. (a) Healthy a
volunteers were inoculated with RSV A M37 via nasal drops and followed up with blood and nasal sampling for 28 day
RSV detection in nasal washes was achieved by quantitative PCR and subjects were deemed infected if PCR+ on ≥2
consecutive days. Common-cold symptoms were determined by a cumulative modified Jackson score. (c) Serum and
nasal lavage were taken at baseline (7-14 days pre-inoculation) and 28 days post-inoculation. Serum neutralizing ant
was determined by plaque reduction neutralization and nasal IgA by ELISA. Baseline serum neutralising (top), nasal I
against whole RSV (middle), and nasal anti-RSV fusion (F) protein (bottom) antibody levels in uninfected (○) vs. infec
(●) subjects. Serum neutralization includes 4 infant samples (∆) and 3 reference serum standards (■ Wyeth 06937, 06
06939, (d) Logistic regression of probability of protection and baseline serum neutralising (top), nasal whole RSV-IgA
(middle), and nasal RSV fusion (F) protein IgA (bottom) antibody levels. Predicted probability (solid line) and pointwis
95% confidence band (shaded) are shown. OR = odds ratio (and 95% confidence interval), P-values for unpaired Man
Whitney-Wilcoxon (U) test or Chi-squared test are shown (ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001).
Complete'data'for'61'subjects'(1'subject'is'excluded'
from'nasal'lavage'as'sample'was'too'dilute'to'
measure'anything'(subject'AW'from'2012))'
b)
a)
c)
d)
Figure 3. Nasal IgA is a direct correlate of protection against RSV
infection. Significant higher (a) anti-RSV IgA and (b) anti-F IgA titres
measured by ELISA were found in uninfected (○) vs. infected (●)
subjects. (c) Logistic regression of probability of protection and baseline
nasal whole RSV-IgA antibody levels. (d) Nasal anti-RSV IgA (log2 titer)
and serum anti-RSV IgA (log2 titer) correlated poorly, indicating
differential production/transport of IgA in the two compartments.