Age Affects Quantity but Not Quality of Antibody
Responses after Vaccination with an Inactivated
Flavivirus Vaccine against Tick-Borne Encephalitis
Karin Stiasny1*, Judith H. Aberle1, Michael Keller2, Beatrix Grubeck-Loebenstein2, Franz X. Heinz1
1Department of Virology, Medical University of Vienna, Vienna, Austria, 2Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria
The impairment of immune functions in the elderly (immunosenescence) results in post-vaccination antibody titers that are
significantly lower than in young individuals. It is, however, a controversial question whether also the quality of antibodies
declines with age. In this study, we have therefore investigated the age-dependence of functional characteristics of
antibody responses induced by vaccination with an inactivated flavivirus vaccine against tick-borne encephalitis (TBE). For
this purpose, we quantified TBE virus-specific IgG and neutralizing antibody titers in post-vaccination sera from groups of
young and elderly healthy adults and determined antibody avidities and NT/ELISA titer ratios (functional activity). In contrast
to the quantitative impairment of antibody production in the elderly, we found no age-related differences in the avidity and
functional activity of antibodies induced by vaccination, which also appeared to be independent of the age at primary
immunization. There was no correlation between antibody avidity and NT/ELISA ratios suggesting that additional factors
affect the quality of polyclonal responses, independent of age. Our work indicates that healthy elderly people are able to
produce antibodies in response to vaccination with similar avidity and functional activity as young individuals, albeit at
Citation: Stiasny K, Aberle JH, Keller M, Grubeck-Loebenstein B, Heinz FX (2012) Age Affects Quantity but Not Quality of Antibody Responses after Vaccination
with an Inactivated Flavivirus Vaccine against Tick-Borne Encephalitis. PLoS ONE 7(3): e34145. doi:10.1371/journal.pone.0034145
Editor: Suryaprakash Sambhara, Centers for Disease Control and Prevention, United States of America
Received December 7, 2011; Accepted February 22, 2012; Published March 26, 2012
Copyright: ? 2012 Stiasny et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by intramural funds of the Medical University of Vienna. No external funding was received for this study. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: Franz X. Heinz is an inventor of patents on flavivirus vaccines
and has consulted Baxter for developing tick-borne encephalitis vaccines. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data
* E-mail: email@example.com
The effects of aging on the immune system – commonly termed
‘immunosenescence’ – are multifaceted and impair both innate
and adaptive responses [1,2,3,4]. Together, these defects lead to
suboptimal immune reactions, associated with an increased
susceptibility to infectious diseases and a lower vaccine-effective-
ness in the elderly [5,6,7]. In many instances, quantitative
measurements of serum antibodies – ideally by the use of
functional assays such as virus neutralization or bacterial
opsonophagocytosis – provide excellent correlates of protection
induced by vaccination  and there is overwhelming evidence
from a large body of immunization studies – both in animals and
in humans – that the amount of antibodies induced is strongly
reduced and that titers decline more rapidly in older compared to
younger individuals [5,9]. Factors such as deficiencies in T-cell
help, diminished germinal center reactions, intrinsic B cell defects,
reduced support for long-lived plasma cells in the bone marrow
and reduced numbers of total and specific memory B cells all
contribute to the quantitative impairment of Ig production in the
elderly [9,10,11,12,13]. However, data on the effects of aging on
antibody avidity and the underlying processes of somatic
hypermutation, affinity maturation and the selection of high-
affinity clones are conflicting [7,10,14]. Since these processes can
have a profound impact on the functionality of the antibodies
induced in the course of active immunization, we have investigated
the effect of age on the quality of antibody responses after
vaccination with an inactivated tick-borne encephalitis (TBE)
vaccine. For this purpose, we performed a quantitative analysis of
avidities and functional activities of TBE virus (TBEV)-specific
antibodies in post-vaccination sera from cohorts of different age
TBEV is closely related to yellow fever, dengue, West Nile and
Japanese encephalitis virus  and represents a significant public
health problem in large parts of Europe and Asia . Like with
other encephalitogenic flaviviruses, a functional humoral immune
response is critically important in controlling infections  and
passively transferred antibodies are fully protective against TBE in
an animal model . In humans, the disease can be effectively
prevented by vaccination with a formalin-inactivated purified
whole-virus vaccine that is in widespread use in countries of
Europe and Russia [16,19]. The immunization schedule consists
of a primary vaccination (two doses at an interval of about one
month), a third vaccination after one year and further booster
vaccinations recommended at varying intervals in different
countries . Consistent with the general decline of immune
functions , previous studies had revealed a significantly lower
antibody response in people more than 60 years of age compared
to that of younger individuals (,30 years) [20,21]. Subsequent
studies, however, have shown that the antibody response in people
PLoS ONE | www.plosone.org1 March 2012 | Volume 7 | Issue 3 | e34145
between 50 to 60 years old was already as impaired as in people
.60 years of age .
Despite a strong quantitative impairment of antibody responses
in healthy elderly adults our study shows that there is no significant
age-dependent difference in the avidities and in the ratios of
neutralizing to ELISA-binding activities (functional activities) of
antibodies induced by TBE vaccination. These factors were also
not affected by the age at primary immunization. Surprisingly,
antibody avidity did not correlate with functional activity in both
young and old. This ratio, however, was highly variable from
person to person and - at least to a certain extent - appears to be
an imprinted individual trait, regardless of age.
Materials and Methods
Two panels of serum samples obtained after vaccination with an
inactivated whole-virus vaccine (FSME-ImmunH 0.5 ml, Baxter)
were used in our study: Panel A was derived from a booster
immunization study in young and old healthy adults conducted at
the Institute of Biomedical Aging Research, Austrian Academy of
Sciences, in Innsbruck and consisted of 79 pre- and post-booster
vaccination sera used previously for determining antibody titers
 as well as 29 additional sera of the same cohort. In total, we
therefore had 108 sera in the age groups of ,30 (n=21) and .50
(n=87; 50–60 years: n=22; 60–70 years: n=30; .69 years:
n=35) for functional analyses. All vaccinees had a history of a
completed basic immunization and varying numbers of booster
vaccinations. Serum was collected prior to booster vaccination and
28 days later. Panel B was derived from a primary immunization
study in young and old healthy adults conducted at the
Department of Virology, Medical University of Vienna and
consisted of sera from 33 individuals in the age groups of ,30
(n=11) and .60 (n=21). Serum was collected 28 days after the
basic immunization and after the first booster immunization.
In both studies, participants had no clinically significant
diseases, acute infections or health conditions known to affect
immune responses and were not under immunosuppressive
The studies were approved by the ethics committees of the
Medical University of Innsbruck and of the Medical University of
Vienna, respectively, and the participants gave their written
TBEV-specific IgG antibodies were analyzed by ELISA using
purified formalin-inactivated TBEV as described previously .
Sera were analyzed in duplicates and quantified in VIE Units
using a standard human anti-TBEV serum arbitrarily set at 1000
VIE Units. ELISA values above 155 VIE Units were considered
Neutralization tests (NTs) were carried out in baby hamster
kidney cells (ATCC BHK-21) as described previously . Serial
dilutions of polyclonal sera (in duplicates) were mixed with 25 pfu
virus and incubated for 1 h at 37uC. Cells were added and
incubation was continued for 3 days. The presence of virus in the
supernatant was assessed by ELISA . Titers were determined
after curve-fitting using a four-parameter logistic regression and a
cut-off of 50% reduction of the ELISA absorbance in the absence
of antibody. NT titers $10 were considered positive.
Avidities of TBEV-specific IgG antibodies were analyzed in an
ELISA using purified formalin-inactivated TBEV as described
previously . Sera were tested in tenfold serial dilutions,
including a wash step with or without 8 M urea in PBS pH 7.4
after serum incubation with antigen. Titers were determined at an
absorbance value of 1.0 (490 nm) after curve-fitting using a four-
parameter logistic regression (GraphPad Prism 5; GrapPad
Software Inc.). The avidity of each serum was calculated with
the following formula: Avidity (%)=(titer at absorbance 1.0 plus
urea/titer at absorbance 1.0 without urea)6100. Three indepen-
dent experiments were performed for each serum to calculate
mean avidities. A post infection serum with low avidity was used as
a control in each assay.
Statistical analyses were performed with GraphPad Prism 5
(GraphPad Software Inc.). Logarithmic transformation of the data
was carried out to obtain approximate normal distribution of
antibody concentrations, avidity values and NT/ELISA ratios. A
sample size of 17 volunteers in each group provides a power of
80% (alpha 0.05) to detect a 10% difference in avidities as well as a
20% difference in NT/ELISA ratios following the formula of
Dallal for two-sided statistical tests . Two-tailed t-tests or one-
way ANOVA (Tukey post test) were applied to the transformed
data for significance testing and correlation coefficients were
determined with the Pearson correlation test. P values,0.05 were
regarded as statistically significant.
Similar IgG antibody avidities in young and elderly
To investigate potential age-dependent differences in the quality
and functional activity of antibodies induced by vaccination, we
first analyzed a serum panel from a TBE booster vaccination study
conducted in young and old adults (panel A, Materials and
Methods) with no underlying diseases or impairment by any
immunosuppressive treatments. Consistent with published data
, pre-and post-booster antibody concentrations as well as
neutralizing antibody titers were significantly lower among those
aged .50 years (range: 51–87 years of age) than in the young
control group aged ,30 years (Figure S1).
Avidity is considered to be a crucial parameter of the quality
and functionality of antibodies [17,25] and reports on its age-
dependent impairment are controversial [7,14]. We therefore
determined the relative avidities of post-booster sera using an IgG
ELISA combined with a urea wash step (Materials and Methods).
As shown in Figure 1A, the avidities ranged from ca. 40 to 90%
which are characteristic values for a booster antibody response and
higher than those observed in patients with a recent TBEV
infection (mean avidity 20%) . With respect to the mean
avidities, no statistically significant difference was observed
between the two age groups (unpaired t-test; P value=0.28). In
addition, we specifically compared the avidities of the oldest
vaccinees (.69 years) to those ,30 years of age and also in that
case no statistically significant difference was found between the
two groups (unpaired t-test; P value=0.26; data not shown).
To assess a possible influence of i) the time period before booster
and ii) the age at primary immunization on IgG avidities, elderly
subjects (range: 51 to 87 years of age at booster vaccination) were
stratified by the interval since the last TBE vaccination (#5 and
.5years) (Figure 1B) as well as the age at primary immunization
(,40, 40–50, 50–60, .60 years) (Figure 1C). As can be seen from
Age and Post-Vaccination Antibody Quality
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the figures, neither the time point of the last vaccination (ANOVA;
P value=0.71) nor the age at primary immunization had an
impact on IgG avidities (ANOVA; P value=82).
Similar functional activities of antibodies in young and
The key quality of protective antiviral antibodies is their
capacity to bind to epitopes involved in virus neutralization.
Infection and vaccination, however, can induce substantial
amounts of antibodies that are measurable in in vitro binding
assays (such as ELISA) but do not contribute to virus neutraliza-
tion and protection [26,27]. To evaluate the functional quality of
all of the virus-reactive antibodies induced by vaccination and to
compare this parameter between the young and the elderly, we
determined the ratios of neutralizing antibody titers (measured in
NT) versus total TBEV-specific ELISA binding IgG antibodies.
Similar to what has been demonstrated for antibody avidity, no
significant differences were detected in the NT/ELISA ratios
between the two age groups (Figure 2A) (unpaired t-test; P
value=0.25) and there was no significant difference between the
oldest group (.69 years of age) and those of ,30 years of age
(unpaired t-test; P=0.42; data not shown). Also, neither the time
point of the last vaccination before the booster (Figure 2B)
(ANOVA; P value=0.61) nor the age at primary immunization
(Figure 2C) (ANOVA; P value=0.29) had an influence on the
NT/ELISA ratios suggesting that the overall functionality of
antibodies is not affected by the age of the vaccinee.
Although age at primary immunization did not appear to
influence the quality of antibody responses, we analyzed whether it
had an effect on the amount of booster-induced TBEV
neutralizing antibodies. As shown in Figure 3, the antibody titers
of the elderly booster groups were similar and no statistical
difference was found (ANOVA; Tukey post hoc test; P
values.0.05). The titers of the elderly, however, were significantly
reduced in comparison to those of the young control group
(ANOVA; Tukey post hoc test; P values,0.05).
No correlation between antibody avidities and functional
quality of antibodies
We could not detect differences in the mean IgG avidities and
NT/ELISA ratios between the young and elderly, but some
degree of variation of both parameters was observed in both age
groups (Figures 1 and 2). Since affinity maturation is regarded as
essential for the generation of highly functional antibodies , we
would have expected co-variation of the two parameters.
Surprisingly, however, analysis of the data did not reveal any
significant correlation (Figure S2; Pearson correlation cofefficients
r=0.06 and r=20.14 in the young and elderly, respectively),
suggesting that other factors in addition to avidity are major
determinants of functional antibody activity in this case.
Tendency of individual imprinting of functional antibody
The strong variation of functional antibody activities (NT/
ELISA ratios) observed (Table 1) raised the question whether this
trait has the potential to change upon revaccination or has a
tendency for individual imprinting. In both age groups, a positive
correlation between the pre- and post-booster NT/ELISA ratios
was found, indicating that this functional parameter is maintained
upon booster vaccination at least to a certain extent (Figure 4A,B).
To determine whether such a phenomenon can already be
detected after primary immunization, we analyzed a second
panel of sera from a recent TBE immunization study in which the
Figure 1. Avidity of TBEV-specific IgG antibodies of young
(, ,30) and elderly (. .50: range 51–87 years of age) TBE
vaccinees after booster immunization. Avidities were determined
by ELISA after a urea-washing step and expressed as percent of the
values obtained in the absence of urea. (A) Groups were based on age
at booster vaccination. (B) Groups were based on age at booster
vaccination (young, elderly) and on the time interval to the last
vaccination before booster. (C) Groups were based on age at booster
vaccination (young, elderly) and the elderly vaccinees were further
stratified according to their age at primary vaccination which was
known for 76 of 108 samples. Data represent mean values +/295%
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age-dependence of the immune response after primary vaccination
(2 doses) and the first booster vaccination (3rd vaccination) was
investigated (panel B, Materials and Methods). Similar to what was
seen after booster vaccinations, the comparison of NT/ELISA
ratios of sera collected 4 weeks after basic immunization and 4
weeks after the third immunization yielded a positive correlation in
the young and elderly groups (Figure 4C,D).
With both serum panels, the correlation in the elderly was
slightly stronger and – in contrast to the young – significant. Since,
especially in the primary immunization study (Figure 3C), fewer
sera of young vaccinees were available for correlation analysis, we
combined the data of the young of both studies (Figure 4A,C).
Again a positive association was observed and the Pearson
coefficient remained in the same range (r=0.39), but now the
correlation was significant (P value=0.03).
Central to our work was the question whether the quality of
antibody responses to an inactivated whole virus vaccine is
impaired in healthy elderly. The data presented demonstrate that
neither the avidity of the polyclonal antibodies present in TBE
post-vaccination sera nor their functional activity (defined as ratio
of neutralizing vs. virion binding antibodies) displayed any
statistically significant differences in healthy young and old
individuals. With respect to avidities, all sera from both age
groups had low avidities consistent with values characteristic of
robust anamnestic immune responses . The NT/ELISA
ratios, however, exhibited strong age-independent individual
variation and it might be possible that subtle differences between
age groups could go unnoticed due to the relatively small sample
size of some groups used in the analyses. The unimpaired quality
of antibodies is in clear contrast to the well-established fact –
reconfirmed in our study (Figure S1; Figure 3) – that the quantity
Figure 2. Functional activities of TBEV-specific IgG antibodies
of young (, ,30) and elderly (. .50: range 51–87 years of age)
TBE vaccinees after booster immunization. The functional activity
was measured by calculating the ratio of neutralizing antibodies to
ELISA binding IgG antibodies according to the following formula: (NT50
titer/ELISA IgG units)6100. (A) Groups were based on age at booster
vaccination. (B) Groups were based on age at booster vaccination
(young, elderly) and on the time interval to the last vaccination before
booster. (C) Groups were based on age at booster vaccination (young,
elderly) and the elderly vaccinees were further stratified according to
their age at primary vaccination which was known for 76 of 108
samples. Data represent mean values +/295% confidence intervals.
Figure 3. TBEV-specific neutralizing antibody titers of young
(, ,30) and elderly (. .50: range 51–87 years of age) TBE
vaccinees after booster immunization. Groups were based on
age at booster vaccination (young, elderly) and the elderly vaccinees
were further stratified according to their age at primary vaccination
which was known for 76 of 108 samples. Data represent mean values +/
295% confidence intervals. Statistically significant differences between
pairs of groups (P values of ,0.05) are indicated by lines and asterisks
(ANOVA; Tukey post test).
Age and Post-Vaccination Antibody Quality
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of post-vaccination antibodies produced in the elderly is severely
diminished [5,6,28]. Even when primary immunization occurred
at a younger age, the antibody titers of elderly vaccinees were
significantly reduced compared to those of the young control
In the case of TBE vaccination, this impairment was already
found for the age group of 50 to 60 year old individuals . In a
recent study with the same vaccine, it was further shown that not
only the number of antigen-specific memory B cells induced by
primary immunization was about 3-fold lower in the elderly but
also the antibody response per memory B cell after revaccination
was strongly reduced . These impairments were associated
with profound functional defects in antigen-specific CD4+helper T
In addition to quantity, however, it is the quality of antibodies
that matters and high avidity binding to the pathogen is
considered to be essential for antibody effector functions [17,25].
Data on the influence of age on affinity maturation are conflicting
[7,14,29,30] and our analyses of post-vaccination sera suggest that
the elderly – despite their overall impairment of immune functions
– are still able to produce high-affinity antibodies that have similar
functional activities as those produced in young age. These
findings are fully congruent with a recent study that analyzed the
avidities of plasmablast-derived antibody-responses to seasonal
influenza vaccination in young and elderly adults . Although
the frequency of vaccine-specific plasmablasts and the amount of
antibodies produced by these cells in vitro were lower in the
elderly, neither the yields of secreted IgG per plasmablast nor the
avidity of these antibodies was different in the two age groups. The
annual influenza infections/vaccinations with a variety of subtypes
and strains, however, make it difficult to differentiate between
primary and booster responses, and exposure history as well as
original antigenic sin phenomena can influence the results
obtained [31,32]. Our data, in contrast, are not confounded by
such problems of antigenic shift and drift and demonstrate in a
non-variable single antigen system that the quality of vaccine-
Table 1. Variation of NT/ELISA ratios within groups of young
and elderly TBE vaccinees.
Age groupsn Range of NT/ELISA ratios
,30 years21 3.5–12.432.5
.50 years87 1.5–20.754
Figure 4. Correlation of NT/ELISA ratios pre- and post-TBE booster vaccination (A,B) as well as after primary and first booster
vaccination (C,D) in the young (A,C) and elderly (B,D). The Pearson correlation coefficients r and the P values are indicated.
Age and Post-Vaccination Antibody Quality
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induced antibodies is not diminished in elderly vaccinees and even
appears to be unaffected when priming occurs at an advanced age.
It is a surprising and intriguing finding of our work that there
was no correlation between antibody avidity and functional
activity of vaccine-induced antibodies, a trait that was character-
istic for both the young and the old age groups. Considering the
importance of high-avidity for antibody effector functions [17,25],
we have to conclude that there are other factors that contribute to
their functional activity. These factors may be related to individual
differences in the antibody subsets of polyclonal immune responses
measured in NT and ELISA. For flaviviruses there is evidence that
the most potent virus-neutralizing antibodies are primarily
directed to one of the three structural domains (Domain III) of
the viral envelope protein E whereas those to other domains are
less effective . In the context of NT/ELISA ratios, it is also
important to note that the ELISA – due to partial denaturation of
the antigen during coating – detects antibodies to antigenic sites
that are not exposed at the surface of infectious virions and
therefore do not contribute to virus neutralization [26,27,34].
Nevertheless, such antibodies can have a profound effect on the
ratio of neutralizing to ELISA binding antibodies in individual
sera. The importance of these considerations is underscored by
studies with monoclonal antibodies obtained after measles and
influenza vaccination which revealed that only a relatively small
subset of the antibodies reactive in ELISA was able to neutralize
the viruses . Therefore, independent of antibody avidity, the
composition of antibody populations in individual sera and their
varying specificities with subsets of antigenic sites may have a
significant impact on their neutralizing activity.
Although our analyses did not reveal an overall difference in the
avidities and functional activities (NT/ELISA ratios) of post-
vaccination antibodies in young and old, we observed a
remarkable degree of individual variation of the ratios in both
age groups. We interpret these findings as evidence for variations
in the composition of antibody subsets as well as their specificities
and functionalities between different individuals. Most interest-
ingly, the functional quality of antibody responses appears to be
imprinted individually, at least to a certain extent, since we found
a positive correlation between the NT/ELISA ratios before and
after booster vaccination and also between primary immunization
and first booster vaccination. This suggests a tendency for
maintaining a ‘specific antibody profile established during initial
priming of the immune response. We have recently shown in a
related flavivirus model that certain prime-boost regimens can
modulate the ratio of post-vaccination antibody specificities and
increase the amount of highly functional antibodies .
Specifically designed studies will be necessary to find out to which
extent the individual variation of vaccine-induced antibody
specificities contributes to the variation in NT/ELISA ratios and
possibly to the protectiveness of the antibody response.
antibody titers (B) of young (,30) and elderly (.50: range 51–87
years of age) TBE vaccinees. Dotted bars: values obtained before
TBE booster immunization; white bars: values obtained 4 weeks
after TBE booster vaccination. IgG antibodies were determined by
ELISA and the functional activity by neutralization tests, using
50% virus neutralization as a cut-off (NT50). Results are expressed
as geometric mean values +/295% confidence intervals. The
statistical analyses are given on top of each panel (unpaired t-tests)
and P values of ,0.05 are significant.
Mean TBEV-specific IgG units (A) and neutralizing
TBE booster vaccination in young (A) and elderly (B) individuals.
The Pearson correlation coefficients r and the P values are
Correlation of avidity and NT/ELISA ratio after
We thank Cornelia Stoeckl and Jutta Hutecek for their excellent technical
assistance throughout the course of this work and Walter Holzer for help
with virus production and inactivation.
Conceived and designed the experiments: KS FXH. Performed the
experiments: KS JA MK BGL. Analyzed the data: KS JA FXH.
Contributed reagents/materials/analysis tools: JA MK BGL. Wrote the
paper: KS FXH.
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Age and Post-Vaccination Antibody Quality
PLoS ONE | www.plosone.org7 March 2012 | Volume 7 | Issue 3 | e34145