Antibody levels against eleven Staphylococcus aureus antigens in a
healthy population
Patricia Colque-Navarro1, Gunnar Jacobsson2, Rune Andersson3, Jan-Ingmar Flock1, Roland
Möllby1
1
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm,
Sweden
2
Department for Infectious Diseases and 3Research and Development Centre, Skaraborg
Hospital, Skövde
Index words: Staphylococcus aureus, antibody levels, colonization, serology
Corresponding author:
Patricia Colque-Navarro
Karolinska Institutet
Department of Microbiology, Tumor and Cell Biology
Box 280
SE 171 77 Stockholm
Sweden
bodies than others, and certain extracellular proteins were more often inducing high levels in the same individuals.
Abstract
Serum samples from 151 healthy individuals aged from 15 to 89 y of age were
investigated regarding IgG levels against
11 different purified antigens from
Staphylococcus aureus by an ELISA that
resulted in quantitative data, which
allowed statistical calculations between
individuals and in relation to nasal
carrier state at the time of sampling.
These findings are important for the
development of improved serological
diagnostics and constitute an important
knowledge for future monitoring of invasive Staphylococcus aureus infections.
There was a great variation in antibody
levels in both young and elder healthy
individuals. Occurrence of S. aureus in
the nares at time of sampling was correlated to higher antibody levels, and ages
over 65 y showed only slightly lower
levels. Certain individuals were more
prone to produce/not to produce anti1
Antibody levels in normal individuals
are stable for years and are functional,
based on neutralizing and opsonocytic
activity (Dryla, Prustomersky et al.
2005).
Patients with complicated S. aureus infections have initially lower levels of
antibodies against some antigens in acute
phase sera in comparison with levels in
the healthy population.(Colque-Navarro,
Palma et al. 2000).
Introduction
Staphylococcus aureus is a pathogen
giving rise to from mild infections in the
skin to deep invasive infections (Lowy
1998). This versatile pathogen is one of
the common causes of both nosocomial
and community aquired infections.
Twenty percent of the population is persistently colonized with S. aureus, and
another 40% are transiently colonized
(Wertheim, Melles et al. 2005)
(Jacobsson, Dashti et al. 2007)( van
Belkum 2009). Treatment is increasingly
more difficult due to the capacity of this
bacterium to develop multidrug resistance (de Lencastre, Oliveira et al.
2007).
The serological diagnosis may contribute
to the choice of treatment of the patient,
e.g. through ascertaining the bacteriological diagnosis, to discriminate between soft tissue and bone infections or
to monitor the progression of the infection. (Persson, Johansson et al. 2009) .
Today S. aureus serology encounters
many problems such as finding the relevant antigens to base the diagnosis on,
the use of different methods and technologies (neutralization, RIA, ELISA,
and Luminex technology), the different
calculation models to express the antibody levels, and uncertainties about the
“normal” antibody levels to compare
with. All these points make the use of
serology difficult in inexperienced
hands. (Colque-Navarro, Soderquist et
al. 1998; Ryding, Espersen et al. 2002;
Verkaik, de Vogel et al. 2009).
The presence of circulating antibodies in
Staphylococcus aureus disease has been
intensively studied. The protective role
of the antibodies, besides the neutralization capacity towards the extracellular
toxins, is still poorly understood. Since
80 % of the patients developing deep
infections are infected with endogenous
strains, an alternative treatment based on
passive and/or active immunoprophylaxis is highly desired. (von Eiff, Becker
et al. 2001)
Individuals carrying S. aureus in the
nose are at higher risk than non-carrying
individuals for developing bacteraemia,
but are at lower risk of S. aureus bacteraemia-related death (Wertheim 2004).
Holtfreter (2006) reported that carriers
neutralize superantigens by antibodies
specific for their colonizing strain, and
this may be is the explanation for the
improved prognosis in severe sepsis for
carriers. It has been demonstrated that
carriers show higher levels of antibodies
against TSST-1, SEA, Clf-A and Clf-B
(Wertheim 2008; Verkaik, de Vogel et
al. 2009).
The aim of this study was to investigate
the antibody levels in a healthy population that agewise mirrors the common
population with invasive S. aureus infections, and to compare the antibody
repertoire between carriers and noncarriers. Possible relevant antigens were
selected and a reproducible ELISA with
calculation methods for quantitative
analysis was chosen. The methods and
the results may be used to improve the
serological diagnosis in clinical practise
2
as well as make up the basis for the development of new immunoprophylactic
and immunotherapeutic tools.
°C. Serum samples diluted in PBS-T
were applied and incubated for 1 h at
37°C; each patient sample was titrated in
4 steps in two-fold dilutions (Table 1).
Alkaline phosphatase conjugated to
monoclonal
mouse
anti-human
antibodies (Sigma Chemical Co., St.
Louis, Mo.) diluted 1/30 000 in PBS-T
was then added, and incubation was
continued for 2 h at 37 °C. After the final
wash, the reaction was developed by the
addition of p-nitrophenylphosphate
substrate (Sigma Chemical Co.). The
enzymatic reaction was measured at
405 nm in a Titertek Multiscan
microplate reader (Flow Laboratories,
Irvine, Scotland) after approximately
20 min incubation. The absorbance
values were transmitted online to a
computer, and calculations were
performed with the Unitcalc software
(PhPlate Stockholm AB, Stockholm,
Sweden).
Material and Methods
Material
Antibody levels against 11 different
antigens were investigated in 151
healthy individuals. The main part of this
material (115 samples) was collected as
reference material (matched ages) in a
prospective study regarding invasive S.
aureus infections (Jacobsson, Dashti et
al. 2007). These individuals attended a
vaccine centre and were screened for
nasal carriage of S. aureus according to
standard laboratory procedures. In order
to compensate the skewed age
distribution of the individuals, we
included another 36 samples from
younger blood donors. The gender
distribution was 90 men, 60 women with
age averages 56 and 50 years. The age
distribution of the total material was 29
% of the ages 15-35 years, 21 % of ages
35-65, and 49 % of ages 65-90 years.
For every two plates, eight twofold
dilutions of a reference serum (Golden
Standard), consisting of pooled sera from
six patients with confirmed S. aureus
sepsis, were included. The first dilution
varied with the respective antigens
(Table 1). Three control sera (two
positive and one negative) were also
included at single dilutions in duplicate
wells to monitor the reaction.
Antibody determination
ELISA
Serum IgG levels were determined by
the enzyme-linked immunosorbent assay
(ELISA) described earlier (ColqueNavarro, Palma et al. 2000) . The
working volume was 100 µl, and after
each step the micro plates were washed
three times with phosphate-buffered
saline (pH 7.4) (PBS) plus 0.05 %
Tween 20 (PBS-T). Briefly, microplates
were coated with the appropriate antigen
diluted in PBS and incubated overnight
at 20 °C. Next day microplates coated
with antigens Clf-B, Eap and Bsp were
blocked with 2 % BSA for one h at 20
Antigens
Eleven highly purified antigens from S.
aureus were used in separately
developed assays.
3
Table 1. Antigen table
Short
Compound
Properties
Coating
conc.
(µg/ml)
Starting serum dilution
in ELISA
1
1/2 500
1
1/250
Prof. JI Flock
1
1/125
Prof. JI Flock
4
1/125
Ass. Prof C Rydén
PhPlate, Stockholm
Supplier
Reference
Surface antigens
Teichoic acid
TA
Clumping factor A
Clf-A
Clumping factor B
Clf-B
Bone sialoproteinbinding protein
Bbp
Ribitol polymer,
native
Cellwall component
Recombinant
protein
Recombinant
protein
Recombinant
protein
Surface-located fibrinogen binding
protein
Surface-located fibrinogen binding
protein
Surface-located protein that binds
bone sialoprotein
PhPlate, Stockholm
(Colque-Navarro,
Soderquist et al. 1998)
(Colque-Navarro, Palma
et al. 2000)
(Verkaik, de Vogel et al.
2009)
(Tung, Guss et al. 2000)
Extra-cellular proteins and toxins
Alpha-toxin
At
Native protein
Hemolytic toxin, cytotoxin
3.5
1/250
Lipase
Lip
Native protein
Hydrolysis of low density
lipoproteins
2
1/2 500
Staphylococcal
enterotoxin-A
SEA
Native protein
Emetic toxin, superantigen
1
1/250
Toxin Technology,
Florida
Toxic shock
syndrom toxin
TSST
Native protein
Pyrogenic-toxin, superantigen
1
1/250
Toxin Technology,
Florida
Scalded skin
syndrom toxin
SSS
Native protein
Exfoliative toxin, epidermolytic
1
1/250
Toxin Technology,
Florida
Efb
Native protein
0.6
1/250
Prof. JI Flock
Eap
Recombinant
protein
1
1/125
Prof. JI Flock
Fibrinogen binding
protein
Extracellular
adherence protein
Extracellular fibrinogen binding
protein, related with wound healing
Interferes with wound healing
mechanisms
4
Prof. S. Tyski
(Colque-Navarro,
Soderquist et al. 1998)
(Tyski, Colque-Navarro
et al. 1991)
(Kanclerski, Soderquist
et al. 1996)
(Kanclerski, Soderquist
et al. 1996)Kanclerski et
al
(Colque-Navarro,
unpublished)
(Colque-Navarro, Palma
et al. 2000)
(Hussain M, Haggar A
2008)
Software, USA) and with Excel,
Microsoft. For comparisons of levels
between individuals the unit values were
normalized into quotients of their
respective mean value, upon which pair
wise correlation coefficients and
clustering according to the UPGMA
method by the PhPWin software was
performed
(PhPlate
Microplate
Techniques AB, Stockholm, Sweden).
Interpretations of ELISA results
into Units (U)
The antibody levels were expressed as
arbitrary units by using the reference line
unit calculation method (Reizenstein,
Hallander et al. 1995). The dilution
curve of each sample (four dilutions)
was adapted into a straight line parallel
to that of the reference serum, after
which the distance, expressed in dilution
steps, between the two lines was
determined. The reference serum was
given the value of 1,000 units (U) and
the antibody levels of the tested serum
were related to these units. E.g., levels of
2,000 U in a sample means that this
sample could be diluted twice to
generate the same dilution curve as the
reference
serum.
(Colque-Navarro,
Palma et al. 2000)
RESULTS
Antibody levels against 11
antigens in healthy individuals.
Staphylococcus aureus develops different virulence factors during various
stages of the infection. The present study
considers antibodies against two main
types of factors: the 11 different antigens
were divided into surface antigens and
extracellular proteins and toxins.
Furthermore, the selected healthy population matched the age of individuals
with deep infections. The distribution of
antibody levels according to age against
the 11 antigens is shown in Figure 1. It is
to be noted that the units are the same on
all y-axes, but different basic serum
dilutions were applied when testing
against different antigens as stated in
Table 1.
Statistical methods
Antibody levels were not normally
distributed, why the non-parametric
method of Mann-Whitney was used.
Comparisons with obtained and expected
numbers of individuals showing high or
low antibody levels were performed with
standard
Chi2-test.
Figures
and
calculations were performed with the
GraphPad Prism software (GraphPad
5
1A
surface antigens
Teichoic acid
Bsp
1000
1000
units
10000
units
10000
100
100
10
10
20
40
60
80
100
20
40
Clf-A
60
80
100
Clf-B
1000
1000
units
10000
units
10000
100
100
10
10
20
40
60
80
100
20
years
40
60
80
100
years
Figure 1
Serum IgG levels against different antigens: 1A shows surface antigens; 1B extracellular
proteins.
Each graphic shows the levels expressed in Units as described in M&M. Circles on the xaxis indicates individuals lacking measurable levels to the respective antigen. A great
variability against different antigens in different ages is observed.
6
1B
extra cellular proteins
alpha-toxin
Lipase
1000
1000
units
10000
units
10000
100
100
10
10
20
40
60
80
100
20
40
100
80
100
TSST-1
10000
10000
1000
1000
100
100
10
10
20
40
60
80
100
20
40
SSS
60
Efb
10000
1000
1000
units
10000
100
100
10
10
20
40
60
80
100
20
years
40
60
years
Eap
10000
1000
units
units
80
units
units
SEA
60
100
10
20
40
60
years
7
80
100
80
100
results were obtained from individuals
above 65 y of age for Clf-B.
Antibody levels in relation to
age
It appears as there were no distinct
differences in antibody levels related to
age. However, mostly the medians were
lower at an increased age; significant
difference was only found for Clumping
factor B. An exception was seen for
antibodies against TST, where the mean
levels were twice as high in individuals
above 65 y compared to those below 65
y. In a number of antibody determinations it was not possible to detect any
antibodies at all; these are marked as
circles on the x-axes. The majority of
these samples originated from subjects
below 65 y of age for ClfA, lipase, SSS
and TST, while most of the negative
Antibody levels in relation to
colonization of the nares
In general individuals that were
colonized with Staphylococcus aureus at
the time of blood sampling showed
higher antibody median levels against all
antigens tested, except for Extracellular
Fibrinogen binding protein (Efb). For
five of the analysed antigens, the
difference was statistically significant
(Table 2). E.g. the antibody levels
against Extracellular adherence protein
were three times higher in healthy individuals colonized with Staphylococcus
aureus than in individuals not colonized.
Table 2. Antibody levels in colonized individuals versus non-colonized (median
values)
Antigen
Colonized
n=26
Non colonized
n=89
P values
Surface antigens
Teichoic acid
912
422
0.013
Clf-A
Clf-B
Bsp
170
191
233
134
163
161
0,239
0,092
0,067
Extracellular proteins
Alpha toxin
Lipase
SEA
TSS-1
SSS
Efb
Eap
325
364
483
1043
161
394
268
165
176
271
378
115
438
85
0.056
0,007
0,006
0,022
0,275
0,402
0,009
8
against one or more antigens. In this
case, a “low level” was arbitrarily designed as a level below the 20 % percentile for the respective antigen. In
comparison with the random distribution
of such individuals, it is seen that there
are more individuals with low or no
reactions against several antigens than
by chance (p=0.001). As most, 3/151,
showed low or no antibody levels
against 9 out of the 11 antigens tested.
This finding might indicate that certain
individuals have a special tendency not
to react to Staphylococcal antigens.
None of the individuals showing low
antibody levels to more than four antigens were colonized (p=0.04). Certain
antigens did not give rise to any antibody
response at all, an event that happened
most often for Bsp, Clf-B and Eap
(Figure 1).
Antibody levels in relation to
sex
There were no differences seen in the
antibody levels as compared to sex.
Tendencies to produce low or
high levels of antibodies to
more than one antigen.
From Figure 1 it is evident that there are
great variations in the antibody levels
against all antigens between individuals,
at least 100-fold and against some antigens 1000-fold. Besides, against some
antigens in some samples it was not
possible to measure any reactions at all,
even at the lowest dilution 1 /125. Some
individuals showed low antibody levels
against several antigens. Figure 2A
presents the distribution of the
individuals showing low antibody levels
9
Figure 2
Observed versus expected numbers of individuals showing low or high levels of antibodies against one or more antigens. X-axis denote number of antigens and Y-axis
number of individuals.
1A: Low levels, defined as levels below the 20th percentile for each antigen.
2B: High levels, defined as levels above the 90th percentile for each antigen.
Similarly, there was a skewed distribution of the number of antigens against
which certain individuals showed high
levels of antibodies. “High level” was
arbitrarily designed as a level above the
90 % percentile for the respective antigen. Again, 12 individuals had high
levels against 4-5 antigens, an event that
is highly unlikely to happen by random
chance (p=0.001). This finding thus
might indicate that certain individuals
have a special tendency to react strongly
to some Staphylococcal antigens. Also,
six out of eleven of these individuals
were colonized with Staphylococcus
aureus (p=0.02).
Correlations between antibody
levels against different antigens
The various levels of antibodies to the
different antigens were compared between individuals (Figure 3). All antigens showed a mean correlation
coefficient of as low as 0.15, but the
extracellular proteins lipase, alpha-toxin,
enterotoxin
A
and
extracellular
adherence protein clustered together
with a mean correlation coefficient of
0.35. Interestingly enough, these levels
also slightly co-varied with those against
teichoic acid (mean cc = 0.29) , the polyribitol compound bound to the cell wall
of S. aureus. The surface bound proteins
(Clumping factors A and B and bone
sialoprotein-binding protein) co-varied
10
with a mean correlation coefficient of
0.24. The least co-varying levels with
any of the others were obtained with the
levels against extracellular fibrinogen
binding protein, which showed a mean
correlation coefficient on 0.11 against all
other antigens.
TA
At
Lip
SEA
Eap
TSS
SSS
Efb
Clf-A
Clf-B
Bsp
Figure 3
Dendrogram depicting the relations of antibody levels to different antigens in the healthy
individuals.
Upper X-axis denote correlation coefficient levels and right y-axis denote the respective
antigen
This study used 11 highly purified antigens in a conventional indirect ELISA to
determine the levels of serum IgG levels
to each of these. The antigens were
selected as to represent surface antigens
that may be relevant for colonization,
Discussion
Antigens
Staphylococcus aureus produces a wide
variety of antigens and virulence factors.
11
virulence factors involved in wound
healing such as fibrinogen binding
proteins and Eap and specific toxins involved in invasive disease. The specific
toxins SEA, TST and SSS are not
produced by all strains, a fact which
might diminish the occurrence of antibodies in the healthy population. The
antibody levels were however of the
same magnitude against these antigens
as against the others. As can be seen in
Figure 1, in fact there were individuals
who did not show any antibody response
against individual antigens, notably Bsp,
Eap, Clf-B and SSS, but generally most
individuals show antibodies against all
antigens tested and all individuals show
antibodies against some Staphylococcal
antigens, although at different levels
(Granstrom, Julander et al. 1983;
Julander, Granstrom et al. 1983;
Christensson, Fehrenbach et al. 1985;
Hollsing, Granstrom et al. 1987;
Christensson, Boutonnier et al. 1991;
Ryding, Renneberg et al. 1992; Dryla,
Prustomersky et al. 2005; Persson,
Johansson et al. 2009)
various amounts of antibodies to the
respective antigen and they are analysed
at three different starting concentrations
in order to obtain optimal dilutions
curves to relate to the standard. E.g.
Dryla et al (Dryla, Prustomersky et al.
2005) stated that most of the antibodies
were produced against surface antigens.
In our study the protein surface antigens
used did not appear to produce particularly high levels, since Clf-A antibodies
had to be tested for at an initial dilution
of 1/250 and Clf-B and Bsp antibodies at
the lowest initial dilution of 1/125. In
contrast, teichoic acid was tested for at
the high initial dilution of 1/2 500 and
still resulted in the same absorbance
values as the other surface antigens.
Ages
Various results are available on the influence of age upon antibody levels. It is
generally agreed that the levels increase
during childhood and adolescence to
reach a steady level in the adult. However, several studies have shown that the
levels in the healthy elderly decrease
with age ((Granstrom, Julander et al.
1983; Julander, Granstrom et al. 1983;
Dryla, Prustomersky et al. 2005). We
noted a tendency towards a decrease in
antibody levels above 65 years of age.
However, this decrease was only significant for the antigen ClfB and the levels
actually increased 100 % against TST,
although not significant due to the large
variation. It could be surmised that
elderly individuals have experienced
longer exposure to the bacterium,
possibly including earlier invasive infections, but on the other hand the
immune system is expected to become
less reactive with age.
Comparisons
The ELISA assay and the reference line
unit method used in this study give
excellent reproducible and quantitative
data on the antibody levels measured,
and make it possible to perform proper
statistical calculations on the units
obtained to compare antibody levels
between
healthy
and
diseased
individuals in investigations on Staphylococcal infections of various kinds.
However, the relative amounts of antibodies produced against the various
antigens in the same individual are
difficult to quantify with the present
method, since they are all related to one
Golden Standard serum containing
12
Colonization
High and low antibody levels vs
colonization
Van Belkum and coworkers (van
Belkum, Verkaik et al. 2009) stated that
only individuals with persistent colonization of S. aureus showed increased
antibody levels. Our study is based upon
one time sampling, why the issue of persistent colonization was not possible to
evaluate. However, there evidently was a
correlation between carrying the bacteria
in the nares at the time of sampling and
having higher antibody levels against all
antigens
investigated,
significantly
against five of eleven (Table 2). It could
be anticipated that the protein surface
antigens, possibly taking part in the
colonization, should have activated the
immune system more than extracellular
proteins and toxins, but this was not the
case. Teichoic acid, which is supposed to
be an adhesive element, showed more
than twice the levels of antibodies in individuals carrying S. aureus, though.
Cole 2001 (Cole, Tahk et al.
2001)demonstrated
that
colonized
persons had defects in their local innate
immunitiy towards S. aureus. However,
Clark 2006 (Clarke, Brummell et al.
2006) found higher levels of reactive
IgG to iron-responsive surface determinant (Isd) A and Isd H from non-carriers.
Carriers have better prognosis than noncarriers in bacteraemia, and Holtfreter (
(Holtfreter, Roschack et al. 2006) have
explained the improved prognosis
through the increased levels of preformed antibodies, and for toxins like
TST and SEA the existence of antibody
levels are clearly protective (Holtfreter,
Roschack et al. 2006; Verkaik, de Vogel
et al. 2009) The view that humoral
immune response do not protect from
colonization can be challenged by the
work of Clark. Clark showed beneficial
effects of vaccination with Isd A or H in
an animal model against nasal carriage.
It is well known that certain individuals
are at risk to develop Toxic shock
syndrome by S. aureus due to their lack
of capacity to produce neutralising antibodies against the TSST. In this study
we could show that certain individuals
had a stronger or weaker tendency to
produce antibodies against some of the
eleven antigens tested. The individuals
that were high producers to more than
three antigens were significantly more
often colonized in the nares, but none of
the individuals that were low producers
against more than four antigens were
colonized. These data could be interpreted as if certain individuals have a
tendency to produce and some not to
produce antibodies against S. aureus
antigens. Individuals that are lacking
antibodies against several antigens might
have been in less contact with Staphylococcus aureus, but this is difficult to
anticipate for healthy individuals. The
relevance of such low levels of antibodies against S. aureus as lowered
protection against invasive S. aureus infections has been recently discussed
(Dryla, Prustomersky et al. 2005;
Holtfreter, Roschack et al. 2006)).
Correlations
in
antibody
production between individuals
Related to the discussion above is the
question whether certain groups of antigens are more likely to induce antibody
production than others. In order to investigate this, the relative quantitative
responses against all antigens were compared between all individuals and
clustered into a dendrogram (Figure 3).
The results clearly indicated that antibodies against the extracellular proteins
alpha-toxin, lipase, enterotoxin A and
13
extracellular adhesive protein more often
were found in the same individuals, most
often together with the surface bound
teichoic acid.
protection against nasal carriage." J
Infect Dis 193(8): 1098-108.
Cole, A. M., S. Tahk, et al. (2001).
"Determinants of Staphylococcus aureus
nasal carriage." Clin Diagn Lab Immunol 8(6): 1064-9.
Conclusions
In conclusion, this study has pointed out
the great variations in antibody levels in
healthy young and elder individuals.
Occurrence of S. aureus in the nares at
time of sampling was correlated to
higher antibody levels but ages over 65 y
showed only slightly lower levels.
Certain individuals were more prone to
produce/not to produce antibodies than
others, and certain antigens were more
often inducing high levels in the same
individuals.
These findings are important for the
development of improved serological
diagnostics and constitute an important
knowledge for future immune prophylaxis
and
therapy of
invasive
Staphylococcus aureus infections.
Colque-Navarro, P., M. Palma, et al.
(2000). "Antibody responses in patients
with staphylococcal septicemia against
two Staphylococcus aureus fibrinogen
binding proteins: clumping factor and an
extracellular fibrinogen binding protein."
Clin Diagn Lab Immunol 7(1): 14-20.
Colque-Navarro, P., B. Soderquist, et al.
(1998). "Antibody response in Staphylococcus aureus septicaemia--a prospective
study." J Med Microbiol 47(3): 217-25.
de Lencastre, H., D. Oliveira, et al.
(2007). "Antibiotic resistant Staphylococcus aureus: a paradigm of adaptive
power." Curr Opin Microbiol 10(5): 42835.
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