Z3
Global antibody response to Staphylococcus aureus live-cell vaccination Tobias
1,* Hertlein ,
1,* Selle ,
1 Oesterreich ,
1 Klemm ,
2 Kloppot ,
3,4 Müller ,
Martina Babett Theresa Peggy Elke 3,4 5 5 6,7 1 Ehricht , Sebastian Stentzel , Barbara M. Bröker , Susanne Engelmann & Knut Ohlsen
Ralf
1) University Würzburg, Institute for Molecular Infection Biology, Würzburg, Germany. 2) University Greifswald, Institute for Microbiology, Greifswald, Germany. 3) Alere Technologies GmbH, Jena, Germany. 4) InfectoGnostics Research Campus Jena, Germany. 5) University Medicine Greifswald, Department of Immunology, Greifswald, Germany. 6) Technical University Braunschweig, Institute for Microbiology, Braunschweig, Germany. 7) Helmholtz-Zentrum für Infektionsforschung, Mikrobielle Proteomik, Braunschweig, Germany. *These authors contributed equally to this work.
Introduction
1
The scope of this work was to study the protectivity of an antibody response in mice against Staphylococcus aureus. We were interested in this topic since several active or passive immunization approaches against this bug failed at different stages of clinical development. This was why we wondered, whether it is in general possible to induce a protective immune response in mice and if yes, what are the essential features of this adaptive immune response. In order to investigate the adaption of the host’s immune system against this pathogen we vaccinated mice three times with sub-lethal doses of live bacteria in intervals of 14 days. After and during this vaccination regimen, we analyzed the amount and specificity of antibodies against S. aureus in the mice. Mice were challenged at the end of the vaccination with a high dose of S. aureus in the thigh muscle to test whether the adaption of the immune system has led to a superior outcome in subsequent severe infection compared to naïve cognates.
Vaccination procedure d0
d14
d28
1st infection
2nd infection
3rd infection
d40
d44
d49
Severe thigh muscle infection Obtaining of serum samples for isotyping and antibody specificity pattern
Recovery of infected thigh muscles, inner organs and blood
(Fig. 1) Time scale for vaccination and subsequent severe infection. Mice were challenged three times with S. aureus Newman wt (2 x 106 CFU) during vaccination and with S. aureus Newman lux (1 x 108 CFU) during severe thigh muscle infection. (From Selle M. et al. 2016)
Results The antibody titers increased during vaccination irrespectively of the vaccination route. Nonetheless, the total level of Ig was higher in intravenously vaccinated mice than in intramuscularly challenged cognates. 15000
3aA
IgG1 ***
8000
**
***
30
***
3b
**
no Cytosolic Proteins
6000
20
4000
5000 2000
10 0 0 d4
0 d3
6 d1
d2
0 d4
1500 1000
L1 78 8
C
O
lip
Is aA
Sa SC ur IN JH 1_ 20 34
L0 98 5 C O L1 16 4
O
5 O
L2 29
9
C
C
O
L1 16
nu c
Pl c
7 SS L
D Sp l
Lu
B2
1000
C 20
10 500
8
i.v. vac.
i.m. vac. 1µm
Tu f
A
L0 12 9 O
C
L0 02 1
O
O C
L1 06 5
Ti g
lp Q G
1 L1 SS
lC Sp
C
i.v. vac.
0 d4
0 d3
6 d1
d2
0 d4
0 d3
6 d1
d2
0 d4
0 d3
6 d1
d2
0 d4
0 d3
d1
6
0
d2
0
lb
la 50
i.v. vac. i.m. vac.
0
0
100
10 5
H
µg/ml
200
**
4 2
150 400
**
re
*
200
600
**
6
Score
Score
IgE
IgA
15
0 d4
0 d3
6 d1
d2
0
0 d3
6
d4
i.m. vac.
i.v. vac.
i.m. vac.
i.v. vac.
d1
d2
0 d4
0 d3
6 d1
d2
0 d4
0 d3
d1
6
0
d2
0
H
500
µg/ml
kF
H 1500
µg/ml
µg/ml
2000
B
***
Sp l
**
2000
lukF-PV
0 lg B
**
Hlb
4
i.m. vac.
i.v. vac.
-P V
0 d3
6
10
IgM
***
*
*
Score
i.m. vac. IgG3
2500
***
***
***
20
d2
0 d4
0 d3
6 d1
d2
d4
d3
d1
0
0
0
0
6
200
d2
100
i.v. vac.
lg A
400
d1
200
***
***
***
30
600
µg/ml
300
B1
C
Sb i_
800
H
Ef bC
IgG2b
**
***
400
I-I V Sb i_ III -IV
0
IgG2a
HlgC
Efb SCIN SaurJH1_2034 SACOL 1169 Sbi I-IV / III-IV HlgA
Mice were challenged at the end of the vaccination with a high dose of luminescent S. aureus. Intravenously but not intramuscularly vaccinated mice showed reduced bacterial burden after 5 days of infection and lower bioluminescent signals throughout the course of infection.
Hly HlgB Geh Lip GlpQ IsaA Extracellular SplA SplC Nuc Plc SspA SplB enzymes SspB SplD SEC-bov SEB Ssl-7 SEK SEC SEM SEI Superantigens SEQ SEL TSST-1 Ssl-11 SACOL 0479 SACOL 0021 SACOL 1065 SACOL 0985 SACOL 0480 SACOL 0129 SACOL 1169 SACOL 1164 SACOL 0669 SACOL 0444 SACOL 2295 SACOL 0755 SACOL 0723 Putatively secreted SACOL 0820 SACOL 0742 SACOL 0908 SACOL 1788 proteins SACOL 1802 SACOL 1870 SACOL 2197 SACOL 2661 red: signal higher in intravenous than in intramuscular model blue: signal higher in intramuscular than in intravenous model
d4
0 d3
6
i.m. vac.
i.v. vac.
i.m. vac.
d1
d2
0 d4
0 d3
6 d1
d2
0 d4
0 d3
6 d1
d2
0 d4
0 d3
6 d1
d2
i.v. vac.
µg/ml
Pore-forming proteins
high
GreA Tuf
Immunomodulators
0
0
Tig
Sbi I-II
Score
µg/ml
µg/ml
10000
CitC PknB PurA Stp
Antibody response low intermediate
G
2
total Ig level in serum *
We identified the specificities of antibodies against S. aureus after the vaccination with the help of a protein array. A clear difference in amount and pattern could be identified between intravenously and intramuscularly vaccinated mice with first ones showing more different specificities and higher levels.
i.m. vac.
(Fig. 2) Total Ig and Ig isotype levels during vaccination regimen in intravenously and intramuscularly challenged mice. Antibody titers were determined with FlowCytomix (eBioscience) and ELISAs. Displayed are the means +/- SEM for each group. Statistically significant differences were determined by one-way ANOVA and Mann-Whitney test, respectively, with a Bonferroni correction and are indicated by asterisks (*p < 0.05, **p < 0.01; ***p < 0.001). (From Selle M. et al. 2016)
Future perspective Having a mouse model which allows us to study the adaption of the immune system to S. aureus, we are interested in the plasticity of the T and B cell populations during vaccination and its influence upon subsequent severe infection. On the other hand, we are interested in the mechanisms of S. aureus to interfere with this adaption.
(Fig. 3) Antibody specificity pattern generated by the vaccination regimen determined with Staph-Toxin-Ag03 array stripes (Alere Technologies). (3a). Each individual mouse was scored based on the signal intensity on the chip (low signal: 1, intermediate signal: 2, high signal: 3). Shown are the sums of each protein and group. Statistical significance was determined based on original signal values from the array and tested using the Mann-Whitney test with Bonferroni correction (*p < 0.05, **p < 0.01; ***p < 0.001). (3b). Tabular representation of the mean antibody signal against each antigen. (From Selle M. et al. 2016)
Conclusion • Vaccination of mice with sub-lethal doses of S. aureus leads to increasing titers of antibodies • The specificities of these antibodies vary significantly based on the route of administration, indicating differences in virulence factor expression • The adaption of the immune system to S. aureus pre-challenge can lead to superior outcome in subsequent severe infection
Tobias Hertlein
[email protected] ++49-(0)931-31 85796
(Fig. 4) Outcome of severe thigh muscle infection with S. aureus Newman lux after vaccination. (4a) Biolumenscent images of one representative mouse of each group during course of infection. (4b) Mean (+/SEM) biolumenscent signals of each group. (4c) The thigh muscles were recovered at day 5 p.i., homogenized and dilutions plated to determine the bacterial burden at the end of the experiment. Shown are individual values and the median for each group. Statistically significant differences between the groups were determined by ANOVA with Dunn’s post test (**p < 0.01;***p < 0.001). (From Selle M. et al. 2016)
Acknowledgements I would like to thank Liane Dreher for her excellent technical support during these experiments and Christina Daumberger for her help with the wellfare of the animals. Furthermore, I wish to thank the SFB/Transregio 34 for funding these project.
