Vaccine 33 (2015) 2207–2212

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis , is a highly protective antigen against serotype 2 Jana Seele a,1 , Lena-Maria Hillermann a,1 , Andreas Beineke b , Maren Seitz a , Ulrich von Pawel-Rammingen c , Peter Valentin-Weigand a , Christoph G. Baums a,d,∗ a

Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany c Department of Molecular Biology and Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden d Institute for Bacteriology and Mycology, Centre of Infectious Diseases, College of Veterinary Medicine, University Leipzig, Leipzig, Germany b

a r t i c l e

i n f o

Article history: Received 3 December 2014 Received in revised form 3 March 2015 Accepted 17 March 2015 Available online 28 March 2015 Keywords: IgM protease Streptococcus suis Pigs Bactericidal assay Neutralizing antibodies

a b s t r a c t Streptococcus suis (S. suis) is a major porcine pathogen causing meningitis, arthritis and several other pathologies. Recently, we identified a highly specific immunoglobulin M degrading enzyme of S. suis, designated IdeSsuis , which is expressed by various serotypes. The objective of this work was to access the immunogenicity and protective efficacy of a recombinant vaccine including IdeSsuis . Vaccination with rIdeSsuis elicited antibodies efficiently neutralizing the IgM protease activity. Importantly, 18 piglets vaccinated with rIdeSsuis alone or in combination with bacterin priming were completely protected against mortality and severe morbidity after S. suis serotype 2 challenge. In contrast, 12 of the 17 piglets either treated with the placebo or primed with the bacterin only, succumbed to S. suis disease. Immunity against IdeSsuis was associated with increased killing of S. suis wt in porcine blood ex vivo leading to a tenfold difference in the bacterial survival factor in blood of placebo-treated and rIdeSsuis -vaccinated piglets. In conclusion, the results of this study indicate that rIdeSsuis is a highly protective antigen in pigs. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Streptococcus suis (S. suis) is one of the most important porcine pathogens causing meningitis, arthritis and other pathologies [1]. In affected herds, problems typically augment between 7 and 10 weeks of age. Although S. suis is a very diverse species [2], at least 30% of S. suis diseases in Europe and China are caused by serotype 2 strains [3–5]. S. suis serotype 2 is also an important zoonotic agent causing meningitis and other diseases in humans [6,7]. In Europe no licensed vaccine is available and autologous bacterins are commonly used for prophylaxis [8]. However, there are major limitations associated with these bacterins, in particular, (i) most often only partial protection against the homologous serotype is observed, (ii) protection against heterologous serotypes is not

∗ Corresponding author at: Universität Leipzig, Zentrum für Infektionsmedizin, Veterinärmedizinische Fakultät, Institut für Bakteriologie und Mykologie, An den Tierkliniken 29, 04103 Leipzig, Germany. Tel.: +49 0 341 9738180; fax: +49 0 341 9733199. E-mail address: [email protected] (C.G. Baums). 1 Contributed equally. http://dx.doi.org/10.1016/j.vaccine.2015.03.047 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

elicited, (iii) differentiation of infected and vaccinated animals is generally not possible, and (iv) interference with maternal immunity is very common [9,10]. The latter makes it very difficult to prime piglets earlier than at an age of 4 to 6 weeks in porcine practice. Several S. suis proteins have been tested as vaccine candidates, but substantial limitations are known. The hemolysin, suilysin (SLY), was found to elicit protection in mice but piglets with high titers of SLY neutralizing antibodies were found to be susceptible to infection [11,12]. A combination of muramidase-released protein (MRP) and extracellular factor (EF) is protective against serotype 2 [13], but other important pathotypes such as MRP* serotype 9 do not express EF. Recently, we identified an immunoglobulin M-degrading enzyme of S. suis, designated IdeSsuis . The protein is homologous to the IgG protease IdeS of S. pyogenes [14], but cleaves solely class M antibodies. IdeSsuis is the first factor described reflecting S. suis adaptation to its main host as the protease exclusively cleaves porcine IgM [15]. It is expressed by all S. suis strains investigated and important for bacterial survival in blood of bacterin-primed piglets [15]. Thus, we designed this study to investigate the immunogenicities and protective efficacies of recombinant IdeSsuis (rIdeSsuis ) vaccination.

2208

J. Seele et al. / Vaccine 33 (2015) 2207–2212

2. Materials and methods 2.1. Bacterial strains and growth conditions S. suis strain 10 (MRP+ EF+ SLY+ ) is a virulent serotype 2 strain that has been used before for experimental infections [10,13,16]. The isogenic mutant 10ideSsuis deficient in IgM cleavage was included in the bactericidal assay to reveal effects mediated by antigen-specific immunity [15]. The unencapsulated mutant 10cpsEF was used as antigen in an ELISA to measure IgMantibodies directed against other antigens but the capsule [17]. Strain I9841/1 is as strain 10 an MRP+ EF+ SLY+ CPS2 wt strain of clonal complex 1 used for bacterin generation [18]. S. suis was grown on Columbia agar plates supplemented with 6% sheep blood or in BactoTM Todd Hewitt broth (THB). Escherichia coli (E. coli) strains were cultured in Luria-Bertani (LB) medium with the addition of 100 ␮g/ml ampicillin.

bacteriological (b) investigations as described previously [10,19,20]: cerebrospinal fluid (b); brain (b, h); tarsal and carpal joints (b, h), peritoneal, pleural and pericardial swabs (b), peritoneum, pleura and pericardium (h); cranial lobe of the left lung (b, h); liver (b, h); spleen (b, h); bicuspidalis (b, h) and tonsil (b, h). The histological screenings were scored as described [19] and briefly mentioned in the footnotes of Table 2. Isolation of the challenge strains was confirmed in a PCR for detection of mrp, epf, sly, arcA, gdh, cps1, cps2, cps7 and cps9 [4]. 2.5. IgM cleavage neutralization assay

The expression and the purification of recombinant IdeSsuis was performed as previously described [15].

Sera of all piglets vaccinated with rIdeSsuis only or treated with the placebo were analyzed with regard to the presence of antibodies neutralizing the IgM cleavage activity of IdeSsuis . For this, sera were diluted 1:5 in PBS, spiked with the indicated concentrations of rIdeSsuis and incubated on a rotator for 45 min at 8 ◦ C. Subsequently, samples were incubated for 2 h at 37 ◦ C to allow cleavage of IgM by non-neutralized IdeSsuis . Serum proteins were separated under non-reducing conditions by SDS-PAGE [15] and analyzed in ␣pig IgM Western blots (Serotec, MCA637 was used as ␣pig IgM antibody as described [15]).

2.3. Immunization of piglets

2.6. Bactericidal assay

Piglets were prime and booster vaccinated with 0.25 mg rIdeSsuis containing 20% [vol/vol] Emulsigen as an adjuvant to generate reference hyperimmune sera independently of the vaccination trial. Vaccination of piglets at our institute is registered under 12A226 at the Lower Saxonian State Office for Consumer Protection and Food Safety (LAVES).

Survival of S. suis in porcine blood ex vivo was determined as previously described [15]. Briefly, 500 ␮l of heparinized blood (16 I. U. heparin/ml) was mixed with 1.5 × 105 CFU of exponentially grown bacteria (OD600 : 0.5–0.6). The samples were incubated for 2 h at 37 ◦ C on a rotator. Blood was drawn from all piglets of the vaccination trial three days before challenge for comparative analysis of survival of S. suis strain 10 and its isogenic mutant 10ideSsuis in the bactericidal assay. The specific bacterial content in CFU/ml were determined by plating serial dilutions at t = 0 min and t = 120 min and the survival factor of S. suis for each sample was calculated by dividing the two values.

2.2. Expression and purification of recombinant IdeSsuis protein

2.4. Animal experiments German Landrace piglets from a herd known to be free of sly+ mrp+ epf+ cps2+ strains were infected experimentally and cared for in accordance with the principles outlined in the EU Directive 2010/63/EU (http://ec.europa.eu/environment/ chemicals/lab animals/legislation en.htm) and the German Animal Protection Law. The animal experiment was approved by the Committee on Animal Experiments of LAVES (permit no. 33.1442502-04-12/0965). At an age of 5 weeks, either a placebo (first and second group) or a vaccine containing rIdeSsuis as antigen (third and fourth group) were applied intramuscularly. Fourteen days later, the four groups were vaccinated as follows: placebo (first group); a serotype 2 bacterin (second group); rIdeSsuis and separately a serotype 2 bacterin (third group); rIdeSsuis (fourth group). One dose of prime and booster rIdeSsuis vaccination contained 0.4 mg and 0.25 mg rIdeSsuis , respectively, supplemented with 20% [vol/vol] Emulsigen as adjuvant. The bacterin was generated with an overnight culture of strain I9841/1 inactivated in 0.2% formaldehyde (application of 1.6 ml with 1.8 × 109 inactivated bacteria/ml and 20% Emulsigen). The placebo consisted of PBS with 20% [vol/vol] Emulsigen. Piglets were challenged intranasally at an age of 9 weeks (14 days after the second vaccination) with 1.2 × 109 CFU of S. suis strain 10 as described [19]. The health status of the animals was monitored every 8 h. A piglet was classified as morbid if a body temperature of ≥40.2 ◦ C or/and severe clinical signs of an acute disease were observed. In case of high fever (≥40.5 ◦ C), apathy and anorexia persisting over 36 h as well as in all cases of central nervous system dysfunction or clinical signs of acute polyarthritis animals were euthanized for reasons of animal welfare. All surviving piglets were sacrificed 14 days post infection (dpi). After euthanasia every animal went through the same procedure of necropsy to collect the following samples for histological (h) and semi-quantitative

2.7. Detection of anti-(˛)IdeSsuis IgG, ˛MRP IgG and ˛S. suis IgM antibodies The detection of ␣MRP IgG was performed as previously described [10] ␣IdeSsuis antibodies were detected using the same protocol except that the plates were coated with rIdeSsuis . Serum of piglets immunized with rIdeSsuis and a truncated derivative (rIdeSsuis homologue, [15]) served as reference serum and positive control in the ␣IdeSsuis ELISA, respectively. ␣S. suis IgM titers were determined in an ELISA using a peroxidase-conjugated goat ␣pig IgM antibody (PA1-84625, Thermo Scientific, Schwerte, Germany) and Maxisorb® plates (Nunc, Rochester, USA) coated either with strain 10 or 10cpsEF (inactivated with 0.2% formaldehyde). Log linear regression analysis was conducted after background submission to calculate antibody concentrations. ELISA units were calculated as the mean of the calculated units for each of the four dilutions of two series. Quality was assessed through comparison to preset values for controls [10]. 2.8. Suilysin-neutralization assay Antibody titers neutralizing SLY were determined in a hemolysis assay as described [11]. 2.9. Statistical analysis Differences between groups were analyzed with the Mann–Whitney U-test. The Wilcoxon test was used for comparison

J. Seele et al. / Vaccine 33 (2015) 2207–2212

Fig. 1. An ␣IdeSsuis hyperimmune serum inhibits the IgM-cleaving activity of rIdeSsuis . Anti-IgM Western blot analysis of porcine sera incubated with the indicated concentration of rIdeSsuis or as a control with PBS. Sera were either drawn before (pre immune) or after prime booster vaccination with rIdeSsuis (␣-rIdeSsuis serum). The marker bands are shown on the left side (sizes in kDA). The arrow indicates the position of the uncleaved IgM polymer. The cleavage products are indicated by asterisks on the left side of each band.

of different time point values within the same group. The data presented in the Kaplan–Meier-diagrams were analyzed with the log rank test. Means and standard deviation of the results are shown. Probabilities lower than 0.05 were considered significant (p < 0.05 *, p < 0.01 ** and p < 0.001 ***). 3. Results 3.1. Vaccination of a piglet with rIdeSsuis elicits antibodies neutralizing IgM protease activity of IdeSsuis In our previous study we demonstrated that IdeSsuis is a highly specific porcine IgM protease [15]. At the beginning of this work, an IdeSsuis hyperimmune serum was generated in a piglet and investigated for inhibition of IgM cleavage activity in a neutralization assay with subsequent Western blot analysis. In accordance with our previous study, treatment of a serum drawn before immunization of this piglet with rIdeSsuis elicited specific IgM-cleavage products (Fig. 1). In contrast, incubation of the porcine anti-IdeSsuis hyperimmune serum with rIdeSsuis did not result in cleavage of IgM (Fig. 1). In summary, immunization of a piglet with rIdeSsuis elicted humoral immunity neutralizing the IgM protease activity of IdeSsuis . 3.2. Vaccination with rIdeSsuis protects growing piglets against morbidity and mortality caused by S. suis serotype 2 infection We hypothesized that neutralization of IdeSsuis in piglets with high IgM titers might lead to IgM-mediated killing of S. suis and thus to protection in bacterin-primed piglets. We combined rIdeSsuis prime booster vaccination with bacterin priming in a vaccination trial to challenge this hypothesis. A group vaccinated with rIdeSsuis only (prime and booster) and another group primed with a bacterin only was also included in order to demonstrate this putative synergistic effect. Vaccination with rIdeSsuis elicited a systemic IgG response against IdeSsuis in all of the 18 piglets (Fig. 2). The mean anti-IdeSsuis titers of the groups vaccinated with rIdeSsuis alone or in combination with bacterin priming were 70.4 (S.D. = 35.5) and 67.6 (S.D. = 28.6), respectively, and thus significantly higher than the titers in the placebo and in the bacterin-primed group which were below the detection limit (p < 0.001; Fig. 2). Noteworthy, S. suis strains isolated from the tonsils prior experimental infection expressed IdeSsuis antigen (Fig. S1). Furthermore, IgM cleavage activity of IdeSsuis is neutralized in sera drawn from piglets of this trial after booster

2209

Fig. 2. Vaccination with rIdeSsuis elicits high ␣IdeSsuis IgG-antibody-titers. AntiIdeSsuis IgG titers in serum were determined directly before vaccination (pre) and 11 days after the last vaccination (post) in porcine serum samples. Mean values are indicated by horizontal lines and statistical analysis was conducted with the Mann–Whitney U-test (comparison of groups) or the Wilcoxon test (comparison of pre and post immune sera). Significant differences are indicated (** p < 0.01; *** p < 0.001).

vaccination with rIdeSsuis in contrast to sera of piglets of the placebo group (Fig. S2). Suilysin neutralization, ␣S. suis IgM and ␣MRP IgG titers were determined to further characterize the humoral immune status of the four groups. Antibodies neutralizing suilysin were not detectable prior challenge except for two piglets having a very low positive titer (Fig. S3). However, the piglets of all four groups with the exception of one piglet had specific IgM titers above 20 ELISA units as determined by ELISA using either the wt strain or an unencapsulated mutant as antigen (Fig. S4). Differences in IgM titers between the four groups were not recorded, while IgG titers against MRP were significantly higher in bacterin-primed piglets (Fig. S5). In conclusion, bacterin priming in these growing piglets did not result in significant differences in IgM titers against S. suis or titers of antibodies neutralizing suilysin but elicited low IgG titers against MRP. All 18 piglets vaccinated with rIdeSsuis either alone or in combination with bacterin priming survived the serotype 2 challenge. In contrast, the placebo and the bacterin-primed group showed significantly higher mortality rates with 67% and 75%, respectively (placebo vs. prime and booster vaccination with rIdeSsuis p = 0.0031; prime bacterin vs. prime and booster rIdeSsuis vaccination plus bacterin priming p = 0.0013) (Fig. 3A). Furthermore, only one piglet of each of the two IdeSsuis vaccinated groups was classified as morbid due to a body temperature > 40.2 ◦ C for one day. Other clinical signs, such as central nervous system dysfunction, lameness and kyphosis were not recorded in IdeSsuis -vaccinated piglets. In contrast, 6 of 9 placebo-treated piglets were euthanized because of signs of central nervous system dysfunction such as convulsions (Table 1). All 8 bacterin-primed piglets and 7 of 9 placebo-treated piglets showed clinical signs of an acute disease after challenge (Fig. 3B). Differences in morbidity between the two IdeSsuis -vaccinated and the two other groups were highly significant (placebo vs. prime and booster rIdeSsuis p = 0.0044; prime bacterin vs. prime and booster rIdeSsuis and prime bacterin p = 0.0002). The histological screening revealed that none of the 18 IdeSsuis vaccinated piglets had fibrinosuppurative lesions in the brain, the four investigated joints, the mitral valve or in any of the investigated samples of the liver, the pleura, the peritoneum and the pericard, in contrast to the placebo group and the bacterin-primed group (Table 2). Only the spleen showed mild focal neutrophilic accumulation in the red pulp in 7 of 18 rIdeSsuis -vaccinated piglets

2210

J. Seele et al. / Vaccine 33 (2015) 2207–2212

of the other two groups (Table 3). However, the challenge strain was isolated from the tonsils of 7 of 18 IdeSsuis -vaccinated piglets indicating that these piglets had not eliminated the challenge strain completely. In conclusion, vaccination with rIdeSsuis protected growing piglets against morbidity, mortality and pathology following S. suis serotype 2 infection. 3.3. IdeSsuis -vaccination elicited antigen-specific immunity leading to efficient killing of S. suis serotype 2 in porcine blood

Fig. 3. Vaccination with rIdeSsuis protects growing piglets against mortality (A) and morbidity (B) caused by S. suis serotype 2 infection. Kaplan–Meier diagrams of vaccinated growing piglets after an intranasal challenge with 1.2 × 109 CFU of S. suis strain 10 (MRP+ EF+ SLY+ CPS2). A piglet was classified as morbid if a body temperature of ≥40.2 ◦ C or/and severe clinical signs of an acute disease were observed. Statistical analysis was conducted with the log-rank test (all p-values are shown below the diagrams).

(Table 2). Accordingly, only the two rIdeSsuis -vaccinated groups received average histological scores ω equal to or smaller than 1 (ω = scoremax /nanimals; scoremax is the highest score obtained by a piglet in any investigated tissue; Table 2) [19]. This indicates protection against fibrinosuppurative inflammation due to S. suis serotype 2 infection. In contrast, the placebo and the bacterin-primed group obtained average histological scores ω of 3.6 and 3.0, respectively (scores ranging from 0 to 5; Table 2). The challenge strain was not detected in the CSF, the brain, the liver, the mitral valve, the different joint fluids and the swabs from different serosae in IdeSsuis -vaccinated piglets in contrast to piglets

As bacteremia is an important part of the pathogenesis of S. suis diseases we determined bacterial survival in blood ex vivo of all piglets three days before challenge (11 days after the second vaccination). This assay included comparative analysis of survival of S. suis wt and 10ideSsuis to specifically assess killing mediated by IdeSsuis -specific immunity, as this should affect only the wild type and not the isogenic mutant. The isogenic mutant 10ideSsuis was found to be attenuated in piglets of the placebo and the bacterinprimed group, both showing titers of specific IgM (Fig. 4, Fig. S4). Importantly, S. suis wt but not the isogenic mutant 10ideSsuis had significantly lower survival factors in blood of IdeSsuis -vaccinated piglets in comparison to placebo treated and solely bacterinprimed piglets (Fig. 4). Specifically, the survival factors of S. suis wt were 7.7 (S.D. = 6.6) and 0.64 (S.D. = 1.1) in blood of placebo-treated and rIdeSsuis -vaccinated piglets, respectively (p = 0.011). In contrast, the mutant 10ideSsuis showed no significant differences in survival factors in blood of placebo-treated and rIdeSsuis -vaccinated piglets (SF = 3.4 with S.D. = 4.1 and 5.5 with S.D. = 5.4, respectively; p = 0.3199). Noteworthy, the mutant 10ideSsuis survived significantly better than the wt in blood of rIdeSsuis -vaccinated piglets (p = 0.0142) but significantly worse in the blood of placebo-treated piglets (p = 0.0029). Very similar results were obtained for the group that was vaccinated with rIdeSsuis and prime-vaccinated with a bacterin (Fig. 4). Killing of S. suis wt was most pronounced in blood of these piglets (SF = 0.32 with with S.D. = 0.8). In conclusion, rIdeSsuis is a highly protective antigen. S. suis wt is efficiently killed in blood of piglets vaccinated with IdeSsuis , at least in the presence of specific IgM. 4. Discussion After many years of experience with S. suis vaccination trials we have become very critical regarding the protective efficacy of single protein antigens. This is mainly because we have observed many piglets with high titers against MRP, SAO and suilysin being highly susceptible to a challenge with S. suis [10,11,20]. Thus, we assumed that vaccination with the IgM protease IdeSsuis might, at its best, improve the protective efficacy of a bacterin by eliciting protection after priming. Accordingly, we designed the vaccination trial of this study to test the putative synergistic effects. Unexpectedly, the clinical and pathological results of the animal experiment did not reveal such a synergism but demonstrated that rIdeSsuis protein is a highly protective antigen by its own. We are not aware of a study demonstrating complete protection against mortality and specific

Table 1 Assessment of the protection induced by the indicated vaccines against morbidity and mortality after intranasal S. suis serotype 2 challenge. Immunization antigen

Placebo Bacterin (prime) rIdeSsuis (prime + booster) + bacterin (prime) rIdeSsuis (prime + booster) a

Morbidity

7/9 8/8 1/9 1/9

Mortality

6/9 6/8 0/9 0/9

Max. body temperature (◦ C)

Clinical signs CNSa

Lameness

Unspecific

40.2

6/9 3/8 0/9 0/9

2/9 3/8 0/9 0/9

0/9 3/8 1/9 1/9

2/9 0/8 8/9 6/9

1/9 1/8 0/9 2/9

6/9 7/8 1/9 1/9

Signs of central nervous system (CNS) dysfunction such as convulsions and opisthotonus.

J. Seele et al. / Vaccine 33 (2015) 2207–2212

2211

Table 2 Scoring of fibrinosuppurative lesions of piglets challenged with S.suis serotype 2 strain 10 (mrp+ epf+ sly+ cps2). Immunization antigen

Placebo Bacterin (prime) rIdeSsuis (prime+booster) + bacterin (prime) rIdeSsuis (prime+booster)

Piglets without lesionsa

Brain

Serosa

Joint

Spleen and liver

Lung

Heart

Meningitis, chorioiditis

Pleuritis or peritonitis

Synovialitis

Splenitisb or hepatitis

Pneumonia

Endocarditis

5c

3d

1e

4c

2d

1e

4c

2d

1e

4c

2d

1e

4c

2d

1e

4c

2d

1e

ωf

1/9 2/9 4/9

4/9 1/8 0/9

0/9 1/8 0/9

0/9 0/8 0/9

0/9 2/8 0/9

0/9 1/8 0/9

0/9 0/8 0/9

1/9 1/8 0/9

0/9 1/8 0/9

0/9 0/8 0/9

2/9 1/8 0/9

6/9 3/8 4/9

0/9 0/8 1/9

1/9 1/8 1g /9

0/9 1/8 0/9

0/9 0/8 0/9

0/9 1/8 0/9

0/9 0/8 0/9

0/9 0/8 0/9

3.6 3.0 1.0

5/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

0/9

3/9

0/9

1/9

0/9

0/9

0/9

0/9

0/9

0.7

a

Only fibrinosuppurative lesions are considered. b Neutrophilic accumulation of the splenic red pulp. c Scoring of 4 and 5 indicates moderate to severe diffuse or multifocal fibrinosuppurative inflammations. d Scoring of 2 and 3 indicates mild focal fibrinosuppurative inflammation. e Individual single perivascular neutrophils received a score of 1. f ω = scoremax /nanimals [19]. g One pig showed a moderate diffuse lymphohistocytic interstitial, partially suppurative to granulomatous pneumonia, not associated with the detection of the challenge strain.

Table 3 Reisolation of the challenge strain from piglets after immunization and intranasal challenge with S. suis serotype 2. Immunization antigen

Placebo Bacterin (prime) rIdeSsuis (prime + booster) + bacterin (prime) rIdeSsuis (prime + booster) a b c d e f

Number of piglets positive for the isolation of the challenge strain

Number of piglets in which the S. suis challenge straina was isolated from

in an inner organb or in serosa or in joint fluid

Tonsils

Lungc

Serosad

Spleen

Liver

Brain, CSFe

Joint fluidf

Endocard

6/9 6/8 1/9

4/9 2/8 2/9

3/9 3/8 0/9

3/9 3/8 0/9

5/9 5/8 1/9

5/9 3/8 0/9

4/9 2/8 0/9

4/9 1/8 0/9

4/9 2/8 0/9

1/9

5/9

1/9

0/9

0/9

0/9

0/9

0/9

0/9

The challenge strain was identified through PCR. Inner organ refers to lung, spleen, liver, brain, CSF or endocard but not the tonsils. One cranial lobe was investigated. Pleural, peritoneal or pericardial cavity. Cerebrospinal fluid. Punctures of both tarsal and carpal joints were investigated in each animal. In case of lameness additional joint punctures of the respective limb were screened.

Fig. 4. Survival of S. suis wt but not of the isogenic mutant 10ideSsuis in porcine blood ex vivo is significantly reduced in piglets vaccinated with rIdeSsuis . Survival of wild type strain 10 (wt) and 10ideSsuis () was determined in porcine blood drawn from the piglets of the animal experiment 3 days prior challenge (11 days after the last vaccination). Bars and error bars represent mean values and standard deviations, respectively. The Mann–Whitney-U-test and the Wilcoxon test were used for comparison of the different animal groups and the survival factors of the two S. suis strains, respectively. Significant differences are indicated (* p < 0.05; ** p < 0.01;*** p < 0.001).

clinical signs of S. suis diseases in piglets using a single S. suis protein antigen for vaccination of at least 6 piglets [21]. Thus, we postulate that IdeSsuis is unmatched in comparison to other described antigens with regard to the protective efficacy elicited in piglets. The piglets of this herd did not have notable antibody titers against IdeSsuis prior vaccination, although their mucosal surfaces were colonized by S. suis strains expressing IdeSsuis (Fig. S1). This finding is in contrast to findings regarding other S. suis antigens such as surface antigen one expressed by many different S. suis serotypes [10,22]. Furthermore, piglets infected with suilysin positive S. suis strains show very often high titers against suilysin [11]. Thus, the very low background of ␣IdeSsuis antibodies in nonvaccinated pigs might be a major advantage for differentiation of IdeSsuis -vaccinated and infected piglets in the future, e. g. by a combination of an ␣IdeSsuis -ELISA and the suilysin neutralization test. Furthermore, the low background of IdeSsuis antibodies in conventional piglets might allow to successfully vaccinate suckling piglets in the field, as inhibition of active immunization by maternal antibodies is not expected under these conditions. However, future studies are needed to investigate IdeSsuis -specific immunity in herds endemically infected with strains of clonal complex 1 expressing higher amounts of IdeSsuis . Antibodies elicited by rIdeSsuis vaccination were found to neutralize the IgM protease activity of IdeSsuis . As all piglets included in this vaccination trial had detectable specific IgM antibodies against S. suis, neutralization of IgM proteolysis might at least in part explain the high protective efficacy of IdeSsuis vaccination. As indicated by the results of this study, it is not necessary to

2212

J. Seele et al. / Vaccine 33 (2015) 2207–2212

specifically induce high IgM titers to elicit protection by IdeSsuis vaccination in growers, the group of piglets most often affected by S. suis meningitis. Results of our ␣S. suis IgM ELISA with serum from colostrum-deprived piglets as negative control indicates that piglets in the field are generally not free of S. suis specific IgM antibodies (results not shown). Thus, we speculate that piglets of different ages might benefit from antibodies neutralizing the IgM protease IdeSsuis . IdeSsuis belongs to the IdeS family of Ig proteases [23]. The protective efficacy and immunogenicity of vaccination with the related IgG proteases has only marginally been investigated. Vaccination of mice with IdeE or IdeE2 elicited protection against S. equi subsp. equi challenge [24], but it is not clear if this protection is associated with neutralization of IgG protease activity. The results of this study are very encouraging with regard to the development of a rIdeSsuis vaccine, as complete protection against mortality was observed in a trial using the natural route of infection in the target species. As IdeSsuis is expressed by all investigated S. suis strains it is not unlikely that rIdeSsuis confers also protection against other serotypes. Experiments are planned to elucidate the protective efficacy of IdeSsuis against other S. suis pathotypes and to better understand the mechanism of protection.

[4]

[5]

[6] [7] [8]

[9]

[10]

[11]

[12]

[13]

Conflict of interest statement IDT Biologika GmbH applied for a European patent concerning an IdeSsuis -based S. suis vaccine (No. 14 170 637.4). Acknowledgements Lena-Maria Hillermann and Jana Seele contributed equally to this work. We thank H. Smith (DLO-Lelystad, Netherlands) for S. suis strains 10 and 10cpsEF. This study was financially supported by IDT Biologika GmbH and by the German Federal Ministry for Research and Education (BMBF) within the Helmholtz–CAS–Joint Research Group ZooStrep (HCJRG-116).

[14]

[15]

[16]

[17]

[18]

Appendix A. Supplementary data [19]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vaccine.2015.03.047. References [1] Gottschalk M. Streptococcosis. In: Zimmerman JJ, Kariker LA, Ramirez A, Schwartz KJ, Stevenson GW, editors. Diseases of swine. 10th ed. Oxford: WileyBlackwell; 2011. p. 841–55. [2] Gottschalk M, Segura M, Xu J. Streptococcus suis infections in humans: the Chinese experience and the situation in North America. Anim Health Res Rev 2007;8:29–45. [3] Wisselink HJ, Smith HE, Stockhofe-Zurwieden N, Peperkamp K, Vecht U. Distribution of capsular types and production of muramidase-released protein (MRP)

[20]

[21]

[22]

[23]

[24]

and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries. Vet Microbiol 2000;74:237–48. Silva LM, Baums CG, Rehm T, Wisselink HJ, Goethe R, Valentin-Weigand P. Virulence-associated gene profiling of Streptococcus suis isolates by PCR. Vet Microbiol 2006;115:117–27. Wei Z, Li R, Zhang A, He H, Hua Y, Xia J, et al. Characterization of Streptococcus suis isolates from the diseased pigs in China between 2003 and 2007. Vet Microbiol 2009;137:196–201. Gottschalk M, Xu J, Calzas C, Segura M. Streptococcus suis: a new emerging or an old neglected zoonotic pathogen. Future Microbiol 2010;5:371–91. Wertheim HF, Nghia HD, Taylor W, Schultsz C. Streptococcus suis: an emerging human pathogen. Clin Infect Dis 2009;48:617–25. Haesebrouck F, Pasmans F, Chiers K, Maes D, Ducatelle R, Decostere A. Efficacy of vaccines against bacterial diseases in swine: what can we expect. Vet Microbiol 2004;100:255–68. Baums CG, Brüggemann C, Kock C, Beineke A, Waldmann KH, Valentin-Weigand P. Immunogenicity of an autogenous Streptococcus suis bacterin in preparturient sows and their piglets in relation to protection after weaning. Clin Vaccine Immunol 2010;17:1589–97. Baums CG, Kock C, Beineke A, Bennecke K, Goethe R, Schröder C, et al. Streptococcus suis bacterin and subunit vaccine immunogenicities and protective efficacies against serotypes 2 and 9. ClinVaccine Immunol 2009;16:200–8. Kock C, Beineke A, Seitz M, Ganter M, Waldmann KH, Valentin-Weigand P, et al. Intranasal immunization with a live Streptococcus suis isogenic ofs mutant elicited suilysin-neutralization titers but failed to induce opsonizing antibodies and protection. Vet Immunol Immunopathol 2009;132:135–45. Jacobs AAC, Loeffen PLW, van den Berg AJG, Storm PK. Identification, purification, and characterization of a thiol-activated hemolysin (suilysin) of Streptococcus suis. Infect Immun 1994;62:1742–8. Wisselink HJ, Vecht U, Stockhofe-Zurwieden N, Smith HE. Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine. Vet Rec 2001;148:473–7. Pawel-Rammingen U, Johansson BP, Bjorck L. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 2002;21:1607–15. Seele J, Singpiel A, Spoerry C, Pawel-Rammingen U, Valentin-Weigand P, Baums CG. Identification of a novel host-specific IgM protease in Streptococcus suis. J Bacteriol 2013;195:930–40. Smith HE, Damman M, van der Velde J, Wagenaar F, Wisselink HJ, StockhofeZurwieden N, et al. Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infect Immun 1999;67:1750–6. Smith HE, de Vries R, van’t Slot R, Smits MA. The cps locus of Streptococcus suis serotype 2: genetic determinant for the synthesis of sialic acid. Microb Pathog 2000;29:127–34. Rehm T, Baums CG, Strommenger B, Beyerbach M, Valentin-Weigand P, Goethe R. Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background. J Med Microbiol 2007;56:102–9. Baums CG, Kaim U, Fulde M, Ramachandran G, Goethe R, Valentin-Weigand P. Identification of a novel virulence determinant with serum opacification activity in Streptococcus suis. Infect Immun 2006;74:6154–62. Büttner N, Beineke A, de Buhr N, Lilienthal S, Merkel J, Waldmann KH, et al. Streptococcus suis serotype 9 bacterin immunogenicity and protective efficacy. Vet Immunol Immunopathol 2012;146:191–200. Baums CG, Valentin-Weigand P. Surface-associated and secreted factors of Streptococcus suis in epidemiology, pathogenesis and vaccine development. Anim Health Res Rev 2009;10:65–83. Li Y, Martinez G, Gottschalk M, Lacouture S, Wilson P, Dubreuil JD, et al. Identification of a surface protein of Streptococcus suis and evaluation of its immunogenic and protective capacity in pigs. Infect Immun 2006;74:305–12. Rawlings ND, Waller M, Barrett AJ, Bateman A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 2014;42:D503–9. Hulting G, Flock M, Frykberg L, Lannergard J, Flock JI, Guss B. Two novel IgG endopeptidases of Streptococcus equi. FEMS Microbiol Lett 2009;298:44–50.