Protection against Streptococcus suis Serotype 2 Infection Using aCapsular Polysaccharide Glycoconjugate Vaccine

Guillaume Goyette-Desjardins,a Cynthia Calzas,a Tze Chieh Shiao,b Axel Neubauer,c Jennifer Kempker,c René Roy,b

Marcelo Gottschalk,d Mariela Seguraa

Laboratory of Immunology, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canadaa; Pharmaqam, Department of Chemistry, Université duQuébec à Montréal, Montreal, Quebec, Canadab; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, Missouri, USAc; Laboratory of Research on Streptococcus suis, Faculty ofVeterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canadad

Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine in-dustry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with otherencapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid,and the immunological features of the postconjugation preparations were evaluated in vivo. In mice, experiments evaluatingthree different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies,while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trialscomparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while25 �g of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosisassay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showedimmunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adju-vants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity invitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proofof concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections.

Streptococcus suis is a Gram-positive encapsulated bacteriumand one of the most important bacterial pathogens in the por-

cine industry, resulting in important economic losses (1). To date,35 S. suis serotypes have been described based on the capsularpolysaccharide (CPS) antigenic diversity. S. suis serotype 2 is con-sidered the most virulent; it is the serotype most frequently iso-lated from clinical samples and associated with disease in pigs inmost countries (2). S. suis, mainly serotype 2, is also an importantemerging zoonotic agent for humans in close contact with pigs orpig-derived products (2). The natural habitat of S. suis is the upperrespiratory tract of pigs, more particularly the tonsils and nasalcavities, as well as the genital and digestive tracts (3). Of the vari-ous manifestations of the disease, septicemia and meningitis areby far the most striking, but other clinical outcomes can also beobserved (4). Although the incidence of disease in swine variesover time and is generally less than 5%, mortality rates can reach20% in the absence of treatment (5). Affected animals are gener-ally between 5 and 10 weeks of age, but infections have also beenreported in newborn piglets to 32-week-old pigs (3).

The thick surface-associated S. suis CPS confers on the bacteriaprotection against the immune system, notably by resistingphagocytosis (6). As with most extracellular encapsulated bacte-ria, protection against S. suis is likely mediated by opsonizing an-tibodies which induce bacterial clearance by opsonophagocytosis.Anti-CPS antibodies have previously been demonstrated to beprotective against S. suis serotype 2 infection following passiveimmunizations (7–9).

Research has been ongoing for years in the hope of developingan efficient commercial vaccine to protect postweaning pigsagainst S. suis disease. Yet, to our knowledge, no such vaccine isavailable. Commercial or autogenous bacterins, which are sus-

pensions of heat-killed or formalin-killed bacteria, are used in thefield with limited success (1, 10–13). Other strategies have beenexperimentally tested, such as live strains and subunit vaccines.The use of live avirulent strains gave inconsistent results and maypresent some safety concerns (zoonosis) (11, 14, 15). After severalyears of research, there is still no proven and commercially avail-able protein-based subunit vaccine using well-characterized viru-lence factors and/or protective antigens (15). While some of thesecandidates failed, others are promising and still in early stages ofclinical investigations. Because this pathogen has a multifactorialvirulence mechanism and presents a relatively high phenotypicheterogeneity, vaccine development is a real challenge (2, 16).

As the CPS is the most external bacterial layer in contact withthe host, antibodies against it are highly opsonizing and protec-tive, as demonstrated for several encapsulated pathogens (17–20).Paradoxically, due to their carbohydrate nature, CPSs are in gen-eral considered poorly immunogenic and do not generate long-

Received 12 February 2016 Returned for modification 9 March 2016Accepted 20 April 2016

Accepted manuscript posted online 25 April 2016

Citation Goyette-Desjardins G, Calzas C, Shiao TC, Neubauer A, Kempker J, Roy R,Gottschalk M, Segura M. 2016. Protection against Streptococcus suis serotype 2infection using a capsular polysaccharide glycoconjugate vaccine. Infect Immun84:2059 –2075. doi:10.1128/IAI.00139-16.

Editor: L. Pirofski, Albert Einstein College of Medicine

Address correspondence to Mariela Segura, mariela.segura@umontreal.ca.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00139-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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lasting adaptive immune responses. Indeed, polysaccharides, un-like proteins and peptides, are generally recognized as T cell-independent (TI) antigens, which illustrate their innate inabilityto stimulate helper T (Th) cells via major histocompatibility com-plex (MHC) class II signaling, resulting in low immune cell pro-liferation, no antibody class switching or affinity/specificity mat-uration, and, more importantly, lack of immunological memory(21). The scientific advancement allowing for the widespread useof carbohydrate-based vaccines was the discovery that when prop-erly conjugated to protein carriers serving as T cell-dependent(TD) epitopes, polysaccharides become potent vaccine antigens(21). These vaccines were named “glycoconjugate vaccines.” Sinceit has been reported that anti-CPS antibodies, if produced, have ahigh protective potential against infection caused by S. suis (22,23), an interesting strategy for the development of an S. suis type 2vaccine would be the use of a glycoconjugate vaccine made ofCPS coupled to an immunogenic carrier protein, such as teta-nus toxoid (TT), diphtheria toxoid, cross-reactive material 197(CRM197), or many more (24). As a matter of fact, glycoconjugatevaccines are very successful in the fight against encapsulated hu-man pathogens such as Haemophilus influenzae (Hiberix), Neisse-ria meningitidis (MenACWY), and Streptococcus pneumoniae(PCV13) (20). Despite the popular use of glycoconjugate vaccinesin human medicine, this strategy has been poorly developed forveterinary practice. A CPS conjugate vaccine has been suggestedfor veterinary use against Actinobacillus pleuropneumoniae, theetiological agent of porcine pleuropneumonia (25–27).

In 2010, Van Calsteren et al. reported the exact structure of therepeating unit for the serotype 2 CPS (28). The CPS repeating unitis composed of a unique arrangement of 1 rhamnose, 1 glucose, 3galactoses, 1 N-acetylglucosamine, and 1 sialic acid (also namedN-acetylneuraminic acid [Neu5Ac]). The sialic acid (Neu5Ac) isfound to be terminal on a branch with an �2,6 linkage to a galac-tose. The precise knowledge of the S. suis serotype 2 CPS structureprovides the chemical bases for the construction of a glycoconju-gate. Thus, the main objective of this study was to explore thefeasibility/potential, immunogenicity, and protection in mice andin pigs of a CPS-TT conjugate vaccine against S. suis serotype 2.The effects of different adjuvants on the immunological featuresof the antibody response against the CPS were also studied.

MATERIALS AND METHODSAll chemical reagents used were of ACS grade or higher and were fromeither Sigma-Aldrich (Oakville, Ontario, Canada) or Acros Organics(Fisher Scientific, Ottawa, Ontario, Canada).

Bacterial strains and growth conditions. S. suis serotype 2 referencestrain S735 (ATCC 43765) was used as the source of type 2 CPS (28), as thetarget strain for in vitro opsonophagocytosis assays (OPA), and to preparethe heat-killed bacteria used to hyperimmunize mice. Isolated colonies onsheep blood agar plates were inoculated in 5 ml of Todd-Hewitt broth(THB) (Oxoid, Nepean, Ontario, Canada) and incubated for 8 h in awater bath at 37°C with agitation at 120 rpm. Working cultures wereprepared by transferring 10 �l of 8-h cultures diluted 1:1,000 with phos-phate-buffered saline (PBS) into 30 ml of THB, which was incubated for16 h. Bacteria were washed once and resuspended in PBS to obtain 5 � 108

CFU/ml. Heat-killed bacterial cultures were obtained as previously de-scribed (29). Briefly, overnight cultures were washed once with PBS andthen resuspended in 30 ml of fresh THB. A sample was taken to performbacterial counts on Todd-Hewitt agar (THA). Bacteria were immediatelykilled by incubating at 60°C for 45 min and then were cooled on ice.Bacterial killing was confirmed by absence of growth on blood agar for 48h. Strains used for the swine challenge model are described below.

Isolation and purification of type 2 S. suis CPS. Bacterial cultureswere performed as described by Calzas et al. (30). CPS extraction andpurification, followed by quality control procedures comprising proteindetermination with the modified Lowry protein assay kit (Pierce, Rock-ford, IL), nucleic acid quantification using an ND-1000 spectrometer(NanoDrop, Wilmington, DE), and one-dimensional (1D)/2D 1H nu-clear magnetic resonance (NMR) analysis to ensure purity and identitywere performed as described by Van Calsteren et al. (28).

Depolymerization of type 2 CPS. Twenty milliliters of a 2-mg/mlsolution of CPS in 50 mM NH4HCO3 was transferred to a 50-ml conicalpolypropylene tube in an ice bath. A titanium 1/8-in. microtip probemounted on a Virsonic 600 sonicator (VirTis, Gardiner, NY) was im-mersed in the CPS solution, and sonication was performed at 20 kHz and24 W for 60 min. After sonication, a sample of CPS was taken to determinethe weight-average molar mass (Mw) by size exclusion chromatographycoupled with multiangle light scattering (SEC-MALS) as previously de-scribed (31), with some modifications. Briefly, the chromatographic sep-aration was performed with two 8- by 300-mm Shodex OHpak gel filtra-tion columns connected in series (SB-806 and SB-804), preceded by aSB-807G guard column (Showa Denko, Tokyo, Japan). Elution was doneat 0.5 ml/min using 0.1 M NaNO3 as the mobile phase. Molar masses weredetermined using a Dawn EOS MALS detector (Wyatt, Santa Barbara,CA), and calculations were performed with the ASTRA software version6.1.1.17 (Wyatt) using 11 detectors from angle 34.8° to 132.2° (detectors 5to 15) for the depolymerized samples. The remaining solution of depo-lymerized CPS was dialyzed against water (Spectra/Por; molecular weightcutoff [MWCO], 3,500 [Spectrum Laboratories, Rancho Dominguez,CA]) and lyophilized. The optimal conditions for sonication were deter-mined in pretests using different time points (see Fig. S1 in the supple-mental material).

Mild periodate oxidation of depolymerized CPS. Depolymerized S.suis serotype 2 CPS (8.8 mg, 6.7 �mol) was incubated with 620 �M so-dium periodate in 1.1 ml of water in the dark with a stirring magnet atroom temperature for 1 h. An excess of two equivalents of triethyleneglycol per periodate was added and left for 1 h to consume any residualperiodate. The mixture was dialyzed against water (Spectra/Por; MWCO,1,000 [Spectrum Laboratories]) and lyophilized. The optimal conditionsfor oxidation were determined in preliminary tests (data not shown).

The degree of oxidation of the sialic acid (Neu5Ac) residues was as-sessed by gas chromatography (GC) analysis of the peracetylated methylglycosides adapted from a previously described method (32). Briefly, ox-idized CPS (0.4 mg) was reduced by treatment with 100 �l of NaBH4 (10mg/ml) in water for 1 h at room temperature. The reaction was quenchedwith 5% acetic acid solution in methanol and evaporated to dryness usinga stream of N2. Evaporations were repeated 3 times by the addition of 250�l of methanol each time. The composition of the residue was determinedby methanolysis. To this end, methanol (465 �l) and acetyl chloride (35�l), which generate HCl, were added to the residue. The solution washeated for 17 h at 75°C and evaporated to dryness, followed by addition of500 �l of tert-butanol and evaporation to dryness again. The methyl gly-cosides were acetylated with 150 �l of pyridine and 150 �l of acetic anhy-dride at 100°C for 20 min. The cooled solution was partitioned with 5 mlof water and 1 ml of CH2Cl2. The organic layer containing the peracety-lated methyl glycosides was analyzed by GC using flame ionization detec-tion (GC-FID). GC-FID analysis was done on a Hewlett-Packard model7890 gas chromatograph equipped with a 30-m-by-0.32-mm (0.25-�mparticle size) HP-5 capillary column (Agilent Technologies, Santa Clara,CA) using the following temperature program: 50°C for 2 min, an increaseof 30°C/min to 150°C, then an increase of 3°C/min to 230°C, and a holdfor 5 min. The temperatures of the injector and the flame ionization de-tector were 225°C and 250°C, respectively.

Purification of TT monomer. Tetanus toxoid (TT) monomer wasobtained by gel filtration chromatography before conjugation. One mil-liliter of a liquid preparation containing 4.5 mg/ml protein (as determinedby the modified Lowry protein assay) was loaded onto a XK16-100 col-

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umn filled with Superdex 200 Prep Grade (GE Healthcare Life Sciences,Uppsala, Sweden) equilibrated in PBS (20 mM NaHPO4 [pH 7.2], 150mM NaCl) and eluted with the same buffer. The protein eluted from thecolumn in two peaks: the earlier-eluting peak contained oligomerizedtoxoid, and the later-eluting peak, corresponding to a Mr of 150,000,contained TT monomer. Fractions corresponding to the later (monomer)peak were pooled, desalted against deionized water, concentrated using aCentricon Plus-70 centrifugal filter device (30K Ultracel PL membrane;Millipore, Billerica, MA), and then lyophilized.

Conjugation of type 2 CPS to TT by reductive amination. Periodate-treated type 2 CPS (3.6 mg, 40 nmol) and purified TT monomer (3.0 mg,20 nmol) were dissolved in 2.2 ml of 0.1 M sodium bicarbonate (pH 8.1)for the 2:1 conjugate ratio. Sodium cyanoborohydride (7.5 mg, 120 �mol)was added, and the mixture was incubated at 37°C with orbital agitationfor 2 days. For the 1:1 conjugate ratio, conjugation was performed in thesame manner as described above except with 1.8 mg (20 nmol) of oxidizedCPS. Sodium borohydride (4.7 mg, 124 �mol) was then added to thereaction mixture to reduce any remaining free aldehyde groups. The pro-duced 2:1 and 1:1 mixed conjugates were extensively dialyzed againstwater (Spectra/Por; MWCO, 3,500 [Spectrum Laboratories]) and lyoph-ilized. Conjugation was controlled by gel shift on SDS-PAGE, immuno-blotting, and high-performance liquid chromatography (HPLC) as de-scribed below. The conditions for conjugation by reductive aminationwere determined in pretests using different CPS-to-TT ratios, differentpercentages of CPS oxidation, and different incubation times (data notshown).

SDS-PAGE and immunoblotting. Samples were diluted in twice-con-centrated SDS-PAGE loading buffer containing 2-mercaptoethanol, de-natured by heating at 100°C for 5 min, and separated by SDS-PAGE on7.5% acrylamide gels. The gels either were stained with Coomassie G-250stain or a silver stain kit (Bio-Rad Hercules, CA) or were transferred tonitrocellulose Western blot membrane (Bio-Rad). The membrane wasthen blocked with a solution of Tris-buffered saline (TBS) (10 mM Tris-HCl [pH 7.4], 150 mM NaCl) containing 2% skim milk for 1 h. Themembrane was washed 3 times with TBS and incubated for 2 h with eithera mouse monoclonal antibody (MAb) against serotype 2 CPS of S. suis(clone Z3) (23) diluted 1:50 or a mouse MAb against TT (clone HYB278-01; Abcam, Toronto, Ontario, Canada) diluted 1:500 in blocking buf-fer. The membrane was then washed 3 times with TBS and incubated for1 h with a goat anti-mouse IgG-IgM(H�L) horseradish peroxidase(HRP)-conjugated antibody (Jackson ImmunoResearch, West Grove,PA) either diluted 1:3,000 for CPS or diluted 1:1,000 for TT in blockingbuffer. The membrane was washed 3 times with TBS and developed witha 4-chloro-1-naphthol solution (Sigma-Aldrich).

HPLC analysis of the conjugates. HPLC analysis of the glycoconju-gate preparations was done by size exclusion chromatography. The chro-matographic separation was performed with three 8- by 300-mm ShodexOHpak gel filtration columns connected in series (two SB-804 and oneSB-803) preceded by a SB-807G guard column (Showa Denko). The gly-coconjugate vaccine was eluted with 0.1 M NaNO3 at a flow rate of 0.4ml/min using a Knauer Smartline system equipped with a differentialrefractometer (RI) detector model 2300 and a UV detector model 2600 ata wavelength of 280 nm. The conjugate preparation (8-mg/ml solution inthe mobile phase) was injected using a 50-�l injection loop. In selectedexperiments (see below), the fractions eluting at the void volume, whichcorrespond to the conjugate fractions, were pooled, dialyzed against water(Spectra/Por; MWCO, 12,000 to 14,000 [Spectrum Laboratories]), andlyophilized. This corresponds to the 2:1 fractionated conjugate.

Mouse immunizations. All experiments involving mice were con-ducted in accordance with the guidelines and policies of the CanadianCouncil on Animal Care and the principles set forth in the Guide for theCare and Use of Laboratory Animals by the Animal Welfare Committee ofthe University of Montreal. Five- to 6-week-old C57BL/6 female mice(Charles River, Wilmington, MA), largely used for studies on S. suispathogenesis (17, 33–35), were immunized subcutaneously with different

doses of the S. suis CPS conjugate preparations (see Results) in 0.1 ml PBSon day 0 and boosted on day 21. In a first set of experiments aimed tocompare different adjuvants, 3 groups (n � 10) received 25 �g of the 2:1mixed conjugate dissolved in PBS adjuvanted with either 20 �g of CpGoligodeoxyribonucleotide (ODN) 1826 (InvivoGen, San Diego, CA),Stimune (Prionics, La Vista, NE), or TiterMax Gold (CytRx Corporation,Norcross, GA) following the manufacturers’ recommendations. Threeplacebo groups (n � 5) received only PBS adjuvanted as described above.In a second set of experiments, a dose-response study was performedusing groups of mice (n � 8) immunized with either 1, 2.5, 5, or 25 �g ofthe 2:1 mixed conjugate emulsified 1:1 (vol/vol) with TiterMax Gold.Mice (n � 8) immunized with similar doses of free (unconjugated) depo-lymerized CPS emulsified with TiterMax Gold were included for compar-ison purposes. A placebo group (n � 5) was also included. In a third set ofexperiments, to compare the efficiencies of different conjugates, groups ofmice (n � 10) received 25 �g of either the 1:1 mixed conjugate, the 2:1fractionated conjugate, or a free (unconjugated) mixture of 2 CPS to 1 TT.All preparations were emulsified with TiterMax Gold, and a placebogroup was also included.

In all experiments, to follow antibody responses, mice were bled (10�l) weekly on days �1, 7, 14, 21, 28, 35, and 41 postimmunization by thetail vein. Diluted blood was directly used in the enzyme-linked immu-nosorbent assay (ELISA) as described below. At day 42 postimmuniza-tion, mice were humanely euthanized and sera collected and frozenat �80°C for ELISA Ig titration and isotyping and for OPA analyses (seebelow).

Immunization and challenge of pigs. Animals were treated in accor-dance with the ethical guidelines of the Institutional Animal Care and UseCommittee of Boehringer Ingelheim Vetmedica, Inc. A total of 44 3-week-old piglets (�5 days) were obtained from a commercial herd in Nebraska,USA, for this study. Tonsil swabs from the study piglets were negative byS. suis serotype 2 PCR prior to study initiation (2). The piglets wereblocked by litter and then randomly assigned to one of four groups by abiostatistician using SAS version 9.3: group 1, n � 14; group 2, n � 10,group 3, n � 15; and group 4, n � 5. Groups 1 to 4 were commingled untilgroup 4 (strict control) was removed at study day 35. Blood samples werecollected on study days 0, 21, and 34 for determination of serum antibodylevels.

The vaccines and the placebo were adjuvanted with Stimune accord-ing to the manufacturer’s instructions. The piglets were injected intra-muscularly twice at a 3-week interval (study days 0 and 21) with 2 ml of thevaccine or placebo: group 1 was vaccinated with formalin-inactivated,adjuvanted S. suis type 2 culture containing 2 � 1010 bacteria (isolate3977B, a virulent field isolate of S. suis serotype 2 previously used in abacterin efficacy study [A. Neubauer, unpublished]), group 2 was injectedwith the adjuvanted 2:1 mixed conjugate vaccine containing either 85 �g(first vaccination) or 56 �g (second vaccination) of the antigen, and group3 was given 2 ml of adjuvanted PBS.

On day 36, groups 1 to 3 were challenged intraperitoneally with 2 ml(3 � 109 CFU/dose) of a late-exponential-phase S. suis type 2 culturegrown in THB with 5% fetal bovine serum. The animals were challengedwith isolate ATCC 700794, which is the established challenge strain in themodel used. Following challenge, pigs were monitored daily over a periodof 7 days for the presence of clinical signs. The individuals observing theanimals were blinded to the treatments. Assessed were behaviors, includ-ing those indicating functional alteration of the central nervous system(CNS), and locomotion. The observations for general behavior were nu-merically scored as follows: 0, physiological; 1, depression; and 2, apathy.Observations for locomotion were scored as follows: 0, physiological; 1,slightly to moderately lame; 2, severely lame/reluctant to stand; and 3,animal partially/completely down, i.e., animals can rise but lie down againwithin 10 s. CNS signs were scored as follows: 0, absent; and 1, present. Inaccordance with the humane endpoints defined in the animal use protocolas well as 9CFR 117.4 (36), animals unresponsive to stimuli, animals ex-hibiting CNS signs, and animals that were assigned a lameness score of 3

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were humanely euthanized. At study day 43, all remaining animals werehumanely euthanized.

All animals found dead or that had been euthanized were necropsied.Meninges, pericardium, and joint swabs were collected and streaked outon Columbia blood agar plates. Alpha-hemolytic colonies present on theplates following overnight incubation were tested with type 2 antisera and,as deemed necessary, by S. suis type 2 PCR. Gross pathology observationswere recorded for the thoracic cavity (presence of fibrin, fluid, congestion,and pericarditis), and for the joints (presence of fibrin and fluid). Theindividuals conducting the necropsies were blinded to the treatments.

Control mouse antiserum. Hyperimmune mice (n � 6) were ob-tained by repeated immunization of 5-week-old female C57BL/6 micewith 7.5 � 108 CFU/ml heat-killed S. suis serotype 2 strain S735 in THB byintraperitoneal injection on days 0, 7, 21, and 28. On day 42, serum wascollected, pooled, aliquoted, and stored at �80°C.

Measurement of antibodies against type 2 S. suis CPS and TT. Tomeasure specific antibodies, 200 ng of either native S. suis serotype 2 CPSor TT in 0.1 M NaCO3 (pH 9.6) were added to wells of an ELISA plate(Nunc-Immuno Polysorp; Canadawide Scientific, Toronto, Ontario,Canada). After overnight coating at 4°C, plates were washed with PBScontaining 0.05% (vol/vol) Tween 20 (PBST) and blocked by treatmentwith PBS containing 1% (wt/vol) bovine serum albumin (BSA) (HyClone,Logan, UT) for 1 h. After washing, mouse blood or mouse/porcine serumsamples diluted in PBST were added to the wells and left for 1 h. Afterwashing, the plates were incubated for 1 h with an HRP-conjugated iso-type specific antibody diluted in PBST as described below. The enzymereaction was developed by addition of 3,3=,5,5=-tetramethylbenzidine(TMB) (Invitrogen, Burlington, Ontario, Canada) and stopped by addi-tion of 0.5 M H2SO4, and the absorbance was read at 450 nm with anELISA plate reader.

To follow the kinetics of total (IgG plus IgM) antibody responses toCPS and TT, mouse blood collected from the tail vein was diluted1:100 or 1:20,000, respectively. Dilution optimization had previouslybeen conducted (data not shown). HRP-conjugated goat anti-mouseIgG-IgM(H�L) at a dilution of 1:2,500 (Jackson ImmunoResearch)was used as a detection antibody.

To perform the titration of mouse Ig isotypes, day 42 serum was seri-ally diluted (2-fold) in PBST, and antibodies were detected using eitherHRP-conjugated goat anti-mouse IgG plus IgM as mentioned above, goatanti-IgM diluted 1:1000, goat anti-IgG1, goat anti-IgG2b, goat anti-IgG2c, or goat anti-IgG3 diluted 1:400 (Southern Biotech). For porcineserum, 2-fold serial dilutions were performed in PBST and antibodieswere detected using HRP-conjugated goat anti-swine total Ig (IgG plusIgM) diluted 1:4,000 (Jackson ImmunoResearch). To detect porcine IgGsubclasses, unconjugated mouse anti-swine IgG1 or mouse anti-swineIgG2 (AbD Serotec, Raleigh, NC) diluted 1:250 was added, followed byincubation with HRP-conjugated goat anti-mouse secondary antibody.For both mouse and pig serum titration, the reciprocal of the last serumdilution that resulted in an optical density at 450 nm (OD450) equal to orlower than 0.2 (as a preestablished cutoff for comparison purposes) wasconsidered the titer of that serum. For representation purposes, negativetiters (less than or equal to the cutoff) were given an arbitrary titervalue of 10.

To control interplate variations, an internal reference positive controlwas added to each plate. For titration of mouse antibodies, this controlwas a pool of sera from hyper-immunized mice (produced as describedabove). For titration of pig antibodies, this control was a serum of a pighyper-immunized with 108 CFU of a killed suspension of S. suis serotype2. Reaction in TMB was stopped when an OD450 nm of 1 was obtained forthe positive internal control. Optimal dilutions of the coating antigen(CPS or TT), the positive internal control sera and the HRP-conjugatedanti-mouse or anti-pig antibodies were determined during preliminarystandardizations.

OPA. Blood was collected by intracardiac puncture from naiveC57BL/6 mice, treated with sodium heparin, and then diluted to obtain

6.25 � 106 leukocytes/ml in RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum, 10 mM HEPES, 2 mM L-glutamine, and 50�M 2-mercaptoethanol. All reagents were from Gibco (Invitrogen, Burl-ington, Ontario, Canada). All blood preparations were kept at room tem-perature. Using washed bacterial cultures grown as described above, finalbacterial suspensions were prepared in complete cell culture medium toobtain a concentration of 1.25 � 106 CFU/ml. The number of CFU/ml inthe final suspension was determined by plating samples onto THA usingan Autoplate 4000 automated spiral plater (Spiral Biotech, Norwood,MA). All bacterial preparations were kept on ice. Diluted whole blood at5 � 105 leukocytes was mixed with 5 � 104 CFU of S. suis (multiplicity ofinfection [MOI] of 0.1) and 40% (vol/vol) of serum from naive or vacci-nated mice in a microtube to a final volume of 0.2 ml. The tube tops werepierced using a sterile 25-gauge needle, and then the microtubes wereincubated for 2 h at 37°C with 5% CO2, with gentle manual agitation every20 min. After incubation, viable bacterial counts were performed on THAusing an Autoplate 4000 automated spiral plater. Tubes with addition ofnaive rabbit serum or rabbit anti-S. suis type 2 strain S735 serum (37) wereused as negative and positive controls, respectively. The bacterial killingpercentage was determined using the following formula: percentage ofbacteria killed � [1 � (bacteria recovered from sample tubes/bacteriarecovered from negative-control tubes with naive mouse sera)] � 100.Final OPA conditions were selected based on several pretrials using dif-ferent incubation times and MOIs (38).

Statistical analyses. All data are expressed as means � standard errorsof the means (SEM). Data were analyzed for significance using analysis ofvariance (ANOVA) from SigmaPlot version 11.0, except for the survivalcurves analysis, which was performed using the log rank test from Graph-Pad version 5.01. Significance is denoted in the figures as follows: *, P 0.05; **, P 0.01; and ***, P 0.001.

Summaries of and data analyses for the pig study were conducted by abiostatistician using SAS version 9.3. Clinical observations (death, lame-ness, CNS signs, and behavioral changes) were summarized as frequenciesby day and treatment. The incidence of normal versus not normal for eachcharacteristic was analyzed, where appropriate, using the GLIMMIX pro-cedure of SAS with binomial error and logit link. The model included thefixed effect of treatment and the random effects of litter and residual. Inaddition, the proportion of the observations for each animal that were notnormal was analyzed. Prior to analysis, the proportion was transformedusing the arcsine square root transformation. The mixed model includedthe fixed effect of treatment and the random effects of litter and residual.Comparisons of interest include the following and were evaluated using atwo-sided test with alpha � 0.05: groups 1 (bacterin) and 2 (conjugate)versus 3 (protection provided against challenge with isolate ATCC700794).

RESULTSPreparation of the conjugate vaccines. Using highly purified CPSfrom S. suis type 2 containing less than 1% (wt/wt) of proteins ornucleic acids (as previously described by Calzas et al. [30]), weinvestigated whether conjugation to a carrier protein, such as TT,would circumvent the TI antigenicity of the CPS and instead in-duce a TD protective humoral response in animals.

Due to its high Mw, found to be between 410,000 and 480,000Da by SEC-MALS (28, 30), the native polysaccharide of S. suis type2 must first be depolymerized into smaller fragments. This re-duces polydispersity and yields higher ratios of polysaccharide toprotein following coupling. To perform this depolymerization, weopted for ultrasonic irradiation as described by Szu et al. (39). Bymonitoring the CPS Mw of samples by SEC-MALS during pretests,it was shown that depolymerization plateaued after 45 min ofsonication (see Fig. S1 in the supplemental material). Based onthese observations, we selected a depolymerization time of 60 minof sonication, at which two different lots were produced with re-

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producible results, giving an average Mw of 115,000 Da (113,000to 118,000 Da) (see Table S1 in the supplemental material). 1HNMR investigations of these two lots found no structural altera-tion of the polysaccharide other than the depolymerization itself(data not shown). These depolymerized CPSs were used in thesubsequent preparation of the conjugate vaccine formulations.

The presence of sialic acid (Neu5Ac) as a constituent in therepeating-unit sequence of S. suis serotype 2 CPS allowed the useof mild conditions in order to achieve an oxidative cleavage be-tween C-8 and C-9 of the glycerol side chain, thus leaving freeterminal aldehydes as reacting groups for subsequent conjugationto TT by reductive amination (40). To preserve CPS immunoge-nicity, a 10% level of oxidation was targeted, leaving 90% of allNeu5Ac untouched. For an 115,000-Da CPS, this resulted in anaverage of 9 oxidized Neu5Ac per chain. Following pretests, 0.1equivalent of sodium periodate per Neu5Ac was selected, and thetwo different CPS lots were oxidized. Reproducible oxidation lev-els at C-8 of 9.2 to 9.4% were obtained as determined by GC-FIDanalysis of the peracetylated methyl glycosides. No oxidation atC-7 was observed under these conditions. 1H NMR investigationsfound no other structural modification of the polysaccharide(data not shown).

The depolymerized-oxidized CPS and purified TT monomerwere conjugated at a molar ratio of 2 chains of CPS to 1 TT or at amolar ratio of 1:1 by reductive amination (41). The optimal incu-bation time for conjugation was found to be 2 days during pretests(data not shown). After incubation, remaining aldehyde groupswere reduced by the addition of sodium borohydride. Reagentswere then eliminated from the conjugate mixes by extensive dial-ysis against water.

Gel shift, Western blotting, and HPLC analysis confirm suc-cessful conjugation of CPS to TT. The presence of conjugates inthe different preparations was verified by gel shift and Westernblot experiments (Fig. 1) and by HPLC analysis (Fig. 2). For the gelshift experiments, both Coomassie blue (Fig. 1A) and silver (Fig.1B) staining showed a considerable shift from the purified TTmonomer at 150 kDa (lanes 2) to a thick band of over 250 kDa inthe conjugates (lanes 3 and 4). This shift resulted from the cova-lent addition of a random number of 115-kDa CPS chains to theprotein. Interestingly, the selected silver stain kit included one stepof dichromate oxidation, favoring a higher-intensity signal fromglycoproteins (42). Accordingly, a strong reaction was observedwith the conjugate preparations (Fig. 1B, lanes 3 and 4). NeitherCoomassie blue (Fig. 1A), silver staining (Fig. 1B), nor Western

FIG 1 SDS-PAGE and Western blot characterization of the glycoconjugates. Separating gels containing 7.5% acrylamide either were stained with Coomassie bluestain (A) or silver stain (B) or were transferred onto a nitrocellulose membrane and revealed by immunoblotting with either an anti-S. suis serotype 2 capsularpolysaccharide (CPS) monoclonal antibody (MAb) (C) or an anti-tetanus toxoid (TT) MAb (D). Lanes 1, 10 �l of SeeBlue Plus 2 prestained protein standard(Ladder; Invitrogen); lanes 2, 10 �g of TT purified monomer; lanes 3, 10 �g of the 2:1 mixed conjugate; lanes 4, 10 �g of the 1:1 mixed conjugate; lanes 5, 10 �gof free depolymerized S. suis serotype 2 CPS.

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blot using an anti-CPS MAb (Fig. 1C) revealed any band for thedepolymerized CPS included as a control in all gels (lanes 5), il-lustrating its weak capacity to migrate through the gels underthese assay conditions. As such, the positive signal observed for thebands of �250 kDa when revealed using an anti-CPS MAb defi-nitely proved the covalent nature of the linkage between CPS andTT in the conjugates (Fig. 1C, lanes 3 and 4). Control stainingusing an anti-TT MAb (Fig. 1D) shows that the epitope to whichthe MAb binds was preserved in the conjugates. Preservation ofTT antigenicity is essential, since it is the key mechanism allowingfor the production of a T cell-dependent anti-CPS humoral re-sponse. It should be noted that differences in signal intensitiesbetween the 2:1 and 1:1 mixed conjugates (Fig. 1, lanes 3 and 4) arelikely related to the total amounts of protein content (4.5 �g ver-sus 6.3 �g, respectively) within the 10 �g of sample loaded perlane.

HPLC analysis (Fig. 2) showed the elution of the conjugate(�250 kDa), of free CPS (100 kDa), and of free TT (150 kDa). Byintegrating the UV signal at 280 nm from the chromatograms, itwas estimated that 48% � 6% (mean � standard deviation [SD])of the protein content from the mixture is indeed found in theconjugate fraction. Taken together, the gel shift and Western blotexperiments combined with HPLC analysis of the two conjugatesamples revealed the presence of conjugates in the 2 CPS:1 TT and1:1 preparations.

Emulsifying adjuvants show higher immunomodulatoryproperties than CpG ODN for a polysaccharide antigen. Usingthe 2:1 mixed conjugate, optimization of the immunization pro-tocol was performed in a murine model using inbred C57BL/6mice. The performances of three different adjuvants were com-pared. CpG ODN is a synthetic version of a bacterial oligonucle-otide with unmethylated CpG motifs and acts as a Toll-like recep-tor 9 (TLR9) ligand with immunostimulatory properties toward aTh1 response (43). Stimune (Specol) is a water-in-oil adjuvantcomposed of purified and defined mineral oil (Markol 52) withSpan 85 and Tween 85 as emulsifiers (44). It has been used as agood alternative to Freund’s adjuvant for weak immunogens inanimals, such as mice and pigs (45). TiterMax Gold is also a water-in-oil adjuvant consisting of squalene as a metabolizable oil, sor-bitan monooleate 80 as an emulsifier, and CRL8300 (a patentedblock copolymer) and microparticulate silica as stabilizers (44).TiterMax Gold has been suggested as a superior alternative to

Freund’s adjuvant, providing comparable titers with fewer injec-tions and less undesired reactivity in mice (46).

Based on the literature (47), a dose of 20 �g of CpG was chosento be added for adjuvanting. In parallel, conjugates were emulsi-fied with recommended ratios of 4 parts aqueous antigen per 5parts adjuvant for Stimune or 1:1 for TiterMax Gold. Doses of 25�g of the 2:1 mixed conjugate vaccine for each adjuvant wereadministered to mice on days 0 and 21. The kinetics of total IgG-plus-IgM antibody responses against CPS or TT were followedweekly in tail vein blood samples (Fig. 3). Overall, CpG ODN 1826(Fig. 3A) gave the lowest anti-CPS and anti-TT responses. Also,anti-CPS Ig isotyping showed a strict IgM isotype response (datanot shown). In contrast, Stimune (Fig. 3B) and TiterMax Gold(Fig. 3C) gave comparably strong anti-CPS and anti-TT total IgG-plus-IgM responses. Furthermore, anti-CPS Ig isotype switchingwas observed with both emulsifying adjuvants (see below). Al-though a memory antibody response against TT was observedwith all three adjuvants, Stimune and TiterMax Gold inducedfaster and higher anti-CPS antibody levels after boosting, suggest-ing that generation of immunological memory against the CPSantigen is favored by these emulsifying adjuvants. Finally, itshould be noted that no placebo mice, injected with only PBS andadjuvant, produced any nonspecific antibody response (data notshown).

As TiterMax Gold is recognized as one of the best adjuvants formice (48, 49), it was selected for further immunizations with thisspecies. Because its response is comparable to that of TiterMaxGold and it has been previously evaluated in large animals (49,50), Stimune was selected as the adjuvant for immunization ofpigs.

While the free polysaccharide is nonimmunogenic, a dose-response effect on antibody levels is observed with the conjugatevaccine. Using TiterMax Gold as the adjuvant, mice were immu-nized on days 0 and 21 with doses of 1, 2.5, 5, or 25 �g of the 2:1mixed conjugate to evaluate the dose-response effect on antibodyproduction. Groups of mice were also immunized with differentdoses of S. suis serotype 2 free (unconjugated) CPS to assess if itcould be immunogenic by itself when adjuvanted with TiterMaxGold.

Even at a high dose (25 �g) of free CPS, no significant totalIgG-plus-IgM primary or memory antibody responses were ob-served throughout the immunization period (Fig. 4). In contrast,a dose-response effect was observed when mice were immunizedwith the 2:1 mixed conjugate, with the 25-�g dose yielding thehighest total IgG-plus-IgM anti-CPS antibody response as mea-sured in blood samples collected weekly.

Conjugation of S. suis type 2 CPS to TT induces antibodyisotype switching in mice. Not only a stronger response followingboosting (as illustrated in Fig. 3 and 4) but also antibody isotypeswitching is a good indicator of conjugate immunogenicity andability to induce a T cell-dependent response. Therefore, titers ofthe different anti-CPS antibody isotypes were determined in miceimmunized with 25 �g of the 2:1 mixed conjugate vaccine adju-vanted with TiterMax Gold. As shown in Fig. 5, not only strongIgM titers but also high levels of all IgG subclasses were observed,including IgG1, IgG2b, IgG2c, and IgG3 specific for the CPS an-tigen. To evaluate if isotype switching was dependent on the ad-juvant, serum samples from mice immunized with 25 �g of the 2:1mixed conjugate adjuvanted with Stimune were analyzed.Stimune also induced isotype switching in mice; however, levels

FIG 2 HPLC elution profile of the 2:1 mixed conjugate. Differential refrac-tometer (RI) (solid line) and UV detection (dotted line) profiles are shown.Similar profiles were observed for the 1:1 mixed conjugate.

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were lower and profiles differed from those observed with Titer-Max Gold, with no production of the IgG2c and IgG3 subclasses(Fig. 6A).

In order to determine the effect of the CPS-to-TT ratio on theconjugate immunogenicity, another mixed conjugate, this timeusing a ratio of 1 CPS to 1 TT, was prepared (Fig. 1) and emulsifiedin TiterMax Gold. Immunized mice showed similar IgM titers,reduced (but not significantly different) IgG1 and IgG3 titers, andsignificantly lower IgG2b titers (P 0.01) than those induced bythe 2:1 mixed conjugate in TiterMax Gold (Fig. 5). Interestingly,the 1:1 mixed conjugate failed to induce significant titers of theIgG2c subclass.

In order to prove that the observed immunogenicity was in factdue to the conjugate present in the vaccine formulation and notonly due to remaining free CPS and TT, two additional controlswere included in the study. A first control was the HPLC-isolatedspecific fraction corresponding to the conjugate from the 2:1mixed conjugate (2:1 fractionated conjugate). The second control

was a mixture of free CPS and free TT in the same ratio of 2:1 asbefore conjugation. In general, no major differences were ob-served between the 2:1 mixed conjugate and the specific 2:1 frac-tionated conjugate, yet higher titers of IgG1, IgG2b, and IgG3 wereobserved with the latter preparation (Fig. 5) (P 0.01). In con-trast, the mixture of unconjugated CPS and TT gave a strong IgMtiter but very low titers of IgG1 compared to those with the 2:1mixed conjugate (P 0.01). In addition, no production of IgG2csubclass was observed in mice immunized with this control un-conjugated preparation.

Finally, the control hyperimmune sera (from mice repeatedlyinjected with heat-killed bacteria) resulted in a high titer of IgM,production of IgG2b and IgG2c, but absence of IgG1 and IgG3subclasses against the CPS antigen (Fig. 5). Similar results wereobtained when mice were hyperimmunized with heat-killed bac-teria adjuvanted in TiterMax Gold (data not shown).

Functional activity of antibodies. The protective capacity ofsera from immunized mice was evaluated using an OPA test, a

FIG 3 Kinetics of total antibody responses of mice immunized with 25 �g of the 2:1 mixed conjugate adjuvanted with either CpG (A), Stimune (B), or TiterMaxGold (C). Mice (n � 10) were immunized on day 0 and boosted on day 21. ELISA plates were coated with either native capsular polysaccharide (CPS) or tetanustoxoid (TT) and incubated with blood samples diluted 1:100 or 1:20,000 to measure anti-CPS and anti-TT antibodies, respectively. Total (IgG plus IgM) antibodylevels are shown for individual mice, with horizontal bars representing means (�SEM) of values for optical density at 450 nm. The arrow at day 21 indicates theboost. To simplify the graph, kinetics for the respective placebo groups (which were all negative) are not shown.

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recognized correlate of immunity for encapsulated Gram-positivebacteria, such as S. pneumoniae (51). Instead of using a cell line ora single cell type, the OPA was standardized using whole bloodfrom naive mice. This model takes into account all blood leuko-cytes and thus represents a more realistic model of the complexinteractions between all immune cells and the bacteria during asystemic infection, as is the case for S. suis. As shown in Fig. 7, serafrom mice immunized with the 2:1 mixed conjugate adjuvantedwith TiterMax Gold induced high bacterial killing levels ranging

from 64 to 77%. Sera from mice immunized with the 2:1 fraction-ated conjugate gave higher, but not significantly different, bacte-rial killing values ranging from 74 to 98% (Fig. 7). The effect of theadjuvant was also evaluated in the OPA test; sera from mice im-munized with the 2:1 mixed conjugate adjuvanted with Stimuneinduced bacterial killing levels ranging from 39 to 74% (Fig. 6B),which were not significantly different from those induced by Ti-terMax Gold (P � 0.05). When the OPA was performed using serafrom mice immunized with the unconjugated CPS and TT mix-

FIG 4 Dose-response effect on total antibody levels of mice immunized with either free depolymerized capsular polysaccharide (CPS) or 2:1 mixed conjugateat 1, 2.5, 5, or 25 �g adjuvanted with TiterMax Gold. Mouse groups (n � 8) were injected on day 0 and boosted on day 21. ELISA plates were coated with nativeCPS and incubated with blood samples diluted 1:100. Total (IgG plus IgM) anti-CPS antibody levels are shown for individual mice, with horizontal barsrepresenting means (�SEM) of values for optical density at 450 nm. The arrow at day 21 indicates the boost.

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FIG 5 Titers of different anti-CPS antibody isotypes in mice immunized with conjugate vaccines adjuvanted in TiterMax Gold. Mouse groups were as follows:placebo (n � 5), 2:1 mixed conjugate (n � 18, animals from the 2 previous immunizations with 25 �g), 1:1 mixed conjugate (n � 10), 2:1 fractionated conjugate(n � 10), and 2 CPS:1 TT unconjugated control mixture (n � 10). All mice were immunized with 25 �g of antigen in TiterMax Gold on day 0 and boosted onday 21, and sera were collected at day 42. A pool of hyperimmune mouse sera from 6 mice was also included for comparative purposes. For the titration, ELISAplates were coated with native CPS and incubated with 2-fold serial dilutions of sera. Isotypes were detected using specific HRP-conjugated anti-mouse IgG plusIgM, IgM, IgG1, IgG2b, IgG2c, or IgG3 antibodies. Titers for individual mice are shown, with horizontal bars representing means � SEM. #, titers significantlydifferent from those of the placebo group (P 0.05). Differences between other groups are denoted as follows: **, P 0.01; ***, P 0.001. Abbreviations: CPS,capsular polysaccharide from S. suis serotype 2; TT, tetanus toxoid.

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ture, significantly lower values (between 0 and 47%) of bacterialkilling were observed compared to those with conjugates (Fig. 7)(P 0.001). Finally, pooled sera from mice hyperimmunized withkilled whole-cell preparations gave bacterial killing values highlysimilar to those with the unconjugated mixture (Fig. 7) (P � 0.05).

Immunogenicity and protection in pigs. Based on previousresults, the 2:1 mixed conjugate was selected to evaluate the im-munogenicity and protection in the S. suis natural host, the pig.The adjuvant Stimune was chosen, as it had been previously in-cluded in S. suis bacterin-based vaccines (12, 52). The perfor-mance of the conjugate was compared to that of an S. suis type 2bacterin adjuvanted with Stimune. To this end, pigs were injectedintramuscularly twice at a 3-week interval, and serum sampleswere collected on days 0, 21, and 34 for titration and isotyping ofanti-CPS antibodies (Fig. 8A). On day 21 postimmunization, totalIgG-plus-IgM anti-CPS titers induced by the 2:1 conjugate vac-cine were significantly higher (P 0.01) than those with the pla-cebo and bacterin. After boosting, on day 34 postimmunization,an increase in anti-CPS titers was observed for both vaccinatedgroups compared to those on day 21. However, only the titers

from pigs vaccinated with the 2:1 mixed conjugate were signif-icantly higher than those for the placebo control group (P 0.001). Titers of the different swine IgG subclasses, namely,IgG1 and IgG2, were also assayed post-boost injection (day 34).Forty percent of pigs immunized with the 2:1 mixed conjugateshowed significant levels of anti-CPS IgG1 subclass (Fig. 8A).No switch to the IgG2 subclass was observed (data not shown).In contrast, vaccination of pigs with the bacterin failed to in-duce an anti-CPS Ig class switch (Fig. 8A and data not shown).

On study day 36, pigs were challenged intraperitoneally with adose of 3 �109 CFU of ATCC 700794, a virulent S. suis serotype 2strain. Most pigs in the placebo group died during the systemicphase of S. suis infection, reaching a mortality of 86.7%. In con-trast, pigs immunized with the bacterin or the 2:1 mixed conjugateshowed mortalities of 28.6% and of 30.0%, respectively. Analysisof survival curves (Fig. 8B) showed a significant difference as soonas day 3 between both immunized groups and the placebo group(P � 0.009). Protection induced by the 2:1 mixed conjugate wassimilar to that for the control type 2 bacterin during the systemicphase of an S. suis challenge infection in pigs.

FIG 6 Isotyping and functional studies of antibodies induced in mice immunized with 2:1 mixed conjugate vaccine in Stimune. Mice (n � 10) were immunizedon day 0 and boosted on day 21 with 25 �g of the 2:1 mixed conjugate adjuvanted with Stimune. Placebo mice (n � 5) were similarly injected with PBS adjuvantedwith Stimune. Sera were collected on day 42. (A) For titration of anti-CPS antibody isotypes, ELISA plates were coated with native CPS and incubated with 2-foldserial dilutions of sera, and isotypes were detected using specific HRP-conjugated anti-mouse IgG plus IgM, IgM, IgG1, IgG2b, IgG2c, or IgG3 antibodies. (B)Opsonophagocytosis killing of S. suis type 2 strain S735 by day 42 sera from mice immunized with 25 �g of the 2:1 mixed conjugate adjuvanted with Stimune.A 40% (vol/vol) sample serum and a bacterial MOI of 0.1 were added to fresh whole blood from naive mice to perform the assay. Viable bacterial counts weredetermined after 2 h of incubation. To determine bacterial killing, viable bacterial counts from tubes incubated with sample sera were compared to thoseincubated with control naive mouse sera. Results are expressed as percent bacterial killing for individual mice, with horizontal bars representing means � SEM.Abbreviations: CPS, capsular polysaccharide from S. suis serotype 2.

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Pigs were also monitored for clinical signs (behavior, locomo-tion problems, or CNS signs) for seven consecutive days afterchallenge. In 31.6% of all observations for the bacterin-vaccinatedgroup and in 28.1% of all observations for the conjugate-vacci-nated group, abnormal behavior was observed. This was signifi-cantly lower than the findings in the placebo group, in which90.7% of the observations revealed abnormal behavior (Table 1)(adjusted P value, 0.05). Lameness was observed in 26.3% of allobservations for the bacterin-vaccinated group and in 33.5% of allobservations for the conjugate-vaccinated group, compared to89.3% for the placebo group (Table 1) (adjusted P value, 0.05).These differences were also observed when the distribution ofclinical scores was analyzed daily for each group (data notshown). CNS signs were observed in only a few pigs, and nostatistically significant differences were observed between thethree challenged groups (Table 1). This can be explained by thefact that animals were observed for only a 7 day-period post-challenge, as the study design focused mainly on the systemicphase of the disease. All pigs found dead as well as all eutha-nized pigs were necropsied. The frequency of gross lesions in

the thoracic cavity (i.e., fibrin, excess fluid, or pericarditis) orin the joints was overall reduced in vaccinated animals com-pared to the placebo group, though the observed differencesdid not reach statistical significance (see Table S2 in the sup-plemental material). The conjugate vaccine significantly re-duced recovery of the challenge strain from joint swabs (P 0.01). The S. suis challenge strain was also less frequently isolatedfrom the meningeal and pericardial swabs than from the placebogroup, yet the differences were not statistically different (see TableS3 in the supplemental material).

DISCUSSION

While glycoconjugate vaccines (made from CPS conjugated to animmunogenic protein carrier) are very effective in the fight againstencapsulated bacteria in human medicine, so far no such vaccineis available for swine veterinary practice. Although some conju-gates have previously been suggested for veterinary medicine use(25–27, 53), so far none have been marketed. For the first time, wereport the feasibility of a glycoconjugate vaccine to protect pigsfrom S. suis type 2 infections. This glycoconjugate, made with type2 CPS coupled to TT, is immunogenic in mice and pigs by induc-ing production of IgG antibodies, which are functional in vitroand protective in vivo.

Capsular polysaccharides are considered TI antigens, as theyare in general unable to stimulate MHC class II-dependent Th cellhelp (30), which results in poor immunogenicity. In contrast toTD antigens, TI antigens do not induce the classical antibody iso-type switch and high-affinity memory B cells (54). Yet, purifiedCPSs from S. pneumoniae (Pneumovax, 23 valent) and fromgroup B Streptococcus (GBS) serotype III can induce not only IgMbut also IgG antibody responses in mice and in adults withoutadjuvant (19, 55–58). In contrast, S. suis serotype 2 CPS alone isunable to induce any significant antibody response, even whenadjuvanted with TiterMax Gold or Stimune or when combinedwith the TLR ligand CpG in mice (unpublished results). Further-more, previous studies using live S. suis serotype 2 infectionshowed modest IgM and no isotype-switched IgG anti-CPS anti-body titers in pigs and in mice even after an experimental reinfec-tion (17). Thus, S. suis serotype 2 CPS seems to be particularlynonimmunogenic, making it an ideal candidate for evaluation ofthe effect of glycoconjugation on immunogenicity. A precedentexists in the literature, where serotype 2 CPS was conjugated tobovine serum albumin with the aim to obtain anti-CPS controlsera for in vitro studies (59). However, neither the biochemicalcharacteristics nor the immunogenicity and functional activity ofthat conjugate were investigated.

To produce the glycoconjugate, it was first decided to depoly-merize the CPS to a smaller size in order to improve the efficacy ofthe conjugation and the residual exposure of the T cell peptideepitopes of the protein carrier. Ultrasonic irradiation (sonication)was chosen over fragmentation by chemical (60–62) or enzymatic(63–66) methods to avoid chemical alterations (67). In addition, itis easy to use, is reliable for labile epitopes, and does not requireelimination of excess reagents. Another great advantage of ultra-sonic irradiation is the reduction in sample polydispersity (39),facilitating biochemical characterization, particularly within theglycoconjugate.

In order to conjugate the CPS to a protein carrier by reductiveamination, aldehydes must be introduced, ideally regioselectively,in the CPS by oxidation with sodium periodate. The presence of

FIG 7 Opsonophagocytosis killing of S. suis type 2 strain S735 by day 42 serafrom mice immunized with different CPS conjugate vaccines adjuvanted withTiterMax Gold. Mouse groups were as follows: placebo (n � 5), 2:1 mixedconjugate (n � 10), 2:1 fractionated conjugate (n � 10), and 2 CPS:1 TTunconjugated control mixture (n � 10). All mice were immunized with 25 �gof antigen in TiterMax Gold on day 0 and boosted on day 21, and sera werecollected at day 42. A pool of hyperimmune mouse sera from 6 mice was alsoincluded for comparative purposes (n � 8 experimental replicates). A 40%(vol/vol) sample serum and a bacterial MOI of 0.1 were added to fresh wholeblood from naive mice to perform the assay. Viable bacterial counts wereperformed after 2 h of incubation. To determine bacterial killing, viable bac-terial counts from tubes incubated with sample sera were compared to thoseincubated with control naive mouse sera. Results are expressed as percentbacterial killing for individual mice, with horizontal bars representing means �SEM. #, values significantly different from those of the placebo group (P 0.01). Differences between other groups: ***, P 0.001. Abbreviations: CPS,capsular polysaccharide from S. suis serotype 2; TT, tetanus toxoid.

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sialic acid offers a unique opportunity to use mild oxidative con-ditions, leaving the remainder of the polysaccharide unmodified,as already described for conjugates against different serotypes ofGBS (41, 68–73). The glycerol side chain of the sialic acid residuesis particularly reactive toward mild periodate oxidation. The de-sired percentage of oxidation is also a critical parameter: too fewreactive groups will yield a poor conjugate, while too many willleave few intact epitopes of the native polysaccharide (40). For S.suis type 2, we found that a low oxidation level (10%) yieldedimmunogenic conjugates and was thus preferred over very highoxidation levels. In accordance, some immunogenic conjugatesfor GBS types Ia, Ib, and II have been described with an oxidationlevel of 10% (68–70). In contrast, conjugates of other GBS types,including type III, routinely use higher oxidation levels of 25%(41, 70, 71, 73) or between 40 and 50% (72) and could go up to66% without loss of immunogenicity for serotype III (74), eventhough the type III GBS CPS conformational epitope is controlledby the sialic acid residue (75, 76).

Two conjugate vaccine formulations with different CPS to TTratios were obtained. The 2:1 mixed conjugate was found to be themost immunogenic, resulting in significantly higher titers ofIgG2b and IgG2c anti-CPS isotypes. This difference in immuno-

FIG 8 Immunogenicity and protection studies in pigs. Animals were blocked by litter and then randomly assigned to one of four groups: group 1, n � 14; group2, n � 10, group 3, n � 15; and group 4, n � 5. Groups 1 to 4 were commingled until group 4 (strict control) was removed at study day 35. Blood samples werecollected on study days 0, 21, and 34 for determination of serum antibody levels. The piglets were injected intramuscularly twice at a 3-week interval (study days0 and 21) with 2 ml of the respective vaccine or placebo adjuvanted with Stimune: group 1 was vaccinated with adjuvanted S. suis type 2 bacterin, group 2 wasinjected with the adjuvanted 2:1 mixed conjugate, and group 3 was given 2 ml of adjuvanted PBS. (A) Kinetics of serum antibody response of immunized pigs.ELISA plates were coated with native capsular polysaccharide and incubated for 1 h with 2-fold serial dilutions of sera, and isotypes were detected using specificHRP-conjugated anti-pig IgG plus IgM or IgG1 antibodies. Antibody titers for individual pigs are shown, with horizontal bars representing means � SEM. Thearrow at day 21 indicates the boost. **, P 0.01; ***, P 0.001 (as determined by one-way ANOVA). (B) Protection study. On day 36, groups 1 to 3 werechallenged intraperitoneally with 3 � 109 CFU/dose of S. suis type 2 isolate ATCC 700794. Following challenge, pigs were monitored daily over a period of 7 daysfor the presence of clinical signs. Note that on day 21, one animal from the bacterin group was euthanized due to complications following serum collection, whichleaves n � 14 at day 34 and for the challenge. **, P 0.01 for both bacterin- and 2:1 mixed conjugate-vaccinated groups compared to placebo (challenge control)group.

TABLE 1 Clinical evaluation of immunized pigs after experimentalchallenge with S. suis serotype 2a

Group

Abnormalbehavior

Abnormallocomotion

CNS clinicalsigns

%b P valuec % P value % P value

Type 2 bacterin 31.6 0.0209 26.3 0.0064 1.2 NSd

2:1 mixedconjugate

28.1 0.0308 33.5 0.0335 0 NS

Placebo (challengecontrol)

90.7 89.3 2.6

a Assessed were behavior, including any behavior indicating an effect of challenge onthe central nervous system (CNS), and locomotion. The observations for behavior werenumerically scored as follows: 0, physiological; 1, depression; and 2, apathy.Observations for locomotion were scored as follows: 0, physiological; 1, slightly tomoderately lame; 2, severely lame/reluctant to stand; and 3, animal partially/completelydown (animal can rise but lies down again within 10 s). CNS signs were scored asfollows: 0, absent; and 1, present.b Percentage of evaluations where behavior, locomotion, or CNS signs gave a value of�0 across days. Assessment was for the cumulative observation period. Data areexpressed as least-squares means (back-transformed).c Adjusted P value (Scheffé’s test). All values are compared to the challenge controlgroup; the strict control group was not included in the assessment.d NS, not significant (P � 0.05).

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genicity may arise from the higher percentage of total CPS in the2:1 than in the 1:1 mixed conjugate, which might influence thecapacity of the conjugate to modulate the immune cells, includingantigen-presenting cells (APCs), presumably through its highermolecular mass/size, which might ease uptake and internalization.In this regard, it has been shown that the polysaccharide sizeused for conjugation, the obtained conjugate size, and the de-gree of polysaccharide-protein cross-linking influence the im-munogenicity and protective efficacy of a GBS type III-TT con-jugate vaccine (74, 77, 78). Further studies evaluating how theaforementioned parameters affect the immunogenicity of an S.suis type 2 CPS conjugate, which can contribute to improve-ments of the design of the conjugate vaccine, are under way.

During pretrials, it was observed that conjugation alone wasnot enough to induce a robust immunological response against S.suis type 2 CPS (data not shown). In this regard, subunit vaccinesare known to induce more potent and durable antigen-specificimmunity if combined with an adjuvant (79). It has been shownthat adjuvants not only can improve the immunogenicity of con-jugate vaccines but also can direct the antipolysaccharide antibodyisotype switch toward the desired IgG subclasses (80, 81). CpGODN 1826 (82) was shown to enhance the isotype switching fromIgM to IgG2a and IgG3 subclasses for pneumococcal conjugates ina serotype- and mouse age-dependent manner (43, 80, 83). In ourstudy, CpG ODN 1826 failed to significantly improve the immu-nogenicity of the S. suis type 2 CPS mixed conjugate or to induceisotype switching. Conditions differing from those used in thisstudy, such as the vaccine and adjuvant doses, might explain theobserved differences. The biochemical properties of differentCPSs might also influence the adjuvant capacity of CpG (83). Assuch, the choice of adjuvant is vital to maximize isotype switching.In the present study, high levels of IgM as well as of both Th1(IgG2b, IgG2c, and IgG3) and Th2 (IgG1) IgG subclasses wereobserved in mice immunized with conjugates in TiterMax Gold.Similarly, Stimune induced high levels of IgM and a mixed Th1/Th2 IgG response. However, the Th1 antibody response was re-stricted to the IgG2b subclass when using Stimune. Our data are inagreement with previous studies suggesting that water-in-oil ad-juvants, such as TiterMax Gold and Stimune, induce strong hu-moral responses by means of the depot effect and coimmunos-timulatory properties, which are associated with mixed Th1 andTh2 antibody responses (46, 84–86). While Stimune is composedprincipally of purified and defined mineral oil, TiterMax Goldincludes metabolizable squalene as the oil phase and also includesblock copolymers in the formulation. This special formulationmight contribute to the increased isotype switching activity ob-served with this adjuvant in mice. These block copolymers havebeen shown to influence the localization and retention of antigenin lymphoid tissues and to recruit and activate APCs and lympho-cytes (48, 87).

However, even when using TiterMax Gold as adjuvant, not allmice developed anti-CPS IgG subclass-switched antibodies, al-though they all exhibited strong anti-CPS IgM titers in addition tohigh antibody levels to TT. Isotype switching from IgM to IgGdepends on the signals (cytokines and costimulatory molecules)provided by APCs to T cells and then by T cells to B cells. Thesesignals are induced by TT and the adjuvant, since the CPS is notimmunostimulatory per se (24, 48). The intensity of these signalsmight vary among individual mice. As such, some of them will not

be able to switch or will present isotype profiles different fromthose of other individuals from the same group.

Compared to the conjugate vaccine, a TiterMax Gold-adju-vanted control mixture of free CPS and TT induced a reduced TDanti-CPS antibody response. This also confirms that the immuno-genicity in the 2:1 mixed conjugate is in fact due to the conjugatepresent in the vaccine formulation. Free polysaccharides in con-jugate vaccines have been shown to have adverse effects on theimmune response (by presenting mixed TI and TD forms of theCPS antigen) (88), as demonstrated with pneumococcal CPStypes 3, 4, 6B, and 14 (89–91). In contrast, based on our results,free S. suis CPS does not seem to influence the immunogenicityof the conjugate vaccine. This is suggested by the facts that nosuppressive effect was observed when using high doses of vac-cine preparations and that the 2:1 fractionated conjugate be-haves similarly to the mixed vaccine conjugate. The 2:1 mixedconjugate vaccine was also sufficient to induce protection inpigs, rendering the purification of the postconjugation prepa-ration by chromatography unnecessary for potential use in thefield.

While ELISA titers are great tools to evaluate the immunoge-nicity of vaccine prototypes, they are not generally consideredreliable correlates of immunity. A strong antibody response doesnot necessarily reflect upon the protection of an individual (38).In this regard, functional assays are preferred, such as the OPA, arecognized correlate of protective immunity against extracellularencapsulated Gram-positive bacteria (18, 92). During the OPA,opsonizing antibodies from the immunized serum will opsonizethe target bacteria, which results in bacterial phagocytosis andbactericidal activity (38, 93–95). Specific cell type activation de-pends on the Ig isotypes/subclasses present in the immune serum,since each isotype/subclass possesses different preferences forbinding to Fc receptors, which differently influences the cell re-sponse (38, 94). Besides IgM, the predominant subclass of protec-tive antibodies to TI antigens in mice is IgG3 (54, 96–98). A studyusing mouse monoclonal antibodies proposed that the type 1 sub-classes (IgG3 �� IgG2b � IgG2a) are superior in both op-sonophagocytosis activity and complement activation to the type2 IgG1subclass. Yet, these functional properties of mouse IgG sub-classes seem to depend on the target antigen (protein versus car-bohydrate), antigen distribution, and susceptibility of the bacteriato antibody/complement attack (99, 100). In our OPA experi-ments, the two groups which obtained the highest bacterial killingvalues were the 2:1 mixed or fractionated conjugate adjuvantedwith TiterMax Gold, both containing the highest titers of all type 1IgG subclasses. They were closely followed by the 2:1 mixed con-jugate adjuvanted with Stimune, although this group lacks pro-duction of IgG3 and IgG2c. It has been shown that mice lackingthe dominant IgG3 subclass made to bacterial CPS are more sus-ceptible to fatal S. pneumoniae sepsis than wild-type mice, yetthese mice can be rescued by induction of IgG1 using an S. pneu-moniae glycoconjugate (100). In the IgG3�/� mouse model, it wasproposed that low titers of IgG1 anti-CPS antibodies in combina-tion with complement deposition mediated by IgM anti-CPS an-tibodies may have been adequate to opsonize pneumococci foruptake by macrophages (100). Thus, in the absence of high levelsof the most opsonizing antibodies (such as IgG3), other Ig iso-types/subclasses might compensate. As such, protection dependson the right balance between functionality, affinity, and quantityof different anti-CPS Ig isotypes/subclasses induced by the vac-

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cine. In this regard, control mouse groups immunized with themixture of free CPS and free TT or mice hyperimmunized withkilled bacteria failed to adequately perform in the OPA test, prob-ably due to the combined absence or low levels of several IgGsubclasses, including IgG1. Together with the absence of protec-tion in placebo groups, the OPA data confirm that anti-CPS anti-bodies generated by the glycoconjugate play a crucial role in op-sonophagocytosis of S. suis and thus in the protective effectobserved with the vaccine.

For an ultimate proof of concept, evaluation of the conjugatewas conducted in the natural host of S. suis, the pig. In the field,pigs usually present clinical signs between 5 and 10 weeks of age(3), which would correspond to approximately 7 to 14 days post-boost. Pigs immunized with the 2:1 mixed conjugate showed sig-nificant protection against a challenge systemic infection. As men-tioned above, the combined action of IgM and IgG1 might besufficient to confer protection in the swine model, yet the func-tional activities of different swine isotypes/subclasses remain to beelucidated. During swine immunization trials, a bacterin adju-vanted with Stimune was used as control. Although the bacterininduced levels of protection similar to those for the conjugatevaccine, this protection was not related to anti-CPS antibodies butprobably was related to antiprotein antibodies. Yet, and in con-trast to CPS, which is a universal antigen for S. suis type 2, proteinantigens vary depending on the strain origin or sequence type (ST)within the same serotype (16, 101–105). Strains belonging to thesame ST (ST1) were used as bacterin and challenge strains, andthus the capacity of such a vaccine preparation to protect against aheterologous challenge (ST25 and ST28 strains) remains to beelucidated (2). It can, however, be expected that protection con-ferred by a bacterin might be strain/ST dependent, while that pro-vided by a CPS conjugate vaccine is serotype specific and thusstrain/ST independent (106).

In conclusion, the conception and laboratory-scale productionof a glycoconjugate vaccine against S. suis serotype 2 are reportedfor the first time. In its unpurified form as a desalted postconju-gation formulation (mixed conjugate), the vaccine was shown tobe efficient when emulsified with water-in-oil adjuvants. In mice,anti-CPS IgM and isotype-switched IgG antibodies were observedand found to be functional in an in vitro opsonophagocytosis as-say. In pigs, anti-CPS IgM and IgG1 antibodies were detected andfound to be significantly protective in an in vivo lethal-dose chal-lenge with virulent S. suis serotype 2. At this stage, these resultsrepresent a proof of concept for the potential of glycoconjugatevaccines in veterinary medicine applications against invasive bac-terial infections.

ACKNOWLEDGMENT

Conflict of interest statement: We certify that all financial and materialsupport for the conduct of this study and/or preparation of this manu-script is clearly described below in the Funding Information section of themanuscript.

FUNDING INFORMATIONThis work was mainly supported by the Natural Sciences and EngineeringResearch Council of Canada (NSERC) through a grant to MS (# 342150)and by Boehringer Ingelheim Vetmedica, Inc. Research Agreement No.16579. Boehringer Ingelheim Vetmedica, Inc. provided support for theconduct of the research and the preparation of the article. The sponsorcontributed to the in vivo study design; in the collection, analysis andinterpretation of swine data; in the writing of the report; and in the deci-

sion to submit the article for publication. Partial contribution was alsoprovided by Canadian Institutes of Health Research grant to MS, RR andMG (# 203549), by a NSERC Canadian Research Chair to RR (# UBR303231), and by a Fonds de recherche du Québec - Nature et technologies(FRQ-NT) team grant to RR, MS, and MG.

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Proof of Concept for S. suis Serotype 2 Conjugate

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