WO2021073778A1 - Compositions and methods for vaccinating piglets against streptococcus - Google Patents

Abstract

The present application relates to methods for treating infection by Streptococcus suis (S. suis) in mammals, particularly in pigs. The application more particularly relates to a prime-boost vaccination approach for treating (in a preventive or curative setting) infections by Streptococcus suis (and/or diseases caused by infection by Streptococcus suis) in mammals. The methods of the invention use an IgM protease of, or (derived) from, Streptococcus suis as an antigen to induce protective immunity in piglets.

Description

Compositions and methods for vaccinating piglets against Streptococcus

The present application relates to methods for treating infection by Streptococcus suis (S. suis) in mammals, particularly in pigs. The application more particularly relates to a prime-boost vaccination approach for treating (in a preventive or curative setting) infections by Streptococcus suis (and/or diseases caused by infection by Streptococcus suis) in mammals. The methods of the invention use an IgM protease of, or (derived) from, Streptococcus suis as an antigen to induce protective immunity in piglets. Introduction

S. suis cps9 has become the most important serotype in some European countries with a huge pig industry such as Spain and the Netherlands [1 ,2]. In the field, prophylaxis against cps9 is very problematic, because bacterin application protects only against mortality but not morbidity [3] and does not reduce colonization and transmission [4] Furthermore, cps9 is a strong biofilm inducer and endocarditis is a main manifestation, which might also occur in vaccinated piglets [3,5,6].

A few S. suis proteins have been shown to provide protection against cps2 challenge in pigs, including a combination of muramidase-released protein (MRP) and extracellular factor (EF) [7], surface antigen one (SAO) [8], HP0197 [9], SsPepO [10] and lde_Ssuis [11]· Interestingly, only SAO has been shown to protect against a further serotype, namely cps1 [12] However, most cps1 strains are closely related to cps2. Strains of both serotypes often belong to clonal complex (CC) 1 [13]. Noteworthy, other independent studies have shown that piglets with high antibody titers against SAO are highly susceptible to S. suis challenge and that these antibodies are not opsonizing [14,15]. Thus, it is currently unknown whether any of the identified protective antigens has the cross-protective potential needed for a universal S. suis vaccine.

Different S. suis serotypes express a highly specific immunoglobulin M-degrading enzyme, designated lde_Ssuis[16]. The protein is homologous to the IgG protease IdeS of S. pyogenes [17], but cleaves solely class M antibodies of swine. Mutants expressing no or a point-mutated lde_Ssuis variant deficient in IgM cleavage show enhanced deposition of C3b on the bacterial surface indicating that IgM cleavage by lde_Ssuis is involved in complement evasion [18,19]. Vaccination of weaning piglets with rlde_Ssuis elicits antibodies neutralizing the IgM protease activity and protects against morbidity and mortality induced by cps2 challenge [11].

Read out parameters in vaccination trials with piglets focused on humoral immunity. However, cellular immunity is known to be crucial to restrict colonization of the respiratory tract by the related pathogen S. pneumoniae [20,21], In mouse model CD4KO were found to be more susceptible to systemic S. suis infection at a low infection dose [22], whereas in pigs the T cell immune response is not well characterized.

Here, we investigated humoral and cellular immunogenicities of lde_Ssuis vaccinated suckling piglets, being the age class of choice for vaccination in the field. Furthermore, we asked if lde_Ssuis protects against S. suis cps9, the most important and troublesome pathotype in Europe.

Summary of the invention

An object of the invention resides in an IgM protease antigen of Streptococcus suis for use in the prophylactic or therapeutic treatment of Streptococcus Suis infection in a piglet, said use comprising a first and a second administrations of IgM protease antigen to said piglet before 35 days of age. Preferably, the use further comprises a third administration of IgM protease antigen to said piglet after 35 days of age.

Another object of the invention resides in the use of an IgM protease antigen of Streptococcus suis for the manufacture of a medicament (such as a vaccine) for the prophylactic or therapeutic treatment of Streptococcus Suis infection in a piglet, comprising a first and a second administrations of IgM protease antigen to said piglet before 35 days of age. Preferably, a third administration of IgM protease antigen is performed to said piglet after 35 days of age.

Another object of the invention resides in an IgM protease antigen of Streptococcus suis for use for protecting a piglet against mortality caused by a Streptococcus Suis strain of serotype 9 infection, said use comprising a first and a second administration of an IgM protease antigen to said piglet before 35 days of age, and a third administration after 35 days of age.

The invention also relates to the use of an IgM protease antigen of Streptococcus suis for the manufacture of a medicament (or vaccine) for protecting a piglet against mortality caused by a Streptococcus Suis strain of serotype 9 infection, comprising a first and a second administration of an IgM protease antigen to said piglet before 35 days of age, and a third administration after 35 days of age.

The invention also relates to a method for the prophylactic or therapeutic treatment of Streptococcus Suis infection in a piglet, said method comprising a first and a second administrations of an IgM protease antigen to said piglet before 35 days of age. Preferably a third administration is performed to the piglet after 35 days of age.

The invention also relates to a method for vaccinating piglets against Streptococcus Suis infection and/or an associated disorder, the method comprising a first and a second administrations of an IgM protease antigen to said piglet before 35 days of age. Preferably a third administration is performed to the piglet after 35 days of age.

The invention also relates to a kit comprising, in a suitable container, a first and a second doses of an IgM protease antigen, and a suitable injection device.

Legend to the figures

Fig. 1 : Strain 16085/3b shows increased survival in porcine blood compared to other cps9 strains. Bactericidal assays were conducted with S. suis cps9 strains A3286/94, 15-3/3, V5402/2 and 16085/3b belonging to the indicated sequence types (ST) with blood drawn from the same S. suis cps9 free piglets (n=5) at the specified ages. The survival factor represents the ratio of CFU at 120 min to CFU at time zero. Bars and error bars represent mean values and standard deviations, respectively. Significant differences were determined using two-way ANOVA and a subsequent Tukey’s multiple comparisons test. Significances are indicated (^* p < 0.05, ^** p < 0.01 , ^*** p < 0.001 ).

Fig. 2: rlde_Ssuis vaccination induces specific IgG antibodies in serum. Time course of □- lde_Ssuis IgG antibodies in rlde_Ssuis-vaccinated and placebo-treated piglets during prime- booster-booster vaccination and after S. suis cps9 infection. IgG levels were determined in the 2^nd week of life (pre immunization) and 7 days after the first booster in rlde_Ssuis- vaccinated (O,●) and placebo-treated (■, □) piglets (n = 20/group). In the 6^th week of life, nine piglets per group were boostered (●) or placebo-treated (□), respectively, for a second time and challenged 17 days later. Blood samples taken from euthanized piglets are indicated as final bleeding and include the time period from 1 until 14 dpi.

Mean values are indicated by horizontal lines, standard deviations by error bars. Statistical analyses were conducted with the Mann-Whitney U-test (placebo-treated vs. rlde_Ssuis-vaccinated), the Wilcoxon test (comparison of pre immunization and 7d post 1^st booster immune sera, n = 20/group) or the one-way ANOVA and subsequently with Dunn’s multiple comparisons test (pre and post immune sera of the 18 piglets challenged with S. suis cps9). Significant differences are indicated (^** p < 0.01 , ^**** p < 0.0001)

Fig. 3: Survival of S. suis cps9 strain 16085/3b is significantly reduced in blood of rlde_Ssuis- vaccinated piglets (n = 20) in comparison to placebo-treated littermates 7 days after the first booster vaccination. Bacterial survival of cps2 strain 10 and its isogenic mutant 10 Aides_suis was also determined in the blood of the same piglets. Bars and error bars represent mean values and standard deviations, respectively. The Mann-Whitney-U-test was used for comparison of rlde_Ssuis-vaccinated versus placebo-treated group. Two-way ANOVA and Tukey^'s multiple comparisons post-hoc tests were used for comparison of survival factors of the three S. suis strains per group. Significant differences are indicated

(^*** p < 0.001 , ^**** p < 0.0001 ).

Fig. 4: rlde_Ssuis vaccination protects against mortality caused by S. suis cps9 challenge. The Kaplan-Meier diagram shows mortality of rlde_Ssuisprime-booster-booster (n=9) and placebo-treated (n=9) growing piglets after infection. Piglets were challenged through intravenous application of 2 x 10⁸ CFU of S. suis strain 16085/3b ( mrp ^* sly⁺ cps9 ) 17 days after 2^nd booster immunization. In case of high fever (³40.5°C), apathy and anorexia persisting over 32 hours as well as in all cases of central nervous system dysfunction or clinical signs of acute polyarthritis, animals were euthanized for animal welfare reasons. All surviving piglets were sacrificed 14 days post infection (dpi). Statistical analysis was conducted with the log-rank test (p value is shown below the diagram).

Fig. 5. Vaccination-induced lde_Ssuis-specific Th cell frequencies correlate with lde_Ssuis- specific IgG levels. Detection of lde_Ssuis-reactive Th cells in rlde_Ssuis-vaccinated piglets 17 d post 2^nd booster immunization (prior cps9 challenge). A) PBMCs from rlde_Ssuis- vaccinated and placebo-treated pigs (n = 9 per group) were antigen-specifically stimulated with 5 μg/ml rlde_Ssuis or the recombinant fibronectin binding domain of Sfbl as a non-relevant control antigen for 18 h in presence of Brefeldin A (2 μg/ml) for the last 14 h to detect intracellular CD154 and cytokine expression. As controls PMA/ionomycin (10 ng/ml and 500 ng/ml, respectively, positive control) and medium (med, negative control) were used. For statistical analysis non-parametric Kruskal-Wallis test and Dunn^'s multiple comparisons post-hoc test was used (^*p<0.05, ^**p<0.01 , ^***p<0.001). B) Pearson correlation between frequencies of CD154+ Th cells of rlde_Ssuis-stimulated PBMCs and lde_Ssuis IgG levels.

Detailed description of the invention

Vaccination of weaning piglets with the IgM protease enzyme of Streptococcus suis (S. suis), lde_Ssuis, elicits protection against disease caused by serotype (cps) 2 infection. In Europe, S. suis cps9 is at least as important as cps2 in causing severe herd problems associated with meningitis, septicemia and arthritis. The objective of this invention is to develop a vaccination for suckling piglets effective against infection by virulent cps9 serotype strains of S. suis. The present invention shows that effective protection and treatment can be obtained with a prime-boost vaccination approach. More specifically, vaccination in the 2^nd and 4^th weeks of life with lde_Ssuis induced seroconversion against lde_Ssuis in 13 of 20 vaccinated piglets. In the 5^th week, survival of the S. suis cps9 strain was significantly reduced in the blood of prime-booster vaccinated piglets. Furthermore, after a 2^nd booster vaccination, lde_Ssuis-reactive T helper (Th) cells partially producing TNF-α, IL-17A or IFNyywere detectable in lde_Ssuis-vaccinated but not in placebo-treated piglets and frequencies of specific Th cells correlated with a-lde_Ssuis-lgG levels. An intravenous challenge, conducted with a cps9 strain of sequence type (ST) 94, lead to 89 % mortality in placebo-treated piglets due to septicemia and meningitis. In contrast, all lde_Ssuis vaccinated according to the invention survived the challenge despite signs of disease such as fever and lameness. In conclusion, the lde_Ssuis vaccination of the invention induces humoral and detectable lde_Ssuis-specific Th cell responses and leads to protection against a highly virulent cps9 strain.

Antigen

The invention uses an IgM protease of, or (derived) from, Streptococcus suis as an antigen.

The antigen is preferably in isolated form, with any level of purification. Preferably the antigen is isolated from its natural medium. More preferably, the antigen does not contain live, intact, Streptococcus suis cells.

Most preferred form is recombinant antigen, produced by expression of a coding nucleic acid in a heterologous recombinant cell or system such as E.coli, yeast cells, insect cells, or mammalian cells. The antigen may be essentially pure, or used as a culture supernatant (which may be enriched prior to formulation).

The IgM protease of, or (derived) from, Streptococcus suis, is also designated in the present application as Ides_suis (or rldes_suis when recombinant). Any such antigen may be used, as well as variants thereof, immunogenic fragments thereof, o immunogenic complexes (or conjugates thereof).

In a particular embodiment, the antigen comprises an amino acid sequence selected from SEQ ID Nos: 1 -2, 5-7 and 10-14.

In a further particular embodiment, the antigen is a protein comprising an amino acid sequence selected from SEQ ID Nos: 1 -2, 5-7 and 10-14.

In another particular embodiment, the antigen consists essentially of, or consists of, an amino acid sequence selected from SEQ ID Nos: 1 -2, 5-7 and 10-14.

In a further particular embodiment, the antigen is a protein consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID Nos: 1 -2, 5-7 and 10-14.

In a further embodiment, the antigen comprises, consists essentially of, or consists of, a variant of a sequence as defined above. More specifically, the variants comprise, consist essentially of, or consist of, a sequence having at least 90% identity, more preferably at least 91%, 92%, 93%, 94%, 95/ identity or more, such as even at least 96%, 97%, 98%, or 99% identity, over the entire length, with anyone of SEQ ID Nos: 1 -2, 5-7 and 10-14.

In a preferred embodiment, the antigen is a protein comprising, or consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 1 , 2, 5, 6 or 7.

Further examples of suitable antigens are disclosed in WO2015181356. They include, for instance, antigens comprising or consisting essentially of, or consisting of amino acid sequence of SEQ ID NO: 1 lacking the amino acids from position 1 to 34.

Fragments of the above antigens are also included for use in the invention. The term "fragment" designates a shorter amino acid sequence than a referenvce sequence. Preferred fragments shall contain or consist of the highly conserved part of the Mac-1 domain (SEQ ID NO: 5) or an amino acid sequence which is at least 95% identical thereto. Further examples of antigens of the invention comprise, in addition to the above equences, additional groups or sequences such as added epitopes, tags, or secretion sequences. For instance, a HIS tag may be included in the sequence, preferably terminally.

Formulation and dosage

The antigen in the invention may be formulated in compositions and vaccines suitable for administration to mammals.

Such compositions (e.g., vaccines) preferably comprise an immunologically effective amount of antigen as described above in a pharmaceutically acceptable vehicle. As a result of the vaccination with a composition of the invention, pigs become at least partially or completely immune to S. suis infections, or resistant to developing moderate or severe diseases/symptoms caused by said pathogen, or protected from mortality caused by said pathogen. In practice, the exact amount required for an immunologically effective dose may vary from subject to subject depending on factors such as the age and general condition of the subject, the nature of the formulation and the mode of administration. Appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. For instance, methods are known in the art for determining or titrating suitable dosages of a vaccine to find minimal effective dosages based on the weight of the non human animal subject, concentration of the vaccine and other typical factors.

In atypical embodiment, each administration comprises between 0,025 and 1 mg of IgM protease antigen, preferably between 0,1 and 0,8 mg of IgM protease antigen.

In a particular embodiment, the vaccine comprises the antigen of the invention in purified form.

In another particular embodiment, the antigen is linked to a carrier material.

Compositions of the invention may comprise other ingredients, known per se by one of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration. Examples of pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as light liquid paraffin oil, or heavy liquid paraffin oil; squalene; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or iso- propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1 ,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the vaccine composition and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.

Examples of adjuvants include, but are not limited to, oil in water emulsions, aluminum hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL- 1 , IL-2, IL-7, IFN-[alpha], IFN-[beta], IFN-y, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like. Other suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat- stable enterotoxin(s) isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanus toxin and pertussis toxin may be inactivated prior to use, for example, by treatment with formaldehyde.

Examples of freeze-drying stabilizer may be for example carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.

Vaccines may additionally comprise at least one immunogen from at least one additional pathogen, eg., a pig pathogen such as Actinobacillus pleuropneunomia; Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Balantidium coli; Bordetella bronchiseptica; Brachyspira spp., preferably B. hyodyentheriae, B.pilosicoli, B. innocens, Brucella suis, preferably biovars 1 ,2 and 3; Classical swine fever virus, African swine fever virus; Chlamydia and Chlamydophila sp. and preferably C. pecorum and C. abortus; Clostridium spp., preferably Cl. difficile, Cl. perfringens types A, B and C, Cl.novyi, Cl.septicum, Cl.tetani; Digestive and respiratory Coronavirus; Cryptosporidium parvum ; Eimeria spp; Eperythrozoonis suis currently named Mycoplasma haemosuis; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1 ,7 and 14; Hemagglutinating encephalomyelitis virus; Isospora suis ; Japanese Encephalitis virus; Lawsonia intracellulars; Leptospira spp., preferably Leptospira australis, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagicae, Leptospira interrogans, Leptospira Pomona and Leptospira tarassovi; Mannheimia haemolytica ; Mycobacterium spp. preferably, M. avium, M. intracellular and M.bovis: Mycoplasma hyponeumoniae; Parvovirus ; Pasteurella multocida; Porcine cytomegolovirus; Porcine parovirus, Porcine reproductive and respiratory syndrome virus: Pseudorabies virus; Rotavirus; Sagiyama virus ; Salmonella spp. preferably, S. thyhimurium and S.choleraesuis; Staphylococcus spp. preferably, S. hyicus; Streptococcus spp., preferably Strep, suis; Swine cytomegalovirus ; Swine herpes virus; Swine influenza virus; Swine pox virus; Toxoplasma gondii ; Vesicular stomatitis virus and virus of exanthema of swine; or other isolates and subtypes of porcine circo virus.

The vaccine compositions of the invention may be liquid formulations such as an aqueous solution, water-in-oil or oil-in-water emulsion, syrup, an elixir, a tincture, a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents.

The route of administration can be percutaneous, via mucosal administration, or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccines compositions according to the present invention may be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. Vaccines of the invention may, in addition, comprise one or several additional protein antigens from different serotypes. A preferred administration route is by injection, particularly intramuscular injection(s). The present invention also relates to methods of immunizing or inducing an immune response in pigs comprising administering to said pig an antigen as defined above.

The present invention also relates to methods of treating and/or preventing S. suis associated diseases in pigs comprising administering to said pigs an antigen as described above.

Protocol

The present invention demonstrates that a prime-boost approach as defined by the inventors allows effective protection against highly virulent S. suis psc9 serotype. Such approach also generates cross-protective immunity against several serotypes, including psc2 and pcs9.

According to the invention, the antigen (or vaccine or composition of the invention) shall be administered at least twice before 35 days of age. Furthermore, in a particular embodiment, a third administration is performed after day 35. For each administration, the antigen may be the same or not. The dosages and administration route are preferably as defined below.

Preferred methods of the invention comprise:

. a first administration of an IgM protease antigen to a piglet between 7 days and 21 days of age, preferably at 14 days of age,

. a second administration of an IgM protease antigen to said piglet when the piglet is between 21 days and 35 days of age, preferably at 28 days of age, and,

. optionally, a third administration of an IgM protease antigen to said piglet, when the piglet is between 35 and 49 days of age, preferably at 42 days of age.

As illustrated in the examples, such administration regimen provides effective protection against virulent serotypes. Such administration regimen also protects the piglets against mortality associated with S. suis infection.

The present invention also provides a container comprising an immunologically effective amount a protein, nucleic acid or vaccine as described above. The invention also provides vaccination kits comprising an optionally sterile container comprising an immunologically effective amount of the vaccine, means for administering the vaccine to animals, and optionally an instruction manual including information for the administration of the immunologically effective amount the composition for treating and/or preventing PCV2 associated diseases.

Further aspects and advantages of the invention will be disclosed in the following experimental section.

Examples

Materials and Methods

Bacterial strains and growth conditions. S. suis strain A3286/94 is an mrp^* sly+ cps9 strain belonging to sequence type (ST) 99 of CC16 originally isolated from a pig with meningitis [3,15,23,24], Strains 15-3/3, V5404/2 and 16085/3b were isolated from inner organs of diseased piglets with meningitis and/or septicemia. S. suis strain 10 ( mrp⁺ epf* sly⁺) is a virulent serotype 2 strain [11 ,15,25]. The isogenic mutant 10Δide_Ssuis deficient in IgM cleavage was included in the bactericidal assay to reveal effects mediated by antigen-specific immunity [11 ,26]. S. suis was grown on Columbia agar plates supplemented with 6 % sheep blood or in Bacto™ Todd Hewitt broth (THB). Escherichia coli (E. coli) strains were cultured in Luria- Bertani (LB) medium including 100 μg/ml ampicillin, if appropriate. Expression and purification of recombinant (r) proteins.

The expression and the purification of rlde_Ssuis [26] and His-tagged fibronectin binding domain of streptococcal fibronection-binding protein I of Streptococcus pyogenes (Sfbl) [27] was performed as described previously [26]. Animal experiments.

Piglets 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. All animal experiments or samplings were conducted by veterinarians and in accordance with the principles outlined in the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes and the German Animal Protection Law (Tierschutzgesetz). The animal experiment of this study was approved by the Landesdirektion Sachsen (permit no. TVV28/16), which includes approval through the registered committee for animal experiments. The collection of blood samples was approved by the Landesdirektion Sachsen (permit no. N01/16 and N19/14).

Five weeks prior to farrowing four pregnant sows of a German Landrace pig herd considered to be free of cps9+ S. suis strains were transported to the Faculty of Veterinary Medicine, Leipzig University. The classification as cps9 free was based on the genotyping results including a described MP-PCR [28] of S. suis isolates from the tonsils of more than 400 animals over the last 14 years. After birth the suckling piglets were treated as follows: Piglets of one litter were equally distributed to the vaccination and the placebo group with a final number of 20 piglets per group. At an age of 2 weeks a vaccine containing rlde_Ssuis as antigen or a placebo was applicated intramuscularly, both supplemented with 20 % [vol/vol] Emulsigen as adjuvant. The piglets were boostered 14 days later. Nine piglets per group were boostered a second time another 14 days later. One dose of rlde_Ssuis vaccination contained 0.4 mg rlde_Ssuis- After the first booster vaccination at an age of 4 weeks, piglets were weaned and moved to the trial pen. All piglets (n=18) used for experimental infection were placed in one pen. The person (CGB) who made the final decision of euthanasia did not know whether a specific piglet was vaccinated with rlde_Ssuis or placebo-treated (partially blinded experiment). Piglets were challenged intravenously at an age of 8 to 9 weeks (17 days after the second booster) with 2 x 10⁸ CFU of S. suis strain 16085/3b grown in Bacto™ Tryptic Soy Broth (TSB) without dextrose. Post infection, the health status of the animals was monitored every 8 h, including measurement of the inner body temperature, assessment of movements and feed intake (piglets were only fed at these time points). Piglets were 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 (³40.5 °C), apathy and anorexia persisting over 32 hours as well as in all cases of central nervous system dysfunction or clinical signs of acute polyarthritis, animals were euthanized for animal welfare reasons. 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 bacteriological (b) investigations as described previously [3,15]: 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); mitral valve (b, h) and tonsil (b, h). The histological screenings were scored as described [29]. Isolation of the challenge strain was confirmed by PCR detecting mrp, epf, sly, arcA, gdh, cps1, cps2, cps7 and cps9 [28]. Bactericidal assay.

Survival of S. suis in porcine blood ex vivo was determined as previously described [11]. Briefly, 500 mI of heparinized blood (16 I. U. heparin/ml) was mixed with 6x10⁵ CFU of exponentially grown bacteria (OD₆oo: 0.5-0.6). The samples were incubated for 2 h at 37 °C on a rotator. Blood for bactericidal assays was drawn from all piglets of the vaccination trial 7 and 17 days after first and second booster vaccination, respectively. The bactericidal assays were conducted within 4 h after blood collection. The specific bacterial contents 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.

Detection of anti (α) -lde_Ssuis IgG. The detection of a-lde_Ssuis IgG was performed as previously described [11]. Sera of piglets immunized with rlde_Ssuis and a truncated derivative (rlde_Ssuis_homologue) in the previous study served as reference serum and positive control in the a-lde_SsuisELISA, respectively.

Detection of Ide_Ssu/s-specific T-cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by density gradient centrifugation on Biocoll (Merck-Biochrom, Berlin, Germany) and cryo-conserved at -80 °C until usage for restimulation. PBMCs were cultivated in 96-well plates (1x10⁶ cells/well) in complete Iscove's Modified Dulbecco's Medium (IMDM, Pan Biotech, Aidenbach, Germany) containing 10 % FCS (Gibco) and penicillin/ streptomycin (100 U/ml and 100 μg/ml, respectively; Merck-Biochrome, Berlin, Germany). Antigen-specific restimulation was conducted with 5 μg/ml rlde_Ssuis or 5 μg/ml His-tagged recombinant (r)Sfbl [27] as non-relevant control antigen. PMA/ lonomycin (10 ng/ml and 500 ng/ml, respectively, both purchased from Merck-SIGMA-Aldrich, Taufkirchen, Germany) were used as positive control added for the last 4 h of incubation time and medium alone was used as negative control. PBMCs were stimulated for 18 h in presence of 2 μg/ml Brefeldin A (Enzo Life Science, Lorrach, Germany) for the last 14 h at 37 °C, 5 % CO2 in humidified atmosphere. To label cells for flow cytometry analysis, the stimulated cells were cropped and stained as previously described [30]. Measurement of samples was conducted at LSR-Fortessa™, BD Bioscience) recording 3-5x10⁵ viable CD3⁺CD4⁺ cells. Frequency of antigen-specific Th cells (CD154+) were determined from the activated Th cell population, described as CD3⁺CD4⁺CD8a⁺ cells. Frequency of lde_Ssuis-induced Th cells was calculated as difference of the of percentage of CD154⁺ cells after Ide_Ssuis re-stimulation minus the percentage of CD154⁺ cells of medium cultivated cells from the same sample.

Statistical analysis.

The evaluation of more than two groups was carried out using two- or one- way analysis of variance (ANOVA) or Kruskall-Wallis with a subsequent Tukey^'s or Dunn^'s multiple comparisons test, respectively. Differences between two groups were analyzed with the Mann-Whitney U-test. Correlation was calculated with the Pearson test. The Wilcoxon test was used for comparison of different time point values within the same group in case of no more than two repeated measures. The data presented in the Kaplan-Meier- diagrams were analyzed with the log rank test. Means and standard deviation of the results are shown. All statistical tests were conducted with GraphPad Prism 7.01 software. Probabilities lower than 0.05 were considered significant (^* p < 0.05, ^** p <

0.01 , ^*** p < 0.001 , ^**** p < 0.0001 ).

Results

S. suis 16085/3b exhibits increased bacterial survival in porcine blood in comparison to other cps9 strains.

In Europe, S. suis cps9 is a major porcine pathogen causing herd problems associated mainly with meningitis, endocarditis and arthritis [2,28]. Recently, a herd experienced an escalating S. suis cps9 problem in growing piglets due to meningitis and septicemia associated with cyanosis and sudden death. As the extent and severity of disease on this farm appeared extreme, the S. suis cps9 strain 16085/3b isolated from the spleen of a pig from this farm was compared to other S. suis cps9 strains by profiling of virulence- associated factors, multi locus sequence typing (MLST) and analysis of bacterial survival in porcine blood. Interestingly, strain 16085/3b is a mrp+ sly+ cps9+ strain of sequence type (ST) 94 and thus genetically distinct to other invasive cps9 strains. As shown in Fig. 1 S. suis cps9 strain 16085/3b shows significantly higher bacterial survival factors than the other three tested cps9 strains of ST15 (V5402/2 and 15-3/3) and ST99 (A3286/94) in blood from cps9 free piglets at an age of 6.5, 7.5 and 8.5 weeks. In addition, for the 16085/3b strain we observed a significant increase of bacterial survival in blood of 8.5 weeks (mean = 2.8, SD = 3.3, n = 5) old piglets in comparison to 4.5 week (mean = 0.9, SD = 0.6, n = 5) and 5.5 week (mean = 1 .4, SD = 0.5, n = 5) old piglets (Fig. 1). Due to this phenotype, we chose strain 16085/3b as challenge strain for this vaccination study, since previous experimental infection studies with other cps9 strains led to rather low rates of mortality making it difficult or even impossible to draw conclusions on protection against mortality [3,4],

Early prime-booster vaccination of piglets in the 2^nd and 4^th week with rlde_Ssuis elicits specific IgG antibody titers and bactericidal immunity against S. suis cps9 strain 16085/3b.

Induction of early protective immunity against S. suis by suckling piglet vaccination would be advantageous over weaning piglet vaccination, as diseases might occur already after weaning. Thus, we investigated humoral immunogenicities of early prime-booster rlde_Ssuis vaccination in the 2^nd and 4^th week of life, respectively. Prior to vaccination in the 2^nd week of life, all suckling piglets had α-lde_Ssuis IgG levels below 5 ELISA units (mean = 1.7; SD = 0.9; n = 20, Fig. 2). Seven days after the 1^st booster vaccination, at an age of 5 weeks, a mean of 20.9 ELISA units (SD = 14.4, n = 20) α-lde_Ssuis IgG was recorded in vaccinated piglets, which is significantly higher than the antibody level found in their placebo-treated littermates (mean = 0.5 ELISA units, SD = 0.2, n = 20; Fig. 2).

A bactericidal assay conducted 7 days after first booster vaccination revealed significantly lower survival factors of the cps9 strain 16085/3b in the blood of the vaccinated piglets (mean = 0.3, SD = 0.2, n=20) compared to the blood of placebo- treated piglets (mean = 0.5, SD = 0.3, n=20; Fig. 3). The cps2 wt strain 10 also had a lower survival factor in the blood of vaccinated piglets (mean = 0.2, SD = 0.3, n = 20) in comparison to placebo-treated piglets (mean = 0.3, SD = 0.5, n = 20), (Fig. 3).

In conclusion, an early prime-booster lde_Ssuis vaccination (in the 2^nd and 4^th week of life) leads to an increase of α-lde_Ssuis-lgG in serum at an age of 5 weeks which is associated with reduced survival of S. suis in blood.

Vaccination with rlde_Ssuis protects growing piglets against mortality caused by S. suis cps9 infection.

The effect of a 2^nd booster on the level of seroconversion against rlde_Ssuis was investigated. The 2^nd booster led to an increase of the α-lde_Ssuis IgG level to a mean of 64.4 ELISA units (SD = 43.9, n = 9) 17 days after the 2^nd booster in the 8^th week of life compared to a mean of 18.2 ELISA units (SD = 15.3, n = 9) 7 days after the 1^st booster in these nine piglets (Fig. 2). These prime-booster-booster vaccinated piglets (n = 9/ group) were used for the challenge experiment. A bactericidal assay conducted after the 2^nd booster immunization suggested killing of the cps9 strain with a mean survival factor of 0.6 (SD = 0.6) in the lde_Ssuis-immunized group in contrast to substantially higher survival in placebo-treated littermates with a mean survival factor of 1.3 (SD = 1.15), respectively (data not shown)). The intravenous challenge with the cps9 strain 16085/3b was conducted on the same day to test protection against this important serotype. Six of nine placebo-treated piglets died or were killed after reaching a clinical score of 25 within 24 h after experimental infection (Fig. 4). Four of them displayed a peracute course of disease with the inability to rise, pain vocalization, vomiting and apathy within less than 8 h after challenge. Further two showed signs of central nervous system dysfunction including convulsions, opisthotonus and tremor the day after challenge. Another placebo-treated piglet showed signs of central nervous system dysfunction as late as 14 dpi. In contrast, none of the nine vaccinated piglets died or showed signs of acute septicemia, central nervous system dysfunction or the inability to rise in the observation period after the cps9 challenge. Only one vaccinated piglet received a clinical score above 10 after experimental infection. Flowever, short time fever and lameness were recorded in seven of nine and four of nine vaccinated piglets, respectively, but these piglets recovered within 24 to 32 h in all cases except for one. Thus, prime-booster- booster rlde_Ssuis vaccination protected piglets against mortality in this intravenous cps9 challenge model.

The histological screening revealed that six of nine placebo-treated piglets had typical fibrinosuppurative lesions in at least two inner organs. Accordingly, S. suis cps9 was detected in seven of nine placebo-treated piglets in two or more internal organs indicating severe bacteremia or infection of multiple organs after challenge. The pathological score w was substantially lower in rlde_Ssuis-vaccinated piglets (2.0 versus 3.0) as moderate and severe fibrinosuppurative lesions were not detected in the brain of these animals. Furthermore, the challenge strain was isolated from an inner organ of only one rlde_Ssuis- vaccinated piglet.

In conclusion, recording of fibrinosuppurative inflammations and bacteriology indicated protection in prime-booster-booster rlde_Ssuis-vaccinated piglets against meningitis and bacterial dissemination.

Prime-booster-booster rlde_Ssuis vaccination elicits a detectable antigen-specific Th cell response

We recently described a method to detect antigen-specific Th cell responses in pigs using the transiently expressed T cell activation marker CD154 [30]. In comparison to piglets of the placebo-group, the re-stimulation of PBMCs of rlde_Ssuis-immunized piglets with rlde_Ssuis induced a significantly increased number of CD154⁺ Th cells (Fig. 5A, upper left) within the activated Th cell population, characterized as CD3⁺CD4⁺CD8a⁺ cells [31]. In contrast, stimulation with a non-relevant control antigen (recombinant fibronectin binding domain of Sfbl) did not induce CD154⁺ Th cells in both groups that confirm the antigen-specificity of the assay. Considering the cytokine profile of the rlde_Ssuis-induced CD154⁺ Th cells, we found a significantly increased number of TNF-a (Fig. 5A, upper right) and IL-17A producer (Fig. 5A, lower left) in the vaccination group in comparison to placebo-treated animals. The same trend was found for IFN-g producing CD154⁺ Th cells.

To analyze, whether the observed lde_Ssuis-specific Th cell response is linked to the B cell- mediated lde_Ssuis-specific IgG response described above (Fig. 2), correlation of the frequencies of rlde_Ssuis-induced CD154⁺ Th cells and lde_Ssuis-specific IgG levels was performed using the Pearson test. In contrast to the placebo group (r² = 0.10), within the rlde_Ssuis-vaccinated group (n = 9) the number of lde_Ssuis-specific CD154⁺ Th cells correlated with the lde_Ssuis-specific IgG level (r² = 0.48) in this manner that animals with higher frequencies of rlde_Ssuis-induced CD154+ Th cells showed higher levels of anti- lde_Ssuis-lgGs (Fig. 5B). Taken together, rlde_Ssuis-prime-booster-booster vaccination of piglets induces a Th cell response that is linked to lde_Ssuis-specific IgG level.

In addition, we analyzed the frequency of lde_Ssuis-specific Th cells after challenge to prove whether vaccination-induced Th cells are re-activated by the challenge with cps9 strain 16085/3b. Therefore, the two placebo-treated animals that survived until day 14 and five rlde_Ssuis-immunized piglets, all with a maximal cumulative clinical score > 4 were selected. We could not detect an increase of lde_Ssuis-specific Th cells after challenge, neither for the frequency of all rlde_Ssuis-induced CD154⁺ cells nor for a cytokine producing subtype of them. Rather, by trend we observed a reduced number of lde_Ssuis-reactive Th. We found a similar result for the α-lde_Ssuis IgG levels, which did not increase after the challenge, but decreased in tendency.

In summary we observed an induction of lde_Ssuis-reactive Th cells by immunization, but no recall upon challenge infection.

DISCUSSION

S. suis cps2 and 9 have an enormous impact on animal health in Europe. A vaccine protecting against both serotypes is needed in the field to ensure return of investments in the pig industry and improve animal health and welfare. Though cps9 is a major porcine pathogen in the field, no recombinant S. suis vaccine has been tested in a challenge experiment with a cps9 strain [32],

The present application revealed protection through prime-boost vaccination with rlde_Ssuis against mortality induced by intravenous application of a S. suis cps9 strain, rlde_Ssuis vaccination has been shown to protect piglets also against S. suis cps2 challenge [11]. Thus, lde_Ssuis is a cross-protective antigen covering at least related cps2 and cps9 strains. No sudden death, recumbency, convulsions and polyarthritis were observed vaccinated piglets. As these signs occurred a few hours after challenge in contrast to the intranasal cps2 challenge, the intravenous application of the cps9 strain 16085/3b with 2x10⁸CFU conducted in this study is regarded as a very hard challenge. Noteworthy, sudden death is not a common sign in experimental infections with S. suis. Based on the rapid progression of disease and the high rate of mortality after challenge as well as the increased survival of this cps9 strain in porcine blood in comparison to the other investigated cps9 strains, strain 16085/3b is considered very virulent. Pathohistological screenings revealed fibrinosuppurative meningitis only in placebo- treated piglets (4/9) and in accordance a substantially lower pathohistological score in rlde_Ssuis-vaccinated piglets (w = 2.0 versus w = 3.0, respectively). Of note, meningitis is the most important pathology of S. suis infection in weaning piglets causing high numbers of losses. Though fibrinous endocarditis was observed in three placebo-treated and also in three rlde_Ssuis-vaccinated piglets, the challenge strain was detected on the respective mitral valve in six placebo-treated but only in one rlde_Ssuis-vaccinated piglet. As S. suis forms biofilms covered with fibrin on heart valves, killing of streptococci by humoral immunity is very limited once this vegetation is formed [6]. However, as intravenous application bypasses mucosal immunity, rlde_Ssuis vaccination might still protect against endocarditis in mucosal infections. Compared to our previous S. suis cps2 study [11], the pathohistological score in placebo-treated piglets was notably lower (w = 3.6 versus w = 3.0, respectively). We suggest that this is due to the sudden death of four piglets on the day of infection. The peracute course of disease did most likely not leave enough time for severe lesions to develop. The piglets only reached pathohistological scores of 3 at the maximum.

We investigated cellular immunogenicities to find out if antigen-specific Th cells are elicited by our vaccination protocol. Since Th cell-mediated B cell activation supports the production of highly affine specific IgGs [33], we investigated the correlation of rlde_Ssuis- specific Th cell frequencies with rlde_Ssuis-specific IgG levels. For this Th cell support especially the activity of TfH cells (follicular T-helper cells) in draining lymph nodes is of importance, as recently also demonstrated in pigs [34], However, lymph node tissue from immunized animals in a vaccination study is usually not accessible. Therefore, it is more beneficial to use readout-parameters for T cell reactivity from blood samples. The results of this study showed that prime-boost vaccination approach of the invention with a vaccine comprising rlde_Ssuis results in induction of antigen-specific Th cells detectable in porcine blood. The antigen-specificity of this read out parameter is confirmed by using a S. pyogenes antigen (the fibronectin-binding domain of Sfbl) as control that was also expressed as His-tagged protein in E. coli and purified via Ni-affinity chromatography [35]. Furthermore, the number of lde_Ssuis-specific CD154⁺ Th cells correlated with the lde_Ssuis-specific IgG level within the immunization group. This is in accordance with our previous comparison of lde_Ssuis-specific CD154⁺ Th cell frequencies and lde_Ssuis-specific IgG levels [30]. In addition, we analyzed lde_Ssuis-specific Th cell response in PBMCs after cps9 challenge (14 dpi.). Obviously, the adaptive immune response to lde_Ssuiswas not recalled by the S. suis cps9 challenge for both, lde_Ssuis-specific-lgGs and -Th cells. In experiments designed to analyse antigen-specific recall of Th cells by using DO11.10/RagKO mice immunized and infected with Ova-expressing S. pneumoniae, Trzcihski et al. demonstrated that vaccination-induced Th17 cells are only protective against S. pneumoniae by antigen-specific recall during challenge infection [21]. Thus, due to the acute and rapid course of disease induced under invasive S. suis infection a vaccination that is designed to activate antigen-specific memory response of Th cells, preferentially Th17 cells in case of streptococci, should include immunodominant T cell antigen(s) under challenge infection.

As we could previously demonstrate, the vaccination with rlde_Ssuis elicits antibodies neutralizing the IgM protease activity of lde_Ssuis[11]. Recently we demonstrated that the activity of complement is important for clearance of S. suis in porcine blood, whereas the complement deposition is reduced by protease activity of lde_Ssuis [19]. Thus, neutralization of IgM protease by a-lde_Ssuis-lgGs should improve the complement- mediated killing of S. suis in porcine blood.

Furthermore, lde_Ssuis is located on the bacterial surface [16] and thus a-lde_Ssuis antibodies are thought to mediate also opsonophagocytosis, as indicated by the lower survival factor of the wt in blood of vaccinated piglets ex vivo in comparison to the respective factor of the mutant 10Δide_Ssuis [11].

In conclusion, the prime-boost vaccination method of the invention provides effective protection against S suis, particularly against very virulent cps9 serotype strains. Our data further suggest that the protective mechanism to control the invasive infection with the virulent cps9 challenge strain 16085/3b is primarily mediated by α- lde_Ssuis IgGs, induced by rlde_Ssuis-vaccination, that is supported by antigen-specific Th cell response.

List of sequences

SEQ1

MNIQERFSLRKSAVGLVSVSLLCAIYTSTVAADTVVTGVNEIIEESQVKDEVSIESEKNESLDGSNI EIVEEIADNIPSPVIAEGEVAVEMKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTES GEQRQIIWAHGITPPAMEQSGGFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAA VSSNMVHWWLEQNSSYVERYLKEKKGTVNVEENYAITDLRRYINSFQNQQNSRVFDMFKTYY GYRTNGFVSDALVDLFINGYKPKAQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERF GEDVRTVLESKGLLGLTYRTLGYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGIT RDASGNPRLNNHMKNNSAGALLDYVHTIRLGQDLWEEYFNPLAKAKETASQTLADTKKALDLS IQGQSELPESMRLIYLEKLNNLYNQGILSIQKAESSEMLSGALENGLNSLKSLDFPISEVGNALAPD LPVGDRSTVSDVDSLSSQETSSTNLEADTENAGIIADGTNQLHFPVEAQTTSSVEAEGDNVFEQE ADTT PTTTENKDEFGSET ,S R NMOTS FTP S T ,VV A VEEDVKNDEV AOVEET J .F.SEK VENOSSET J ,SDT LIVES ANDKEEDRVEAVVSEQPDSIPHQNVEISLVEPTNVETETVVTPINDAATPHGSPTYIDNSVT ESVATPLEKDSIQAGETEIAEPTSSESTNVETETVVTPVNDVATPHGSPTYIDNSVTESVATPLEKD SIQAGETEIAEPTSSESTNVETETVVTPVNDVATPHGSPTYIDNSVTESVATPLEKDSIQAGETEIAE PTSSESTSVEAELVDNSEIHAATSSVTPCGSSAYADGSTTESVATPLEKDSIQTGNTEIAEPTSSKST NVEAASVDNSEIHADASLTAVSSVNLDNPVIEPVAISLIGSKRDTNAEVEVSSLSKREVRKTNTDG LISVQSKVIKKELLESSLAEAGSPLLEATIAQSSNSNSTEIGMSYQNTVLLESNNTERQVSKAEIVM EHKETELVETVSSASEPVVLVENISQTSNNTIESGKNMGVQSQAGAKQILGVEQSSKVSTPTSRQI MGVGLLTLVLGSALGLLKKRRK

SEQ2

MAHHHHHHVGTGSNDDDDKSPDPVVTGVNEIIEESQVKDEVSIESEKNESLDGSNIEIVEEIADNI PSPVIAEGEVAVEMKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTESGEQRQIIW AHGITPPAMEQSGGFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVH WWLEQNSSYVERYLKEKKGTVNVEENYAITDLRRYINSFQNQQNSRVFDMFKTYYGYRTNGFV SDALVDLFINGYKPKAQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVL ESKGLLGLTYRTLGYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPR LNNHMKNNSAGALLDYVHTIRLGQDLWEEYFNPLAKAKETASQTLADTKKALDLSIQGQSELPE SMRLIYLEKLNNLYNQGILSIQKAESSEMLSGALENGLNSLKSLDFPISEVGNALAPDLPVGDRST VSDVDSLSSQETSSTNLEADTENAGIIADGTNQLHFPVEAQTTSSVEAEGDNVFEQEADTLPIIIEN KDEFGSELSRNMQTSETDSLVVAVEEDVKNDEVAQVEELLESEKVENQSSELLSDTLIVESANDK EEDRVEAVVSEQPDSIPHQNVEISLVEPTNVETETVVTPINDAATPHGSPTYIDNSVTESVATPLEK DSIQAGETEIAEPTSSESTNVETETVVTPVNDVATPHGSPTYIDNSVTESVATPLEKDSIQAGETEI AEPTSSESTNVETETVVTPVNDVATPHGSPTYIDNSVTESVATPLEKDSIQAGETEIAEPTSSESTS VEAELVDNSEIHAATSSVTPCGSSAYADGSTTESVATPLEKDSIQTGNTEIAEPTSSKSTNVEAAS VDNSEIHADASLTAVSSVNLDNPVIEPVAISLIGSKRDTNAEVEVSSLSKREVRKTNTDGLISVQS KVIKKELLESSLAEAGSPLLEATIAQSSNSNSTEIGMSYQNTVLLESNNTERQVSKAEIVMEHKET ELVETVSSASEPVVLVENISQTSNNTIESGKNMGVQSQAGAKQILGVEQSSKVSTPTSRQIMGVG LLTLVLGSALGLLKKRRK

SEQ3

ATGAACATTCAAGAACGATTTTCTTTGAGAAAATCCGCGGTTGGCTTGGTCTCAGTCTCTTTG CTATGTGCTATTTATACATCCACTGTTGCTGCCGATACAGTTGTTACAGGAGTGAATGAAAT AATTGAAGAATCACAAGTCAAGGATGAGGTATCTATTGAATCAGAAAAAAATGAATCCCTA GATGGTTCTAATATTGAAATTGTAGAGGAAATAGCAGACAACATCCCATCACCTGTTATCGC TGAAGGGGAAGTAGCGGTAGAGATGAAAGTTGACAGAGGGACCGAGAATGTAGTTTCTAGA AATGATACAGAAGTTACGACGAGCGAGCAAAATCAGATAGAGGTTACTGAGACAAAAGAA ATTTTGAATCAGACCAGTTATCAAACGGAGAGTGGCGAGCAACGACAAATTATATGGGCCC ATGGAATTACTCCTCCTGCAATGGAACAAAGCGGTGGTTTTGTAAAGGAAAAGTATGGAGA

CTATTTAAACTATACAGCGCCATTTGAGGCTGGAAAAGGCTACTATGATACCAATAAGAGTC

TGAATGCTTCATTTATTGACCTTAATCTTTGTTTTGCAGCTGTGTCTTCAAACATGGTACATTG

GTGGTTGGAACAGAATAGTTCCTATGTTGAGCGATATCTCAAAGAAAAAAAGGGTACAGTA

AATGTTGAAGAAAACTACGCAATAACGGACTTACGGCGCTATATTAATTCATTCCAAAATCA

ACAAAATAGTCGAGTTTTTGATATGTTCAAAACTTACTATGGTTATCGTACAAATGGTTTTGT

ATCAGATGCCTTGGTTGACTTGTTTATTAACGGATATAAACCTAAGGCACAGGGCGGTGTCA

ATCTGGAAGATAGCCAGTTAGTACCAGATAGTAGGGGTGGCTTTTTCTACGACGTTTTCAAA

GAGAAAAAACTGACAAATCGAATTTTTAGTGGTAGTTATGAGCGGTTTGGTGAGGATGTTCG

AACTGTTTTGGAAAGCAAAGGATTACTCGGCTTAACTTATAGAACATTAGGTTATGCAACGC

ATATTGTGACGGTATGGGGTGCTGAGTACGACAATCAAGGTAAGATTAAGGCTGTCTATATC

ACAGATTCTGATGATCAACAAGAACAAATTGGTTTGAAGCGTATGGGAATCACTCGTGATGC

TTCCGGAAATCCACGTTTGAATAATCATATGAAAAATAATTCAGCTGGAGCGCTTTTGGATT

ATGTCCATACAATCCGTCTGGGTCAAGACTTATGGGAAGAATATTTCAATCCGCTTGCAAAA

GCCAAAGAAACAGCTAGTCAGACATTAGCCGATACAAAGAAGGCGTTGGATTTGTCTATTC

AAGGACAATCTGAATTGCCAGAATCAATGCGACTGATTTATCTTGAAAAACTAAATAATCTC

TATAATCAAGGAATTCTATCTATTCAAAAGGCAGAAAGTTCTGAGATGCTAAGTGGTGCATT

GGAAAATGGTTTAAATAGTTTAAAGAGTTTAGATTTTCCTATTTCAGAAGTTGGAAATGCTTT

GGCACCAGATTTACCAGTAGGTGATCGCTCAACGGTTTCAGATGTTGATTCTCTATCATCTCA

AGAAACAAGTTCCACAAATTTGGAAGCAGACACAGAGAATGCAGGTATTATTGCAGATGGT

ACCAATCAATTGCATTTTCCAGTGGAGGCCCAAACGACATCTTCAGTAGAGGCTGAGGGAG

ATAATGTTTTTGAACAAGAGGCAGATACATTACCAATAATTATTGAAAACAAGGATGAATTT

GGTTCAGAACTATCAAGAAACATGCAAACGTCAGAAACGGATTCGCTAGTAGTAGCTGTTG

AAGAAGATGTGAAAAATGATGAGGTAGCCCAAGTTGAAGAGCTTCTTGAATCAGAAAAAGT

TGAAAATCAGAGTTCGGAACTTCTGTCAGACACCCTAATCGTAGAGAGTGCAAATGACAAA

GAAGAAGATAGAGTGGAGGCGGTTGTTTCTGAACAACCAGACTCAATACCACATCAAAATG

TAGAAATCTCTCTTGTAGAACCAACGAATGTCGAAACTGAAACTGTGGTCACTCCTATTAAT

GATGCAGCTACTCCTCATGGTTCCCCGACGTATATAGATAATTCCGTAACTGAATCTGTAGCT

ACTCCACTTGAAAAAGACTCCATTCAAGCCGGGGAGACAGAGATTGCAGAACCAACCTCGA

GCGAATCAACGAATGTCGAAACTGAAACTGTGGTCACTCCTGTTAATGATGTAGCTACTCCT

C ATGGTTCCCCG ACGT AT AT AG AT A ATTCCGT A ACTG A ATCTGT AGCTACTCC ACTTG A A A A

AGACTCCATTCAAGCCGGAGAGACAGAAATTGCAGAACCAACCTCGAGCGAATCAACGAAT

GTCGAAACTGAAACTGTGGTCACTCCTGTTAATGATGTAGCTACTCCTCATGGTTCCCCGAC

GTATATAGATAATTCCGTAACTGAATCTGTAGCTACTCCACTTGAAAAAGACTCCATTCAAG

CCGGGGAGACAGAGATTGCAGAACCAACCTCGAGCGAATCAACTAGTGTTGAAGCTGAACT

TGTCGACAATTCTGAAATTCATGCAGCTACCTCTTCAGTTACTCCCTGTGGCTCCTCGGCATA

TGCAGATGGTTCCACAACTGAATCTGTAGCCACTCCGCTTGAAAAAGACTCCATTCAGACTG

GA A AT AC AG A A ATTGC AG A ACC A ACCTCG AGC A A ATC A ACT A ATGT AG A AGCTGC ATCTGT

CGACAATTCTGAAATTCATGCAGATGCCTCTCTAACTGCTGTTTCATCAGTTAATCTGGATAA

TCCAGTGATTGAACCAGTAGCTATCTCCCTTATCGGTTCTAAGAGGGACACGAATGCAGAAG

TAGAAGTTTCTTCATTATCGAAAAGAGAGGTTAGAAAAACAAATACTGACGGGCTAATCTCT

GTTCAATCAAAAGTTATTAAGAAAGAATTGCTAGAATCAAGCTTAGCAGAAGCAGGGTCTC

CATTGCTAGAAGCCACCATTGCTCAGTCTTCAAACTCAAATAGTACTGAGATAGGTATGAGC

TATCAGAATACTGTGTTATTAGAGTCTAATAATACAGAGCGTCAGGTGTCTAAAGCAGAAAT

TGTTATGGAACACAAGGAGACAGAGTTAGTTGAAACGGTTTCATCTGCTTCTGAGCCTGTAG

TGCTCGT AG A A A AT ATCTC AC A A ACCTC A A ATA AT ACTATTG A ATCTGGT A AG A AT ATGGG A

GTTCAATCTCAAGCAGGTGCAAAACAAATTTTAGGCGTAGAACAATCTTCGAAAGTAAGTAC

ACCTACTTCAAGACAGATTATGGGAGTCGGTCTATTGACTCTTGTTCTTGGTAGTGCTTTAGG

TTT GTTAAAGAAAAGACGTAAGTAA

SEQ4 ATGGCACATCACCACCACCATCACGTGGGTACCGGTTCGAATGATGACGACGACAAGAGTC

CGGATCCAGTTGTTACAGGAGTGAATGAAATAATTGAAGAATCACAAGTCAAGGATGAGGT

ATCTATTGAATCAGAAAAAAATGAATCCCTAGATGGTTCTAATATTGAAATTGTAGAGGAAA

TAGCAGACAACATCCCATCACCTGTTATCGCTGAAGGGGAAGTAGCGGTAGAGATGAAAGT

TGACAGAGGGACCGAGAATGTAGTTTCTAGAAATGATACAGAAGTTACGACGAGCGAGCAA

AATCAGATAGAGGTTACTGAGACAAAAGAAATTTTGAATCAGACCAGTTATCAAACGGAGA

GTGGCGAGCAACGACAAATTATATGGGCCCATGGAATTACTCCTCCTGCAATGGAACAAAG

CGGTGGTTTTGTAAAGGAAAAGTATGGAGACTATTTAAACTATACAGCGCCATTTGAGGCTG

GAAAAGGCTACTATGATACCAATAAGAGTCTGAATGCTTCATTTATTGACCTTAATCTTTGTT

TTGCAGCTGTGTCTTCAAACATGGTACATTGGTGGTTGGAACAGAATAGTTCCTATGTTGAG

CGATATCTCAAAGAAAAAAAGGGTACAGTAAATGTTGAAGAAAACTACGCAATAACGGACT

TACGGCGCTATATTAATTCATTCCAAAATCAACAAAATAGTCGAGTTTTTGATATGTTCAAA

ACTTACTATGGTTATCGTACAAATGGTTTTGTATCAGATGCCTTGGTTGACTTGTTTATTAAC

GGATATAAACCTAAGGCACAGGGCGGTGTCAATCTGGAAGATAGCCAGTTAGTACCAGATA

GTAGGGGTGGCTTTTTCTACGACGTTTTCAAAGAGAAAAAACTGACAAATCGAATTTTTAGT

GGTAGTTATGAGCGGTTTGGTGAGGATGTTCGAACTGTTTTGGAAAGCAAAGGATTACTCGG

CTTAACTTATAGAACATTAGGTTATGCAACGCATATTGTGACGGTATGGGGTGCTGAGTACG

ACAATCAAGGTAAGATTAAGGCTGTCTATATCACAGATTCTGATGATCAACAAGAACAAATT

GGTTTGAAGCGTATGGGAATCACTCGTGATGCTTCCGGAAATCCACGTTTGAATAATCATAT

GAAAAATAATTCAGCTGGAGCGCTTTTGGATTATGTCCATACAATCCGTCTGGGTCAAGACT

TATGGGAAGAATATTTCAATCCGCTTGCAAAAGCCAAAGAAACAGCTAGTCAGACATTAGC

CGATACAAAGAAGGCGTTGGATTTGTCTATTCAAGGACAATCTGAATTGCCAGAATCAATGC

GACTGATTTATCTTGAAAAACTAAATAATCTCTATAATCAAGGAATTCTATCTATTCAAAAG

GCAGAAAGTTCTGAGATGCTAAGTGGTGCATTGGAAAATGGTTTAAATAGTTTAAAGAGTTT

AGATTTTCCTATTTCAGAAGTTGGAAATGCTTTGGCACCAGATTTACCAGTAGGTGATCGCTC

AACGGTTTCAGATGTTGATTCTCTATCATCTCAAGAAACAAGTTCCACAAATTTGGAAGCAG

ACACAGAGAATGCAGGTATTATTGCAGATGGTACCAATCAATTGCATTTTCCAGTGGAGGCC

CAAACGACATCTTCAGTAGAGGCTGAGGGAGATAATGTTTTTGAACAAGAGGCAGATACAT

TACCAATAATTATTGAAAACAAGGATGAATTTGGTTCAGAACTATCAAGAAACATGCAAAC

GTCAGAAACGGATTCGCTAGTAGTAGCTGTTGAAGAAGATGTGAAAAATGATGAGGTAGCC

CAAGTTGAAGAGCTTCTTGAATCAGAAAAAGTTGAAAATCAGAGTTCGGAACTTCTGTCAGA

CACCCTAATCGTAGAGAGTGCAAATGACAAAGAAGAAGATAGAGTGGAGGCGGTTGTTTCT

GAACAACCAGACTCAATACCACATCAAAATGTAGAAATCTCTCTTGTAGAACCAACGAATGT

CGAAACTGAAACTGTGGTCACTCCTATTAATGATGCAGCTACTCCTCATGGTTCCCCGACGT

ATATAGATAATTCCGTAACTGAATCTGTAGCTACTCCACTTGAAAAAGACTCCATTCAAGCC

GGGGAGACAGAGATTGCAGAACCAACCTCGAGCGAATCAACGAATGTCGAAACTGAAACTG

TGGTCACTCCTGTTAATGATGTAGCTACTCCTCATGGTTCCCCGACGTATATAGATAATTCCG

TAACTGAATCTGTAGCTACTCCACTTGAAAAAGACTCCATTCAAGCCGGAGAGACAGAAATT

GCAGAACCAACCTCGAGCGAATCAACGAATGTCGAAACTGAAACTGTGGTCACTCCTGTTA

ATG ATGT AGCT ACTCCTC ATGGTTCCCCG ACGT ATATAG AT A ATTCCGT A ACTG A ATCTGT AG

CTACTCCACTTGAAAAAGACTCCATTCAAGCCGGGGAGACAGAGATTGCAGAACCAACCTC

GAGCGAATCAACTAGTGTTGAAGCTGAACTTGTCGACAATTCTGAAATTCATGCAGCTACCT

CTTCAGTTACTCCCTGTGGCTCCTCGGCATATGCAGATGGTTCCACAACTGAATCTGTAGCCA

CTCCGCTTGAAAAAGACTCCATTCAGACTGGAAATACAGAAATTGCAGAACCAACCTCGAG

CAAATCAACTAATGTAGAAGCTGCATCTGTCGACAATTCTGAAATTCATGCAGATGCCTCTC

TAACTGCTGTTTCATCAGTTAATCTGGATAATCCAGTGATTGAACCAGTAGCTATCTCCCTTA

TCGGTTCTAAGAGGGACACGAATGCAGAAGTAGAAGTTTCTTCATTATCGAAAAGAGAGGT

TAGAAAAACAAATACTGACGGGCTAATCTCTGTTCAATCAAAAGTTATTAAGAAAGAATTGC

TAGAATCAAGCTTAGCAGAAGCAGGGTCTCCATTGCTAGAAGCCACCATTGCTCAGTCTTCA

AACTCAAATAGTACTGAGATAGGTATGAGCTATCAGAATACTGTGTTATTAGAGTCTAATAA

TACAGAGCGTCAGGTGTCTAAAGCAGAAATTGTTATGGAACACAAGGAGACAGAGTTAGTT GAAACGGTTTCATCTGCTTCTGAGCCTGTAGTGCTCGTAGAAAATATCTCACAAACCTCAAA

TAATACTATTGAATCTGGTAAGAATATGGGAGTTCAATCTCAAGCAGGTGCAAAACAAATTT

TAGGCGTAGAACAATCTTCGAAAGTAAGTACACCTACTTCAAGACAGATTATGGGAGTCGGT

CTATTGACTCTTGTTCTTGGTAGTGCTTTAGGTTTGTTAAAGAAAAGACGTAAGTAA

SEQ5

MKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTESGEQRQIIWAHGITPPAMEQSG

GFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVHWWLEQNSSYVER

YLKEKKGTVNVEENYAITDLRRYINSFQNQQNSRVFDMFKTYYGYRTNGFVSDALVDLFINGYK

PKAQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVLESKGLLGLTYRTL

GYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPRLNNHMKNNSAGA

LLDYVHTIRLGQDLWE

SEQ6

MAHHHHHHVGTGSNDDDDKSPDPVVTGVNEIIEESQVKDEVSIESEKNESLDGSNIEIVEEIADNI

PSPVIAEGEVAVEMKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTESGEQRQIIW

AHGITPPAMEQSGGFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVH

WWLEQNSSYVERYLKEKKGTVNVEENYAITDLRRYINSFQNQQNSRVFDMFKTYYGYRTNGFV

SDALVDLFINGYKPKAQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVL

ESKGLLGLTYRTLGYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPR

LNNHMKNNSAGALLDYVHTIRLGQDLWEEYFNPLAKACRSTSLRPHSSLVKKPLLRNLNASTW

TRLLAQLN

SEQ7

MAHHHHHHVGTGSNDDDDKSPDPVVTGVNEIIEESQVKDEVSIESEKNESLDGSNIEIVEEIADNI

PSPVIAEGEVAVEMKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTESGEQRQIIW

AHGITPPAMEQSGGFVKEKYGDYLNYTAPFKAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVH

WWLEQNSSYVERYLKEKKGTVNVGENYAITDLRRYIDSFQDQQNSRVFDMFKTYYGYRTNGFV

SDALVDLFINGYKPKVQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVL

ESKGLLGLTYRTLGYATHIVTVWGAEYDNQGKIRAVYITDSDDQQEQIGLKRMGITRDASGNPR

LNNHVKNNSAGALLDYVHTIRLGQDLWEEYFNPFAKAKEIASQILADRKKALVLSIQGQSELPES

MRLIYLEKLNNLYNQGILSIQKTESSEMLSGALENGLNSLKSLDFPISEVGNALAPDLSVGDRSTV

SDVDSLSSQETSSTNLEADTENAGIIADGTNQLHFPVEAQTTSSVEAEGDNVFEQEADTLPIIIENK

DEFGSELSGNMQTSETDSLVVAVEEDVKNDEVDQVEKLLESEKVENQSSELLSDTLIVEGANDK

EEDRVEAVVSEQPDSIPHQNVEISPVEPMNVETESVVTPINDAATPHGFPMYIDNSVTESVATPLE

KDSIQAGETEIAEPTSSESTSVEAELVDNSEIHSATSSVTPRGSSAYADSSTTESVATLLEKDSIQAG

ETEIAEPTSSKSTNVEAASVDNSEIHADTSLTAVSSVNLDNPVIEPVAIPLIGSKRDTNAEVEVSSLS

KREVRKPNTEGLISVQSKVIKKELLESSLVEAGSPLLEATIAQSSNSNSTEIGMSYQNTVLLESNNT

ERQVSKAEIVIEHKETELVETVSSASEPVVLVENISQTSNNTIESGKNMGVQSQAGAKQILGIEQSS

KVSTPTSRQIMGVGLLTLVLGSALGLLKKRRK

SEQ8

ATGGCACATCACCACCACCATCACGTGGGTACCGGTTCGAATGATGACGACGACAAGAGTC

CGGATCCAGTTGTTACAGGAGTGAATGAAATAATTGAAGAATCACAAGTCAAGGATGAGGT

ATCTATTGAATCAGAAAAAAATGAATCCCTAGATGGTTCTAATATTGAAATTGTAGAGGAAA

TAGCAGACAACATCCCATCACCTGTTATCGCTGAAGGGGAAGTAGCGGTAGAGATGAAAGT

TGACAGAGGGACCGAGAATGTAGTTTCTAGAAATGATACAGAAGTTACGACGAGCGAGCAA

AATCAGATAGAGGTTACTGAGACAAAAGAAATTTTGAATCAGACCAGTTATCAAACGGAGA

GTGGCGAGCAACGACAAATTATATGGGCCCATGGAATTACTCCTCCTGCAATGGAACAAAG

CGGTGGTTTTGTAAAGGAAAAGTATGGAGACTATTTAAACTATACAGCGCCATTTGAGGCTG

GAAAAGGCTACTATGATACCAATAAGAGTCTGAATGCTTCATTTATTGACCTTAATCTTTGTT TTGCAGCTGTGTCTTCAAACATGGTACATTGGTGGTTGGAACAGAATAGTTCCTATGTTGAG

CGATATCTCAAAGAAAAAAAGGGTACAGTAAATGTTGAAGAAAACTACGCAATAACGGACT

TACGGCGCTATATTAATTCATTCCAAAATCAACAAAATAGTCGAGTTTTTGATATGTTCAAA

ACTTACTATGGTTATCGTACAAATGGTTTTGTATCAGATGCCTTGGTTGACTTGTTTATTAAC

GGATATAAACCTAAGGCACAGGGCGGTGTCAATCTGGAAGATAGCCAGTTAGTACCAGATA

GTAGGGGTGGCTTTTTCTACGACGTTTTCAAAGAGAAAAAACTGACAAATCGAATTTTTAGT

GGTAGTTATGAGCGGTTTGGTGAGGATGTTCGAACTGTTTTGGAAAGCAAAGGATTACTCGG

CTTAACTTATAGAACATTAGGTTATGCAACGCATATTGTGACGGTATGGGGTGCTGAGTACG

ACAATCAAGGTAAGATTAAGGCTGTCTATATCACAGATTCTGATGATCAACAAGAACAAATT

GGTTTGAAGCGTATGGGAATCACTCGTGATGCTTCCGGAAATCCACGTTTGAATAATCATAT

GAAAAATAATTCAGCTGGAGCGCTTTTGGATTATGTCCATACAATCCGTCTGGGTCAAGACT

TATGGGAAGAATATTTCAATCCGCTTGCAAAAGCCTGCAGGTCGACAAGCTTGCGGCCGCAC

TCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACT

AGCGC AGCTT A ATT A A

SEQ9

ATGGCACATCACCACCACCATCACGTGGGTACCGGTTCGAATGATGACGACGACAAGAGTC

CGGATCCAGTTGTTACAGGAGTGAATGAAATAATTGAAGAATCACAAGTCAAGGATGAGGT

ATCTATTGAATCAGAAAAAAATGAATCCCTAGATGGTTCTAATATTGAAATTGTAGAGGAAA

TAGCAGACAACATCCCATCACCTGTTATCGCTGAAGGGGAAGTAGCGGTAGAGATGAAAGT

TGACAGAGGGACCGAGAATGTAGTTTCTAGAAATGATACAGAAGTTACGACGAGCGAGCAA

AATCAGATAGAGGTTACTGAGACAAAAGAAATTTTGAATCAGACCAGTTATCAAACGGAGA

GTGGCGAGCAACGACAAATTATATGGGCCCATGGAATTACTCCTCCTGCAATGGAACAAAG

CGGTGGTTTTGTAAAGGAAAAGTATGGAGACTATTTAAACTATACAGCGCCATTTAAGGCAG

GA A A AGGCT ATT ATG AT ACC A ATA A A AGTCTC A ATGCTTC ATTT ATTG ACCTT A ACCT ATGTT

TTGCAGCCGTGTCTTCCAACATGGTACATTGGTGGTTGGAACAGAATAGTTCCTATGTTGAG

CGATATCTCAAAGAAAAAAAGGGTACAGTAAATGTTGGGGAAAACTATGCAATAACGGACC

TACGTCGCTATATTGATTCGTTCCAGGATCAGCAAAATAGTCGAGTCTTTGATATGTTCAAA

ACTT ACT ACGGTT ATCGT AC A A ATGGTTTTGTGTC AG ATGCCCT AGTTG ACTTGTTT ATT A AT

GGATATAAACCTAAGGTACAGGGTGGTGTCAATCTGGAAGATAGCCAGTTAGTACCAGATA

GTAGGGGTGGCTTTTTCTACGACGTTTTCAAAGAGAAAAAACTGACAAATCGTATTTTTAGT

GGTAGCTATGAGCGTTTTGGTGAGGATGTTCGAACTGTTTTGGAGAGCAAAGGATTACTCGG

TCT A ACTT AT AG A AC ATT AGGCT ATGC A ACGC AT ATTGTG ACGGT ATGGGGTGCTG AGTACG

ATAATCAAGGTAAGATTAGGGCTGTCTATATCACTGATTCCGATGATCAACAAGAACAAATT

GGTTTGAAGCGTATGGGAATCACTCGTGATGCTTCTGGAAATCCGCGTTTGAATAATCATGT

GAAAAATAATTCAGCTGGGGCGCTTTTGGATTATGTCCATACAATCCGTCTTGGTCAAGACT

TATGGGAAGAATATTTCAATCCGTTCGCAAAAGCCAAAGAAATAGCTAGTCAGATACTAGCT

GATAGAAAGAAGGCGTTGGTTCTGTCTATTCAAGGACAATCTGAATTGCCAGAATCAATGCG

GCTGATTTATCTTGAAAAACTAAATAATCTCTATAATCAAGGGATTCTATCTATTCAAAAGA

CAGAAAGTTCTGAGATGCTAAGTGGTGCATTGGAAAATGGTTTAAATAGTTTAAAGAGTTTA

GATTTTCCTATTTCAGAAGTTGGAAATGCTTTGGCACCAGATTTATCAGTAGGTGATCGCTCA

ACGGTTTCAGATGTTGATTCTCTATCATCTCAAGAAACAAGTTCCACAAATTTGGAAGCAGA

CACAGAGAATGCAGGTATTATTGCAGATGGTACCAATCAATTGCATTTTCCAGTGGAGGCCC

AAACGACATCTTCAGTAGAGGCTGAGGGAGATAATGTTTTTGAACAAGAGGCAGATACATT

ACCAATAATTATTGAAAACAAGGATGAATTTGGTTCAGAACTATCAGGAAACATGCAAACG

TCAGAAACGGATTCGCTAGTAGTAGCTGTTGAAGAAGATGTGAAAAATGATGAGGTAGACC

AAGTTGAAAAGCTTCTTGAATCAGAAAAAGTTGAAAATCAGAGTTCGGAACTTCTGTCAGAC

ACCCTAATCGTAGAGGGTGCAAATGACAAAGAAGAAGATAGAGTGGAGGCGGTTGTTTCTG

AACAACCAGACTCAATACCACATCAAAATGTAGAAATCTCTCCTGTAGAACCAATGAATGTC

GAAACTGAATCTGTGGTCACTCCTATTAATGATGCAGCTACTCCTCATGGTTTCCCGATGTAT

ATAGATAATTCCGTAACTGAATCTGTAGCTACTCCACTTGAAAAAGACTCCATTCAAGCCGG AGAGACAGAAATTGCAGAACCAACCTCGAGCGAATCAACTAGTGTTGAAGCTGAACTTGTC GACAATTCTGAAATCCATTCAGCTACCTCTTCAGTTACTCCCCGTGGTTCCTCGGCATATGCA GATAGTTCCACAACTGAATCTGTAGCTACTCTGCTTGAAAAAGACTCCATTCAGGCTGGAGA GACAGAAATTGCAGAACCAACCTCGAGCAAATCAACTAATGTCGAAGCTGCATCTGTCGAC AATTCTGAAATTCATGCAGATACCTCTCTAACTGCTGTTTCATCAGTCAATCTGGATAATCCA GTGATTGAACCAGTAGCTATCCCCCTTATCGGTTCTAAGAGGGACACGAATGCAGAAGTGGA AGTTTCTTC ATT ATCG A A A AG AG AGGTT AG A A A ACC A A AT ACTG A AGGGCT A ATCTCTGTTC AATC A A A AGTT ATT A AG A A AG A ATTGCT AG A ATC A AGCTTAGT AG A AGC AGGGTCTCC ATT GCTAGAAGCCACCATTGCTCAGTCTTCAAACTCAAATAGTACTGAGATAGGTATGAGCTATC AG A AT ACTGTGTT ATT AG AGTCT A AT A AT AC AG AGCGTC AGGTGTCT A A AGC AG A A ATTGTT ATAGAACACAAGGAGACAGAGTTAGTTGAAACGGTTTCATCTGCTTCTGAGCCTGTAGTGCT CGTAGAAAATATCTCACAAACCTCAAATAATACTATTGAATCTGGTAAGAATATGGGAGTTC AATCTC A AGC AGGTGC A A A AC A A ATTTT AGGC AT AG A AC A ATCTTCG A A AGT AAGT AC ACC TACTTCAAGACAGATTATGGGAGTCGGTCTATTGACTCTTGTTCTTGGTAGTGCTTTAGGTTT GTTAAAGAAAAGACGTAAGTAA

SEQ10

MKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTESGEQRQIIWAHGITPPAMEQSG GFVKEKYGDYLNYTAPFKAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVHWWLEQNSSYVER YLKEKKGTVNVGENYAITDLRRYIDSFQDQQNSRVFDMFKTYYGYRTNGFVSDALVDLFINGY KPKVQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVLESKGLLGLTYRT LGYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPRLNNHMKNNSAG ALLDYVHTIRLGQDLWE

SEQ11

MKVDRGTENVVSRNDTEVTTSEQNQIEVTETKEILNQTSYQTESGEQRQIIWAHGITPPAMEQSG GFVKEKYGDYLNYTAPFKAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVHWWLEQNSSYVER YLKEKKGTVNVGENYAITDLRRYIDSFQDQQNSRVFDMFKTYYGYRTNGFVSDALVDLFINGY KPKVQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVLESKGLLGLTYRT LGYATHIVTVWGAEYDNQGKIRAVYITDSDDQQEQIGLKRMGITRDASGNPRLNNHVKNNSAG ALLDYVHTIRLGQDLWE

SEQ12 MKVDRGTENVVSRNDKEVTTSEKNQIEVTETKEILNQTSYQTESGEQRQIIWAHGITPPAMEQSG GFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVHWWLEQNSSYVER YLKEKNSTVNVGENYAITDLRRYINSFQNQQNSRVFDMFKTYYGYRTNGFVSDALVDLFINGYK PKAQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVLESKGLLGLTYRTL GYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPRLNNHVKNNSAGA LLDYVHTIRLGQDLWE

SEQ13

MKSDNGDENAVSRDDSEVTTNEQNQIEVTETKEILNQTSYQTESGEQRQIIWAHGITPPAMEQSG GFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVHWWLEQNSSYVER YLKEKKGTVNVEENYAITDIRRYINSFQNQQNSRVFDMFKTYYGYRTNGFVSDALVDLFINGYK PKSQGGVNLEDSHLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVLESKGLLGLTYRTL GYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPRLNNHMKNNSAGA LLDYVHTIRLGQDLWE SEQ14 MNSDNGDENVVSRDDSEVTTNEQNQIEVTETKEILNYTSYQTESGEQRQIVWAYGITPPVMEQK GGFVKEKYGDYLNYTAPFEAGKGYYDTNKSLNASFIDLNLCFAAVSSNMVHWWLEQNSSYVE RYLKEKKGTVNVEENYAITDLRRYINSFQNQQNSRVFDMFKTYYGYRTNGFVSDALVDLFINGY KPKAQGGVNLEDSQLVPDSRGGFFYDVFKEKKLTNRIFSGSYERFGEDVRTVLESKGLLGLTYRT LGYATHIVTVWGAEYDNQGKIKAVYITDSDDQQEQIGLKRMGITRDASGNPRLNNHMKNNSAG ALLDYVHTIRLGQDLWE

References

[1] Goyette-Desjardins G, Auger J-PP, Xu J, Segura M, Gottschalk M. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing. Emerg Microbes Infect 2014;3:e45. doi:10.1038/emi.2014.45.

[2] Wisselink HJ, Smith HE, Stockhofe-Zurwieden N, Peperkamp K, Vecht U. Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries. Vet Microbiol 2000;74:237-48. doi:10.1016/S0378-1135(00)00188-7.

[3] Buttner N, Beineke A, de Buhr N, Lilienthal S, Merkel J, Waldmann K-H, et al. Streptococcus suis serotype 9 bacterin immunogenicity and protective efficacy. Vet Immunol Immunopathol 2012;146. doi:10.1016/j.vetimm.2012.03.012.

[4] Dekker CNT, Bouma A, Daemen AJJM, Van Leengoed LAMG, Jonker FH, Wagenaar JA, et al. Homologous whole bacterin vaccination is not able to reduce Streptococcus suis serotype 9 strain 7997 transmission among pigs or colonization. Vaccine 2011 ;30:1379-87. doi:10.1016/j. vaccine.2011.12.035.

[5] Dawei G, Liping W, Chengping L. In vitro biofilm forming potential of Streptococcus suis isolated from human and swine in China. Brazilian J Microbiol 2012;43:993-1004. doi:10.1590/S1517-83822012000300021.

[6] Rieckmann K, Muller K, Moter A, Baums CG, Seydel A. Streptococcus suisserotype 9 endocarditis and subsequent severe meningitis in a growing pig despite specific bactericidal humoral immunity. JMM Case Reports 2017. doi:10.1099/jmmcr.0.005093.

[7] 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. doi:10.1136/VR.148.15.473.

[8] Li Y, Gottschalk M, Esgleas M, Lacouture S, Dubreuil JD, Willson P, et al. Immunization with recombinant Sao protein confers protection against Streptococcus suis infection. Clin Vaccine Immunol 2007;14:937-43. doi:10.1128/CVI.00046-07.

[9] Zhang A, Chen B, Li R, Mu X, Han L, Zhou H, et al. Identification of a surface protective antigen, HP0197 of Streptococcus suis serotype 2. Vaccine 2009;27:5209-13. doi:10.1016/j.vaccine.2009.06.074.

[10] Li J, Xia J, Tan C, Zhou Y, Wang Y, Zheng C, et al. Evaluation of the immunogenicity and the protective efficacy of a novel identified immunogenic protein, SsPepO, of Streptococcus suis serotype 2. Vaccine 2011 ;29:6514-9. doi:10.1016/j.vaccine.2011.07.010.

[11] Seele J, Hillermann L-MM, Beineke A, Seitz M, von Pawel-Rammingen U, Valentin-Weigand P, et al. The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, is a highly protective antigen against serotype 2. Vaccine 2015;33:2207-12. doi:10.1016/j.vaccine.2015.03.047.

[12] Hsueh KJ, Lee JW, Hou SM, Chen HS, Chang TC, Chu CY. Evaluation on a Streptococcus suis Vaccine Using Recombinant Sao-L Protein Manufactured by Bioreactors as the Antigen in Pigs. Transbound Emerg Dis 2014;61 :e35-43. doi:10.1111 /tbed.12067.

[13] King SJ, Leigh JA, Heath PJ, Luque I, Tarradas C, Dowson CG, et al. Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis: Identification of virulent clones and potential capsular serotype exchange. J Clin Microbiol 2002;40:3671-80. doi:10.1128/JCM.40.10.3671 -3680.2002.

[14] Li Y, Martinez G, Gottschalk M, Lacouture S, Willson 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. doi:10.1128/IAI.74.1.305-312.2006.

[15] Baums CG, Kock C, Beineke A, Bennecke K, Goethe R, Schroder C, et al. Streptococcus suis bacterin and subunit vaccine immunogenicities and protective efficacies against serotypes 2 and 9. Clin Vaccine Immunol 2009;16:200-8. doi:10.1128/CVI.00371 -08.

[16] Seele J, Singpiel A, Spoerry C, Pawel-Rammingen UV, Valentin-Weigand P, Baums CG. Identification of a Novel Host-Specific IgM Protease in Streptococcus suis. J Bacteriol 2013;195. doi:10.1128/JB.01875-12.

[17] Von Pawel-Rammingen U, Johansson BP, Bjorck L. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 2002. doi:10.1093/emboj/21.7.1607.

[18] Seele J, Beineke A, Hillermann LM, Jaschok-Kentner B, Von Pawel-Rammingen U, Valentin-Weigand P, et al. The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, is involved in complement evasion. Vet Res

2015;46:45. doi:10.1186/s13567-015-0171 -6. [19] Rungelrath V, WeiBe C, Schutze N, Muller U, Meurer M, Rohde M, et al. IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism. Virulence

2018;9:21505594.2018.1496778. doi:10.1080/21505594.2018.1496778.

[20] Wilson R, Cohen JM, Jose RJ, De Vogel C, Baxendale H, Brown JS. Protection against Streptococcus pneumoniae lung infection after nasopharyngeal colonization requires both humoral and cellular immune responses. Mucosal Immunol 2015;8:627-39. doi:10.1038/mi.2014.95.

[21] Trzcihski K, Thompson CM, Srivastava A, Basset A, Malley R, Lipsitch M. Protection against nasopharyngeal colonization by Streptococcus pneumoniae is mediated by antigen-specific CD4+T cells. Infect Immun 2008;76:2678-84. doi:10.1128/IAI.00141-08.

[22] Lecours M-P, Letendre C, Clarke D, Lemire P, Galbas T, Benoit-Biancamano M- O, et al. Immune-responsiveness of CD4+ T cells during Streptococcus suis serotype 2 infection. Sci Rep 2016. doi:10.1038/srep38061 .

[23] Beineke A, Bennecke K, Neis C, Schroder C, Waldmann K-H, Baumgartner W, et al. Comparative evaluation of virulence and pathology of Streptococcus suis serotypes 2 and 9 in experimentally infected growers. Vet Microbiol 2008;128. doi:10.1016/j.vetmic.2007.10.028.

[24] 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. doi:10.1099/jmm.0.46616-0.

[25] Smith HE, Damman M, van der Velde J, Wagenaar F, Wisselink HJ, Stockhofe- Zurwieden 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.

[26] Seele J, Singpiel A, Spoerry C, von 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. doi:10.1128/JB.01875-12.

[27] Molinari G, Talay SR, Valentin-Weigand P, Rohde M, Chhatwal GS. The fibronectin-binding protein of Streptococcus pyogenes, Sfbl, is involved in the internalization of group A streptococci by epithelial cells. Infect Immun 1997.

[28] Silva LMG, 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. doi:10.1016/j.vetmic.2005.12.013. [29] 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. doi:10.1128/IAI.00359-06.

[30] Ebner F, Schwiertz P, Steinfelder S, Pieper R, Zentek J, Schutze N, et al. Pathogen-reactive T helper cell analysis in the pig. Front Immunol 2017;8. doi:10.3389/fimmu.2017.00565.

[31] Gerner W, Kaser T, Saalmuller A. Porcine T lymphocytes and NK cells - An update. Dev Comp Immunol 2009. doi:10.1016/j.dci.2008.06.003.

[32] Segura M. Streptococcus suis vaccines: candidate antigens and progress. Expert Rev Vaccines 2015;0584:1-22. doi:10.1586/14760584.2015.1101349.

[33] Martins KAO, Cooper CL, Stronsky SM, Norris SLW, Kwilas SA, Steffens JT, et al. Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity. EBioMedicine 2016. doi:10.1016/j.ebiom.2015.11.041.

[34] Ugolini M, Gerhard J, Burkert S, Jensen KJ, Georg P, Ebner F, et al. Recognition of microbial viability via TLR8 drives TFHcell differentiation and vaccine responses. Nat Immunol 2018. doi:10.1038/s41590-018-0068-4.

[35] Medina E, Talay SR, Chhatwal GS, Guzman CA. Fibronectin-binding protein I of Streptococcus pyogenes is a promising adjuvant for antigens delivered by mucosal route. Eur J Immunol 1998. doi:10.1002/(SICI)1521 - 4141 (199803)28:03<1069::AID-IMMU1069>3.0.CO;2-P.

Claims

1. An IgM protease antigen of Streptococcus suis for use in the prophylactic or therapeutic treatment of Streptococcus Suis infection in a piglet, said use comprising a first and a second administrations of IgM protease antigen to said piglet before 35 days of age.

2. An IgM protease antigen of Streptococcus suis for use according to claim 1 , further comprising a third administration of IgM protease antigen to said piglet after 35 days of age.

3. An IgM protease antigen of Streptococcus suis for use according to claim 1 or 2, wherein the Streptococcus Suis infection is caused by a serotype 2 and/or a serotype 9 strain.

4. An IgM protease antigen of Streptococcus suis for use according to claim 3, for treating Streptococcus Suis infection by a serotype 9 strain.

5. An IgM protease antigen of Streptococcus suis for use according to any one of claims 1 to 4, wherein the first administration is performed when the piglet is between 7 days and 21 days of age, preferably at 14 days of age, and the second administration is performed when the piglet is between 21 days and 35 days of age, preferably at 28 days of age.

6. An IgM protease antigen of Streptococcus suis for use according to any one of claims 2 to 5, wherein the third administration is performed when the piglet is between 35 and 49 days of age, preferably at 42 days of age.

7. An IgM protease antigen of Streptococcus suis for use according to any one of claims 1 to 6, wherein the IgM protease antigen comprises a sequence selected from anyone of SEQ ID NOs : 1-2, 5-7 and 10-14 or a sequence having at least 90% identity over the entire length, preferably at least 95% identity over the entire length, with any one of SEQ ID NOs : 1-2, 5-7 or 10-14.

8. An IgM protease antigen of Streptococcus suis for use according to claim 7, wherein the IgM protease antigen is a protein comprising sequence SEQ ID NO : 1 , 2, 5, 6 or 7 or a protein comprising a sequence having at least 90% identity over the entire length, preferably at least 95% identity over the entire length, with any one of SEQ ID NO : 1 , 2, 5, 6 or 7.

9. An IgM protease antigen of Streptococcus suis for use according to claim anyone of the preceding claims, wherein the antigen is a recombinant protein.

10. An IgM protease antigen of Streptococcus suis for use according to any one of claims 1 to 9, for protecting said piglet against mortality caused by the Streptococcus Suis infection.

11. An IgM protease antigen of Streptococcus suis for use according to any one of claims 1 to 10, wherein each administration comprises between 0,025 and 1 mg of IgM protease antigen, preferably between 0,1 and 0,8 mg of IgM protease antigen.

12. An IgM protease antigen of Streptococcus suis for use according to any one of claims 1 to 11 , wherein each administration is by injection, preferably by intramuscular injection.

13. An IgM protease antigen of Streptococcus suis for use according to any one of the preceding claims, wherein the same antigen is used for each administration.

14. An IgM protease antigen of Streptococcus suis for use according to any one of the preceding claims, wherein the antigen is formulated with an adjuvant.

15. An IgM protease antigen of Streptococcus suis for use for protecting a piglet against mortality caused by a Streptococcus Suis strain of serotype 9 infection, said use comprising a first and a second administration of an IgM protease antigen to said piglet before 35 days of age, and a third administration after 35 days of age.