Vaccines to protect against various serotypes of Streptococcus suis

Abstract

The present invention relates to a vaccine comprising an IgM protease antigen of Streptococcus suis serotype (7) in combination with Streptococcus suis bacterin serotype (9), sequence type (16) and a pharma- ceutically acceptable carrier. The present invention also relates to a combination of the IgM protease antigen of Streptococcus suis serotype (7) and Streptococcus suis bacterin serotype (9), sequence type (16) for use in a method for protecting pigs against pathogenic infections with Streptococcus suis, and a method for protecting pigs against pathogenic infections with Streptococcus suis by administering to the pig the IgM protease antigen of Streptococcus suis serotype (7) and Streptococcus suis bacterin serotype (9), sequence type (16).

Description

[Technical field]

[0001] General Field of the Invention The present invention relates to the protection of pigs against pathogenic infections with Streptococcus suis bacteria of various serotypes, in particular the most prevalent serotypes 1, 2, 7 and 9. [Background technology]

[0002] 2. Background of the Invention Streptococcus suis (S. suis) is one of the main causative agents of infectious bacterial diseases in pigs. This pathogen can cause a variety of clinical syndromes, including meningitis, arthritis, pericarditis, polyserositis, septicemia, pneumonia and sudden death. S. suis is a Gram-positive, facultative anaerobic coccus originally defined as Lancefield groups R, S, R / S or T. Later, a new classification system was proposed based on type-specific capsular polysaccharide antigens located in the cell wall. This resulted in a system that includes 35 serotypes (Rasmussen and Andresen, 1998, “16S rDNA sequence variations of some Streptococcus suis serotypes”, Int. J. Syst. Bacteriol. 48, 1063-1065), of which serotypes 1, 2, 7 and 9 are currently the most prevalent, especially in Europe. However, it is recognized that capsular serotypes are not good markers of virulence. Therefore, an alternative system was developed to help understand the epidemiology of S. suis infection and the biological relevance of serotyping approaches, namely the so-called multilocus sequence typing (MLST) as described by King et al. in the Journal of Clinical Microbiology, October 2002, p. 3671-3680 (Development of a Multilocus Sequence Typing Scheme for the pig pathogen Streptococcus suis: Identification of virulent clones and potential capsular serotype exchange). In that study, 92 sequence types were identified, among which the ST complexes ST1, ST27 and ST87, each containing multiple sequence types, are predominant in the population.See also the Streptococcus suis MLST website (https: / / pubmlst.org / ssuis / ) hosted at the University of Oxford (Jolley et al. Wellcome Open Res 2018,3:124 (funded by the Wellcome Trust) which references the paper by King et al. and allows easy identification of the sequence type of any Streptococcus suis strain.

[0003] Control of S. suis in pig herds appears to be difficult. S. suis is a commensal and opportunistic pathogen of pigs. Apparently, not every outbreak of infection triggers the immune system. Secondly, S. suis is a well-encapsulated pathogen that uses abundant virulence factors to evade the host immune system. These characteristics combined have been obstacles to the development of an effective vaccine to combat this important pathogen. A review article was published several years ago, which provides an overview of existing and experimental vaccines against S. suis (Mariela Segura: “Streptococcus suis vaccines: candidate antigens and progress, in Expert Review of Vaccines, Volume 14, 2015, Issue 12, pages 1587-1608). In this review, clinical and experimental data were compiled and compared, providing an overview of the status of vaccine development against S. suis, as outlined herein below.

[0004] Currently commercially available vaccines are mainly whole-cell bacterins. However, field reports document difficulties in controlling and managing the disease, and “vaccine failures” are common, especially when using bacterin vaccines, due to extremely poor heterologous protection. Carrier pigs are the primary source of infection, and both vertical and horizontal transmission are involved in the spread of the disease within herds. Clinical disease is usually brought about by mixing carrier animals with susceptible animals under stressful conditions such as weaning and transportation. Medicated early weaning and early weaning-separation housing practices do not eliminate Streptococcus suis infection. Therefore, effective control measures to prevent the disease rely on prophylactic / transmission prophylaxis (when possible) and vaccination. Currently, field immunization efforts focus on the use of commercially available bacterins or autologous bacterins. These vaccine strategies have been applied to either piglets or sows. From weaning onwards, piglets are more susceptible to S. suis infections due to the stress associated with weaning and the general subsequent transport. Therefore, pre-partum immunization of sows is often used to try and transfer passive immunity to piglets and to confer protection against S. suis early in life in these stressful environments. Furthermore, vaccination of sows is an economical alternative to vaccination of piglets as it is less costly and labor intensive. However, available results seem to indicate that vaccination of sows with bacterins is also controversial. In many cases, vaccinated sows respond poorly or not at all to vaccination, even when vaccinated twice before parturition, resulting in low maternal immunity transferred to their litters. Also, even if sufficient levels of maternal immunity are transferred, in many cases maternal antibodies are too low to confer protection during the most critical period of 4–7 weeks of age.

[0005] In piglets, autologous bacterins are frequently used in the field, especially in Europe. They are prepared from virulent strains isolated in farms with clinical problems and are applied in the same farms. One drawback of autologous bacterins is that vaccine safety data is lacking and severe adverse reactions can occur. Sampling errors (by using only one or two pigs or samples) can result in failure to identify strains or serotypes associated with recent outbreaks. This failure can be particularly problematic in endemic herds. Finally, the most important problem with autologous bacterins is that their actual efficacy has not been well studied. As the application of autologous vaccines is empirical, it is not surprising that the results obtained with these vaccines are inconsistent and often disappointing.

[0006] Other experimental vaccines have also been described in the art. Kai-Jen Hsueh et al. show that bacterin + subunits can be the basis for successful vaccination of sows to confer protective immunity to their piglets ("Immunization with Streptococcus suis bacterin plus recombinant Sao protein in sows conveys passive immunity to their piglets", in: BMC Veterinary Research, BMC series-open, inclusive and trusted, 13:15, 7 January 2017).

[0007] Live attenuated vaccines have also been envisaged in the art. Non-encapsulated isogenic mutants of Streptococcus suis serotype 2 have been clearly shown to be avirulent. Nevertheless, live vaccine formulations based on non-encapsulated serotype 2 mutants induced only partial protection against death and failed to prevent the development of clinical signs in pigs challenged with wild-type strains (Wisselink HJ, Stockhofe-Zurwieden N, Hilgers LA, et al. “Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2” in: Vet Microbiol. 2002, 84: 155-168.).

[0008] During the past few years, an extensive list of antigenic or immunogenic Streptococcus suis molecules has been reported, most of which were discovered through immunoproteomics using either convalescent sera from infected pigs or humans and / or immune sera produced in the laboratory. WO 2015 / 181356 (IDT Biologika GmbH) showed that the IgM protease antigen (either the whole protein or the highly conserved Mac-1 domain, which represents only about 35% of the complete protein) was able to induce a protective immune response in piglets in a vaccination scheme administering two doses of the IgM protease antigen, which may be combined with a priming vaccination containing a bacterin. The '356 patent application suggests that due to the fact that the IgM protease antigen is highly conserved across most, if not all, S. suis serotypes, particularly the most prevalent serotypes 1, 2, 7 and 9, the IgM protease antigen may be used to achieve broad cross-protection among S. suis serotypes, particularly serotypes 1, 2, 7 and 9.

[0009] WO 2017 / 005913 (Intervacc AB) confirms the fact that the IgM protease is highly conserved across the various Streptococcus suis serotypes and therefore the expected broad protection that can be reached using this antigen.

[0010] Recently, patent applications have been published that relate to the use of IgM protease antigens to protect against other serotypes, in particular IgM protease antigens of serotype 2. These applications confirm the cross-protective properties of the IgM protease antigens.

[0011] In particular, WO 2020 / 094762 describes the use of an IgM protease antigen of serotype 2 against a challenge with serotype 14. It appears that very good protection can be obtained.

[0012] In WO 2019 / 115741, it has been shown that IgM protease antigens are effective in protecting against pathogenic infections with Streptococcus suis of serotype 9. However, protection appears to be modest, at best at the level obtainable with common bacterin vaccines, i.e. a reduction in mortality and positive blood isolates in artificial challenge experiments of about 50% (in practice, it is not excluded that many are challenged less vigorously, resulting in higher levels of protection). At first glance, this rather disappointing protection seems at odds with the high level of protection conferred by IgM protease antigen against infection with serotype 9 S. suis in an artificial challenge experiment reported by Rieckmann et al. in Vaccine, 3 (2019) 100046 ("Vaccination with the immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, leads to protection against a highly virulent serotype 9 strain"), and against serotype 14 S. suis as shown in WO 2020 / 094762. Based on the art, the relatively low level of protection against common serotype 9 bacteria cannot be understood.

[0013] Although at least some protection would be expected, there are no data available in the art regarding protection of IgM protease serotype 2 against challenge with serotypes 1 and 7 Streptococcus suis. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2015 / 181356 [Patent Document 2] International Publication No. 2017 / 005913 [Patent Document 3] International Publication No. 2020 / 094762 [Patent Document 4] International Publication No. 2019 / 115741 [Non-patent literature]

[0015] [Non-Patent Document 1] Rasmussen and Andresen, 1998, “16S rDNA sequence variations of some Streptococcus suis serotypes”, Int.J.Syst.Bacteriol.48, 1063-1065 [Non-Patent Document 2] King et al. the Journal of Clinical Microbiology,Oct.2002,p.3671-3680(Development of a Multilocus Sequence Typing Scheme for the pig pathogen Streptococcus suis:Identification of virulent clones and potential capsular serotype exchange) [Non-Patent Document 3] Jolley et al.Wellcome Open Res 2018,3:124 [Non-Patent Document 4] Mariela Segura:“Streptococcus suis vaccines:candidate antigens and progress,in Expert Review of Vaccines,Volume 14,2015,Issue 12,pages 1587-1608 [Non-Patent Document 5] Kai-Jen Hsueh et al. “Immunization with Streptococcus suis bacterin plus recombinant Sao protein in sows conveys passive immunity to their piglets”, in:BMC Veterinary Research,BMC series-open, inclusive and trusted,13:15,7 January 2017 [Non-Patent Document 6] Wisselink HJ, Stockhofe-Zurwieden N, Hilgers LA, et al. “Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.” in:Vet Microbiol.2002,84:155-168 [Non-Patent Document 7] Rieckmann et al.Vaccine,3(2019)100046(“Vaccination with the immunoglobulin M-degrading enzyme of Streptococcus suis,IdeSsuis,leads to protection against a highly virulent serotype 9 strain”) Summary of the Invention [Problem to be solved by the invention]

[0016] Object of the invention The aim of the present invention is to find an improved vaccine to provide (cross-)protection in pigs against Streptococcus suis, in particular against various serotypes including serotypes 1, 2, 7 and 9. Preferably the vaccine comprises antigens from fewer than these four serotypes, but is still able to provide sufficient protection against at least all of these four serotypes, at least against representative strains of these serotypes present in the field. [Means for solving the problem]

[0017] In order to fulfill the objectives of the present invention, a vaccine has been devised that includes, in combination, an IgM protease antigen of Streptococcus suis serotype 7, Streptococcus suis bacterin serotype 9, sequence type 16, and a pharma- ceutically acceptable carrier.

[0018] The present invention was based on two unexpected findings. First, the heterologous protection conferred by the IgM protease of serotype 2 did not seem to be as good as expected based on the teachings of the prior art, especially since the IgM protease is highly conserved among the different serotypes of Streptococcus suis. In particular, as best understood, the Mac-1 domain is present with a very high level of identity in all Streptococcus suis serotypes known today. Nevertheless, although the homologous protection conferred by the IgM protease antigen of a serotype against serotype 2 attack is excellent, the protection against Streptococcus serotypes 1 and 7 could still be significantly improved. Another unexpected finding was that the heterologous protection conferred by the IgM protease antigen of serotype 7, in turn, is extremely good, especially against serotypes 1 and 2. In particular, the fact that the level of heterologous protection conferred against serotype 2 is significantly better than the heterologous protection conferred by serotype 2 against serotype 7 is totally unexpected.

[0019] A further quite unexpected finding was that the IgM protease antigen of serotype 2, or indeed of any serotype, offers barely sufficient protection against the most prevalent type of Streptococcus serotype 9, namely Streptococcus serotype 9, sequence type 16 (some protection is given, but the level is not sufficient for a commercially successful vaccine). At first glance, this finding seems to be at odds with the results reported in Rieckmann. However, on closer inspection, it appears that in the Rieckmann study a Streptococcus suis strain of serotype 9, sequence type 94 is used. In WO 2019 / 115741, a Streptococcus suis strain of sequence type 16 is used, although not shown. This was found later by determining the type of the challenge strain used according to multilocus sequence typing, as described by King et al. (see above). Apparently, for the latter type (S. suis serotype 9, sequence type 16), the IgM protease antigen provides protection at a substantially lower level. The reasons for this are not entirely clear, but it is highly unfavourable, since in many countries, especially European countries such as the Netherlands, S. suis serotype 16 is the most prevalent (up to about 95%) pathogenic type of S. suis serotype 9 bacteria (Willemse et al. Scientific Reports, 2019, 9:15429, “Clonal expansion of a virulent Streptococcus suis serotype 9 lineage distinguishable from carriage subpopulations”). Thus, although IgM protease can confer protection across serotypes, gaps in effective protection were found to exist, particularly with respect to S. suis serotype 9, sequence type 16.

[0020] Only after realizing all the above discoveries, it was possible to conclude that it was possible to devise an improved vaccine, the present vaccine, which provides sufficient protection against serotypes 1, 2, 7 and 9 Streptococcus suis in the field (and therefore against the most prevalent strain types). It was found that using an IgM protease antigen of serotype 7, sufficient protection against serotypes 1, 2 and 7 can be obtained at a level superior to that obtained by the IgM protease of serotype 2 used in the art. It was also found that the gap in protection against serotype 9, sequence type 16 can be closed by using a serotype 9, sequence type 16 Streptococcus suis bacterin to protect pigs against pathogenic infection with Streptococcus suis serotype 9, sequence type 16. The IgM protease present in the combination, which is not itself suitable to give sufficient protection against serotype 9, sequence type 16 Streptococcus suis, may even improve the protection of the bacterin.

[0021] The present invention allows to obtain sufficient protection against the most prevalent Streptococcus suis bacteria by using only two Streptococcus suis antigens of two different serotypes (7 and 9), closing a gap or shortcoming in protection against Streptococcus suis bacteria when using an IgM protease of serotype 2 bacteria. The present invention not only makes it possible to reach the best possible protection against Streptococcus suis bacteria of serotype 9, with sequence type 16 as an important representative, but also makes it possible to reach a method to reach a very broad and high level of protection across all prevalent serotypes, especially serotypes 1, 2, 7 and 9.

[0022] The present invention also relates to a combination of an IgM protease antigen of Streptococcus suis serotype 7 and a Streptococcus suis bacterin serotype 9, sequence type 16 for use in a method of protecting pigs against pathogenic infection with Streptococcus suis.

[0023] Following this, the present invention relates to the use of an IgM protease antigen of Streptococcus suis serotype 7 and a Streptococcus suis bacterin serotype 9, sequence type 16 for the manufacture of a vaccine for protecting pigs against pathogenic infections caused by Streptococcus suis, as well as a method for protecting pigs against pathogenic infections such as Streptococcus suis by administering to pigs an IgM protease antigen of Streptococcus suis serotype 7 and a Streptococcus suis bacterin serotype 9, sequence type 16.

[0024] definition The IgM protease antigen of Streptococcus suis is an enzyme that specifically degrades porcine IgM (but not porcine IgG or porcine IgA; Seele at al, Journal of Bacteriology, 2013, 195 930-940; and Vaccine 33:2207-2212; 5 May 2015), a protein designated IdeSsuis, or an immunogenic portion thereof (typically having a length of at least about 30-35% of the full-length enzyme). The full enzyme has a mass of about 100-125 kDa, corresponding to about 1000-1150 amino acids, with the size depending on the serotype of S. suis. WO 2015 / 181356 gives several sequences corresponding to the IgM protease antigen of Streptococcus suis, namely SEQ ID NO:1 (also incorporated in the present application), SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:5, the latter being an immunogenic part of the full-length enzyme (represented as the Mac-1 domain, i.e. amino acids 80 to 414 of SEQ ID NO:7). Other examples of immunogenic parts of the full-length enzyme are given in WO 2017 / 005913. A particular example of an IgM protease is a protease according to SEQ ID NO:1 of WO 2015 / 1818356, or a protein having at least 90%, or even 91, 92, 93, 94, 95, 96, 97, 98, 99%, up to 100% sequence identity in the overlapping regions. Amino acid sequence identity can be established with the BLAST program using the blastp algorithm with default parameters. IgM proteases of various serotypes of Streptococcus suis are expected to have a sequence identity higher than 75%, in particular 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, up to 100%.For example, artificial proteins created to optimize yield in recombinant production systems of antigens may result in lower amino acid sequence identity, such as 85%, 80%, 75%, 70%, 65, 60, 55 or even 50%, compared to the complete enzyme while maintaining the required immunogenic function, and are understood to be Streptococcus suis IgM protease antigens within the meaning of the present invention.

[0025] A complete IgM protease antigen of Streptococcus suis is one that contains at least the Mac-1 domain, the region associated with structural function, the CNV region, and may contain the cell adhesion region (see Example 1 for the identification of these regions in the Streptococcus suis genome). In any case, since the naturally occurring (wild-type) secreted enzyme is thought to lack a signal peptide and the cell adhesion region is thought to be dispensable for function as a protease, it can be considered as a complete IgM protease antigen.

[0026] A vaccine is a composition suitable for application to a subject, typically in combination with a pharma- ceutically acceptable carrier, that contains one or more antigens in an immunologically effective amount (i.e., capable of sufficiently stimulating the target subject's immune system to at least reduce the negative effects of a wild-type microbial attack) and, when administered to a subject, induces an immune response to treat an infectious disease, i.e., to help prevent, ameliorate, or cure the infectious disease or any disease or disorder resulting from that infection.

[0027] A repeat in a genome or corresponding amino acid sequence is a copy (either exactly the same or highly similar, e.g., homologue) that is repeated one or more times in the genome or corresponding amino acid sequence of an organism. Repeats are part of the phenomenon of copy number polymorphism, in which sections of a genome are repeated. Typically, the number of repeats varies between different strains of the same organism. Copy number polymorphism is a type of structural polymorphism. Copy number polymorphism is a type of duplication event that typically affects a significant number of base pairs, e.g., anywhere between 30-400 base pairs, corresponding to 10-130 amino acids.

[0028] Protection against pathogenic infection by a microorganism is equivalent to achieving protective immunity, i.e. helping to prevent, ameliorate or cure a pathogenic infection by that microorganism or a disorder resulting from that infection, for example to prevent or reduce one or more clinical signs resulting from an actual infection or pathogenic infection by a pathogen.

[0029] Bacterins are suspensions of killed bacteria for use as vaccines.

[0030] Antigen combination refers to the use of separate antigens together in one vaccination strategy, either by combining these antigens in one vaccine formulation or by using separate antigen formulations for co-administration of the separate formulations.

[0031] A combination vaccine (i.e. a vaccine containing a combination of antigens) is one (single) formulation that contains different antigens at the same time. These different antigens can be mixed in a factory to provide a so-called ready-to-use combination vaccine, or they can be mixed just before or during administration (e.g. using a device with two separate chambers for the separate antigens, the contents of these chambers being mixed when using the device for administration), as long as the antigens end up in the same formulation.

[0032] A pig is any animal belonging to the family Suidae.

[0033] A pharma- ceutically acceptable carrier is a biocompatible medium, i.e. a medium that, after administration, does not induce significant adverse reactions in the treated subject and is capable of presenting an antigen to the subject's immune system after administration of a composition containing the carrier. Such a pharma- ceutically acceptable carrier may be, for example, a liquid containing water and / or any other biocompatible solvent, or a solid carrier, such as those commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), which may contain immune stimulants (also called adjuvants). Depending on the intended use or desired properties of the corresponding vaccine, other substances such as stabilizers, viscosity modifiers or other ingredients may be added. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Further aspects of the invention In a further embodiment of the vaccine according to the invention, the IgM protease antigen of Streptococcus suis serotype 7 is a complete IgM protease antigen having at least 90% sequence identity with the corresponding naturally occurring (i.e. wild type) IgM protease of Streptococcus suis serotype 7 bacteria. From the art it is known that only the Mac-1 domain of the IgM protease (about 35%) is sufficient to provide protection, but it is believed that the complete antigen provides a more effective immune response. In particular, a sequence identity of 90% or more, for example 91, 92, 93, 94, 95, 96, 97, 98, 99 or even 100% with the naturally occurring IgM protease is preferred to reach sufficient homologous and heterologous protection.

[0035] In yet a further embodiment of the vaccine according to the invention, the IgM protease antigen of Streptococcus suis serotype 7 comprises less than four repeats in its amino acid sequence. Structural analysis of the genome of Streptococcus suis reveals that the genome of this bacterium is prone to the phenomenon of copy number variation (CNV), in which sections of the genome are repeated. In particular, the repeats have similarity to known protein sequences with hydrolase activity. It was found that the IgM protease of serotype 2 differs mainly from those that provide better heterologous protection (such as serotypes 1 and 7) in that serotype 2 comprises four repeats. It is therefore considered advantageous to reach the best possible (heterologous) protection if the number of repeats is less than 4, or even less than 3, such as 2.

[0036] Most preferred is the IgM protease antigen of Streptococcus suis serotype 7 of sequence type 29, which contains two repeats.

[0037] The vaccine may contain additional S. suis antigens, such as the IgM protease of serotype 2, but it has been found to be sufficient for the vaccine to contain no other S. suis antigens than the IgM protease antigen of S. suis serotype 7 and the S. suis bacterin serotype 9, sequence type 16, or at most the IgM protease antigen of S. suis serotype 1. More antigens means higher costs and higher risk of side effects due to higher antigen load.

[0038] In a further embodiment of the combination for use in protecting against Streptococcus suis, the protection is against pathogenic infection by any of serotypes 1, 2, 7 and 9 Streptococcus suis.

[0039] In a further embodiment of the combination for use according to the invention, the method comprises administering an IgM protease antigen of Streptococcus suis serotype 7 and Streptococcus suis bacterin serotype 9, sequence type 16 to pigs up to 35 days of age.

[0040] In another embodiment, the method comprises administering to a sow an IgM protease antigen of Streptococcus suis serotype 7 and a Streptococcus suis bacterin serotype 9, sequence type 16 to protect the piglet through ingestion of the sow's colostrum. It is known that the IgM protease (see WO 2019 / 193078) confers sufficient and long-lasting protection to the piglet when the piglet consumes colostrum from a vaccinated sow. It is also generally known that the protection conferred by the bacterin is transferred to the piglet via the colostrum.

[0041] In one embodiment, the IgM protease antigen of Streptococcus suis serotype 7 and the Streptococcus suis bacterin serotype 9, sequence type 16 are administered twice to the sow before the piglet receives the colostrum.

[0042] The invention will now be further illustrated by the following specific examples.

[0043] [Example] Example 1 Structural analysis of the Streptococcus suis genome.

[0044] Example 2 examines the cross-protection of IgM protease serotype 2 against serotype 1.

[0045] Example 3 examines cross-protection of IgM protease serotype 2 against serotype 7.

[0046] Example 4 examines cross-protection of IgM protease serotype 2 against serotype 9, sequence type 16.

[0047] Example 5 examines the protection conferred by serotype 1 and 7 IgM proteases against challenge with serotype 1.

[0048] Example 6 examines the protection conferred by serotype 1 and 7 IgM proteases against serotype 2 challenge.

[0049] Example 7 examines the protection conferred by serotype 1 and 7 IgM proteases against challenge with serotype 7.

[0050] Example 8 examines the protection afforded by the bacterin against challenge with serotype 9, sequence type 16.

[0051] [Example 1] In this example, an analysis of the genome of Streptococcus suis, i.e. the part that codes for IgM protease, is provided in order to show how the part of the genome that codes for IgM protease is structured. For this, we use the genome of the Streptococcus suis bacterium of serotype 2, known from WO 2015 / 181356 and published in that patent application under SEQ ID NO:1. This sequence is also included in the sequence listing of this patent under SEQ ID NO:1. A sequence similarity search using protein annotations (PDBSum and InterPro) plus Needleman-Wunsch alignments (see Needleman et al 1970, Laskowski et al 1997, Apweiler et al 2000; default settings) reveals the structure of the IgM protease genome in which five regions can be identified.

[0052] Region 1 (Met1 to Thr34): signal sequence from position 1; Region 2 (Val35-Glu426): Mac-1 domain with predicted hydrolase activity; Region 3 (Thr427-Pro687): A region involved in structural functions (eg, involved in proper folding) and substrate binding. Region 4 (Thr688~Ser919): a region consisting of four repeats (1*{Thr688~Ser744}, 2*{Thr745~Ser801}, 3*{Thr802~Ser858}, 4*{Thr859~Ser919}) with similarity to known protein sequences with hydrolase activity; Region 5 (Thr920-Lys1141): Contains a predicted transmembrane domain that exhibits cell wall anchoring function.

[0053] The structure of the other serotypes of Streptococcus suis bacteria is largely the same, but for serotype 9, sequence type 16, substantial differences exist (shown here below): -The signal peptide is highly conserved among Streptococcus suis strains; -Mac-1 domain is always present and highly conserved among all known strains, including serotype 9 and sequence type 16 strains; - Region 3, which is related to structural functions, is always present and is also highly conserved, but is only about half the length of serotype 9 and sequence type 16; -For the CNV region, the repeats are highly similar between the different serotypes, but the number varies, typically between 2 and 6. Serotype 9, sequence type 16, has 12 repeats of a completely different type, which are much shorter compared to the repeats of the other serotypes (i.e., 12 AA versus approximately 60) and can be subdivided into three significantly different repeats; - The cell adhesion region is also highly conserved among the different serotypes, but there is virtually no amino acid sequence identity with this region in serotype 9, sequence type 16 strains.

[0054] Briefly, the genomes are largely identical in structure among most serotypes and sequence types, with the most notable differences being the number of repeats in the CNV regions. The IgM protease portions of the genomes of serotype 9, sequence type 16 are highly similar as far as the Mac-1 domain is concerned, but differ significantly over the remainder of the genome.

[0055] [Example 2] Research Objective From the prior art it is known that the complete IgM protease of Streptococcus suis serotype 2 (SEQ ID NO:1) provides good protection against a homologous challenge. Also some cross-protection against serotypes 9 and 14 is known from the art. In this example we evaluate the actual level of protection provided by this antigen against a serotype 1 challenge. For this purpose we used a strain of sequence type 13, which is a common variety of bacteria and a good representative of this serotype in the field.

[0056] Study design First, to evaluate protection against challenge with serotype 1 bacteria, the only challenge model available is one in which piglets at 3 weeks of age are challenged. This means that the piglets themselves cannot be vaccinated to evaluate the protective effect induced by the IgM protease antigen, since the time to develop an effective immune response is expected to be too short. Therefore, to evaluate the protection conferred by the vaccine, sows are vaccinated before farrowing so that the induced antibodies are transferred to the piglets via ingestion of colostrum. It is known from the art (US Pat. No. 10,751,403) that if the IgM protease antigen provides protection to the vaccinated animal itself, it also confers excellent protection to the progeny of the vaccinated sow. In other words, the protection seen in this (indirect) challenge model naturally indicates the protection conferred to the vaccinated animal itself, secondary to the protection conferred to the piglets via ingestion of the colostrum of the vaccinated sow.

[0057] For this study, 10 pregnant sows were used and divided into two groups of 5 sows each. Six and two weeks before expected farrowing, one group was vaccinated with a subunit vaccine (Seele et al: Vaccine 33:2207-2212;5 May 2015, par. 2.2.) containing recombinant rIdeSsuis IgM protease antigen of serotype 2 at 80 μg / dose in an oil-in-water adjuvant (μDiluvac Forte, MSD Animal Health), and one group was left as a non-vaccinated control group. After farrowing, at 3 weeks of age, 10 piglets from the vaccinated sows and 10 piglets from the control sows were selected for challenge (each group contained 2 piglets per sow). Using a catheter, 10 ml of the challenge inoculum (5.0 × 10 10 Piglets (2 × 10, vaccinated and control) were challenged intratracheally with 1000 sera (targeting 100 CFU / ml) or (if this was not possible) by using transtracheal injection as an alternative. After challenge, piglets were observed daily for clinical signs of S. suis infection such as depression, motor impairment and / or neurological signs and scored using a routine scoring system ranging from 0 (no signs) to 3 for severe cases. Animals that reached a humane end point were euthanized. Serum blood was taken for antibody determination at regular times before and after vaccination (10 sows) as well as immediately before challenge (20 piglets). Heparinized blood was taken for reisolation of the challenge strain at regular times before and after challenge (20 piglets). At the end of the study (i.e. 11 days after challenge), all surviving piglets were euthanized.

[0058] result None of the vaccines induced unacceptable site (i.e., local) or systemic reactions and could therefore be considered safe. Post-challenge data for the period prior to euthanasia are shown in Table 1. [Table 1]

[0059] conclusion Serotype 2 IgM proteases do not protect against attack by serotype 1 Streptococcus suis bacteria.

[0060] [Example 3] Research Objective In this example, the actual level of protection by the same antigen (serotype 2 IgM protease) used in Example 2 against serotype 7 challenge is evaluated. For this, a strain of sequence type 29 was used, which is a common variety of bacteria and representative of this serotype in the field.

[0061] Study design Similar to serotype 1, the only challenge model available to evaluate protection against challenge with serotype 7 bacteria is one in which piglets are challenged at 3.5 weeks of age. Therefore, in this study, sows are vaccinated before farrowing so that induced antibodies are transferred to the piglets via ingestion of colostrum.

[0062] For this study, 10 pregnant sows were used and divided into two groups of 5 sows each. Six and two weeks before expected farrowing, one group was vaccinated with a subunit vaccine (Seele et al: Vaccine 33:2207-2212;5 May 2015, par. 2.2.) containing recombinant rIdeSsuis IgM protease antigen of serotype 2 at 80 μg / dose in an oil-in-water adjuvant (μDiluvac Forte, MSD Animal Health) and one group was left as a non-vaccinated control group. After farrowing, at 3.5 weeks of age, 10 piglets from the vaccinated sows and 10 piglets from the control sows were selected for challenge (each group contained 2 piglets per sow). The piglets (2 × 10, vaccinated and control) were vaccinated with 10 ml of challenge inoculum (1.0 × 10 9Piglets were challenged intratracheally with 1000 mg / mL of S. suis (targeting 1000 CFU / ml). After challenge, piglets were observed daily for clinical signs of S. suis infection, such as depression, motor impairment and / or neurological signs, and scored using a routine scoring system ranging from 0 (no signs) to 3 for severe cases. Animals that reached a humane endpoint were euthanized. Serum blood was taken for antibody determination at regular times before and after vaccination (10 sows) and immediately prior to challenge (20 piglets). Heparinized blood was taken for re-isolation of the challenge strain at regular times before and after challenge (20 piglets). At the end of the study (i.e., 11 days after challenge), all surviving piglets were euthanized.

[0063] result None of the vaccines induced unacceptable site or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 2. [Table 2]

[0064] conclusion Serotype 2 IgM protease confers no protection against attack by serotype 7 Streptococcus suis bacteria.

[0065] [Example 4] Research Objective The aim of this study was to test the level of actual protection provided by the same antigen used in Examples 2 and 3 (i.e., serotype 2 IgM protease) against serotype 9 challenge, in particular against challenge with serotype 9, sequence type 16 bacteria.

[0066] Study design Twenty-four 3-week-old seronegative SPF piglets were used. The piglets were assigned to two groups (evenly distributed across different litters) of 10 piglets each. Group 1 was vaccinated twice intramuscularly at 3 and 5 weeks of age as described in Examples 2 and 3, while group 2 remained as a non-vaccinated challenge control group. At 7 weeks of age, the pigs were challenged intratracheally with a virulent culture of S. suis serotype 9 as described herein above. After challenge, the pigs were observed daily for clinical signs of S. suis infection, such as depression, motor impairment and / or neurological signs, for 10 days. Animals that reached the humane end point after showing specific clinical signs (i.e., motor or neurological) were euthanized without necropsy. Animals that reached the humane end point without showing specific clinical signs were euthanized and necropsy, including bacteriological examination, was performed to confirm S. suis infection. Heparinized blood was collected at regular times before and after challenge for reisolation of the challenge strain. On the day of first vaccination (5 weeks of age), pigs were seronegative for IgM proteases from serotype 2.

[0067] result None of the vaccines induced unacceptable site or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 3. [Table 3]

[0068] conclusion Serotype 2 IgM protease confers no protection against attack by serotype 9, sequence type 16 Streptococcus suis bacteria.

[0069] [Example 5] Research Objective In this example, the protection against serotype 1 challenge is evaluated for IgM protease antigens of serotype 1 and serotype 7 Streptococcus suis strains. For this, antigens corresponding to serotype 2 IgM protease were generated as used in examples 2, 3 and 4, i.e. using the E. coli expression system as described in the art (Seele et al., see above). The sequence used for the serotype 7 IgM protease antigen is shown in the attached SEQ ID NO:2, whereas the sequence used for the serotype 1 IgM protease antigen is shown in the attached SEQ ID NO:3. Both sequences contain a CNV region next to the Mac-1 region, with two repeats in this region. The challenge strain was the same as that used in example 2.

[0070] Study design The study design was the same as in Examples 2 and 3, but using 3.5 week old piglets in each case for challenge, and groups of 10 piglets. The challenges for each of the serotypes corresponded to the challenges in Examples 2 and 3. Group 1 was vaccinated with serotype 1 IgM protease, group 2 was vaccinated with serotype 7 IgM protease, and group 3 remained as challenge control.

[0071] result None of the vaccines induced unacceptable site or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 4. [Table 4]

[0072] conclusion From the data it can be concluded that the IgM proteases of serotype 1 as well as of serotype 7 protect against virulent challenge with serotype 1 strains. The homologous protection conferred by serotype 1 antigens appears to be slightly better than the heterologous protection conferred by serotype 7 antigens.

[0073] [Example 6] Research Objective In this example, IgM protease antigens of serotype 1 and serotype 7 Streptococcus suis strains are evaluated for protection against a serotype 2 challenge. For this, the same antigens were used as in Example 5. The challenge strain was a serotype 2, sequence type 1 strain representative of field strains.

[0074] Study design The study design was generally the same as that of Example 4. Thirty 3-week-old piglets were used. The piglets were assigned to three groups (evenly distributed across different litters) of 10 piglets each. Groups 1 and 2 were vaccinated intramuscularly twice with the respective subunit vaccines at 3 and 5 weeks of age, while group 3 remained unvaccinated. At 7 weeks of age, the pigs were challenged intratracheally with a virulent culture of S. suis serotype 2 strain. For 11 days after challenge, the pigs were observed daily for clinical signs of S. suis infection, such as depression, motor impairment and / or neurological signs. Animals that reached the humane endpoint (HEP) were euthanized. Heparinized blood was collected for reisolation of the challenge strain immediately before challenge, 2 days after challenge, and, if applicable, on the day of HEP (just before euthanasia).

[0075] On the day of the first vaccination, the piglets were either seronegative or had very low titers in the specific IgM antibody ELISA. After vaccination, groups 1 and 2 showed good antibody responses against the IgM protease, whereas the controls remained at very low levels.

[0076] result None of the vaccines induced unacceptable site or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 5. One animal in group 1 had to be euthanized after challenge for reasons not specific to S. suis. [Table 5]

[0077] conclusion From the data it can be concluded that the IgM protease of serotype 1 as well as that of serotype 7 protects against virulent challenge with serotype 2 strains.

[0078] [Example 7] Research Objective In this example, IgM protease antigens of serotype 1 and serotype 7 Streptococcus suis strains are evaluated for protection against serotype 7 challenge. For this, the same antigens were used as in Examples 5 and 6. The challenge strain was serotype 7, sequence type 29 strain representative of field strains.

[0079] Study design The study design was the same as in Example 5 (except for the challenge strain). The challenges for each of the serotypes corresponded to the challenges in Examples 2 and 3. Group 1 was vaccinated with serotype 1 IgM protease, group 2 was vaccinated with serotype 7 IgM protease, and group 3 remained as the challenge control.

[0080] result None of the vaccines induced unacceptable site or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 6. [Table 6]

[0081] conclusion Although the challenge appeared to be less virulent as in previous studies, it can be concluded from the data that the serotype 1 IgM protease as well as the serotype 7 IgM protease protect against a virulent challenge with a serotype 7 strain.

[0082] [Example 8] Research Objective The aim of this study was to find protective antigens against serotype 9 challenge, particularly against challenge with serotype 9, sequence type 16 bacteria representative of strains circulating in the field. The options evaluated were bacterin alone and bacterin in combination with an IgM protease, which is understood in the art to improve bacterin efficacy (see Seele et al, Journal of Bacteriology, p. 930-940 March 2013, Volume 195 Number 5, "Identification of a Novel Host-Specific IgM Protease in Streptococcus suis"; reviewed in WO 2015 / 181356).

[0083] Study design The study design was the same as that used in Example 4, but non-SPF piglets were used, assigned to three groups of 12 piglets each (evenly distributed across different litters). Group 1 contained 2 × 10 inactivated Streptococcus suis bacteria of serotype 9, sequence type 16. 9 Pigs were vaccinated intramuscularly twice at 3 and 5 weeks of age with a bacterin vaccine containing the IgG at the cell level. Group 2 additionally contained 80 μg / dose of the IgM protease of Example 2. Both vaccines were formulated in the oil-in-water adjuvant used in other examples. Group 3 remained as an unvaccinated challenge control group. At 7 weeks of age, pigs were challenged intratracheally with a virulent culture of S. suis serotype 9 as described herein above. After challenge, pigs were observed daily for clinical signs of S. suis infection, such as depression, motor impairment and / or neurological signs, for 10 days. Animals that reached a humane end point after showing specific clinical signs (i.e., motor or neurological) were euthanized without necropsy. Animals that reached a humane end point without showing specific clinical signs were euthanized and necropsy, including bacteriological examination, was performed to confirm S. suis infection. Heparinized blood was collected at regular times before and after challenge for reisolation of the challenge strain. On the day of first vaccination (5 weeks of age), pigs were seronegative for IgM proteases from serotype 2.

[0084] result None of the vaccines induced unacceptable sitel or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 7. One animal in group 2 had to be euthanized after challenge for reasons not specific to S. suis. [Table 7]

[0085] conclusion Protection against virulent attack by Streptococcus suis of serotype 9, sequence type 16, could be provided by a bacterin of that serotype and by a bacterin in combination with an IgM protease. The two types of antigens do not adversely interfere, which is in agreement with what would be expected on the basis of the prior art.

[0086] Based on the above examples, the objectives of the present invention can be met by combining the IgM protease antigen of Streptococcus suis serotype 7 or 1 with Streptococcus suis bacterin serotype 9, sequence type 16 in a combined vaccination strategy. It is also considered that the two IgM protease antigens can be combined if necessary to reach better protection against both serotype 1 and 7 challenge. Also, since the CNV region is where there is a difference between the IgM protease molecules of serotypes 1 and 7 when compared to serotype 2, and since the IgM proteases of serotypes 1 and 7 each have two repeats while serotype 2 has four repeats, it is reasonable to assume that the level of cross-protection is related to the number of repeats in the CNV region of the IgM protease. The reason for the difference in cross-protection is not cleat, but it seems that a smaller number of repeats is advantageous to reach a better level of cross-protection.

Claims

1. A vaccine comprising a combination of an IgM protease antigen of Streptococcus suis serotype 7, a bacterin of Streptococcus suis serotype 9, sequence type 16, and a pharmaceutically acceptable carrier.

2. The vaccine according to claim 1, wherein the IgM protease antigen of Streptococcus suis serotype 7 is a complete IgM protease antigen having at least 90% sequence identity with the corresponding naturally occurring IgM protease of Streptococcus suis serotype 7 bacteria.

3. The vaccine according to claim 1 or 2, wherein the IgM protease antigen of Streptococcus suis serotype 7 is a complete IgM protease antigen having at least 95% sequence identity with the corresponding naturally occurring IgM protease of Streptococcus suis serotype 7 bacteria.

4. The vaccine according to claim 1 or 2, wherein the IgM protease antigen of Streptococcus suis serotype 7 contains less than 4 repeats in its amino acid sequence.

5. The vaccine according to claim 1 or 2, wherein the IgM protease antigen of Streptococcus suis serotype 7 contains less than 3 repeats in its amino acid sequence.

6. The vaccine according to claim 1 or 2, wherein the IgM protease antigen of Streptococcus suis serotype 7 contains 2 repeats in its amino acid sequence.

7. The vaccine according to claim 1 or 2, wherein the IgM protease antigen of Streptococcus suis serotype 7 is of sequence type 29.

8. The vaccine according to claim 1 or 2, wherein the vaccine does not contain the IgM protease antigen of Streptococcus suis serotype 7 and other Streptococcus suis antigens other than the bacterin of Streptococcus suis serotype 9, sequence type 16, or contains at most the IgM protease antigen of Streptococcus suis serotype 1.

9. A combination of an IgM protease antigen of Streptococcus suis serotype 7 and a bacterin of Streptococcus suis serotype 9, sequence type 16, for use in a method for protecting pigs against pathogenic infections caused by Streptococcus suis.

10. The combination for use according to claim 9, characterized in that said protection is against pathogenic infections caused by Streptococcus suis of any of serotypes 1, 2, 7 and 9.

11. The combination for use according to any of claims 9 and 10, characterized in that said method comprises administering said IgM protease antigen of Streptococcus suis serotype 7 and said Streptococcus suis bacterin serotype 9, sequence type 16 to said piglets up to 35 days old.

12. The combination for use according to any of claims 9 and 10, characterized in that said method comprises administering said IgM protease antigen of Streptococcus suis serotype 7 and said Streptococcus suis bacterin serotype 9, sequence type 16 to said sow in order to protect the piglets through ingestion of the sow's colostrum.

13. The combination for use according to claim 12, characterized in that said IgM protease antigen of Streptococcus suis serotype 7 and said Streptococcus suis bacterin serotype 9, sequence type 16 are administered twice to said sow before said piglets ingest the colostrum.

14. Use of an IgM protease antigen of Streptococcus suis serotype 7 and a Streptococcus suis bacterin serotype 9, sequence type 16 for the manufacture of a vaccine for protecting pigs against pathogenic infections caused by Streptococcus suis.

15. A method for protecting pigs from pathogenic infections caused by Streptococcus suis by administering an IgM protease antigen of Streptococcus suis serotype 7 and a Streptococcus suis bacterin serotype 9, sequence type 16 to the pigs.