Vaccines for protection against various serotypes of Streptococcus suis

Abstract

The present invention relates to a vaccine comprising an IgM protease antigen of Streptococcus suis serotype 1 in combination with a Streptococcus suis bacterin serotype 9, sequence type 16 and a pharma- ceutically acceptable carrier. The present invention also relates to a combination of an IgM protease antigen of Streptococcus suis serotype 1 and a Streptococcus suis bacterin serotype 9, sequence type 16 for use in a method of protecting pigs against pathogenic infection with Streptococcus suis, and to a method for protecting pigs against pathogenic infection with Streptococcus suis by administering to said pig the IgM protease antigen of Streptococcus suis serotype 1 and a Streptococcus suis bacterin serotype 9, sequence type 16.

Description

[Technical field]

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

[0002] Streptococcus suis (S. suis) is one of the main causative agents of infectious bacterial diseases in pigs. This pathogen can cause various clinical syndromes such as 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 typing system was proposed based on type-specific capsular polysaccharide antigens located in the cell wall. This led to 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 common, especially in Europe. However, it has been recognized that capsular serotypes are insufficient markers of virulence. Therefore, an alternative system to help understand the epidemiology of S. suis infection and the biological relevance of serotyping approaches has been developed, namely the so-called multilocus sequence typing (MLST) described by King et al. in Journal of Clinical Microbiology, October 2002, pp. 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 sequence type (ST) complexes ST1, ST27, and ST87 each contain multiple sequence types and dominate the population.See King et al., University of Oxford (Jolley et al., Wellcome Open Res 2018, 3:124 (a Wellcome Trust-funded site) and the Streptococcus suis MLST website (https: / / pubmlst.org / ssuis / ), which allows easy sequence typing of any Streptococcus suis strain.

[0003] Control of S. suis in pig herds seems to be difficult. S. suis is a commensal and opportunistic pathogen of pigs. Apparently, the immune system is not triggered every time of infection. S. suis is also a well-encapsulated pathogen, evading the host immune system by using a wide range of virulence factors. Together, these characteristics have made the development of an effective vaccine to combat this important pathogen a challenge. A review was published several years ago, which summarized the existing and exploratory vaccines against S. suis (Mariela Segura: "Streptococcus suis vaccines: candidate antigens and progress", Expert Review of Vaccines, Vol. 14, No. 12, 2015, pp. 1587-1608). This review summarized and compared information from clinical practice and experimental data to provide an overview of the state of vaccine development against S. suis, which is outlined below.

[0004] Currently commercially available vaccines are mainly whole cell bacterins. However, field reports document difficulties in controlling and managing the disease, and "vaccine failure" is common, especially when using bacterin vaccines, as heterologous protection is very weak. Carrier pigs are the main source of infection, and both vertical and horizontal transmission are involved in the spread of the disease within herds. Mixing of carrier and susceptible animals under stressful conditions, such as weaning and transportation, most often results in clinical disease. Early dosing and weaning, or early weaning practices with isolation, do not eliminate S. suis infection. Effective control measures to prevent the disease therefore rely on prophylaxis / mass prophylaxis (when possible) and vaccination. Currently, field immunization efforts are focused on the use of commercial or autologous bacterins. These vaccine strategies are applied to either piglets or sows. Piglets from weaning age onwards are susceptible to S. suis infections due to the stresses associated with weaning and the transport that usually follows. Therefore, pre-partum immunization of sows is often used to try and transfer passive immunity to piglets and provide protection against S. suis during these stressful conditions early in life. Furthermore, vaccination of sows is less expensive and less labor intensive, making it an economical alternative to vaccination of piglets. However, the results obtained 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 farrowing, 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 provide protection during the most critical period of 4–7 weeks of age.

[0005] For piglets, autologous bacterins are frequently used in the field, especially in Europe. Autologous bacterins are prepared from virulent strains isolated in a feedlot with clinical problems and are applied in the same feedlot. 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 lead to failure to identify strains or serotypes associated with recent outbreaks. This failure can be particularly problematic in endemic herds. Finally, the most important dilemma of autologous bacterins is that their real efficacy has been scarcely studied. Since autologous vaccines are applied empirically, it is not surprising that the results obtained with this vaccine are inconsistent and often disappointing.

[0006] Other experimental vaccines have been described in the literature. Kai-Jen Hsueh and colleagues show that subunit-loaded bacterins 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": BMC Veterinary Research, BMC series-open, inclusive and trusted, 13:15, January 7, 2017).

[0007] Live attenuated vaccines have also been contemplated in the literature. Non-encapsulated isogenic mutants of S. suis serotype 2 have been clearly shown to be avirulent. However, live vaccine formulations based on non-encapsulated serotype 2 mutants induced only partial protection against mortality and failed to prevent the development of clinical signs in pigs challenged with the wild-type strain (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." Vet Microbiol. 2002, 84:155-168).

[0008] In the last few years, an extensive list of antigenic or immunogenic Streptococcus suis molecules has been reported, most of which have been discovered by immunoproteomics using either convalescent sera from infected pigs or humans and / or immune sera produced in the laboratory. In WO 2015 / 181356 (IDT Biologica) it has been shown that an IgM protease antigen (either the whole protein or the highly conserved Mac-1 domain, which represents only about 35% of the complete protein) may be combined with a prime vaccination containing a bacterin, and in a vaccination scheme with two doses of IgM protease antigen, can elicit a protective immune response in piglets. The '356 patent application suggests that the fact that the IgM protease antigen is highly conserved across most, if not all, S. suis serotypes, particularly the most common serotypes 1, 2, 7 and 9, may allow for broad cross-protection between S. suis serotypes, particularly serotypes 1, 2, 7 and 9, using the IgM protease antigen.

[0009] WO 2017 / 005913 (Intervacc AB) confirms that the IgM protease is highly conserved across the various Streptococcus suis serotypes, thus confirming the broad protection that can be expected to be achieved using this antigen.

[0010] Recently, patent applications have been published that relate to the use of IgM protease antigens for protection against other serotypes, in particular IgM protease antigens of serotype 2. In these applications the cross-protective properties of the IgM protease antigens have been confirmed.

[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 a very reasonable protective effect 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, the protection is not very high and at best appears to be at the level obtained with conventional bacterin vaccines, i.e. a reduction of about 50% in mortality and positive blood isolates in artificial challenge experiments (which does not exclude that in practice protection could be at a higher level, usually in the face of a less severe challenge). At first glance, this somewhat disappointing protection seems to contradict the high level of protection obtained with the IgM protease antigen against infection with serotype 9 S. suis 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"), as well as the high level of protection in artificial challenge experiments and the protection against serotype 14 S. suis shown in WO 2020 / 094762. Based on the literature, the relatively low level of protection against common serotype 9 bacteria is incomprehensible.

[0013] Although at least some protection would be expected, there are no data available in the literature on the protective effect 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., Journal of Clinical Microbiology, October 2002, pp. 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] Mariela Segura: "Streptococcus suis vaccines: candidate antigens and progress", Expert Review of Vaccines, Vol. 14, No. 12, 2015, pp. 1587-1608 [Non-Patent Document 4] Kai-Jen Hsueh et al., “Immunization with Streptococcus suis bacterin plus recombinant Sao protein in sows conveys passive immunity to their piglets”: BMC Veterinary Research, BMC series-open, inclusive and trusted, 13:15, January 7, 2017. [Non-Patent Document 5] 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.” Vet Microbiol., 2002, 84:155–168 [Non-Patent Document 6] 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] The aim of the present invention is to find an improved vaccine to provide (cross-)protection for pigs against Streptococcus suis, in particular against various serotypes of Streptococcus suis including serotypes 1, 2, 7 and 9. Preferably, the vaccine comprises antigens from fewer than four of these serotypes and is still able to provide reasonable protection against at least all four of these serotypes, at least against representative strains of these serotypes present in the field. [Means for solving the problem]

[0017] In order to achieve the objectives of the present invention, a vaccine has been devised which comprises an IgM protease antigen of Streptococcus suis serotype 1 in combination with a bacterin serotype 9, sequence type 16 of Streptococcus suis and a pharmaceutically acceptable carrier.

[0018] The present invention is based on two unexpected findings. Firstly, the heterologous protection conferred by the IgM protease of serotype 2 did not seem to be as good as would be 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. Although the homologous protection conferred by the IgM protease antigen of a serotype against a serotype 2 challenge 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 1 was also very good, especially against serotypes 2 and 7. 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 1 is totally unexpected.

[0019] A further, highly unexpected finding was that the IgM protease antigen of serotype 2, or indeed of any serotype, hardly offers any reasonable protection against the most common type of Streptococcus serotype 9, namely Streptococcus serotype 9, sequence type 16 (some protection is obtained, but the level is not sufficient for a commercially successful vaccine). At first glance, this finding seems to contradict the results reported in Rieckmann. However, on closer inspection, it appears that the Rieckmann study used a Streptococcus suis strain of serotype 9, sequence type 94. In WO 2019 / 115741, a Streptococcus suis strain of sequence type 16 is used, although not indicated. This was found later by typing the challenge strains used according to the multilocus sequence typing described by King et al. (see above). Apparently, against the latter type (S. suis serotype 9, sequence type 16), the IgM protease antigen provides a substantially lower level of protection. The reasons for this are not entirely clear, but are highly unfavourable, as in many countries, particularly in European countries such as the Netherlands, S. suis sequence type 16 is the most common (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 subpopulation”). Thus, it was found that while IgM proteases can provide protection across all serotypes, there is a gap in the available protection, particularly with regard to S. suis serotype 9, sequence type 16.

[0020] Only after realizing all the above discoveries could it be concluded that an improved vaccine could be devised, the vaccine of the present invention, which adequately protects in the field against serotypes 1, 2, 7 and 9 S. suis (and therefore against the most common strain types). It was found that when using a serotype 1 IgM protease antigen, reasonable protection against serotypes 1, 2 and 7 could be obtained at a better level than that obtained with the serotype 2 IgM protease used in the art. It was also found that the gap in protection against serotype 9, sequence type 16 could be filled by using a serotype 9, sequence type 16 S. suis bacterin to protect pigs against pathogenic infection with S. suis serotype 9, sequence type 16. The IgM protease present in this combination is not suitable in itself to provide reasonable protection against serotype 9, sequence type 16 S. suis, but may further improve the protection of the bacterin.

[0021] The present invention allows to obtain reasonable protection against the most common S. suis bacteria by using only two S. suis antigens of two different serotypes (1 and 9) and to fill any gaps or shortcomings in protection against S. suis bacteria when using the IgM protease of bacteria of serotype 2. It is possible by the present invention to reach not only the best possible protection against S. suis serotype 9, including the important representative sequence type 16, but also a way to reach a very broad and high level of protection across all the common 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 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis for use in a method of protecting pigs against pathogenic infection with Streptococcus suis.

[0023] The present invention also relates to the use of an IgM protease antigen of Streptococcus suis serotype 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis for the preparation of a vaccine for the protection of pigs against pathogenic infection with Streptococcus suis, as well as a method for protecting pigs against pathogenic infection with Streptococcus suis by administering to the pig an IgM protease antigen of Streptococcus suis serotype 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis.

[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 et al., Journal of Bacteriology, 2013, 195, 930-940; and Vaccine 33:2207-2212, May 5, 2015), a protein designated IdeSsuis, or an immunogenic portion thereof (generally at least about 30-35% in length of the full-length enzyme). The full enzyme has a mass of about 100-125 kDa, corresponding to about 1000-1150 amino acids, the size depending on the serotype of S. suis. WO 2015 / 181356 gives several sequences that represent 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 (these four sequences 2, 6, 7 and 5 are not incorporated in the present application), the latter being an immunogenic part of the full-length enzyme (designated as the Mac-1 domain, i.e. amino acids 80 to 414 of SEQ ID NO: 7). Other examples of immunogenic parts of full-length enzymes are given in WO 2017 / 005913. Particular examples of IgM proteases are the proteases according to SEQ ID NO: 1 of WO 2015 / 1818356 or proteins having at least 90%, and even 91, 92, 93, 94, 95, 96, 97, 98, 99% 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. The IgM proteases of the various serotypes of Streptococcus suis are predicted to have sequence identities greater than 75%, particularly 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99% to 100%.For example, artificial proteins created to optimize yield in recombinant production systems of antigens may have lower amino acid sequence identity compared to the whole enzyme, such as 85%, 80%, 75%, 70%, 65, 60, 55 or even 50%, while maintaining the required immunogenic function, and are understood to be Streptococcus suis IgM protease antigens in the sense of the present invention.

[0025] The full IgM protease antigen of Streptococcus suis is an antigen that contains at least the Mac-1 domain, regions associated with structural function, CNV regions, and may contain a cell adhesion region (see Example 1 for the identification of these regions in the genome of Streptococcus suis). It can be considered as a full IgM protease antigen, since the signal peptide would in any case be lacking in the naturally occurring (i.e. wild type) secreted enzyme, and the cell adhesion region would not be essential for function as a protease.

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

[0027] A repeated sequence in a genome or corresponding amino acid sequence is a copy (either identical or very similar, e.g., homologue) that is repeated one or more times in the genome or corresponding amino acid sequence of an organism. This is part of the phenomenon of copy number polymorphism, where parts of a genome are repeated. Typically, the number of repeated sequences varies between different strains of the same organism. Copy number polymorphism is a type of structural polymorphism. It 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 between 10-130 amino acids.

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

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

[0030] Combining antigens means using these (individually distinct) antigens together in one vaccination regimen, either by adding different antigens to 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 simultaneously. 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 are 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 does not induce significant adverse reactions in the treated subject after administration and that can present the 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), and may also contain immunostimulants (also called adjuvants). Depending on the intended use or required properties of the corresponding vaccine, other substances may be added, such as stabilizers, viscosity adjusters, or other ingredients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] In a further embodiment of the vaccine according to the invention, the IgM protease antigen of Streptococcus suis serotype 1 is a whole IgM protease antigen having at least 90% sequence identity with the corresponding naturally occurring (i.e. wild type) IgM protease of the bacterium Streptococcus suis serotype 1. It is known from the literature that only the Mac-1 domain of the IgM protease (about 35%) is sufficient to provide a protective effect, but the whole antigen is believed to provide 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 in order to reach a reasonable homologous and heterologous protective effect.

[0035] In yet a further embodiment of the vaccine according to the invention, the IgM protease antigen of Streptococcus suis serotype 1 contains less than four repeats in its amino acid sequence. Structural analysis of the genome of Streptococcus suis has revealed that the genome of this bacterium is prone to the phenomenon of copy number variation (CNV), in which parts of the genome are repeated. In particular, the repeats have similarity to known protein sequences with hydrolase activity. It has been found that the IgM proteases of serotype 2 are very different from those that provide better heterologous protection (such as serotypes 1 and 7), in that serotype 2 contains four repeats. Therefore, a number of repeats less than four, or even less than three, for example two, seems to be advantageous to reach the best possible (heterologous) protection.

[0036] Most preferred is the IgM protease antigen of Streptococcus suis serotype 1 of sequence type 13, which contains two repeat sequences.

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

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

[0039] In yet a further embodiment of the combination for use according to the invention, the method comprises administering an IgM protease antigen of Streptococcus suis serotype 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis to a pig up to 35 days of age.

[0040] In an alternative embodiment, the method includes administering to a sow an IgM protease antigen of Streptococcus suis serotype 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis to protect the sow (usually a piglet) through ingestion of the sow's colostrum. IgM protease (see WO 2019 / 193078) is known to provide reasonable and long-lasting protection to piglets when they ingest colostrum from a vaccinated sow. It is also generally known that the protection provided by the bacterin is transferred to the piglet via the colostrum.

[0041] In one embodiment, the IgM protease antigen of Streptococcus suis serotype 1 and the bacterin serotype 9, sequence type 16 of Streptococcus suis are administered twice to the sow before the piglets receive the colostrum.

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

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

[0044] In Example 2, the cross-protective effect of IgM protease serotype 2 against serotype 1 is investigated.

[0045] In Example 3, the cross-protective effect of IgM protease serotype 2 against serotype 7 is studied.

[0046] In Example 4, the cross-protective effect of IgM protease serotype 2 against serotype 9, sequence type 16 is studied.

[0047] In Example 5, the protection conferred by IgM proteases of serotypes 1 and 7 against a challenge with serotype 1 is studied.

[0048] In Example 6, the protection conferred by IgM proteases of serotypes 1 and 7 against a challenge with serotype 2 is studied.

[0049] In Example 7, the protection conferred by IgM proteases of serotypes 1 and 7 against serotype 7 challenge is studied.

[0050] In Example 8, the protection conferred by the bacterin against a serotype 9, sequence type 16 challenge is studied.

[0051] [Example 1] In this example, the genome of Streptococcus suis, i.e. the part that codes for the IgM protease, is analyzed in order to show how this part of the genome is structured. For this, we use the genome of the bacterium Streptococcus suis of serotype 2, known from WO 2015 / 181356 and published in that patent application as SEQ ID NO: 1. The sequence is also included in the sequence listing of this patent as SEQ ID NO: 1. In addition to protein annotation (PDBSum and InterPro), a sequence similarity search using the Needleman-Wunsch alignment (Needleman et al. 1970; Laskowski et al. 1997; Apweiler et al. 2000, see default settings) reveals the structure of the IgM protease genome, allowing the identification of five regions.

[0052] Region 1 (Met 1-Thr 34): signal sequence from position 1 Region 2 (Val 35-Glu 426): Mac-1 domain with predicted hydrolase activity Region 3 (Thr 427-Pro 687): A region involved in structural functions (e.g., involved in proper folding) and substrate binding Region 4 (Thr 688-Ser 919): A region consisting of four repeat sequences (1*{Thr 688-Ser 744}, 2*{Thr 745-Ser 801}, 3*{Thr 802-Ser 858}, 4*{Thr 859-Ser 919}) that show similarity to known protein sequences with hydrolase activity. Region 5 (Thr 920-Lys 1141): Contains a predicted transmembrane domain that exhibits cell wall anchoring function.

[0053] The structure of the other serotypes of Streptococcus suis bacteria is nearly identical, but for serotype 9 and sequence type 16, substantial differences exist (as shown below).

[0054] The -signal peptide is highly conserved among Streptococcus suis strains.

[0055] The -Mac-1 domain is always present and highly conserved among all known strains, including serotype 9 and sequence type 16 strains.

[0056] - Region 3, which is related to structural function, is always present and highly conserved, but is only about half the length of serotype 9 and sequence type 16.

[0057] With regard to the -CNV region, the repeats are very similar between the different serotypes, but the number varies, generally between 2 and 6. Serotype 9, sequence type 16, has 12 repeats of a completely different type that are much shorter compared to those of the other serotypes (i.e., 12 amino acids versus approximately 60) and can be subdivided into three substantially different repeats.

[0058] - The cell adhesion region is also highly conserved among the different serotypes, but shares little amino acid sequence identity with the region in serotype 9, sequence type 16 strains.

[0059] In summary, the genomes are almost identical in structure among most serotypes and sequence types, with the most notable difference being the number of repeats in the CNV regions. The IgM protease portions of the genomes of serotype 9, sequence type 16 are very similar as far as the Mac-1 domain is concerned, but differ substantially in the remainder of the genome.

[0060] [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 homologous challenge. Also some cross-protection against serotypes 9 and 14 is known from the literature. In this example the actual level of protection provided by this antigen against a serotype 1 challenge is evaluated. For this purpose a strain of sequence type 13 was used, which is the usual type of the bacterium and is a good representative of this serotype in the field.

[0061] Study design Firstly, the only available challenge model for evaluating the protective effect against challenge with serotype 1 bacteria is the one in which piglets are challenged at 3 weeks of age. This means that vaccination of the piglets themselves is not possible in order to evaluate the protective effect induced by the IgM protease antigen, since the time for the subsequent development of an effective immune response is expected to be too short. Therefore, in order to evaluate the protection provided by the vaccine, sows are vaccinated before farrowing, which transfers the induced antibodies to the piglets via the ingestion of colostrum. It is known from the literature (US Pat. No. 10,751,403) that the protection provided by the IgM protease antigen to the vaccinated animal itself also provides a good protection to the progeny of the vaccinated sow. In other words, the protection seen in this (indirect) challenge model naturally represents the protection provided to the vaccinated animal itself in addition to the protection provided to the piglets via the ingestion of the colostrum of the vaccinated sow.

[0062] In this study, 10 pregnant sows were used and divided into two groups of 5 sows each. Six and two weeks before the expected farrowing, one group was vaccinated with a subunit vaccine containing recombinant rIdeSsuis IgM protease antigen of serotype 2 (Seele et al., Vaccine 33:2207-2212, May 5, 2015, item 2.2.) at 80 μg per dose in an oil-in-water adjuvant (μDiluvac Forte, MSD Animal Health), and the other 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 (each group contained 2 piglets per sow) were selected for challenge. The piglets (2 × 10, vaccinated and control) were catheterized and immunized with 5.0 × 10 10 The piglets were challenged intratracheally with 10 ml of challenge inoculum (targeting 100 CFU / ml) or (if this was not possible) by transtracheal injection. 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 systematic scoring system ranging from 0 (no signs) to 3 for severe cases. Animals reaching 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 reisolation of the challenge strain at regular times before and after challenge (20 piglets). At the end of the study (i.e. 11 days post-challenge), all surviving piglets were euthanized.

[0063] result None of the vaccines induced any unacceptable site (i.e. local) or systemic reactions and could therefore be considered safe. Post-challenge data for the period before euthanasia are shown in Table 1. [Table 1]

[0064] conclusion Serotype 2 IgM proteases do not confer protection against attack by serotype 1 Streptococcus suis bacteria.

[0065] [Example 3] Research Objective In this example, the actual level of protection provided by the same antigen used in Example 2 (serotype 2 IgM protease) against a challenge with serotype 7 is evaluated. For this, a strain of sequence type 29 was used, which is the common type of the bacterium and representative of this serotype in the field.

[0066] Study design As with serotype 1, the only available challenge model to evaluate the protective effect against challenge with serotype 7 bacteria is a challenge model in piglets at 3.5 weeks of age. Therefore, also in this study, sows were vaccinated before farrowing, which allows the transfer of induced antibodies to the piglets via ingestion of colostrum.

[0067] In 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 containing recombinant rIdeSsuis IgM protease antigen of serotype 2 (Seele et al., Vaccine 33:2207-2212, May 5, 2015, item 2.2.) at 80 μg per dose in an oil-in-water adjuvant (μDiluvac Forte, MSD Animal Health), and the other 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 (each group contained 2 piglets per sow) were selected for challenge. The piglets (2×10, vaccinated and control) were administered 1.0×10 9An intratracheal challenge was given with 10 ml of challenge inoculum (targeting 100 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 systematic scoring system ranging from 0 (no signs) to 3 for severe cases. Animals reaching a humane endpoint were euthanized. Serum blood was taken for antibody determination at regular times before and after vaccination (10 sows) and 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.

[0068] result None of the vaccines induced any 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]

[0069] conclusion Serotype 2 IgM proteases do not confer protection against attack by serotype 7 Streptococcus suis bacteria.

[0070] [Example 4] Research Objective The aim of this study was to test the actual level of protection provided by the same antigen used in Examples 2 and 3 (i.e. serotype 2 IgM protease) against a serotype 9 challenge, and in particular a challenge with serotype 9, sequence type 16 bacteria.

[0071] Study design Twenty-four seronegative specific pathogen-free (SPF) 3-week-old piglets were used. The piglets were assigned to two groups of 10 piglets each (evenly distributed among different litters). The first group was vaccinated intramuscularly twice at 3 and 5 weeks of age as described in Examples 2 and 3, while the second group 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 above. After challenge, the pigs were observed daily for 10 days for clinical signs of S. suis infection, such as depression, motor impairment and / or neurological signs. Animals that reached a humane endpoint after showing specific clinical signs (i.e., motor or neurological) were euthanized without necropsy. Animals that reached a humane endpoint without showing specific clinical signs were euthanized and necropsied, including bacteriological examination, to confirm S. suis infection. Heparinized blood was collected at regular times before and after challenge for reisolation of the challenge strain.Pigs were seronegative for serotype 2-derived IgM protease on the day of first vaccination (5 weeks of age).

[0072] result None of the vaccines induced any 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]

[0073] conclusion Serotype 2 IgM proteases do not confer protection against challenge with serotype 9, sequence type 16 Streptococcus suis bacteria.

[0074] [Example 5] Research Objective In this example, the protective effect against serotype 1 challenge is evaluated for the IgM protease antigens of Streptococcus suis strains of serotype 1 and serotype 7. For this, antigens were produced corresponding to the IgM protease of serotype 2 used in examples 2, 3 and 4, i.e. using the E. coli expression system described in the literature (Seele et al., see above). The sequence used for the IgM protease antigen of serotype 7 is given in SEQ ID NO: 2 attached, whereas the sequence used for the IgM protease antigen of serotype 1 is given in SEQ ID NO: 3 attached. Both sequences contain a CNV region in addition to the Mac-1 region, which has two repeats. The challenge strain was the same as that used in example 2.

[0075] Study design The study design was the same as in Examples 2 and 3, but in each case piglets aged 3.5 weeks were used for challenge, and groups of 10 piglets were used. The challenge 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.

[0076] result None of the vaccines induced any 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]

[0077] 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 by strains of serotype 1. The homologous protection conferred by serotype 1 antigens appears to be slightly better than the heterologous protection conferred by serotype 7 antigens.

[0078] [Example 6] Research Objective In this example, the protective effect against serotype 2 challenge is evaluated for the IgM protease antigens of Streptococcus suis strains of serotype 1 and serotype 7. For this purpose, the same antigens were used as in Example 5. The challenge strain was a strain of serotype 2, sequence type 1, which is representative of strains in the field.

[0079] Study design The study design was similar to that of Example 4. Thirty three-week-old piglets were used. The piglets were assigned to three groups of 10 piglets each (evenly distributed among the different litters). Groups 1 and 2 were vaccinated intramuscularly twice with the respective subunit vaccines at 3 and 5 weeks of age, while group 3 was not vaccinated. At 7 weeks of age, pigs were challenged intratracheally with a virulent culture of a strain of S. suis serotype 2. For 11 days after challenge, 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 on the day of HEP (just before euthanasia) if applicable.

[0080] The piglets were either seronegative or had very low titers in enzyme-linked immunoassay (ELISA) of specific IgM antibodies on the day of the first vaccination. After vaccination, groups 1 and 2 showed good antibody responses against IgM protease, whereas controls remained at very low levels.

[0081] result None of the vaccines induced any 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]

[0082] 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 by serotype 2 strains.

[0083] [Example 7] Research Objective In this example, the protective effect against serotype 7 challenge is evaluated for the IgM protease antigens of Streptococcus suis strains of serotype 1 and serotype 7. For this purpose, the same antigens were used as in examples 5 and 6. The challenge strain was a strain of serotype 7, sequence type 29, which is representative of strains in the field.

[0084] Study design The study design was the same as in Example 5 (apart from the challenge strain). The challenges for each of the serotypes corresponded to those 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.

[0085] result None of the vaccines induced any 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]

[0086] conclusion Although the challenge appeared to be less virulent as in previous studies, the data allow us to conclude that the serotype 1 IgM protease as well as the serotype 7 IgM protease protect against a virulent challenge with a serotype 7 strain.

[0087] [Example 8] Research Objective The aim of this study was to find protective antigens against serotype 9 challenge, particularly against challenge with bacteria of serotype 9, sequence type 16, which are representative of strains circulating in the field. The options evaluated were bacterin alone and in combination with an IgM protease, which is understood in the literature to improve the efficacy of bacterins (see Seele et al., Journal of Bacteriology, pp. 930-940, March 2013, Vol. 195, No. 5, "Identification of Novel Host-Specific IgM Protease in Streptococcus suis", reviewed in WO 2015 / 181356).

[0088] Study design The study design was the same as that used in Example 4, but with non-SPF piglets, which were assigned to three groups of 12 each (evenly distributed among the different litters). The first group contained 2 × 10 inactivated Streptococcus suis bacteria of serotype 9, sequence type 16. 9 The pigs were vaccinated intramuscularly twice at 3 and 5 weeks of age with a bacterin vaccine containing the cellular level. Group 2 additionally contained the IgM protease of Example 2 at 80 μg per dose. Both vaccines were formulated in the oil-in-water adjuvant used in the other examples. Group 3 remained as a non-vaccinated challenge control. At 7 weeks of age, the pigs were challenged intratracheally with a virulent culture of S. suis serotype 9 as described above. After challenge, the pigs were observed daily for 10 days for clinical signs of S. suis infection, such as depression, motor impairment and / or neurological signs. Animals that reached a humane endpoint after showing specific clinical signs (i.e., motor or neurological) were euthanized without necropsy. Animals that reached a humane endpoint without showing specific clinical signs were euthanized and necropsied, including bacteriological examination, to confirm S. suis infection. Heparinized blood was collected at regular times before and after challenge for reisolation of the challenge strain.Pigs were seronegative for serotype 2-derived IgM protease on the day of first vaccination (5 weeks of age).

[0089] result None of the vaccines induced any unacceptable site 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]

[0090] conclusion Protection against virulent attack by serotype 9, sequence type 16 S. suis could be provided by a bacterin of that serotype and by a bacterin in combination with an IgM protease. The two types of antigens did not negatively interfere, which is consistent with what would be expected on the basis of the prior art.

[0091] Based on the above examples, the object of the present invention can be achieved by combining the IgM protease antigen of Streptococcus suis serotype 7 or 1 with the bacterin serotype 9, sequence type 16 of Streptococcus suis in a mixed vaccination plan. It is also considered that, if necessary, two IgM protease antigens may be combined in the same way to reach a better protection against both serotype 1 and 7 challenges. It is also reasonable to consider that the level of cross-protection is related to the number of repeat sequences in the CNV region of the IgM protease, because there is a difference between the IgM protease molecules of serotypes 1 and 7 when compared with serotype 2. That is, the IgM proteases of serotypes 1 and 7 each have two repeat sequences, while serotype 2 has four repeat sequences. Although the reason for the difference in cross-protection is not cleats, a smaller number of repeat sequences seems to be favorable for reaching a better level of cross-protection.

Claims

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

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

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

4. 2. The vaccine of claim 1, wherein the Streptococcus suis serotype 1 IgM protease antigen contains fewer than four repeat sequences in its amino acid sequence.

5. 2. The vaccine of claim 1, wherein the Streptococcus suis serotype 1 IgM protease antigen contains fewer than three repeat sequences in its amino acid sequence.

6. 2. The vaccine of claim 1, wherein the IgM protease antigen of Streptococcus suis serotype 1 contains two repeat sequences in its amino acid sequence.

7. 2. The vaccine of claim 1, wherein the Streptococcus suis serotype 1 IgM protease antigen is of sequence type 13.

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

9. A combination of an IgM protease antigen of Streptococcus suis serotype 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis for use in a method of protecting pigs from pathogenic infection with Streptococcus suis.

10. 10. The combination for use according to claim 9, characterized in that the protection is against pathogenic infection with Streptococcus suis of any of serotypes 1, 2, 7 and 9.

11. 11. A combination for use according to any of claims 9 and 10, characterized in that the method comprises the administration of the Streptococcus suis serotype 1 IgM protease antigen and the Streptococcus suis bacterin serotype 9, sequence type 16 to the pigs up to 35 days of age.

12. 11. A combination for use according to any of claims 9 and 10, characterized in that the method comprises administering to a sow the IgM protease antigen of Streptococcus suis serotype 1 and the bacterin serotype 9, sequence type 16 of Streptococcus suis for protecting the pig through ingestion of the sow's colostrum.

13. 13. The combination for use according to claim 12, characterized in that the Streptococcus suis serotype 1 IgM protease antigen and the Streptococcus suis bacterin serotype 9, sequence type 16 are administered twice to the sow before the pig receives the colostrum.

14. Use of the IgM protease antigen of Streptococcus suis serotype 1 and the bacterin serotype 9, sequence type 16 of Streptococcus suis for the manufacture of a vaccine for the protection of pigs against pathogenic infection with Streptococcus suis.

15. A method for protecting pigs from pathogenic infection with Streptococcus suis by administering to said pigs an IgM protease antigen of Streptococcus suis serotype 1 and a bacterin serotype 9, sequence type 16 of Streptococcus suis.