Stable vaccine against streptococcus suis

Abstract

The invention pertains to a stable vaccine formulation comprising a protein denoted as IdeSsuis and the vaccine formulation comprising reduced cysteine. The invention also pertains to a vaccine formulation comprising a protein denoted as IdeSsuis and the vaccine formulation comprising reduced cysteine for use in a method for protecting pigs against a pathogenic infection of S. suis bacteria. The invention also pertains to methods of preparing the vaccine formulation, preferably wherein the method comprises a step of lyophilizing the vaccine formulation.

Description

[0001] STABLE VACCINE AGAINST STREPTOCOCCUS SUIS

[0002] TECHNICAL FIELD

[0003] The invention pertains to stable vaccines that provides protection of pigs against a pathogenic infection with Streptococcus suis bacteria of various serotypes. In particular, the invention pertains to a vaccine formulation comprising a protein denoted as IdeSsuis and a reductant that acts as a stabiliser.

[0004] BACKGROUND OF THE INVENTION

[0005] Streptococcus suis (S. suis) is one of the principal etiologic agents of contagious bacterial disease in pigs. The pathogen can cause a variety of clinical syndromes including meningitis, arthritis, pericarditis, polyserositis, septicaemia, pneumonia and sudden death. S. suis is considered one of the most important bacterial swine pathogens leading to important economic losses to the porcine industry worldwide. S. suis is a gram-positive facultatively anaerobic coccus, originally defined as Lancefield groups R, S, R / S or T. Later, a new typing system based on the type-specific capsular polysaccharide antigens located in the cell wall was proposed. This led to a system comprising 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 recognised that the capsular serotype is a poor marker of virulence. Therefore, an alternative system to helping understand the epidemiology of S. suis infection and the biological relevance of the serotyping approach was developed, i.e. the so called multilocus sequence typing (MLST), as described by King et al. in 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"). In that study 92 sequence types were identified, of which ST complexes ST1, ST27 and ST87, each containing multiple sequence types, dominate the population. See also the Streptococcus suis MLST website (https: / / pubmlst.org / organisms / streptococcus-suis / ) sited at the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 (site funded by the Wellcome Trust), which refers to the King et al. paper and allows for easy identification of the sequence type for any S. suis strain. Control of S. suis in pig herds appears to be difficult. S. suis is a commensal and opportunistic pathogen of swine. Apparently, the immune system is not triggered in each and every occasion of an infection. Next to this, S. suis is a well-encapsulated pathogen and uses an arsenal of virulence factors to evade the host immune system. Together, these characteristics have challenged the development of efficacious vaccines to fight this important pathogen. An overview article has been published a few years ago, the article reviewing existing and explorative 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 field information and experimental data have been compiled and compared to give an overview of the status of vaccine development against S. suis.

[0006] In the last couple of years, an extensive list of antigenic or immunogenic S. suis molecules has been reported, and most of these have been discovered through immunoproteomics using either convalescent sera from infected pigs or humans and / or laboratory-produced immune sera. WO2015 / 181356 (IDT Biologika GmbH) has shown that a protein denoted in the art as IdeSsuis, also described as an IgM protease, can elicit a protective immune response in piglets through vaccination with this protein (i.e. the IgM protease) as an antigen, optionally in combination with a prime vaccination containing a bacterin. It is suggested in the ‘356 patent application that the IdeSsuis, due to the fact that it is highly conserved throughout S. suis of various serotypes is able to protect across various S. suis serotypes. W02017 / 005913 (Intervacc AB) confirms the fact that the IgM protease is highly conserved throughout various S. suis serotypes.

[0007] Over the past years several vaccines providing protection of pigs against a pathogenic infection of S. suis have been commercialized, such as Porcilis® Strepsuis (MSD Animal Health), with even more pending in R&D pipelines of manufacturers of animal vaccines.

[0008] Recently, patent applications regarding the use of the IdeSsuis protein, for protection against S.suis of various serotypes, have been published. These applications confirm the cross- protective nature of the IgM protease antigen. 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 adequate protection can be obtained. In line with these developments, W02023 / 011811A1 provides a vaccine providing protection of pigs against S. suis, in particular against S. suis of various serotypes including serotype 1 , 2, 7 and 9.

[0009] However, an occurring problem with vaccines for protecting pigs against a pathogenic infection of S. suis is that they rapidly diminish in potency over time. As a consequence, the protective effect (e.g., potency) of such vaccines can be impaired when stored too long. In addition, these vaccines typically cannot be kept, not even for a few days, at room temperature without a significant loss of titre. Therefor these vaccines are typically stored at 2-8°C. Deviating (upwards) from storing conditions at 2-8°C may have a similar potency-reducing effect on the vaccine as storing the vaccine too long.

[0010] It is generally recommended that deviation from recommended storage conditions for vaccines necessitates that the vaccine products involved are removed from use and destroyed. Typically, when such a deviation of the recommended storage conditions occurs, the long-term quality and (continued) shelf-life of the affected vaccines cannot be guaranteed. When a deviation of the recommended storage conditions occurs, for example during transport of the vaccines, such an event is labeled as a ‘cold chain breach’ rendering the respective vaccine batch unusable.

[0011] It is beneficial for manufacturers and pig-breeders alike that vaccines against S. su / s better retain their potency when stored for prolonged periods of time, e.g. for a longer period of time than current vaccines, and / or retain their potency when (temporarily) stored at temperatures other than 2-8°C, e.g. at room temperature. In other words, there are benefits to improve stability features of vaccines for pigs against S. su / s.

[0012] Therefore, it is an object of the invention to provide for a vaccine that has improved stability for providing protection of pigs against a pathogenic infection of S. su / s.

[0013] SUMMARY OF INVENTION

[0014] In order to meet the object of the invention a vaccine formulation has been devised comprising a protein denoted as IdeSsuis, which is the antigen, a pharmaceutically acceptable carrier and a reductant having a redox potential of -80 mV or less (i.e. lower than -80 mV such as -100 mV).

[0015] The current invention is based on a surprising finding of the inventors to improve the stability of a vaccine for protecting pigs against a pathogenic infection of S. su / s. The inventors found that the addition of a reductant such as reduced cysteine (in this application also denoted simply as “cysteine”) to vaccine formulations comprising a protein denoted as IdeSsuis, improved the stability of the vaccine (i.e. the stability of the antigen).

[0016] It was theorized by the inventors that a strong enough reductant, i.e. a reductant having a redox potential of at least -80 mV, is able to prevent and / or reduce the increase of the amount of molecular complexes of a protein denoted as IdeSsuis in the vaccine formulation whilst also maintaining and / or decreasing the reduction of the amount of non-complexed proteins denoted as IdeSsuis in the vaccine formulation. The inventors contemplate that the formation of molecular complexes of a protein denoted as IdeSsuis in the vaccine formulation may be caused by oxidative crosslinking, which may be irreversible once occurring in the vaccine formulation. Said formation of molecular complexes of the protein denoted as IdeSsuis likely reduces the amount of non-complexed proteins denoted as IdeSsuis in the vaccine formulation. It is contemplated that the molecular complexes of a protein denoted as IdeSsuis are likely contributory, if not causal, for a reduction in potency of the vaccine formulations, therewith reducing the protective effect.

[0017] The inventors hypothesized that the formation of the protein complexes in the vaccine formulation comprising a protein denoted as IdeSsuis is attributable to the formation of disulfide bridges between cysteine amino acid residues in neighbouring IdeSsuis protein molecules. Next, the inventors hypothesized that a reducing agent may improve the stability of the vaccine formulation by preventing the formation of these disulfide bridges. The inventors found that several reducing agents, in particular reduced cysteine, appeared to be able to improve the stability of the vaccine. The inventors found that a reductant is in principle able to maintain the potency of the vaccine (as exemplified in Example 2 of this disclosure, wherein vaccine potency is measured by determining and comparing the antigenic mass of the vaccine at different timepoints). It is hypothesized by the inventors that the addition of a reductant to a vaccine formulation comprising a protein denoted as IdeSsuis and a pharmaceutically acceptable carrier could potentially extend the shelf life of said vaccine formulation to about 2-3 years and / or could enable (temporary) storage of the vaccine formulation at temperatures higher than refrigerator temperatures, e.g., room temperatures, for at least 14 days. During the storage, the reductant will counter any oxidation of the IdeSsuis and is believed to therewith (slowly) attain any of its oxoforms. However, as long as reductant is present in the formulation (thus the corresponding molecule in its reduced form), there will be a stabilising effect. Therefore, in a first aspect of the invention, there is provided for a vaccine formulation comprising a protein denoted as IdeSsuis, a reductant and a pharmaceutically acceptable carrier. A practical minimum amount of the reductant to provide a significant stabilising effect in practice is believed to be 0.01% (w / v) in the vaccine formulation.

[0018] Further it was found that the addition of the reductant such as cysteine to the vaccine formulation did not affect the efficacy or safety of a vaccine formulation comprising a protein denoted as IdeSsuis for use in a method to protect a pig against a pathogenic infection with S. su / s (as exemplified in Example 1 of this disclosure). Therefore, in a second aspect, the invention also pertains to the vaccine formulation according to the invention for use in a method to protect a pig against a pathogenic infection with S. suis.

[0019] Also found was that the addition of a reductant, in particular cysteine, to a vaccine formulation comprising a protein denoted as IdeSsuis did not affect the use of said vaccine formulation in a combination vaccine comprising a further porcine vaccine. In other words, the combination of a vaccine formulation of the invention comprising cysteine and a further porcine vaccine did not result in any impairment of the safety and / or efficacy of that further porcine vaccine nor of the vaccine formulation of the invention. Therefore, in a third aspect, the invention also pertains to a combination vaccine comprising the vaccine formulation of the invention and a further porcine vaccine.

[0020] Finally, in a fourth aspect, the invention pertains to a method of preparing the vaccine formulation according to the invention, wherein the method comprises the steps of: a) recombinantly expressing a protein denoted as IdeSsuis in Escherichia coli (E. coli) bacteria; b) subjecting the E. coli bacteria to a homogenisation operation to induce lysis of the E. coli bacteria and release of IdeSsuis into a supernatant of the lysate; c) separating the supernatant from a pellet of the lysate; and d) mixing the supernatant comprising IdeSsuis with a pharmaceutically acceptable carrier, a reductant having a redox potential of -80m V or less and optionally a buffering agent to constitute the vaccine formulation.

[0021] DEFINITIONS

[0022] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0023] For purposes of the present invention, the following terms are defined below.

[0024] A “protein denoted as IdeSsuis" as used herein is an IgM protease of S. suis. IdeSsuis, also referred to in the art as “Ide-Ssuis". The IgM protease is an enzyme that specifically degrades porcine IgM and not porcine IgG or porcine IgA (Seele et al, in Journal of Bacteriology, 2013, 195 930-940; and in Vaccine 33:2207-2212; 5 May 2015). The enzyme has a weight of about 95-150 kDa, corresponding to about 900-1350 amino acids, the size depending mainly on the strain and in particular on the number of repeats (see W02023 / 011812). IdeSsuis can be used as naturally expressed or recombinantly expressed, for example in E. coli (Seele, 2013). IdeSsuis proteins have been described in the art, such as in WO 2015 / 181356 several sequences are given, viz. SEQ ID NO:1 , 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 (denoted as the Mac-1 domain, i.e. amino acids 80-414 of SED ID NO:7). Examples of IdeSsuis proteins are disclosed in public databases such as GenBank® at NCBI (National Center for Biotechnology Information, Bethesda, MD, USA). A particular example of an IgM protease is the protease according to SEQ ID NO:1 of WO2015 / 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. The amino acid sequence identity may be established with the BLAST program using the blastp algorithm with default parameters. In particular, a protein denoted as IdeSsuis, as used herein is an antigen that comprises at least 1) the Mac-1 domain, 2) the region linked to structural functions, and 3) the CNV region, and optionally the cell adhesion region (see WO 2023 / 011812). A protein comprising these items 1), 2) and 3) can be regarded as a “protein denoted as IdeSsuis”, which thus is “an IgM protease of Streptococcus suis”. Although Streptococcus suis also encodes for a signal peptide, the signal peptide is believed to be missing in the naturally occurring (wild-type) secreted enzyme anyway, and the cell adhesion region is not believed to be essential for its function as a protease. Therefore, a protein comprising these items 1), 2) and 3) can be regarded as a “protein denoted as IdeSsuis”. Optionally, the protein denoted as IdeSsuis comprises a His-tag or other conjugated molecule.

[0025] A reductant in the sense of the present invention is a compound able to prevent the formation of a disulfide bridge between cysteine and another amino acid, in particularly another cysteine molecule. For this, the redox potential needs to be below -80mV, since this is the median redox potential (Eh) of the Cys / CySS reaction (Smita S. et al, “Cysteine Redox Potential... " in PLOS One, Volume 4, Issue 3, March 27, 2009, https: / / doi.org / 10.1371 / journal.pone.0005017). Preferably the redox potential Eh of the reductant is -100 mV or less, in particular - 150 mV or less, more preferably -200, mV or less etc. The redox potential Eh is measured under standard conditions (T = 25 °C, 1 atm) and defined relative to the standard hydrogen electrode (SHE) used as reference electrode, which is arbitrarily given a potential of 0.00 V.

[0026] Reduced cysteine is cysteine in its reduced form, having free SH (thiol) groups. Under normal atmospheric conditions cysteine is typically present in oxidised form, in one of its so called oxoforms. These include disulphide bonds (S-S), S-glutathionylation (S-SG), S-nitrosylation (SNO) and S- sulfenylation (SOH). However, these bonds are reversible and can be reduced to thiol to obtain reduced cysteine. The term reduced cysteine also includes salts of this compound. Correspondingly “reduced glutathione” is glutathione in its reduced form and also includes salts of this compound.

[0027] As used herein, a “combination vaccine” (i.e. a vaccine comprising a combination of antigens) is one (unitary) formulation that at the same time comprises different antigens. These different antigens can be mixed in a factory to provide a so-called ready-to-use combination vaccine, or mixed right before administration or during administration (e.g. using a device having two separate chamber for the separate antigens, the content of these chambers being mixed upon using the device for administration), as long as the antigens do end up in the same formulation.

[0028] As used herein, a “pharmaceutically acceptable carrier” is a biocompatible medium, viz. a medium that after administration does not induce significant adverse reactions in the treated subject, capable of presenting the antigen to the immune system of the subject after administration of the composition comprising the carrier. Such a pharmaceutically acceptable carrier may for example be a liquid containing water and / or any other biocompatible solvent or a solid carrier such as commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), optionally comprising immunostimulating agents (also called an adjuvant). Optionally other substances such as stabilisers, viscosity modifiers or other components are added depending on the intended use or required properties of the corresponding vaccine.

[0029] As used herein, a “pig”, herein interchangeably referred to as “porcine", is any of the animals that belong to the family of Suidae.

[0030] As used herein, “protection” when used in relation to a pathogenic infection of a micro-organism (for example “protecting against a pathogenic infection of the micro-organism S. suis”) is the same as arriving at protective immunity, i.e. aiding in preventing, ameliorating or curing the pathogenic infection with that micro-organism or a disorder arising from that infection, for example to prevent or reduce the actual infection or one or more clinical signs resulting from the pathogenic infection with the micro-organism.

[0031] As used herein, a “vaccine formulation” is a constitution suitable for application to a subject, comprising one or more antigens in an immunologically effective amount (i.e. capable of stimulating the immune system of the target subject sufficiently to protect against a challenge of the wild-type micro-organisms), typically combined with a pharmaceutically acceptable carrier, which upon administration to the subject induces protection against an infection, i.e. aiding in preventing, ameliorating or curing the infection or any disease or disorder arising from that infection. A vaccine typically comprises an adjuvant, i.e. an immune stimulating material such as an oil, a saponine, alum, etc.

[0032] DESCRIPTION OF EMBODIMENTS

[0033] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0034] It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0035] In embodiments, the invention pertains to the vaccine formulation consisting of a protein denoted as IdeSsuis, a reductant having a redox potential of at least -80 mV (i.e. -80 mV or less) and a pharmaceutically acceptable carrier. In further embodiments, the reductant has a redox potential of -100 mV or less, such as -150 mV or less, preferably -200 mV or less.

[0036] In yet other embodiments, the reductant is chosen from the group consisting of reduced cysteine, methionine, sodium thiosulfate, B-mercaptoethanol, tris-2-carboxyethyl-phosphine-hydrochloride (TCEP), reduced glutathione, and dithiothreitol (DTT).

[0037] In embodiments, the protein denoted as IdeSsuis is a recombinant protein that may be obtained by obtaining a supernatant from a culture of recombinant cells, preferably recombinant bacteria, more preferably recombinant E.coli bacteria. Genes expressing the protein denoted as IdeSsuis are cloned into cells, preferably bacteria, more preferably E.coli bacteria, using methods known and described in the art to obtain the recombinant forms thereof.

[0038] In embodiments, the invention pertains to the vaccine formulation comprising a protein denoted as IdeSsuis, a reductant as indicated here above and a pharmaceutically acceptable carrier, wherein the concentration of the reductant in the vaccine formulation is at least 0.01 % w / v. A technically reasonable maximum for the amount of reductant is 20-50% w / v, depending e.g. on type of reductant and the type of continuous phase. Some reductants for example might not be safe for the vaccinated host in higher amounts. Preferably, the vaccine formulation comprises at least 0.05% w / v of the reductant, such as between 0.1% w / v and 0.3% w / v of the reductant.

[0039] The concentration of the reductant, such as for example cysteine, preferably on the basis of its salt, e.g. the HCI salt, is determined prior to lyophilization of the vaccine formulation of the invention. It will be appreciated that lyophilization of the vaccine formulation according to the invention increases the relative concentration of components, including the reductant, in the vaccine formulation due to the loss of the water.

[0040] In some embodiments, the concentration of the reductant is determined on the basis of the volume of the supernatant comprising IdeSsuis that has been mixed with a pharmaceutically acceptable carrier. For example, if 100 mL of vaccine formulation, i.e., a protein denoted as IdeSsuis mixed with a pharmaceutically acceptable carrier, is provided, then the amount of reductant added for a concentration of between 0.01% to 1% w / v is between 0.01 and 1 grams.

[0041] It was surprisingly found by the inventors that the loss in antigenic mass of the vaccine formulation of the invention, when stored for two weeks at 25°C, compared to the antigenic mass of said vaccine formulation on the day of production could be reduced to be a value as low as 20%. This indicates that the concentration of the antigen (II I dose) only decreased by about 20% compared to the concentration (II I dose) on the day of manufacture of said vaccine formulation. It was even more surprisingly found that in some instances the loss in antigenic mass of the vaccine formulation of the invention under the aforementioned conditions was at most 15%, even at most 10%, or even at most just 3%. In some preferred instances it was even found that there was no loss in antigenic mass of the vaccine formulation of the invention.

[0042] In some preferred embodiments, the vaccine formulation comprises a concentration of between 0.01 % to 3% w / v reductant, such as 0.05% w / v, 0.1% w / v, 0.15% w / v, 0.2% w / v, 0.25% w / v, 0.5% w / v, 1 % w / v, 1.5% w / v, 2% w / v, 2.5% w / v and any one other % w / v in between the range of 0.01% to 1 % w / v. More preferably, the vaccine formulation comprises between 0.05% to 0.5% w / v reductant, such as 0.1% w / v, 0.15% w / v, 0.2% w / v, 0.25% w / v or 0.3% w / v. Even more preferably, the vaccine formulation comprises between 0.1 % to 0.3% w / v reductant, most preferably 0.2% w / v reductant. It will be appreciated that any of the lower values for the concentration of reductant mentioned herein may be combined with any of the upper values for the concentration of reductant.

[0043] In a preferred embodiment, there is provided for a vaccine formulation comprising a protein denoted as IdeSsuis, a reductant as defined here above and a pharmaceutically acceptable carrier, wherein the vaccine formulation is lyophilized. The lyophilized vaccine formulation can be reconstituted to provide for a vaccine formulation that is ready-to-administer to a subject. Preferably, reconstitution is by dissolving the lyophilized vaccine formulation in a solvent, such as an adjuvant formulation. In further embodiments, the reconstituting of the lyophilized vaccine formulation of the invention is by mixing with a further vaccine composition, such as a further porcine vaccine, wherein said further vaccine composition is in the form of an emulsion or solution suitable for injection. Lyophilization of the vaccine formulation can be performed by using a lyophilization method as described in the art, such as described by P. Wen et al. (2014) Lyophilization in Vaccine Processes in Vaccine Development and Manufacturing (eds E. P. Wen, R. Ellis and N. S. Pujar).

[0044] In embodiments of the vaccine formulation comprising a protein denoted as IdeSsuis for use in a method to protect a pig against a pathogenic infection with S. su / s the protein denoted as IdeSsuis is of a S. su / s bacterium of serotype 1 , 2 or 7. Although the recombinant expression for the antigen as such does not depend on the serotype, and also, cross protection is an inherent property of IdeSuis as an antigen given the fact that it is highly conserved across serotypes, it was previously described (see, e.g., WO2023 / 088988 or W02023 / 011811) that antigen of these three serotypes provides adequate protection throughout the most common serotypes of S. su / s. The vaccine formulation may thus provide adequate protection against the most prevalent S. su / s bacteria. It is contemplated that the protein denoted as IdeSsuis as comprised in the vaccine formulation of the invention may provide adequate protection against a very broad and high level protection across all prevalent serotypes, in particular serotypes 1 , 2, 7 and 9.

[0045] In further embodiments of the vaccine formulation for use in a method to protect a pig against a pathogenic infection with S. su / s, the method comprises administering the vaccine formulation to the pig at an age of at most 35 days. In some preferred embodiments, the vaccine formulation is administered to the pig at an age of at most 35 days (i.e. , a piglet) by administering the vaccine to a female pig, e.g., a sow, in order to protect a pig (i.e., a piglet) against S. su / s through the intake of colostrum of the vaccinated female pig. Colostrum typically is taken within 48 hours, in particular within 24 hours after birth of the piglet in order to make sure high amounts of the maternally derived antibodies reach the circulatory system of the piglet by uptake of these antibodies through the intestinal walls. In embodiments of the vaccine formulation for use in a method to protect a pig against a pathogenic infection with S. suis, the method comprises administering the vaccine formulation to a sow in order to protect a pig through the intake of colostrum of the said sow. Said vaccine formulation can be administered to a sow by using methods known in the art. Preferably, the vaccine formulation is administered by injection (e.g., intramuscular, intradermal or subcutaneous injection), preferably intramuscular injection. It is known that the administration of a vaccine formulation comprising a protein denoted as IdeSsuis provides adequate and long protection for piglets when they take colostrum from a vaccinated sow (see, WO2019 / 193078). It is known that the administration of a vaccine formulation comprising a protein denoted as IdeSsuis can protect piglets in the period of 2- 3 weeks after weaning (i.e. in the period when the piglets have an age of between 4 -10 weeks) (see WO2019 / 193078). In particular, it has been shown to be useful to vaccinate the female pig in a period of 0-8 weeks before parturition, in particular in a period of 0-6 weeks before parturition, in both cases typically receiving the vaccine at least 1 , 2 or 3 weeks before parturition (see WO2019 / 193078).

[0046] It is recognized that it has been shown inter alia in European patent applications 17184626.4 and 17207758.8 (filed in the name of Intervet International BV) that a vaccination method consisting of administering only one shot of a vaccine comprising an IgM protease antigen of S. suis is sufficient to confer protective immunity in the vaccinated animal and thus, that a booster vaccination may be omitted to arrive at the said protective immunity. It is therefore understood that also in the present method, one shot of the vaccine is sufficient to induce protective levels of anti-S. suis antibodies in the female animal. However, vaccinating a female animal twice may increase the levels of protective antibodies that ultimately arrive in the piglets through uptake of colostrum, and such strategy does not pose serious problems in the everyday practice of keeping adult animals. Therefore, in further embodiments of the vaccine formulation for use in a method to protect a pig against a pathogenic infection with S. suis, the method comprises that the vaccine formulation is administered at least twice to the said sow before the pig takes the said colostrum. Preferably, the sow is administered twice before the pig takes the said colostrum. In such a two-shot regimen, the first (prime) vaccination is typically boosted within 8 weeks from the first administration, commonly within 6, 4 or even within 2 weeks from the first administration.

[0047] In one aspect there is provided for a method for protecting pigs against a pathogenic infection of S. suis, by administering to the pigs the vaccine formulation as provided by the current invention. Preferably, in embodiments of the method for protecting pigs against a pathogenic infection of S. suis, the vaccine formulation as provided by the current invention is administered through the intake of colostrum by said pigs. In embodiments of the combination vaccine provided herein, the further porcine vaccine comprises at least one or more E. coli antigens and / or one or more Clostridium perfringens (C. perfringens) antigens. Preferably, the further porcine vaccine comprises at least one or more E. coli antigens and one or more C. perfringens antigens. In preferred embodiments, the E. coli antigens are selected from fimbrial adherence factors and / or heat-labile toxoids, preferably wherein the fimbrial adherence factors are selected from one or more of: F4ab fimbrial adherence factor, F4ac fimbrial adherence factor, F5 fimbrial adherence factor and F6 fimbrial adherence factor, heat-labile enterotoxin (LT) and / or the C. perfringens antigens are selected from C. perfringens Type C antigens, preferably Type C beta toxoid.

[0048] In some preferred embodiments, the combination vaccine comprises the vaccine formulation comprising a protein denoted as IdeSsuis, a reductant as defined here above and a pharmaceutically acceptable carrier and a further porcine vaccine, wherein said further porcine vaccines comprises the E. coli antigens F4ab fimbrial adherence factor, F4ac fimbrial adherence factor, F5 fimbrial adherence factor, F6 fimbrial adherence factor and heat-labile enterotoxin (LT), and comprises C. perfringens antigen Type C beta toxoid.

[0049] Preferably, the combination vaccine is obtained by providing a first container comprising a lyophilized vaccine formulation according to the invention and a second container comprising the further porcine vaccine and the first container and second container are mixed right before administration to arrive at associated mixed use.

[0050] In an alternative embodiment, the vaccine formulation according to the invention and the further porcine vaccine are administered to a pig in associated non-mixed use, that is the vaccines are administered sequentially or concomitantly to the pig, but as separate formulations.

[0051] In embodiments of the method of preparing the vaccine formulation of the invention the method comprises that the homogenisation operation is a high pressure homogenization. High pressure homogenization is a mechanical operation wherein a liquid is pushed with high pressure through a narrow gap (typically in the micrometer range), thereby creating an acceleration in the liquid over a very short distance and by such establishing high shear stress, for example to reduce particle size or to lyse cells. Typical pressures use are 100-2000 bar (Dumont et al in the International Journal of Pharmaceutics 541 (2018) 117-135). The higher the amount of energy applied during the homogenisation process, the smaller the particle size or the more complete the cell lysis.

[0052] Preferably, the cells undergo a high pressure homogenisation operation at a pressure of at least 500 bar. This allows the release of adequate amounts of the antigen into the supernatant. In more preferred embodiments, the pressure during the high pressure homogenisation operation is at least 1000 bar. High pressure homogenization is a mechanical operation wherein a liquid is pushed with high pressure through a narrow gap (typically in the micrometer range), thereby creating an acceleration in the liquid over a very short distance and by such establishing high shear stress, for example to reduce particle size or to lyse cells. Typical pressures use are 100-2000 bar (Dumont et al in the International Journal of Pharmaceutics 541 (2018) 117-135). The higher the amount of energy applied during the homogenisation process, the smaller the particle size or the more complete the cell lysis. As described in WO203088988A1 (Intervet International B.V.), the higher the pressure, the more of the IgM protease antigen is released into the supernatant. Preferably, the pressure during the high pressure homogenisation operation is at least 1300 bar, such as 1400, 1500, 1600, 1700, 1800, 1900 or even at least 2000 bar.

[0053] The method of preparing the vaccine formulation of the invention comprises that in step d) a reductant having a redox potential of -80mV or less, such as reduced cysteine (which includes a cysteine salt, preferably cysteine HCI), is mixed with the supernatant comprising IdeSsuis with a pharmaceutically acceptable carrier. It will be appreciated that in preferred embodiments a sufficient amount of a reductant is added to arrive at a vaccine formulation comprising between 0.01% to 1% weight per volume (% w / v) of the reductant.

[0054] In some embodiments, the reductant is mixed sequentially with the supernatant comprising IdeSsuis with a pharmaceutically acceptable carrier. Thus, in other words, first the supernatant comprising IdeSsuis is mixed with a pharmaceutically acceptable carrier, thus obtaining a mixture of the supernatant comprising IdeSsuis and the pharmaceutically acceptable carrier, before a reductant is mixed with said obtained mixture, thereby constituting the vaccine formulation of the invention.

[0055] In alternative embodiments, the reductant is mixed concomitantly with the supernatant comprising IdeSsuis with a pharmaceutically acceptable carrier, thus obtaining a mixture of the supernatant comprising IdeSsuis, the pharmaceutically acceptable carrier and the reductant, thereby constituting the vaccine formulation of the invention.

[0056] Optionally, preferably in case a salt of a reductant is used in the method of preparing the vaccine formulation of the invention, a buffering agent is added to stabilize the pH of the vaccine formulation. Preferably, the buffering agent is added in an equivalent amount to the reductant. The pH may be set using sodium or potassium hydroxide (in case a HCI salt was used), or an equivalent agent. A skilled person is aware of suitable agents. In embodiments of the method of preparing the vaccine formulation of the invention the method further comprises: e) Lyophilizing the vaccine formulation obtained in step d).

[0057] By lyophilizing the vaccine formulation obtained in step d) a lyophilized vaccine formulation according to the invention is obtained. Optionally, prior to lyophilization, a volume of the vaccine formulation in step d) is introduced in a container, preferably a container suitable for lyophilization of the contents of said container, e.g., a glass vial. Preferably said container comprises at least 1 dose, at least 5 doses, at least 10 doses, at least 25 doses or at least 50 doses.

[0058] In one final aspect, the invention provides for kit of parts comprising the vaccine formulation of the invention. In one embodiment, the kit of parts comprises one container comprising vaccine formulation of the invention. In another embodiment, the kit of parts comprises at least two containers, one comprising the vaccine formulation of the invention and the other comprising a further porcine vaccine or a solvent, preferably an adjuvant such as an emulsion of water and oil (such as W / O, O / W, W / O / W etc).

[0059] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0060] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0061] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art. It will be understood that all details, embodiments and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments and preferences for all aspect separately.

[0062] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0063] EXAMPLES

[0064] Example 1 : Safety and efficacy test of a vaccine formulation according to the invention formulated in associated mixed use with the commercial vaccine Porcilis® ColiClos (a commercially available vaccine against E. coli and C. perfringens type C) (MSD Animal Health).

[0065] Example 2 : Stability test of several vaccine formulations formulated with various reducing agents at different concentrations.

[0066] Example 3 : Further stability tests of several vaccine formulations formulated with various reducing agents at different concentrations.

[0067] Example 1

[0068] Aim of the study

[0069] In this example, the safety and efficacy of a vaccine formulation according to the invention, formulated with and without reduced cysteine, in pregnant gilts was determined.

[0070] Study design

[0071] Thirty (pregnant) gilts were divided into 2 groups of 15 animals (see Table 1). Vaccinations (2 ml) were administered intramuscularly in the neck at 6 weeks and 2 weeks before anticipated parturition. The gilts of group 1 were vaccinated with the vaccine comprising a protein denoted as IdeSsuis without cysteine in associated mixed use with Porcilis® ColiClos (a commercially available vaccine against E. coli and C. perfringens type C) (MSD Animal Health) (right side). Four weeks later the gilts were vaccinated again using the same vaccine (left side). The gilts of group 2 were vaccinated as described above for group 1 except that the vaccine comprising a protein denoted as IdeSsuis formulated with 0.2% cysteine was used.

[0072] The IdeSsuis protein in this vaccine (as use throughout the entire examples section) is the protein as described in example 5 of WO 2023 / 011812 (according to SEQ ID NO;2 in that application), which is substantially the whole IdeSsuis protein, including the Mac-1 domain, the region linked to structural functions, the CNV region and the cell adhesion region, excluding only the signal peptide.

[0073] Table 1 : Treatment schedule

[0074] Blood samples (serum) were collected for antibody determination just before each vaccination and shortly after parturition. Within 24 hours after delivery, colostrum samples (approximately 1-2 ml) were collected from the gilts for antibody determination. The serum and colostrum samples were tested using antibody ELISA for antibody titers against the recombinant lde-14009-1 antigen (a S. su / s antigen), for antibody titers against E.coli antigens K88ab, K88ac, K99, 987P and LT and for antibody titers against C. perfringens beta toxin.

[0075] During the experiment, the gilts were observed daily for general health and behavior and for site- and systemic reactions caused by the vaccinations.

[0076] Results

[0077] No clinical abnormalities that could be attributed to the vaccines were observed. None of the vaccines induced any unacceptable site- or systemic reactions. Further, the vaccines did not induce unacceptable effects on the reproductive performance of the gilts. No differences in safety of the vaccines between the two groups were observed.

[0078] Also, the vaccinations resulted in nearly identical antibody responses in the two groups for all the antigens (see, Table 2), indicating that the addition of cysteine has no negative effect on the efficacy of the vaccine comprising a protein denoted as IdeSsuis and on the efficacy of Porcilis® ColiClos when administered to gilts in associated mixed use. Table 2: Antibody titers as determined by ELISA

[0079] Conclusion

[0080] From the data it can be concluded that there is no safety risk associated with the addition of cysteine to a vaccine comprising a protein denoted as IdeSsuis. Further, the data concerning the nearly identical antibody responses in the two separate groups for all 7 antigens indicates that the addition of cysteine does not negatively affect the efficacy of a vaccine comprising a protein denoted as IdeSsuis, nor of the efficacy of Porcilis® ColiClos. There even looks to be a tendency for a positive effect on the immune response.

[0081] Example 2

[0082] Aim of the study

[0083] In this example, the stability of a vaccine formulation according to the invention, formulated with and without several reducing agents, was determined.

[0084] Study design

[0085] Several different reducing agents (i.e., cysteine, methionine, sodium thiosulfate and vitamin E acetate) were added in different concentrations to formulations of a vaccine comprising a protein denoted as IdeSsuis. The different concentrations of reducing agents were added during formulation, just before freeze-drying, of the vaccines. The resulting vaccine formulations were tested for their respective antigenic mass in a sandwich Elisa, and were subsequently stored for 14 days at 25°C. After 14 days at 25°C the antigenic mass was tested again and compared to the antigenic mass as measured on day 0.

[0086] Results

[0087] The results of the stability test are shown in Table 3.

[0088] Table 3: Stability data of vaccine formulations

[0089] Conclusion

[0090] From the data it can be concluded that the addition of a reductant that is able to reduce di-sulfide bond formation reduces the decline of antigenic mass (as determined by a sandwich Elisa, picking up only the monomeric IgM protease molecules). In particular, cysteine is able to ensure that the formulated vaccine is stable in terms of antigenic mass for at least 2 weeks at 25°C. Based on these results it is expected that the vaccine of the invention can remain stable for 2-3 years at 2 - 8°C.

[0091] Example 3

[0092] Aim of the study

[0093] In this example, the stability of further vaccine formulations according to the invention, formulated with and without several reducing agents, was determined.

[0094] Study design

[0095] Several different reducing agents (i.e., cysteine, B-mercaptoethanol (MCE), tris-2-carboxyethyl- phosphine-hydrochloride (TCEP), reduced glutathione, and dithiothreitol (DTT) were added in different concentrations to formulations of a vaccine comprising a protein denoted as IdeSsuis. The different concentrations of reducing agents were added during formulation, just before freeze-drying, of the vaccines. The resulting vaccine formulations were tested for their respective antigenic mass and were subsequently stored for 14 days at 25°C. After 14 days at 25°C the antigenic mass was tested again and compared to the antigenic mass as measured on day 0.

[0096] Results

[0097] The results of the stability test are shown in Table 4.

[0098] Table 4: Stability data of vaccine formulations

[0099] Conclusion

[0100] It appears that it was confirmed in these further experiments that the addition of a reductant that is able to reduce di-sulfide bond formation reduces the decline of antigenic mass. In particular, cysteine, glutathione and TCEP are able to ensures that the formulated vaccine is stable in terms of antigenic mass for at least 2 weeks at 25°C. DTT also has significant stabilising properties. The perceived low stabilising effect of B-mercaptoethanol is believed to be due to a loss of the majority of this volatile compound during the freeze-dry process.

Claims

CLAIMS1. A vaccine formulation comprising a protein denoted as IdeSsuis, a pharmaceutically acceptable carrier, characterised in that the vaccine formulation comprises a reductant having a redox potential of -80 mV or less.

2. A vaccine formulation according to claim 1, characterised in that the reductant has a redox potential of -100 mV or less, such as -150 mV or less, preferably -200 mV or less.

3. A vaccine formulation according to any of the preceding claims, characterised in that the reductant is chosen from the group consisting of reduced cysteine, methionine, sodium thiosulfate, B-mercaptoethanol, tris-2-carboxyethyl-phosphine-hydrochloride (TCEP), reduced glutathione, and dithiothreitol (DTT).

4. A vaccine formulation according to any of the preceding claims, characterised in that the Vaccine formulation comprises at least 0.01% w / v of the reductant.

5. A vaccine formulation according to any of the preceding claims, characterised in that the vaccine formulation comprises at least 0.05% w / v of the reductant.

6. A vaccine formulation according to any of the preceding claims, characterised in that the vaccine formulation comprises between 0.1% w / v and 0.3% w / v of the reductant.

7. A vaccine formulation according to any of the preceding claims, characterised in that the vaccine formulation is lyophilized.

8. A vaccine formulation according to any of the preceding claims for use in a method to protect a pig against a pathogenic infection with Streptococcus suis.

9. A vaccine formulation for use according to claim 8, characterised in that the method comprises administering the vaccine formulation to the pig at an age of at most 35 days.

10. A vaccine formulation for use according to claim 8, characterised in that the method comprises administering the vaccine formulation to a sow in order to protect a pig through the intake of colostrum of the said sow.

11. A vaccine formulation for use according to claim 10, wherein the method comprises that the vaccine formulation is administered twice to the said sow before the pig takes the said colostrum.

12. A method for protecting pigs against a pathogenic infection of Streptococcus suis, by administering to the pigs the vaccine formulation of any one of claims 1 - 7.

13. A combination vaccine comprising the vaccine formulation according to any one of claims 1 - 7 and a further porcine vaccine.

14. A combination vaccine according to claim 13, wherein the further porcine vaccine comprises one or more Escherichia coli antigens and one or more Clostridium perfringens antigens, preferably wherein the Escherichia coli antigens are selected from fimbrial adherence factors and / or heat-labile toxoids, preferably wherein the fimbrial adherence factors are selected from one or more of: F4ab fimbrial adherence factor, F4ac fimbrial adherence factor, F5 fimbrial adherence factor and F6 fimbrial adherence factor and / or wherein the Clostridium perfringens antigens are selected from Clostridium perfringens Type C antigens, preferably Type C beta toxoid.

15. A method of preparing the vaccine formulation according to any one of claims 1 - 7, characterised in that the method comprises the steps of: a) recombinantly expressing a protein denoted as IdeSsuis in E. coli bacteria; b) subjecting the E. coli bacteria to a homogenisation operation to induce lysis of the E. coli bacteria and release of IdeSsuis into a supernatant of the lysate; c) separating the supernatant from a pellet of the lysate; and d) mixing the supernatant comprising IdeSsuis with a pharmaceutically acceptable carrier, a reductant having a redox potential of -80mV or less, and optionally a buffering agent to constitute the vaccine formulation.

16. The method according to claim 15, characterised in that the method further comprises: e) lyophilizing the vaccine formulation obtained in step d).

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