Method for producing a vaccine against streptococcus suis and the vaccine

Abstract

The present invention relates to a method for producing a vaccine for protecting pigs against pathogenic infection with Streptococcus suis, comprising recombinantly expressing an IgM protease antigen in E. coli, subjecting the E. coli to a high pressure homogenization operation at a pressure of at least 500 bar to induce cell lysis of the E. coli and release of the IgM protease antigen into a lysate supernatant, separating the supernatant from the pellet, and mixing the supernatant containing the IgM protease antigen with a pharma- ceutically acceptable carrier to produce a vaccine. The present invention also relates to a vaccine produced by the method.

Description

[Technical field]

[0001] General Field of the Invention The present invention relates generally to a method for producing a vaccine for protecting pigs against pathogenic infection with Streptococcus suis bacteria, the vaccine being based on an IgM protease antigen of Streptococcus suis recombinantly expressed in E. coli. [Background technology]

[0002] 2. Background of the Invention Streptococcus suis (S. suis) is one of the major pathogens of infectious bacterial diseases in pigs. The pathogen can cause a variety of clinical syndromes, including meningitis, arthritis, pericarditis, polyserositis, septicemia, pneumonia, and sudden death. Streptococcus suis is a Gram-positive facultative anaerobic coccus that was originally defined as Lancefield groups R, S, R / S, or T. A new classification system was subsequently proposed based on type-specific capsular polysaccharide antigens present in the cell wall. This resulted in a system containing 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 is recognized that capsular serotypes are poor virulence markers. Therefore, alternative systems have been developed to aid in understanding the epidemiology of S. suis infections and the biological relevance of serotyping approaches. This is so-called multilocus sequence typing (MLST) and has been described by King et al., 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. Among them, the ST complexes ST1, ST27, and ST87, each of which contains multiple sequence types, are predominant in the population.See also the Streptococcus suis MLST website at the University of Oxford (https: / / pubmlst.org / ssuis / ) (Jolley et al., Wellcome Open Res 2018,3:124) (funded by the Wellcome Trust), which references King et al. and allows easy sequence typing of any Streptococcus suis strain.

[0003] Control of S. suis in pig populations appears to be difficult. S. suis is a resident opportunistic pathogen of pigs. The immune system does not appear to be activated at every opportunity for infection. Moreover, S. suis is a well-encapsulated pathogen, evading the host immune system by using offensive and defensive virulence factors. These characteristics together make it difficult to develop an effective vaccine to combat this important pathogen. A review article reviewing existing and experimental vaccines against S. suis was published some years ago (Mariela Segura: “Streptococcus suis vaccines: candidate antigens and progress, in Expert Review of Vaccines, Volume 14, 2015, Issue 12, pp. 1587-1608). In this review, clinical information and experimental data are summarized and compared, and an overview of the current status of vaccine development against S. suis is presented.

[0004] In the past few years, a comprehensive list of antigenic or immunogenic S. suis molecules has been reported, most of which were found by immunoproteomics using convalescent sera from infected pigs or humans and / or immune sera generated in the laboratory. WO2015 / 181356 (IDT Biologika GmbH) shows that the IgM protease antigen (either the entire protein or the highly conserved Mac-1 domain, which represents only about 35% of the entire protein) can induce a protective immune response in piglets by vaccination with the IgM protease antigen, optionally combined with a primary vaccination containing a bacterin. In the '356 patent application, it is suggested that since the IgM protease antigen is highly conserved across most, if not all, S. suis serotypes, especially the most prevalent serotypes 1, 2, 7 and 9, it could be used to achieve broad cross-protection between S. suis serotypes, especially serotypes 1, 2, 7 and 9. WO2017 / 005913 (Intervacc AB) confirms that the IgM protease is highly conserved across the different Streptococcus suis serotypes.

[0005] In the art, IgM protease antigens have been produced using the technique published in Seele et al., Journal of Bacteriology p. 930-940, March 2013, Volume 195, Number 5 ("Identification of a Novel Host-Specific IgM Protease in Streptococcus Suis"), in which the IgM protease antigen is recombinantly expressed in Escherichia coli and stimulated with Ni under native conditions. 2+ -nitrilotriacetic acid affinity chromatography. In this way, the IgM protease antigen can be obtained in a highly purified form, but the yield is relatively low. The purified antigen can be mixed with a pharma- ceutical acceptable carrier to obtain a vaccine that protects against pathogenic infections with Streptococcus suis. Summary of the Invention

[0006] Object of the invention It is an object of the present invention to provide an alternative method for producing vaccines containing IgM protease antigens.

[0007] Overview of the Invention In order to achieve the object of the present invention, a method for preparing the vaccine described in the above "General Field of the Invention" section has been devised in the present invention, which comprises recombinantly expressing an IgM protease antigen of Streptococcus suis in E. coli, subjecting the E. coli to a high pressure homogenization operation at a pressure of at least 500 bar to induce cell lysis of the E. coli and release of the IgM protease antigen into the resulting lysate supernatant, separating the supernatant from the lysate pellet, and mixing the supernatant containing the IgM protease antigen with a pharma- ceutically acceptable carrier to produce the vaccine.

[0008] It was found that cell lysis of E. coli is necessary to obtain a large amount of IgM protease. Apparently, the IgM protease antigen is not released by the bacteria into the supernatant. However, it was also found that the cells must be subjected to a high-pressure homogenization operation at a pressure of at least 500 bar in order for a sufficient amount of antigen to actually be released into the supernatant and not remain bound to the E. coli cell debris. Releasing a large amount of antigen into the supernatant has the important advantage that this supernatant, free of (significant amounts of) cell debris, can be very conveniently formulated as a source of antigen, without the need for high affinity purification on columns, after filtration, further clarification, inactivation, concentration, etc. as necessary. Although such (or other) purification can be performed, it was found that the presence of other proteins and small molecules derived from E. coli itself is not a major problem for the safety and efficacy of the vaccine. Therefore, when using the method of the present invention, the purification step can be omitted.

[0009] The method of the present invention is easy to carry out and produces suitable vaccines in high yields. The present invention is also embodied in a vaccine containing the IgM protease antigen of Streptococcus suis obtained using this method. This vaccine differs from known vaccines in that a significant amount of E. coli proteins (particularly 5%, 10%, 15%, 20%, 25%, 30%, 50% or even more of the total amount of proteins, i.e., total weight) and other E. coli compounds (e.g., polysaccharides) may be present in the vaccine.

[0010] definition A vaccine is a composition suitable for application to a subject, comprising an immunologically effective amount (i.e., an amount capable of stimulating the target subject's immune system sufficiently to at least reduce the negative effects of a wild-type microbial challenge) of one or more antigens, typically in combination with a pharma- ceutically acceptable carrier, which, upon administration to a subject, induces an immune response to treat the infection, i.e., to help prevent, ameliorate, or cure the infection or a disease or disorder resulting from that infection.

[0011] Protecting against pathogenic infection by a microorganism is the same as achieving protective immunity, i.e., helping to prevent, ameliorate or cure pathogenic infection by that microorganism or a disorder resulting from that infection, e.g., preventing or reducing actual infection or one or more clinical signs resulting from pathogenic infection by a pathogen.

[0012] The IgM protease antigen of Streptococcus suis is an enzyme that specifically degrades porcine IgM (not porcine IgG or porcine IgA; Seele et al., Journal of Bacteriology, 2013, 195 930-940; and Vaccine 33:2207-2212; 5 May 2015), a protein designated as IdeSsuis, or an immunogenic portion thereof (typically having a length of at least about 30-35% of the full-length enzyme). The entire enzyme has a mass of about 100-125 kDa, which corresponds to about 1000-1150 amino acids, the size depending on the serotype of Streptococcus suis. In WO2015 / 181356, several sequences are presented that represent the IgM protease antigen of Streptococcus suis, namely 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 (designated as the Mac-1 domain, i.e. amino acids 80 to 414 of SEQ ID NO:7). Other examples of immunogenic parts of the full-length enzyme are described in WO2017 / 005913. In particular, the IgM protease may be a protease according to SEQ ID NO:1 of WO2015 / 1818356 or a protein having at least 70%, in particular 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% to 100% sequence identity in the overlapping regions. Amino acid sequence identity can be confirmed by the BLAST program using the blastp algorithm with default parameters. The IgM proteases of various serotypes of Streptococcus suis are expected to have sequence identities of more than 70%, particularly 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% to 100%.For example, artificial proteins produced to optimize the yield in recombinant production systems of the antigen may have a lower amino acid sequence identity compared to the whole enzyme, for example 85%, 80%, 75%, 70% or even 60% amino acid sequence identity, while maintaining the required immunogenic function, and are understood to be Streptococcus suis IgM protease antigens within the meaning of the present invention.

[0013] A pharmaceutically acceptable carrier is a biocompatible medium, i.e., a medium that can present an antigen to the immune system of a subject after administration of a composition containing the carrier, and that does not induce significant adverse reactions in a treated subject after administration. Such a pharmaceutically acceptable carrier can be, for example, a liquid containing water and / or any other biocompatible solvent, or a solid carrier, for example, a solid carrier commonly used to obtain lyophilized vaccines (based on sugars and / or proteins), which may optionally contain an immune stimulant (adjuvant). Optionally, other substances such as stabilizers, viscosity adjusters, adjuvants or other ingredients are added depending on the intended use or required properties of the corresponding vaccine.

[0014] The supernatant is the liquid present above the solid residue after crystallization, precipitation, centrifugation or other treatment.

[0015] A pellet is an insoluble precipitate, for example, after centrifugation or other sedimentation methods.

[0016] High pressure homogenization is a mechanical operation that forces a liquid through a narrow gap (typically in the micrometer range) at high pressure, which causes the acceleration of the liquid over a very short distance, which generates high shear stresses that, for example, reduce particle size or lyse cells. Typical pressures used are 100-2000 bar (Dumont et al., International Journal of Pharmaceutics 541 (2018) 117-135). The greater the amount of energy applied during the homogenization process, the smaller the particle size and the more complete the cell lysis.

[0017] Microfluidization is a form of high pressure homogenization, which works by sending liquid through a microchannel into an interaction chamber so that two fine jets oriented at right angles to each other are generated, typically at high pressures of up to 2000 bar. When these two microstreams collide, a sudden pressure drop occurs, and homogenization occurs as a result of the turbulence, cavitation and shear effects that occur upon collision. Increasing the number of passes through the microfluidizer improves homogenization, but more than three passes has little effect. Equipment for microfluidization is available from Microfluidics™, Westwood, MA, USA.

[0018] The French Pressure Cell Press (also called the "French Press") is a device used in biological experiments to break the cell membrane of cells by forcing the cells through a narrow valve under high pressure. It can break the cell wall without disturbing the cell nucleus. The French Press was invented by Charles Stacy French at the Carnegie Institution of Washington. The press uses an external hydraulic pump to drive a piston inside a larger cylinder that contains the liquid sample. The highly pressurized sample is then squeezed through a needle valve. As the sample passes through the valve, the fluid is subjected to shear stress and decompression, resulting in cell disruption.

[0019] The complete IgM protease antigen of Streptococcus suis is an antigen that includes at least the Mac-1 domain, the structure-function related regions, the CNV region, and optionally the cell adhesion region (see Example 1 for the identification of these regions in the Streptococcus suis genome). It can be considered a complete IgM protease antigen because the signal peptide is not believed to be present even in the naturally occurring (i.e., wild-type) secreted enzyme, and the cell adhesion region is not believed to be essential for its function as a protease.

[0020] Additional Embodiments of the Invention In a first additional embodiment of the method of the present invention, the pressure during the high pressure homogenization operation is at least 1000 bar. In this way, more IgM protease antigens are released into the supernatant. Preferably, the pressure during the high pressure homogenization operation is at least 1300 bar, such as 1400, 1500, 1600, 1700, 1800, 1900 or even at least 2000 bar.

[0021] In another embodiment of the method according to the invention, the device used to perform the high pressure homogenization operation is a French pressure cell press or a microfluidizer. These devices have been found to be particularly suitable for carrying out the method according to the invention. Preferably, a microfluidizer is used.

[0022] In yet another embodiment of the method according to the invention, the IgM protease antigen is a complete IgM protease antigen. It has been found that antigens of this type, which contain at least the Mac-1 domain, the structure-function related regions, the CNV region and optionally the cell adhesion region (see Example 1 for the identification of these regions in the genome of S. suis), can be expressed at high levels, are easily released into the supernatant and are very suitable as vaccine antigens. Preferably, the complete IgM protease antigen is of S. suis bacteria of serotype 1, 2 or 7. Although the recombinant expression of the antigen itself is not serotype dependent, it has been found that antigens of these three serotypes provide moderate protection across the various serotypes of S. suis. In this regard, please refer to European patent application EP21189283.1 (title: “A vaccine for protection against Streptococcus suis of various serotypes”), filed on August 3, 2021 as a priority application with the European Patent Office in the name of Intervet International BV.

[0023] The invention will now be described in more detail by way of the following specific examples.

[0024] Working Example Example 1: Structural analysis of the genome of Streptococcus suis Example 2: Preparation of a vaccine containing a whole IgM protease antigen Example 3: Protective Efficacy of Vaccines

[0025] Example 1 In this example, an analysis of the genome of Streptococcus suis, i.e. the part that codes for IgM protease, is described to show how this part of the genome is structured. For this purpose, the genome of the serotype 2 bacterium Streptococcus suis is used, which is known from WO2015 / 181356 and published in that patent application as SEQ ID NO: 1. Said sequence is reproduced in the sequence listing of this patent as SEQ ID NO: 1. A sequence similarity search using protein annotations (PDBSum and InterPro) plus Needleman-Wunsch alignments (Needleman et al., 1970; Laskowski et al., 1997; Apweiler et al., 2000; see default settings) shows the structure of the IgM protease genome, in which five regions are identified.

[0026] Region 1 (Met1 to Thr34): signal sequence from position 1; Region 2 (Val35-Glu426): Mac-1 domain with putative hydrolase activity; Region 3 (Thr427-Pro687): a region related to structural functions (e.g., those involved in proper folding) and substrate binding; Region 4 (Thr688-Ser919): Four repeats (1 * {Thr688~Ser744}, 2 * {Thr745~Ser801}, 3 * {Thr802~Ser858}, 4 * {Thr859~Ser919}), a so-called CNV region (copy number variation region) where a section of the genome is repeated; Domain 5 (Thr920 to Lys1141): Contains a putative transmembrane domain (cell adhesion domain) that exhibits cell wall anchoring function.

[0027] The structures of other serotypes of Streptococcus suis bacteria are nearly identical, with the most notable difference being the number of repeats in the CNV region.

[0028] Example 2 In this example, we demonstrate a method for producing a vaccine containing an IgM protease antigen. Using the method described by Seele et al. (2013; see above), the entire IgM protease genes of serotype 2 and serotype 7 Streptococcus suis strains were cloned into E. coli using the BL21-AI(DE3) plasmid. Arabinose in combination with lactose was used as inducer.

[0029] E. coli cells were cultured in an animal component-free medium containing lactose, glucose, glycerol, yeast extract, yeastolate, NaCl, KH2PO4 and Na2HPO4 x 2H2O. First, cells were grown to mid-exponential growth phase using a preculture in a shake flask with gentle shaking at 37°C. The same medium was then used to inoculate a 15L culture-scale laboratory-scale fermenter with cells at an inoculum rate of 1%. pH was controlled at 7.0 using 4M NaOH and 4M acetic acid solutions. Dissolved oxygen (pO2) was controlled at 50% using cascade control of agitation speed, airflow and pure oxygen. Temperature was controlled at 37°C. Arabinose was added as an inducer as soon as glucose in the medium was exhausted (determined using a CEDEX analyzer). Cultivation was continued for another 3 hours to obtain a reasonable IgM protease protein concentration.

[0030] After this, E. coli cells were harvested from the culture and stored unconcentrated in refrigerated medium or 0.04M PBS buffer or concentrated up to 4-fold using centrifugation. The cells were then disrupted using a Microfluidics at 30.000 PSI (2068 bar) or a French Press Homogenizer (range 600-2000 bar). Finally, all fractions after cell disruption were centrifuged and the supernatant and pellet were separated and inactivated using BPL (beta-propiolactone). Protein yields were measured using SDS gels with a known BSA series as a reference. The IgM protease was not further purified.

[0031] To investigate the effect of pressure during cell disruption, several experiments were performed to disrupt E. coli cells (serotype 2) at various pressures in a microfluidizer or French press. Table 1 shows the relationship between the pressure applied during cell disruption and the recovery of IgM protease protein in the supernatant after centrifugation following cell disruption. The recovery is calculated by dividing the IgM protease protein concentration in the supernatant after cell disruption and centrifugation by the IgM protease protein concentration after cell disruption and before centrifugation (i.e., total percentage) and multiplying by 100%. No significant effect of the type of device was found, only the effect of the applied pressure. [Table 1]

[0032] Example 3 In this Example 3, the protective efficacy of a vaccine produced using an antigen obtained using the method of Example 2 is shown.

[0033] Study design Vaccines were formulated by mixing the supernatant obtained according to Example 2 with the adjuvant X-Solve (oil-in-water) available from MSD Animal Health to a concentration of 35 μg IgM protease per ml for the first vaccine and 3.5 μg IgM protease per ml for the second vaccine (with the same amount of oil adjuvant). Thirty three-week-old piglets were used in the study. The piglets were allocated to three groups of 10 piglets each (different litters were distributed equally in each group). Groups 1 and 2 were vaccinated intramuscularly twice with different vaccines at 3 and 5 weeks of age. Group 1 was vaccinated with 2 ml of the first vaccine per vaccination (i.e. 70 μg antigen per vaccination) and group 2 was vaccinated with 2 ml of the second vaccine per vaccination (i.e. 7 μg IgM protease antigen per dose). Group 3 remained as unvaccinated challenge controls. At 7 weeks of age, piglets were transferred to the challenge room and challenged immediately. No acclimation period was allowed between transfer and challenge to simulate natural stress. After challenge, piglets were observed daily for clinical signs of S. suis infection (e.g. depression, motor impairment and / or neurological signs) and scored using a conventional scoring system ranging from 0 (no signs) to 3 (severe cases). Severely affected animals were euthanized and examined post-mortem. At the end of the study (11 days after challenge), all surviving pigs were euthanized and examined post-mortem. Serum was taken for antibody measurements immediately prior to vaccination and challenge. Heparinized blood was taken for re-isolation of the challenge strain at regular time points before and after challenge.

[0034] result None of the vaccines induced unacceptable local or systemic reactions and therefore can be considered safe. On the day of vaccination (3 weeks of age), most pigs had low to moderate maternal antibody titers. After vaccination, all vaccine groups showed antibody responses (data not shown). The results for different parameters after challenge are shown in Table 2 below (survival time is shown in days). [Table 2]

[0035] conclusion The results demonstrate that the vaccine induced protection in piglets against challenge with virulent S. suis two weeks after the second vaccination. A vaccine dose of as little as 7 μg was able to provide sufficient protective efficacy.

Claims

1. 1. A method for producing a vaccine for protecting pigs against pathogenic infection with Streptococcus suis, comprising: Recombinant expression of the IgM protease antigen of Streptococcus suis in E. coli; subjecting the E. coli to high pressure homogenization at a pressure of at least 500 bar to induce cell lysis of the E. coli and release of the IgM protease antigen into the lysate supernatant; Separating the supernatant from the lysate pellet; - mixing the supernatant containing the IgM protease antigen with a pharmaceutically acceptable carrier to produce the vaccine; The manufacturing method as described above.

2. 2. The process of claim 1, wherein the pressure during the high-pressure homogenization operation is at least 1000 bar.

3. 2. The process of claim 1, wherein the pressure during the high-pressure homogenization operation is at least 1300 bar.

4. 2. The process of claim 1, wherein the pressure during the high-pressure homogenization operation is at least 2000 bar.

5. 2. The method of claim 1, wherein the apparatus used to perform the high-pressure homogenization operation is a French pressure cell press or a microfluidizer.

6. The method of claim 1, wherein the IgM protease antigen is a complete IgM protease antigen.

7. 7. The method of claim 6, wherein the complete IgM protease antigen is from serotype 1, 2 or 7 Streptococcus suis bacteria.

8. A vaccine comprising an IgM protease antigen of Streptococcus suis obtained by the method of claim 1.

9. 9. The vaccine of claim 8, wherein the vaccine comprises at least 5% native E. coli proteins relative to the IgM protease antigen.