Method for obtaining vaccine against streptococcussuis and said vaccine

Abstract

FIELD: biotechnology.SUBSTANCE: method for producing a vaccine for protecting pigs from the pathogenic infection Streptococcus suis is described, comprising: recombinantly expressing an IgM protease-based antigen of Streptococcus suis in E. coli bacteria; homogenizing the E. coli bacteria under high pressure of at least 500 bar to induce lysis of the E. coli bacteria and release the IgM protease-based antigen into the lysate supernatant; separating the supernatant from the lysate sediment; mixing the supernatant containing the IgM protease-based antigen with a pharmaceutically acceptable carrier to produce a vaccine. Also a vaccine containing an IgM protease-based antigen from Streptococcus suis obtained by the described method is disclosed. According to the invention it contains at least 5% of native E. coli proteins in relation to the IgM protease-based antigen.EFECT: expanding the range of means for producing vaccines to protect pigs from the pathogenic infection Streptococcus suis.8 cl, 2 tbl, 3 ex

Description

[0001] FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The invention generally relates to a method for producing a vaccine for protecting pigs from a pathogenic infection caused by the bacterium Streptococcus suis, which is based on an antigen that is an IgM-specific protease (IgM protease) of Streptococcus suis, recombinantly expressed in E. coli bacteria.

[0003] STATE OF THE ART

[0004] Streptococcus suis (S. suis) is one of the major etiologic agents of infectious bacterial diseases in pigs. The pathogen can cause a variety of clinical syndromes, including meningitis, arthritis, pericarditis, polyserositis, sepsis, pneumonia, and sudden death. S. suis is a Gram-positive, facultatively anaerobic coccus, previously classified as Lancefield groups R, S, R / S, or T. A new typing system based on type-specific capsular polysaccharide antigens located in the cell wall was later proposed. This has led to the development of a system comprising 35 serotypes (Rasmussen and Andresen, 1998, "165 rDNA sequence variations of some Streptococcus suis serotypes", Int. J. Syst. Bacteriol. 48, 1063-1065), of which the most common at present, especially in Europe, are serotypes 1, 2, 7 and 9. However, it is recognized that the capsular serotype is a poor marker of virulence.Therefore, an alternative system was developed to understand the epidemiology of S. suis infection and the biological significance of the serotyping approach, i.e., the so-called multilocus sequence typing (MLST), described by King et al. in the Journal of Clinical Microbiology, Oct. 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 this study, 92 sequence types were identified, of which the ST complexes ST1, ST27, and ST87, each containing several sequence types, were dominant in the population. See also the MLST Streptococcus suis website (https: / / pubmlst.org / ssuis / ) maintained by the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 (Wellcome Trust-funded site), which cites the King et al. paper., allowing easy identification of the sequence type for any strain of Streptococcus suis.

[0005] Streptococcus suis control in swine herds is challenging. Streptococcus suis is a commensal opportunistic pathogen in pigs. Activation of the immune system does not appear to occur with each infection. Furthermore, Streptococcus suis is a well-encapsulated pathogen and utilizes an arsenal of virulence factors to evade the host immune system. Taken together, these characteristics have questioned the feasibility of developing effective vaccines against this important pathogen. Several years ago, a review article was published examining existing and research-based vaccines against Streptococcus suis (Mariela Segura: "Streptococcus suis vaccines: candidate antigens and progress," in Expert Review of Vaccines, Volume 14, 2015, Issue 12, pages 1587-1608).The analysis and comparison of clinical information with experimental data presented in this review provide an overview of the state of the art in the development of vaccines against Streptoccus suis.

[0006] Over the past few years, an extensive list of antigenic or immunogenic molecules of Streptococcus suis has been published, most of which were identified by immunoproteomics using sera obtained from convalescent infected pigs or humans and / or immune sera generated in vitro. International application WO2015 / 181356 (IDT Biologika GmbH) demonstrated that IgM protease-based antigens (either the whole protein or the highly conserved Mac-1 domain, which constitutes only approximately 35% of the full-length protein) can elicit a protective immune response in piglets when vaccinated with an IgM protease-based antigen, optionally in combination with a primary vaccine containing a bacterin.The '356 patent application suggests the possibility of using an IgM protease-based antigen, due to its high conservation across most, if not all, Streptococcus suis serotypes, in particular the most common serotypes 1, 2, 7 and 9, to achieve broad cross-protection among Streptococcus suis serotypes, in particular among serotypes 1, 2, 7 and 9. The high conservation of IgM protease across different Streptococcus suis serotypes was confirmed in the international application WO2017 / 005913 (Intervacc AB).

[0007] In the present art, an IgM protease-based antigen is produced by the method published by Seele et al. in the Journal of Bacteriology p. 930-940, March 2013, Volume 195, Number 35 ("Identification of a Novel Host-Specific IgM Protease in Streptococcus Suis"). In this method, an IgM protease-based antigen is recombinantly expressed in E. coli bacteria and purified by Ni affinity chromatography. 2+-nitrilotriacetic acid under native conditions. This allows for the production of a highly purified IgM protease-based antigen, albeit in relatively low yield. The purified antigen can be mixed with a pharmaceutically acceptable carrier to produce a vaccine to protect against the pathogenic infection Streptococcus suis.

[0008] PURPOSE OF THE INVENTION

[0009] The aim of the invention is to create an alternative method for producing a vaccine containing an antigen based on IgM protease.

[0010] SUMMARY OF THE INVENTION

[0011] To achieve the objective of the invention, a method for producing a vaccine was developed as described above in the section "Background of the invention." The method comprises recombinantly expressing an IgM protease-based antigen of Streptococcus suis in E. coli bacteria, high-pressure homogenizing the E. coli bacteria at a pressure of at least 500 bar to cause lysis of the E. coli bacteria and releasing the IgM protease-based antigen into the supernatant of the resulting lysate, separating the supernatant from the lysate pellet and mixing the supernatant containing the IgM protease-based antigen with a pharmaceutically acceptable carrier to obtain a vaccine.

[0012] It was found that lysis of E. coli cells was necessary to obtain high amounts of IgM protease. Apparently, the IgM protease-based antigen was not released by the bacteria into the supernatant. However, it was also found that the cells must be homogenized under high pressure, at least 500 bar, to ensure that sufficient antigen was released into the supernatant and that it did not remain bound to E. coli cell debris. An important advantage of releasing large amounts of antigen into the supernatant is that vaccine preparation is very simple, thanks to the use of this supernatant directly as the antigen source, which does not contain (significant amounts) of cellular debris, optionally after filtration, further clarification, inactivation, concentration, etc., without the need for purification using high-affinity columns.While this (or other) purification method could be used, it was found that the presence of other proteins and small molecules originating from the E. coli bacteria themselves does not pose a fundamental problem for the safety and efficacy of the vaccine. Therefore, the purification step can be omitted when using the method of the present invention.

[0013] The method of the present invention is simple to implement, provides high yields, and ensures the production of an adequate vaccine. The invention also relates to a vaccine containing an antigen based on the IgM protease of Streptococcus suis, obtained by this method. This vaccine differs from the known vaccine in that it contains a significant amount of E. coli proteins (in particular, more than 5%, 10%, 15%, 20%, 25%, 30%, 50%, or even more of the total amount, i.e., protein weight), while other E. coli compounds (e.g., polysaccharides) may also be present in the vaccine.

[0014] DEFINITIONS

[0015] A vaccine is a structure suitable for use by a subject that contains one or more antigens in an immunologically effective amount (i.e., capable of stimulating the immune system of the target subject sufficiently to at least reduce the adverse effects of infection with wild-type microorganisms) usually in combination with a pharmaceutically acceptable carrier and which, when administered to the subject, induces an immune response to treat an infection, i.e., helps prevent, alleviate, or cure an infection or any disease or disorder resulting from that infection.

[0016] Protection against a pathogenic infection caused by a microorganism is similar to achieving protective immunity, i.e., helping to prevent, alleviate, or treat a pathogenic infection caused by that microorganism or a disorder resulting from that infection, such as to prevent or alleviate an actual infection or one or more clinical signs resulting from infection with the pathogen.

[0017] 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, in Journal of Bacteriology, 2013, 195 930–940; and in Vaccine 33:2207–2212; 5 May 2015), a protein designated IdeSsuis, or an immunogenic portion thereof (usually at least approximately 30–35% of the length of the full-length enzyme). The mass of the full-length enzyme is approximately 100–125 kDa, corresponding to approximately 1000–1150 amino acids; the size varies with the serotype of S. suis. WO 2015 / 181356 discloses several sequences representing an antigen based on the IgM protease 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 representing the immunogenic portion of the full-length enzyme (designated as the Mac-1 domain, i.e., amino acids 80-414 of SEQ ID NO:7). Other examples of immunogenic portions of the full-length enzyme are disclosed in WO2017 / 005913.In particular, the IgM protease may be a protease with SEQ ID NO: 1 of WO2015 / 1818356 or a protein with a sequence identical in overlapping regions by 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%. The identity of the amino acid sequence can be established using the BLAST program using the blastp algorithm with default parameters. The IgM protease sequences of Streptococcus suis of different serotypes are expected to be more than 70% identical, in particular they are expected to be 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% and up to 100% identical.An artificial protein, for example, designed to optimize yield in a recombinant system for producing an antigen, may have an amino acid sequence with a lower sequence identity to the full-length enzyme, for example 85%, 80%, 75%, 70% or even 60%, provided that the necessary immunogenic function is retained, and in the context of the present invention is considered to be an antigen based on the IgM protease of Streptococcus suis.

[0018] A pharmaceutically acceptable carrier is a biocompatible medium, i.e., a medium that, upon administration, does not cause significant adverse reactions in the subject receiving the treatment and that is capable of presenting the antigen to the subject's immune system after administration of a composition containing 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 one commonly used for the production of lyophilized vaccines (based on sugars and / or proteins), optionally containing immunostimulating agents (adjuvants). Other substances, such as stabilizers, viscosity modifiers, adjuvants, or other components, are optionally added, depending on the intended use or the desired properties of the respective vaccine.

[0019] Supernatant is the liquid remaining above the solid precipitate after crystallization, sedimentation, centrifugation or other process.

[0020] The precipitate is an insoluble residue, such as after centrifugation or other sedimentation method in a liquid.

[0021] High-pressure homogenization is a mechanical operation in which a liquid is forced under high pressure through a narrow gap (usually in the micrometer range), thereby creating acceleration in the liquid over a very short distance and high shear stress, for example, to reduce particle size or lyse cells. Typical pressures used range from 100 to 2000 bar (Dumont et al. in International Journal of Pharmaceutics 541 (2018) 117–135). The higher the amount of energy applied during homogenization, the smaller the particle size or the more complete the cell lysis.

[0022] Microfluidization is a form of high-pressure homogenization that involves forcing liquid through microchannels into an interaction chamber, creating two thin jets directed at each other at right angles at high pressures, typically up to 2000 bar. When the two microstreams collide, the pressure drops sharply, and homogenization occurs as a result of the turbulence, cavitation, and shear effects generated by the impact. Increasing the number of passes through the microfluidizer improves homogenization, but after three passes, there is virtually no improvement. Microfluidization devices are available from Microfluidics. ТМ , Westwood, MA, USA.

[0023] The French cell disruption press (also called a "French press") is a device used in biological experiments to disrupt the plasma membrane of cells by forcing them through a narrow valve under high pressure. It is capable of rupturing cell walls while leaving the cell nucleus intact. The French press was invented by Charles Stacy French of the Carnegie Institution of Washington. The press uses an external hydraulic pump to drive a piston inside a larger cylinder containing a liquid sample. The sample is then forced through a needle valve under high pressure. As the sample passes through the valve, the liquid experiences shear stress and decompression, causing cell disruption.

[0024] The full-length IgM protease-based antigen of Streptococcus suis is an antigen containing at least the Mac-1 domain, the structural function-associated region, the CNV region, and, optionally, the cell adhesion region (see Example 1 for identification of these regions in the Streptococcus suis genome). It can be considered a whole IgM protease-based antigen, since the signal peptide is almost always absent from the naturally occurring (i.e., wild-type) secreted enzyme, and the cell adhesion region is not considered essential for its function as a protease.

[0025] ADDITIONAL EMBODIMENTS OF THE INVENTION

[0026] In a first additional embodiment of the method according to the invention, the pressure during high-pressure homogenization is at least 1000 bar. This achieves a higher release of the IgM protease-based antigen into the supernatant. Preferably, the pressure during high-pressure homogenization is at least 1300 bar, such as 1400, 1500, 1600, 1700, 1800, 1900, or even at least 2000 bar.

[0027] In another embodiment of the method of the invention, the device used to perform high-pressure homogenization is a French press for cell disruption under pressure or a microfluidization device. These devices have been found to be particularly suitable for carrying out the method of the invention. A microfluidization device is preferred.

[0028] In another embodiment of the method of the invention, the IgM protease-based antigen is a whole IgM protease-based antigen. It has been found that this type of antigen, which includes at least the Mac-1 domain, the region associated with structural functions, the CNV region, and, optionally, the cell adhesion region (see Example 1 for the identification of these regions in the Streptococcus suis genome), can be expressed at high levels, is easily released into the supernatant, and is excellent for use as a vaccine antigen. Preferably, the IgM protease-based antigen belongs entirely to Streptococcus suis serotype 1, 2, or 7. Although recombinant expression of the antigen as such is not serotype-dependent, it has been found that the antigen of these three serotypes provides adequate protection against various serotypes of Streptococcus suis. For more information, see European Patent Application EP21189283.1, filed on 3 August 2021 in the name of Intervet International BV as a priority application with the European Patent Office, under the title “A vaccine for protection against Streptococcus suis of various serotypes”.

[0029] The invention is now described in more detail using the following specific examples.

[0030] EXAMPLES

[0031] Example 1: Structural analysis of the genome of Streptococcus suis.

[0032] Example 2: Production of a vaccine containing a whole antigen based on IgM protease.

[0033] Example 3: Protective effect of the vaccine.

[0034] Example 1

[0035] This example presents an analysis of the Streptococcus suis genome, i.e. the part encoding IgM protease, to show the structure of this part of the genome. For this purpose, the genome of Streptococcus suis serotype 2, known from application WO 2015 / 181356 and presented in this patent application as SEQ ID NO: 1, was used. This sequence is also included in the sequence listing of the present patent as SEQ ID NO: 1. A search for sequence similarity by alignment with the Needleman-Wunsch method (see Needleman et al 1970, Laskowski et al 1997, Apweiler et al 2000; default settings) in addition to protein annotation (PDBSum and InterPro) revealed the structure of the IgM protease genome, in which 5 regions can be distinguished:

[0036] Region 1 (Met1 - Thr34): signal sequence, starts from position 1;

[0037] Region 2 (Val35 - Glu426): Mac-1 domain with predicted hydrolase activity;

[0038] Region 3 (Thr427 - Pro687): A region that is associated with structural functions (e.g., involved in proper folding) and substrate binding.

[0039] Region 4 (Thr688 - Ser919): a region consisting of 4 repeats (1*{Thr688 - Ser744}, 2*{Thr745 - Ser801}, 3*{Thr802 - Ser858}, 4*{Thr859 - Ser919}), which are similar to known protein sequences with hydrolase activity. It is the so-called CNV (copy number variation region) region, in which regions of the genome are repeated; and

[0040] Region 5 (Thr920 - Lys1141): Contains a putative transmembrane region, indicating an anchoring function in the cell wall (cell adhesion region).

[0041] The structures of Streptococcus suis bacteria and other serotypes are largely similar, the most notable difference being the number of repeats in the CNV region.

[0042] Example 2

[0043] This example demonstrates a method for producing a vaccine containing an IgM protease-based antigen. Full-length IgM protease genes from Streptococcus suis serotype 2 and serotype 7 were cloned into Escherichia coli (E. coli) using the method described by Seele et al. (2013; see above) using the BL21-AI(DE3) plasmid. Arabinose in combination with lactose was used as an inducer.

[0044] E. coli cells were cultured in an animal component-free medium containing lactose, glucose, glycerol, yeast extract, yeast tolate, NaCl, KH2PO4, and Na2HPO4×2H2O. The pre-grown culture was first used in a shake flask, which was gently shaken at 37°C to grow the cells to the mid-exponential growth phase. Then, the cells were inoculated with 1% of the inoculum in a 15 L laboratory fermenter with the same medium. The pH was maintained at 7.0 using 4 M NaOH and 4 M acetic acid solution. The dissolved oxygen (pO2) content was maintained at 50% by cascade control of stirrer speed, air flow, and pure oxygen. The temperature was controlled at 37°C. Once the glucose content in the medium was depleted (determined using a CEDEX analyzer), arabinose was added as an inducer. To obtain an adequate concentration of IgM protease protein, cultivation was continued for an additional 3 hours.

[0045] After this, E. coli cells were collected from the culture and stored cold either unconcentrated in medium or in 0.04 M PBS buffer, or concentrated up to 4-fold by centrifugation. The cells were then disrupted using either a microfluidizer (Microfluidics) at 30,000 psi (2068 bar) or a homogenizer such as a French press (range 600-2000 bar). Finally, the entire fraction after cell disruption was centrifuged, the supernatant and pellet were separated and inactivated with BPL (beta-propiolactone). Protein yield was determined using SDS gels with a known series of BSA as a standard. IgM protease was not subjected to further purification.

[0046] To study the effect of pressure on cell disruption, several experiments were performed in which E. coli (serotype 2) cells were disrupted either in a microfluidizer or a French press at different pressures. Table 1 shows the relationship between the applied pressure during cell disruption and the release of IgM protease protein into the supernatant after centrifugation following cell disruption. The recovery was 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 before centrifugation (i.e., the total fraction) and multiplying by 100%. Only the applied pressure was found to be significant; the device type had no significant effect.

[0047] Table 1. IgM protease-based antigen retrieval into supernatant

[0048] Pressure (bar) Allocation (%) 100 5%* 600 30% 1300 60% 2000 100%

[0049] *Determined by extrapolation

[0050] Example 3

[0051] Example 3 shows the protective effect of a vaccine obtained using an antigen obtained by the method described in Example 2.

[0052] Research Plan

[0053] The vaccines were prepared by mixing the supernatant obtained according to Example 2 with X-Solve oil-in-water adjuvant, available from MSD Animal Health, to obtain a concentration of 35 μg IgM protease / ml for the first vaccine and 3.5 μg IgM protease / ml for the second vaccine, using the same amount of oil adjuvant. Thirty 3-week-old piglets were used in the study. The piglets were divided into three groups (different litters were evenly distributed among the groups), with 10 piglets in each group. Groups 1 and 2 were vaccinated twice intramuscularly at 3 and 5 weeks of age with different vaccines. Group 1 received 2 ml of the first vaccine per vaccination (i.e., 70 μg of antigen per vaccination), and Group 2 received 2 ml of the second vaccine per vaccination (i.e., 7 μg of IgM protease-based antigen per dose). Group 3 served as an unvaccinated challenge control. At 7 weeks of age, piglets were transported to the challenge room and immediately challenged.There was no acclimatization period between transport and the natural stress simulation test. After infection, pigs were examined daily for clinical signs of S. suis infection (such as depression, motor problems, and / or neurological signs) and scored using a standard scoring system from 0 (no signs) to 3 for severe cases. Severely affected animals were euthanized and subjected to postmortem examination. At the end of the study (11 days after infection), all surviving pigs were euthanized and subjected to postmortem examination. Serum was collected for antibody determination immediately before vaccination and infection. At specified intervals before and after infection, heparinized blood was collected for repeat isolation of the control strain.

[0054] Results

[0055] None of the vaccines caused any unacceptable local or systemic reactions and could therefore be considered safe. On the day of vaccination (at 3 weeks of age), most piglets had low to moderate maternal antibody titers. After vaccination, antibody responses were observed in all vaccine groups (data not shown). The results for various post-challenge parameters are shown below in Table 2 (survival times are presented in days).

[0056] Table 2. Post-infection data

[0057] Group Average survival time Average clinical status score Number of positive blood samples The number of deaths after infection 1 9,4 14,9 3 / 10 3 / 10 2 8,6 21,5 3 / 10 3 / 10 3 3,4 64,9 9 / 10 8 / 10

[0058] Conclusion

[0059] The results show that the vaccines provided protection to piglets against infection with pathogenic Streptococcus suis two weeks after the second vaccination. A vaccine dose of only 7 μg provided adequate protection.

Claims

1. A method for producing a vaccine to protect pigs from the pathogenic infection Streptococcus suis, comprising: - recombinant expression of antigen based on IgM protease Streptococcus suis in E. coli bacteria; - high pressure homogenization of E. coli bacteria at a pressure of at least 500 bar to induce lysis of E. coli bacteria and release of IgM protease-based antigen into the lysate supernatant; - separation of the supernatant from the lysate sediment; - mixing the supernatant containing the IgM protease-based antigen with a pharmaceutically acceptable carrier to produce a vaccine.

2. The method according to claim 1, characterized in that the pressure during high-pressure homogenization is at least 1000 bar.

3. The method according to claim 1 or 2, characterized in that the pressure during high pressure homogenization is at least 1300 bar.

4. The method according to any one of claims 1 to 3, characterized in that the pressure during high pressure homogenization is at least 2000 bar.

5. The method according to any one of paragraphs 1-4, characterized in that the device used to carry out high-pressure homogenization is a French press for high-pressure cell disruption or a microfluidization device.

6. The method according to any one of claims 1-5, characterized in that the IgM protease-based antigen is a whole IgM protease-based antigen.

7. The method according to claim 6, characterized in that the whole antigen based on IgM protease belongs to the bacterium Streptococcus suis serotype 1, 2 or 7.

8. A vaccine containing an IgM protease-based antigen of Streptococcus suis obtained by the method according to any one of claims 1 to 7, characterized in that it contains at least 5% of native proteins of E. coli in relation to the IgM protease-based antigen.