Vaccine for protection against serotype 9, sequence type 16 streptococcus suis

Abstract

The present invention relates to a vaccine comprising a combination of an IgM protease antigen of Streptococcus suis and a Streptococcus suis bacterin of serotype 9, sequence type 16, for use in a method of protecting a pig against a pathogenic infection with Streptococcus suis of serotype 9, sequence type 16.

Description

TECHNICAL FIELD

[0001] The present invention relates to the protection of pigs against pathogenic infection by Streptococcus suis bacteria of serotype 9, sequence type 16. BACKGROUND

[0002] Streptococcus s (S. suis) is one of the main causative agents of contagious bacterial disease in pigs. The pathogen can cause a variety of clinical syndromes, including meningitis, arthritis, pericarditis, polyserositis, septicemia, pneumonia and sudden death. S. suis is a Gram-positive, facultative anaerobic coccus, initially defined as Lancefield group R, S, R / S or T, based on the presence of type-specific capsular polysaccharide antigens located in the cell wall. Later, a new typing system was proposed based on the type-specific capsular polysaccharide antigens located in the cell wall. This resulted in 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 2, 9, 1, 7 and 1 / 2 are the most prevalent. However, the recognized capsular serotypes are poor markers of virulence. Thus, an alternative system was developed to help understand the epidemiology of S. suis infection and the biological relevance of serotyping methods, i.e. the so-called multilocus sequence typing (MLST), as described in King et al. in 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 this study, 92 sequence types were identified that were dominant in the human population, of which ST complex ST1, ST27 and ST87 each comprised a plurality of sequence types. See also the Streptococcus suis MLST website at the University of Oxford (https: / / pubmlst.org / ssuis / ) (funded by the Wellcome Trust) which refers to King et al., supra and enables easy identification of the sequence type of any S. suis strain.

[0003] Control of S. suis in pig populations seems to be very difficult. S. suis is an opportunistic commensal of pigs. Apparently, the immune system is not triggered at every infection. Following this, S. suis is a fully capsulated pathogen and uses a repertoire of virulence factors to evade the host immune system. These features have collectively posed challenges to the development of an effective vaccine against this important pathogen. Recently, a review article on vaccines against S. suis has been published (Mariela Segura: "Streptococcus suis vaccines: candidate antigens and progress, in Expert Review of Vaccines, vol. 14, 2015, no. 12, pages 1587-1608). In this review, clinical field information and experimental data have been gathered and compared to outline the current status of vaccine development against S. suis, as outlined below.

[0004] Currently used vaccines are mainly whole-cell bacterins. However, field reports describe difficulties in disease control and management, and in particular "vaccine failures" are common. Carrier pigs are the main source of infection and both vertical and horizontal transmission are associated with the spread of the disease in herds. Mixing of carrier animals with susceptible animals under stressful conditions, such as weaning and transport, often results in clinical disease. Early medication of weaning and early weaning practices do not eliminate S. suis infection. Therefore, the key to an effective control measure to prevent disease will be in prophylactic / metaphylactic programs (where allowed) and vaccination. Currently, field immunization efforts have focused on the use of commercial or autogenous bacterins. These vaccine strategies have been applied to piglets or sows. Piglets are more susceptible to S. suis infection after weaning and onwards due to the stress associated with weaning and later on, transport. Therefore, prepartum immunization of sows is often used to try and deliver passive immunity to piglets and to provide protection against S. suis at these stressful situations early in life. In addition, sow vaccination is less costly and less labor intensive, and thus represents an economical alternative to piglet vaccination. However, available results seem to indicate that sow vaccination with bacterins is also a matter of debate. In many cases, vaccinated sows do not respond well or at all to vaccination, even when vaccinated twice prepartum, which results in low maternal immunity delivered to the piglets. Moreover, even if maternal immunity is delivered at sufficient levels, in many cases, the maternal antibodies are still too low to provide protection during the most critical period of 4-7 weeks of age.

[0005] In piglets, autogenous bacterins are frequently used in the field, especially in Europe. They are prepared from virulent strains isolated from a farm with clinical problems and applied to the same farm. One of the drawbacks of autogenous bacterins is the lack of vaccine safety data and the possibility of serious adverse reactions. Sampling errors (due to the use of only one or two pigs or one or two samples) can lead to the inability to identify the strain or serotype associated with the most recent outbreak. This failure can be especially problematic in endemic herds. Finally, the most important dilemma of autogenous bacterins is the lack of studies on their actual efficacy. Since the use of autogenous vaccines is empirical, it is not surprising that the results obtained with these vaccines are inconsistent.

[0006] Other experimental vaccines are also described in the art. Kai-Jen Hsueh et al. ( “Immunization with Streptococcus suis bacterin plus recombinant Sao protein in sows conveys passive immunity to their piglets”, BMC Veterinary Research, BMC series - open, inclusive and trusted, 13:15, January 7, 2017) suggest that bacterin + subunit can be the basis for successfully vaccinating sows to give their piglets protective immunity.

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

[0008] In the last two years, a broad list of antigenic or immunogenic S. suis molecules has been reported and most of these have been discovered by immunoproteomic approaches using immune sera from infected pigs or humans in convalescence and / or laboratory generated immune sera. WO2015 / 181356 (IDT Biologika GmbH) has shown that an IgM protease antigen (either the complete protein or only the highly conserved Mac-1 domain corresponding to about 35% of the total protein) can elicit a protective immune response in piglets in a vaccination regimen of two doses of IgM protease antigen, optionally combined with an initial prime vaccination with bacterin. It is noted that WO2017 / 005913 (Intervacc AB) also describes the use of an IgM protease antigen (in particular an IgM protease polypeptide fused to a nuclease). However, only the property to be able to elicit a seroresponse has been shown. The protective effect of the IgM protease antigen is not shown in this international patent application.

[0009] A recent WO 2019 / 115741 (assigned to the current applicant) has been published. In this patent application, it is shown that an IgM protease antigen can effectively prevent pathogenic infection with S. suis of serotype 9. However, the protection is not very high and seems to be comparable to the level obtainable with a common bacterin vaccine, i.e. about 50% reduction in mortality and positive blood isolations in an artificial challenge experiment (not excluding that in practice the protection will be at a higher level). At first sight, this somewhat disappointing protection seems to contradict the high level of protection against S. suis of serotype 9 infection obtained with an IgM protease antigen reported by Rieckmann et al., Vaccine, 3 (2019) 100046 (“Vaccination with the immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, leads to protection against a highly virulent serotype 9 strain”) also in an artificial challenge experiment.

[0010] Object of the invention

[0011] It is an object of the present invention to find a composition and therapy that is more effective in protecting pigs against S. suis of various serotypes including serotype 9. SUMMARY

[0012] It is well known that very good protection can be obtained in various serotypes using IgM protease antigens, including serotypes 1, 2, 9 and 14. In this respect, the results reported in WO 2019 / 115741 regarding the level of protection obtainable against serotype 9 seem to be in conflict with the results reported in Rieckmann. However, upon closer inspection, it appears that in the Rieckmann study a Streptococcus suis strain of sequence type 94 was used. In WO 2019 / 115741, although not specified, a Streptococcus suis strain of sequence type 16 was used. This was discovered later by typing the challenge strains used according to the multilocus sequence typing described by King et al. (see above). Apparently, the level of protection provided by the IgM protease antigen is significantly lower against the latter type (S. suis serotype 9, sequence type 16). The reason for this is not clear, but is very disadvantageous because in many countries, especially European countries such as the Netherlands, Streptococcus suis of sequence type 16 is the most prevalent pathogenic type of Streptococcus suis serotype 9 bacteria (Willemse et al., Scientific Reports, 2019, 9: 15429, “Clonal expansion of a virulent Streptococcus suis serotype 9 lineage distinguishable from carriage subpopulations”). Therefore, although IgM protease can produce broad protection across serotypes, a gap in effective protection was found, especially against Streptococcus suis serotype 9, sequence type 16.

[0013] Upon discovering this difference, it was discovered that this difference could be made up for by using a combination of the IgM protease antigen of S. suis and the S. suis bacterin of serotype 9, sequence type 16 to protect pigs against pathogenic infection with S. suis of serotype 9, sequence type 16 and to achieve a higher level of protection than is possible with the IgM protease alone (in particular, in the artificial challenge model, the number of deaths and the blood separation scores were far below 50% relative to the unvaccinated control animals). This is very unexpected, as the two antigens overlap (the bacterin itself also contains all of its subunits) and each antigen alone provides less protection, while the combination of the antigens provides a level of protection that can reach the level of protection against other S. suis strains that is possible with the most effective known vaccines, i.e. the IgM protease antigen-based vaccine is able to provide a certain level of protection against various different (non-serotype 9, sequence type 16) S. suis strains. Thus, it was shown that the antigens used in combination provide a better protection than expected. It was shown that this effect is produced by the simultaneous administration of the two antigens, either as separate single vaccines (by two separate administrations) or when formulated in one (single) vaccine for one administration to administer both antigens at once.

[0014] In other words, the present invention also relates to an IgM protease antigen of S. suis for use in a method in which the IgM protease is administered simultaneously with a S. suis bacterin of serotype 9, sequence type 16 for protecting pigs against pathogenic infection with S. suis of serotype 9, sequence type 16. Alternatively, in the alternative, the present invention relates to a S. suis bacterin of serotype 9, sequence type 16 for use in a method in which the bacterin is administered simultaneously with an IgM protease antigen of S. suis for protecting pigs against pathogenic infection with S. suis of serotype 9, sequence type 16. The simultaneous administration can be in the form of two separate administrations of two separate formulations (one containing the IgM protease antigen and the other containing the bacterin) or one single administration of one single combination vaccine containing both antigens in one single formulation.

[0015] With the present invention, protection against S. suis can be made up. The present invention not only enables the best possible protection against S. suis of serotype 9, including sequence type 16 as an important representative, but also enables a method to achieve very broad protection against all serotypes. It has been proven that the IgM protease antigen can provide broad protection across serotypes and that this protection will inherently be present when using the current combination of antigens.

[0016] The present invention also relates to a kit comprising a combination of an IgM protease antigen of S. suis, a S. suis bacterin of serotype 9, sequence type 16, and a pharmaceutically acceptable carrier.

[0017] The present application also relates to a single vaccine composition comprising a combination of an IgM protease antigen of Streptococcus suis, a Streptococcus suis bacterin of serotype 9, sequence type 16, and a pharmaceutically acceptable carrier.

[0018] The present application also relates to the use of an IgM protease antigen of Streptococcus suis and a Streptococcus suis bacterin of serotype 9, sequence type 16, for the manufacture of a vaccine for protecting a pig against a pathogenic infection with Streptococcus suis of serotype 9, sequence type 16.

[0019] Finally the present application relates to a method for protecting a pig against a pathogenic infection with Streptococcus suis of serotype 9, sequence type 16, by administering to said pig an IgM protease antigen of Streptococcus suis and a Streptococcus suis bacterin of serotype 9, sequence type 16.

[0020] Definitions

[0021] The IgM protease antigen of S. suis is an enzyme, termed IdeSsuis, or an immunogenic portion thereof (typically having at least about 30-35% of the length of the full-length enzyme) that specifically degrades porcine IgM (but not porcine IgG or porcine IgA; Seele et al., Journal of Bacteriology, 2013, 195 930-940; and Vaccine 33:2207-2212; 5 May 2015). The full enzyme has a weight of about 100-125 kDa, corresponding to about 1000-1150 amino acids, the size depending on the serotype of S. suis. In WO 2015 / 181356 several sequences are given that represent IgM protease antigens of S. 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 portion of the full-length enzyme (represented as the Mac-1 domain, i.e., amino acids 80-414 of SEQ ID NO: 7). Further examples of immunogenic portions of the full-length enzyme are given in WO 2017 / 005913. In particular, the IgM protease can be the protease of SEQ ID NO: 1 according to WO 2015 / 1818356 or a protein having at least 90%, or even 91, 92, 93, 94, 95, 96, 97, 98, 99% up to 100% sequence identity in the overlapping regions. Amino acid sequence identity can be established with the BLAST program using the blastp algorithm with default parameters. The IgM protease of S. suis of various serotypes is expected to have a sequence identity higher than 90%, in particular 91, 92, 93, 94, 95, 96, 97, 98, 99% up to 100%. For example, an artificial protein prepared for optimal yield in an antigen recombinant production system can result in a lower amino acid sequence identity compared to the full-length enzyme, e.g., 85%, 80%, 75%, 70% or even 60%, while maintaining the desired immunogenic function and is understood to be an IgM protease antigen of S. suis in the sense of the present application.

[0022] A vaccine is a construct suitable for application to a subject comprising one or more antigens in an immunologically effective amount (i.e., capable of sufficiently stimulating the immune system of the target subject to at least reduce the negative impact of a wild-type microorganism attack), typically in combination with a pharmaceutically acceptable carrier, which upon administration to the subject induces an immune response to treat an infection, i.e., to help prevent, ameliorate or cure an infection or any disease or disorder caused by said infection.

[0023] Protection against a pathogenic infection of a microorganism is the same as acquiring protective immunity, i.e. helping to prevent, ameliorate or cure the pathogenic infection of the microorganism or a condition caused by the infection, e.g. preventing or reducing the actual infection or one or more clinical signs caused by the pathogenic infection of the pathogen.

[0024] A bacterin is a suspension of killed bacteria used as a vaccine.

[0025] A combination of antigens is the joint use of these (individually different) antigens in one vaccination strategy, either by combining different antigens into one vaccine preparation or by simultaneous administration of separate preparations using separate antigen preparations.

[0026] A single vaccine composition is a vaccine that forms a single entity, such as a single liquid comprising all components of the vaccine combined in a single mixture.

[0027] A pharmaceutically acceptable carrier is a biocompatible medium, i.e. does not cause significant adverse reactions in the treated subject after administration, is able to present the antigen to the immune system of the subject after administration of the composition comprising the carrier. Such pharmaceutically acceptable carriers can be, for example, liquid or solid carriers containing water and / or any other biocompatible solvent, such as are usually used to obtain lyophilized vaccines (based on sugars and / or proteins), optionally comprising immunostimulants (adjuvants). Depending on the intended use or desired properties of the respective vaccine, other substances are optionally added, such as stabilizers, viscosity regulators or other components. DETAILED DESCRIPTION

[0028] While the two antigens of the combination of the present application can be administered separately, in a further embodiment the IgM protease antigen of Streptococcus suis and the Streptococcus suis bacterin are comprised in a single composition for administration to a pig. It was found that the effect of the combination is improved when both antigens are present in a single composition that can be administered by a single administration.

[0029] In another embodiment, the composition for use according to the application comprises an IgM protease antigen of Streptococcus suis serotype 2. While it is known that IgM protease is able to induce cross-protection between serotypes 2 and 9 (see WO 2019 / 115741 for IgM protease), it was found that the combination of IgM protease of serotype 2 and bacterin of serotype 9 is advantageous to obtain protection against Streptococcus suis of serotype 9, sequence type 16. The accompanying advantage is that this combination provides broad cross-protection between various serotypes, as is known for IgM protease antigen of serotype 2, 9, 7 and 14. Accordingly, all of the above in the summary section are identified as further embodiments of the same inventive concept, the antigen is advantageously selected from IgM protease of serotype 2, also for the use of the kit, the single vaccine, the antigen for the preparation of a vaccine and the method of treatment.

[0030] In yet another embodiment, the antigen is administered to the pig at most 35 days of age. As mentioned above, Streptococcus suis is a commensal and opportunistic pathogenic bacterium of pigs. In particular under stress, the bacterium can elicit a pathogenic infection and induce disease. Under modern pig production conditions, a major stress is induced when or after the pig reaches 35 days of age, for example induced by weaning of the piglets (3-4 weeks) and transport of the young piglets shortly thereafter. In order to protect against pathogenic infection with Streptococcus suis, the pig thus needs to be vaccinated at a very young age, generally before they reach 28 days of age. It was found that by using the combination antigen, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days of age in the pig at most 28 days of age, sufficient protection can be obtained. As is known in the art, in particular from WO2017 / 005913, positive immune responses against IgM protease antigens (WO2017 / 005913) and bacterins (as is typically applied in piglets before weaning) can be obtained in young pigs from the day of birth and onwards. This means that, by the actual protection shown in pigs at 3-5 weeks of age, it is understood that protection can be obtained even at a younger age.

[0031] In another embodiment, the antigen is administered between the age of weaning of the pig. In other words, the antigen is administered before the actual weaning of the piglets (typically at 3-4 weeks of age). It has been shown that by administering the antigen at this early stage, protection against pathogenic infection with S. suis serotype 9, sequence type 16, which is induced by stress in a short window of 2-3 weeks after weaning, can be obtained. It is acknowledged that WO2015 / 181356 shows successful vaccination using IgM protease as antigen. However, this vaccine was used for piglets of 5-7 weeks of age and challenged at 9 weeks of age, thus well after the risk period of 2-3 weeks after weaning / transportation (i.e. 5-7 weeks of age), i.e. after the peak of pathogenic S. suis infection. Therefore, without any proof of effectiveness in actual circumstances (i.e. challenge infection in the window of 2-3 weeks after weaning and transportation stress), it remains doubtful whether the vaccine strategy described in WO2015 / 181356 is effective in actual circumstances.

[0032] All of the above embodiments are also respective other representative embodiments of the application as defined above in the summary section, i.e. the respective uses, the methods of preparation and the methods of protection of the IgM protease antigen and the bacterin, the kit and the single vaccine.

[0033] The application will now be further explained on the basis of the following examples.

[0034] Example

[0035] The aim of this study was to test whether the combined administration of an IgM protease antigen and a bacterin of serotype 9, sequence type 16 (in this example, an IgM protease antigen of S. suis serotype 2) could provide protection against challenge with S. suis serotype 9, sequence type 16, such that in the artificial challenge model used in WO2019 / 115741, the level of protection would be superior to the level of protection obtained with the known IgM protease or the commonly used bacterin alone (i.e. more than about 50% reduction in the number of deaths after challenge and in the blood re-isolation).

[0036] The antigens used were the ones described in the prior art. The IgM protease corresponds to the antigen as described in WO2019 / 115741, i.e. the E. coli expressed rIdeSsuis IgM protease antigen as described by Seele et al. in Vaccine 33:2207-2212; 5 May 2015, paragraph 2.2, but without the His-tag. The bacterin was a common chloroform killed S. suis.

[0037] Study design

[0038] The study design was identical to that in WO2019 / 115741, but in the current setting the dose of IgM protease was significantly lower (80 pg vs 230 pg). In addition, vaccination and challenge occurred two weeks earlier to better fit the critical period of pathogenic S. suis infection. This means that in the current setting it will be more difficult to achieve the same vaccine efficacy as available in the study described in WO2019 / 115741, if at all.

[0039] In this study, 36 piglets of 3 weeks of age were used. The piglets were assigned to three groups (evenly distributed over different litters), each group of 12 piglets. Groups 1 and 2 were twice intramuscularly inoculated at 3 and 5 weeks of age with each of two different combination vaccines. The first group was inoculated with a single vaccine composition (2 mL per dose) comprising IgM protease antigen of S. suis serotype 2 (at 80 pg per dose) and S. suis bacterin of serotype 9, ST16 (at 2xl0 9 cells per dose) in combination in an oil-in-water adjuvant. The second group was inoculated twice with two separate vaccines, one on the right side of the neck containing IgM protease (same dose) and one on the left side of the neck containing bacterin (same dose), both formulated in the same oil-in-water adjuvant as used in group 1. Group 3 remained unvaccinated. At 7 weeks of age, pigs were challenged intratracheally with a virulent culture of S. suis serotype 9, sequence type ST16.

[0040] After challenge, pigs were observed daily for clinical signs of S. suis infection, such as depression, locomotion problems and / or neurological symptoms, for 11 days and scored using a conventional scoring system from 0 (no symptoms) to 3 for severe cases. Animals reaching a humane endpoint were euthanized. Prior to vaccination and challenge, sera were collected for antibody determination. Heparin blood was collected periodically before and after challenge to re-isolate the challenge strain.

[0041] Results

[0042] None of the vaccines caused any unacceptable local or systemic reactions and can therefore be considered safe. Prior to challenge, one animal in group 1 became lame and received corresponding treatment. This animal did not recover and was therefore euthanized and excluded from the study.

[0043] Post challenge data at pre-euthanasia period (day 11) are shown in Table 1. The average clinical score, survival time, number of animals that died after challenge and number of animals from which the pathogen could be re-isolated from blood seemed to be improved for both vaccination regimes compared to the control. The level of protection against challenge with serotype 9, ST16 strain seemed to be comparable to the level of protection against homologous challenge with serotype 2 strain available with IgM protease as reported in WO2019 / 115741 and seemed to be significantly recognized for the protection against serotype 9, ST16 found in the ‘741 patent. This indicates a synergistic effect between the two antigens. This effect seemed to be more pronounced when used in combination in a single vaccine, especially giving lower clinical score and higher average survival time.

[0044] Table 1: Post challenge data

[0045] Group Clinical score Survival time (days) Death after challenge Positive blood separation 1 14 9.4 2 / 11 2 / 11 2 22 8.5 3 / 12 3 / 12 3 52 5.0 8 / 12 9 / 12

[0046] Conclusions

[0047] The study design was more critical than the one used in WO2019 / 115741. Therefore, even when comparable effectiveness was reached with the alternative vaccines, this indicates that the alternative vaccines are improved over the vaccines described in WO2019 / 115741 (i.e. monovalent IgM protease or bacterin vaccine). However, it was found that with the new combination vaccine, a significantly improved vaccine effectiveness could be reached, indicating a synergistic interaction of the antigens. These effects seemed to be higher when the antigens were combined in separate (single) formulations.

Claims

1. A combination of a Streptococcus suis IgM protease antigen and a Streptococcus suis serotype 9, sequence type 16 vaccine, for use in a method of protecting pigs against pathogenic infection by Streptococcus suis serotype 9, sequence type 16.

2. The combination according to claim 1, characterized in that... The IgM protease antigen of Streptococcus suis and the Streptococcus suis vaccine are contained in a single composition for administration to the pigs.

3. The combination according to claim 1 or 2, characterized in that... The IgM protease antigen of Streptococcus suis is the IgM protease of serotype 2 of Streptococcus suis.

4. The combination according to claim 1 or 2, characterized in that The method includes administering the IgM protease antigen of Streptococcus suis and the Streptococcus suis vaccine to the pigs at a maximum age of 35 days.

5. The combination according to claim 1 or 2, characterized in that... The method includes administering the IgM protease antigen of Streptococcus suis and the Streptococcus suis vaccine to the pigs before weaning age.

6. A kit comprising a combination of Streptococcus suis IgM protease antigen, serotype 9 and sequence type 16 Streptococcus suis vaccines, and a pharmaceutically acceptable vector.

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

8. The use of Streptococcus suis IgM protease antigen and Streptococcus suis serotype 9 and sequence type 16 vaccines in the preparation of vaccines to protect pigs against pathogenic infections of Streptococcus suis serotype 9 and sequence type 16.