Novel vaccine against heamophilus parasuis

Abstract

To provide a vaccine for protecting a pig against Haemophilus parasuis, and a method to protect a pig against Haemophilus parasuis.SOLUTION: Provided is an immunogenic composition comprising a protein having at least 69% sequence identity with the protein according to a specific sequence or an immunogenic fragment of this protein, for use in a prophylactic method to protect a pig against an infection with Haemophilus parasuis. Also provided are a vaccine for protecting a pig against an infection with Haemophilus parasuis comprising the immunogenic composition and a pharmaceutically acceptable carrier, and a method to protect a pig against an infection with Haemophilus parasuis by administering the vaccine to the pig.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention generally relates to a method for treating the pathogenic bacterium Haemophilus parasuis (Haemohilus p In particular, the present invention relates to the treatment of pigs against infection with this bacterium. The present invention relates to a novel vaccine for the prophylactic treatment of pigs against infection by B. difficile. [Background technology]

[0002] Haemophilus parasuis is one of the most important bacteria affecting pigs. The disease caused by this pathogen is characterized by polyserositis and is known as Glaesser's disease. M. parasuis is present in all major pig-raising countries and is a major risk factor in modern pig production systems. Haemophilus parasuis not only causes disease but also remains a significant pathogen. It is frequently isolated from the upper respiratory tract of healthy pigs. There are several serotypes, each of which can be identified using immunodiffusion techniques (Kiel stein et al., J. Clin. Microbiol. 30:862-865;1992, and Rapp-Gabrielson et al., AJVR, 53:659-664;199 2) The success of vaccination in reducing mortality has been achieved through a variety of vaccines. It has been completed.

[0003] Inactivated H. parasuis (i.e., bacterin) vaccines are widely used today. All commercially available H. parasuis vaccines are inactivated vaccines. Most vaccines are produced by growing virulent H. parasuis strains after inactivating the strain. The bacterial culture is pelleted by high-speed centrifugation and resuspended in sterile phosphate-buffered saline. The suspension is then added to a suitable adjuvant (e.g., mineral oil, aluminum hydroxide, carbohydrate, etc.). Formulated with squalene, saponin, vitamin E acetate, squalene, etc. In addition to monovalent vaccines, bivalent, trivalent, or tetravalent H. parasuis vaccines containing various serotypes are available. Vaccines are available. These generally provide low levels of cross-protection and are often used against homologous serotypes. These inactivated vaccines, such as Porcillis Glaesser (MSD, Boxmeer, the Netherlands) is important in controlling Glasgow disease outbreaks around the world. It plays a vital role.

[0004] A stably attenuated H. parasuis strain could theoretically serve as a safe and effective vaccine. However, the development of an attenuated H. parasuis vaccine is expected to be effective against the main H. parasuis strains. Limited by a lack of knowledge about the key virulence factors, Generating potentially useful H. parasuis mutants has been difficult. Currently, there are no genetically engineered live-attenuated or inactivated H. parasuis vaccine candidates. stomach.

[0005] Several subunit vaccines are being investigated, but existing data suggest that a small number of subunit vaccines Unit vaccines were associated with high levels of anti-H. parasuis vaccine neutralizing antibodies and H. parasuis challenge However, it has been suggested that it may be effective in preventing and protecting pigs against Glaesser's disease. No commercially available subunit vaccines are currently available for administration. heng Liu et al., Veterinary Immunology and Immu nopathology, Vol. 180, November 1, 2016, pp53-58), Newly identified protective antigens, including recombinant transferrin-binding protein B (TbpB) A subunit vaccine containing TbpB, OMP2 and OMP5-enhanced outer membrane proteins Protein (OMP) preparation, transferrin-binding protein A (TbpA), trimeric autoantibody transporter (VtaA), six secreted proteins (PflA, Gcp, HsdS, RnfC, and HAPS_0017), three glyceraldehyde-3-phosphate dehydes ATPase (GAPDH), OapA, and HPS-675 fusion proteins, various catabolic Replacement OMPs (SmpA, YgiW, and FOG), 6-phosphogluconate dehydrogenase enzyme, cytolethal distending toxin subunits A, B and C, neuraminidase or lipoproteinase It has been demonstrated that ampaku provides partial protection against H. parasuis challenge.

[0006] To date, DNA vaccines have shown some degree of partial protection against H. parasuis. This vaccine contains the gene encoding H. parasuis GAPDH. Contains DNA.

[0007] However, despite the availability of vaccines, the main Due to limited efficacy, antibiotics are still widely used to treat H. parasuis infections. Pigs given antibiotics early in H. parasuis infection usually survive systemic infection. However, there is much pressure to reduce the amount of antibiotics used in growing pigs. stomach. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2015 / 181356 [Non-patent literature]

[0009] [Non-Patent Document 1] J.Clin.Microbiol.30:862-865;1992, [Non-patent document 2] AJVR, 53:659-664;1992 [Non-patent document 3] Veterinary Immunology and Immunopathology, Vol.180, November 1, 2016, 53-58 [Non-patent document 4] Bioinformatics 23:1073-79, 2007 [Non-Patent Document 5] Nat.Biomed.Res.Found., Washington DC,1978, vol.5, suppl.3 [Non-patent document 6] Science 227, 1435-1441, 1985

[0010] Subject of the invention To provide an alternative vaccine for the prophylactic treatment of pigs against H. parasuis infection. The vaccine is preferably at least as effective as a conventional bacterin vaccine. provides equally good protection. Summary of the Invention [Problem to be solved by the invention]

[0011] Summary of the Invention For purposes of the present invention, a protein having at least 69% sequence identity with the protein set forth in SEQ ID NO:1 is preferred. A protein having sequence identity or an immunogenic fragment of the protein is used to vaccinate pigs. A prophylactic method for protecting pigs from infection with Haemophilus parasuis by administering and the vaccine may be used in a method comprising administering to a subject the protein or its immunogen. Contains the epitoxigenic fragment as an antigen.

[0012] This protein or its immunogenic fragments can be used to treat pigs against H. parasuis infection. The fact that it can be used against various H. parasuis serovars (pathogenic serovars 4, 5, 12, 1 3, 15) are conserved in the natural proteins, which are putative serine proteases. Based on the surprising finding that β-amyloid plays a key role in the transmission of parasuis. This suggests that vaccination with (part of) naturally occurring proteins may be effective in preventing the spread of pathogenic H. pylori. It provides very good protection against parasuis and is superior to conventional, well-established bacterial vaccines. This is confirmed by the fact that the defensive effect is even better than that which can be achieved by the chin. This suggests that this serine protease plays a key role in bacterial pathogenicity. Neutralizing the function of this protein reduces infection, including the clinical disease that results from it. In this respect, the inventors' advantage is that this It is recognized that protease plays an important role in the virulence of H. parasuis. Once this is recognized, it is possible to induce antibodies against this protein in H. parahaemolyticus. The study was followed by a study in which the vaccine was found to be effective as a treatment for the Swiss infection. A more direct method involves administering a protein or polypeptide similar to the wild-type protein. This is what we should do.

[0013] Regarding the protein itself, the full-length protein was identified across various serotypes. The natural variation is about 69% for proteins having an amino acid sequence according to SEQ ID NO: 1. Therefore, the use of proteins meeting this level of identity is comparable to the corresponding protein in nature. used to achieve protection against various wild-type H. parasuis strains of different serotypes producing It can be used.

[0014] In order to discover the gist of the present invention, the natural protein was used as an antigen to induce antibodies against the natural protein. Although the protein of SEQ ID NO: 1 (which is complete) was used, antibodies were used to identify specific (naturally occurring) ) When a protein needs to be produced, typically the whole protein is used. It is generally known that it is not necessary to use immunoreactive fragments of the corresponding protein. It is possible that the antibody itself is capable of binding to the corresponding protein. It is possible to use antibodies that are conjugated to a carrier, such as KLH, that induce a response. This is particularly true for the serine protease of the present invention. The proteins are already part of naturally occurring proteins containing autotransporter β-barrels. Secondly, the function of H. parasuis in the pathogenicity of H. parasuis is still unknown, but The protein was found to be homologous to the human Mac-1 protein. Cobalt alignments: RPS-BLAST, BLASTP, and PHI-BLA ST is used to search for conserved domain databases, protein motif databases, and a collection of pairwise constraints derived from sequence similarity. The pairwise constraints can then be established via a progressive multiple alignment tool. (Papadopoulos JS and Agarwala R, Bioinformatics 23:1073-79, 2007; PMID: 17 (See 332019 for more information.) This suggests that the Mac-1 family of Haemophilus bacteria A search of the NCBI database for the "Lee" protein revealed that H. parasuis contains Mac-1 This was confirmed by the presence of proteins with domain homologs. The protein is disclosed in WO2015 / 181356 (IDT Biologika GmbH). As described, the IgM protease of the swine pathogenic bacterium Streptococcus suis It is known to be homologous to ze. As taught in WO2015 / 181356, Vaccines against the full-length protein or a fragment containing only the highly conserved Mac-1 domain The antibody can induce antibodies against the full-length naturally occurring protein, thereby H. parasuis is completely unrelated to S. suis. However, both bacteria are known to have immunoglobulin peptidase activity. The fact that the protein having the -1 domain homologue is produced (WO2015 / 1813 56 confirmed cases (see the European Molecular Biology Laboratory PFAM database) and the disease Clinically similar (both diseases cause polyserositis and typically occur only in piglets) Combined with the fact that H. In parasuis, as in Streptococcus suis, the corresponding natural protein Furthermore, in both pathogenic bacteria, the Mac-1 domain is involved in evasion. The genotype is highly conserved among serovars, whereas the remainder is similar to that of H. parasuis and strains of The fact that the Putococcus suis protein is completely unrelated (Mac-1 domain) The overall protein identity level is low (13%), including H. parasuis serovars 3 and 4. , 5, 9, 12, 13 and 15, the identity of the Mac-1 domains is further expanded by 10 0% (while for the rest of the protein, this is much higher at 69% This, combined with the low level of IgG, suggests that the Mac-1 domain contains the major immunogenic epitopes. Evidence suggests that this is itself a naturally occurring serine protease that contains this so-called Mac-1 domain. Preferably, the fragment is capable of inducing antibodies that neutralize H. parasitism. The naturally occurring Mac-1 domain of the gene (i.e., the sequence according to SEQ ID NO: 2) is included. However, as is generally known, small variations of 10-20% in sequence identity Even up to 30% of the heterologous fraction can still serve as an effective antigen and induce neutralizing antibodies. These variations can be amino acid(s) differences across the entire sequence or within the sequence. This may be reflected by the deletion, substitution, insertion, inversion or addition of amino acid(s) in the organism. Amino acid substitutions that do not essentially alter the biological and immunological activity of the antibody may be used, for example, in the Neurat "The Proteins" by H. et al., Academic Press New The amino acid substitution or evolution between related amino acids is described in York (1979). Frequent substitutions in the ribosomal amino acid sequence are Ser / Ala, Ser / Gly, Asp / Gl, among others. y, Asp / Asn, Ile / Val (Dayhof, MD, Atlas o f protein sequence and structure, Nat.Bio med.Res.Found., Washington DC, 1978, vol. 5, suppl. 3). Other amino acid substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / Ile, Leu / Val and Al Based on this information, Lipman and Pearson quickly We developed a method for rapid and sensitive protein comparison (Science 227, 143 5-1441, 1985) to determine the functional similarity between homologous proteins. In particular embodiments, such amino acid substitutions, as well as mutations having deletions and / or insertions, are within the scope of the present invention. Thus, fragments for use in the present invention include those having SEQ ID NO: 2 and at least 70%, preferably at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical It should contain a certain polypeptide.

[0015] Overall, the use of the proteins of the invention in vaccines reduces the risk of naturally occurring H. It has been found that antibodies against the parasuisse serine protease can be induced. This means that H. parasuis infection can be treated after vaccination. Human Mac-1 protein and IgM proteases containing the Mac-1 domain were identified. Based on the homology with the Streptococcus suis strain that produces it, (at least) the current The Mac-1 domain of the protease (e.g., optionally linked to an immunogenic carrier such as KLH) When used as an antigen in a vaccine, the polypeptide containing the naturally occurring It is understood that the amount of the antibody produced is sufficient to induce antibodies against the protein.

[0016] The present invention also provides a vaccine for protecting pigs against infection with Haemophilus parasuis. The vaccine is embodied in a vaccine comprising a protein of SEQ ID NO: 1 and a nucleotide sequence of at least 69%. A protein having sequence identity or an immunogenic fragment of this protein, and a pharmaceutically acceptable The composition includes an acceptable carrier.

[0017] The present invention also provides a vaccine for protecting pigs from infection with Haemophilus parasuis. At least 69% sequence identity with the protein of SEQ ID NO: 1 as an antigen for the production or an immunogenic fragment of this protein.

[0018] Following this, the present invention aims to prevent the spread of Haemophilus spp. by administering a vaccine to pigs. In a method for protecting pigs from infection with R. suis, the vaccine comprises, as an antigen, the A protein having at least 69% sequence identity with the protein described in 1 or this protein. It contains an immunogenic fragment of a protein. DETAILED DESCRIPTION OF THE INVENTION

[0019] definition Antigens are antigenic molecules derived from microorganisms, even though the antigens are ultimately produced artificially. An antigen is a molecule that binds to a corresponding naturally occurring compound (typically a protein). Initiates and mediates the formation of antibodies that work against bacteria, viruses, protozoa, and other microorganisms. These are important sources of antigens, e.g., on the outer surface of cells (envelope antigens), inside cells (envelope antigens), (somatic or O antigen), flagella (flagellar or H antigen), or enzymes and toxins e.g. The protein may be a protein or polysaccharide derived from excretory products, including

[0020] A vaccine is a composition suitable for application to an animal and contains an immunologically effective amount of one or more antigens. i.e., to sufficiently stimulate the immune system of the target animal to react against and with the antigen. It is capable of inducing an immune response, such as antibodies, against the corresponding naturally occurring protein. , usually in a pharmaceutically acceptable carrier (i.e., a biocompatible medium, i.e., a medium that is compatible with the subject after administration) presenting antigens to the immune system of the host animal after administration of the vaccine without inducing significant adverse reactions in the animal a liquid containing water and / or other biocompatible solvents, is commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins) in combination with a solid carrier as described above, optionally including an immunostimulant (adjuvant), This allows the vaccine to protect animals from infection (after vaccination) when administered to them. Induce an immune response that can

[0021] A preventative method is a method that prevents infection or a corresponding disease by acting before infection actually occurs. A method designed to protect against diseases that the target animal is typically expected to be infected with. This is done by prior treatment of the subject with the vaccine.

[0022] Protection of pigs against H. parasuis infection includes prevention of pathogenic infection with H. parasuis, To help improve or cure, or to prevent, improve or cure disorders resulting from the infection. to help, for example, prevent or alleviate one or more clinical signs resulting from infection with H. parasuis. It means to reduce or mitigate.

[0023] The immunogenic fragment still retains its ability to induce an immune response in the host. , i.e., a fragment of a protein that contains a B-cell or T-cell epitope. A variety of techniques are commonly used to readily identify determinants, particularly immunogenic fragments of proteins. The method described by Geysen et al. (patent application WO 84 / 035 64, Patent Application WO 86 / 06487, U.S. Patent No. 4,833,092, Proc. .Natl Acad.Sci.81:3998-4002(1984), J.Imm. Meth., 102, 259-274 (1987), the so-called PEPSCAN method A simple, rapid and well-established method for detecting immunogenic epitopes of proteins. This method is used worldwide and is well known to those skilled in the art. Experimental methods are particularly suitable for detecting B cell epitopes. Given the sequence of a gene to be read, a computer algorithm can specific proteins based on their sequential and / or structural correspondence with the epitope Fragments can be designated as immunologically significant epitopes. The determination of these regions is , Hopp and Woods (Proc. Natl. Acad. Sci. 78:3824 8-3828 (1981)) and the hydrophilicity criteria by Chou and Fasman ( Advances in Enzymology 47:45-148(1987) and and U.S. Patent No. 4,554,101). Epitopes can also be identified by Berzofsky's amphipathic criteria (Science, 235, 1059-1062 (1987) and U.S. Patent Application No. NTIS US07 / 005,88 5) can be predicted from the sequence by computer. Found in: Shan Lu on Common Principles: Tibtech 9:238-2 42 (1991), Good et al. on malaria epitopes; Science 235: 1059-1062(1987), reviewed by Lu; Vaccine 10:3-7(1 992), Berzofsky on HIV-epitopes; The FASEB Joint Urnal 5:2412-2418(1991). To be immunogenic, MHC For MHC I receptor binding, the peptide must be a minimum length of 8-11 aa and must be MHC I It is common knowledge that the length must be 11 to 15 aa for receptor binding (e.g., RNGermain & D.H. Margulies, 1993, Annu.Re. v.Immunol., vol.11, p403-450:The biochemis try and cell biology of antigen processing ng and presentation).

[0024] Sequence identity between two polypeptides (or nucleic acids) is determined using default parameters. Polypeptides were identified as established in the BLAST program using the blastp algorithm. The percentage of identical amino acids (or nucleotides) in overlapping regions of peptides (or nucleic acids) (Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174:247-250;1999 (See

[0025] Further embodiments of the present invention In a further embodiment, the protein has at least one identical sequence to the protein set forth in SEQ ID NO:1. and has at least 90% sequence identity with the protein set forth in SEQ ID NO: 1 and at least 95% sequence identity with the protein set forth in SEQ ID NO: 1. There is even sequence identity, with up to 100% sequence identity.

[0026] In another embodiment, the protein or immunogenic fragment thereof is from Haemophilus pallas. The present invention is used in a method for protecting pigs from an increased risk of death due to infection with flu.

[0027] In yet another embodiment, the protein or immunogenic fragment thereof is from Haemophilus Method for protecting pigs against one or more clinical signs of infection with Parasuis Used.

[0028] The following specific examples are used to further illustrate the invention. [Example]

[0029] Example 1 the purpose The purpose of this alignment experiment was to identify sequences across various H. parasuis strains of various serotypes. The sequence identity level of the phosphoproteases was found to be higher than that of Mac-1 in serine proteases. The aim was to identify the domain.

[0030] result The following table lists the corresponding serine proteases in other H. parasuis strains: The level of identity is shown in Table 1. The highest known pathogenic and most prevalent strains of For strains within the serogroups 4, 5, 12, and 13, which are involved, the level of identity is even greater. Furthermore, it is over 90%.

[0031] [Table 1]

[0032] Following the above, the Mac-1 domain of H. parasuis was identified and presented in the present specification as SEQ ID NO:2. The protein according to SEQ ID NO: 1 is a hemoglobin having a length of 780 amino acids. Viral parasuis serotype 5 strain SH0165 (Genbank No. ACL32961 .1(Link)) is a putative extracellular serine protease. The -domain (AA521-780) was removed and the HMMR identification (www.hmmer.org g;Robert Finn et al., Nucleic Acids Research, 20 See 11 Jul 1;39, Web Server issue, W29-W37 When performing this procedure, the Mac-1 family domain begins at AA130 and ends at AA221. It indicates that it is finished.

[0033] The Mac-1 domain of H. parasuis was compared with the Mac-1 domain of Streptococcus suis. When compared to the domain, there is only 17% identity (using Blastp), which is showing that only a small fraction of the proteins is essential for antibody binding and / or protease activity This in turn suggests that the Mac-1 domain is involved in the expression of various H. parasuis serovars. Although it has 100% identity (as shown here; in other strains or serotypes Despite the low level of identity, the variation in naturally occurring proteins is at least Variations within the range of 70% identity level or higher relative to the native protein This indicates that there is a high possibility of inducing effective antibodies.

[0034] Example 2 the purpose The objective of this study was to evaluate the efficacy of a subunit vaccine against H. parasuis serotype 5 challenge. The purpose of this study was to verify the efficacy of subunit vaccines by comparing them with conventional bacterin vaccines. comprises a polypeptide according to SEQ ID NO: 1, wherein the corresponding DNA is Serotype 5, cloned from strain SH0165 (Genbank no. ACL32961.1) The vector was expressed in an E. coli expression vector system (pET22b, containing the pelB signal sequence and HIS The bacterin vaccine was expressed in a serotype 5 strain of Haemophilus parasitism. It contained inactivated cells of the bacteria.

[0035] Study design In this study, 30 healthy 4-week-old piglets were used. The piglets were divided into three groups (different groups) with 10 piglets in each group. Group 1 received 75 μL of bronchodilators at 4 and 6 weeks of age. 2 ml of vaccine containing 100 mg / ml of subunits suspended in oil in water adjuvant Group 2 was vaccinated intramuscularly with 100 mg of HIV-1 vaccine suspended in an oil-in-water adjuvant. Group 3 received two doses of a bacterin vaccine containing inactivated cells intramuscularly. At 8 weeks of age, pigs were vaccinated with H. pneumoniae. A virulent culture of La Suis serotype 5 was administered intratracheally.

[0036] For 10 days after challenge, pigs showed no signs of H. parahaemolyticus, such as depression, motor impairment, and / or neurological signs. Clinical signs of Swiss infection were observed daily and scored using a standard scoring system. Serum blood was collected for antibody measurements immediately prior to each vaccination and challenge. Heparinized blood samples were collected at regular intervals before and after the outbreak for reisolation of the challenge strain. was performed on all animals sacrificed before the scheduled necropsy date as well as on all surviving animals.

[0037] result Before the challenge, no abnormalities were observed, and no intercurrent deaths were observed. The core, the number of animals that had to be euthanized before the end of the study, and the blood re-isolation results are shown in Table 2 below. Shown below.

[0038] [Table 2]

[0039] conclusion The results show that the recombinant subunit vaccine is highly effective against H. parasuis challenge. The induced protection was comparable to that induced by the bacterin vaccine. It was better than the defense we were led to.

Claims

1. Protecting pigs from Haemophilus parasuis infection by vaccinating them for use in a preventative manner to A protein having at least 69% sequence identity with the protein set forth in SEQ ID NO: 1 or an immunogenic fragment of this protein, The vaccine comprises the protein or an immunogenic fragment thereof as an antigen. or an immunogenic fragment of this protein.

2. The protein has at least 90% sequence identity with the protein of SEQ ID NO:

1. A protein or immunogenic fragment thereof for use according to claim 1, characterized in that it is

3. The protein has at least 95% sequence identity with the protein of SEQ ID NO:

1. A protein for use according to claim 1 or 2, or its immunogenicity, characterized in that piece.

4. The protein according to any one of claims 1 to 3 is a protein set forth in SEQ ID NO:

1.

3. A protein or immunogenic fragment thereof for use according to any one of 3.

5. The method protects pigs from an increased risk of death due to infection with Haemophilus parasuis. A tank for use according to any of the preceding claims, characterized in that it is for protecting Protein or immunogenic fragment thereof.

6. The method protects pigs from one or more clinical signs of infection with Haemophilus parasuis. A protein or a compound for use according to any one of claims 1 to 4, characterized in that it protects against is its immunogenic fragment.

7. A protein having at least 69% sequence identity with the protein set forth in SEQ ID NO: 1 or an immunogenic fragment of this protein, and a pharmaceutically acceptable carrier. A vaccine to protect pigs against infection with Rus parasuis.

8. For producing a vaccine to protect pigs against infection with Haemophilus parasuis As an antigen, it has at least 69% sequence identity with the protein set forth in SEQ ID NO:

1. Use of the protein or an immunogenic fragment of this protein.

9. A protein having at least 69% sequence identity with the protein of SEQ ID NO: 1 or by administering to pigs a vaccine containing an immunogenic fragment of the protein as an antigen. Methods for protecting pigs from infection with Morphilus parasuis.

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