A NOVEL VACCINE AGAINST HEAMOPHILUS PARASUIS

Abstract

The invention pertains to a protein having at least 69% sequence identity with the protein according to SEQ ID No: 1 or an immunogenic fragment of this protein, for use in a prophylactic method to protect a pig against an infection with Haemophilus parasuis by administering a vaccine to the pig, the vaccine comprising the protein or the immunogenic fragment thereof as an antigen. The invention also pertains to a vaccine, a method to manufacture such a vaccine and a method to protect a pig against H. parasuis.

Claims

1. (canceled)

2. The method of claim 9, wherein the protein antigen comprises an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 1, or an immunogenic fragment thereof.

3. The method of claim 2, wherein the protein antigen comprises an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 1, or an immunogenic fragment thereof.

4. The method of claim 3, wherein the protein antigen comprises the amino acid sequence of SEQ ID NO: 1, or an immunogenic fragment thereof.

5. The method of claim 9, wherein said administering of the vaccine protects the pig against an increased risk of mortality due to the infection with Haemophilus parasuis.

6. The method of claim 9, wherein said administering of the vaccine protects the pig against one or more clinical signs due to the infection with Haemophilus parasuis.

7. A vaccine to protect a pig against an infection with Haemophilus parasuis, the vaccine comprising a protein antigen comprising an amino acid sequence that has at least 69% sequence identity with SEQ ID NO: 1, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

8. (canceled)

9. A method to protect a pig against an infection with Haemophilus parasuis comprising administering a vaccine to the pig, wherein the vaccine comprises a protein antigen that comprises an amino acid sequence that has at least 69% sequence identity with SEQ ID NO: 1, or an immunogenic fragment thereof.

10. The vaccine of claim 7, wherein the protein antigen comprises an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 1, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

11. The vaccine of claim 10, wherein the protein antigen comprises an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 1, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

12. The vaccine of claim 7, wherein the protein antigen comprises the amino acid sequence of SEQ ID NO: 1, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

Description

EXAMPLES

Example 1

[0029] Objective

[0030] The objective of this alignment experiment was to find the sequence identity level of the serine protease across various H. parasuis strains of various serotypes, and to identify the Mac-1 domain in the serine protease.

[0031] Results

[0032] In the table here below, it is indicated what the sequence identity is with respect to SEQ ID No:1 for the corresponding serine proteases in other H. parasuis strains. It appears that the level of identity is at least 69% among various strains of common serotypes. For strains within the group of known highly pathogenic and most prevalent serotypes 4, 5, 12 and 13, the level of identity is even 90% or higher.

TABLE-US-00001 TABLE 1 Sequence identity with SEQ ID No: 1 for various H. parasuis strains Strain Start End % Identity Serotype 3-strain SW1 1 491 69 Serotype 4-strain GX0 1 471 90 Serotype 4-strain HPS 1 523 97 Serotype 5-strain 297 1 523 99 Serotype 5-strain Nag 1 523 98 Serotype 5-strain SH0 1 523 99 Serotype 9-strain D74 1 727 70 Serotype 12-strain ZJ 1 523 98 Serotype 13-strain MN 1 503 95 Serotype 15-strain 84 1 523 99

[0033] Next to the above, the Mac-1 domain of H. parasuis was identified, and herewith disclosed as SEQ ID No:2. The protein according to SEQ ID No:1 is derived from the putative extracellular serine protease of Haemophilus parasuis serotype 5, strain SH0165 (Genbank No ACL32961.1), having a length of 780 amino acids. When removing the autotransporter domain (AA's 521 to 780), and performing a HMMR identification (see www.hmmer.org; Robert Finn et al in Nucleic Acids Research, 2011 Jul 1; 39, Web Server issue, W29—W37), the Mac-1 family domain is indicated to start at AA 130 and ending at AA 221.

[0034] When comparing the Mac-1 domain of H. parasuis with the Mac-1 domain of Streptococcus suis there is only a 17% identity (using Blastp), which is an indication that only a small part of the domain is essential for the antibody binding and/or protease activity. Next to this, this is an indication that although the Mac-1 domain is present with 100% identity in various H. parasuis serotypes (as indicated here above; notwithstanding that in other strains or serotypes the level of identity is lower), variations of the naturally occurring protein are likely to induce effective antibodies against the natural protein, at least variations within 70% identity level or more.

Example 2

[0035] Objective

[0036] The objective of this study was to test the efficacy of a subunit vaccine compared to a conventional bacterin vaccine against H. parasuis serotype 5 challenge. The subunit vaccine comprised the polypeptide according to SEQ ID No:1, wherein the corresponding DNA was cloned from H. parasuis serotype 5, strain SH0165 (Genbank no. ACL32961.1), expressed in an E. coli expression vector system (pET22b, with pelB signal sequence and a HIS tag). The bacterin vaccine contained inactivated cells of Haemophilus parasuis bacteria of serotype 5.

[0037] Study Design

[0038] For this study thirty healthy piglets at 4 weeks of age were used. The piglets were allotted to three groups (evenly distributed over the different litters) of 10 piglets each.

[0039] Group 1 was vaccinated twice intramuscularly at 4 and 6 weeks of age with 2 ml of a vaccine containing the subunit at 75 μg/ml, suspended in an oil in water adjuvant. Group 2 was vaccinated twice intramuscularly with the bacterin vaccine, comprising the inactivated cells suspended in an oil in water adjuvant, and group 3 was left unvaccinated as challenge control. At 8 weeks of age the pigs were challenged intra-tracheally with a virulent culture of H. parasuis serotype 5.

[0040] During 10 days after challenge the pigs were observed daily for clinical signs of H. parasuis infection such as depression, locomotory problems and/or neurological signs and scored using a standard scoring system. Just before each vaccination and challenge, serum blood was collected for antibody determination. At regular times before and after challenge heparin blood was collected for re-isolation of challenge strain. Necropsy was performed on all animals that were culled before the scheduled day of necropsy as well as on all surviving animals.

[0041] Results

[0042] Before challenge, no abnormalities were observed and no intercurrent death occurred. Average survival time in days, average clinical scores, the number of animals that needed to be euthanized before the end of the study as well as the blood re-isolation results are indicated here below in table 2.

TABLE-US-00002 TABLE 2 Results of vaccination-challenge study Average Average # blood survival clinical # culture Group time (days) score euthanised positive Subunit 7.7 27.1 3/10 3/10 Bacterin 4.9 47.4 6/10 3/10 Control 2.0 71.0 9/10 6/10

CONCLUSION

[0043] The results indicate that the recombinant subunit vaccine induced very good protection against H. parasuis challenge. Protection induced was superior to that induced by a bacterin vaccine.