VACCINE COMPOSITION AGAINST STREPTOCOCCUS SUIS INFECTION

Abstract

Described is a vaccine composition comprising an effective amount of at least one polypeptide selected from the group of IdeSsuis, rIdeSsuis, an analogue or a fragment thereof, or a polynucleotide encoding the same. This vaccine composition is used in the prophylactic, metaphylactic or therapeutic treatment of a Streptococcus suis infections in pigs or humans.

Claims

1. A method for protecting a pig against S. suis infection comprising administering to the pig an effective amount of a vaccine composition to induce neutralizing antibodies in a pig and thereby protecting the pig against S. suis infection, wherein the vaccine composition comprises an IdeS.sub.Suis antigen and at least a pharmaceutical carrier, a diluent or an adjuvant.

2. The method according to claim 1, wherein said IdeS.sub.suis antigen comprises Mac1 domain of IdeS.sub.suis protein where the active center of the protease has been inactivated by mutagenesis of the Cys-residue.

3. The method of claim 1 wherein said vaccine composition is administered to the pig by intramuscular, intravenous, subcutaneous or dermal injection or mucosal application.

4. The method of claim 1 wherein said IdeS.sub.Suis antigen is administered in a dose within a range of 0.05 to 2 mg/pig.

5. The method of claim 1 wherein said vaccine composition is administered to the pig by intramuscular, intravenous, subcutaneous or dermal injection or mucosal application.

6. The method of claim 1 wherein said vaccine composition is administered at least once to the pig.

7. The method of claim 1 wherein said vaccine composition is administered twice to the pig.

Description

[0115] The present invention is further described with reference to the following figures, where

[0116] FIG. 1 shows a time table representing the vaccination challenge experiments with S. suis in pigs,

[0117] FIG. 2 shows a graph representing the results gained with the vaccination challenge experiments of FIG. 1,

[0118] FIG. 3 shows a diagram of a bactericidal assay involving vaccination with placebo vs. rIdeSsuis, and

[0119] FIG. 4 depicts a diagram of a bactericidal assay involving vaccination with rIdeSsuis or rIdeSsuis analogues vs. a control group.

[0120] Further, a Sequence Listing is attached to this description.

[0121] SEQ ID NO: 1 shows the amino acid sequence of the protein IdeSsuis;

[0122] SEQ ID NO: 2 shows the amino acid sequence of the recombinant protein rIdeSsuis without the signal peptide;

[0123] SEQ ID NO: 3 shows the nucleotide sequence coding for IdeSsuis and SEQ ID NO: 4 shows the nucleotide sequence coding for rIdeSsuis.

[0124] SEQ ID NO: 5 shows the amino acid sequence of the highly conserved part of the Mac-1 domain of IdeSsuis.

[0125] SEQ ID NO: 6 shows the amino acid sequence of the rIdeSsuis analogue rIdeSsuis_homologue. SEQ ID NO: 7 shows the amino acid sequence of the rIdeSsuis analogue rIdeSsuisB2.

[0126] SEQ ID NO: 8 shows the nucleotide sequence coding for rIdeSsuis analogue rIdeSsuis_homologue.

[0127] SEQ ID NO: 9 shows the nucleotide sequence coding for rIdeSsuis analogue rIdeSsuisB2.

[0128] SEQ ID NO: 10 shows amino acid sequence of amino acids 91 to 425 of WP_044671938.

[0129] SEQ ID NO: 11 shows amino acid sequence of amino acids 91 to 425 of WP_002935529.

[0130] SEQ ID NO: 12 shows amino acid sequence of amino acids 92 to 426 of WP_015647040.

[0131] SEQ ID NO: 13 shows amino acid sequence of amino acids 92 to 426 of WP_023370787.

[0132] SEQ ID NO: 14 shows amino acid sequence of amino acids 92 to 426 of WP_044678723.

[0133] The following examples shall explain the present invention. The examples shall be understood only as one preferred embodiment of the invention. It is not intended to limit the present invention to the scope of the given examples.

EXAMPLE 1

[0134] The following example provides experimental data after performing vaccination challenge experiments in piglets infected by S. suis.

[0135] Establishment of vaccination challenge experiments in piglets for different S. suis serotypes (serotype 2 and 9) and infection routes (intravenous and intranasal).

[0136] Briefly as shown in FIG. 1, piglets at an age of five weeks were prime vaccinated with a rIdeSsuis vaccine. At an age of 7 weeks these piglets were boostered with rIdeSsuis and in one group also prime vaccinated with a bacterin by intramuscular injection (given in the table in FIG. 1). One group of piglets was only prime vaccinated with a bacterin at an age of 7 weeks and a last group of animals was vaccinated twice with a placebo consisting of buffer and adjuvant. The piglets were challenged two weeks after the second immunizations intranasally. Animals were further monitored every eight hours. For reasons of animal welfare, animals were euthanized in any case in which a piglet exhibits high fever in combination with apathy and anorexia as well as in the case of clinical signs of acute polyarthritis or severe meningitis.

[0137] All surviving piglets were sacrificed at 14 days post infection.

[0138] The experiment revealed that the immunization of piglets by using the recombinant protein rIdeSsuis by itself is enough to protect piglets from infections by S. suis serotype 2 (FIG. 2).

EXAMPLE 2

[0139] In this example, the bactericidal assay has been used to evaluate the effectiveness of a given vaccine. This test involves the determination of the survival of S. suis bacteria of a certain serotype after adding the same to the blood of a test animal. If antibodies protective against a certain serotype are present in the blood of this test animal, the bacteria will be killed during an incubation time of 2 hours efficiently. The extent of protection is designated as “survival factor” (SF) and is the ratio of the colony count after 120 min. and the colony count directly after adding the bacteria to the blood of the test animal. A low survival factor means an efficient killing of the bacteria in the blood and, therefore, an effective protection of the test animal.

[0140] The expression and purification of recombinant IdeSsuis (SEQ ID NO: 2), recombinant IdeSsuisB2 (SEQ ID NO: 7) and recombinant IdeSsuis_homologue (SEQ ID NO: 6) was performed by growth of the appropriate strains in LB broth plus ampicillin. Protein expression was induced by adding IPTG. The purification of the recombinant proteins by Ni.sup.2+-nitrilotriacetic acid affinity chromatography under native conditions was carried out as recommended by the manufacturer (Macherey-Nagel).

[0141] Immunization of piglets: Piglets were prime and booster vaccinated with 1.5 ml vaccine containing 0.25 mg rIdeSsuis or 0.25 mg rIdeSsuisB2 or 0.5 mg rIdeSsuis_homologue containing 20% [vol/vol] Emulsigen as an adjuvant.

Trial 1:

[0142] Two or four litter mates each are randomly distributed into two trial groups (n=9/group), group 1 control (placebo), group 2 immunized with rIdeSsuis (SEQ ID NO: 2). The animals were immunized and tested according to the following test scheme:

TABLE-US-00002 number group of pigs 1. immunization 2. immunization bactericidal assay 1 9 Placebo (PBS Placebo (PBS Serotype 2 Serotype 9 plus adjuvant) plus adjuvant) (St. 10) (A3286/94) 2 9 rldeSsuis rldeSsuis Serotype 2 Serotype 9 (0.25 mg/piglet) (0.25 mg/piglet) (St. 10) (A3286/94)

[0143] FIG. 3 shows the results which were achieved.

[0144] The control group (placebo) showed a much higher survival factor than the vaccinated group. The recombinant antigen rIdeSsuis (group vaccinated), containing the complete sequence of IdeSsuis proteins of serotype 2 strain (SEQ ID NO: 2) induces antibodies effecting an efficient killing of S. suis bacteria of strain 2 as well as of strain 9.

Trial 2:

[0145] Four litter mates each are randomly distributed into four trial groups (n=6/group with the exception of group 4, where n=S), group 1 control (not immunized), group 2 immunized with rIdeSsuis_homologue (SEQ ID NO: 6), group 3 immunized with IdeSsuis derived from serotype 7 strain (rIdeSsuisB2; SEQ ID NO: 7), group 4 immunized with SEQ ID NO: 2. The animals were immunized and tested according to the following test scheme:

TABLE-US-00003 number bactericidal group of pigs 1. immunization 2. immunization assay 1 6 no immunization no immunization S. suis serotype 9 strain A3286/94 2 6 rideSsuis_homologue rldeSsuis_homologue S. suis (0.5 mg/piglet) (0.5 mg/piglet) serotype 9 strain A3286/94 3 6 rldeSsuisB2 rldeSsuisB2 S. suis (0.25 mg/piglet) (0.25 mg/piglet) serotype 9 strain A3286/94 4 5 rldeSsuis rldeSsuis S. suis (0.25 mg/piglet) (0.25 mg/piglet) serotype 9 strain A3286/94

[0146] FIG. 4 shows the results which were achieved.

[0147] The results show a considerably higher survival factor in the control group (group 1) than in the three vaccination groups. The recombinant antigen rIdeSsuis_homologue (group 2), only containing the N-terminal fragment including the highly conserved Mac-1 domain (IgM-Protease-domain), induced antibodies which result in a much better killing of bacteria compared to the control group. The result of group 2 can be compared with those obtained for group 4. Also antigen rIdeSsuis, containing the complete amino acid sequence of mature IdeSsuis protein of a S. suis serotype2 strain induces antibodies which reduce the survival of S. suis serotype9 strain in the blood considerably. The survival of serotype9 strain is even more compromised by antibodies which have been induced by antigen rIdeSsuisB2 (group 3).

[0148] rIdeSsuisB2 (SEQ ID NO: 7) contains the complete amino acid sequence of the mature IdeSsuis protein of a S. suis serotype7 strain and differs in the C terminal half of the protein since it lacks a sequence of 114 aa compared to SEQ ID NO: 1. Aa 80 to 414 of SEQ ID NO: 7 (the highly conserved part of the so-called Mac-1 domain) correspond in 97.9% to the sequence of SEQ ID NO: 5. The identity between remaining (-terminal part of SEQ ID NO: 7 and 1 is 96.4%.

[0149] The conclusions which can be drawn from the experimental results are as follows: [0150] rIdeSsuis (SEQ ID NO: 2) provides immune protection across different serotypes of S. suis, [0151] An amino acid at least containing the highly conserved Mac-1 domain is sufficient to provide immune protection, [0152] Also IdeSsuis proteins of other serotypes, at least containing the highly conserved Mac-1 domain induce protection, even if their overall sequence outside this domain differs from that of the serotype 2 strains (even if certain sequence segments are entirely absent).