Vaccine comprising a PCV2 ORF2 protein of genotype 2b

Abstract

The present invention pertains to a vaccine comprising an ORF2 encoded protein of porcine circo virus 2 (PCV2) and a pharmaceutically acceptable carrier, for use in a method to protect a pig against an infection with porcine circo virus type 2 by administering the vaccine to the pig, wherein the vaccine comprises less than 20 μg per dose of the ORF2 encoded protein, the protein being of a porcine circo virus of genotype 2b.

Claims

1. A method of protecting a pig against an infection with porcine circo virus type 2, comprising administering to the pig a vaccine comprising an ORF2 encoded protein of porcine circo virus 2 (PCV2) and a pharmaceutically acceptable carrier, wherein the vaccine comprises less than 20 μg per dose of the ORF2 encoded protein, wherein the protein is of a porcine circo virus of genotype 2b; and wherein the vaccine provides protection against PCV2 of genotype 2d at a level corresponding to the protection conferred when the pig is vaccinated with an ORF2 encoded protein of PCV2d.

2. The method of claim 1, wherein the vaccine comprises less than 5 μg per dose of the ORF2 encoded protein.

3. The method of claim 1, wherein the vaccine is for protecting pigs that have antibodies against PCV2.

4. The method of claim 3, wherein the antibodies against PCV2 are of maternal origin.

5. The method of claim 1, wherein the pig is 4 weeks old or less.

6. The method of claim 1, wherein the vaccine provides protection after one single administration of the vaccine.

7. The method of claim 4, wherein the vaccine comprises less than 5 μg per dose.

8. The method of claim 4, wherein one single dose of the vaccine is administered to arrive at the protection.

Description

(1) FIG. 1 schematically shows the IHC score after PCV2b challenge.

(2) FIG. 2 schematically shows the IHC score after PCV2d challenge.

EXAMPLES

Example 1: Methods for Determining the Amount of ORF2 Protein in a Sample

(3) The amount of ORF2 encoded protein can be determined by any art known method. EP 1926496, in example 4 describes a common method based on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and coloration with Coomassie Briliant Blue. This type of method for determining the protein content of a sample is commonly known and for example described in The Protein Protocols Handbook, 2.sup.nd edition, September 2002, edited by J. M. Walker, Humana Press Inc., Totowa, N.J. Chapter 29, pp 237-242. The full length protein has a length of about 26-27 kDa. Common truncated versions have length typically 2-4 kDa lower. If deemed necessary (for example when other proteins of comparable length are also present in the sample), the correct bands with the ORF2 proteins can be identified using anti-ORF2 antibodies that can be visualised using labelled antibodies.

(4) In an alternative method, the amount of ORF2 encoded protein (also capable of detecting full length protein as well as truncated versions thereof), is established via multiple reaction monitoring (MRM) mass spectrometry using purified ORF2 protein as a standard. For this, the protein is firstly digested, for example by using trypsin. Thereafter the peptides are separated, for example by using liquid chromatography, and then the peptides are subjected to mass spectrometry using a signal peptide (in this case “VEFWPCSPITQGDR”) for normalising variations in signal. The signal peptide observed is unique to PCV2 ORF2 digested with trypsin. An elaborate description of the method is given in a declaration of Merill Schaeffer, Ph. D, as sent to the European Patent Office as document “D60” on 23 Aug. 2016 by Boehringer Ingelheim Vetmedica GmbH, represented by Hoffmann Eitle, in its opposition against EP 1926496.

(5) Applying these methods to Porcilis PCV and CircoFLEX reveal that in each case the amount of ORF2 encoded protein is well above 20 μg per dose.

Example 2: Establishment of Cross Protection of ORF2 Protein of PCV2a, 2b and 2d

(6) As is known from the art, at a 100% level of existing vaccines, the ORF2 protein of PCV2a and other genotypes provides adequate cross protection, all at comparable levels. In the following experiment, vaccines corresponding to Porcilis PCV were used, diluted either 4 times (“25%” version) or 16 times (“6.25%”) version when compared to the vaccine at its full (“100%”) dose. The vaccine was made in line with the teaching of EP1926496, having a full dose of about 80 μg of ORF2 encoded protein.

(7) The study was performed to evaluate the efficacy of veterinary vaccines with different PCV2 strains (PCV2a, PCV2b and PCV2d) against PCV2b and PCV2d challenge to investigate whether at some low dosis there is a preference for an ORF2 protein of a particular genotype, in particular when assessing cross protection properties.

(8) For this study 2 separate experiments were performed, each experiment involving 70 animals. The 140 animals were derived from 14 sows and were allotted to 14 treatment groups of 10 animals each. The piglets had detectable levels of anti-ORF2 antibodies ranging from low to very high, the average value for all animals being substantially the same for both experiments (about 5.8 at time of vaccination). The piglets were vaccinated intramuscularly with 2 ml when they were approximately three weeks old. In each experiment of 70 animals, groups 1 through 6 were vaccinated with a PCV2 vaccine with different concentrations of ORF2 encoded PCV2 and strains of different genotypes PCV2, as follows:

Experiment 1

(9) Piglets from group 1 were vaccinated with a 25% dose of PCV2a strain (about 20 μg);

(10) Piglets from group 2 were vaccinated with a 6.25% dose of PCV2a strain (about 5 μg);

(11) Piglets from group 3 were vaccinated with a 25% dose of PCV2b strain (about 20 μg);

(12) Piglets from group 4 were vaccinated with a 6.25% dose of PCV2b strain (about 5 μg);

(13) Piglets from group 5 were vaccinated with a 25% dose of PCV2d strain (about 20 μg);

(14) Piglets from group 6 were vaccinated with a 6.25% dose of PCV2d strain (about 5 μg).

(15) Piglets from control group 7 were not vaccinated.

Experiment 2

(16) Piglets from group 1 were vaccinated with a 25% dose of PCV2a strain (about 20 μg);

(17) Piglets from group 2 were vaccinated with a 6.25% dose of PCV2a strain (about 5 μg);

(18) Piglets from group 3 were vaccinated with a 25% dose of PCV2b strain (about 20 μg);

(19) Piglets from group 4 were vaccinated with a 6.25% dose of PCV2b strain (about 5 μg);

(20) Piglets from group 5 were vaccinated with a 25% dose of PCV2d strain (about 20 μg);

(21) Piglets from group 6 were vaccinated with a 6.25% dose of PCV2d strain (about 5 μg).

(22) Piglets from control group 7 were not vaccinated.

(23) Two weeks post vaccination all animals were transported to Intervet International B.V. challenge facilities. At three weeks post vaccination (6 weeks of age) all animals were challenged using 5.0 log 10 TCID50/mL of wild-type PCV2b challenge virus strain (Experiment 1) or a wild type PCV2d strain (Experiment 2) applied intranasally at 3 mL per nostril. Three weeks post challenge, all animals were necropsied and mesenteric lymph node, tonsil and inguinal lymph node were sampled for the detection of PCV2 virus via an immunohistochemistry (IHC) analysis, using a scoring ranging from 0 (no specific positive staining) to 3 (more than 50% of the lymphoid follicles contain cells with positive staining).

(24) The results are indicated in FIGS. 1 and 2. As becomes clear from FIG. 1 homologous protection of a PCV2b vaccine, as expected, is better than heterologous protection. PCV2a seems to provide a slightly better cross protection against PCV2b than PCV2d, although at the lowest dose there is no difference.

(25) Surprisingly, as FIG. 2 shows, the ORF2 encoded protein of PCV2b provides heterologous protection at the low doses tested at a level which is the same as the homologous protection of the ORF2 encoded protein of PCV2d. This is in contrast with the heterologous protection offered by the ORF2 encoded protein of PCV2a which provides less good protection at the low doses tested. So in contrast with common knowledge that ORF2 protein of PCV2a and PCV2b genotypes provides an equal level of cross protection, it appears that below 20 μg, a dosis which is known to be needed in order to effectively break through MDA against PCV2 (see EP 1926496), the ORF2 encoded protein of PCV2b provides significantly better cross protection that the ORF2 encoded protein of PCV2a. Also, despite the presence of MDA's, adequate protection could be arrived at when compared to control at these low dosis levels, in particular when using the ORF2 encoded protein of PCV2b.