VACCINE TO PROTECT A PIG AGAINST ACTINOBACILLUS PLEUROPNEUMONIAE

Abstract

The present invention pertains to a vaccine to protect a pig against an infection with Actinobacillus pleuropneumoniae, the vaccine comprising an RTX toxin of Actinobacillus pleuropneumoniae recombinantly expressed by a baculovirus, and a pharmaceutically acceptable carrier.

Claims

1. A vaccine to protect a pig against an infection with Actinobacillus pleuropneumoniae, the vaccine comprising an RTX toxin of Actinobacillus pleuropneumoniae recombinantly expressed by a baculovirus, and a pharmaceutically acceptable carrier.

2. The vaccine of claim 1, wherein the RTX toxin is ApxI.

3-6. (canceled)

7. A method of protecting a pig against an infection with Actinobacillus pleuropneumoniae, by administering a vaccine comprising an RTX toxin of Actinobacillus pleuropneumoniae recombinantly expressed by a baculovirus and a pharmaceutically acceptable carrier, to the pig.

8. The method of claim 7, wherein the RTX toxin is ApxI.

9. The method of claim 8, wherein the vaccine is administered systemically.

10. The method of claim 8, wherein the vaccine is administered intramuscularly

11. The method of claim 8, wherein the vaccine is administered intradermally.

12. The method of claim 7, wherein the vaccine is administered systemically.

13. The method of claim 7, wherein the vaccine is administered intramuscularly

14. The method of claim 7, wherein the vaccine is administered intradermally.

Description

EXAMPLES

Example 1: Recombinant Expression of ApxI

[0021] Construction of transfervector pFastbac-ApxIA

[0022] The rApxIA gene was synthesized based on the ApxIA amino acid sequence of the Actinobacillus pleuropneumoniae strain 4074, Swiss prot accession number: P55128. The gene was codon optimized for baculovirus polyhedrin usage and a Kozak sequence (TATAAAT) and a 3′ hexahistidine tag were included. The rApxIA-His gene was cloned behind the polyhedrin promoter of plasmid pFastbacl (Life Technologies, Carlsbad,

[0023] USA) as a BamHI fragment, resulting in plasmid pFastbac-ApxIA TAT.

[0024] Generation of recombinant baculovirus BacdCCApxIA-TAT.

[0025] Recombinant baculovirus was generated using the plasmid as described here above in the Bac to Bac system (Life Technologies, Carlsbad, USA) according to manufacturer's protocol. E. coli cells used for transformation contained the parental baculovirus with a deletion of the chitinase and v-cathapsin genes (Kaba SA, Salcedo AM, Wafula PO, Vlak JM, van Oers MM. J Virol Methods. 2004 Dec. 1;122(1):113-8.) E. coli bacteria were grown in an animal component free (ACF) medium.

[0026] BacdCCApxIA-TAT DNA was isolated from E. coli and used for transfection into Spodoptera frugiperda (Sf)9-900 cells. Sf9-900 cells were grown in an animal compound free medium. Expression of the 110 kDa ApxIA protein was confirmed using SDS-PAGE gels, Western blots and Immune Fluorescence Assays using an anti-histidine-tag monoclonal antibody. Transfection supernatants were once amplified and the resulting virus stock was used for all further virus cultures.

[0027] Histag purification baculovirus produced ApxIA.

[0028] Sf9-900 cells were infected with the BacdCCApxIA-TAT baculovirus with a multiplicity of infection of 0.1, followed by culturing for 4 to 5 days at 27.5° C. From those cells rApxIA-His protein was purified using the AKTA Avant protein purification system (GE Healthcare Life Sciences, Cleveland, USA) using a HIStrap FF column. Lysates were made from insect cells infected with BacdCCApxIA-TAT using Lysis buffer Triton X114 (0.15 M NaCl, 10 mM Tris-HC1 pH8, 2.5 mM CaC12, 1 mM DTT, 1% triton X114). After centrifugation the pellet was resuspended in wash buffer (50 mM Tris pH=8, 300mM NaCl, 6 M Ureum, 2.5 mM CaC12, 1 mM DTT) and filtered using a 0.45 μM filter before applying to the AKTA Avant. After equilibration of the column with denaturing wash buffer the sample was applied twice to the column at a rate of 3 ml/min. Protein bound to the column was renatured with redox buffer (50 mM Tris pH=8, 300mM NaCl, 2.5 mM CaC12, 0.1% oxidized glutathione, 0.01% reduced glutathione, 1mM DTT) and eluted from the column with a linear imidazole gradient, using elution buffer (50mM Tis pH=8, 300 mM NaCl, 2.5 mM CaC12, 1mM DTT, 500 mM Imidazole). Purified ApxI protein was dialyzed against dialysis buffer (50mM Tris pH=8, 300mM NaCl, 2.5 mM CaC12) using Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, Waltham, USA) with several buffer changes.

Example 2: Vaccine Efficacy

[0029] Vaccine formulation

[0030] Two different vaccines were made for the study. A first vaccine comprised purified baculovirus expressed ApxI as obtained with a method described under Example 1. A second vaccine for use as a positive control was comparable with the commercially available vaccine Porcilis® APP, comprising ApxI and ApxII purified from the culture supernatant of A. pleuropneumoniae (and thus complexed with LPS), also denoted as “native ApxI +ApxII”. The study vaccine different from the commercially available vaccine Porcilis® APP in that it did not contain the ApxIII toxin. However, for the challenge with a serotype 10 field isolate, this is not relevant (serotype 10 does not produce ApxIII). The antigens were were mixed with a mineral oil-containing adjuvant (XSolve, available from MSD Animal Health, Boxmeer, The Netherlands) at a final concentration of 25 μg/m1 for each antigen.

[0031] Vaccination protocol

[0032] Three groups of eight piglets from an A. pleuropneumoniae free herd were used. The two vaccines were administered intramuscularly as a 2 ml dose at five and nine weeks of age.

[0033] The remaining eight piglets were injected with PBS and used as non-vaccinated negative control group. At regular intervals, blood samples were taken for serology.

[0034] A. pleuropneumoniae serotype 10 challenge infection

[0035] At approximately 12 weeks of age, all 24 piglets received a challenge infection.

[0036] The challenge compound was a serotype 10 field isolate (strain HV211) of A. pleuropneumoniae. The challenge culture was freshly prepared prior to challenge. The piglets were challenged with A. pleuropneumoniae by the aerosol route. The aerosol was given by means of a Devilbis Nebulizer (total amount 30 ml). The challenge dose was determined by plate counting and the suspension was found to contain 7.0×10.sup.8 CFU/ml.

[0037] After challenge, respiratory disease and other abnormalities were scored daily for a period of seven days, after which the surviving animals were necropsied.

[0038] The scoring system used was as follows:

TABLE-US-00001 0 = normal 1 = shivering 2 = depressed 3 = increased respiration rate 4 = vomiting 5 = diarrhoea 6 = coughing 7 = abdominal respiration 8 = dyspnoea

[0039] For animal welfare reasons, animals that were found to be moribund were euthanized. Pigs that were found dead or had been euthanized were inspected for typical Actinobacillus pleuropneumoniae lesions, of which the extent per lung lobe was scored on a 0-5 scale (max score per animal: 35). Also, the lungs of the surviving animals were scored at day seven post challenge.

[0040] Results

[0041] All pigs were serologically negative at the start of the experiment, and at the time of challenge the vaccinated animals had seroconverted for ApxI, as measured by ELISA with native ApxI as the coating antigen. The main antibody titres were loge 12.9±1.6 and 13.1±1.1 for the Baculo-ApxI and native ApxI+ApxII groups, respectively. Table 1 provides a summary of the challenge results, and Table 2 shows the clinical abnormalities observed for the individual pigs.

TABLE-US-00002 TABLE 1 Protection of the piglets Mortality Mean lung Vaccine [n/n.sub.tot] lesion score Baculo-ApxI 2/8* 1.5 ± 3.1* Native ApxI + 1/8* 0.4 ± 1.1* ApxII PBS 8/8  20.3 ± 5.7  *significantly different from controls (p < 0.05, Fischer's exact-test for mortality rate and Mann-Whitney U-test for lesion scores)

TABLE-US-00003 TABLE 2 Clinical abnormalities per group Baculo-ApxI Native ApxI + ApxII PBS Dead Dead Dead pig Abnorm.sup.1 day.sup.2 Abnorm.sup.1 day.sup.2 Abnorm.sup.1 day.sup.2 1 2 — 2 — 2, 3, 7, 8 1 2 2, 7 3 2, 3, 7 1 2, 3, 7, 8 1 3 0 — 0 — 2, 3, 7, 8 1 4 2, 5 — 0 — 2, 3, 7, 8 1 5 0 — 0 — 2, 3, 7, 8 1 6 2, 3, 7 3 0 — 2, 3, 7, 8 1 7 0 — 2, 7 — 2, 3, 7, 8 1 8 0 — 0 — 2, 3, 7, 8 1 .sup.1Clinical abnormalities observed (scored as described under challenge infection) .sup.2Died/euthanized on indicated day post challenge

[0042] Significant reductions of clinical signs, mortality and lung lesion were observed for both vaccinated groups. The difference between the two vaccine groups was not statistically significant. It can therefore be concluded that the vaccine containing RTX toxin recombinantly expressed by baculovirus provides protection that is similar to the protection provided the commercial vaccine Porcilis® APP.