VACCINE FOR MYCOPLASMA BOVIS

Abstract

Currently, there is no effective vaccination against M. bovis on the market, and treatment options become increasingly limited due to restrictions in the use of, and resistance to antibiotics. This is complicated by results that demonstrate the induction of vaccine-enhanced disease, upon the use of certain M. bovis proteins as a vaccine. Thus, there is an urgent need for an effective and safe M. bovis vaccine. A novel vaccine composition was found that comprises one or more recombinant proteins which (combined) contain one or more epitopes from each of a set of specific M. bovis proteins. Vaccines based on these recombinant proteins were found to be safe, and were effective in protecting ruminants against infection and disease resulting from a severe challenge infection with M. bovis, as was apparent from a strong reduction of lung damage and colonisation of the trachea.

Claims

1. Composition comprising one or more recombinant proteins, characterised in that the recombinant protein or the combination of recombinant protein comprises at least one epitope from each of the Mycoplasma bovis (M. bovis) proteins with the GenBank accession number: WP_014829937, WP_075271052, WP_013456547, SBO45938, WP_013455936, WP_075271207, WP_013954974, WP_013954588, WP_013456028, WP_013954511, WP_075271115, WP_013456252, and WP_041309176, or from a homologue of said M. bovis proteins.

2. The composition according to claim 1, characterised in that the recombinant protein or the combination of recombinant proteins also comprise at least one epitope from each of the M. bovis Vsp proteins with the GenBank accession number: SBO46569, SBO46572, SBO46576, and SBO46580, or from a homologue of said M. bovis Vsp proteins.

3. The composition according to claim 1, characterised in that the recombinant protein or the combination of recombinant proteins also comprise one or more additional sequences selected from: signal-, transmembrane-, anchor-, linker-, spacer-, marker-, and cleavage sequences.

4. The composition according to claim 1, characterised in that the recombinant protein or the combination of recombinant proteins comprise each of the epitopes from SEQ ID NO: 8 through 25, or a homologue of said epitopes.

5. The composition according to claim 4, characterised in that the recombinant protein or the combination of recombinant proteins also comprise each of the epitopes from SEQ ID NO: 26 through 30, or a homologue of said epitopes.

6. Recombinant vector capable of expressing the recombinant protein or the combination of recombinant proteins as defined in claim 1, and wherein said vector is selected from a nucleic acid, a replicon particle (RP), a virus, and a bacterium.

7. The recombinant vector according to claim 6, characterised in that: a. the nucleic acid is a DNA expression plasmid or an RNA molecule; b. the RP is an Alphavirus RP; c. the virus is selected from the group consisting of: a Herpesvirus, a Poxvirus, a d. Retrovirus, a Paramyxovirus, a Rhabdovirus, a Baculovirus and an Adenovirus; or d. the bacterium is selected from the group consisting of the genera: Escherichia, Bacillus, Salmonella, Caulobacter, Lactobacillus, and Mycoplasma.

8. Host cell comprising the recombinant vector according to claim 6.

9. A method for the manufacture of a composition comprising one or more recombinant proteins, the method comprising obtaining the recombinant protein or the combination of recombinant proteins from a vector according to claim 6 wherein the recombinant protein or the combination of recombinant protein comprises at least one epitope from each of the Mycoplasma bovis (M. bovis) proteins with the GenBank accession number: WP_014829937, WP_075271052, WP_013456547, SBO45938, WP_013455936, WP_075271207, WP_013954974, WP_013954588, WP_013456028, WP_013954511, WP_075271115, WP_013456252, and WP_041309176, or from a homologue of said M. bovis proteins.

10. A vaccine for reducing infection or disease caused by M. bovis, the vaccine comprising the composition according to claim 1, and a pharmaceutically acceptable carrier.

11. The vaccine according to claim 10 characterised in that the vaccine comprises an adjuvant.

12. A Method for the manufacture of a vaccine, the method comprising the admixing of the composition according to claim 1, with a pharmaceutically acceptable carrier.

13. (canceled)

14. (canceled)

15. A Method for reducing infection or disease caused by M. bovis in a target, the method comprising administering to said target the vaccine according to claim 10.

Description

LEGEND TO THE FIGURES

[0271] FIG. 1: Results from vaccination-challenge experiment testing different compositions with recombinant proteins according to the invention. Displayed on the vertical axis are the relative lung lesion scores per animal. The short horizontal bars indicate the median value found for each group. Details are described in Example 2.

EXAMPLES

Example 1: Preparation of Recombinant Proteins of M. bovis Epitopes

[0272] Recombinant proteins comprising the epitopes as defined for the invention were constructed, whereby the epitopes to be expressed were grouped in a few sets as follows (epitope numbers as indicated in Tables 3 and 4 above): [0273] Set 1: epitope numbers 1-10 (SEQ ID NO: 8-17), [0274] Set 2: epitope numbers 11-18 (SEQ ID NO: 18-25), and [0275] Set 3: epitope numbers Vsp1-Vsp5 (SEQ ID NO: 26-30).

[0276] In the nucleotide sequence constructs encoding the epitope-containing recombinant protein(s) for the invention, these epitope sequences were interspersed with linker sequences Link1 through LinkS (SEQ ID NO: 2-6), in an alternating way as described herein.

[0277] Further, the epitope sets 1 and 2 were each prepared in two ways numbered a and b, whereby the order of the epitopes was mixed-up; thus resulting in sets 1a, 1b and 2a, 2b respectively. These are the encoding nucleotide sequence constructs described in SEQ ID NO: 36-40, encoding the recombinant proteins SEQ ID NO: 31-35, respectively.

[0278] These sets of epitopes were then expressed by a recombinant vector, specifically an M. pneumoniae (Mpneumo) bacterium. For the expression by the Mpneumo vector each construct was given a signal sequence to target expression to the bacterial surface. The signal sequence was put at some distance by two more copies of Linker 1 (SEQ ID NO: 2). Also, to be able to detect expression and isolate the recombinant proteins, the different constructs were provided with a nucleotide sequence encoding a marker sequence, to be expressed at their C-terminus. Constructs 1a, 2a and 3 were provide with a Flag-tag (SEQ ID NO: 7), and constructs 1b and 2b, with a 6x His-tag.

[0279] This resulted in constructs encoding recombinant proteins for the invention, with the following schematic lay-outs:

[0280] Construct 1a: [0281] Signal-3xlink1-Ep1-link2-Ep2-link3-Ep3-link4-Ep4-link5-Ep5-link1-Ep7-link2-Ep6-link3-Ep8-link4-Ep9-link5- Ep10-link1-FlagTag

[0282] Construct 1b: [0283] Signal-3xlink1-Ep5-link2-Ep7-link3-Ep6-link4-Ep9-link5-Ep10-link1-Ep8-link2-Ep1-link3-Ep2-link4-Ep4-link5-Ep3-link1-HisTag

[0284] Construct 2a: [0285] Signal-3xlink1-Ep11-link2-Ep12-link3-Ep12-link4-Ep14-link5-Ep15-link1-Ep16-link2-Ep17-link3-Ep18-link1-FlagTag

[0286] Construct 2b: [0287] Signal-3xlink1-Ep17-link2-Ep18-link3-Ep14-link4-Ep15-link5-Ep16-link1-Ep13-link2-Ep12-link3-Ep11-link1-HisTag

[0288] Construct 3: [0289] Signal-3xlink1-Vsp4-link2-Vsp5-link3-Vsp2-link4-Vsp3-link5-Vsp1-link1-FlagTag

[0290] The nucleic acids encoding the various constructs for the invention were designed and codon optimized towards the codon usage table of Mpneumo (as described by Weber et al., 2020, supra) and prepared by a commercial supplier. The sequences were verified, and the synthetic genes were inserted in a pMTn series transposon cloning plasmid; the plasmids were electroporated into Mpneumo resulting in the random transposon insertion of the epitope constructs into its genome, essentially as described in Pich et al. (2006, Microbiology, vol. 152, p. 519-527).

[0291] The encoding nucleotide sequences for these constructs were inserted into the Mpneumo vector in combinations: 1a and 1b in one; 2a and 2b in another, and 3 in yet another Mpneumo bacterial expression vector.

[0292] The promoter used to drive the expression of the double- or single epitope-construct inserts was a 22 nucleotide section from the putative Mg438 gene of M. genitalium, as described in Pich et al., 2006 (supra).

[0293] The resulting recombinant Mpneumo bacteria were then cultured in a growth medium resembling Friis medium with 10% porcine serum added, and passaged once in the same medium for expansion before harvest. Expression of the fusion constructs was checked by Western blot, by detecting the Flag- or His-tag respectively using monoclonal antibodies.

[0294] The bacteria were inactivated with binary ethylenimine (BEI) at 0.8% v/v concentration for 24 hours at 37 C., with constant shaking. This was repeated once more. Next the BEI was neutralised with thiosulfate by incubation for 24 hours at 37 C. with constant shaking.

[0295] The inactivated bacteria were quantified by protein content using a BCA assay. Next the mixture was formulated with the described aluminium-hydroxidesaponin adjuvant, by first mixing the antigen with aluminium hydroxide under constant stirring at room temperature, followed by the addition of a solution containing saponin, so that the final composition had 1.7% w/v of aluminium hydroxide, and 0.03 v/v % saponin. After complete mixing, the pH was adjusted to 7.3. This adjuvated vaccine was administered to calves.

Example 2: Vaccination-Challenge Experiment with Recombinant Proteins Comprising M. bovis Epitopes

[0296] 2.1. Introduction

[0297] This experiment was done to determine the safety and efficacy of an M. bovis vaccine based on recombinant proteins comprising epitopes from specific M. bovis proteins. The main way to determine efficacy of protection against an M. bovis challenge, is by scoring the lung lesions induced and the bacterial load of challenge bacteria in the trachea. As vector for expression of the recombinant proteins for the invention served Mpneumo bacteria, which were inactivated and mixed with adjuvant prior to vaccination, as described in Example 1.

[0298] 2.2. Material and Methods

[0299] 2.2.1. Experimental Set-Up

[0300] Fifteen calves were included in this study, divided over 3 groups of 5 animals. Two groups received different mixtures of the inactivated Mpneumo bacteria expressing the recombinant proteins for the invention with M. bovis epitopes. These were formulated with aluminium-hydroxidesaponin adjuvant. Mix 1 contained 200 g of each of 2 different inactivated bacterial cultures, one culture expressing constructs 1a and 1b, and one culture expressing constructs 2a and 2b, as described in Example 1. Mix 2 had the same compounds as in mix 1, but in addition also had 200 g of bacteria expressing construct 3. A negative control group was also included, that group received a mock vaccine of PBS in adjuvant.

[0301] All calves were vaccinated according to a prime-boost schedule with a 3 week interval, and received a 2 ml dose given intra-muscularly in the neck. Blood samples were taken prior to vaccination, throughout the study, and at necropsy after euthanisation, to allow serology testing. Additionally after vaccination, health assessments were done, to judge the systemic response of the calves and the occurrence of local reactions.

[0302] Two weeks after boost, the calves were given a challenge infection using virulent M. bovis, on three consecutive days. Necropsy was performed at about two weeks after challenge.

[0303] 2.2.2.Treatments of Experimental Animals

[0304] Holstein-Frisian calves of 4-6 weeks old, identified individually by ear tag, were assembled on one farm, and given a primo vaccination, and a booster vaccination 3 weeks later. After two more weeks they were transported to containment facilities, where they were given the challenge inoculations. At 16 days after the last challenge inoculation, the calves were sedated, euthanised and necropsied.

[0305] The calves were given examinations for general health by a veterinarian, both before vaccination and before challenge, and only calves that were clinically healthy at start, were used in the experiment. The calves were fed standard rations, and water was available ad libitum. Checks for any anti-M. bovis antibodies prior to vaccination was done on blood samples taken 1 week before the primo vaccination, which were tested using the BIO K260Monoscreen AbELISA Mycoplasma bovis kit (BIO-X Diagnostics S.A.).

[0306] Division over the groups was such that the average age in each group was the same.

[0307] Before- and 3 days after each vaccination, animals were palpated at the injection site to determine any local reactions. Also rectal temperature readings were taken before and after vaccinations.

[0308] Blood samples (serum and heparin) were taken from the jugular vein, on the day of- and prior to the vaccinations, at day of first challenge inoculation, and at the end of the experiment.

[0309] At necropsy, percentage lung lesion score was recorded for each calf individually using the procedure described by Jericho & Langford (supra). Also two samples were taken from the lungs for histology, one from the left- and one from the right cranial lobe. Tracheal swabs were taken from an area of 1 cm.sup.2 just before the bifurcation point, using a mould for consistent sampling areas between animals. The samples were taken by single swipe. Further, the right cranial lung lobe of animals from group 1 was flushed using 10 ml SP4 medium with 100 g/ml ampicillin.

[0310] 2.2.3. Challenge

[0311] 200 l of M. bovis strain JF4278, low passage stock, was inoculated into 20 ml of SP4 medium with 100 g/ml ampicillin, and incubated at 37 C. for 24 hours. Subsequently 8 ml of the overnight culture was subcultured into 800 ml SP4 medium with 800 g/ml ampicillin, and incubated at 37 C. for 24 hours. The challenge material was plated on Mycoplasma agar (Mycoplasma Experience) plates, for quantification and to test the viability and absence of contaminations. Plates were incubated at 37 C. in 5% CO.sub.2 for five days.

[0312] All animals were challenged intratracheally once daily on three consecutive days, by way of a tube via the nose, and reaching to the bifurcation of the trachea. For each inoculation 30 ml challenge culture was administered having about 10{circumflex over ()}9 M. bovis JF4278 cells/ml.

[0313] 2.2.4.Laboratory Procedures

[0314] Plain blood- and heparin blood samples were taken to determine the reaction of the animals to the vaccines. After euthanising the animals, several samples and swabs were taken during necropsy. Lung lesion scores were assessed and compared between the groups. Nasal and tracheal swabs were taken as well during necropsy, to measure the M. bovis load in the animals. These results were used to determine if there was a good vaccine take, to check if there was no co-infection, and to determine the efficacy of the vaccines.

[0315] 2.3. Results

[0316] Vaccine-take was confirmed by the detection of antibodies against the vector bacteria, in blood samples from the experimental animals. The animals in group 1 (PBS vaccine) showed only background serological response against the Mpneumo vector, with an average Log2 titre of 7.6 at day of challenge. However both vector-vaccinated groups 2 and 3 showed a significant anti-vector response, as both groups had an average antibody titre of 12 Log2, at day of challenge.

[0317] This demonstrates that all the vaccinated groups had indeed received the bacterial vector-expressed antigen, and this had mounted an immune response.

[0318] After the vaccinations, no clear signs of vaccination-reactions, local or systemic, such as swelling, fever, malaise, etc., were observed.

[0319] Trachea Swabs

[0320] The trachea swabs taken at 16 days post challenge were plated on Mycoplasma agar and quantified, and results are presented in Table 5. Numbers given are the averages per group in CFU/ml, with their standard deviation (SD).

[0321] As is clear from these results, the vaccination as applied to Group 2 already strongly reduced the colonisation by the M. bovis challenge bacteria by 96%. The vaccine of Group 3 was even more effective, and reduced colonisation by 99%.

TABLE-US-00005 TABLE 5 Results of challenge bacteria re-isolation CFU/ml Group Average SD 1 - PBS 73847 152365 2 - constr. 1a-1b and 2a-2b 2550 625 3 - constr. 1a-1b, 2a-2b, and 3 53 74

[0322] Lung Lesion Scores

[0323] The total relative lung lesion scores (LLS) at 16 days after challenge were determined as described. Results are presented in FIG. 1. Because of the nature of these experiments, some variability in the individual scores is inevitable. Therefore it is best to focus on the median LLS per group; these were: [0324] Group 1 (PBS): 3.1 [0325] Group 2 (mix of 1a, 1b, 2a, and 2b): 1.4 [0326] Group 3 (mix of 1a, 1b, 2a, 2b, and 3): 0.7

[0327] These results present the same trend as was found for the trachea swabs: the vaccine administered to Group 2 (the mixture of recombinant proteins 1a-1b and 2a-2b) was already capable of strongly reducing the lung lesions induced by challenge infection by 55%, as compared to the mock vaccinated-challenged calves. Remarkably, the vaccine administered to Group 3 (the mixture of recombinant proteins 1a-1b, 2a-2b, and 3) was even more effective, and reduced LLS by 77%. Also this reduced the spread among the group 3 calves.

[0328] Such strong reduction of lung lesions scores, after a severe M. bovis challenge infection, has not been reported before.

[0329] 2.4. Conclusions

[0330] No indications of vaccine-enhanced disease were observed in the vaccinated groups, nor any local reactions or undesired systemic response. This indicated that the vaccines of the invention are safe.

[0331] Group 2 and in particular group 3 had clearly lower lung lesion scores, than did the mock-vaccinated-challenged group 1. In addition, group 2 and in particular group 3 also had lower average loads of challenge bacteria in the trachea, than the calves of group 1 had.

[0332] Taken together these findings strongly support the safety and the efficacy of the vaccines according to the invention, against infection and disease caused by M. bovis infection.