Mycobacterium avium subsp. paratuberculosis, the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. The first generation vaccines, composed of whole mycobacteria (killed or live-attenuated) in oily adjuvant confer a partial protection, by delaying the excretion of the mycobacteria in the faeces and by reducing the number of animals progressing to the clinical phase. However, they interfere with the diagnostic test of bovine tuberculosis making their use in cattle problematic. It is important to develop a new generation of vaccines which will protect better from the infection and which will not interfere with the screening tests of tuberculosis and paratuberculosis. M. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. This particularity hampers infection, immunity and vaccination studies. In order to facilitate the screening of new vaccine candidates we have developed a luminescent M. paratuberculosis expressing luxAB genes of Vibrio harveyi and we have described its use for vaccine testing in an experimental mouse model, replacing fastidious and costly enumeration of CFU on agar by easy and rapid luminometry. Using this luminescent isolate, we have re-evaluated the effect of murine Slc11a1 (formerly called Nramp1) polymorphism on susceptibility to M. paratuberculosis. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results confirm that, as for M. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c H-2d, congenic BALB.B10 H-2b (BALB/c background; H-2b), C57BL/6 H-2b, and beige C57BL/6bg/bg mice (all four homozygous for the susceptible Slc11a1s allele) bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection. In DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both homozygous for the resistant Slc11a1r allele) and in (C57BL/6 × DBA/2)F1 mice (heterozygous Slc11a1s/r), bacterial numbers decreased more than 10-fold during the first 4 weeks of infection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6bg/bg mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice. In our screening of new vaccine candidates, we evaluated the immunogenicity and the protective potential of ten M. paratuberculosis specific proteins identified by two differents approaches. The first consisted in the systematic proteomic identification of proteins present in M. paratuberculosis culture filtrate (CF), followed by the selection of M. paratuberculosis-specific proteins by BLAST query on available mycobacterial genome and in the immunoproteomic analysis of both M. paratuberculosis extract and CF, using sera from M. paratuberculosis-infected and M. bovis-infected cattle. The second “in silico” approach consisted of the comparison of M. paratuberculosis sequences with all mycobacterial genomes and only sequences which not have homology were selected. A third selection was carried out using three different T cell epitope prediction programs, ‘‘Tsites’’, ‘‘BIMAS’’ and ‘‘SYFPEITHI’’, in order to select the most promising antigenic sequences, with respect to future vaccine development. The ten candidates selected were MAP0586c, MAP4308c, MAP1693c, Ag3 (CF036), MAP2677c, (identified by post-genomic and immuno-proteomic analysis of M. paratuberculosis secretome) and Ag5, Ag6, MAP1637c, MAP0388 and MAP3743 (identified by bioinformatic in silico screening of the M. paratuberculosis genome). Each antigen was cloned into the eukaryotic expression vector V1.Jns-tPA and plasmids were used to vaccinate BALB/c and C57BL/6. Plasmid DNA vaccination is a powerful and easy tool for the high throughput screening of the vaccine potential of protein antigens and DNA vaccines are particularly promising for the prevention of infections caused by intracellular pathogens, precisely because of the ease with which they induce strong Th1 biased cellular immune responses without the use of additional adjuvants. Each candidate was also cloned into the prokaryotic expression vector pQE-80L and purified as hexa-histidine taggedrecombinant protein by Immobilized Metal Affinity Chromatography (IMAC). Strong, antigen-specific IFN- and IL-2 responses were induced in mice vaccinated with plasmid DNA encoding MAP0586c, MAP4308c, MAP1693c, MAP1637c, MAP0388 and MAP3743. In contrast, T cell responses in M. paratuberculosis infected mice were directed preferentially against MAP0586c and Ag5 and to a lesser extent against MAP3743. Interestingly, vaccination with plasmid DNA encoding MAP0586c induced only very weak levels of IgG1 antibodies in C57BL/6 and BALB/c mice. Vaccination with DNA coding for MAP4308c, MAP1693c and MAP2677c stimulated the production of antibodies in both strains of mice. DNA vaccinated BALB/c mice showed also lower antibody responses against three in silico identified antigens i.e. Ag5, Ag6 and MAP1637c. Finally, one of the tested DNA vaccines, i.e. DNA vaccine encoding MAP0586c, the putative trasnglycocylase, could protect BALB/c mice against subsequent challenge with luminescent M. paratuberculosis to the same extent as the Mycobacterium bovis BCG vaccine, indicating that this protein is an interesting vaccine candidate that warrants further investigation.
| Date of Award | 16 Mar 2012 |
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| Original language | French |
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| Awarding Institution | |
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| Supervisor | JEAN-JACQUES LETESSON (Supervisor), Benoit Muylkens (President), Kris Huygen (Jury), Ruddy Wattiez (Jury) & Lieve HERMAN (Jury) |
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