In the present study we demonstrate that immunization of C57BL/6 mice using a fusion construct of four MAP genes delivered by differing routes in combinations of plasmid and viral vectors can achieve significant attenuation of pre-existing experimental MAP infection as well as some prophylaxis against subsequent challenge ().
Summary of mean fold reductions in MAP loads in spleen and liver in response to vaccination
The 4 MAP genes within the 95 kDa polypeptide fusion construct were selected on the basis of known constitutive secreted expression or predicted presence at the mycobacterial cell surface. AhpC is a secreted virulence factor constitutively expressed in MAP and is shared by other pathogenic mycobacteria 
. AhpC is up-regulated during transition of mycobacteria to a state of chronic persistence 
and is involved in oxidative stress defence within host macrophages. Gsd is a predicted cell surface fucosyl transferase 
involved in peripheral oligosaccharide biosynthesis of serovar-2-specific glycopeptidolipid (GPL) in M. avium
. However, the genes for the biosynthesis of the intermediate portion of M. avium
GPLs between the lipopeptide core and peripheral oligosaccharides are not present in MAP and GPLs are reported to be absent from these chronic enteric pathogens 
. The structure and functions of the acceptor substrates for peripheral fucosylation by Gsd in MAP have yet to be elucidated. Preliminary work in our lab using microarray analysis has suggested that gsd
is up-regulated when MAP enters the intracellular environment.
p12 is the carboxyterminal 17 kDa fragment of p43 encoded by the MAP specific insertion sequence IS900 
. This fragment is released into media from MAP cultures as well as from rec.E.coli
expressing IS900 
probably by proteolytic cleavage of membrane bound p43. Mpa is a putative acetyltransferase present together with Gsd in the pathogenicity associated GS element. The 10 predicted transmembrane regions within the structure of Mpa also suggest a pore function 
. An intact Mpa gene is unique to MAP. It has homologues in Shigella flexneri
and Salmonella typhimurium
that are cell wall associated and determine serotype specificity and virulence 
. Only the extracellular and intracellular loop regions of Mpa were included in the fusion construct to maximise the solubility of the fusion polypeptide removing hydrophobic transmembrane sequences. Stable full length expression of the 95 kDa fusion polypeptide HAV was demonstrated from all three vectors. Full length expression in vivo was evidenced by consistent ELISPOT recognition of the strong murine T cell epitope GFAEINPIA located near the C-terminal end of HAV.
The profiles of T-cell recognition of vaccine-specific recombinant antigen and synthetic peptides in ELISPOT assays were different between the DNA-prime/MVA-boost and Ad5-prime/MVA-boost vaccinated mice, probably reflecting differences in CD4+ and CD8+ responses to different vaccination protocols 
. With plasmid priming in naïve mice () there was T-cell recognition of both rec.AhpC and rec.Mpa with limited recognition of synthetic peptides. With Ad5.HAV-prime/MVA.HAV-boost in naïve mice (), no T-cell response to either recombinant antigen was seen but there was substantial recognition of synthetic peptides representing several domains throughout the fusion protein. This T-cell response to synthetic peptides was also seen in vaccination of MAP infected mice (). Of particular note was the consistent identification of the strong murine T-cell epitope GFAEINPIA (peptide F9.1) sited near the carboxy-terminal end of the HAV polyprotein representing the fifth and smallest predicted extracellular peptide loop of Mpa 
. It is interesting to note that T-cell reactivity to GFAEINPIA was a specific consequence of vaccination and did not occur in any sham vaccinated MAP infected mice.
By contrast, the Ad5.HAV-prime/MVA.HAV-boost protocol in naïve mice () resulted in substantial IgG antibody recognition of rec.AhpC and rec.Mpa. This was also seen in response to vaccination in MAP infected animals (). Antibody responses to GFAEINPIA were consistently negative in all experiments. None of the recombinant proteins or synthetic peptides comprising the vaccine was recognised in any ELISPOT and ELISA tests on unvaccinated MAP infected animals.
MAP infection in unvaccinated mice caused substantial enlargement of the spleen. Prophylactic vaccination significantly reduced splenic enlargement following MAP challenge compared with vector only and saline control groups. However once splenic enlargement had been established by previous infection it was not reduced by subsequent vaccination within the 12 week time period of observation.
Natural MAP infection in animals can persist in a subclinical state for months or years. Despite the fact that many of the mice in the present study carried a huge infective load of these pathogens, none of the animals showed any visible deterioration in their clinical condition or significant diminution of their body weights even though the livers of some of them contained more than 108
organisms. This illustrates the relatively benign host-pathogen relationships in experimental MAP infection in mice in sharp contrast to that seen with M. tuberculosis
. It is consistent with the long recognised differences in pathogenicity and efficacy of anti-microbial drug treatments between MAP and M. tuberculosis
infections in humans 
the genetic basis for which is beginning to emerge 
There were substantial differences in the prophylactic and therapeutic effects of vaccination on the burden of MAP infection in spleen and liver between individual mice within groups. Despite this, both prophylactic and therapeutic vaccination protocols using the HAV insert delivered in both plasmid and viral vectors could achieve up to a 4 log reduction in the tissue abundance of these resilient and versatile intracellular pathogens. Experimental MAP infections in mice are not models of natural Johne's disease in ruminants or of MAP infection in Crohn's disease in humans. Although experimental infection in mice does involve the intestine and mesenteric lymph nodes the infection is general and disseminated 
. Experimental infection using laboratory cultures of bovine strains of MAP in animals is uniformly pluribacillary with the organisms in their classical ZN-positive mycobacterial phenotype. In humans with Crohn's disease MAP infection is paucimicrobial with the organisms in a uniformly ZN-negative phenotype with accompanying florid granulomatous inflammatory disease similar to tuberculoid leprosy 
. The ability of the fusion polypeptide HAV to confer some protection against challenge and attenuate pre-existing MAP infection when delivered in combinations of DNA or viral vectors suggests that further studies of the present vaccine in naturally infected animals and humans are indicated.