Proceedings of the 10th International Colloquium on Paratuberculosis

#199 Development <strong>of</strong> DNA constructs expressing Mycobacterium avium subsp. paratuberculosis
proteins in mammalian cell line and study on <strong>the</strong>ir immunogenicity in murine model
P. P. Goswami, S. Chakravarti, S. Chandra Sekar, Rajib Deb
Indian Veterinary Research Institute, Izatnagar India
Several antigens <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) were studied as vaccine candidate
and <strong>the</strong>ir immunogenicity has been evaluated. Previous studies in our laboratory revealed that 35 kDa protein
(P35) expressed in prokaryotc expression vector could induce significant T cell immune response as well as
secretion <strong>of</strong> Th1 associated cytokine interferon gamma (IFNγ). We have fur<strong>the</strong>r cloned P35 (35 kDa), PE (10
kDa) and PPE (34.9 kDa) protein genes <strong>of</strong> Map alone and with murine IFNγ into <strong>the</strong> eukaryotic expression
plasmid pIRES 6.1 and transfected in mammalian cell line which showed eukaryotic expression <strong>of</strong> recombinant
proteins detected with hyperimmune sera raised against respective recombinant proteins in western blot
and immun<strong>of</strong>luorescent assay. These monocistronic and bicistronic constructs were used as DNA vaccine in
mice and <strong>the</strong>ir immunogenicity was studied by delayed type hypersensitvity (DTH), lymphocyte proliferation,
nitric oxide (NO) determination and IFNγ assay. Significant DTH responses were evoked in mice immunized
with bicistrionic constructs than <strong>the</strong> monocistrionic constructs. Also higher proliferation <strong>of</strong> <strong>the</strong> splenocytes and
enhanced production <strong>of</strong> NO exposed to respective antigen was found in bicistronic groups. Significantly higher
amount <strong>of</strong> IFNγ was also released in bicistronic groups. Flow cytometry analysis revealed higher CD4 + and
CD8 + T cell response to <strong>the</strong> recombinant antigens. Studies also showed that co-expression <strong>of</strong> IFNγ with Map
genes enhanced <strong>the</strong> immunogenicity. These results indicate <strong>the</strong> T cell epitopic nature <strong>of</strong> <strong>the</strong> antigens which
could potentially be used in <strong>the</strong> development <strong>of</strong> effective DNA vaccine against paratuberculosis infection.
#208 MAP –Epi<strong>the</strong>lial cross-talk induces phagosome acidification and Nod2 expression to
Enlist IL-1β processing
Elise Angele Lamont, Srinand Sreevatsan
University <strong>of</strong> Minnesota, USA
It is well recognized that <strong>the</strong> initial site <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) infection occurs
at <strong>the</strong> epi<strong>the</strong>lium; however, few studies have investigated this interaction. Prior to residing in <strong>the</strong> macrophage,
MAP must first be processed by <strong>the</strong> host epi<strong>the</strong>lium. Processing <strong>of</strong> MAP by <strong>the</strong> host epi<strong>the</strong>lium involves a
dynamic innate immune response initiated by MAP-Epi<strong>the</strong>lial cross-talk, which may also be augmented by
fur<strong>the</strong>r cross-talk between host pathways as well as cell types (epi<strong>the</strong>lial-macrophage). We hypo<strong>the</strong>size that
chronic stimulation <strong>of</strong> Nod2, a pathogen recognition receptor, by MAP or its major cell wall components will
result in prolonged inflammation that is sustained by toll-like receptors (TLRs) 2 and 4 in JD. We investigated
Mac-T cell (bovine mammary epi<strong>the</strong>lial cell line) responses to MAP strain K-10 (pWes4) and its cell wall components
at 10-240 minutes. Data show early phagosome acidification by epi<strong>the</strong>lial cells in response to MAP
K-10 (pWes4). Phagosome acidification coincides with a strong host upregulation <strong>of</strong> IL-1β, which may serve
as a chemoattractant for macrophages. The Nod2-caspase 1 pathway has previously been reported to be an
essential signaling cascade required for IL-1β expression. Our findings indicate that MAP is capable <strong>of</strong> inducing
Nod2 expression with 240 min. Nod2 expression appears to be preferentially upregulated by ManLAM.
We propose that MAP allows phagosome-acidification in epi<strong>the</strong>lial cells to induce IL-1β processing in order to
cause host migration <strong>of</strong> macrophages. Thus, MAP guidance <strong>of</strong> phagosome-acidification and Nod2 may enlist
IL-1β processing in order for it to easily transverse through <strong>the</strong> epi<strong>the</strong>lium and into its niche—<strong>the</strong> macrophage.
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