Capacity to elicit Cytotoxic CD8 T cell activity against Mycobacterium avium subsp. paratuberculosis is retained in a vaccine candidate 35 kD peptide modified for expression in mammalian cells

Authors: FRANCESCHI, VALENTINA - University Of Parma; ASMMA, MAHMOUD - Washington State University; ABDELLRAZEQ, GARBER - Washington State University; TEBALDI, GIULIA - University Of Parma; MACCHI, FRANCESCA - University Of Parma; RUSSO, LUCA - University Of Parma; Fry, Lindsay; ELNAGGAR, MAHMOUD - Washington State University; Bannantine, John; PARK, KUN-TAEK - Inje University; HULUBEI, VICTORIA - Washington State University; CAVIRANI, SANDRO - University Of Parma; DAVIS, WILLIAM - Washington State University; DONOFRIO, GAETANO - University Of Parma

Submitted to: Frontiers in Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2019
Publication Date: 12/11/2019
Citation: Franceschi, V., Asmma, M.H., Abdellrazeq, G.S., Tebaldi, G., Macchi, F., Russo, L., Fry, L.M., Elnaggar, M.M., Bannantine, J.P., Park, K., Hulubei, V., Cavirani, S., Davis, W.C., Donofrio, G. 2019. Capacity to elicit Cytotoxic CD8 T cell activity against Mycobacterium avium subsp. paratuberculosis is retained in a vaccine candidate 35 kD peptide modified for expression in mammalian cells . Frontiers in Immunology. https://doi.org/10.3389/fimmu.2019.02859.
DOI: https://doi.org/10.3389/fimmu.2019.02859

Interpretive Summary: The development of a vaccine to protect cattle from Johnes Disease, caused by Mycobacterium avium subsp. paratuberculosis, is of urgent importance to reduce animal losses and improve meat and milk yields from cattle. Protective immunity to this organism requires the development of a functional CD8+ cytotoxic T lymphocyte (CTL) response. In this study, we show that a candidate vaccine antigen, known as MMP, can elicit a functional CTL response when made in mammalian cells. This finding will help in vaccine development because it paves the way for the development of a virus-vectored, MMP-based vaccine for use in cattle.

Technical Abstract: Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/'relA, with cells from a vaccinated steer demonstrated a 35 kD membrane peptide (MMP) is one of the targets of the response. This observation suggested it might be possible to develop a peptide- based vaccine. Stimulation of PBMC with antigen presenting cells pulsed with MMP alone or incorporated into a nanoparticle vector elicited the same CTL response as the Map/'relA mutant. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace 5 potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. Ex vivo analysis demonstrated the full immunogenic activity of native MMP was preserved in the modified MMPs, MMP and MMP-2mut, and lost in MMP-5mut. The data show the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious vaccine against Ptb.