Use of antimicrobial peptides as adjuvants for vaccines against Mycoplasma bovis
Mycoplasma bovis is known as a major contributing etiological agent in chronic pneumonia and arthritis in cattle. Since the neonatal calf is at the highest risk of exposure to M. bovis, we proposed to develop a vaccine for the newborn that can be administered at the time of tagging followed by a boost vaccination before shipping to the feedlot. We chose the M. bovis Gapdh protein as our test antigen and we proposed to combine this antigen with antimicrobial peptides (AMP) that are known to be effective as adjuvants. We first tested several AMP for their ability to stimulate in-vitro expression of several chemokines and cytokines by bovine peripheral-blood mononuclear cells (PBMC) and we chose indolicidin, BMAP28 and TAP for the construction of chimeric proteins.
As part of the characterization of indolicidin, BMAP28 and TAP, we tested their antimicrobial activity on M. bovis cultures. The three peptides were able to reduce the number of attached cells to the surface. M. bovis has the capability of forming biofilms. Adherent cells in biofilms are resistant to antibiotics and can evade the immune system surveillance. We tested whether the three peptides could prevent biofilm formation and the results indicated that the peptides decrease the number of viable counts in biofilms but this decrease was not statistically significant. We also tested whether the peptides could kill bacteria in a previously formed biofilm and the results indicated that at the concentrations used, there was a reduced number of viable M. bovis in biofilms after treatment with BMAP28 or indolicidin.
Three chimeric proteins were constructed, sGapI, sGapM and GapT. The genes encoding sGapI and sGapM were optimized for efficient expression in E. coli hosts. We also optimized the gene encoding the wild-type M. bovis GAPDH protein (sGap). These four proteins possessed GAPDH activity and the three chimeric proteins sGapI, sGapM and GapT showed moderate antimicrobial activity on formed biofilms suggesting that the antimicrobial peptide moiety on the chimeras was active. We conducted an animal vaccine trial to compare the immune response to the Gapdh-AMP chimeras with the response to the Gapdh protein and AMP included in the same vaccine formulation. The results suggested that there was a marginal improvement when the chimeras were used with the Gap-I protein being the best candidate. We also tested if the chimeric proteins retained the properties of their original components, i.e. Gapdh and antimicrobial activities. The results indicated that the Gapdh activity was still present in the all the chimeras albeit reduced and that the chimeric protein composed of Gapdh and BMAP28 retained the antimicrobial activity of the peptide on previously formed M. bovis biofilms. We chose the Gapdh-indolicidin (Gap-I) chimera for our DNA immunization trial and thus we optimized the codon usage of the genes encoding the wild-type Gapdh and the Gap-I proteins for expression in bovine species. Expression of the proteins was observed in-vitro on Mac-T and EVM-1 bovine cell lines.
The proof of concept trial was carried out by vaccinating young cattle with DNA/protein or protein/protein combinations. The animals from a cow-calf operation were kept in the farm and transported to VIDO before the challenge. We developed a co-challenge model that combines exposure to Bovine Herpes Virus 1 (BHV-1) followed by a challenge with M. bovis. The animals were challenged and monitored for signs of disease. The weight losses and elevated temperatures were evident in all the vaccinated and in the control group. The survival rate was not different between the groups and the gross lung lesions were present in all the groups. Bacteriology analyses indicated that after challenge, M. bovis was isolated from most of the tissues tested. Together with M. bovis, several animals were infected with other bovine respiratory pathogens such as Mannheimia haemolytica and Pasteurella haemolytica. These findings may help to explain the severity of the disease observed.
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