CpG Motif in Phage Genome DNA Enhanced the Efficacy of Phage Therapy by Immunostimulation

Abstract

Lytic phages for multiple-drug resistant E. coli and Pseudomonas aeruginosa were isolated. They belong to Siphoviridea and Myoviridea families, respectively. Lytic life cycle of the phage has therapeutic implications. Concurrent immune-stimulation of adaptive immunity against the etiologic agent, the MDR bacteria in the victim would revolutionize the field of phage therapy. In this pretext, methylated and unmethylated CpG motifs were identified in genome DNA of these phages by MspI and HpaII restriction fragmentation. To test the immune-stimulatory effects, two groups of BALB/c mice and two groups of rabbits were vaccinated with soluble antigen of Salmonella typhi with or without fragmented genome DNA of these phages. In addition, adjuvant efficacy of CpG motifs was compared with mineral oil. In antisera, types of antibodies were determined by immunoelectrophoresis and Western blotting. ELISA was done for quantitative analysis and has shown IgG immunoglobulin was produced by BALB/c and rabbit when fragmented DNA was used as adjuvant. Strong and specific immunogenicity has been attributed to the presence of CpG motifs. However, IgG antibody titer in antiserum raised by Pseudomonas' Phage DNA was high as compared to E. coli . The plausible reason for the variation in immune response is that this DNA has additional AACGAT motifs to enhance immunopotential of its CpG motifs. Strong and discrete bands were highlighted due to IgG Fab on the Western blots. We predict that efficacy of phage therapy will be enhanced by concurrent immune stimulation with CpG motif DNA injection with the respective MDR pathogen’s antigen for the control of disease.