Neutralizing antibody response against subcutaneously injected bacteriophages in rabbit model.

Bacteriophage therapy is currently experiencing a renaissance. Therapeutic efficacy of bacteriophages depends on phage-bacterial and phage-host interactions. The appearance of neutralizing anti-phage antibody has been speculated to be one of the few reasons for bacteriophage therapy's failure. This study aimed to know whether there is a rise in the neutralizing antibody on the parenteral injection of bacteriophages in an animal model. This study included bacteriophages against five different bacteria, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella Typhi and Staphylococcus aureus. These bacteriophages were isolated, propagated and purified. Bacteriophage specificity was confirmed by spot testing on the respective bacterial lawn. Weekly subcutaneous injection of purified bacteriophages (109PFU) was given to five rabbits for six weeks. Blood samples were collected before administering the next dose every week. The antibody response was tested by phage neutralization followed by plaque assay by using double agar overlay method. The rise in anti-phage neutralizing antibodies was observed usually after the 3rd week after immunization. Complete neutralization of bacteriophages could be seen between 3 and 5 weeks after immunization. A further rise in bacteriophage counts (PFU), especially on 1:1000 and 1:2000 serum dilutions, could be noticed by the end of 6th week against most bacteriophages injected. Background anti-phage neutralizing antibodies were observed against bacteriophage specific to Escherichia coli. However, it was absent against bacteriophages specific to other four bacteria. Bacteriophage interacts with mammalian host and induces anti-phages neutralizing antibody production. However, neutralization of phage depends on repeated administration and duration of therapy. The significant rise in neutralizing antibody could be seen at the end of 3rd week. Therefore, bacteriophage can be effectively used in acute cases where therapy duration is less than 2 weeks. However, for prolonged therapy, bacteriophage cocktail of different antigenicity may be suggested.