Novel Phage Display-Based Subtractive Screening To Identify Vaccine Candidates ofBrugia malayi

Abstract

This study describes a novel phage display method based on an iterative subtraction strategy to identify candidate vaccine antigens of Brugia malayi. A cDNA library of the infective larval stage of B. malayi expressed on the surface of T7 phage was sequentially screened with sera samples from human subjects showing different manifestations of the disease. Antigens that selectively and specifically bind to immune sera were then enriched using a multi-step panning procedure. This strategy identified five antigens, four of which were previously reported (ALT-2, TPX-2, VAH and COX-2) and the other one was a novel cuticular collagen (Col-4). Sera from immune individuals specifically recognized all the five antigens. However, ALT-2 appeared to be the most predominantly recognized antigen by the immune sera. Therefore, it was decided to evaluate the vaccine potential of recombinant ALT-2 (rALT-2) in a mouse and jird model. The results presented show that immunization with rALT-2 conferred over 73% protection against a challenge infection in the jird model and over 64% protection in the mouse model. The present study suggests that phage display-based cDNA screening may be a powerful tool to identify candidate vaccine antigens of infectious agents.

Cloning and characterization of a novel immunogenic protein 3 (NIP3) from Brugia malayi by immuno screening of a phage-display cDNA expression library

“…Circulating microfilariae in the blood samples were identified as described previously (Gnanasekar et al 2004) and the circulating filarial antigens were detected using a commercially available Og4C3 kit (Lalitha et al 1998) and a WbSXP-based ELISA (Rao et al 2000). Individuals with no circulating antigen or microfilariae were considered as EN, whereas, subjects with circulating microfilariae and/or circulating antigen as detected by Og4C3/WbSXP ELISA were considered as MF.…”

“…malayi L3 cDNA was cloned into T7 select 1-1 phage-display vector as described previously (Gnanasekar et al 2004). Briefly, B. malayi L3 cDNA library constructed in Uni-ZAP XR vector was PCR amplified with T3 and T7 promoter primers.…”

“…The strategy used for biopanning the T7 BmL3 phage display library to select EN-specific clones was similar to those described previously (Gnanasekar et al 2004) with slight modification. Briefly, 96-well plates (Pierce Biotechnology, Rockford, IL) were coated with 1:100 diluted pooled EN serum sample (from 10 individuals) overnight at 4°C.…”

“…The gene inserts in these plaques were amplified by PCR as described previously (Gnanasekar et al 2004). Briefly, the inserts were amplified using vectorspecific primers, T7SelectUP and T7SelectDown.…”

“…Therefore, the gene that codes for the displayed protein can be easily cloned from the phages. Recently, we used this technique to identify potential vaccine candidates of B. malayi (Gnanasekar et al 2004). The phage-display screening is a simple, efficient and sensitive method.…”