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

Abstract

Iterative screening of a phage display cDNA expression library of the third-stage larvae (L3) of Brugia malayi with sera from putatively immune individuals (endemic normal, EN) identified a novel clone with insert showing significant homology to Onchocerca volvulus novel immunogenic protein-3 (Ov-NIP3) gene and Caenorhabditis elegans NIP3-like protein and hence the gene was designated Brugia malayi NIP3-like protein (BmNIP3). EST database analysis showed that ESTs from several gastrointestinal nematodes such as Ancylostoma caninum, Teladorsagia circumcincta, Haemonchus contortus and Strongyloides stercoralis has BmNIP3 homologues, but the gene has not been described from these parasites. Sequence analyses showed that BmNIP3 has three potential mucin-type O-glycosylation sites and several serine/threonine phosphorylation sites. As expected, BmNIP3 protein isolated from the parasite was serine/threonine phosphorylated. Further analyses showed that BmNIP3 is differentially transcribed, with highest level of expression present in the larval (L3 and L4) stages. Mice immunized with rBmNIP3 developed strong antibody responses predominantly of the IgG1 and IgG2a subtype. A similar analyses of the sera samples from EN individuals showed that they also carry high levels of IgG1 and IgG2 antibodies against BmNIP3, whereas, chronically infected patients carry largely IgG3 antibodies and MF individuals carry high levels of IgG1 antibodies against BmNIP3. This study thus describes a novel protein from B. malayi that appears to be highly immunogenic in both humans and mice.

Comparative Analysis of the Secretome from a Model Filarial Nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites

“…5A). However, AM ESP also contained several previously described but uncharacterized proteins, such as RAL-2 (52), nematode secreted protein 22U (61), and immunogenic protein 3 (62). A novel KH (RNAbinding) domain protein had homologs in other filarial species, but also displayed weak homology to the Vasa DEAD-box helicase GLH-2 from Caenorhabditis elegans (supplemental Table S2), which is associated with spermatogenic chromatin (63).…”

Selection of high affinity peptide ligands for detection of circulating antibodies in neurocysticercosis

“…Since the proteins displayed on the surface of phage is physically linked to the genetic material that codes for it, the gene coding for the displayed protein can be easily cloned from the phages. The uniqueness of the technique is that even the few protein molecules displayed on the surface of bacteriophages could be used as bait to screen and subsequently clone the protein of interest with sufficient efficiency [31].…”

Immunization with Brugia malayi Calreticulin Protein Generates Robust Antiparasitic Immunity and Offers Protection during Experimental Lymphatic Filariasis

“…In the past, a number of secretory proteins of B. malayi have been identified as potential vaccine candidates against LF, and some of them have even shown a significant degree of protection in animal models. , However, none of them could significantly impact the clearance of adult worms or lower the Mf burden to the extent that they may be considered for preclinical development. Interestingly, B. malayi secretory protein calreticulin (BmCRT) has been identified as a potent virulence factor in a broad range of parasitic organisms from different phyla, − and the CRT of the human hookworm Necator americanus has been reported to strongly induce type 2 effector functions. , …”