Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin inSaccharomyces cerevisiaeand Oral Administration of It for Protective Immune Responses against Challenge byActinobacillus pleuropneumoniae

Abstract

A neutralizing epitope fragment of ApxIIA toxin (ApxIIA#5) of the Korean Actinobacillus pleuropneumoniae serotype 2 strain was expressed and immobilized on the cell surface of Saccharomyces cerevisiae for efficient vaccine development. Expression of ApxIIA#5 was confirmed by Western blot analysis using cell-wall proteins, and the surface display of ApxIIA#5 was further visualized under confocal microscopy. Quantitative ELISA revealed that the recombinant ApxIIA#5 directed to the cell surface consisted of approximately 16% cell-wall proteins, estimated to be 35 mg of ApxIIA#5 protein per liter of cultured cells. An immunoassay revealed that antigen-specific antibodies against ApxIIA#5 were present in the sera of mice fed recombinant ApxIIA#5-expressing yeast, but not in mice fed the wild-type nor the vector-only transformant. Moreover, the mice fed the recombinant epitope-expressing yeast were protected from injection of a lethal dose of A. pleuropneumoniae.

Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

“…The efficacy of the coexpression of LTB and the LTB::ApxIIA#5 fusion protein as an oral antigen was significantly increased due to the heteropentameric assembly, which can be ascribed to the binding capability of the antigen to the GM1 ganglioside for an efficient mucosal entry. These better efficacies in protection are consistent with the results of (26), improved strategies for efficient antigen presentation and immune responses are still needed for enhanced protection against pathogen infection. Thus, the pentameric formation itself is a good strategy for better delivery to achieve improved mucosal immunization, and it is expected that coexpression allowing a large fusion protein to assemble into the pentameric formation for biological function can be applied in several other biotechnological approaches, including enhanced oral vaccine development.…”

“…The ApxIIA epitope gene (ApxIIA#5) encoding amino acid (aa) residues 439 to 801 (ApxIIA; GenBank accession no. AF363362) was amplified from the plasmid pApxIIA5 (26). The resulting two PCR amplicons were fused by overlap extension PCR to incorporate an amino acid hinge (GPGP; underlined sequence) to generate a degree of flexibility between the LTB and ApxIIA#5 using the overlap primers overlap-forward (5Ј-CAATCAGTATGGAAAACGGACC AGGACCACAAGGTTATGATTCTCGT-3Ј) and overlap-reverse (5Ј-CGAGA ATCATAACCTTGTGGTCCTGGTCCGTTTTCCATACTGATTG-3Ј).…”

“…The wells were then loaded with 250 ng of protein preparations of CFE and then incubated overnight at 4°C. The wells were washed three times in PBST, loaded with 100 l per well of either a 1:4,000 dilution of rabbit anti-LTB primary antibody (Immunology Consultants Laboratory, Inc.) or mouse anti-ApxIIA antiserum (26), and incubated for 3 h at room temperature, followed by three washes with PBST. The remaining steps were conducted as described in the ELISA quantification section above.…”

“…First, A. pleuropneumoniae gains access to its host through mucosal surfaces of the respiratory tract. Second, we previously reported that orally administered ApxIIA or an antigenic fragment containing its neutralizing epitopes could induce an immune response against A. pleuropneumoniae infection (26,33,43,55,56). However, improved strategies for efficient antigen presentation and immune responses are still needed for enhanced protection against pathogen infection.…”

Detection of Actinobacillus pleuropneumoniae in drinking water from pig farms

“…A. pleuropneumoniae can be divided in two biotypes based on their NAD requirement. Serotypes 1, 5 and 7 are the predominant serotypes found in North America while serotype 2 is the predominant serotype in many European countries (Chiers et al, 2010;Kim et al, 2010). The clinical signs of the disease can be hyperacute, acute or chronic Ohba et al, 2010).…”