Induction of antigen-specific immune responses by oral vaccination withSaccharomyces cerevisiaeexpressingActinobacillus pleuropneumoniaeApxIIA

Abstract

An effective way of inducing both mucosal and systemic immune responses to protect against Actinobacillus pleuropneumoniae serotype 2 Korean isolate was examined in mice by oral immunization using Saccharomyces cerevisiae expressing the ApxIIA protein. The immunogenicity of the yeast-derived ApxIIA antigen was confirmed by the challenge test and ApxIIA-specific IgG antibody response assay. The group subcutaneously immunized with the protein extracted from the yeast expressing ApxIIA showed a higher survival rate after challenging with A. pleuropneumoniae serotype 2 isolate and IgG antibody level in serum than the group injected with that prepared from the yeast harboring vector only. Feeding the yeast expressing ApxIIA to mice induced both systemic and mucosal immune responses against the antigen. ApxIIA-specific IgA antibody titers and the number of IgA-secreting cells of mice vaccinated with S. cerevisiae expressing ApxIIA dose-dependently increased from the third immunization in both intestine and lung (P<0.01). A similar tendency of ApxIIA-specific IgG antibody responses was observed in the sera. The protective efficacy of the oral immunization was then evaluated by a challenge with a minimal lethal dose (MLD, 4.5 x 10(7) CFU/ml) of the A. pleuropneumoniae serotype 2 isolate. Fifty percent of the 30 mg administered group and 30% of the 15 mg administered group survived while none of the mice in the control groups survived after 36 h. These results suggest that feeding animals the yeast expressing the antigen can be an effective strategy to induce protective immune responses against A. pleuropneumoniae infection.

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

“…The amylase 1A (Ramy1A) signal peptide (ASP) and the ApxIIA#5 gene encoding aa residues 439-801 (GenBank accession no. AF363362) for the neutralizing epitope, as described previously, 13,20,21) were fused by overlap extension PCR 22) to create BamHI and EcoRI restriction sites at the 5 0 and 3 0 ends respectively, using primers forward 5 0 -GGATCCGCATGCAGGTGC-3 0 and reverse 5 0 -GGGAATTCTG-TAATAGAATCATTTCC-3 0 , and overlap-forward 5 0 -TCTAACTT-GACAGCCGGGCAAGGTTATGATTCTCGT-3 0 and overlap-reverse 5 0 -ACGAGAATCATAACCTTGCCCGGCTGTCAAGTTAGA-3 0 . The amplified gene was cloned in pGEM-T Easy Vector (Promega, Madison, WI), analyzed by restriction enzymes digestion, and confirmed by DNA sequencing.…”

“…They were suspended in the same solution, and 100 ml of cell suspension (10 7 cells/ml) was placed on a glass slide and air-dried for immunostaining and observation. Anti-ApxIIA#5 mouse polyclonal antibody, obtained from a previous study, 12,13) was used as the primary antibody at a dilution of 1:50. The cells and primary antibody were incubated at room temperature for 2 h. After the cells were washed, the second antibody, fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (1:50; Sigma) was reacted with them at room temperature for 2 h. After washing, the cells were observed by confocal laser scanning microscopy (Carl Zeiss, Zena, Germany).…”

“…Color was developed using BCIP/NBT (USB, Cleveland, OH) in TMN buffer (100 mM Tris, pH 9.5, 5 mM MgCl 2 , and 100 mM NaCl), by methods described previously. 12,13) ELISA quantification of the recombinant ApxIIA#5 protein. The quantity of ApxIIA#5 expressed in CWPs was determined by quantitative ELISA, as described previously.…”

“…13) In brief, CWPs at a 10-fold dilution were utilized to coat a 96-well microtitration plate at a concentration of 100 ml/well in bicarbonate buffer (15 mM Na 2 CO 3 and 35 mM NaHCO 3 ) and incubated overnight at 4 C. The wells were washed 3 times in PBS containing 0.05% Tween-20 (PBST), blocked for 5 h at 25 C with the addition of 300 ml 1% BSA in PBS, and washed again 3 times in PBST. The plates were incubated with a 1:5,000 dilution of anti-ApxIIA antiserum, as in our previous study, 13) and in 0.01 M PBS containing 0.5% BSA for 2 h at 25 C, and then washed 3 times with PBST. The plate was then incubated with a 1:5,000 dilution of anti-mouse IgG conjugated with horseradish peroxidise (Promega) in 0.01 M PBS containing 0.1% BSA, and then washed 3 times with PBST buffer.…”

“…Secondly, we have reported that oral administration of Saccharomyces cerevisiae expressing full-length ApxIIA and a transgenic plant expressing the full-length ApxIIA toxin of the Korean strain are capable of inducing an antigenspecific immune response, and have suggested that orally administered ApxIIA can induce an effective immune response against A. pleuropneumoniae infection. [12][13][14][15] However, improved strategies for efficient y To whom correspondence should be addressed. Tel: +82-63-270-3440; Fax: +82-63-270-4312; E-mail: dhkim@chonbuk.ac.kr antigen presentation and immune responses are still needed for enhanced protection against pathogen infection.…”