Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant
Introduction
Riemerella anatipestifer is a non-motile, non-spore forming, Gram-negative bacillus that causes septicemia and infectious serositis in domestic ducks [1]. Infected birds show clinical signs that include fibrinous pericarditis, airsacculitis, and perihepatitis. R. anatipestifer infection can result in major economic losses in duck farms because of poor feed conversion, high treatment costs, and high mortality rates. In infected farms, the mortality rate can reach as high as 75% for ducklings under eight weeks of age [2].
Various antibiotics are currently used to prevent and control R. anatipestifer infection in ducks, but the emergence of drug-resistant and multi-drug resistant strains poses serious challenge. Recent studies have found antibiotic resistance to chloramphenicol [3], florfenicol [4], fluoroquinolones and a number of other antibiotics [5]. The widespread practice of antimicrobial prophylaxis in duck farms [5] and the detection of residual antibiotics in duck-related products all necessitate other methods of R. anatipestifer control.
Immunization of ducks with bacterin vaccine provides a viable alternative to antibiotics, but no significant cross-protection was reported for different R. anatipestifer serotypes. At least twenty one serotypes of R. anatipestifer are identified [6], [7] and several attempts have been made to immunize ducks using live [6], inactivated bacterin [8], [9], or cell-free culture filtrate vaccines [10]. In these cases, while significant protection was observed for homologous challenges, lack of cross-protection was consistently observed for heterologous challenges.
To achieve cross-protection, the use of conserved antigens in a subunit vaccine is a promising approach. The conserved outer membrane protein A (OmpA) of R. anatipestifer is a virulence factor important for adhesion and invasion [11]. All R. anatipestifer serotypes contain OmpA but some minor genetic heterogeneity can be found among different serotypes [12]. OmpA induces strong antibody response [13]. Furthermore, while searching for promising vaccine candidates using the immunoproteomics approach, studies have consistently identified OmpA as one of the most cross-reactive antigens[14], [15], with protective indices reaching 50% and 60% against serotypes 1 and 2, respectively [16]. Therefore, OmpA is a suitable candidate protein for further vaccine development.
Most protein-based vaccines require the use of adjuvants and studies indicate that synthetic CpG oligodeoxynucleotides (mimics bacterial DNA) is worthy of investigation. Bacterial DNA contains CG dinucleotides and unmethylated cytosines that are rare in mammals, thus acting as an agonist that triggers mammalian Toll-like receptor (TLR) 9 [17], fish TLR9 [18], and chicken TLR21[19], [20]. Through these TLRs, CpG ODN triggers an innate immune response that leads to pro-inflammatory cytokine production and potent Th1-biased immunity [21], [22], [23]. When used as vaccine adjuvants, CpG ODN often helps produce a balanced and more effective immune response [24]. CpG ODN can be classified into A-, B-, or C-class based on different stimulation profiles [25] and the effect of CpG ODN can also be species-specific. Of the B-class CpG ODN, the optimal motif is GTCGTT for humans and chickens [21], [26], [27], and GACGTT for mice, cows [28], and ducks [29]. In addition, recent development of plasmids containing CpG ODN motifs has made the use of CpG ODN economically feasible, especially for veterinary use [30].
In this study, R. anatipestifer OmpA will be combined with plasmids containing 12 copies of the GACGTT motif to formulate a subunit vaccine. Immune response and protective efficacy of the vaccine will be evaluated in ducklings.
Section snippets
Bacterial strain and DNA extraction
R. anatipestifer was isolated from field cases in southern Taiwan and its identify was confirmed using PCR with 16S rRNA primers F: (5′-CAGCTTAACTGTAGAACTGC-3′) AND R: (5′-TCGAGATTTGCATCACTTCG-3′). The strain was sent to the Animal Health Research Institute (Danshui District, New Taipei city, Taiwan) for serotyping using the slide agglutination test and was determined to be of serotype 2. The isolated strain was grown at 37 °C in Tryptic Soy Broth (BD, MD, USA) supplemented with 3% Chicken Serum
Production of a recombinant R. anatipestifer OmpA vaccine with CpG ODN adjuvant
This study aimed to evaluate the immune response and protective efficacy of a recombinant R. anatipestifer OmpA vaccine containing CpG ODN as the adjuvant. To produce recombinant OmpA, the gene was amplified from a field strain of R. anatipestifer (serotype 2) in Taiwan. The resulting 1173 bp PCR product was cloned into the pET-32a plasmid for protein expression in E. coli. Induced protein expression of the rOmpA showed a 65 kDa polypeptide (Fig. 1A) and its immunogenicity was confirmed by
Discussion
Our study found that the addition of CpG ODN plasmid constructs enhanced the immune response induced by recombinantly expressed OmpA. The significant boost of cellular immunity by CpG ODN is particularly helpful for protein-based vaccines. Furthermore, the constructed plasmid has made the use of CpG ODN as an adjuvant much more economically feasible.
The cytokine expression profiles observed in this study are consistent with published data. CpG ODN, by involving lymphocytes, natural killer cells
Conflict of interest
All authors have no financial or personal relationships with other people or organizations that could inappropriately influence or bias their work.
Acknowledgement
We thank Dr. Tsung-Chou Chang of the Department of Veterinary Medicine, NPUST, for pathological evaluation of the ducks.
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