Development and immunogenicity of recombinant GapA+ Mycoplasma gallisepticum vaccine strain ts-11 expressing infectious bronchitis virus-S1 glycoprotein and chicken interleukin-6
Introduction
Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide. It is the aetiological agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys and can cause severe losses in poultry enterprises [1], [2]. The chronic respiratory disease it causes is characterised by exfoliation of ciliated epithelial cells, accumulation of inflammatory exudates in the trachea, and a severe inflammatory response in the air sacs [3]. Antibiotics have proven ineffective in clearing MG infections [4], and current control practices use intense biosecurity and serological monitoring of flocks [4], [5]. Regulatory measures have been largely successful at minimising MG outbreaks in broiler and turkey industries, primarily due to their “all-in, all-out” production cycle. However, within the layer industry eradication of infected flocks is usually not feasible [6]. To address this, live attenuated MG vaccines have been developed as an alternative approach to control [7].
The MG vaccine strain ts-11 is a temperature sensitive mutant generated by chemical mutagenesis of a moderately virulent Australian field isolate (strain 80083). It grows normally at 33 °C but has reduced growth at 39.5 °C, and a single dose by eye-drop application results in colonisation of the upper respiratory tract and induction of long-term immunity [8], [9]. However, the efficacy of this vaccine is highly dose dependent [10] and serological monitoring of vaccinated flocks has been difficult because of the low level of antibodies induced in some vaccinated flocks [11].
Studies of gene expression in mycoplasmas have been difficult mainly because of the lack of understanding of gene regulatory elements and uncertainty in identifying promoter sequences. The first foreign gene to be functionally expressed in a mycoplasma was the β-galactosidase (lacZ) gene of Escherichia coli. The lacZ gene was used as a reporter gene in mollicutes in experiments with Acholeplasma oculi [12] and M. gallisepticum using a transposon Tn4001 derivative (Tn4001lac) within the plasmid pISM2062lac. This derivative had a promoterless lacZ gene inserted into one of the IS256 elements [13]. The expression of lacZ has also been tested under the control of various mollicute promoters in M. gallisepticum [14], Mycoplasma pulmonis [15] and M. capricolum [16].
Infectious bronchitis virus (IBV) is a member of the family Coronaviridae. The virion is enveloped, with club-shaped surface projections (spikes) [17]. The spike glycoprotein (approximately 1145 amino acids in length) consists of the amino terminal S1 (approximately 520 amino acid residues) and carboxyl terminal S2 (approximately 625 amino acid residues), which are generated by post-translational cleavage. S1 and S2 associate to form the viral envelope, in which S1 is exposed on the virion surface, anchored by S2 [18]. The S1 subunit induces neutralising, serotype-specific and haemagglutination-inhibiting antibodies [19]. Thus the best candidate gene for inclusion in a recombinant vector to protect against IBV would seem to be the one encoding S1.
Cytokines play an important role in the functional diversity of immune responses. Interleukin-6 (IL-6) is a multifunctional cytokine produced by T and B lymphocytes, monocytes/macrophages and endothelial cells, and has been found to be potent in promoting systemic and mucosal immunity when co-administered with protein antigens by improving Th2 responses [20]. Additionally, it is involved in the initial activation of T lymphocytes [21] and is able to switch the differentiation of dendritic cells to macrophages [22] and to induce an acute phase response. Thus the gene encoding chicken IL-6 would seem to be an appropriate choice for expression as an adjuvant.
For successful colonisation of the respiratory tract MG must first establish a specific and firm attachment to its target cell to avoid rapid clearance by innate host defence mechanisms [23]. GapA, a 105 kDa protein, is considered the primary cytadhesin molecule in MG. Studies have found that GapA and CrmA co-expression is essential for MG cytadherence and virulence [24]. Absence of GapA but not CrmA has been observed in the MG vaccine strain, ts-11 [25], and the expression of CrmA is not dependent on expression of GapA [26].
In this study, we developed a transposon-based vector construct that enabled the expression and release of a foreign protein in MG and used this to develop strains of GapA+ MG ts-11 that expressed the IBV-S1 glycoprotein with ChIL-6 either fused to the S1 protein or secreted from the bacterial cell. We examined the capacity of these recombinants to generate protective immunity in the respiratory tract of chickens.
Section snippets
Mycoplasma gallisepticum strain and culture
The GapA+ MG ts-11 strain was used in this study. This strain was originally isolated from a MG ts-11 working seed culture and selected for expression of GapA by plating the culture, picking single colonies and examining them by PCR and immunoblotting using specific antisera against GapA [26]. Culture of the organism was performed in mycoplasma broth (MB) (7.5 g trypticase peptone, 2.5 g phytone peptone, 0.5 g thiotone peptone, 5 g yeast extract, 0.25 g benzyl penicillin, 5 g NaCl, 0.4 g KCl, 0.35 g
Development and characterisation of MG ts-11 C3 (+CS) and MG ts-11 C2 (−CS)
The presence of gentamicin resistance genes, the presence or absence of the cleavage signal sequence, and the absence of a 20 bp insertion in the gapA gene were confirmed by PCR, and expression of S1, ChIL-6 and GapA were detected by Western blotting (results not shown). Although the presence of ChIL-6 in the culture supernatant of the recombinant MG ts-11 C3 (+CS) was unable to be detected by Western blotting, release of ChIL-6 into the culture supernatant was detectable using the 7TD1 bioassay
Discussion
In this study, recombinant strains based on GapA+ MG ts-11 were generated by transposon mutagenesis. This is only the second report of successful expression of a chicken cytokine in any mycoplasma species [34]. The recombinant MG ts-11 C3 (+CS) expressed S1 and released ChIL-6 into the extracellular milieu and the recombinant MG ts-11 C2 (−CS) expressed a fusion of S1 and ChIL-6. Both the recombinants retained their temperature-sensitive phenotype after transposon mutagenesis. PCR analysis
Acknowledgements
The authors gratefully acknowledge the assistance of Associate Professor Amir H. Noormohammadi, Dr. Marc Marenda, Cheryl Colson, June Daly, Josie Wilson, Rebecca Agnew, Faye Docherty, Shukriti Sharma and Shaiful Islam. This work was supported by an International Postgraduate Research Scholarship (IPRS) from the Australian Government and a Melbourne International Research Scholarship (MIRS) from The University of Melbourne.
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