Elsevier

Vaccine

Volume 27, Issue 10, 4 March 2009, Pages 1571-1578
Vaccine

Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection

https://doi.org/10.1016/j.vaccine.2009.01.002Get rights and content

Abstract

Bacterial ghosts may be generated by the controlled expression of the phiX174 lysis gene E in Gram-negative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The Edwardsiella tarda ghost (ETG) vaccine was prepared using this technology and tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+ T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease.

Introduction

Edwardsiella tarda (E. tarda) is an intracellular, rod-shaped Gram-negative, non-capsulated, motile, facultative anaerobic bacteria that was first isolated from a pond-cultured eel by Hoshina [1]. E. tarda is widely distributed in aquatic environments [2], [3] and is infectious to variety of animals including humans [4], [5], [6], [7], fish [8], [9], [10], [11], [12], [13], amphibians [14], [15], reptiles [14], [16], [17] and birds [18], [19]. This organism's versatility with respect to the broad-range of hosts highlight the importance of developing strategies for the protection of both animals and humans from E. tarda infections. In recent years, chemotherapy has been used effectively in controling fish infections [20], however, there is significant concern regarding food safety following chemotherapeutic interventions in addition to the danger of selecting for antibiotic-resistant E. tarda isolates which have been reported worldwide [21], [22], [23]. These concerns have prompted the development of novel vaccination strategies for the control of E. tarda infections.

Over the last decade, vaccination has become an important prevention strategy against numerous infectious agents affecting humans and farm animals [24], [25]. Although, the development of E. tarda vaccines has been attempted, their efficacy against challenge has been inconsistent [26], [27], [28], [29]. The commercial vaccines presently available consist of heat- or formalin-inactivated E. tarda formulations, however, these strategies can affect the physio-chemical/structural properties of surface antigens thereby negatively affecting the development of protective immunity [30], [31].

Bacterial ghosts are empty cell envelopes that are produced, for example, by the controlled expression of the phiX174 lysis gene E in Gram-negative bacteria. Expression of lysis gene E leads to the formation of trans-membrane tunnels which consequently lead to the loss of cytoplasmic contents [31], [32]. The resulting bacterial ghosts have been demonstrated to retain functional and antigenic determinants of the envelope [33]. Even highly sensitive and fragile structures such as pili are well protected following ghost formation as demonstrated during the generation of Vibrio cholerae ghosts expressing the toxin-coregulated pilus [34]. These data suggested that ghosts could be used in place of the traditional live-attenuated vaccine preparations to elicit immunity [25], [35], [36], [37].

During the development of conventional, nonviable whole-cell vaccines formulations antigenic epitopes can be potentially altered as a result of the inactivation process that is absent in the generation of bacterial-ghosts [30], [31]. Furthermore, inactivated whole-cell vaccine preparations and subunit vaccines are often less immunogenic, necessitating the use of adjuvants which may have significant negative side effects on the host [25], [30], [31]. In contrast, bacterial ghost formulations possess inherent adjuvant-like properties thereby negating the need of additional immunostimulatory formulations [25].

Because economic losses due to E. tarda infections of fish are significant and human infections can result in gastroenteritis, various vaccine candidates have been developed and tested in fish and mouse models of disease [38], [39], [40]. Although, E. tarda ghosts vaccine have been reported to confer protection in Oreochromis mosambicus (tilapia) and the Paralichthys olivaceus (olive flounder) infection models [32], [41], the fish immune system is not conducive to testing cellular immunity. In this study we demonstrate that ETG vaccination stimulated both cellular and humoral immunity and conferred significant protection against E. tarda infection in a mouse model of disease.

Section snippets

The pLysis E plasmid

The plasmid pElysis [42] carrying the E lysis gene and the PBV220 (Fig. 1A) plasmid carrying the PR/CI857 regulatory system were used in the generation of the E. tarda ghosts. For the construction of the lysis plasmid, the E gene was amplified by PCR from the pElysis plasmid with oligonucleotides E-Forward (5′-GGATCCATGGTACGCTGGACTTTG-3′) and E-Reverse (5′-CTGCAGTCACTCCTTCTGCACGTA-3′) containing the BamHI and PstI restriction enzyme sites, respectively (italicized). PCR amplification steps

Production and characterization of bacterial ghosts

Lysis plasmid pLysis E (Fig. 1B) was successfully constructed and cotransformed into E. tarda Strain CD and ghosts produced by protein E-mediated lysis of bacterial cells. Production of ghosts in E. tarda transformants was carried out using temperature increments to activate the pLysis E gene (up to 42 °C). The OD of the transformed E. tarda cultures decreased during the first hour after induction of gene E expression as a function of cell lysis and remained constant for the next 8 h until the

Discussion

Although E. tarda ghost vaccines have been reported to protect tilapia (Oreochromis mosambicus) and the olive flounder (Paralichthys olivaceus) against E. tarda infections [32], [41], determining the capacity of this vaccination strategy to elicit T cell-mediated immunity cannot be carried out in fish models. We therefore developed an infection model in mice that allowed us to examine both humoral and cellular response to E. tarda and this study is the first to determine immune response in

Acknowledgements

We thank Y.Y. Wang and R.B. Cao for providing the E. tarda strain CD, and the plasmid PBV220, respectively.

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