Elsevier

Vaccine

Volume 31, Issue 5, 21 January 2013, Pages 820-826
Vaccine

Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate

https://doi.org/10.1016/j.vaccine.2012.11.064Get rights and content

Abstract

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

Highlights

Reverse vaccinology has been used to select vaccine candidates for the treatment of fish pasteurellosis. ► A new gene of PDP for a surface-exposed lipoprotein has been identified as a vaccine candidate. ► The lipoprotein PDP_0080 is involved in the adherence of the bacterium. ► Immunization with PDP_0080 recombinant antigen conferred protection against PDP challenge. ► PDP_0080 can be a promising vaccine candidate against infection by PDP in sea bass.

Introduction

Photobacterium damselae subsp. piscicida (PDP) is a facultative intracellular, halophilic Gram-negative bacterium and is the aetiological agent of fish pasteurellosis. This disease is a bacterial septicaemia reported in a wide variety of marine fish, notably the yellowtail (Seriola quinqueradiata) in Japan, gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax) in Europe, striped bass (Morone saxatilis) and white perch (Morone americana) in the USA and hybrid striped bass [Morone saxatilis (Morone chrysops)] and cobia (Rachycentron canadum) in Taiwan [1], [2]. Fish pasteurellosis is considered one of the most threatening diseases in world aquaculture due to high mortality, broad host range and ubiquitous distribution [3]. Chemotherapy is usually ineffective due to widespread antibiotic resistance and intracellular parasitism of PDP within macrophages during infection [1]. Vaccination has proven to be the best way to control fish diseases and the use of vaccines in aquaculture is an important measure to reduce economic losses and to limit antibiotic use in fish farming [4]. To date, several types of PDP vaccines have been reported, including inactivated bacteria, LPS formulations and ECP-enriched bacterin preparation; however the effect of vaccination seems to be poor and protective vaccines to prevent pasteurellosis are not available [3]. Recently, three PDP antigens have been shown to induce a protective effect in cobia and therefore have been reported as potential vaccine candidates for the development of a subunit vaccine against PDP [2].

The genomic approach to predict putative antigens, independent of their abundance and immunogenicity during infection, is a useful tool in isolating new genes and identifying promising targets for recombinant subunit vaccine development [5].

In this study, the reverse vaccinology approach was applied for the first time to identify vaccine candidates against PDP in the European sea bass Dicentrarchus labrax, a species of high economic importance. Since the complete genome sequence of PDP was not available, a sequencing strategy was planned to obtain new genomic sequences, which were analysed in silico to select vaccine candidates. In order to screen for proteins with vaccine potential, we performed the inhibition adherence assay that allowed us to select a protein that was predicted to be a lipoprotein. We investigated the immunoprotective potential of this antigen as a purified recombinant subunit vaccine in a laboratory-scale vaccination-challenge trial.

Section snippets

DNA sequencing and annotation

Genome data of PDP NCIMB 2058 were obtained by sequencing a genomic cosmid library constructed in pWEB vector (Epicentre Biotechnologies) and with average insert size of 30–40 kb. Cosmid overlapping was investigated by sequencing insert ends and by PCR. Non-overlapping cosmids were subcloned into pCR4Blunt-TOPO plasmid vector (Invitrogen) for shotgun sequencing with universal primers. Random sequences were assembled into contigs with the Staden Package (http://staden.sourceforge.net/) and gaps

PDP genomic sequencing and selection of vaccine candidates

To obtain new genetic information on PDP, 10–12% genome of NCIMB 2058 strain was sequenced from the cosmid library using the shotgun approach. While the sequencing project was in progress, about 470,000 bp, corresponding to 13 contigs, were analysed to select potential vaccine candidates. In silico analysis led to the identification of 370 ORFs, 26 of which were interrupted by insertion elements or by premature stop codons.

The cellular localization prediction by PSORTb identified 172 cytoplasmic

Discussion

Potential vaccine candidates can be selected by the reverse vaccinology approach, using bioinformatic algorithms to identify new proteins localized on the bacterial surface [5]. Protein localization, transmembrane domains and protein conservation were the main criteria for selection of possible vaccine candidates against PDP from novel genomic sequences. With this approach, eight potential vaccine immunogens were identified and produced as recombinant proteins. As a preliminary in vitro

Acknowledgements

We would like to thank Irene Bianconi, Francesca Gorini and Giulia Maricchiolo for their contribution to this work and Mirco Fanelli for his precious help and discussion. We are grateful to the technical staff of IAMC-CNR Messina: Paolo De Francesco and Francesco Soraci, for assistance during farming of specimens in the experimental plant. This work was partially supported by the Programma Operativo Nazionale “Ricerca, Sviluppo Tecnologico, Alta Formazione” 2000–2006 (Siracusa, Italy) and

References (33)

  • K. Gravningen et al.

    The efficacy and safety of an oil-based vaccine against Photobacterium damsela subsp. piscicida in yellowtail (Seriola quinqueradiata): a field study

    Fish Shellfish Immunol

    (2008)
  • R. van Gelderen et al.

    Experimental vaccination of Atlantic salmon (Salmo salar L.) against marine flexibacteriosis

    Aquaculture

    (2009)
  • J.L. Romalde

    Photobacterium damselae subsp. piscicida: an integrated view of a bacterial fish pathogen

    Int Microbiol

    (2002)
  • A.C. Barnes et al.

    Update on bacterial vaccines: Photobacterium damselae subsp. piscicida

    Dev Biol (Basel)

    (2005)
  • T. Hastein et al.

    Bacterial vaccines for fish—an update of the current situation worldwide

    Dev Biol (Basel)

    (2005)
  • A.L. Delcher et al.

    Improved microbial gene identification with GLIMMER

    Nucleic Acids Res

    (1999)
  • Cited by (23)

    • Protective efficacy of four heat-shock proteins as recombinant vaccines against photobacteriosis in Asian seabass (Lates calcarifer)

      2021, Fish and Shellfish Immunology
      Citation Excerpt :

      However, the immunoprotective effect of whole-cell inactivated vaccines has been reported to vary in different fish species [9,17]. Therefore, many studies have been carried out in recent years in an effort to develop new types of vaccine against Phdp, including live attenuated vaccines [26,27], DNA vaccines [28,29], and subunit vaccines [17,30–32]. Recombinant subunit vaccine is a vaccine in which the gene sequence encoding a specific antigen is inserted into a heterologous system for expressing and purifying the protein antigen.

    • Vaccines and immune protection of principal Mediterranean marine fish species

      2019, Fish and Shellfish Immunology
      Citation Excerpt :

      The great majority of commercially-available vaccines confers protection against bacterial pathologies provoked by Gram-negative bacteria such as Listonella anguillarum, Photobacterium damselae, Streptococcus spp., Tenacibaculum maritimum. These vaccines all have whole inactivated pathogens as main antigen, and the use of isolated bacterial components as active antigens is marginally reported [2]. Whole inactivated pathogens as vaccines represent a complex antigenic mixture and have a value in terms of cost/effectiveness; however, they may induce a limited protection time-wise as short as 5 weeks (Table 1).

    • Outer membrane protein FrpA, the siderophore piscibactin receptor of Photobacterium damselae subsp. piscicida, as a subunit vaccine against photobacteriosis in sole (Solea senegalensis)

      2019, Fish and Shellfish Immunology
      Citation Excerpt :

      For this reason, many efforts were done in the last years to develop new vaccines against Pdp. They included works focused on the identification of immunogenic proteins that could be used as antigens in subunit vaccines [26–28,41,42] and the development of a DNA vaccine [43]. Subunit vaccines are considered a much safer alternative than traditional bacterins since they allow a rapid production of focused vaccines based on a single antigen [44], avoiding possible problems related to bacterins toxicity.

    • Immunogenicity of inactivated formalin-killed Photobacterium damselae subsp. piscicida combined with Toll-like receptor 9 agonist in Cobia Rachycentron canadum

      2018, Aquaculture
      Citation Excerpt :

      The R. canadum farming industry has been suffering a growing damage from various infectious diseases mainly from Photobacterium damselae subsp. piscicida (Chang et al., 2006; Guo et al., 2006). Photobacteriosis in its acute form, can cause multifocal necrosis in the liver and spleen and bacteria can accumulate freely in phagocytes, capillaries and interstitial spaces (Andreoni et al., 2013). To date, several types of P. damselae subsp. piscicida vaccines have been reported in different fishes, including inactivated bacteria (Romalde and Magarinos, 1997), LPS formulations in yellowtail Seriola quinqueradiata (Temminck & Schlegel, 1845) (Fakuda and Kusuda, 1985), ECP-enriched bacterin preparation in gilthead seabream Sparus aurata L. 1758 (Magarinos et al., 1994); water-in-oil emulsion vaccine in S. quinqueradiata (Gravningen et al., 2008), subunit vaccine in R. canadum and seabass Dicentrarchus labrax L. 1758 (Andreoni et al., 2013; Ho et al., 2011).

    View all citing articles on Scopus
    1

    Present address: Department of Life and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche-Monte Dago, 60131 Ancona, Italy.

    View full text