Abstract
Listeria monocytogenes (LM) is a gram-positive facultative intracellular pathogen that could stimulate host to produce inflammatory response, cell-mediated immunity, and humoral immunity. In this study, an attenuated live vector vaccine for Aeromonas hydrophila (AH) named EGDeABdd-dat-ompW was successfully constructed using an attenuated vector named EGDeABdd, in which dal, dat, actA, and inlB genes were deleted from wild-type LM-EGDe. To construct EGDeABdd-dat-ompW, a recombinant plasmid pERL3-dat-ompW obtained by inserting the dat gene from EGDe and outer membrane protein gene ompW from AH into pERL3 plasmid was transformed into EGDeABdd cell. The safety and immunogenicity of EGDeABdd-dat-ompW as an attenuated vector vaccine for delivery of OMPW were assessed through analyzing invasion to Caco-2 cells and mice, cytokine production of macrophagocyte and mouse splenocytes, and T-cell proliferation of mouse splenocytes. Serum titers against AH and the immunoprotective effect of the vaccine to mice were also measured after intravenous injection with vaccine for four times. The results showed that the live vector vaccine EGDeABdd-dat-ompW for AH exhibited high attenuation in invading Caco-2 cells and mice than did EGDe. Real-time PCR (RT-PCR) showed that cytokines (e.g., TNF-α, IL-6, and IL-1β from macrophages; and IL-6 and IFN-γ from mouse splenocytes) had significantly increased after immunization by EGDeABdd-dat-ompW. Meanwhile, the vaccine could induce the production of CD3+CD4+ and CD3+CD8+ T-cell proliferation of mice and generate effective immunoprotection against lethal challenge of 20 × LD50 AH. All these results indicated that the attenuated EGDeABdd-dat could be used as a live vector for the delivery of the exogenous gene, not only possessing safety but also providing high immunogenicity. The successful application in the AH vaccine further showed that it could be used in other fields such as vaccines in cancer or infectious diseases.
Highlights
Listeria monocytogenes (LM), a gram-positive facultative intracellular pathogen, usually causes listeriosis
Upon invading a host cell, LM could escape from the phagocytic cells such as macrophages and dendritic cells (Alberti-Segui et al, 2007) by producing a pore-forming protein, listeriolysin O (LLO), which lyses the vesicular membrane, allowing LM to enter the cytoplasm, where the secreted proteins by LM are degraded to peptides accessed to major histocompatibility complex (MHC) class I molecules of antigen processing and presentation to CD8+ T cells
Protein Expression of the Vaccine Strain EGDeABdd-dat-ompW The Brain heart infusion (BHI) supernatant of the vaccine strain was collected after overnight culture, and the protein in the supernatant was precipitated using pre-cooled ethanol
Summary
Listeria monocytogenes (LM), a gram-positive facultative intracellular pathogen, usually causes listeriosis. Upon invading a host cell, LM could escape from the phagocytic cells such as macrophages and dendritic cells (Alberti-Segui et al, 2007) by producing a pore-forming protein, listeriolysin O (LLO), which lyses the vesicular membrane, allowing LM to enter the cytoplasm, where the secreted proteins by LM are degraded to peptides accessed to major histocompatibility complex (MHC) class I molecules of antigen processing and presentation to CD8+ T cells. The majority of engulfed bacteria are killed by phagolysosomes, and the degraded proteins access directly to MHC class II molecules of antigen presentation to CD4+ T cells (Chen et al, 2014). The preferred way is the use of antibiotic to control the disease in aquaculture, which is presently not recommended owing to the risk involved in the development of resistance in pathogens and the transfer of resistance genes to other animals and human pathogens (Karunasagar et al, 1994). One of the major problems in the development of vaccine is how to select an effective antigen
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