Pleurocidin Research Articles

Protective Immunity against Vibrio harveyi in Grouper Induced by Single Vaccination with Poly (Lactide-co-glycolide) Microparticles Releasing Pleurocidin Peptide and Recombinant Glyceraldehyde-3-phosphate Dehydrogenase

The peptide adjuvant, pleurocidin (PLE), and the Vibrio harveyi antigen, recombinant glyceraldehyde-3-phosphate dehydrogenase (rGAPDH) protein, were encapsulated with poly (lactide-co-glycolide) (PLG) polymers in our previous study to produce PLG-encapsulated PLE plus rGAPDH microparticles (PLG-PLE/rGAPDH MPs) that sustained stable release of both PLE and rGAPDH as well as, after two-time vaccination with MPs, generated long-term protective immunity against V. harveyi in grouper. Stable controlled-release of PLE plus rGAPDH from PLG-PLE/rGAPDH MPs is an attractive feature for developing an effective single-dose vaccine. In the present study, therefore, we aim to evaluate whether single administration with PLG-PLE/rGAPDH MPs in grouper would result in protective immunity against V. harveyi. Peritoneal vaccination of grouper with one dose of PLG-PLE/rGAPDH MPs raised serum titers over a long 12-week period. Moreover, twelve weeks after vaccination, significant lymphocyte proliferation and maximum TNF-α production were found in grouper immunized with a single dose of PLG-PLE/rGAPDH MPs. More importantly, immune responses elicited by single vaccination with PLG-PLE/rGAPDH MPs protected 80% of fish against a lethal peritoneal challenge of the highly virulent V. harveyi (Vh MML-1). In conclusion, our data truly reveal the feasibility of the development of a single-dose vaccine against V. harveyi based on PLG-PLE/rGAPDH MPs.

Amidated pleurocidin peptide encapsulated in biodegradable poly(lactide-co-glycolide) microparticles sustains its antibacterial activity against Photobacterium damselae subsp. piscicida in cobia

Photobacterium damselae subsp. piscicida (PDP), the causative agent of fish pasteurellosis, has become a serious threat leading to high mortalities in cage-cultured juveniles of cobia (Rachycentron canadum). However, overuse of conventional antibiotics to control the infection raises the emergence of resistant bacteria as well as the presence of residual drugs in animals and the environment. Further efforts are therefore needed to look for new and effective antibacterial agents, such as antimicrobial peptides (AMPs), for use on the fish farm to replace conventional antibiotics before commercial vaccines against PDP are available. In the present study, the fish-derived AMP, pleurocidin (PLE), was used as an anti-PDP agent. After C-terminal amidation, the resulting amidated PLE peptide, PLE(am), was further encapsulated with the poly (lactide-co-glycolide) (PLG) polymer to generate PLG-PLE(am) microparticles that would sustain release of antibacterial PLE(am) and elicit effective protection against PDP in cobia. PLG-PLE(am) microparticles, 4.25–5.39 μm in diameter, contained 14.69–18.47 μg of entrapped PLE(am) per milligram of dry weight of microparticles and showed 81%~84% entrapment efficiency. The amount of released PLE(am) from PLG microparticles gradually increased over a 28-day triphasic controlled-release period and the released PLE(am) still retained its antibacterial activity. Peritoneal injection with PLG-PLE(am) microparticles further protected all cobia from a lethal peritoneal infection of 2 × 103 CFU PDP (MML-3) and allowed cobia to survive relatively longer (14 days post infection). In conclusion, the sustained release of antibacterial PLE(am) peptide from PLG-PLE(am) microparticles can preserve the antibacterial activity to result in effective protection in cobia. Our study provides notable evidence to indicate the feasibility of using PLG-PLE(am) microparticles as an antibacterial agent against PDP in cobia.

Pleurocidin Peptide Enhances Grouper Anti-Vibrio harveyi Immunity Elicited by Poly(lactide-co-glycolide)-Encapsulated Recombinant Glyceraldehyde-3-phosphate Dehydrogenase

Outer membrane proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are considered immunodominant antigens for eliciting protective immunity against Vibrio harveyi, the main etiological agent of vibriosis in fish. Cationic antimicrobial peptides (AMPs), such as pleurocidin (PLE), play important roles in activating and recruiting immune cells, thereby contributing to subsequent innate and adaptive immune responses. In the present study, we aimed to use PLE peptide as a potent adjuvant to improve the immunogenicity of V. harveyi recombinant GAPDH (rGAPDH). In order to prepare a controlled-release vaccine, PLE peptide and rGAPDH protein were simultaneously encapsulated into polymeric microparticles made from the biodegradable poly(lactide-co-glycolide) (PLG) polymer. The resulting PLG-encapsulated PLE plus rGAPDH (PLG-PLE/rGAPDH) microparticles, 3.21–6.27 μm in diameter, showed 72%–83% entrapment efficiency and durably released both PLE and rGAPDH for a long 30-day period. Following peritoneal immunization in grouper (Epinephelus coioides), PLG-PLE/rGAPDH microparticles resulted in significantly higher (p < 0.05, nested design) long-lasting GAPDH-specific immunity (serum titers and lymphocyte proliferation) than PLG-encapsulated rGAPDH (PLG-rGAPDH) microparticles. After an experimental challenge of V. harveyi, PLG-PLE/rGAPDH microparticles conferred a high survival rate (85%), which was significantly higher (p < 0.05, chi-square test) than that induced by PLG-rGAPDH microparticles (67%). In conclusion, PLE peptide exhibits an efficacious adjuvant effect to elicit not only improved immunity, but also enhanced protection against V. harveyi in grouper induced by rGAPDH protein encapsulated in PLG microparticles.