PLGA and PLA particles as vaccine delivery systems for Atlantic salmon : a study on formulation and use with an emphasis on immune responses

Abstract

Vaccines are regarded as the safest and most cost-effective strategy to prevent infectious diseases. For some diseases, vaccine improvements are required as protection levels are still inadequate. The key to solving this challenge might lie in the development of more efficacious vaccine delivery systems and adjuvants. Poly (lactide-co-glycolide) (PLGA) is a biodegradable polymer which has an extensive safety record in biological systems and possesses immunological adjuvant properties as injectable particles. In the present work, micro- and nanoparticles of PLGA and PLA were explored as a vaccine delivery system in Atlantic salmon (Salmo salar). The overall objectives were to investigate their adjuvant abilities in provoking innate and adaptive immune responses, forming antigen depots and inducing protective immunity in a challenge test with infectious pancreatic necrosis virus (IPNV). Formulation parameters in preparation of polymeric particles were systematically optimized (paper IV) to achieve stable PLGA particle products containing co-entrapped model antigens and β-glucan (paper I and II), or virus particles of infectious pancreatic necrosis virus (IPNV) (paper III). Post immunization potency of nanoparticles (300-400 nm) was demonstrated by their ability to induce early innate responses (day 2, 4 and 8) at transcription levels equal to or higher than the oil-adjuvanted formulation (paper I). Temporal differences in expression levels of innate markers were observed, suggesting rapid systemic distribution of particles (paper I). By tracing of isotope labelled proteins, nanoparticles (˂ 1000 nm) were found to localize antigens in the head kidney while micro-sized (~ 8 µm) particles generally retained antigen at the injection site. Irrespective of size, particles made of polymers with high molecular weight (MW) generally had superior depot capabilities compared to their low MW counterparts (paper II). Adaptive immune responses to immunization were assessed by QPCR and ELISA. T cell markers were not differentially expressed at the selected early time points (paper I), but at day 60 and 75 antibody responses were found to be elevated (paper II and III). In a dose-response study, micro- but not nanoparticles were demonstrated to be equally potent compared to the oil-adjuvanted control group with regard to induction of antibody responses. Long-term antibody responses induced by particles were generally less robust and therefore declined towards the end of the experimental period (120 days), while responses induced by the oil-adjuvanted formulation progressively increased. Following immunization, antibody responses were not related to polymer qualities or the ability of particles to depot or distribute antigens. Scoring of side effects demonstrated excellent safety profiles for the particle formulations (paper II and discussed in paper V). In paper III, vaccine efficacy was tested in a cohabitation challenge with IPN. Survival rates for the nanoparticle vaccinated groups were comparable to the non-vaccinated control fish and demonstrated that their ability to induce protection against IPN was inferior to the oil-adjuvanted vaccines. Virus re-isolation from head kidney and blood during the challenge period did however demonstrate some level of protection as the nanoparticle vaccinated groups were able to delay the IPNV infection.

In the presented studies, the principal adjuvant properties of PLGA particles in Atlantic salmon have been demonstrated to include their capacity to induce strong innate responses and provide antigen depots for long-term delivery of antigens. In addition, indication of particle presence in lymphoid organs was an interesting finding that could suggest a certain targeting effect to phagocytic cells. To achieve a better understanding of how PLGA particles may be used to direct immune responses in salmon, more detailed studies on particle qualities-cell interactions/responses are required.