Mapping Uncertainties in the Upstream: The Case of PLGA Nanoparticles in Salmon Vaccines

The diversity of nanotechnologies and of the governance challenges that their applications raise calls for exploration and learning across different cases. We present an Upstream Oversight Assessment (UOA) of expected benefits and potential harms of nanoparticles made of a synthetic polymer (PLGA) to improve vaccines for farmed salmon. Suggested by Jennifer Kuzma and colleagues, an UOA may help identify and prioritise research needs, and it may support evaluations of the adequacy of relevant existing regulatory frameworks. In this work, the UOA approach is modified and supported with elements from the uncertainty analysis framework developed by Warren Walker and colleagues. Empirically, we draw on relevant available published literature and insights generated in an ongoing nanoparticle salmon vaccine project, in which one of the authors participates. Nanotechnologies have not previously been encountered in the regulatory context of fish vaccines, which in part raises unique challenges due to prospective large scale vaccine use in semi-open aquatic systems. Strengthened through cooperation between ELSA and technology researchers we found the UOA useful for an early mapping of benefits and concerns, and for identifying areas in need of further research prior to a nanoparticle based salmon vaccine is developed and taken into use. We consider our approach to represent one among several complementing initiatives that seek to contribute to early stage evaluations of possible negative side effects, broadly conceived, in order to facilitate a more robust nanotechnology development

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 107.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality

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

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

Differences in smolt status affect the resistance of Atlantic salmon (Salmo salar L.) against infectious pancreatic necrosis, while vaccine-mediated protection is unaffected

In today's aquaculture of Atlantic salmon (Salmo salar L.), a majority of viral disease outbreaks occur after seawater transfer. A relevant question is how the parr–smolt transformation influences the efficacy of viral vaccines and the innate resistance against viral diseases. In this study, vaccinated and unvaccinated A. salmon parr were exposed to different photoperiodic regimens (1‐, 3‐ or 6‐week continuous light—WCL). Fish groups at different stages in the smoltification process were induced, as demonstrated by differences in morphological and physiological smolt parameters. At the time of seawater transfer, the 6‐WCL group had reached a more pronounced stage in the smoltification process than the 1‐WCL group. In unvaccinated fish, the subsequent cohabitation challenge with infectious pancreatic necrosis virus (IPNV) gave a significantly higher accumulated mortality in the 6‐WCL group (87%) compared to the 1‐WCL group (39%). In the vaccinated groups, this effect was not apparent and there were no differences in accumulated mortality between the 1 WCL, 3 WCL and 6‐WCL groups. These data suggest that the resistance to IPN in A. salmon was negatively influenced by smoltification, while vaccine‐mediated protection to IPN was maintained equally well irrespective of smolt status

A Systematic Approach towards Optimizing a Cohabitation Challenge Model for Infectious Pancreatic Necrosis Virus in Atlantic Salmon (Salmo salar L.).

A cohabitation challenge model was developed for use in evaluating the efficacy of vaccines developed against infectious pancreatic necrosis virus (IPNV) in Atlantic salmon (Salmo salar L) using a stepwise approach. The study involved identifying a set of input variables that were optimized before inclusion in the model. Input variables identified included the highly virulent Norwegian Sp strain NVI015-TA encoding the T217A221 motif having the ability to cause >90% mortality and a hazard risk ratio of 490.18 (p<0.000) for use as challenge virus. The challenge dose was estimated at 1x10(7) TCID50/mL per fish while the proportion of virus shedders was estimated at 12.5% of the total number of fish per tank. The model was designed based on a three parallel tank system in which the Cox hazard proportional regression model was used to estimate the minimum number of fish required to show significant differences between the vaccinated and control fish in each tank. All input variables were optimized to generate mortality >75% in the unvaccinated fish in order to attain a high discriminatory capacity (DC) between the vaccinated and control fish as a measure of vaccine efficacy. The model shows the importance of using highly susceptible fish to IPNV in the optimization of challenge models by showing that highly susceptible fish had a better DC of differentiating vaccine protected fish from the unvaccinated control fish than the less susceptible fish. Once all input variables were optimized, the model was tested for its reproducibility by generating similar results from three independent cohabitation challenge trials using the same input variables. Overall, data presented here show that the cohabitation challenge model developed in this study is reproducible and that it can reliably be used to evaluate the efficacy of vaccines developed against IPNV in Atlantic salmon. We envision that the approach used here will open new avenues for developing optimal challenge models for use in evaluating the efficacy of different vaccines used in aquaculture

Post challenge survival proportions and hazard ratios of different IPNV isolates used for selecting the challenge virus strain.

<p>Post challenge survival proportions and hazard ratios of different IPNV isolates used for selecting the challenge virus strain.</p

Comparison of post challenge survival proportions and risk ratios of the high and low Atlantic salmon susceptible strains to NVI015-TA.

<p>Comparison of post challenge survival proportions and risk ratios of the high and low Atlantic salmon susceptible strains to NVI015-TA.</p

High and low virus challenge dose.

<p>Kaplan Meyer’s (KM) survival analysis of Atlantic salmon challenged using a high and low challenge dose carried out using strain NVI015-TA (Study II). (A) KM survival analysis for the high challenge dose (HC_dose), 1x10⁷ TCID₅₀/fish. (B) KM survival analysis for the low challenge dose (LC_dose), 1x10⁵ TCID₅₀/fish. Fig 1A shows a wider discriminatory capacity (DC>58%) compared to Fig 1B (DC = 40%) between the vaccinated and unvaccinated control fish.</p

Proportion of virus shedders.

<p>KM survival analysis for cohabitees challenged with different (12.5%, 20% and 30%) proportions of virus shedders of the total number of fish per tank (<i>N</i> = 90; Study III). All virus shedders were injected with 1x10⁷ TCID₅₀/fish with strain NVI015-TA. Mortality of shedders in the 10% and 30% proportion groups started on day 18 after challenge while for the 20% group day 21 after challenge was the first day of mortality. Overlaps in the KM survival curves for the cohabitees show that there was no statistical difference between the 10% and 20% (p>0.368), as well as between the 10% and 30% (p>0.1051) proportion of virus shedders of the total number of fish per tank.</p