Sophie A M Elliott
email: sophie_elliott@yahoo.com

Daniel Bearup

Alexandre Carpentier

Angela Larivain

Thomas Trancart

Eric Feunteun

Evaluating the effectiveness of management measures on skates in a changing world

Keywords: climate change, elasmobranchs, IUCN red list species, management measures, population dynamic model, species conservation

Global declines in elasmobranchs have been observed. Conservation measures such as area closures and fisheries prohibitions have been put in place to support the recovery of vulnerable species. However, the effectiveness of such measures is rarely evaluated in the context of other factors that may affect population abundance. This study investigates the effectiveness of management measures using 1) General additive mixed model derivative changes, taking into account environmental factors that may affect population stochasticity and 2) an age-structured density dependent population dynamic model. The Raja undulata (undulate ray) 2009 targeted fisheries prohibition was used as a case study.

Potential beneficial responses on sympatric species Raja clavata (thornback ray) were modelled.

A significant increase in abundance was observed in both IUCN red list species during the ban. Surface seawater temperature had a marginal effect on the abundance of both species. The prohibition was in place for an insufficient length of time for long lasting effects to be detected on skate length. The population dynamic model indicated that the increase in abundance was only possible when combining the fisheries ban with increased juvenile discard survival. Our results indicate that species conservation measures may not only have positive effects on the species in question, but also on species with a niche overlap. Nonetheless, due to ongoing fishing for other species, the full potential of fisheries prohibitions may not be realised. For real benefits to be assessed, evaluation of bans should take place once a steady state is observed.

Introduction

The rise in demand for fish and fish products has led to increased fishing pressure, declines and collapses in both targeted and non-targeted species.

Pressures from fishing, alongside climate change effects, have been attributed as the most important reasons for declines in populations and changes in species distribution [START_REF] Halpern | Evaluating and ranking the vulnerability of global marine ecosystems to anthropogenic threats[END_REF][START_REF] Heath | Review of climate change impacts on marine fish and shellfish around the UK and Ireland[END_REF][START_REF] Perry | Sensitivity of marine systems to climate and fishing: Concepts, issues and management responses[END_REF]. Pressures from fishing activity not only have an impact on the size and structure of fish populations and their habitats, but also their life-history traits. Fisheries induced pressures have been observed to modify size and age at maturation, reproductive effort and growth rates, among other effects [START_REF] Enberg | Implications of fisheries-induced evolution for stock rebuilding and recovery[END_REF][START_REF] Heino | Fisheries-induced selection pressures in the context of sustainable fisheries[END_REF][START_REF] Stevens | The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosystems[END_REF]. Effects from climate change on fish, can include modifications to their distribution, loss of coastal habitats, changes in productivity, etc. [START_REF] Dulvy | Extinction vulnerability in marine populations[END_REF][START_REF] Perry | Potential impacts of climate change on marine wild capture fisheries: an update[END_REF][START_REF] Perry | Sensitivity of marine systems to climate and fishing: Concepts, issues and management responses[END_REF].

Larger, slower maturing and less fecund species, such as elasmobranchs, are more vulnerable to anthropogenic pressures than most teleost fish [START_REF] Dulvy | Fishery stability, local extinctions, and shifts in community structure in skates[END_REF][START_REF] Stevens | The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosystems[END_REF][START_REF] Ellis | Preliminary observations on the life history and movements of skates (Rajidae) around the Island of Jersey, western English Channel[END_REF]. Their life history traits lead to low rates of reproduction, and therefore low rates for population recovery (Dulvy et al., 2000;[START_REF] Stevens | The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosystems[END_REF][START_REF] Walker | Sensitive skates or resilient rays? Spatial and temporal shifts in ray species composition in the central and north-western North Sea between 1930 and the present day[END_REF]. As a result of severe global declines observed during the late twentieth century [START_REF] Dulvy | Challenges and Priorities in Shark and Ray Conservation[END_REF][START_REF] Stevens | The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosystems[END_REF], conservation management measures were set up to protect elasmobranch species (e.g. added to IUCN red list, prohibited from commercial exploitation, country specific national action plans, etc.) [START_REF] Dulvy | Challenges and Priorities in Shark and Ray Conservation[END_REF].

However, few studies have evaluated the effectiveness of such measures (Dulvy et al., 2017;[START_REF] Hutchings | Collapse and recovery of marine fishes[END_REF][START_REF] Shiffman | Shark conservation and management policy: a review and primer for non-specialists[END_REF].

Before after control impact studies [START_REF] Underwood | Beyond BACI: the detection of environmental impacts on populations in the real, but variable, world[END_REF][START_REF] Underwood | On beyond BACI: Sampling designs that might reliably detect environmental disturbances[END_REF] are widely accepted as the most appropriate method to assess the effectiveness of spatial management measures [START_REF] Ahmadia | Integrating impact evaluation in the design and implementation of monitoring marine protected areas[END_REF]Clarke et al., 2015;[START_REF] Claudet | Improving assessments of marine protected areas[END_REF]. Nonetheless, to evaluate spatial management measures using before after control impact methods, control areas are required. Other possible methods to assess population recovery following the implementation of management measures include evaluating biomass time series trends, as undertaken within demographic analysis and stock assessments [START_REF] Dowling | Generic solutions for data-limited fishery assessments are not so simple[END_REF][START_REF] Hutchings | Collapse and recovery of marine fishes[END_REF]. For data deficient species, as is the case for most elasmobranchs, such assessments often lack reference points at which populations trends should be compared against (ICES, 2018a-d;[START_REF] Dowling | Generic solutions for data-limited fishery assessments are not so simple[END_REF]. The study of surrogate species is also increasingly used in conservation when species of conservation focus cannot be used, or to study the response of a given species from a disturbance, to predict the response of another species to a similar disturbance [START_REF] Caro | Use of Substitute Species in Conservation Biology[END_REF].

In this study, we used long-term fisheries dependent (2003 to 2018) and independent (1995 to 2018) data on skate abundance (the mean number of skate individuals observed per haul) and length. We analysed significant generalised additive mixed model derivative changes according to the implementation of a species-specific fisheries ban. We used the Raja undulata (undulate ray) fisheries ban, set up in 2009, prohibiting targeted fishing and bycatch landing (EC 43/2009;[START_REF] Ellis | An overview of the biology and status of undulate ray Raja undulata in the north-east Atlantic Ocean[END_REF]. Due to declines in R. undulata abundance, it was categorised under the IUCN red list as 'Endangered' (IUCN, 2019). The imposition of this ban was controversial with certain fishers due to high capture rates observed (ICES, 2018a & b). Consequently, R. undulata was removed from the prohibition list in 2015, and a small but annually increasing quota was permitted in the English As a result of missing control data on R. undulata to undertake a before after control impact study, sympatric species Raja clavata (thornback ray; classified as 'Near threatened') (IUCN, 2019;[START_REF] Ellis | The Distribution of Chondrichthyan Fishes Around the British Isles and Implications for Conservation[END_REF][START_REF] Martin | Modelled distributions of ten demersal elasmobranchs of the eastern English Channel in relation to the environment[END_REF], was analysed as a surrogate species which may benefit from the ban. To understand whether changes in skate abundance were due to the fisheries ban rather than climate change, the trend in abundance was modelled with seawater temperature. French fishing effort changes (fished days at sea) were also evaluated. To assess recovery of populations from potential fisheries induced effects, length changes over time were modelled. Finally, a density dependent agestructured Leslie matrix population dynamic model was developed to determine historical trends in exploitation and assess the compensatory ability of such a kstrategy species to a fisheries ban.

It was hypothesised that an increase in mean abundance would be observed in both species some time after the fisheries ban, as a result of their sympatric nature. It was also hypothesised that climate change would accentuate the increase in mean abundance of both species, and, in particular R. undulata given its warmer water preference [START_REF] Sguotti | Distribution of skates and sharks in the North Sea: 112 years of change[END_REF]. An immediate slight increase in size of both species was expected due the removal of targeted fishing of larger individuals. However, no overall long-term increase in mean size was expected due to the reintroduction of the quotas. Given the fishing effort data analysed was measured in days at sea for all species catch, by the gear types which caught skates (partly as bycatch), it was expected that the overall fishing effort would have remained more or less constant.

Method

Survey data

Fisheries independent Channel Ground Fish Survey (CGFS) data and French fisheries dependent observer (ObsMer) data were analysed from North-Eastern Atlantic waters (Fig. 1). CGFS surveys have been carried out annually within the Eastern English Channel (International Council for the Exploration of the Sea (ICES) division 7.d) between September and November since 1988 using a fixed sampling design. Only data from 1995 were used due to standardisation in the survey locations from this date. The sampling gear for CGFSs are Grande Ouverture Verticale trawls with a 10 m horizontal by 3 m vertical opening and a codend of 20 mm [START_REF] Bourdaud | Inferring the annual, seasonal, and spatial distributions of marine species from complementary research and commercial vessels' catch rates[END_REF]. Scientific bottom trawl survey data from the North Sea (International Bottom Trawl Survey) and the west coast of Europe, were not used for R. undulata due to excessively high zero counts (i.e. 99%).

ObsMer data provides targeted and bycatch, landed and discarded data from fishing vessels throughout the year from the Greater North Sea, Celtic Sea, the Bay of Biscay and the Mediterranean. Only data from ICES divisions which contained the two species skates were considered (ICES divisions 4.c,7.d,7.e,7.g and 7.h,8.a and 8.b).The ObsMer programme begun in 2003. The procedure for ObsMer data collection is summarised in [START_REF] Fauconnet | Characterizing catches taken by different gears as a step towards evaluating fishing pressure on fish communities[END_REF].

For both datasets, species caught were identified, the number of individuals of each species per haul recorded, and their length to the nearest cm. Catch per unit effort was not calculated due to a large number of unavailable information in both datasets. In addition, for the ObsMer data, insufficient survey information was available to calculate catch per unit effort for each gear type. Hauls that did not contain skate landings were included to account for zeros.

Skate abundance variations

All statistical modelling and mapping were undertaken within R CRAN free software (version 3.3, http://cran.r-project.org). The mean number of skates per haul was modelled against year rather than the total number per haul per year to reduce the stochasticity of population trends. Due to the large spatio-temporal differences between the fisheries dependent and independent datasets, the datasets were analysed independently. Potential outliers were identified with boxplots and mapping aberrant values. Generalised additive mixed models were implemented to observe non-linear trends over the years. Negative binomial distributions were implemented to account for over dispersion. The abundances were square root transformed to reduce right skewness and improve the model fit.

To reduce counting false zeros within the ObsMer dataset, only complete observations from each haul were used, and both landed and discarded individuals were considered. To reduce zero inflation, spatial and temporal bias, and presence over estimations, a skate catch of more than one percent, and only gear types with an even spatial-temporal coverage were used for analysis. For both skates, these included trammel nets, otter beam and otter twin trawls. Data outside the known ranges of the skates depth distribution (>100 m for R. undulata and >150 m for R. clavata; accessed from ObsMer, CGFSs and published literature) [START_REF] Ellis | An overview of the biology and status of undulate ray Raja undulata in the north-east Atlantic Ocean[END_REF][START_REF] Serra-Pereira | Morphometric ratios of six commercially landed species of skate from 29 the Portuguese continental shelf, and their utility for identification[END_REF] were removed to reduce zero inflation. To account for gear and area (ICES divisions) effects on skate abundance from the ObsMer data, both variables were incorporated into the model as random effects. To account for varying trawl durations using the CGFSs, swept surface area (km 2 ) was included in the model as an offset.

For both CGFS and ObsMer abundance models, violations assumptions of homogeneity of variance were observed, and refitted with a varPower variance structure [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology with R[END_REF]). Backward stepwise model selection was implemented [START_REF] Bolker | Generalized linear mixed models: a practical guide for ecology and evolution[END_REF][START_REF] Zuur | Mixed Effects Models and Extensions in Ecology with R[END_REF] and a log likelihood ratio test was used to test model significance against the null hypothesis in addition to checking residual plots.

To interpret the fitted trends and identify whether there were any changes in abundance over year, significant derivative changes from zero, along with the 95% confidence intervals were calculated. The model's fitted values were calculated at 200 equally spaced points and were calculated again along the trend line and the model refitted. The difference between the two sets of fitted values was divided by the difference in year to give a predictor matrix of the slope of the spline. The predictor matrix was then multiplied by the coefficients of 10,000 random simulations from the posterior distribution of the model. From this method, 95% confidence intervals of the derivatives were calculated by taking the two extreme quantiles of the distribution. When the 95% confidence intervals of the derivative did not include zero, a significant increase or decrease in the abundance was recorded (Clarke et al., 2015;[START_REF] Simpson | gavinsimpson's gists[END_REF].

Climate change effects

To explore whether climate change effects were responsible for the increase in skate abundance, monthly mean sea surface temperature data were downloaded To explore which variable was more important, generalised additive mixed models with just 'years' as an explanatory variable, just 'temperature' ( • C), and 'temperature' and 'years' were implemented (Table 1). The Akaike information criterion score with the lowest value was used as the factor discriminating the variable of greatest influence (Table 1).

Length variations

Changes in length over time were explored using generalised additive mixed models. The aforementioned derivative analysis was then applied to explore potential changes in mean length per haul over time. Kernel smoothed probability density length plots were also analysed to understand potential changes in length distribution according to the different management regimes.

Other parameters to investigate potential fisheries induced evolution effects were not taken into consideration, due to insufficient biological information on maturity and age. Life history trait information is scarce in elasmobranchs and the baseline information which exists for these species varies greatly between regions (e.g. [START_REF] Ellis | Assessing the status of demersal elasmobranchs in UK waters: A review[END_REF]Mccully et al., 2012).

Fishing effort changes

To infer fishing effort change over the years, aggregated fishing effort data (days at sea per year by gear type per region) were provided by the Institut Français de Recherche pour l'Exploitation de la MER (an oceanographic institution in France -IFREMER). The aggregated fishing effort data was analysed between 2006 and 2018 using general and linear mixed effect models. Only gear types (trammel nets, otter beam and otter twin trawls) and regions (Bay of Biscay and the English Channel) used in the abundance models were taken into consideration for comparative purposes.

The Population Dynamic Model

To understand the compensatory ability of R. undulata to a fishery ban and identify parameters that most affected population dynamics, an age-structured population dynamic model, with density dependent fecundity was developed from the von Bertalanffy growth function calculation. The population dynamic model was developed from similar fishing scenarios to those experienced by R. undulata (a set fishing mortality prior to the ban, followed by zero fishing mortality during the ban (2008 to 2015) and set fishing mortality after the relax in measures in 2015). A 'recovered' population was calculated from 1945, after the second world war, when skate populations were supposed to have recovered from pre-war fishing pressure [START_REF] Walker | Sensitive skates or resilient rays? Spatial and temporal shifts in ray species composition in the central and north-western North Sea between 1930 and the present day[END_REF]).

Length at age was calculated from Von Bertalanffy Growth Function (VBGF) (Equation 1), using length information from the ObsMer dataset. ObsMer data were used since it is collected throughout the year and can therefore provide information on skate growth. The VBGF was performed using electronic length frequency analysis with genetic algorithm used for estimating growth parameters in the TropFish R package [START_REF] Mildenberger | TropFishR : an R package for fisheries analysis with length-frequency data[END_REF]. Isles. Portuguese R. undulata populations have been observed to be smaller than those found in the Channel and the Bay of Biscay.

= (1 -exp ( -( -)) (1)

Density dependent Leslie matrix model

A density dependent Leslie matrix model for R. undulata was developed (equation 2).

= , = ⋮ , = … ℎ … 0 0 ⋮ ⋱ ⋮ ⋮ 0 0 ℎ 0 (2)
the system, and ! , ! , , and ℎ ! denote the fecundity, natural survival rate, and fishing (or discard) survival rate for each age class " respectively (Table 2 and3).

As far as possible, we parameterised the model (natural survival, fecundity, maturity, fishing and discard survival) from existing literature [START_REF] Frisk | Estimation and analysis of biological parameters in elasmobranch fishes: a comparative life history study[END_REF][START_REF] Froese | A new approach for estimating stock status from length frequency data[END_REF][START_REF] Hordyk | Comment on "A new approach for estimating stock status from length frequency data[END_REF][START_REF] Serra-Pereira | Using biological variables and reproductive strategy of the undulate ray Raja undulata to evaluate productivity and susceptibility to exploitation[END_REF] and by comparison to stock records (ICES, 2018a & b). However, the precise parameter values are uncertain so we performed a sensitivity analysis, centred on the chosen parameter set, to assess the effect of variation in these parameters (Fig. S1; Table 2 and3).

Originally fishing survival and discard survival were taken equal to 1 in an unfished scenario. In a fished scenario, fishing survival was fixed for all age-classes.

However, it was observed that model predictions of population recovery after the ban did not match empirical data. Since the sensitivity analysis highlighted the importance of juvenile survival (age-1 to age-3) (Fig. S1), we introduced a survival rate for juveniles in the fished scenario. This represents the probability that a juvenile survived having been caught and discarded (juvenile discard survival (Table 3; Fig. S2).

Following [START_REF] Levin | Analysis of an age-structured fishery model[END_REF], we assumed that fecundity varied with total population # = ∑ ! !% according to a Ricker equation (equation 3).

! = & 0, " < ( ) exp( -*# ) , " ≥ ( ) (3)
where is the maximum fecundity (taking into account sex ratio), ( ) the age of maturity and * scales the density dependent effect.

Results

Skate abundance variations

Using both datasets, a significant increase in the number of R. undulata and R. clavata was observed during the fisheries ban, which tailed off shortly after quotes for R. undulata was reintroduced in 2015 (Fig. 2). From the CGFS data, the significant increase in R. undulata occurred from 2010 (Fig. 2

Climate change effects

For both species the model of best fit was that of an interaction between temperature and year (Table 1). Both species had a non-linear preference for warmer water (Fig. 3). The increased abundance observed during in the later years with increased temperature will most likely as a result of the ban and other external factors. When comparing predictor variables independently, year was the stronger predictor variable for both species (Table 1).

Length variations

For both species and both datasets, there was little if any mean length variation from the derivative plots in relation to the different management measures (Fig. S3). However, using both datasets a higher probability density function of small R. undulata (~20-40 cm) was observed before the ban than during or after, whereas the probability density function of larger individuals (~30-50 cm) evened out after the ban (Fig.

Fishing effort changes

A slight decrease in fishing effort (fishing days per gear type and region) was observed from 2006 to 2018 (log likelihood ratio = -1594.81, degrees of freedom = 4, p < 0.001; Fig. 5).

The population dynamic model

The long-term behaviour of the population was predicted by analysis of the Leslie matrix model. Population growth occurs when the leading Leslie matrix eigenvalue (lambda, ,) is greater than one [START_REF] Leslie | On the use of matrices in certain population mathematics[END_REF]. We found that, for an unfished population, the best-case scenario, , is 1.06; so, the population is sustainable. When fishing was included in the model, reducing survival, , decreased. In particular, using this model, we identified that a fishing survival rate of less than 88% would reduce , below 1, resulting in the population declining to extinction. Consequently, this model predicts that sustainable fishing is only possible when fishing mortality is less than 12% (Fig. 6).

Fishing survival and juvenile discard survival were varied in order to match the behaviour of the CGFS and ObsMer data (Fig. 2.a.i-ii; Fig 6). Similar recovery dynamics to the real data were only observed for R. undulata when the fishing ban increased juvenile discard survival (Table 3; Fig. 6iii.b-c). However, the rapid decline after the end of the ban predicted by the population dynamic model (Fig. 6), was not observed within the real data. The sensitivity analysis demonstrated that the recovery dynamics were highly sensitive to juvenile survival rates and fishing survival, and somewhat sensitive to fecundity, while being relatively insensitive to the other model parameters (Fig. S1).

Discussion

Given our increasing human population causing growing pressures on our natural resources [START_REF] Lotze | Depletion, Degradation, and Recovery Potential of Estuaries and Coastal Seas[END_REF], the implementation and monitoring of management measures are of ever greater importance [START_REF] Hutchings | Marine Fish Population Collapses: Consequences for Recovery and Extinction Risk[END_REF][START_REF] Ward-Paige | Recovery potential and conservation options for elasmobranchs[END_REF]. However, the effectiveness of management measures is rarely evaluated in the context of other factors that may affect populations. In this study, we combined statistical and theoretical models to distinguish the effect of a fisheries ban from other external factors that may affect the two species of skate.

Abundance changes

Using the derivative model, we were able to detect a slight but significant increase in the skate abundance in both the fisheries dependent and independent data. The generalised additive mixed models suggest that the increase in abundance was due to the implementation of the R. undulata fisheries ban. Similar An increase in sympatric R. clavata was also observed. The increase in R.

clavata detected in both datasets may have been as a result of beneficial effect of the fisheries ban on R. clavata, given the overlap in niche of these species [START_REF] Elliott | Distribution and life history trait models indicate vulnerability of skates[END_REF][START_REF] Ellis | The Distribution of Chondrichthyan Fishes Around the British Isles and Implications for Conservation[END_REF][START_REF] Martin | Modelled distributions of ten demersal elasmobranchs of the eastern English Channel in relation to the environment[END_REF]. The increase in the number of R. clavata began slightly before the R. undulata ban was implemented. The latter is most likely a result of a natural increase in abundance beginning prior to the knock-on benefits of the ban, as observed within stock assessments (ICES, 2018c).

Once the R. undulata quota was reintroduced in 2015, the increase in abundance of both species tailed off. Albeit, for R. clavata, this curtail was not so prominent.

From looking at ICES stock assessments for both species (ICES, 2018a & c) similar trends can be observed.

The use of surrogate species is cautioned against since each species has its own specific life history and ecological traits [START_REF] Caro | Use of Substitute Species in Conservation Biology[END_REF][START_REF] Henry | Do substitute species help or hinder endangered species management?[END_REF]. Nonetheless, the analysis of management measures on sympatric species has benefits to better understand potential ecosystem effects of species-specific conservation measures and anthropogenic pressures [START_REF] Henry | Do substitute species help or hinder endangered species management?[END_REF].

Climate change effects

Although statistically, the implementation of the fisheries ban, had the most important effect on abundance variations in the skates studied, the increase in mean seawater temperature was also of consequence. An increase in seawater temperature may initially be of benefit to R. undulata. However, given R.

undulata's patchy and shallow water habitat occupancy [START_REF] Elliott | Distribution and life history trait models indicate vulnerability of skates[END_REF]Ellis et al., 2012), in future, temperatures may increase beyond its tolerance threshold.

The latter may lead to R. undulata becoming unable to undertake range shifts [START_REF] Heath | Review of climate change impacts on marine fish and shellfish around the UK and Ireland[END_REF][START_REF] Perry | Sensitivity of marine systems to climate and fishing: Concepts, issues and management responses[END_REF], exposing it to greater ecological risk [START_REF] Musick | Criteria to Define Extinction Risk in Marine Fishes: The American Fisheries Society Initiative[END_REF][START_REF] Simpfendorfer | The importance of research and public opinion to conservation management of sharks and rays: a synthesis[END_REF].

From the two datasets, different temperature ranges were observed for R.

clavata, possibly as a result of distinct populations between the Channel and the Bay of Biscay (ICES, 2018d & e). Distinct species population temperature preferences have previously been observed [START_REF] Heath | Review of climate change impacts on marine fish and shellfish around the UK and Ireland[END_REF]. These results demonstrate the importance of taking into account potential effects of global warming when studying long-term distribution and abundance changes in species [START_REF] Barausse | The role of fisheries and the environment in driving the decline of elasmobranchs in the northern Adriatic Sea[END_REF], and in particular over their entire geographic extent. It should be noted that the adjusted R 2 value for the models were relatively low.

Other factors will therefore have contributed to abundance changes over the years.

Length changes

Given our long history of fishing pressure on commercially important species, fisheries-induced evolution effects have been recorded on a number of species (e.g. [START_REF] Hunter | Fishery-induced changes to age and length dependent maturation schedules of three demersal fish species in the Firth of Clyde[END_REF][START_REF] Walker | Sensitive skates or resilient rays? Spatial and temporal shifts in ray species composition in the central and north-western North Sea between 1930 and the present day[END_REF][START_REF] Wright | Fishery-induced demographic changes in the timing of spawning: consequences for reproductive success[END_REF].

Although a reverse in such effects have not been recorded, they have been discussed [START_REF] Sguotti | Distribution of skates and sharks in the North Sea: 112 years of change[END_REF]. The lack of obvious mean length changes from the derivative model was most likely due to the short period of time the ban was in place relative to the life cycle of the skates. Time to maturation for R. undulata is between five and seven years [START_REF] Coelho | Reproductive aspects of the undulate ray, Raja undulata, from the south coast of Portugal[END_REF]Mccully et al., 2012;[START_REF] Moura | The use of caudal thorns for ageing Raja undulata from the Portuguese continental shelf, with comments on its reproductive cycle[END_REF]. The higher probability density function of larger R. undulata observed within the density plots during and after the ban, and the larger discarded individuals during the ban, will have likely been due to decreased fishing effort during the ban. Such length variation changes according to the ban in R. clavata

were not so prominent.

Use of fisheries dependent and independent data

The coherent results, across both data sets strengthen the outcomes observed. The slight differences detected will have been residual effects from spatial and gear variations between the two datasets. Fisheries dependent data are not often used to assess abundances due to their targeted nature. The comparable results between the fisheries dependent and independent data highlight the potential utility of such abundant and opportunistic data. [START_REF] Bourdaud | Inferring the annual, seasonal, and spatial distributions of marine species from complementary research and commercial vessels' catch rates[END_REF] similarly compared abundance trends of a range of commercially fished species using fisheries dependent data with fisheries independent data, and found coherence between datasets in a number of species.

Fishing effort

From the fishing effort data obtained, a slight decrease in days at sea was observed for the gear types analysed. These results are in line with France AgriMer information, where the number of small, medium and large fishing vessels fishing across France Metropolitan have been recorded to decrease between 1995 and 2014 (https://www.franceagrimer.fr/). It should be noted that the cessation of targeted fishing for demersal species rarely eliminates fishing mortality. The latter is because of the low selectivity of fishing gear, the ongoing habitat destruction from demersal fishing [START_REF] Dulvy | Extinction vulnerability in marine populations[END_REF][START_REF] Hutchings | Collapse and recovery of marine fishes[END_REF], and illegal fishing which takes place [START_REF] Davidson | Why have global shark and ray landings declined: improved management or overfishing?[END_REF][START_REF] Hutchings | Marine Fish Population Collapses: Consequences for Recovery and Extinction Risk[END_REF][START_REF] Worm | Global catches, exploitation rates, and rebuilding options for sharks[END_REF].

The population dynamics model

The population dynamic model sensitivity analysis, indicate that the factors most affecting R. undulata's ability to recover from fishing are early years survival rates and fishing survival. Developing a detailed understanding of the effect of individual parameters can inform conservation measures. In particular, our results

suggest that protecting juveniles through measures such as minimum landing sizes and nursery areas could have important benefits to species survival. [START_REF] Brander | Disappearance of common skate Raia batis from Irish Sea[END_REF] and [START_REF] Ward-Paige | Recovery potential and conservation options for elasmobranchs[END_REF], came to similar conclusions regarding the importance of juvenile survival to elasmobranch population recovery.

Little data regarding age specific natural survival rates for these elasmobranchs exist. However, the sensitivity analysis suggests that most of these parameters are relatively unimportant. Instead, it is more important to characterise early years survival of R. undulata in both unfished and fished regimes, and its fecundity. Further research on skate bycatch survival is essential and gear specific mortality should be incorporated into future population dynamic models. Furthermore, at present we are unsure of skate mortality within their nursery habitats. For R. undulata this is particularly important, given juveniles inhabit coastal and estuarian areas which are subject to a range of anthropogenic pressures [START_REF] Elliott | Survival of the fittest: Explanations for gadoid imbalance in heavily fished seas[END_REF].

Implications for conservation and management

To be able to manage our natural resources sustainably and minimise impacts on industry, monitoring of management measures is key to the long-term viability of populations [START_REF] Hilborn | Moving to sustainability by learning from successful fisheries[END_REF][START_REF] Parma | The good, the bad, and the ugly: learning from experience to achieve sustainable fisheries[END_REF][START_REF] Pauly | Anecdotes and the shifting baseline syndrome of fisheries[END_REF].

Surprisingly, very few case studies have been undertaken to explore the effect of fisheries management measures beyond stock assessments (e.g. Clarke et al., 2015;[START_REF] Fernandes | Reversal of Fish Stock Decline in the Northeast Atlantic[END_REF][START_REF] Hutchings | Collapse and recovery of marine fishes[END_REF]. Until recently, fisheries management measures are managed to "squeeze the last 'sustainable' fish" [START_REF] Hilborn | Moving to sustainability by learning from successful fisheries[END_REF], instead of applying extinction risk criteria [START_REF] Fernandes | Coherent assessments of Europe's marine fishes show regional divergence and megafauna loss[END_REF][START_REF] Musick | Criteria to Define Extinction Risk in Marine Fishes: The American Fisheries Society Initiative[END_REF] and placing measures to prevent 'shifting baselines' [START_REF] Pauly | Anecdotes and the shifting baseline syndrome of fisheries[END_REF]. Although the reformed Common Fisheries Policy and other measures such as the Marine Strategy

Framework Directive have been working towards improving the state of fished stocks [START_REF] Fernandes | Coherent assessments of Europe's marine fishes show regional divergence and megafauna loss[END_REF][START_REF] Fernandes | Reversal of Fish Stock Decline in the Northeast Atlantic[END_REF], many species are still fished above scientific advice or deemed as data deficient [START_REF] Fernandes | Coherent assessments of Europe's marine fishes show regional divergence and megafauna loss[END_REF][START_REF] Fernandes | Reversal of Fish Stock Decline in the Northeast Atlantic[END_REF].

The analysis of both fisheries dependent and independent data enabled life history information to be evaluated, which could contribute to specific management measures with minimum landing size thresholds based on maturity [START_REF] Barausse | The role of fisheries and the environment in driving the decline of elasmobranchs in the northern Adriatic Sea[END_REF][START_REF] Frisk | Estimation and analysis of biological parameters in elasmobranch fishes: a comparative life history study[END_REF]. Furthermore, given the apparent high discard survival rate of skates, their exemption from the CFP discard ban could help population recovery. Although an increase in R. undulata was observed during the ban, the mean number observed per trawl remains low and historic abundances for this species is unknown. The results from the population dynamic model indicate that the apparently steady population detected prior to the ban using the CGFS and the ObsMer data, may instead have been a heavily depleted population in slow decline.

To minimise impacts on the fishing industry whilst ensuring recovery of an endangered species, we suggest that protected populations should be permitted to reach a (relatively) steady state before evaluating appropriate quotas required.

This would allow potentially recovered stock levels to be estimated and stochastic effects of environmental and anthropogenic pressures to be disentangled from the implementation of management measures. A comprehensive understanding of the dynamics of a population is essential in designing appropriate management schemes. The fitted line is black, and 95% confidence intervals are shaded in grey.

1 column fitting figure 1945 -2030 and 1995 -2018).

double column fitting figure

  Channel (112 in 2016 to 180 tonnes in 2018) and the Bay of Biscay (25 in 2016 to 30 tonnes in 2018) (EC 2015/960; ICES 2018a & b).

  from Copernicus (1995 to 2016; http://marine.copernicus.eu/) and Pathfinder (2003 to 2018; https://www.nodc.noaa.gov/SatelliteData/pathfinder4km/) websites. For the years that temperature data were accessible from both datasets (2003 to 2016), the mean monthly temperature was used. Copernicus temperature data are based on NEMO v3.6 ocean general circulation model and is run at 1/12 (+/-6-9 km) horizontal resolution. Pathfinder v5.2 is a satellite based temperature estimate with a resolution of ~ 4 km.

  here(cm) is total length expected at age, is time (year), (cm) is the theoretical asymptotic length, exp is the exponential function, is the growth rate and 0 is the theoretical age when length equals zero. The VBGF parameters were L∞ = 108 cm, K = 0.25 -0.27, tanchor = 0.45. tanchor replaces t0 in the VBGF, and refers to the time of year where a new cohort is identified in each year.Age at maturity was estimated from[START_REF] Coelho | Reproductive aspects of the undulate ray, Raja undulata, from the south coast of Portugal[END_REF], McCully et al, (2012) and Moura et al, (2007). More emphasis was placed on calculations by McCully et al, (2012), given she studied R. undulata populations around the British

  .a.i), whereas the significant increase in R. clavata began just before 2009 (Fig. 2.b.i). Using the ObsMer dataset, for both species, a decline in individuals was observed at the beginning of data collection (Fig. 2.ii). R. undulata abundance increased as of 2008 (Fig. 2.a.ii), whereas the number of R. clavata increased between 2005 and 2007 and again as of 2011 (Fig. 2.b.ii).

  4.a). Fig S2, also shows larger R. undulata discarded during the ban than before, albeit landings still took place during the ban. For R. clavata, the results between the different dataset was mixed (Fig. 4.b).

  trends were observed within ICES stock assessment, albeit with stochastic variation (ICES 2018a & b). The results from CGFS and ObsMer data indicate slightly different patterns, most likely due to different geographical coverage (Eastern Channel and North Sea, Channel and Bay of Biscay, respectively).

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .

 1234 Fig. 1. Presence points of Raja undulata and Raja clavata fisheries dependent

Fig. 6 .

 6 Fig. 6. Raja undulata population dynamic results from the age-structured density

Table 1 .

 1 Details of statistical models used to understand the effect of time and temperature on Raja undulata and Raja clavata mean abundance using both the Channel Ground Fish Survey (CGFS) and ObsMer data. Akaike Information Criterion (AIC), Sea Surface Temperature (SST, • C), Swept Surface Area (SSA, km 2 ), Random Effect (RE).

	Dataset

Table 2 .

 2 Density dependent population dynamic model parameters.

	Model
		Value	Age	Size	Survival	Age Size Survival
	parameter
	Max fecundity	70 eggs 0-1	13	0.25	6-7	93	0.9
	Maturity	Age-5	1-2	35	0.35	7-8	96	0.9
	Density effect	1.5x10 -6 2-3	65	0.4	9-10	99	0.9
	Age-4+ fishing
		0.7-0.9 3-4	74	0.5	10-11 101	0.8
	survival
	Age-0-3 discard
	survival (see	0.8-1	4-5	82	0.6	11-12 103	0.0
	table 3)
			5-6	88	0.9

Table 3 .

 3 Raja undulata age-0-3 discard survival during the periods of time when fishing was permitted relating to Figure 5.b.i-iii).

		Discard survival by age
	Fishing survival	Age-0-1	Age-1-2	Age-2-3
	0.7	1	0.9	0.80
	0.8	1	0.95	0.9
	0.9	1	0.975	0.95

! the abundance of individuals between age (" -1) and ", is the Leslie matrix for

Acknowledgements

This study was funded by the French marine fisheries and aquaculture administration (DPMA). We are grateful to DPMA, the 'Systeme d'Information Halieutique', and all those who were involved in collecting and compiling on-board fisheries observer data (ObsMer) and Channel Ground Fish Data. We would like to thank the reviewers for their valuable contribution. The authors would also like to thank Gavin Simpson for his advice with regard to generalised additive mixed models derivative analysis and Philippe Bryère (ARGANS) for access to high resolution PATHFINDER temperature data.

Web reference

IUCN. 2019. https://www.iucnredlist.org/. Downloaded on 09 January 2019. Downloaded on 09 January 2019. European Commission. 2009. EC 43/2009. https://eur-lex.europa.eu/legalcontent/EN/TXT/?uri=CELEX%3A32009R0043 ICES. 2018a. Undulate ray (Raja undulata) in divisions 7.d-e (English Channel). https://doi.org/10.17895/ices.pub.450.


Laurent Souci

Hervé Jaunet

Gérald Le Diguerher

Jean-Marie Guionnet

Véronique Béven

Frédéric Paboeuf

Tristan Montier

Daniel Dory
email: daniel.dory@anses.fr

Gérald Le Diguerher

Daniel Dory Anses

Intranasal inoculations of naked or PLGA-PEI nanovectored DNA vaccine induce systemic and mucosal antibodies in pigs: A feasibility study

Keywords: DNA vaccination, swine, intranasal, antibodies, salivary IgA

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Respiratory pathogens of livestock animals are responsible for high economic losses for farms due to high morbidity and sometimes lethal respiratory diseases. In some rare cases, animal pathogens can be transmitted and emerge in humans, as exemplified with the 2009 H1N1 influenza pandemic of swine origin [START_REF] Hass | The role of swine as "mixing vessel" for interspecies transmission of the influenza A subtype H1N1: a simultaneous Bayesian inference of phylogeny and ancestral hosts[END_REF]. It is therefore necessary to develop strategies to control animal infections, and vaccination represents one of the most powerful tools available. Vaccines administered via the systemic route are often effective in inducing broad and heterologous protective immunity, but there are exceptions. In these cases, like for vaccination against swine influenza or porcine reproductive and respiratory syndrome [START_REF] Dhakal | Nanoparticle-based vaccine development and evaluation against viral infections in pigs[END_REF], mucosal vaccination may be beneficial.

Because respiratory pathogens enter the body through the respiratory mucosa, induction of mucosal protective immune responses may limit entry of the pathogens and associated infection [START_REF] Kraehenbuhl | Mucosal vaccines: where do we stand?[END_REF]. Mucosae are located in various cavities of the body, at the interface between the external and internal environments (e.g respiratory or gastrointestinal tracts). They contribute to essential functions such as respiration, nutrition, and reproduction, while playing a protective role by preventing the physical entry of pathogens [START_REF] Liebler-Tenorio | MALT structure and function in farm animals[END_REF], and by mobilizing the innate and specific local immune systems [START_REF] Lawson | Defending the mucosa: adjuvant and carrier formulations for mucosal immunity[END_REF][START_REF] Liebler-Tenorio | MALT structure and function in farm animals[END_REF][START_REF] Lycke | Recent progress in mucosal vaccine development: potential and limitations[END_REF][START_REF] Pabst | New concepts in the generation and functions of IgA[END_REF]. Moreover, a key part of mucosal immunity is mucosa-associated lymphoid tissues (MALTs), that are functionally interconnected despite being anatomically separated, thus allowing protection through IgA secretion can reach a broad range of distant mucosal surfaces [START_REF] Mcghee | Inside the mucosal immune system[END_REF].

Most mucosal vaccination strategies involve the oral or nasal routes. Most mucosal vaccines consist of attenuated pathogens, with no or very low virulence [START_REF] Lycke | Recent progress in mucosal vaccine development: potential and limitations[END_REF][START_REF] Pavot | New insights in mucosal vaccine development[END_REF]) that mimic as much as possible the wild-type pathogens they are derived from and can therefore enter the organism through the mucosa. Because these viruses remain alive, reversion to virulence or interactions with other pathogens present at the time of inoculation have been reported [START_REF] Eclercy | A Field Recombinant Strain Derived from Two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) Modified Live Vaccines Shows Increased Viremia and Transmission in SPF Pigs[END_REF][START_REF] Liu | High reversion potential of a cell-adapted vaccine candidate against highly pathogenic porcine reproductive and respiratory syndrome[END_REF]. Therefore, as a safety measure, it is recommended to develop subunit vaccines. For example, vaccine antigens can be inserted into viral or bacterial vectors, be administered as recombinant proteins [START_REF] Wang | Intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies[END_REF] or as DNA vaccines [START_REF] Farris | Micro-and nanoparticulates for DNA vaccine delivery[END_REF][START_REF] Oh | Nasal absorption and biodistribution of plasmid DNA: an alternative route of DNA vaccine delivery[END_REF]. Among those, the DNA vaccine strategy presents many advantages, the main one being its simplicity. DNA vaccination consists in inoculating plasmid DNA encoding the vaccine antigen. These antigens are then produced in the vaccinated animals, inducing humoral and cellular immune responses [START_REF] Dufour | DNA vaccines: new applications for veterinary medicine[END_REF]. In addition, DNA vaccines are non-infectious, easy to develop and to produce in high quantities, and they induce an immune response different from that of natural infection allowing differentiation of infected and vaccinated animals (DIVA). DNA vaccination has successfully been achieved in the veterinary field and several vaccines are available on the market (Clynav ® , West Nile Innovator ® , Apex-IHN ® and Oncept ® vaccines) [START_REF] Dalmo | DNA vaccines for fish: Review and perspectives on correlates of protection[END_REF]. For intranasal (IN) DNA vaccination, the vaccine is either be inoculated in its naked form [START_REF] Gomes | Intranasal delivery of naked DNA encoding the LACK antigen leads to protective immunity against visceral leishmaniasis in mice[END_REF] or combined with vectors (liposomal or nano/microparticulate structures) that serve as mucosal delivery systems [START_REF] Csaba | Nanoparticles for nasal vaccination[END_REF].

It was previously shown in rodents that such nanovectors can protect the vaccine DNA from mucosal degradation and favor interaction with mucosal cells, antigen-presenting cells (APCs) or inductive sites of MALTs [START_REF] Kraehenbuhl | Mucosal vaccines: where do we stand?[END_REF]. The two most common biopolymers, described for IN DNA vaccination purposes, are chitosan [START_REF] Khatri | Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B[END_REF] and poly(lactic-co-glycolic) acid (PLGA) [START_REF] Du | Adsorption of a porcine reproductive and respiratory syndrome virus DNA vaccine candidate onto biodegradable nanoparticles improves immunogenicity in mice[END_REF]. In particular, PLGA has been optimized for a more efficient delivery and internalization of the DNA vaccine by mucosal surfaces (e.g. nasal cavity). For example, the combination of polyethylenimine (PEI) with PLGA enhances electrostatic interactions of PLGA with DNA plasmids (thanks to cationic nature of the PEI) and promotes its interaction with mucosal cells, and no cell cytotoxicity was reported [START_REF] Shau | A one-step process in preparation of cationic nanoparticles with poly(lactide-co-glycolide)-containing polyethylenimine gives efficient gene delivery[END_REF]. However, it is important to point out that the vast majority of IN DNA vaccine studies were performed using mice or guinea pigs [START_REF] Ai | Enhanced protection against pulmonary mycobacterial challenge by chitosan-formulated polyepitope gene vaccine is associated with increased pulmonary secretory IgA and gamma-interferon(+) T cell responses[END_REF][START_REF] Du | Adsorption of a porcine reproductive and respiratory syndrome virus DNA vaccine candidate onto biodegradable nanoparticles improves immunogenicity in mice[END_REF][START_REF] Iqbal | Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus (RSV) induces protective CTL responses in BALB/c mice[END_REF][START_REF] Khatri | Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B[END_REF][START_REF] Nanda | Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs[END_REF]. Given the importance of respiratory pathogens in the veterinary field, and associated risks for zoonotic diseases, there is a strong need to conduct new studies to determine the efficacy of IN DNA vaccination for livestock animals such as pigs.

Given that the respiratory tract is the most common route of viral entry, IN DNA vaccination strategies represent a novel and promising vaccination strategy. The pseudorabies virus (PrV) that uses nasal cells as a route of entry into the pig body [START_REF] Babic | Propagation of pseudorabies virus in the nervous system of the mouse after intranasal inoculation[END_REF] is an ideal viral model to test IN DNA vaccination in pigs. Also, intramuscular DNA vaccination is effective and can be used as positive control [START_REF] Dory | Effective protection of pigs against lethal Pseudorabies virus infection after a single injection of low-dose Sindbis-derived plasmids encoding PrV gB, gC and gD glycoproteins[END_REF][START_REF] Gerdts | Protection of pigs against Aujeszky's disease by DNA vaccination[END_REF]. Among the glycoproteins encoded by the PrV DNA vaccine, the glycoprotein B (gB) is of particular interest because it is highly immunogenic [START_REF] Dory | Pseudorabies virus glycoprotein B can be used to carry foot and mouth disease antigens in DNA vaccination of pigs[END_REF] and could be used to validate new DNA vaccine strategies. This protein has previously been used in our laboratory to evaluate the potential of electroporation for porcine DNA vaccination [START_REF] Le Moigne | Electroporation improves the immune response induced by a DNA vaccine against pseudorabies virus glycoprotein B in pigs[END_REF].

In the present study, the plasmid encoding PrV-gB (pcDNA3-gB) associated with a plasmid pcDNA3-GM-CSF encoding granulocyte macrophage colony stimulating factor (GM-CSF used as an adjuvant [START_REF] Somasundaram | Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus[END_REF]) was administered intranasally in its naked form or associated with the nanovector PLGA-PEI. Naked DNA IN inoculation resulted in mucosal salivary IgA antibodies production. Furthermore, the vectored DNA vaccine appeared to extend the duration of IgA production compared to naked DNA alone. These promising results justify further studies to optimize IN DNA vaccination in pigs to prevent infectious respiratory diseases.

Materials and Methods

Plasmids

Plasmids pcDNA3-gB (encoding PrV-gB) [START_REF] Dory | Pseudorabies virus glycoprotein B can be used to carry foot and mouth disease antigens in DNA vaccination of pigs[END_REF], pcDNA3-GM-CSF (encoding granulocyte macrophage colony stimulating factor) [START_REF] Somasundaram | Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus[END_REF], and pTG11033 (encoding luciferase) [START_REF] Lindberg | Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure[END_REF] were produced in E. coli and purified using a Maxiprep plus endotoxin-free DNA extraction kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. The plasmids were resuspended in endotoxin-free tris-EDTA buffer. Plasmid concentrations and purities were assessed spectrophotometrically by measuring OD ratios at 260 nm/280 nm, and 260 nm/230 nm, respectively (Plate reader infinite 200 Pro, TECAN Ltd, Switzerland).

Preparation of plasmid-PLGA-PEI nanoparticles

PLGA-PEI nanoparticles were purchased from NanoVex Biotechnologies (Spain). The sizes of the particles were 200 ± 12 nm and the zeta potentials were 38.9 ± 0.4 mV. plasmid-PLGA-PEI nanoparticles were obtained by the adsorption of plasmid DNA onto PLGA-PEI surfaces, as previously described [START_REF] Shau | A one-step process in preparation of cationic nanoparticles with poly(lactide-co-glycolide)-containing polyethylenimine gives efficient gene delivery[END_REF]. Briefly, plasmid DNA and PLGA-PEI were diluted into ultrapure water and mixed at a weight DNA/weight PEI ratio of 12, vortexed for 30 sec and incubated for one hour at room temperature (RT). Freshly prepared nanoparticles were inoculated or studied in vitro within hours.

Assessment of humoral immune responses after IM injection or IN inoculation of a naked PrV-gB-based DNA vaccine

All the experimental protocols described in this paper were approved by the ethics committee for animal experimentation of ANSES/National Veterinary School of Alfort/University of Paris-Est Créteil (France) (Notice number 10/07/18-3), and by the French Ministry of Research under reference 15614-201806210855647_v2.

Two groups of 8-9-week-old specific pathogen-free (SPF) pigs were inoculated three times at 10-day intervals (Fig. 1A). They weighed 24.5 ± 2.9 kg at the time of the first inoculation. Pigs in group #1 (n=3 pigs) were injected with 4 mL of the DNA vaccine composed of 400 µg of pcDNA3-gB and 100 µg of pcDNA3-GM-CSF by the IM route in the neck muscle. pcDNA3-GM-CSF was used as an adjuvant, as previously described [START_REF] Somasundaram | Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus[END_REF]. Pigs in group #2 (n=3 pigs) were inoculated with the same DNA vaccine by the IN route (2 mL in each nostril) using an MAD100 Nasal TM Intranasal Mucosal Atomization Device (Teleflex Medical, Wayne, USA). Non-inoculated control pigs made up an additional group, group #3 (n=2 pigs). All pigs were observed daily to detect any adverse reactions. Body temperatures were measured daily and pigs were weighed weekly.

Blood sera were collected on days 0, 10, 20, 29 and 37 post-inoculation (pi). Salivary samples were collected on days 29 and 37 pi. On day 38 pi, pigs were anesthetized with an auricular intravenous injection of thiopental (1 g/50 kg body weight) and euthanized. All the organs and the snout of each pig were observed during the autopsies to detect any problem linked to inoculations.

Assessment of humoral immune responses after IN inoculation of naked or nanovectored PrV-gB-based DNA vaccines

Four groups of 7-8-week-old SPF pigs were inoculated three times at 10-day intervals (Fig. 1B). They weighed 25.9 ± 5.7 kg at the time of the first inoculation. Three groups of three pigs were inoculated by the IN route using an MAD100 Nasal TM Intranasal Mucosal Atomization Device (Teleflex Medical, Wayne, USA) with 300 µg of pcDNA3-gB + 100 µg of pcDNA3-GM-CSF (2 mL corresponding to half of the DNA vaccine in each nostril). Pigs in groups #1 and #2 were inoculated with naked pcDNA3-gB and PLGA-PEI-pcDNA3-gB, respectively. One control pig (group #3) was injected three times at the same time points with the same naked DNA vaccine (4 mL in total) by the IM route. Finally, an additional group of four non-inoculated pigs served as negative controls (group #4). Clinical observations and euthanasia were performed as described above, except that euthanasia was performed on day 50 pi. Blood sera were collected the day before the first inoculation and on days 9, 20, 27, 37, 44 and 50 pi. Salivary samples were collected the day before the first inoculation and on days 10, 20, 30, 37 and 44 pi.

Determination of serum anti-PrV IgG and IgA antibody titers

An indirect ELISA for the determination of serum anti-PrV-specific IgG and IgA antibody titers was adapted from the protocol previously described [START_REF] Le Moigne | Electroporation improves the immune response induced by a DNA vaccine against pseudorabies virus glycoprotein B in pigs[END_REF].

Briefly, maxiSorp 96-well plates (Nunc, ThermoFisher, USA) were coated overnight with 1.5 µg/well of PrV glycoproteins diluted in 100 µL of 0.05 M carbonate buffer (Sigma-Aldrich, USA) at 4°C. The plates were washed twice with phosphate buffered saline (PBS) buffer pH 7.4 containing 0.05% tween 20 (PBS-T, Sigma-Aldrich, USA) prior to being blocked with 300 µL/well of blocking buffer [1% BSA (Eurobio, France) in PBS-T] for two hours at 37°C. After two washes with PBS-T, the plates were incubated for 1 h at 37°C with 100 µL/well of two-fold serial diluted serum samples in PBS-T. Each serum sample was analyzed in triplicate. After three washes, the plates were incubated for 1 h at 37°C with 100 µL/well of 1:20,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-pig IgG (H/L) (Bio-Rad, USA) or 1:100,000 diluted HRP-conjugated goat anti-pig IgA AAI40P antibody (Bio-Rad, USA) for IgG or IgA determinations, respectively. These antibodies were diluted in 1% BSA/PBS-T. Finally, after three washes, 100 µL/well of tetramethylene benzidine (TMB) substrate (Pierce TMB substrate kit, ThermoFischer Scientific, USA) was added to the plates for 5 or 10 min for IgG or IgA determinations, respectively. The reactions were stopped by adding 100 µL/well of 2 M sulphuric acid (H2SO4, Sigma-Aldrich, USA). The plates were shaken for 1 min and the absorbance at 450 nm of each well was read on an Infinite 200 Pro plate reader (TECAN Ltd, Switzerland).

Antibody titers were expressed as the highest dilution giving an OD value higher than the threefold OD of the control sera from the non-vaccinated and non-infected pigs.

Determination of salivary anti-PrV IgA antibody titers

An indirect ELISA was performed on salivary samples in triplicate as described above for the serum samples, with minor modifications. 1:150,000 diluted AAI40P HRP-conjugated goat anti-pig IgA antibody and 15 min of incubation with the TMB-substrate were used.

In vitro characterization of the nanovectored plasmids

The plasmids were characterized as previously described, with some modifications [START_REF] Berchel | Cationic lipophosphoramidates containing a hydroxylated polar headgroup for improving gene delivery[END_REF][START_REF] Gall | Arsonium-containing lipophosphoramides, poly-functional nano-carriers for simultaneous antibacterial action and eukaryotic cell transfection[END_REF]. Twenty thousand cells/well of human bronchial epithelial cells (HBE-16) were incubated in 96-well plates overnight (37°C, 5% CO2) with Eagle's minimal essential medium (EMEM) supplemented with 1% penicillin/streptomycin antibiotics, 1% glucosamine (Gibco, ThermoFisher, USA) and 10% fetal bovine serum.

Then, the cells were incubated in triplicate for 72 hours with 40 µL/well of either naked pTG11033 plasmid or PLGA-PEI-pTG11033. Controls consisted of EMEM or of PLGA-PEI without DNA. Thereafter, the cultures were assessed for cytotoxic effects and the transfection potential of each DNA or nanovector preparation.

Cytotoxic effect evaluation

Each preparation described above was evaluated for cytotoxic effects on cell supernatants using a CytoTox 96 ® Non-Radioactive Cytotoxicity Assay kit, according to the manufacturer's instructions (Promega, USA).

Transfection potential evaluation

After removing the remaining culture medium, 75 µL/well of passive lysis buffer (PLB buffer, Promega, USA) were added. Then, 25 µL of each cell lysate were transferred to two separates plates. The first plate (a transparent MaxiSorp 96-well plate, Nunc, Naperville, USA) was used to determine total protein content using a BC Assay kit, according to the manufacturer's instructions (Pierce, ThermoFisher, USA). The other plate (a white MaxiSorpLumiNunc 96-well plate, Nunc, USA) was used to measure luciferase activities using the Promega Firefly Luciferase Assay System (Promega, USA), according to the manufacturer's instructions. An amount of 65 µL per well of Promega Luciferase Assay Reagent was added. After 2 min incubation at RT in the dark, the luminescence was measured with a luminescence Infinite 200 Pro plate reader (TECAN Ltd, Switzerland), with reading and delay times of one second each. The results were expressed as relative light units (RLUs) per milligram of total proteins.

Statistical analyses

For the in vitro experiments, a Mann-Whitney-Wilcoxon (U) test (non-directional test) was used. Comparisons were performed at a limit of significance of 0.05.

Results

PrV-specifc systemic and mucosal humoral responses induced after IN inoculation of the naked PrV-gB-based DNA vaccine (pig experiment #1)

Two groups of three pigs were inoculated three times at ten-day intervals with a naked pcDNA3-gB-based DNA vaccine either through the IM or IN route, respectively (Fig. 1A).

A third group of two pigs was not inoculated and served as a control group. None of the DNA inoculated pigs had fever, showed side effects, and they all grew similarly to the non-inoculated animals for the duration of the experiment. At necropsy, no lesions related to the inoculation or injection procedures were observed.

Determination of serum anti-PrV IgG and IgA following IM or IN inoculation of the naked PrV-gB-based DNA vaccine

In previous studies, we showed that pcDNA3-gB when inoculated intramuscularly induced strong production of systemic anti-PrV IgG antibodies [START_REF] Dory | Pseudorabies virus glycoprotein B can be used to carry foot and mouth disease antigens in DNA vaccination of pigs[END_REF][START_REF] Le Moigne | Electroporation improves the immune response induced by a DNA vaccine against pseudorabies virus glycoprotein B in pigs[END_REF]. Here, as expected, the three pigs injected with the PrV-gB-based DNA vaccine by the IM route exhibited an elevated serum anti-PrV IgG response that started to be detected on day 20 pi (10 days after the second injection), and the response remained high or increased after the third injection until the end of the pig experiment (Fig. 2A). Maximum titers expressed in log2 base were between 13.5 (pigs #2 and #3) and 15.2 (pig #1). Serum anti-PrV IgA antibodies started to be detected on day 10 pi for pig #1, and on day 20 pi for the other two pigs (data not shown). The highest titers were observed on day 29 pi (between 6.3 and 7.3 log2). The two non-inoculated pigs produced no anti-PrV IgG and IgA (Fig. 2A and data not shown).

For the group inoculated by the IN route with the naked PrV-gB-based DNA vaccine, pig #3 produced IgG antibodies against PrV from 20 days pi to the end of the pig experiment (Fig. 2B). On day 37 pi, the titer was 9.3 log2 for this pig. Pig #2 in this group also exhibited an increasing level of PrV-specific serum IgG response, but it started only after the third inoculation (day 29 pi). It reached a titer of 6.3 log2 at the end of the pig experiment (37 dpi).

For the third pig (pig #1), a transient and very low response was observed only on day 29 pi (4.4 log2). Concerning the induction of systemic anti-PrV IgA, pig #1 elicited such a response from 20 dpi (highest titer of 6.3 log2 on day 20 pi) (data not shown). The other two pigs showed only a slight increase of the titer on day 37 pi (3.1 log2) (data not shown).

Determination of anti-PrV sIgA in the saliva of pigs following IM or IN inoculation of the naked PrV-gB-based DNA vaccine

Thereafter, the presence of anti-PrV sIgA was determined in pig saliva collected on days 29 and 37 pi (Fig. 3). No antibodies were detected in the samples from the two non-inoculated pigs. Antibodies were detected in two out of three pigs injected by the IM route with the naked DNA vaccine. The titers of antibodies were 3.4 and 4.3 log 2, respectively. More interestingly, all the pigs inoculated through the IN route with the naked PrV-gB-based DNA vaccine had sIgA titers against PrV at least at the same order of magnitude as those of the pigs injected through the IM route with the same vaccine.

Furthermore, pig #2 presented higher titers than those detected in the IM group (titers of 7.3 and 6.3 log2 on days 29 and 37 pi, respectively).

PrV

In vitro characterization of the nanovectored DNA plasmids

The PLGA-PEI nanovectored DNA plasmids encoding luciferase were prepared as described above and incubated for 72 hours with the bronchial epithelial cell line HBE-16.

Cytotoxic analyses performed on the supernatants of these cell cultures revealed that the preparation with PLGA-PEI had no toxicity for the cells (Fig. 4A). Furthermore, only the association of PLGA-PEI with the plasmid DNA encoding luciferase enabled the expression of luciferase within the cells (Fig. 4B). These results indicate that the nanovectors composed of PLGA-PEI are functional and can be safely administered to pigs.

Determination of serum anti-PrV IgG and IgA following IN inoculation of the

PLGA-PEI-nanovectored PrV-gB-based DNA vaccine

Two groups of three pigs were intranasally inoculated three times at ten-day intervals with the naked PrV-gB-based DNA vaccine or PLGA-PEI PrV-gB-based DNA vaccine, respectively. As a control, an additional pig was intramuscularly injected at the same time points with the naked DNA vaccine. Finally, a fourth group of four non-inoculated pigs was added. No adverse reactions following any of the inoculations were observed throughout the pig experiment. Humoral immune responses against PrV were measured until day 50 pi (Fig. 1B).

Specific IgG and

Determination of anti-PrV sIgA in the saliva of pigs inoculated with nanovectored pcDNA3-gB through the IN route

Compared to experiment #1, saliva was collected for a longer period from day 0 until day 44 pi (instead of only days 29 and 37 pi as in the first pig experiment). sIgA against PrV was detected in most of the pigs inoculated with the DNA vaccine after the second or third inoculation (naked DNA vaccine injected through the IM route (Fig. 6A) or inoculated through the IN route (Fig. 6B) and nanovectored PLGA-PEI-DNA vaccine (Fig. 6C)).

However, for the majority of the pigs, the detection of these antibodies was transient since on day 44 pi, these antibodies were no longer detected (Fig. 6A

, 6B and pig #2 of Fig 6C).

There was two notable exceptions: sIgA against PrV was still detected in saliva in pigs #1 and #3 in the PLGA-PEI DNA vaccine group on day 44 pi (titers of 3.3) (Fig. 6C).

Discussion

There is a need to develop new vaccine strategies against respiratory pathogens in pigs.

Very promising results for IN DNA vaccination have been obtained using mice or guinea pigs [START_REF] Ai | Enhanced protection against pulmonary mycobacterial challenge by chitosan-formulated polyepitope gene vaccine is associated with increased pulmonary secretory IgA and gamma-interferon(+) T cell responses[END_REF][START_REF] Du | Adsorption of a porcine reproductive and respiratory syndrome virus DNA vaccine candidate onto biodegradable nanoparticles improves immunogenicity in mice[END_REF][START_REF] Iqbal | Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus (RSV) induces protective CTL responses in BALB/c mice[END_REF][START_REF] Khatri | Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B[END_REF][START_REF] Nanda | Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs[END_REF]. Of specific interest, IN inoculation to mice of a naked plasmid encoding the same PrV-gB as the one used in the present study resulted in the induction of vaginal PrV-specific sIgA, but not in protection [START_REF] Yoon | Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus[END_REF]. Unfortunately, there are very few data on mucosal DNA vaccination in large animals, and none on IN DNA vaccination in pigs. Furthermore negative results are common. For example, a DNA vaccine against the foot-and-mouth disease virus (FMDV) was not efficient in sheep when administered through the IN route, regardless of whether the vaccine was naked or not [START_REF] Niborski | Efficacy of particle-based DNA delivery for vaccination of sheep against FMDV[END_REF].

The present study aims at generating preliminary results on the development of IN DNA vaccination in pigs. To do this, we used a DNA vaccine against PrV that induces immune responses after IM injection: a DNA vaccine encoding PrV gB [START_REF] Dory | Pseudorabies virus glycoprotein B can be used to carry foot and mouth disease antigens in DNA vaccination of pigs[END_REF][START_REF] Le Moigne | Electroporation improves the immune response induced by a DNA vaccine against pseudorabies virus glycoprotein B in pigs[END_REF]. This model is especially valuable for such study because (1) the virus enters the body through the nasal route (and therefore it is beneficial to generate protective immunity at this entry site), and ( 2) it has been shown that a plasmid encoding PrV-gB induced the production of mucosal sIgA against PrV in the vagina after IN inoculation of mice [START_REF] Yoon | Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus[END_REF]. It can thus serve as a marker of induction of systemic and mucosal humoral immune responses to study new strategies to inoculate DNA vaccines in pigs. At this stage, it has to be pointed out that PrV-gB alone is not able to induce strong protective immunity against PrV-infection in pigs [START_REF] Van Rooij | A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection[END_REF], and this was also the case after IN inoculation in mice [START_REF] Yoon | Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus[END_REF]. Therefore, PrV-gB is fully adapted to the study we wanted to perform here since the main aim of the present study was first to evaluate if IN inoculation of a DNA vaccine can induce the production of immune responses, and more particularly mucosal immune responses. For this reason, and because they are beyond our objective, protective studies were not conducted. Before a PrV-challenge with a protective DNA vaccine encoding PrV-gB, -gC and -gD [START_REF] Dory | Effective protection of pigs against lethal Pseudorabies virus infection after a single injection of low-dose Sindbis-derived plasmids encoding PrV gB, gC and gD glycoproteins[END_REF][START_REF] Gerdts | Protection of pigs against Aujeszky's disease by DNA vaccination[END_REF][START_REF] Van Rooij | Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant[END_REF] could be designed in pigs, IN administration of DNA vaccine must first be optimized.

In the present study, the IM route of immunization served (1) to show that the PrV-gB DNA vaccine is functional and induces the production of systemic humoral immune response, and (2) to study whether sIgA against PrV are detected in the saliva of intramuscularly injected pigs. In all cases, anti-PrV IgG and IgA were found in the sera of these pigs; anti-PrV sIgA antibodies were detected in the saliva of 3 out of 4 pigs. To our knowledge, this is the first time that such sIgA antibodies have been detected in the saliva of pigs intramuscularly vaccinated with a DNA vaccine against PrV. Previously, such antibodies were also detected in nasal secretions of pigs intramuscularly injected with a commercial inactivated PrV vaccine (Geskypur ® ) [START_REF] Le Luduec | Intradermal vaccination with un-adjuvanted sub-unit vaccines triggers skin innate immunity and confers protective respiratory immunity in domestic swine[END_REF]. Unfortunately, the comparisons of the levels of antibodies obtained after the IM injection of our DNA vaccine and Geskypur ® are not possible since the sIgA levels were measured in two different fluids.

IN inoculation of naked DNA vaccine has mainly been described as ineffective in inducing immune responses or protection, with some notable exceptions. For example, Svanholm et al reported that significant clinical protection against Chlamydophila pneumonia was obtained after IN immunization of mice with a naked plasmid encoding HSP-60 [START_REF] Svanholm | Protective DNA immunization against Chlamydia pneumoniae[END_REF]. Several other studies have reported induction of specific systemic (serum IgG or IgA) or mucosal (sIgA) immune responses against β-galactosidase [START_REF] Csaba | PLGA:poloxamer and PLGA:poloxamine blend nanostructures as carriers for nasal gene delivery[END_REF][START_REF] Cui | Intranasal administration of plasmid DNA-coated nanoparticles results in enhanced immune responses[END_REF], hepatitis B virus [START_REF] Khatri | Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B[END_REF] or Streptococcus pneumoniae [START_REF] Xu | Intranasal vaccination with chitosan-DNA nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by Streptococcus pneumoniae[END_REF] following IN naked DNA immunizations in mice. Herein, we observed the induction of immune responses after IN inoculation of the naked form of the PrV-gB DNA vaccine in pigs. This inoculation induced both systemic and mucosal humoral immune responses in all the vaccinated pigs (six pigs if we take into account both pig experiments). Even though the number of individuals used was too low to allow significance testing, it seems that systemic anti-PrV IgG and IgA levels is lower following IN inoculation than those measured in the IM group. In contrast, levels of sIgA in the saliva were around the same order of magnitude in the IM and IN groups. In fact, the highest levels of sIgA in the saliva were found for individuals of the IN groups in both pig experiments. But we observed a high inter-individual variations, especially in the IN groups that further limits our ability to interpret the data. That being said, it is noteworthy that all the 6 pigs inoculated with the naked DNA vaccine induced the production of sIgA against PrV in the salivary (instead of 3 out of 4 for the IM group), providing a strong evidence that IN DNA vaccination induces the production of respiratory mucosal immune responses against the targeted pathogen. Cao et al that inoculated pigs intranasally with an the attenuated PrV vaccine PrV K61 measured the sIgA titers in saliva around 8 log2 [START_REF] Cao | CpG oligodeoxynucleotide synergizes innate defense regulator peptide for enhancing the systemic and mucosal immune responses to pseudorabies attenuated virus vaccine in piglets in vivo[END_REF], i.e. slightly above the results we obtained here with the naked PrV-gB-based DNA vaccine, which shows that the titers that we obtained are common for the species.

Further studies with a higher number of individuals are now necessary to determine if the IN route has a higher potency at inducing sIgA production. In addition, follow-up studies will need to include a thorough optimization procedure to reduce inter-individual variations and enhance the efficacy of this intranasally inoculated DNA vaccine by testing other adjuvants or modifying several other parameters (use of nanovectors, ways to inoculate, etc.).

Most researchers studying IN DNA vaccination combine their DNA vaccines with nanovectors to protect the DNA molecule from mucosal environment degradation until it reaches the cells involved in the development of immune responses. There are many types of nanovectors (for reviews see [START_REF] Bernocchi | Nasal nanovaccines[END_REF][START_REF] Xu | Intranasal DNA Vaccine for Protection against Respiratory Infectious Diseases: The Delivery Perspectives[END_REF]). For our study, we selected PLGA-PEI nanoparticles. Indeed, PLGA and/or PEI were previously successfully used in IN DNA vaccination in mice [START_REF] Du | Adsorption of a porcine reproductive and respiratory syndrome virus DNA vaccine candidate onto biodegradable nanoparticles improves immunogenicity in mice[END_REF][START_REF] Torrieri-Dramard | Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses[END_REF].

In the present proof of concept study, we selected a DNA/nanovector ratio previously found to be effective to transfect the bronchial lineage HBE-16 cell line in vitro. It did not ensure success of in vivo IN inoculation but we assumed it would increase chances of success [START_REF] Ragelle | Chitosan nanoparticles for siRNA delivery: optimizing formulation to increase stability and efficiency[END_REF]. In association with PLGA-PEI, the duration of mucosal immune response in the saliva induced by the DNA vaccine was longer for two out of three pigs, compared to the three pigs inoculated intranasally with the naked DNA vaccine. This result may be anecdotal, but it reveals that mucosal humoral immune responses might be improved. Clearly, further studies are necessary to optimize the IN DNA vaccine to induce a high and sustained mucosal immune responses. We suggest that such studies should compare the efficiency of PLGA-PEI with other nanovectors, and optimize the other parameters such as the DNA/nanovector ratios, total quantity of DNA, adjuvants and inoculation conditions; inoculation conditions include anesthetized pigs [START_REF] Janakova | Influence of intravenous anesthesia on mucosal and systemic antibody responses to nasal vaccines[END_REF] or non-anesthetized pigs [START_REF] Khatri | Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B[END_REF], the volume of vaccine, number of doses, intervals between the doses, speed of inoculation, and specific devices for inoculation). Only such a thorough and comprehensive approach will allow us to reliably assess the protective immunity induced by IN DNA vaccine strategies.

Conclusion

The current study clearly showed that IN inoculation of a naked DNA vaccine encoding PrV-gB induces both a systemic and a mucosal humoral immune response against PrV in

  -specifc systemic and mucosal humoral responses induced after IN inoculation of the PLGA-PEI-nanovectored DNA vaccine encoding PrV-gB (pig experiment #2) In previous studies, researchers have used nanovectors to protect DNA vaccines from mucosal degradation. In the second part of this study, the PrV-gB-based DNA vaccine was associated with PLGA-PEI already known to enhance DNA vaccine efficacy, mainly in mice experiments.

  IgA titers against PrV were measured in the sera of all the pigs every 7-10 days throughout the pig experiment. Confirming results from experiment #1 described above, anti-PrV IgG and IgA were detected in the sera of the pigs inoculated with the naked DNA vaccine through the IM (data not shown) and IN routes (Fig. 5Afor IgG and data not shown for IgA). For the pigs inoculated with the PLGA-PEI-DNA vaccine, the systemic anti-PrV IgG and IgA immune responses was low for all three pigs (Fig. 5B and data not shown, respectively).

Figure 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2: ELISA determination of systemic IgG antibody levels against PrV after

Figure 3 :

 3 Figure 3: ELISA determination of salivary sIgA antibody levels against PrV after

Figure 4 :

 4 Figure 4: In vitro toxicity and cell transfection potentials of naked and PLGA-PEI

Figure 5 :

 5 Figure 5: ELISA determination of systemic IgG antibody levels against PrV after

Figure 6 :

 6 Figure 6: ELISA determination of salivary sIgA antibody levels against PrV after

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: ELISA determination of systemic IgG antibody levels against PrV after inoculation with a naked PrV-gB-based DNA vaccine. Pigs were inoculated three times with a naked pcDNA3-gB DNA vaccine, as indicated by the arrows. Pig sera were collected every 8-10 days throughout the pig experiment. IgG titers of the two non-inoculated pigs and the three pigs inoculated by the IM route are indicated in (A). IgG titers of the three pigs inoculated by the IN route are indicated in (B). Dilutions were performed in triplicate and the threshold of positivity was established as the OD mean of the negative controls ± 3 SD. Antibody titers are presented in log2 scales. Arrows topped with a number indicate the inoculations with DNA vaccine.

Figure 5 :Figure 6 :

 56 Figure 5: ELISA determination of systemic IgG antibody levels against PrV after intranasal inoculation with a naked or PLGA-PEI associated PrV-gB-based DNA vaccine. Pigs were inoculated three times with the DNA vaccine as indicated by the arrows. Pig sera were collected every 8-10 days throughout the pig experiment. Results for the three pigs inoculated with naked pcDNA3-gB are indicated in (A). Results for the three pigs inoculated with PLGA-PEI-pcDNA3-gB are indicated in (B). Dilutions were performed in triplicate and the threshold of positivity was established as the OD mean of the negative controls ± 3 SD. Antibody titers are presented in log2 scales. Arrows topped with a number indicate the inoculations with DNA vaccine.

pigs. To the best of our knowledge, this is the first report showing the induction of immune responses following IN inoculation of a naked DNA vaccine in pigs. Some immune response parameters appeared enhanced when the DNA vaccine was combined to PLGA-PEI nanovectors. Even though the inter-individual variability observed indicate that the optimized conditions were not reached in the present study, these results suggest that it is possible to improve DNA vaccine efficacy by varying its composition and inoculation conditions. Based on the promising results presented herein, we encourage future studies to build upon the present results and improve IN DNA vaccination in pigs.

Funding

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.