Superiority of PLGA microparticle-encapsulated formalin-killed cell vaccine in protecting olive flounder against Streptococcus parauberis

Aquaculture 509 (2019) 67–71

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Short communication

Superiority of PLGA microparticle-encapsulated formalin-killed cell vaccine in protecting olive flounder against Streptococcus parauberis

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Jin Woo Juna,1, Jeong Woo Kangb,1, Sib Sankar Girib, Saekil Yunb, Hyoun Joong Kimb, Sang Guen Kimb, Sang Wha Kimb, Se Jin Hanb, Jun Kwonb, Woo Taek Ohb, Dalsang Jeonga, ⁎ Se Chang Parkb, a

Department of Aquaculture, Korea National College of Agriculture and Fisheries, Jeonju 54874, Republic of Korea Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea

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A R T I C LE I N FO

A B S T R A C T

Keywords: Poly(D,L-lactide-co-glycolic acid) Vaccine Streptococcus parauberis Paralichthys olivaceus Aquaculture

Olive flounder (Paralichthys olivaceus) is a valuable maricultured fish in Asian countries, especially Korea, Japan, and China. Streptococcosis caused by Streptococcus parauberis has resulted in significant economic loss of olive flounder aquaculture in these countries. Since the conventional formalin-killed cell vaccine (FKC vaccine) is reportedly effective only for short period, the main objective of this study was to develop new vaccine of protecting olive flounders against streptococcosis for longer period. To assess the protective effect of poly(D,L-lactide-co-glycolic acid) (PLGA) microparticle-encapsulated formalin-killed cell vaccine (PLGA vaccine), it was compared to the FKC vaccine. A bacterial agglutination test was performed using the serum of olive flounders after intraperitoneal administration of PLGA vaccine, FKC vaccine, or sterile phosphate-buffered saline (PBS). The PLGA vaccinated group showed higher agglutination titers than the FKC vaccinated group. Additionally, the percentage survival of the olive flounders was monitored after an S. parauberis challenge 10 weeks post-vaccination. In the first bioassay (challenge with 4.0 × 104 CFU/fish), percentage survival 15 days post-challenge (dpc) were 100% for the PLGA vaccine, 80% for the FKC vaccine, and 65% for PBS. In the second bioassay (4.0 × 105 CFU/fish), percentage survival 15 dpc were 100% for the PLGA vaccine, 55% for the FKC vaccine, and 0% for PBS. The results of this study showed that PLGA vaccination of olive flounder led to greater protection against streptococcosis, compared with conventional FKC vaccination.

1. Introduction Olive flounder (Paralichthys olivaceus) is a temperate marine species of large-tooth flatfish and natively inhabits the north-western Pacific Ocean. It is one of the most valuable aquacultured fish in Asian countries, especially Korea, Japan, and China (Seikai, 2002). In 2016, it accounted for 51.9% of the total marine fish aquaculture production in Korea (Korean Statistical Information Service, 2016). Most olive flounder farms are located along the southern coast of Korean Peninsula, where 80% of Korea's olive flounder are produced (Kikuchi, 2017; Seikai, 2002). In particular, almost half of the olive flounder in Korea are produced on Jeju Island because of its natural condition: free access to abundant water and optimum water temperature (18 °C) for olive flounder aquaculture (Kikuchi, 2017; Seikai, 2002).

Streptococcosis is recognized as a serious aquaculture disease in many countries, including Israel (Eldar et al., 1994, 1995), Italy (Eldar et al., 1996), Japan (Kitao, 1993; Kusuda et al., 1976), Spain (Nieto et al., 1995), the USA (Baya et al., 1990; Eldar et al., 1995; Rasheed and Plumb, 1984), and Korea (Baeck et al., 2006; Park et al., 2009; Shin et al., 2006). The etiological agents of streptococcosis are warm-water streptococcosis-associated pathogens, and the outbreaks caused by streptococcosis usually occur in areas above 15 °C (Baeck et al., 2006; Múzquiz et al., 1999). The main bacteria responsible for streptococcosis are Lactococcus garviae, Streptococcus difficilis, S. iniae, and S. parauberis. S. parauberis (formerly known as S. uberis genotype II) causes streptococcosis in turbot (Scophthalmus maximus) and olive flounder (Baeck et al., 2006; Doménech et al., 1996; Mata et al., 2004; Nho et al., 2009). Antibiotics are gradually becoming less effective in aquaculture due

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Corresponding author at: Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea. E-mail address: [email protected] (S.C. Park). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.aquaculture.2019.04.079 Received 23 May 2018; Received in revised form 26 April 2019; Accepted 30 April 2019 Available online 04 May 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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particle size analyzer (LDPSA) used in this study was a BeckmanCoulter LS 13 320 (Beckman Coulter, CA, USA). The measurement time used was 51 s, with 8% of obscuration. The LDPSA was applied with a theoretical measuring range from 0.017 to 2000 μm.

to the increasing incidence of antibiotic resistance (Romero et al., 2012), and vaccination is recognized as the most effective means for disease prevention (Tonheim et al., 2008). Various applications of poly (D,L-lactide-co-glycolic acid) (PLGA) have been documented thus far (Behera and Swain, 2013; Dubey et al., 2016; Fredriksen et al., 2011; Rauta and Nayak, 2015). PLGA nanoparticles have shown potential usefulness as antigen carriers in rohu (Labeo rohita) (Dubey et al., 2016; Rauta and Nayak, 2015) and Atlantic salmon (Salmo salar L.) (Fredriksen et al., 2011). Alginate-chitosan-PLGA composite microspheres induced protection from Aeromonas hydrophila infection in rohu (Behera and Swain, 2013). In our previous study, the PLGA microparticle-encapsulated formalin-killed cell vaccine (PLGA vaccine) protected both cyprinid loach (Misgurnus anguillicaudatus) and common carp (Cyprinus carpio) from A. hydrophila infection (Yun et al., 2017). The aim of the present study is to assess the efficacy of the PLGA vaccine in protecting olive flounders from Streptococcus infection by comparing it to the formalin-killed cell (FKC) vaccine.

2.3.2. Morphological analysis The morphological characterization was performed using a field emission scanning electron microscope (FESEM, Sigma, Carl Zeiss, UK). PLGA microparticles were prepared and mounted onto stubs. Before being scanned, the specimens were sputter-coated with gold for 180 s using a vacuum coater (EM ACE 200, Leica, Austria). 2.4. Assessment of efficacy of PLGA vaccine 2.4.1. Fish and ethics statement Four hundred and sixty healthy olive flounders (average length, 19.49 ± 1.38 cm; average weigt, 68.35 ± 11.27 g) were procured from a fish farm in Taean-gun, South Korea. Prior to the experiments, 20 fish were randomly collected and subjected to polymerase chain reaction (PCR) analysis, as previously described (Baeck et al., 2006), in order to confirm that the fish were free of S. parauberis infection. Four hundred forty fish were acclimatized to laboratory conditions in 2.5-ton tanks at 20–22 °C for 1 week. The fish were fed once a day with commercial food (Cargil Agri Purina Inc., Korea). Approximately 20% of the water was exchanged daily, and 50% of the water was exchanged weekly. Among the 440 olive flounders, 140 fish were used in an experimental challenge to determine the median lethal dose (LD50) of S. parauberis SNUFPC-050803; 300 fish were designated for the vaccination experiment. All animal care and experimental protocols were performed according to the guidelines of the Animal Ethical Committee at Seoul National University.

2. Materials and methods 2.1. Bacterial strain and growth condition S. parauberis SNUFPC-050803 was isolated from diseased olive flounder obtained from an olive flounder aquaculture farm in Jeju Island, South Korea in May of 2005 (Baeck et al., 2006). S. parauberis SNUFPC-050803 proved to be pathogenic to olive flounder via an intraperitoneal (IP) challenge on healthy olive flounders (Kim et al., 2006). The bacterial strain was cultured in brain-heart infusion (BHI) broth or BHI agar (supplemented with 1.5% NaCl) at 25 °C. 2.2. Vaccine preparation 2.2.1. Preparation of FKC vaccine The FKC vaccine for streptococcosis was prepared as previously described (Yun et al., 2017), with some modifications. S. parauberis SNUFPC-050803 was grown to the early-exponential phase (OD600 ~1.0), corresponding to ~108 CFU/ml. Ten ml of this suspension was treated with 100 μl of formalin (1% of bacterial suspension volume) and incubated at 25 °C for 12 h, with a shaking speed of 50 rpm. The prepared solution was then centrifuged at 10,000 ×g for 20 min, washed twice with sterile phosphate-buffered saline (PBS), and re-suspended in 10 ml of sterile PBS.

2.4.2. Vaccination Fish were divided into three groups (I, II, and III) in 2.5-ton tanks at 20–22 °C. Each group consisted of 100 fish. The fish in group I, the control group, were given an IP injection of 0.1 ml of sterile PBS. The fish in group II and III were administered IP injections of 0.1 ml of the PLGA vaccine or the FKC vaccine, respectively. The total antigen content in 0.1 ml of each vaccine was adjusted to 4.0 × 107 colony-forming units (CFU). 2.4.3. Sample collection and bacterial agglutination test Three fish were randomly collected from each of the three groups every two weeks. Blood samples were collected from the caudal vein using a 1-ml syringe following anesthetization with MS-222 (SigmaAldrich, MO, USA). The blood samples were transferred to centrifuge tubes (Eppendorf, Hamburg, Germany). Serum was collected after centrifugation at 6500 ×g at 4 °C for 10 min. Serum was incubated at 44 °C for 20 min for complement inactivation. The serum agglutination experiment was performed using a 96-well U-bottom plate (SigmaAldrich, St. Louis, MO, USA). The serum was serially diluted two-fold in PBS, and then the same volume of heat-killed S. parauberis SNUFPC050803 (ca. 107 CFU/ml) was added to each well. Plates were incubated overnight at 25 °C. Agglutination activity was determined according to the lowest dilution with no agglutination, and the resulting value was considered the reciprocal of that dilution rate.

2.2.2. Preparation of PLGA vaccine The PLGA vaccine was prepared as previously described (Yun et al., 2017), with some modifications. As the first step, inactivation was carried out using the same protocol as for the FKC vaccine. Ten ml of the bacterial suspension S. parauberis SNUFPC-050803 (ca. 108 CFU/ ml) was inactivated with the addition of formalin (1% [v/v]), washed twice with sterile PBS, and re-suspended in 500 μl of sterile PBS. Separately, 210 mg of PLGA was dissolved in 3 ml of dichloromethane with a vortex mixer. These two solutions were combined and emulsified at room temperature in an HG-15D homogenizer (DAIHAN Scientific CO., Ltd. Korea) at 12,000 rpm for 1 min. The preparation was then poured into 50 ml of 4% PVA solution and homogenized at 6000 rpm for 1 min. Fifty ml of distilled water was then slowly added to the suspension, which was stirred at 300 rpm for 8 h to evaporate the organic solvent. The resulting PLGA microparticles were washed twice with sterile PBS and centrifuged for 20 min at 2000 ×g, then lyophilized for 48 h and stored at −20 °C for further study.

2.4.4. Experimental challenge test S. parauberis SNUFPC-050803 was used for the challenge test after it grew to early-exponential phase (OD600 ~ 0.5) and was serially diluted ten-fold with PBS. To determine the LD50 concentration of the strain, duplicate groups of fish (n = 10, per group) were administered 100 μl of the bacterial suspension by IP injection. The final doses of injection ranged from 102 to 107 CFU/fish. The control groups were injected with 100 μl of sterile PBS. After injection, fish were monitored for 15 days. Dead fish were sampled every day; the bacteria were isolated from their

2.3. Characterization of PLGA vaccine 2.3.1. PLGA microparticle size distribution analysis Particle-size analysis was performed using the laser diffraction method as previously described (Yun et al., 2017). The laser diffraction 68

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kidneys and then identified using PCR, as previously described (Baeck et al., 2006). After 10 weeks post-vaccination (wpv), fish from the three groups were administered IP injections containing 100 μl of the bacterial suspension. The concentration of bacteria injected was 104 CFU/fish for the first bioassay and 105 CFU/fish for the second. The challenge experiments included two different doses and were performed at the same time at 10 wpv, and for each dose two parallel tanks were included. The challenged fish were maintained at 21–22 °C in 60-L fiberglass-reinforced plastic aquaria supplied with flow-through seawater. Clinical signs of disease and cumulative mortalities were monitored twice a day for 24 days after injection. Bacterial samples were obtained from the kidneys of the dead fish, then cultured on BHI agar at 25 °C for 24 h. Isolates were identified using PCR, as described above. The percentage survival of each group was monitored to assess vaccine efficacy. 2.4.5. Statistical analysis Bacterial agglutination data were analyzed by one-way ANOVA or two-way ANOVA with the SPSS statistical software package, version 19.0 (IBM Corp., Armonk, NY, USA). Percentage survival of olive flounder after S. parauberis challenge was analyzed by using a chisquare test (p = .05) with SPSS version 19.0.

Fig. 2. Bacterial agglutination test using the serum of olive flounder after administration of the poly(D,L-lactide-co-glycolic acid) vaccine (PLGA vaccine), the formalin-killed cell vaccine (FKC vaccine), or sterile phosphate-buffered saline (PBS) by intraperitoneal injection. Results are shown as the mean ± standard deviation (n = 3). An asterisk denotes a significant difference (p < .05) at that time point in all groups: between PLGA and FKC, FKC and PBS, and PLGA and PBS.

3. Results and discussion 3.1. Characteristics of PLGA vaccine

number of FKCs must be encapsulated inside each particle (Yun et al., 2017). Furthermore, larger particles generally release antigens earlier than smaller particles (Kumari et al., 2010), which is expected to be a strength of the PLGA vaccine; notably, this altered release can elicit a more rapid immune response after vaccination. Our study supports this assumption, because PLGA vaccination resulted in higher agglutination titer than FKC vaccination at 2 wpv. In the initial phase of this study, we attempted to enlarge the particle size. However, larger particle size is sometimes problematic since it can obstruct the bore of the needle used to inject fish. This is likely to occur on a larger scale and is time-consuming because vaccine administrators need to change needles as required. We endeavored to determine the optimal methodology, particularly with respect to stirring rate during the emulsion process, which greatly affects the final particle size (Arshady, 1991; Chaisri et al., 2009). The PLGA vaccine prepared in the current study did not cause this issue, although the preparation was applied to hundreds of fish. The FESEM revealed morphological and distributive patterns of the PLGA microparticle (Fig. 1b): it is spherical and bumpy, and consists of a variety of particle sizes. The speculation that this variety may result in multiple releases of antigen and prolong the activity of the vaccine is

The microparticles produced in this study were formed to different sizes. LDPSA revealed that the microparticles exhibited a mean diameter of 54.42 μm and a median diameter of 33.46 μm (Fig. 1a). The difference of these two values is a double-edged sword. PLGA has been used in the pharmaceutical and medical fields since it is able to regulate drug release by controlling the degradation rate (Makadia and Siegel, 2011). It is essential to uniformize the particle size of PLGA for drug delivery, since the time of its degradation and drug release should be estimated for a desired application. It may require a more exact manufacturing process and cause an increase in expense, which may be impractical in aquaculture. On the other hand, various particle sizes could be desirable trait in the PLGA vaccine. The differences of particle size in PLGA cause differences in its degradation, resulting in multiple antigen releases. As a result, it prolongs the activity of the vaccine; the activity of one PLGA vaccine was proved to last as long as multiplevaccinations with the FKC vaccine. Large size formulations of PLGA particles are known to act as a reservoir for drug that can be delivered over a longer interval (Makadia and Siegel, 2011). The larger PLGA particle size is advantageous in vaccine production because a large

Fig. 1. Characteristics of the PLGA vaccine. (a) poly(D,L-lactide-co-glycolic acid) (PLGA) microparticle size distribution analysis performed by the laser diffraction particle size analyzer and (b) morphological characterization performed by a field emission scanning electron microscope (bar = 2 μm). 69

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Fig. 3. Percentage survival of olive flounder after Streptococcus parauberis challenge after 10 weeks post-vaccination with the poly(D,L-lactide-co-glycolic acid) vaccine (PLGA vaccine), the formalin-killed cell vaccine (FKC vaccine), or sterile phosphate-buffered saline (PBS) by intraperitoneal injection. The bacterial challenge was performed using 4.0 × 104 CFU/fish (1st bioassay, left) or 4.0 × 105 CFU/fish (2nd bioassay, right) of S. parauberis SNUFPC-050803 by intraperitoneal injection. Results are presented as the mean of two parallel tanks.

group continued to produce high levels of agglutination titers at 10 wpv, while the FKC vaccinated group did not; these titers were significantly different (p < .05). The experimental challenge test to determine the LD50 concentration of the strain showed that the percentage survival of the groups injected with 4.0 × 102, 4.0 × 103, 4.0 × 104, 4.0 × 105, 4.0 × 106, and 4.0 × 107 CFU/fish were 100, 97.5, 65, 0, 0, and 0, respectively (data not shown). It was concluded that the LD50 concentration of the strain was between 104 and 105 CFU/fish, and two bioassays were performed by experimental challenge using two different bacterial doses: 4.0 × 104 and 4.0 × 105 CFU/fish. The experimental challenge test was performed at 10 wpv to determine if, at that point, the discernible difference in agglutination titers between groups II and III generated a significant difference in percentage survival. Consistent with the result of the agglutination test, a significantly (p < .05) higher percentage survival was observed in group II than that in group III after the bacterial challenge by IP method (Fig. 3). The fish in the control group (I), which were not vaccinated, began to die on the 1st day post-challenge, and percentage survival over 15 days were 65 ± 7% (1st bioassay, 4.0 × 104 CFU/fish) and 0% (2nd bioassay, 4.0 × 105 CFU/fish) (Fig. 3). In the first bioassay, both of the vaccinated groups showed 100% percentage survival until 9 days postchallenge (dpc), but the difference in percentage survival was observed thereafter in both groups (100% on 15 dpc, group II; 80% on 15 dpc, group III) (Fig. 3). In the second bioassay, a significant difference (p < .05) was observed in percentage survival between the two groups (100% 15 dpc, group II; 55 ± 7% 15 dpc, group III) (Fig. 3). In every group that completed the experimental challenge test, no dead fish were observed after 14 dpc.

the main strength of the PLGA vaccine presented in this study. In the mid-1990s, streptococcosis prevention with the FKC vaccine was considered successful; this was previously reported by Eldar et al. (1997), who noted that FKC-vaccinated rainbow trout were protected for 4 months from S. iniae infection. However, the success of FKC vaccination was limited because of the re-emergence of infection, often within weeks of vaccination (Agnew and Barnes, 2007). Re-emergence of infection has been reported predominately at high water temperatures, in which primary antibody titers subside within 30–40 days (Agnew and Barnes, 2007). Long-lasting protection was achieved when adjuvants were included with inactivated vaccines; the utilization of oil as an adjuvant reduced outbreaks in the early 1990s (Brudeseth et al., 2013). However, an oil-based vaccine may cause adverse effects, such as inflammation at the injection site, melanization, organ adhesions, and autoimmunity (Koppang et al., 2008; Midtlyng, 1997; Midtlyng et al., 1996). Development of a new vaccine that is able to protect for longer periods is needed, as the main drawback of the FKC vaccine is that it is only effective for a short period (Gravningen et al., 2008; Kawakami et al., 1997). For this reason, the FKC vaccine for streptococcosis seems to be losing its credibility in the field of olive flounder aquaculture. Although FKC vaccines produced by several companies have been commercialized, more and more aquaculturists in South Korea question its efficacy.

3.2. Efficacy of PLGA vaccine Prior to vaccination with the three different preparations, sterile PBS (group I), the PLGA vaccine (group II), and the FKC vaccine (group III), serum titers indicated no detectable antibodies in any of the groups. In the control group (I), serum titers showed no detectable antibodies throughout the experiment (Fig. 2). In the FKC vaccinated group (III), agglutination titers increased at 2 wpv and continued to increase until 6 wpv (Fig. 2). The titers decreased thereafter until the end of the experiment: from 5.33 ± 0.58 (Log2) to 2.33 ± 0.58 (Log2) (Fig. 2). In contrast, titers in the PLGA vaccinated group (II) increased throughout the experiment: from an undetectable point (at 0 wpv) to 7.33 ± 0.58 (Log2, 10 wpv) (Fig. 2). Thus, both vaccinated groups (II and III) showed similar increases in agglutination titers from 2 to 6 wpv, relative to those of the control group (I); furthermore, both vaccines elicited higher titers than those observed in the control group at 8 wpv, although the agglutination titer of the FKC vaccinated group began to decrease. There was a trend suggestive of a higher agglutination titer in the PLGA vaccinated group, compared with that of the FKC vaccinated group at 2–8 wpv; however, there was no statistically significant difference between the two groups. The PLGA vaccinated

4. Conclusions In summary, PLGA vaccination protected olive flounder against S. parauberis infection, without any mortalities being observed after experimental challenge, and PLGA vaccination gave higher survival than the conventional FKC vaccine. Furthermore, there were minimal additional expenses involved in the production of the PLGA vaccine, compared with production of the FKC vaccine: approximately 0.4 dollars per 100 fish. Despite the protective effect of the PLGA vaccine against S. parauberis infection, further studies of the PLGA vaccine are still required. Investigations should focus on the adaptive immune responses as well as duration of immunity, since the bacterial agglutination titer kept increasing until the end of the experiment. Further research is now in progress to compare the immune-inducing ability of the PLGA vaccine to that of a commercial vaccine product for the long-term use.

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