Journal Pre-proof

Chitosan nanoparticle immersion vaccine offers protection against tilapia lake virus in
laboratory and field studies

Puntanat Tattiyapong, Sirikorn Kitiyodom, Teerapong Yata, Krittayapong Jantharadej,

Mikolaj Adamek, Win Surachetpong

PII: S1050-4648(22)00739-2
DOI: https://doi.org/10.1016/j.fsi.2022.10.063
Reference: YFSIM 8406

To appear in: Fish and Shellfish Immunology

Received Date: 15 August 2022

Revised Date: 27 October 2022
Accepted Date: 30 October 2022

Please cite this article as: Tattiyapong P, Kitiyodom S, Yata T, Jantharadej K, Adamek M, Surachetpong
W, Chitosan nanoparticle immersion vaccine offers protection against tilapia lake virus in laboratory and
field studies, Fish and Shellfish Immunology (2022), doi: https://doi.org/10.1016/j.fsi.2022.10.063.

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Puntanat Tattiyapong: Conceptualization; Methodology; Investigation; Data curation;
Writing original draft; Visualization
Sirikorn Kitiyodom: Investigation; Data curation; Writing-Original draft preparation;
Visualization
Teerapong Yata: Conceptualization; Methodology; Writing-Review and Editing;
Krittayapong Jantharadej: Methodology; Investigation; Writing-Original draft preparation
Mikolaj Adamek: Conceptualization; Writing-Review and Editing; Visualization
Win Surachetpong: Conceptualization; Methodology; Data curation; Validation; Reviewing
and Editing; Writing-Review and Editing; Visualization; Project administration; Funding
acquisition; Supervision

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Title: Chitosan nanoparticle immersion vaccine offers protection against tilapia lake virus in

laboratory and field studies

Puntanat Tattiyapong1,2, Sirikorn Kitiyodom3, Teerapong Yata3, Krittayapong Jantharadej2, Mikolaj

Adamek4, Win Surachetpong1,2*

1
Interdisciplinary Program in Genetic Engineering and Bioinformatics, Graduate school, Kasetsart

University, Bangkok, Thailand

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Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart

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University, Thailand.

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Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Thailand.
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Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine Hannover,
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Hannover, Germany
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Abstract

Tilapia lake virus (TiLV), an enveloped negative-sense single-stranded RNA virus, causes tilapia

lake virus disease (TiLVD), which is associated with mass mortality and severe economic impacts in

wild and farmed tilapia industries worldwide. In this study, we developed a chitosan nanoparticle TiLV

immersion vaccine and assessed the efficacy of the vaccine in laboratory and field trials. Transmission

electron microscopy showed that the inactivated vaccine had a particle size of 210.3 nm, while the nano

inactivated vaccine had a spherical shape with a diameter of 120.4 nm. Further analysis using fluorescent

staining and immunohistochemistry analysis revealed the mucoadhesive properties of the nanovaccine

(CN-KV) through fish gills. We assessed the efficacy of an immersion-based TiLV nanovaccine using a

cohabitation challenge model. The fish that received the nanovaccine showed better relative percent
survival (RPS) at 68.17% compared with the RPS of the inactivated virus vaccine (KV) group at 25.01%.

The CN-KV group also showed a higher TiLV-specific antibody response than the control and KV

groups (p < 0.05). Importantly, under field conditions, the fish receiving the CN-KV nanovaccine had

better RPS at 52.2% than the nonvaccinated control group. Taken together, the CN-KV nanovaccinated

fish showed better survival and antibody response than the control and KV groups both under laboratory

control challenge conditions and field trials. The newly developed immersion-based nanovaccine is easy

to administer in small fish, is less labor-intensive, and allows for mass vaccination to protect fish from

TiLV infection.

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Keywords: tilapia lake virus, nanovaccine, immunity, immersion, tilapia
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1. Introduction

Tilapia lake virus (TiLV) or Tilapia tilapinevirus is an enveloped, negative-sense single-strand

RNA virus with 10 genomic segments [1-3]. Infection with TiLV causes severe morbidity and mortality

in tilapia and other freshwater fish [4-8]. Due to the wide distribution of TiLV and the severity of the

disease and its economic impacts on affected fish farms [9], preventing and limiting the spread of TiLV

is necessary for sustainable tilapia aquaculture. Previous studies have suggested that during TiLV

infection, tilapia develop an early innate immune response associated with the high expression of

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inflammatory cytokines and antiviral genes in the gills, liver, brain, spleen, and intestines [10-12].

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Moreover, rapid antibody production specifically against TiLV has been detected in fish exposed to TiLV

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[13, 14]. Indeed, fish that survive TiLV infection do not show signs of morbidity and mortality even after
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intraperitoneal exposure to the virulent strain of TiLV [14]. This evidence supports the principle of the
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application of vaccines to prevent TiLV.

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Fish possess an immune system containing both innate and adaptive immunity, along with a

memory and anamnestic response against specific pathogens [15]. The application of vaccines to
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stimulate the fish immune system and develop protective immunity against pathogens is thus a valid
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approach to protect them from infectious diseases. This strategy is currently implemented in modern

aquaculture [15-18]. In fact, there are several vaccines commercially available to prevent viral infections

in finfish [19, 20]. In tilapia, various types of vaccines are available to prevent bacterial diseases,

including streptococcosis, francisellosis, and columnaris diseases, as well as viral diseases such as

infectious spleen and kidney necrosis virus [21-24]. However, acquiring a highly efficacious vaccine to

control emerging pathogens needs substantial time and effort due to the limited knowledge and tools

available for vaccine development.

In the process of vaccine development, understanding the pathogenesis of, and how the host

responds to, a specific pathogen is critical to designing an effective vaccine. Although TiLV can cause
disease at all stages of the tilapia development [25-27], the virus mainly—and severely—affects fry,

juveniles, and fingerlings [6, 28]. A promising TiLV vaccine should therefore be practical for

administration in young (small) fish, easy to apply in large quantities without being labor intensive, and

provide good mucosal immunity. All such goals could be achieved by developing an immersion vaccine;

however, recent TiLV vaccines have been developed in injection form [29-34], and the application of an

immersion vaccine has not yet been investigated. This highlights the importance of ongoing TiLV

vaccine research and the urgent need to acquire new technologies in this area. Research has shown that

the nanoparticle-based delivery system can overcome the technical limitations of applying an immersion

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vaccine in fish by enhancing antigen uptake, allowing the attachment of the vaccine to the mucosal

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surface, and facilitating the delivery of specific antigens to the target immune cells. For example,

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Kitiyodom et al., [22] applied a chitosan-complexed nano-immersion vaccine to protect tilapia against
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columnaris disease. This mucoadhesive polymer chitosan-complexed nanovaccine facilitates antigen
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uptake, increases the expression of immune-related genes, and provides an antibody response against
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Flavobacterium columnare (F. columnare) in tilapia [35, 36]. Notably, the small particle sizes of
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nanovaccines can promote antigen uptake and its delivery to the target cells, thus magnifying the immune
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response. In this study, we developed an immersion TiLV vaccine based on a chitosan nanoparticle

encapsulated with inactivated TiLV virus. The efficacy of the nanovaccine was assessed under both

laboratory and field conditions by measuring improvements in the survival rate and antibody response

of tilapia during infection by TiLV.

2. Materials and methods

2.1 Fish samples

Eight hundred thirty red hybrid tilapia (Oreochromis spp.) with an average body weight of 5 ± 1

g were used in this vaccine efficacy study. The fish were acquired from a tilapia hatchery in Phetchaburi
province, Thailand, with no history of TiLV outbreaks. The fish were acclimated in 400 L fiber tanks at

the animal research facility in the Faculty of Veterinary Medicine, Kasetsart University, Bangkok, for

1 week. Five fish were randomly selected and examined for ectoparasites by gill biopsy and skin scrapes,

bacterial isolation from the anterior kidney, and TiLV screening by a reverse transcription quantitative

polymerase chain reaction (RT-qPCR) assay, as previously described [37]. The fish were fed twice a day

with commercial feed at a rate of 4% total body weight/day. The water quality parameters, namely,

temperature, dissolved oxygen, and ammonia, were measured daily. The animal use protocol was

approved by the Institutional Animal Care and Use Committee, Kasetsart University (protocol number

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ACKU62-VET-063).

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2.2 Cells culture and virus propagation -p
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The TiLV strain VET-KUTV08 previously isolated from moribund red tilapia from Ang-thong
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province, Thailand, in 2019 was used to prepare the vaccine. The virus was propagated in the E-11 cell
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line, which was purchased from the European Collection of Authenticated Cell Cultures (England). The
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E-11 cells were grown in Leibowitz’s (L-15) media supplemented with 5% fetal bovine serum and 2 mM
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L-glutamine and cultured at 25°C without CO2. The virus stock was grown in confluent E-11 cells with

a titer of 103 tissue culture infectious dose (TCID)50/mL and incubated at 25°C for 1 h to allow for viral

infection. The cells were then maintained at 25°C for 4–7 days with an L-15 medium containing 2% fetal

bovine serum and 2 mM L-glutamine until 80% cytopathic effects (CPE) were observed. Thereafter, the

virus was harvested by the freeze–thaw process to facilitate cell breakage. The lysate was then transferred

to a 50 mL tube and centrifuged for 10 minutes at 4°C at 3,000 × g. The supernatant containing the viral

particle was collected and stored at −80°C until use. A TCID50/mL assay was used to quantify the virus

titer, as described by Reed and Muench method [38].

2.3 Chitosan-complexed nanovaccine preparation

 The virus suspension described in section 2.2 was diluted with phosphate buffer saline (PBS) to

a final concentration with a titer of 2×105 TCID50/mL. The virus was incubated with 0.1% formalin for

24 h at 4°C to inactivate the viral infectivity. The inactivation process was confirmed by inoculating 100

µL of inactivated virus into a flask containing confluent E-11 cells. CPE was observed daily for 10 days.

A chitosan-complexed nanovaccine killed virus (CN-KV) was prepared by mixing formalin-killed virus

with 6% (w/w) polyoxyethylene (20) sorbitan monolaurate, 2% (w/w) medium-chain triglycerides

(Miglyol), and 62% (w/w) water, which was then sonicated at 40% amplitude for 5 min. The vaccine

was subsequently mixed with 1% chitosan dissolved in a 1% acetic acid solution (Sigma Aldrich, USA)

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at a ratio of 1:1 (v/v). Thereafter, the vaccine was stirred with a sonicator probe for 1 h at room

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temperature.

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2.4 Characterization of the CN-KV
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The CN-KV was diluted in deionized water at a dilution of 1:1,000 and the size distribution and
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zeta potential at 25°C were determined using the Malvern Instruments Zetasizer Nano ZX (Malvern
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Panalytical Inc., USA). The samples were diluted in distilled water at a ratio of 1:50 on carbon tape. The
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structure and morphology of the CN-KV were examined at magnifications of 5,000–20,000 times with a

20 kV electron beam using an environmental scanning electron microscope (S-3400, Horiba, Japan).

2.5 Assessing the mucoadhesion of the CN-KV on fish gills

In total, 180 red hybrid tilapia (Oreochromis spp.) with an average body weight of 10 ± 1 g were

divided into four groups: control (PBS), PBS containing chitosan nanoparticles (CN), formalin-killed

virus vaccine (KV), and CN-KV. For each group, 45 fish were divided equally into three tanks (three

replicates per group) with 15 fish per tank. To assess the vaccine adherence to the gills, 1 µg/mL of Nile

red dye dissolved in PBS was mixed with PBS, CN, KV or CN-KV at a dilution ratio of 1:1000. Then,

the fish were immersed in the Nile red-incorporated PBS, CN, KV or CN-KV in water at a ratio of 1:100
dilution for 30 min in the darkroom. After 30 min, three fish from each tank were randomly selected and

euthanized with a Eugenol solution (Betagro, Thailand) at 5 mL/L for 5 min. The gills were immediately

removed and examined under a Nikon Eclipse TE2000-U fluorescence microscope (Nikon Instruments

Inc., USA). The accumulation of the Nile red-stained vaccines on the mucosal membranes was examined

using a bioluminescence imaging instrument (Bruker, USA) with 4× magnification and a fluorescent

setting to create fluorescence images, which demonstrated the uptake of the vaccine formulation in the

mucosal membranes. To demonstrate the presence of the virus antigen in fish gills, after the immersion

in PBS, CN, KV, or CN-KV for 30 min, gills were collected from six fish per group and were fixed in

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10% neutral buffered formalin for 48 hr. The tissues were embedded in paraffin, cut at 4 µm thick

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sections, and processed for immunohistochemistry (IHC) analysis using a specific antibody against TiLV

as previously described [46]. -p

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2.6 Immunization, challenge study, and sample collection

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For the mortality test, 180 red hybrid tilapia with an average body weight of 5 ± 1 g were divided
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into three groups designated as the control (CN), KV, and CN-KV groups. The fish were immersed in 1
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L of water containing 10 mL of the PBS containing chitosan nanoparticles, KV, or CN-KV vaccine at a

1:100 dilution for 30 min with aeration. The calculated TiLV concentration in immersion water in KV

and CN-KV groups was 103 TCID50/mL. Following immersion, the fish from each group were divided

equally, transferred to two 30 L glass tanks, and kept for 28 days prior to the TiLV challenge. Twenty-

eight-day post vaccination (dpv), 10 inducer fish were injected intraperitoneally with 50 µL of TiLV

strain VET-KUTV08 at a titer of 105 TCID50/mL (designated as viral inducers) and placed in each tank

at a ratio of inducer fish to cohabitation fish of 1:3. The cohabitation challenge model has previously

been described in detail [14]. The mortality data were collected from two independent experiments with

180 fish/experiment. The mean cumulative mortality with the standard error of the mean (SEM) of each

group was generated from both data sets.

 For the sample collection, 120 fish (10 ± 1 g) were distributed into three groups (40 fish per

group) and then immersed with PBS containing chitosan nanoparticles (CN), KV, or CN-KV at the same

concentration used in the mortality test. The fish were kept for 28 days to allow them to develop an

immunological response to the vaccine. At 28 dpv (0 day post TiLV challenge; dpc), 15 inducer fish

were injected intraperitoneally with 50 µL of TiLV strain VET-KUTV08 at a titer of 105 TCID50/mL and

placed in each tank at a ratio of inducer fish to cohabitation fish of 1:3. At 0, 7, and 0, 7 and 14 dpc, five

fish were randomly selected from each group at each time point for blood and tissue collection (Fig 1).

The fish were anesthetized using a Eugenol solution of 3 mL/L for 5 min. Blood was drawn from the

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caudal vein using a 1 mL syringe with a 24-gauge needle and stored at 4°C overnight to allow for serum

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separation, before being centrifuged at 1,500 × g for 25 min. The serum was heat-inactivated at 56°C for

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30 min and stored at −20°C until further analysis. At 14 dpc, five fish each from the control, KV, and
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CN-KV groups were randomly euthanized. Liver samples were aseptically collected and tested for TiLV
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using RT-qPCR, as previously described [37].

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Fig 1. Experimental design. (A) The chitosan-complexed nanovaccine killed virus (CN-KV) was
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prepared from a formalin-inactivated virus and then formulated with a chitosan complex. (B) Efficacy
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test of the CN-KV in the laboratory trial. Fish were divided into the control (CN), KV, and CN-KV
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groups with two tanks containing 30 fish each for the mortality study and another tank containing 40 fish

for the sample collection. At day 28 post vaccination (0 dpc), the fish were challenged with TiLV by a

cohabitation model. Blood was collected from five fish at 0 and 7 dpv and 0, 7, and 14 dpc. At 14 dpc,

five fish were collected from each group for TiLV quantification using RT-qPCR. (C) Efficacy test of

the CN-KV in field conditions. Red hybrid tilapia (n = 10,000) were divided into the CN and CN-KV

groups comprising 5,000 fish each. After inoculation with the immersion vaccination, the fish were

reared in an earthen pond for 28 days and then transferred to river cages.

2.7 Measurement of the antibody response in the TiLV-challenged fish

 The antibody level specifically against TiLV was evaluated via an indirect enzyme-linked

immunosorbent assay (ELISA) [14]. Briefly, purified TiLV was diluted in 1× KPL coating solution

(Seracare, USA) and coated with a concentration of 0.25 µg per well in 96-well plates. The plates were

incubated for 18 h at 4°C and then blocked with 3% bovine serum albumin in a PBS containing 0.05%

Tween 20 (PBST) at 37°C for 1 h. The plates were washed three times with the PBST before adding the

serum (1:100 dilution) into each well. The plates were incubated at room temperature for 1 h before

washing three times with the PBST and incubated with monoclonal IgM antibody against tilapia (1:1,000

dilution) for 1 h at room temperature. Thereafter, the samples were washed three times with the PBST

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and incubated with HRP-conjugated anti-mouse IgG antibody (SeraCare, USA) at 1:2,000 dilution at

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room temperature for 1 h. After washing three times with the PBST, the samples were incubated with 50

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µL of tetramethylbenzidine for 25 min under no light exposure. The reaction was terminated using 50
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µL of 2M H2SO4, and the samples were analyzed at an optical density of 450 nm using a microplate
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reader (BioTek™ H1, FisherScientific, UK).

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2.8 Field trial

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The field trial was carried out at a tilapia farm with a history of multiple TiLV outbreaks in

Pranakorn Sri Ayutthaya province, Thailand. Red hybrid tilapia (Oreochromis spp.) with an average size

1 g ± 0.5 g (n = 10,000) were acquired from a hatchery and kept in 5×5×2 m (H×W×D) cages in an

800 m3 earth pond. The fish were divided equally into the control (CN) and CN-KV groups with 5,000

fish per group. After which, 600 mL of PBS containing chitosan nanoparticles (CN) or CN-KV were

dispensed in 60 L water and transferred to 4 round plastic basins with the diameter of 50 cm. The dilution

ratio of the vaccine and water was 1:100. The fish were divided into 1,250 fish/basin and immersed in

the CN-KV nanovaccine or PBS containing chitosan nanoparticles for 30 min under constant aeration.

To confirm the health status, 10 fish were randomly collected prior to vaccination for bacterial screening

from the anterior kidney, ectoparasite examination by gill biopsy and skin scraping and examined under
light microscope, and TiLV detection. The fish were kept in an experimental earth pond for 28 days with

a constant paddle wheel aerator and then transferred into the Noi River, Ayutthaya province, for a grow

out period of six months. The daily mortality and clinical signs were recorded. Five moribund fish were

collected from each cage (two cages from the control group and two cages from the CN-KV group) and

checked for TiLV using an RT-qPCR assay, bacterial isolation, and ectoparasite examination.

2.9 Statistical analysis

The mortality of the control (CN), KV, and CN-KV groups under laboratory trials and the CN

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and CN-KV groups under field study were calculated to relative percent survival (RPS) and analyzed

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using a log-rank (Mantel–Cox) test. All graphs were generated using GraphPad Prism software version

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8.0 (GraphPad Software, Inc., USA). The specific antibody levels were compared using two-way analysis
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of variance with Tukey’s multiple comparisons test. All the data were considered significant at p ˂ 0.05.
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The relative percent survival (RPS) was calculated using the formula (1 – [% mortality of vaccinated fish
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/ % mortality of control fish]) × 100.

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3. Results

3.1. Morphology and mucoadhesive property of the chitosan-complexed nanovaccine

The TiLV strain VET-KUTV08 isolated from red hybrid tilapia was propagated in E-11 cells.

After the virus reached a titer of 105 TCID50/mL, the cell culture media was collected and inactivated in

0.1% (v/v) formalin for 24 h at 4°C. The safety of the inactivated virus was tested by inoculation into E-

11 cells without CPE formation (data not shown). The inactivated virus was mixed with an oil emulsion

containing chitosan to form the CN-KV. An analysis of the CN-KV morphology and size using a

scanning electron microscope and a transmission electron microscope revealed the uniform spherical

shapes of the external surface (Fig 2A) and smooth surface (Fig 2B) particles. In addition, the CN-KV

had a mean diameter of 120.4 nm, which was 1.7 fold smaller than the non-formulated KV at 210.3 nm.
The zeta potential of the KV and CN-KV was shifted from a negative value at −32.9 mV to a positive

value at +22.0 mV in the CN-KV.

The mucoadhesive property of the nanovaccine was demonstrated on the gills of the red hybrid

tilapia immersed in antigen labelled with fluorescent Nile red dye. Specifically, accumulation of the

nanomaterial was found on the gill lamellae of the CN and CN-KV groups, while no fluorescence signal

was found in KV and control (PBS) groups (Fig 3). The control fish were used to compare the auto-

fluorescence of the fluorescent dye on the tilapia gills. Further analysis of the mucoadhesive properties

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of the CN-KV on the fish gills revealed that the fish exposed to CN-KV showed higher fluorescence

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intensity than the KV, and control fish (Supplementary Fig 1). Notably, immunohistochemistry analysis

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revealed a specific detection of TiLV on the gills of the KV group with a higher accumulation of TiLV
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antigen on the gill lamellar in CN-KV group after the direct immersion for 30 min (Fig 3).
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Fig 2. Characterization of a chitosan-complexed nanovaccine killed virus (CN-KV) particle. (A) A

representative micrograph of the external surface of the CN-KV particles examined by scanning electron

microscopy (bar = 10 µm). (B) Cross-section of the CN-KV captured using transmission electron

microscopy (bar = 200 nm). Arrows indicated the CN-KV particles.

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Fig 3. Mucoadhesive properties of the TiLV nanovaccine on tilapia gills after direct immersion. Top
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panel: Representative brightfield micrographs of the gills from control (PBS), PBS containing chitosan
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nanoparticles (CN), killed virus vaccine (KV), and chitosan-complexed nanovaccine killed virus (CN-

KV) groups, Middle panel: Fluorescence images of the fish gills after the direct immersion for 30 min in

PBS, CN, KV, and CN-KV groups stained with Nile red dye (scale bar = 500 µm). Bottom panel:

Immunohistochemistry (IHC) analysis of the gills section probed with TiLV-specific antibody (scale bar

= 50 µm)

3.2 Vaccine efficacy test under laboratory conditions

 After the immersion of the fish in the TiLV vaccine (KV or CN-KV), no abnormal signs or

mortality were observed. At 28 dpv, the fish were challenged with TiLV using the cohabitation challenge

model, as previously described [14]. The clinical signs of TiLV infection, including anorexia, lethargy,

fin erosion, skin congestion, and hemorrhages, developed in the inducer fish at 3–5 days, while the

cohabitation fish showed clinical signs of TiLV infection at 7–10 days. Further, all the inducer fish had

a cumulative mortality of 69.17%–74.45% at 14 dpc. The mortality of the cohabitation fish from both

the control and vaccinated groups started at 10–12 days and continued until 21 days after the challenge.

In fact, the cumulative mortality in the control group ranged between 23.33%–50.00%, while the

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cumulative mortality in the KV group and CN-KV groups was 23.33%–33.33% and 6.67%–16.67%,

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respectively (Supplementary table 1). Notably, the mean cumulative mortality calculated from two

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experiments was 36.67% in the control group, while the mean mortality in the KV and CN-KV groups
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was 27.50% and 11.67%, respectively (Fig 4). Hence, the mean RPS of the KV and CN-KV groups was
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25.01% and 68.17%, respectively, relative to the control group. Five liver samples were collected from
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each group during the onset of clinical signs (at 14 dpc) to determine the TiLV concentration using an
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RT-qPCR assay. The results confirmed that all the experimental groups were infected with TiLV. Indeed,
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the mean viral copy numbers of the CN-KV, KV and control groups were 3.31 log10 ± 0.15, 3.65 log10 ±

0.14, and 4.46 log10 ± 2.0 per µg of total RNA, respectively (Supplementary table 2). No significant

differences in viral concentrations were found in any of the groups (p > 0.05).
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Fig 4. Average cumulative mortality of the control (CN), killed virus vaccine (KV), and chitosan-
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complexed nanovaccine killed virus (CN-KV) fish after TiLV infection during the cohabitation
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challenge. On day 0, 10 inducer fish were injected intraperitoneally (dashed lines) with 50 µL of TiLV
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at a titer of 105 TCID50/mL and kept with 30 cohabitation fish (solid lines) for 28 days. Cumulative
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mortality from the CN, KV, and CN-KV groups was collected from two tanks per group with 30 fish per
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tank. The mean cumulative mortality with the standard error of the mean (SEM) of each group was

generated from two independent experiments.

3.3 Analysis of TiLV specific antibody in vaccinated fish

To assess the TiLV-specific immune response based on antibody production, serum samples from

five fish per group were randomly collected on days 0, 7 post vaccination and days 0, 7 and 14 post TiLV

challenge and analyzed using an ELISA assay. In particular, after vaccination, no differences were found

between the TiLV-specific antibody levels in any of the studied groups or any of the time points post

vaccination (p > 0.05; Fig 5). Hence, the TiLV-specific antibody increased rapidly at 14 dpc in the CN-
KV group with a mean OD450 value of 0.26 ± 0.08 compared to the mean OD450 value of the control and

KV groups at 0.12 ± 0.07 and 0.08 ± 0.03, respectively (p < 0.05). Notwithstanding, no differences in

antibody levels were observed between the groups at 7 dpc.

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Fig 5. Levels of TiLV-specific antibodies in the serum of red hybrid tilapia measured by ELISA. The
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levels of antibodies were examined from the serum of the control (CN), killed virus vaccine (KV), and
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chitosan-complexed nanovaccine killed virus (CN-KV) groups with five fish per group at 0 and 7 days
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post vaccination (dpv) and 0, 7, and 14 days post TiLV challenge (dpc). The differences in antibody

levels between the groups were compared using two-way analysis of variance and Tukey’s multiple

comparisons test. p ˂ 0.05 was considered a significant value (*).

3.4 CN-KV efficacy test under field conditions

The efficacy of the CN-KV was further tested under field conditions in an area with a high

prevalence of TiLV. Red hybrid tilapia (n = 10,000) were divided equally into two groups (two cages per

group) as the mock-vaccinated control (CN) and CN-KV groups. During immunization, the fish were
kept in an earthen pond for 28 days and then transferred to grow out cages in the river to allow for natural

exposure to TiLV. The water temperature ranged from 28°C–31°C ± 2°C, the dissolved oxygen was 4.4

± 0.5 mg/L, and ammonia 0.1 ± 2 TAN. At 12 and 14 days post transfer, some fish from the control and

CN-KV groups showed clinical signs of TiLV infection, and mortality was observed (Fig 6). The

mortality peaked between 14 and 18 days post transfer and was recorded until 33 days post transfer with

cumulative mortality in the control and CN-KV groups at 55.45% and 26.50%, respectively. Hence, the

RPS of the CN-KV was 52.22% compared to the control group (p < 0.05). Interestingly, the mortality

pattern in the CN-KV-vaccinated fish occurred faster and stopped before the mortality of the control fish

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(Fig 6). Five moribund fish were collected from each cage (two cages from the control group and two

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cages from the CN-KV group) and confirmed positive for TiLV infection using an RT-qPCR assay with

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the viral copy number between 2.8 and 5.4 log10 per µg of total RNA. Furthermore, the bacterial and
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parasite screenings of the moribund fish revealed the presence of Aeromonas hydrophila in the anterior
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kidney and Trichodina spp. infestation in the gills and mucus of both groups, with more severe infection
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in the unvaccinated fish.

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Fig 6. Cumulative mortality of the control (CN) and chitosan-complexed nanovaccine killed virus (CN-

KV) vaccinated red hybrid tilapia under field conditions. The control and CN-KV-vaccinated fish (5,000

fish per group) were divided into two floating cages and raised in cages in rivers with a previous history

of TiLV outbreaks. The daily mortality was recorded until 33 days post transfer. (•) Distinct clinical signs

of TiLV infection with progressive mortality were observed in the control and CN-KV groups. (⬥) Ten

moribund fish (five fish/cage) were collected and examined for the presence of bacteria, viruses, and

parasites. The differences in relative percent survival between the groups were calculated and compared

using a log-rank (Mantel–Cox) test with a significant value (p ˂ 0.05).

of
ro
Discussion
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re
Until now, TiLV, a causative agent of an emerging viral disease, has had a substantial economic
lP

impact on global tilapia aquaculture [9] as there are no effective vaccine or prevention strategies available
na

for tilapia farms that are at risk of being affected by TiLV outbreaks. In this study, we developed an
ur

inactivated TiLV vaccine combined with nanotechnology to deliver a TiLV vaccine in small particles
Jo

that can adhere to fish gills. The immersion-based TiLV nanovaccine was easy to administer in small

fish, stimulated a higher antibody response, and improved survival during TiLV infection. Previous

efforts to develop a TiLV vaccine have included an inactivated viral vaccine [39, 40], a DNA vaccine

[41], and a recombinant protein vaccine [42]. For instance, Zeng et al., [40] developed a formalin and β-

propiolactone inactivated TiLV vaccine mixed with a montanide adjuvant, which had an RPS of 85.7%

in the vaccinated group. Another study used formalin- and heat-killed TiLV vaccines administered by

intraperitoneal injection, and survival in the vaccinated fish increased by 79.6% and 71.1%, respectively

[39]. Moreover, the passive transfer of serum from the fish receiving the inactivated vaccines to the naive

recipient improved survival between 85% and 90% [43]. A DNA vaccine based on segment 10 of TiLV

(TiLV-ORF10) provided an 85% RPS with significantly elevated immune-related gene expression,
which included IgM, TLR2, MyD88, IL-8, and TNFα, in the vaccinated fish [41]. Indeed, a prime-boost

vaccination strategy using a DNA and recombinant protein-based vaccine showed a 72.5% RPS and

elicited both cellular and humoral immunity [42]. However, all these TiLV vaccines were based on

traditional vaccine development and intraperitoneal delivery without the application of nanotechnology.

Interestingly, nanotechnology has been applied to enhance vaccine efficacy in terrestrial and aquatic

animals [44, 45]. Nanotechnology has been applied in aquatic vaccines to improve the efficacy of

bacterial vaccines in tilapia. Specifically, a chitosan-coated mucoadhesive vaccine reportedly increased

the effectiveness of immersion vaccination in tilapia with an RPS of more than 70% against

of
Flavobacterium columnare [22]. A subsequent study of this mucoadhesive F. columnare vaccine

ro
demonstrated the upregulation of IgM and IgT genes and a specific antibody response in vaccinated fish

at 14 and 21 dpv [36]. -p

re
Recently, a nanovaccine generated by mannose coated on a chitosan-complexed DNA vaccine of
lP

TiLV segment 2 improved fish survival (76.9% RPS) and provided a significantly elevated antibody
na

response and immune-related gene expression [34]. Nonetheless, this nanovaccine needs to be
ur

administered by intramuscular injection, which is a time-consuming, labor-intensive, and costly process.

Jo

In contrast, our immersion-based nanovaccine offers many advantages as it saves time and money and

can be administered to younger (smaller) fish, which are highly susceptible to TiLV [26]. Notably, a key

highlight of this study is the cohabitation challenge model of inoculating inducer fish with a virus and

allowing them to spread the virus to mimic natural infection. Previous studies have shown that TiLV can

enter gills and intestinal epithelial cells rapidly (i.e., within 1 day) [11, 46], so the cohabitation model

should be applied when testing vaccine efficacy. In addition to the increasing survival rate, the fish that

received the immersion-based TiLV nanovaccine developed a rapid antibody response after the TiLV

challenge. Although, no significant difference of TiLV viral concentration were observed among groups

at 14 dpc, the viral concentration may be different between the control and vaccinated fish at other time

courses. Thus, further investigation of the viral concentration in the control and vaccinated fish at the
earlier or later times should be examined. Our results further revealed that the immersion vaccine

improved fish survival during the first month in the tilapia farms with a history of severe TiLV outbreaks.

To the best of our knowledge, this is the first field study of a TiLV nanovaccine, and it delineates

important steps that demonstrate through both theoretical and practical results that the TiLV nanovaccine

is feasible for TiLV prevention. Notably, differences of vaccine efficacy between the laboratory and field

trials may be due to different size of the fish leading to the individuals being exposed to different number

of vaccine particles, however despite, the vaccine concentration in water for both laboratory and field

trials used in this study was initially adjusted to the same concentration (103 TCID50/mL). Despite these

of
discrepancies, successful immunization and improved survival were achieved after exposure to the TiLV

ro
in both experimental and field conditions. Although we did not monitor the efficacy of the immersion-

-p
based TiLV nanovaccine under field condition throughout the grow out period (6–8 months), our
re
previous study confirmed that fish develop a solid immunological response and memory to prevent them
lP

from subsequent infection [14]. The immersion-based TiLV nanovaccine developed in this study will
na

therefore improve the survival of fish while allowing them to develop natural immunity against TiLV.
ur

Although we measured the vaccine efficacy based on survival, antibody levels, and viral loads in fish
Jo

tissues, other aspects of vaccination immunology still need to be addressed, namely, the induction of

mucosal immunity and cell-mediated immunity such as the T cell response as well as alteration of the

immune-related genes in the vaccinated fish.

Remarkably, the immersion vaccine stimulated both systemic and mucosal immunity. In this

study, we showed that the nanotechnology improved the attachment of the TiLV antigen onto fish gills,

which is consistent with previous studies [36, 45, 47]. Comparisons of the adhesion properties using

fluorescent staining and immunohistochemistry analysis in the PBS, CN, KV, and CN-KV groups

revealed that the chitosan-complexed nanoparticles enhance the accumulation of TiLV antigen on fish

gills. The application of chitosan provided the CN-KV with a positive charge property and improved the

attachment of the nanovaccine to the negatively charged fish mucosal surface membrane [48].
Importantly, making the vaccine smaller and increasing the surface adhesion property improved the

antigen delivery and ultimately stimulated a better immune response. Nevertheless, other factors,

including temperature, age, water quality, and the density of the fish, as well as modifications to the

vaccination preparation or application (like the use of adjuvants) to boost or cause intermittent mucosa

disruption during vaccination to increase antigen uptake could affect the efficacy of immersion vaccines

[49-51]. These factors require further evaluation when using the immersion-based TiLV nanovaccine,

especially if some modifications are able to improve the levels of protection. What is important at the

current level of study is that we were able to conclude that there were no safety concerns in relation to

of
the immersion-based TiLV nanovaccine as no fish mortality occurred during or after vaccination, and

ro
the vaccine did not cause infection in either the cell culture or fish.

-p
In conclusion, we successfully applied an innovative chitosan nanoparticles technology to
re
develop an immersion vaccine against TiLV. The use of nanotechnology reduced the size of the antigen,
lP

formed suitable electrostatic properties, and increased the permission and deposition on fish mucosa,
na

which together improved the delivery of the vaccine and stimulated a better immune response. The novel
ur

immersion-based TiLV nanovaccine developed in this study improved fish survival against TiLV
Jo

infection in laboratory and field studies. Importantly, immersion vaccines offer advantages over injection

vaccines by reducing the risk of fish handling, stress, practicality in small fish, and mass vaccination.

However, the large-scale application of chitosan nanoparticles technology to prevent TiLV in tilapia

farms requires further evaluation and additional feasibility studies.

ACKNOWLEDGEMENTS

This work was financially supported by the Office of the Ministry of Higher Education, Science,

Research and Innovation and the Thailand Science Research and Innovation through the Kasetsart

University Reinventing University Program 2021. The project was funded by National Science and

Technology Development Agency, Thailand under the projects P-18-52277 and P-19-50187. Puntanat
Tattiyapong received financial support from the National Research Council of Thailand (NRCT) through

the Royal Golden Jubilee PhD Program (Grant no. NRCT5-RGJ63002-037), Thailand. We would like to

thank Associate Professor Somporn Techangamsuwan and Dr. Chutchai Piewbang for their technical

support on immunohistochemistry.

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Supplementary Fig 1. Fluorescence images and intensity of Nile red dye on fish gills after direct
lP

immersion in the PBS (control), killed virus vaccine (KV), and chitosan-complexed nanovaccine killed
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virus (CN-KV). The side bar indicates the fluorescence intensity.

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Highlights:

• An immersion based chitosan complex nanovaccine was developed for tilapia lake

virus.

• The nano-delivery system improves mucoadhesive properties through fish gills.

• Upon TiLV challenge, better antibody response was observed in fish received the

nanovaccine.

• The immersion-based nanovaccine improves fish survival both under laboratory and

f
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field trials.

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