A published article was reviewed for it content on developing a novel technology for effective fish oral vaccine against vibriosis.

1. Development of a Novel Delivery
Technology for
Oral Vaccine in Fish: A Review
D.D.T.T. Darshana Senarathna
Aquaculture Health Management
AARM/FAB, School of Environment, Resources and Development
Asian Institute of Technology

2. Outline
• Approach
• Introduction
• Objectives
• Materials and methods
• Results & Discussion
• Conclusion
• Recommendations and suggestions
• Acknowledgement
• References
• Supplementary details

3. Approach
Aquaculture
Economic losses
Antibiotics
Environmental issues
Diagnosis, Treatment, Biosecurity
• Inactivated vaccine
• Live attenuated vaccine
• Toxoids (inactivated toxins)
• Subunit vaccine
• DNA vaccine or mRNA vaccine
Immersion, Injection, Oral
ORAL VACCINE
1

4. pH-Controlled Release of Antigens Using
Mesoporous Silica Nanoparticles Delivery
System for Developing a Fish Oral Vaccine
Zhang W, Zhu C, Xiao F, Liu X, Xie A, Chen F, Dong P, Lin P, Zheng C, Zhang H,
Gong H and Wu Y (2021) pH-Controlled Release of Antigens Using Mesoporous
Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine. Front.
Immunol. 12:644396. doi: 10.3389/fimmu.2021.644396
Present as a review by: D.D.T.T. Darshana Senarathna, as an assignment
for Aquaculture Health Management at Asian Institute of Technology, 2021.11. 20
2

5. 1. Introduction
Vibriosis
Vibrio parahaemolyticus
Vibrio alginolyticus
Vibrio harveyi
largest mariculture
fish in China (2017):
177,640 tons
Convenient, safe,
efficient vaccine and
delivery system
High-performance
Cost-effective
Stable vaccines
Better release kinetics
Whole-pathogen vaccines
Single proteins and
peptides as antigens
Antigens have a greatly
reduced immunogenicity
Not achieve the desired level
of immune protection
Necessary to develop
adjuvants and effective
delivery systems
Larimichthys polyactis
3
Xu
and
Lu,
2018
Mahboubeh
et
al.,
2017

6. Adjuvant
Activate antigen-presenting cells
Strong immune response
Less toxicity and side effects
Long-term protection
Carrier systems
Low antigen degradation
Controlled antigen release
Enhance bioavailability
Suitable size
Absorb by endocytosis
Promote antigen absorption
Enhance Antigen presentation
Nanoliposomes
Macromolecule
Inorganic nanoparticles
Immunostimulatory
Complexes
Mesoporous silica
nanoparticles
(MSN)
Humoral and Cell-mediated
immune responses
4
Smith
et
al.,
2015
Gregory
et
al.,
2013
Shaalan
et
al.,
2016

7. Hypothesis
Objectives
Methylcellulose phthalate (HP55) coated Mesoporous
silica nanoparticles (MSN) might act as an effective
delivery system for an oral vaccine developed from
purified antigen, Dihydrolipoamide dehydrogenase
(DLDH) of Vibrio alginolyticus.
• To develop DLDH loaded MSN coated with HP55.
• To analyze the antigen releasing behavior of developed nanoparticles.
• To analyze the toxicity of nanoparticles.
• To analyze the effectiveness of nanoparticle based oral vaccine against vibrio.
5

8. Conceptual Framework
6
Gene Clone and Protein Expression
Characterization of Materials
Acid-Base Release Characteristics
In Vitro Cytotoxicity Analysis of Vaccine
Relative Percent Survival Assays
Analysis of Serum Antibody Levels
Expression Levels of Cytokines
Vibrio alginolyticus
DLDH
DLDH loaded MSN DLDH loaded MSN mixed feed
Processing and antigen
presenting cells
T cells
IFNγ
B cells
Antibodies

9. 2. Materials
• Large yellow croaker
• Kidney cells of large yellow croaker
• Vibrio alginolyticus
• Monoclonal antibody 2H5F4
• HP55 and span 80
• N, N, N-trimethylhexadecan-1-aminium 4- methylbenzenesulfonate (CTATos)
• Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG
• Triethanolamine (TEAH3), tetraethyl orthosilicate (TEOS), polyvinyl alcohol
(PVA) and triethyl citrate
• Luminescent cell viability assay kit
7

10. 3. Method 3.1 Gene Cloning and Protein Expression
DLDH
Centrifugation (6,720
g for 10 min) +
ultrasonic cell disruptor
Remove cell fragments
(centrifugation at 98,900 g)
nickel-nitrilotriacetic acid (Ni2+-NTA)
pET-28a
Recombination
3h in LB +
IPTG 0.3mM
at 16°C,12h
PCR
E.coli
Tag removal
1
2
3
4
5
6
7
8
9
10
8

11. Swiss-model on line server used for homology modeling to build the three-dimensional structure
of DLDH.
The DLDH protein from Colwellia psychrerythraea 34H (PDB ID 3IC9, 60.17% identity) has
used as a template for modeling, and the three-dimensional structure diagram was performed using
PyMOL software
9
Rigsby
and
Parker,
2016

12. CTATos (4.80 g)
TEAH3 (0.7 mL)
H2O (250 mL)
Stir 1h, 80⁰C
3.2 Preparation of Nanoparticles & Feed
Stir 2h, 80⁰C
Extract using
NH4NO3 methanol
solution, 60⁰C
Buffer solution
1:1.28
(MSN/DLDH)
DLDH
MSN
DLDH load(%) = DLDHload× 100%.
(DLDHload + MSNmass)
MSN-DLDH
HP55 enteric
coatings
Double emulsion
Chitosan solution (3s)
+ H2O (5s) + Dry
Commercial feed + binder
1 2
3
4
5
MSN-DLDH@HP55
10

13. 3.3 Acid-Base Release Characteristics 11

14. 3.4 In Vitro Cell Cytotoxicity Assay
Cell Titer-Lumi™ plus luminescent cell viability assay kit
96-well plates (104/well) in M199
media supplemented with 10% fetal
bovine serum for 24 h at 27°C.
Yellow croaker
kidney cells
Culture
DLDH proteins &
MSN-DLDH@HP55
(0, 4, 8, 10, 20, 40, 60, 80, 100,
120, 140, 160, 180, 200 μg/ml)
Incubate for 24 h
100 ml Cell Titer-
Lumi™ plus solution
shake at room temperature for 2 min
and incubate at 25°C for 10 min
microplate
reader
Cell viability (%) = (Sample/Control) × 100%.
12

15. 3.4 Relative Percent Survival Assay
One week after the second stimulation
intraperitoneal inoculated
with 0.2 ml PBS
10-fold median lethal dose (1.1 × 107 CFU/ml) of
Vibrio alginolyticus
RPS =
[1 - (mortality in immunized group /
mortality in control group)] x 100%
Kole
et
al.,
2019
13

16. 3.5 Analysis of Serum Antibody Levels
Coated with 20 mg/ml of
purified DLDH protein
Primary antibody:
Antiserum of large
yellow croaker
(1:50)
Secondary antibody:
Monoclonal antibody
2H5F4 against large
yellow croaker (1:500)
Third antibody:
Goat anti-mouse IgG
conjugated to HRP
(1:5,000)
PI (%) = (1-OD490 of sample serum/OD490 of control serum) ×100%.
Absolute value of the PI >18.4% = POSITIVE
(OD) value of each well was determined
by microplate spectrophotometer
Gaps
0,7,14,21
days
Blocking ELISA
14

17. 3.5 Analysis of the Expression Levels of Cytokines
Total RNA
Trizol reagent
cDNA
Reverse transcription:
PrimeScript™ RT reagent
kit with gDNA Eraser
expression of
IFNγ, IL-1β,
IL-2, IL-4,
and IL-13
genes
RT-qPCR
TB
GreenTM
Premix
Relative expression
2−ΔΔCt
primers used for RT-qPCR assay.
A
B
1
2
3
4
15

18. 4.Results and Discussion
4.1 Gene Clone and Protein Expression
(A) PCR amplification of the DLDH gene. Lane M: 2 KB DNA marker; Lane 1: 1500
bp DLDH gene product. (B) SDS-PAGE analysis of DLDH purification by Ni2+-NTA
column.
16
Lane 1: After removal of
His-tag
Lane M: Protein marker
Lane 2: Precipitation
Lane 3: Supernatant
Lane 4: Flowthrough
Lanes 5–9: Fractions
eluted with 0, 10, 20, 30,
and 40 mM imidazole
Lane 10: Target protein
eluted with 300 mM
imidazole.

19. 4.2 Characterization of Materials
Transmission electron microscopy (TEM)
Energy dispersive X-ray spectroscopy (EDX)
A B C
A B
hydrodynamic particle size (81.6 nm)
A. MSN
71.39 ± 8.00 nm
B. MSN-DLDH
72.49 ± 8.07 nm
C. MSN-DLDH
coated with HP55
A. MSN
B. MSN-DLDH
15

20. Dynamic light scattering
analysis of MSN and MSN-DLDH.
Particle size analysis of
(A) MSN and (B) MSN-DLDH.
Zeta Potential analysis of
(C) MSN and (D) MSN-DLDH.
(ddH2O at 25°C)
16

21. Thermo gravimetric analysis (TGA) Nitrogen adsorption-desorption analysis
11.26%
53.41%
Loading degree of DLDH
protein in MSN = 42.15%
MSN MSN-DLDH
Specific surface area 679.90 m2/g 29.38 m2/g
Pore volume 1.13 cm3/g 0.06 cm3/g
Pore diameter 7.83 nm 6.67 nm
17

22. 4.3 Acid-Base Release Characteristics
In vitro assay of nanoparticles: Acid-base triggered release of MSN-DLDH@HP55.
Enteric-coated MSN-DLDH@HP55
nanoparticles are stable in acidic
conditions and release the loaded
DLDH protein in weak alkaline
conditions
18

23. 4.4 In Vitro Cytotoxicity Analysis of Vaccine
Analysis of cytotoxicity of MSN, DLDH and MSN-DLDH@HP55 in vitro by
CellTiter-Lumi™ plus luminescent cell viability assay kit
71%
79%
19

24. 4.5 Relative Percent Survival Assays
76.92%
15.39%
20

25. 4.6 Analysis of Serum Antibody Levels
Serum antibody level of large yellow croaker was assessed at 7, 14, and 21 days after immunization
with MSN-DLDH@HP55 vaccine
65.05%
89.61%
<6%
21

26. 4.7 Analysis of the Expression Levels of Cytokines 22
IFNγ
IL-1β
Fredriksen
et
al.,
2011

27. 23
IL-2
IL-4
IL-13

28. 5. Conclusion
Theoretical foundation for
industrialization of oral vaccine
against Vibrio species.
New directions for developing
vaccines with good stability and
biocompatibility under a
gastrointestinal environment.
24

29. 6. Recommendation and suggestions
Further optimization is required to refine the vaccine formula ratio, the most suitable
does of oral administration feeding, and the best immunization periods prior to
clinical application.
DLDH is the common protective antigen with cross-protection effect for the pathogen
of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi. The antigenic
epitopes were similar, thus providing the theoretical basis for the future
development
Additional clinical tests are required for vaccine biosafety evaluation.
Systematic exploration of the effects of various factors on the MSN nanomaterials
and evaluation of its long-term in vivo mechanism will be the focus of future work.
25

30. Acknowledgement
All the authors of the original research article: Zhang W, Zhu C, Xiao F, Liu X, Xie
A, Chen F, Dong P, Lin P, Zheng C, Zhang H, Gong H and Wu Y and institutions
where the research was carried out.
All the journal reviewers and editors of the original research article.
26

31. Reference
Xu JL, Lu YH. China fishery statistics yearbook. Beijing: China Agriculture Press (2018).
Mahboubeh E, Maryam H, Mohsen M, Gholamreza H, Khalil A, Mohammad R, et al. Co-delivery of dual toll-Like
receptor aagonists and antigen in poly(lactic-co-glycolic) acid polyethylenimine cationic hybrid nanoparticles
promote efficient in vivo immune responses. Front Immunol (2017) 8:1077. doi: 10.3389/fimmu.2017.01077
Smith JD, Morton LD, Ulery BD. Nanoparticles as synthetic vaccines. Curr Opin Biotech (2015) 34:217–24.
doi: 10.1016/j.copbio.2015.03.014
Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Mi (2013) 3:13.
doi: 10.3389/fcimb.2013.00013
Shaalan M, Saleh M, El-Mahdy M, El-Matbouli M. Recent progress in applications of nanoparticles in fish
medicine: a review. Nanomed-Nanotechnol (2016) 12:701–10. doi: 10.1016/j.nano.2015.11.005
Mahony D, Cavallaro AS, Mody KT, Xiong L, Mahony TJ, Qiao SZ, et al. In vivo delivery of bovine viral diahorrea
virus, E2 protein using hollow mesoporous silica nanoparticles. Nanoscale (2014) 6:6617–26.
doi: 10.1039/c4nr01202j

32. Rigsby RE, Parker AB. Using the PyMOL application to reinforce visual understanding of protein
structure. Biochem Mol Biol Edu (2016) 44:433–7. doi: 10.1002/bmb.20966
Kole S, Qadiri SSN, Shin SM, Kim WS, Lee J, Jung SJ. Nanoencapsulation of inactivated-viral vaccine using
chitosan nanoparticles: evaluation of its protective efficacy and immune modulatory effects in olive flounder
(Paralichthys olivaceus) against viral haemorrhagic septicaemia virus (VHSV) infection. Fish Shellfish
Immun (2019) 91:136–47. doi: 10.1016/j.fsi.2019.05.017
A
B
RNA extraction using trizol/tri, https://openwetware.org/wiki/RNA_extraction_using_trizol/tri
https://www.takarabio.com/learning-centers/real-time-pcr/overview/one-step-rt-qpcr-kits

33. Thank you!

34. Supplementary Details
ELISA plate are
coated with antigen
Antigen-coated
plates are washed
with PBS
Nonspecific binding sites are
blocked with 100 µL of blocking
buffer (PBS containing 5%
skim milk)
Diluted serum were
added to each well
and incubated
washed 3 times with PBST
Incubate with
Monoclonal antibody
washed 3 times with PBST
goat anti-mouse IgG
(Sigma, USA)
conjugated to HRP is
added and incubate
washed 3 times with PBST
3,3′,5,5′-tetramethyl
benzidine was added and
cells were incubated
reaction was then
stopped by adding 0.1
N sulfuric acid
The optical density (OD)
was measured at 450 nm
Blocking ELISA
Xuesong
et
al.,
2012

35. Transmission electron microscopy (TEM), Energy dispersive Xray spectroscopy
(EDX)
Dynamic light scattering
Thermo
gravimetric analysis
(TGA)