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MINISTRY OF EDUCATION AND TRAINING
MINISTRY OF DEFENSE
VIETNAM MILITARY MEDICAL UNIVERSITY

HOANG TRUNG KIEN

RESEARCH ON PRODUCTION AND EVALUATING
THE EFFECTIVENESS OF IgY AGAINST VIBRIO
CHOLERAE AND IgY AGAINST CHOLERATOXIN
IN EXPERIMENTAL ANIMAL

SPECIALTY: BIOMEDICAL SCINENCE
CODE NO.: 9720101

SUMMARY OF Ph.D THESIS

HA NOI – 2019


THE THESIS WAS FULFILLED AT VIETNAM MILITARY
MEDICAL UNIVERSITY


Names of supervisors:
1.

Ass. Prof. Nguyễn Đặng Dũng Ph.D

2.

Ass. Prof. Lê Thu Hồng Ph.D

Opponent member 1:
Opponent member 2:
Opponent member 3:

This thesis will be defensed :

Hour

date

month

year

The dissertation can be found at:
1. National Library.
2. Library of Vietnam Military Medical University


1
INTRODUCTION
Acute diarrhea caused by Vibrio cholerae infection, also known
as cholera, is one of the cause of death globally, mainly in chindren,
and especially in developing countries. Pathogenesis of the disease
involves choleratoxin secreted by V. cholerae after its infection in
patient's intestine, and patient may die within 12-14 hours from the
onset if not being promptly treated [1]. Among different therapies for
cholera, specific antiserum is proved to be effective in treating and
preventing the disease. Conventional technology in production of
antiserum have long history, which mainly involves immunization on
big mammalials, such as horse, sheep. However, this technology has
some disadvantages in immunization procedure and in collecting
antiserum. Yolk immunoglobulin (IgY) production, meanwhile,
showed some advantages over the conventional one, being high
antibody titration, easy to perform, and low cost, just to name a few
[2], [3], [4], [5], [6], [7], [8].
In this study, entitled "Research on production and evaluating
the effectiveness of IgY against Vibrio cholerae and IgY against
choleratoxin in experimental animal", we aim at:
1. Producing IgY against V.cholerae and IgY against
choleratoxin by immunizing egg laying hen and isolating antibodies
from egg yolk.
2. Evaluating the protective effect of the IgY antibodies against
V. cholerae and against choleratoxin on experimental animals.


2
Summary on new findings and contributions of the
dissertation:
By immunizing egg laying hens and isolating IgY from the eggs,
we have successfully produced IgY against V. cholerae and IgY
against choleratoxin.
The IgY antibodies exerted protective effect on mice
gastrointestinally infected with V. cholerae and mice intoxicated with
choleratoxin, respectively. The IgY antibodies might be used to form
a passive immunity with cholera.
Organization of the thesis:
The thesis consists of 108 pages, including:
- Introductions: 2 pages

- Review of literature: 25 pages
- Materials and Methods: 27 pages
- Results: 25 pages
- Discussion: 26 pages
- Conclusions: 3 pages
There are 11 tables, 14 graphs, 1 scheme, 28 figures, 109
reference papers (of which 23 are in Vietnamese, and 86 are in
English).


3

Chapter 1.
Literature review
1.1. Microbiological characteristics and pathogenicity of V.
cholerae
1.1.1 . Microbiological characteristics of V. cholerae
Vibrio cholerae is a Gram-negative, comma shaped bacteria,
which is 0.3 x 3 µm in size, has a flagellum at one cell pole and is
highly motile.
1.1.2. Pathogenicity of V. cholerae
1.1.2.1. Pathogenesis of cholera
Under natural conditions, V. cholerae is pathogenic for
human only when there is a gastrointesinal infection of the
bacterium. Upon access to the intestinal wall, V.cholerae secretes
choleratoxin (CT), which is composed of two subunits: subunit A and
subunit B. The CT-B subunit binds to the GM1 ganglioside receptor
on surface of epithelial cells (known as enterocytes), facilitating the
endocytosis of the toxin. Once inside the enterocyte, the enzymatic
A1 fragment of the toxin A subunit stimulates adenylate cyclase to
continuously produce cAMP. The high cAMP level then activates the
"pumping" system of the cells, causing a dramatic efflux of ions (Na +
and Cl) and water from infected enterocytes, leading to watery
diarrhea. Dehydration and loss of electrolytes are the most typical
features of cholera.
1.1.2.2. Epidemiology of cholera
Up to now, there have been 7 cholera epidemics in the world
[18] causing tens of millions of deaths. According to statistics from
the World Health Organization (WHO, 2017), 132,121 cases of
cholera have been reported, with 2,420 deaths in 38 countries
worldwide, making mortality rates of 1.8%. Cholera is one of the
most important causes of acute diarrhea in Vietnam, for more than a


4
century, with more than 2 million cholera cases reported. Vietnam is
one of the coutries with cholera in recent years. In Vietnam, in
October 2007 to July 2009, a large cholera epidemic occurred in
northern Vietnam with 8,064 patients and one death [29].
1.2. PREVENTION AND TREATMENT OF CHOLERA
1.2.1. Cholera prevention
After Robert Koch isolated the cholera bacteria in 1884 [33],
scientists around the world started to develop preventive vaccines for
cholera. Oral cholera vaccines have been tried, which induced
immune responses in the gastrointestinal tract against one or more of
the major antigens of the bacteria. Vietnam is the first and only
country in the world to regularly use oral vaccines (OCVs) in cholera
control programs. Between 1998 and 2012, more than 10.9 million
doses of OCVs were deployed in 16 provinces and cities through
expanded vaccination in Vietnam.
1.2.2. Treatment of cholera
With the increase of antibiotic-resistant Vibrio cholerae [46],
[47], [48], it is necessary to introduce new therapies to replace
antibiotics, and immunotherapy using IgY antibodies is one of the
promissing approach [49]. Research on the production of antibodies
against cholera for use as a passive immunotherapy for prevention
and / or treatment of cholera has been carried out by several research
groups over the years [52].
1.3. EGG YOLK IMMUNOGLOBULIN AND ITS MEDICAL
APPLICATIONS
1.3.1. Characteristics of egg yolk immunoglobulins


5
Yolk immunoglobulin is originally an antibody in bird
species, including chickens. The antibody has the similar structure
and function as IgG antibody in humans and mammals. The
molecular weight of IgY is 180 kilo-dalton (kDa), larger than
mammalian IgG (160 kDa) [56]. The antibody can be transferred
from hen's plasma to egg yolk laid of the hen, and hence the name
IgY.
1.3.2. Technology of IgY antibody production
Technique for extracting IgY from egg yolk is relatively
simple. IgY is in the water-soluble part. There are several ways to
extract, depending on the requirements of the purity of the antibody
and the purpose used to select the method. After immunizing egg
laying hen with antigen, IgY antibodies specific to the antigen will
appear in the blood and egg yolk laid by the immunized hen.
1.3.3. Potential of IgY antibody applications in medicine
Some researchers have shown a number of remarkable
advantages in the production of IgY antibody in egg yolk compared
to the production of antisera from IgG antibodies in mammals. It is
easier to immunize chicken (to stimulate an IgY antibodies response)
compared to that in mammals. Thanks to these characteristics, IgY
has been widely used in the treatment of infectious diseases in
humans and animals.
1.4. OVERVIEW OF RESEARCHES AND APPLICATIONS OF
IgY

AGAINST

V.

CHOLERAE

AND

IgY

AGAINST

CHOLERATOXIN
In 2010, Kazuyuki Hirai et al found that suckling mice
infected with V. cholerae and treated by IgY against V. cholerae had
significantly longer survival time compared to controls. Akbari M. R
et al., 2018, published a study on the use of IgY antibody extracted
from chicken egg yolk immunized with a mixture of V.cholerae
(1x109 CFU/ml) and LPS (100 μg), showing that the antibody was
specifically reactive with V. cholerae antigen, and hence, can be used
by oral route to provide passive immunity for cholera.


6
In 2017, Nguyen Hoang Ngan et al evaluated the preventive
and therapeutic effects of a mixture of 2 IgY antibodies against
cholera and against choleratoxin in mice infected with V. cholerae. It
was revealed by the study that both antibodies was effective in
prevention and treatment of 4-day old suckling mice infected with V.
cholerae.


7

CHAPTER 2
SUBJECTS, MATERIALS AND METHODS
2.1. SUBJECTS
2.1.1. Animal for immunization to produce specific IgY
24 egg-laying hens (Tam Hoang breed) at 20 weeks old of
age, weighing 1.5 - 2 kilograms
2.1.2. Animals for experimental infection with V.cholerae and
with choleratoxin
Suckling mice (3-5 days old), weighing 1.8 - 1.84 grams.
2.2.1. Vibrio cholerae strain
Vibrio cholerae O1 strain isolated from stool sample of
cholera patients at Military Hospital 103.
2.2.2. Choleratoxin
Choleratoxin (code number C0852, Sigma) in freeze-dried
form, stored at 2- 80C until use.
2.2.3. Other reagents
Reagents for immunizing hens, for bacterium culture, and for
ELISA.
2.2.4. Equipments
Common laboratory equipment, at Center of AppliedBiomedical and Pharmaceutical Research, and at Dept. of
Immunology, Vietnam Military Medical University.
2.3. METHODS


8

Figure 2.1. The structure of the research
2.3.1. Producing V.cholerae antigen from selected V.cholerae
isolates
Bacterial culture and identification testing of the V.cholerae
isolates; generating inactivated bacterial suspension (served as
whole-cell bacterial antigen), and testing for sterility of the antigen.
2.3.2. Producing choleratoxin antigen
Mix choleratoxin with complete Freund adjuvant (for the
first immunization) or with incomplete Freund adjuvant (for
subsequent immunization) at the volume ratio of 1:1.
2.3.3. Immunizing egg laying hens to induce antibody response
against V. cholerae and against choleratoxin


9
Antigen was injected intramascularly into pectoral muscles of
the hens, 4 times with interval of 3 weeks between the injections.
2.3.4. Extraction and purification of IgY antibody from chicken
eggs
Extracting IgY from egg yolk in 3 steps: lipid removal, precipitation
with ammonium sulfate, and membrane dialysis for salt removal.
Purification of IgY: using anion exchange column on the Biologic-LP
Low Pressure Biologic System supplied by BioRad with
corresponding buffer.
2.3.5. IgY confirmation by SDS-PAGE
Sodium dodecyl sulfate - polyacrylamide gel electrophoresis
(SDS-PAGE) was preformed according to method described by
Laemmli on Mini-PROTEIN II Cell (BioRad).
2.3.6. Testing specificity of IgY by ELISA
- ELISA for IgY against V.cholerae: the V.cholerae antigen
solution (5 µg/ml) was incubated in ELISA wells at 40C overnight .
Adding sample, incubating at 370C for 1hr; after washing the wells,
adding peroxidase-conjugated anti-IgY and incubating for 1 hr at
370C; washing the wells again and adding OPD substrate, then
measuring optical density (OD) of the wells.
- ELISA for choleratoxin: adding 100 µl GM1 into the wells and
incubating at room temperature ovenight; adding choleratoxin (CT),
incubating at 370C for 1hr; washing the well and adding sample
(supposed to contain IgY specific to CT ), then incubating for 1 hr at
370C; washing the wells, and adding peroxidase-conjugated anti-IgY;
incubating the wells at 370C for 1hr; finally, washing the wells again
and adding OPD, then measuring OD of the wells.
2.3.7. V. cholerae bacterial agglutination test


10
Bacterial suspension was mixed with antibody in U-shaped test
tube, the let for spontaneous sedimentationat 40C overnight. The test
was positive when an obvious agglutination was observed.
2.3.8. Investigation of binding activity of CT-B to GM1 in vitro
Firstly, GM1 was immobilized on ELISA wells. Adding 50
µl horse radish peroxidase (HRP)-conjugated choleratoxin B subunit
(CTB) into each well, and incubating at RT for 15 mins in a dark
chamber, then measuring OD of the wells.
2.3.9. Inhibition effect of IgY on the binding of CT-B to GM1
IgY was mixed with HRP-conjugated CTB before adding to
ELISA wells precoated with GM1.
2.3.10. Establising animal models infected with V.cholerae and
intoxicated with choleratoxin
2.3.10.1. Animal model of suckling mice infected with V. cholerae
White suckling mice (3-5 days old) were given 50 µl of live
V.cholerae suspension (1x108; 7.5x108 and 1x109 CFU/ml), through
esophageal intubation; number of mice died at different time points
was recorded, until no mice was alive.
2.3.10.1. Animal model of suckling mice intoxicated with
choleratoxin
White suckling mice (3-5 days old) were given 50 µl of
choleratoxin (0.008; 0.04; 0.2; 1; and 5mg/ml in NaCl 0,9%), through
esophageal intubation; number of mice died at different time points
was recorded, until no mice was alive.
2.3.11. Protective effect of IgY against V.cholerae and IgY against
choleratoxin in V.cholerae-infected suckling mice
White suckling mice (3-5 days old) were infected with V.
cholerae (50l of 1x108CFU/ml), and then treated by IgY against


11
V.cholerae or IgY against choleratoxin. Number of survival mice was
recorded every 2 hours, until no mice was alive.
2.3.12. Protective effect of IgY against choleratoxin in suckling
mice intoxicated with choleratoxin
White suckling mice (3-5 days old) were intoxicated with
choleratoxin (50l of 1mg/ml), and then treated by IgY against
choleratoxin. Number of survival mice was recorded every 2 hours,
until no mice was alive.
2.3.13. Immunohistochemistry analysis
Immunohistochemitry technique was adopted to determine the
presence of choleratoxin on intestine's epithelial cells of the mice
intoxicated with choleratoxin gastrointestinally.
2.3.14. Statistical data analysis
Data collected in the study were analyzed by Microsoft Excel
and SPSS 20.0 softwares.
2.4. Place of research
All experiments were performed at Center of Applied
Biomedical and Pharmaceutical Research and Dept of Immunology,
Vietnam Military Medical University
2.5. Ethical issues of the study
Protocols of immunization, blood sampling and collecting
were performed in accordance with regulations regarding animal
welfare.


12

OD (450nm)

CHAPTER 3
RESEARCH RESULTS
3.1. Results of IgY antibody anti-cholerae and IgY antibody
against choleragen.
3.1.1. The resulting immunizations to produce IgY antibodies to
Vibrio cholerae
3.1.1. 1. IgY antibody activity against vibrio cholerae in chicken
blood

OD (450nm)

Graph 3.1. ELISA response when detecting IgY
antibodies to Vibrio cholera in chicken serum
3.1.1.2. IgY antibody activity against Vibrio cholera in chicken eggs


13
Figure 3.2. ELISA response when detecting IgY
against Vibrio cholerae in chicken eggs

OD (450nm)

3.1.2. Immune response to IgY antibodies against choleratoxin
3.1.2.1. IgY antibody activity against choleratoxin in chicken blood

OD (450nm)

Figure 3.3. ELISA response to choleratoxin IgY against chicken
serum
3.1.2.2. IgY antibody activity against Vibrio cholera in chicken eggs

Figure 3.4. ELISA response to choleratoxin IgY against
chicken eggs
3.2.Results of isolation and purification of IgY antibody against
Vibrio cholera and choleratoxin resistance from egg yolk


14

mg/1 eggs

mg/1 egg yolk

3.2.1. Effective IgY antibody extraction from egg yolk

Figure 3.5. Average IgY antibody /
1 egg after extraction

Figure 3.6. Average IgY
antibody/1 ml of egg yolk
after extraction

3.2.2 Analysis and purification of IgY products by ion exchange
chromatography

Figure 3.1. Purification of antibodies by ion exchange
chromatography technique
3.2.3. Result of SDS-PAGE electrophoresis of IgY after
extraction and purification


15

Figure 3.2. Result of SDS-PAGE electrophoresis
under IgY denaturation
3.3 Results of evaluation of mechanism of IgY antibody specific
for Vibrio cholera and choleratoxin in vitro
3.3.1. The agglutination activity of Vibrio cholera of IgY antibody

Figure 3.3. Vibrio cholera agglutination by IgY antibody after
ammonium sulphate crystallization in liquid medium
3.3.2. Results of the effect of anti-choleratoxin IgY antibody
3.3.2.1. Binding of CTB with GM1


OD (450nm)

16

OD (450nm)

Figure 3.7. Binding of CTB with GM1 in vitro
3.3.2.1. IgY inhibits binding of CTB with GM1

OD (450nm)

Figure 3.8. IgY inhibits binding of CTB with GM1 in vitro (GM1:
5pmol/well; CTB: 5ng/well)

Figure 3.9. IgY inhibits binding of CTB with GM1 in vitro (GM1:
5pmol/well; CTB: 1ng/well)


OD (450nm)

17

Figure 3.10. IgY inhibits binding of CTB with GM1 in vitro in vitro
(GM1: 5pmol/well; CTB: 0,2ng/well
3.4. Results of evaluation of protective effect of anti-Vibrio
cholerae

antibody

IgY

and

choleratoxin

resistance

on

experimental model
3.4.1. The pattern of Vibrio cholerae infection in newborn mice

Figure 3.11. Survival time of mice after Vibrio cholerae infection
3.4.2.The results of experiments on choleratoxin in newborn mice

Figure 3.12. Additional survival time of mice after vibrio cholerae
infection


18
3.4.3. Protective effects of anti-vibrio cholerae IgY antibody and
choleratoxin antibody in white mice infected with Vibrio cholerae

Figure 3.13. The survival time of rats infected with Vibrio cholerae
was oral IgY antibody against Vibrio cholerae and cholera IgY
3.4.4. Protective effect of choleratoxin-resistant IgY antibody in
neonatal mice infected with choleratoxin

Figure 3.14. The survival time of mice after choleratoxin
infection was protected by choleratoxin-resistant IgY antibodies
3.5. Results of immunohistochemistry to determine the presence
of choleratoxin in the gut of mice

Figure 3.5. Intestinal mucosa of normal mice
A. Dye H.E x 400


19

A
B
Figure 3.6. Intestinal mucus of rats infected with vibrio

cholerae don’t drink anti-vibrio cholerae IgY antibody

Figure 3.7. Intestinal mucus of rats infected with choleratoxin don’t
drink anti-vibrio choleratoxin IgY antibody

Figure 3.8. Intestinal mucus of
rats infected with vibrio
cholerae drink anti-vibrio
cholerae IgY antibody

Figure 3.9. Intestinal mucus of
rats infected with choleratoxin
drink anti-vibrio cholerae IgY
antibody


20
CHAPTER 4
DISCUSSION
4.1.

IgY antibody production against Vibrio cholerae and

choleratoxin from egg yolk
The research of the immunogenicity of the vibrio cholerae
antigen showed that the intensity of the antibody response tended to
be correlated with the antigen concentration used to immunize the
viral vibrio cholerae ( chart 3.1). IgY antibodies from chickens were
immunized with a dose of 0.1 mg / ml choleratoxin antigen with a
higher antibody titer compared to antibodies from immunized
chickens with antigen doses of 0.05 mg / ml.
4.2.

Extraction and purification of antibodies against Vibrio

cholerae and choleratoxin from egg yolk
The results shown in Fig. 3.5 and Figure 3.6 show that the
antibody extraction method was 200 mg / egg. High antibody IgY
concentration. The collected IgY product exhibited high purity
(Figure 3.2).
4.3 Effects of IgY antibody against vibrio cholerae and
choleratoxin in vitro
In this research, we found the biding of CTB with GM1 on
the surface of intestinal epithelial cells and the results in Figure 3.11
show that with GM1 concentrations of 5 pmol / CTB (from 1.25 ng /
gill to 5 ng / gil) both exhibit specific biding with GM1 in vitro. The
results of ELISA shown in Figure 3.8, Figure 3.9, Figure 3.10 show
that IgY antibody derived from chicken egg yolk induced by the


21
toxins of vibrio cholerae specific to CTB. The graph also shows the
inhibition effect of CTB with GM1.
4.4. Protective effect of anti-vibrio cholerae of IgY antibody and
choleratoxin-resistant of IgY antibody on animal infected with
Vibrio cholerae
To assess the protective effect of the antibody IgY specific to
Vibrio cholerae and choleratoxin in vivo, we used a vibrio cholerae
infection model with a bacterial density of 1x108CFU/ml. Protective
effects of specific IgY antibodies will be demonstrated during the
extra life span of mice after exposure to vibrio cholerae without
specific IgY antibody responses compared to mice after exposure to
Vibrio cholerae. IgY specificity compared with that of non-Vibrio
cholerae mice. Figure 3.13 shows the protective effect of IgY
specific antibody to Vibrio cholerae and choleratoxin in virally
infected white mice with Vibrio cholerae.
In addition to evaluating the protective effect of the antibody
IgY against Vibrio cholerae and choleratoxin in mice infected with
vibrio cholerae, the researchers also found the protective effect of
IgY antibody in white mice which were infected with choleratoxin,
the dose of choleratoxin infection was 50 μl choleratoxin at 1 mg /
ml. Figure 3.14 shows the difference in survival time between groups
of choleratoxin-infective mice but using IgY antibodies and
choleratoxin-infective mice without using IgY antibodies.
4.5. Protective effect of IgY antibody against Vibrio cholerae and
choleratoxin with immunohistochemistry


22
The protective activity of rats infected with Vibrio cholerae
and choleratoxin infection of IgY antibody was also demonstrated by
immunohistochemical results, evaluating lesions of gut and intestinal
epithelial cells after rats died in the groups. research. The mice that
died from Vibrio cholerae did not receive the antibody against Vibrio
cholerae (Figure 3.6) and the mice that died from choleratoxin were
not given anti-choleratoxin antibodies (Figure 3.7). intestinal mucosa
and damaged ducts. In contrast, mice that died with Vibrio cholerae
were treated with specific antibodies against Vibrio cholerae (Fig.
3.8) and mice that died of choleratoxin in the presence of
choleratoxin antibodies (Figure 3.8). Epithelial and intestinal
epithelial cells were not damaged and nearly intact compared to
intestinal mucosa and ductal glands of normal mice (Figure 3.5).


23
CONCLUSION
It was concluded from the data collected, that:
1. By immunizing egg laying hens with Vibrio cholerae
and choleratoxin antigens, we have successfully produced IgY
antibodies against Vibrio cholerae and against choleratoxin:
- Immunization protocol: repeated immunization (4 times
with 3-week interval) by intramuscular injection into pectoral
muscle, with antigen dose of 1 ml of V.cholerae suspension (3x1010
CFU ml), or 1 ml of choleratoxin (0.1 mg / ml).
- IgY antibodies against V.cholerae and against choleratoxin
were specifically reactive to V.cholerae and to choleratoxin antigens
at 1/64,000 and 1/200,000, respectively.
2. IgY antibody against V.cholerae and against
choleratoxin showed protective effect on animals infected with
V.cholera and intoxicated with choleratoxin.
- IgY antibody against V.cholerae and IgY antibody against
choleratoxin have been shown to protect white suckling mice
gastrointestinally infected with V.cholerae.
- IgY antibody against choleratoxin protected white suckling
mice gastrointestinally intoxicated with choleratoxin.


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