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 BiologicLP 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 (50l 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 (50l 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
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