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: Ass Prof Nguyễn Đặng Dũng Ph.D 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: National Library Library of Vietnam Military Medical University 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: Producing IgY against V.cholerae and IgY against choleratoxin by immunizing egg laying hen and isolating antibodies from egg yolk Evaluating the protective effect of the IgY antibodies against V cholerae and against choleratoxin on experimental animals 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: pages - Review of literature: 25 pages - Materials and Methods: 27 pages - Results: 25 pages - Discussion: 26 pages - Conclusions: pages There are 11 tables, 14 graphs, scheme, 28 figures, 109 reference papers (of which 23 are in Vietnamese, and 86 are in English) Chapter 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 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 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 century, with more than 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 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 In 2017, Nguyen Hoang Ngan et al evaluated the preventive and therapeutic effects of a mixture of 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 CHAPTER 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 - 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 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 11 V.cholerae or IgY against choleratoxin Number of survival mice was recorded every 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 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 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 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 / 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 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 pmol / CTB (from 1.25 ng / gill to 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 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: 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 ml of V.cholerae suspension (3x1010 CFU ml), or 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 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 24 RECOMMENDATIONS Evaluation of the preventive and / or therapeutic effect of combined anti-V.cholerae and anti-choleratoxin IgYs in mice infected with V.cholerae and/or intoxicated with choleratoxin Testing the safety of anti-V.cholerae and anti-choleratoxin IgYs on volunteers before widely used in prevention and control of cholera LIST OF RESEARCHES WHOSE RESULTS METIONED IN THE DISSERTATION Hoang Trung Kien, Le Thu Hong, Le Thu Ha et al (2013), “Study on production of yolk immunoglobilin (IgY) against Vibrio Cholerae by egg-laying Hens”, Journal of Military Pharmaco-medicine, 38(1):46-51 Hoang Trung Kien, Nguyen Dang Dung (2014), “Study on quality and mechanism ò action of yolk immunoglobulin (IgY) specific to Cholerae Toxin”, Journal of Military Pharmacomedicine, 39(1):7-11 Hoang Trung Kien, Le Thu Hong, Nguyen Dang Dung (2015), “Protective effect of IgY anti-V.cholerae and anti-choleratoxin IgYs in Suckling Mice challenged with live Vibrio Cholerae”, Journal of Military Pharmaco-medicine, 40(2):26-37 Hoang Trung Kien, Nguyen Anh Tuan, Le Thu Hong et al (2017), “Study on protective effect of specific IgY antibody on cholera toxin-intoxicated suckling mice”, Journal of Military Pharmaco-meddicine, 43(1):151-157 ... 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 IgY antibody activity against vibrio... 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... 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