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We aim to develop the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression in silkworm. By using this system, the recombinant cholera toxin B subunit (CTB) protein will be expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method will provide the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.
The nontoxic B subunit of cholera toxin (CTB) can significantly increase the ability of proteins to induce immunological tolerance after oral administration, when it was conjugated to various proteins. Recombinant CTB offers great potential for treatment of autoimmune disease.In this study, we propose to firstly investigate the feasibility of silkworm baculovirus expression vector system for the cost-effective large-scale production of CTB under the control of a strong polyhedrin promoter by introducing the partial non-coding and coding sequences (ATAAAT and ATGCCGAAT) of polyhedrin to the 5' end of the native CTB gene and analyzing the amount of protein produced in the hemolymph. We hope to find that the silkworm bioreactor will produce this protein vaccine as the glycoslated pentameric form, which will retain the GM1-ganglioside binding affinity and the native antigenicity of CTB. We also hope to further find that mixing with silkworm derived CTB will increase the tolerogenic potential of insulin.The data from this study will demonstrate the potential of silkworm bioreactor to be an ideal production and delivery system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes and that CTB will function as an effective mucosaladjuvant for oral tolerance induction.
Problem and justification
According to the National Institutes of Health, an estimated 850,000 to 1.7 million Americans have Type 1 diabetes. Of those, about 125,000 are kids 19 and under (1).The diagnosis and classification with type 1 diabetes occurs within the first two decades of life, but an increasing number of cases are being recognized in older individuals (2). The geographic incidence varies widely from 1.7/100,000 per year in Japan to more than 35/100,000 in Finland. In the US the lifetime prevalence approaches 0.4%, but in high-incidence countries, such as Finland and Sweden, it may be as high as 1% (2). Bangladesh is one of the poorest countries in the world and has the lowest health care spending per capita. The main focus is on infections and maternal child health (reduction in infant mortality). The prevalence of diabetics is increasing rapidly in the developing countries including Bangladesh with an estimated 5.2% among the adult population. It is estimated that almost 3 million people have diabetes in Bangladesh. The majority have type 2 diabetes. Specialised diabetes care is virtually non-existent in the public sector, but in recent years some private sector hospitals have established diabetes clinics (3). According to the study, published in the May issue of Diabetes Care, the incidence of people with diabetes in Bangladesh is 3.2 million in 2000 which is predicted to go up to 11.1 million by 2030. It is a chronic disease which is never cured, but a diabetic's patient can lead a normal life by controlling the disease through balanced diet, taking appropriate drug, and exercising regularly4. In 2007, the International Diabetes Federation (IDF) estimates that 3.8 million or 4.8% of people living in Bangladesh will have diabetes. By 2025, that number is expected to grow to 7.4 million or 6.1% of the population (4). This explosion in diabetes prevalence will place Bangladesh among the top ten countries in terms of the number of people living with diabetes in 2025. At that date, 80% of all diabetes cases will be in low-and-middle income countries5. The increase in diabetes in Bangladesh is expected to follow global gender patterns, whereby more women than men will live with diabetes. IDF and WHO predict that the number of women in the world with diabetes will double in less than 20 years. In Bangladesh the number of women with diabetes will grow from the current 2 million to 4 million by 2025. During the same period, men with diabetes will rise from 1.8 million to 3.4 million. From a study done in a Rajshahi population it was found that 22.7% people are suffering from type 1 diabetes (4).
Type 1 diabetes is due to a deficiency of insulin as a result of destruction of the pancreatic Î² cells. At the time of clinical symptoms, 60-80% of the Î² cells are destroyed. Cells secreting glucagon, somatostatin, and pancreatic polypeptide are generally preserved but may be redistributed within the islets. Insulitis, an inflammatory infiltrate (Figure 1) containing large numbers of mononuclear cells and CD8 T cells, typically occurs around or within individual islets.
Figure 1.Inflammatory infiltrate of mononuclear cells in an islet from a 2-year-old patient with type 1 diabetes of short duration.
The cause of Î² cell destruction remained an enigma for years, but two discoveries in the 1970s provided the basis for our current thinking about the disease. The first was a strong linkage of type 1 diabetes to the highly polymorphic HLA class II immune recognition molecules - DR and, later, DQ - located on chromosome 6. Over the years, extensive studies have revealed a large number of high- and low-risk HLA alleles (2). The second discovery, providing direct evidence for autoimmunity, came by incubating sera from type 1 diabetic patients with frozen tissue sections of normal blood group 0 pancreas (2). Sera from type 1 diabetic patients with polyendocrine disease were found, by immunofluorescence, to stain pancreatic islets. These antibodies, which came to be known as islet cell antibodies (ICAs), have been widely used to study the clinical course and pathogenesis of type 1 diabetes, although the nature of the islet antigens involved remained unclear for a number of years.
In the 1980s and early 1990s the principal two autoantigens recognized by ICA were identified. The first was a new isoform of glutamic acid decarboxylase (GAD65) (2) and the second was a protein tyrosine phosphatase-like molecule (IA-2) (12). A third antigen, insulin, also was identified in the 1980s (2).
Approximately 70-80% of newly diagnosed type 1 diabetes patients have autoantibodies to GAD65. Nearly the same number or slightly less have autoantibodies to IA-2. Overall, fewer patients appear to have insulin autoantibodies, but this is due to a pronounced age effect: children with newly diagnosed type 1 diabetes have a markedly higher frequency of auto-antibodies to insulin than teenagers or young adults (2). Some patients carry auto-antibodies to only one of the major autoantigens, but others may react to all three. In newly diagnosed subjects, up to 90% have autoantibodies to one or more of these antigens. The percent positivity depends on a variety of factors, including not only the age of the subjects, but also the duration of the disease and, in some cases, their ethnic origins. Some intrinsic variability is also seen in the assay, particularly at the limit of its range of detection. In general, GAD65 autoantibody positivity tends to be stable, whereas IA-2 autoantibodies tend to decrease with duration of disease, and insulin autoantibodies cannot be usefully measured after initiation of insulin therapy. Extensive analyses of these autoantibodies in normal controls suggest that about 1.0% have autoantibodies to IA-2, GAD65, or insulin (2).
The problem that arises due to autoimmunity in type 1 diabetes can be solved by the help of cholera toxin B subunit (CTB). CTB is the pentameric non-toxic portion of cholera toxin (CT), responsible for the holotoxin binding to the GM1 ganglioside receptor present on most nucleated cells (5). When conjugated to autoantigens, the CTB dramatically increases their tolerogenic potential after oral administration (6,7,8,9,10,11,12). This effect is probably mediated by the ability of CTB to act as a mucosal carrier system (7), although CTB might also have direct effects on the immune system (13,14). Recent studies have showed that CTB is an effective mucosal adjuvant in potentiating immune responses or increasing immunological tolerance to corresponding antigens (15,16, 17,8,18,19,20) These investigations indicate that CTB is a powerful edible vaccine if expressed in large-scale production in an edible tissues or organism.
Up to now, the CTB gene has been expressed in Escherichia coli (21), Swiss 3T3 cells (22), transgenic tobacco (23,24,25), potato (26), and tomato (27). However, the recombinant CTB expressed in Escherichia coli was in an insoluble form, which required extensive purification. In addition, bacteria-derived CTB is inappropriate as an edible vaccine because of the contamination of endotoxin. Despite the suitability of plant expression systems for the production of CTB as functional oligomers, the expresssion levels in transgenic plants are not satisfied with the therapeutic applications in humans.
Hence, we aim to develop recombinant protein production in the silkworm baculovirus expression vector system (BEVS) which may overcome this limitation and thus may facilitate the use of genetically modified edible proteins for the production and delivery of vaccine antigens (28,29).
There is the potential for not only low-cost but also high capacity production with the capability to scale-up to agricultural levels. We propose using the Bombyx mori nuclear polyhedrosis virus (BmNPV) of the baculovirus expression system as it also eliminates concerns regarding pathogens that could potentially be transmitted to humans. The baculovirus is non-infectious to vertebral animals, and the system itself is safe (30,31). Collectively, these features make the silkworm system an ideal expression and delivery package for producing oral vaccines. For a developing country such as Bangladesh, where the cost of 50% of diarrheal infection occurs due to cholera, cholera vaccine using the silkworm can decrease the cost of vaccine from the regular price of US$60. This study will also contribute to one of the Millennium Development Goals of reducing the burden of infectious diseases due to cholera.
Generally, silkworm is a high-level expression system (28). The production levels of foreign genes are very different in the silkworm baculovirus system. Several extra techniques are used to improve the production level in this system, such as selecting appropriate codon, introducing 5'-untranslated sequences and coexpressing chaperones (32).
In our research, we aim to specially fuse the partial non-coding and coding sequences of polyhedrin to the 5'-end of the native CTB gene and this modified CTB gene will then be expressed in BmN cells and silkworm larvae with BEVS. We predict that this will lead to production of encouragingly high production of recombinant CTB protein in the hemolymph of silkworm larvae.
Since female population in Bangladesh is a vulnerable social group due to their dietary uptake leading to severe malnutrition especially in the low-income group and type 1 diabetes occurs at large in children , we aim to use female non-obese dieabetic (NOD) four week old mice to investigate oral tolerance of silkworm-derived CTB by using different concentrations of insulin as autoantigen.
Results from this experiment can open up venues to fight other autoimmune diseases and establish the use of CTB as an edible vaccine to decrease the incidence of cholera in Bangladesh. The successful implementation of this research will also establish the potential use of silkworm as a "bioreactor" for production of antigenic proteins for clinical use offers several advantages. It can be used for (i) large-scale production of foreign proteins, (ii) as an edible vaccine if expressed in an edible silkworm pupa or (iii) as a delivery system for oral protein drugs, to either enhance mucosal immunity or induce oral tolerance to the products of these peptides. In contrast to the conventional prokaryotic expression system, large-scale production of purified CTB in bacteria involves the use of expensive fermentation techniques and stringent purification protocols (33), making this a prohibitively expensive technology for developing countries such as Bangladesh. As compared with other eukaryotic expression systems, the expression of heterologous proteins in the silkworm bioreactor is under the control of the strong polyhedrin promoter, allowing levels of expression of up to 20% of total cell protein (34). It is obvious that the protein production capacity of silkworms predominates over that of any other industrial system in use today. In addition, the cost of producing 1 kg of recombinant protein in silkworm is much lower than the cost of producing the same amount by E. coli fermentation or transgenic plants.