The Expression Of Oral Ag85A DNA Vaccine In The Intestine Of Mice

PrefaceAg85A is one of the components of Ag85 complex, a kind of protein ingredient secreted by BCG, Mycobacterium tuberculosis and other genus Mycobacterium. The Antigen 85A protein is coded by the gene called fibronectin-binding protein-A (fbpA) gene.In Mycobacterium tuberculosis, this fibronectin-binding protein-A (fbpA) gene is 1014bp long and has a molecular weight of 32KD. Recent animal studies have shown that vaccination with Recombinant Antigen 85A DNA or the protein has powerful immunological properties, resulting in significant secretion of Th1 cytokines, particularly interleukins and interferon-y which are important in regulating the activities of a number of immune cells, especially the cytotoxic cells. These cytotoxic cells play significant roles in the elimination of viral infected and transformed cells.Increasing evidence suggests that vaccination at the mucosal site is superior to vaccination at other sites in eliciting protection from mucosal infectious diseases. This is partially explained by the observation that memory T and B cells generated upon mucosal vaccination acquire mucosa-homing receptors and preferentially accumulate at the mucosal site of induction. Thus, it is believed that greater immune protection may be achieved if TB vaccine is given mucosally via oral route.The cationic liposome acting as an adjuvant can greatly enhance the expression of recombinant plasmid due to the function of preventing delivered DNA from digested by Dnase and can also promote the absorbance of cellular level.In addition,liposome has several advantages over traditional adjuvant.,such as,easy preparated and ready to use,most important ,with the high efficiency in gene transfection.Ag85A DNA vaccine mainly causes Th1 type immune response after intramuscular injection, while mainly induces Th2 type immune response after the gene gun injection.The gene of Naked DNA vaccine generally expresses in the muscle cells after intramuscular injection, but there is no information about the expression site of oral DNA vaccine in the intestinal tract. We plan to detect the distribution of the product of Ag85A gene in the intestinal tract of C57BL/6 mouse.Material and methods1. Animalfemale C57BL/6 mice, 6～8 weeks old(Academia Sinica Shanghai experimental animal center).2. AgentsDNA vaccine pCDNA3.1+/Ag85A(made in our experiment);Lipofectamine TM 2000 (Invitrogen Corporation);Chicken anti-Ag85A IgY (Prosci Corporation), goat-anti-chicken IgY HRP conjugated, goat-anti-chicken IgY FITC conjugated (Gene Corporation), Purified Armenian Hamster-anti-mouse CD11c (BD Pharmingen Corporation), Goat Anti-Armenian Hamster IgG Texas Red conjugated (Jackson ImmunoResearch Laboratories), UEA-I TRITC conjugated (Vector Laboratories); Endotoxin free Pure Yield Plasmid extraction kit (Promega Corporation); the BSA and DAB kit (Zhongshan company,Beijing).3. Experimental procedureAmplify Ag85A gene sequence with genetic engineering techniques, after the sequence homology analysis, subclone it into the eukaryotic expression vector pCDNA3.1+, then transform it into Escherichia coli DH5α.Extract the recombinant plasmid with endotoxin free kit, further parcel with liposomes to make oral recombinant DNA vaccine. C57BL / 6 mice are randomly divided into two groups, PBS control and the recombinant plasmid parceled with liposome, respectively intragastric administration. The mice are immuned every 14 days, three times altogether. After another 14 days, kill the mice and prepare the intestine to detect the expression of Ag85A in intestinal epithelial cells, Peyer's patches, intestinal M cells and dendritic cells with immunohistochemistry and immunofluorescence.ResultsAg85A recombinant DNA vaccines expressed in the small intestinal mucosa epithelium, Peyer's patches and the spleen widely. The intensity of Ag85A expression in the epithelial cells nearing to lamina propria is higher than in the epithelial cells approaching the enteric cavity side, the intensity of Ag85A expressed in M cell was very high. Ag85A was also expressed in the DCs of small intestinal Peyer's patches, but the quantity of Ag85A+ DCs was obviously less than M cells.DiscussionThe intestine encounters more antigen than any other part of the body and so is the largest component of the immune system. The gut-associated lymphoid tissues (GALT) comprise organised tissues such as the Peyer's patches (PP) and mesenteric lymph nodes (MLN) that are generally considered to be inductive sites, while the effector cells are distributed throughout the mucosa itself. The reason for such a large and complex immune apparatus is to generate strong protective immunity against pathogenic infections. However, much of the antigen load in the intestine consists of harmless or even beneficial materials, such as food proteins in the small intestine and the commensal bacteria, which make up the bulk of the mass of the colon. Although normal individuals may generate low levels of intestinal and even serum antibody responses against these harmless antigens, active T cell responses do not occur under physiological circumstances. Indeed, such responses underlie intestinal disorders such as coeliac disease and Crohn's disease. For these reasons, the default response to harmless antigens in the gut is the induction of a state of immunological hyporesponsiveness, known as oral tolerance. In addition to its physiological importance, the propensity of the intestinal immune system to generate tolerance to non-invasive antigens presents a formidable challenge to the development of novel orally active vaccines comprising purified or recombinant antigens. M cells are considered to be the most effective cells for the transport of antigens from the intestinal lumen into the gut-associated lymphoid tissue. M cells in the FAE provide an entry site for pathogens, such as S. typhimurium, Mycobacterium bovis, Shigella flexneri, Y. enterocolitica and retroviruses. M cells have been identified and documented only in the FAE-associated epithelium and occasionally on villi immediately adjacent to the lymphoid follicle. A previous study indicated that isolated M cells were found in the villous epitheliumnear the PP of the rabbit small intestine. A recent study has provided new evidence that villous M cells are located quite a distance from PP. There is also report that DCs inside the small intestinal mucosa can uptake antigen.Our experiments identified that the villous M cells far away from PP could also take up the DNA vaccine encapsulated with liposome, but we didn't see the expression of DNA vaccine in the small intestines DCs inside the mucosa.DCs are professional antigen presenting cells, the main function of them is to uptake, process and present antigen. DCs are divided into two types: mature DC and immature DC. The immature DCs mainly distribute in non-lymphatic organ and the blood to execute "sentinel function". When the pathogens come in, the immature DCs can uptake and present them to phagocytes. The ability of uptaking, processing and presenting antigen of immature DC is stronger but the ability of arousing immune response is weaker than that of mature DC. In our experiment we didn't observe the expression of DNA vaccine in the DCs of small intestinal mucosa, but observed the expression of DNA vaccine in partial DCs of PP.In summary, our observations indicate that taking orally the DNA vaccine pCDNA 3.1+/ Ag85A encapsulated with liposome can express in the small intestinal mucosa epithelium, Peyer's patches and the spleen widely. The DNA vaccine encapsulated with liposome taken orally can induce a specific mucosa immune response, the mechanism may be: after getting into small intestine, it is uptaken by M cells firstly, then is transported to DCs, and present to T cells after processing to execute specific cellular immune response. The DCs can secrete IL-10, TGF-βand other cytokines which induce Ig, secreted by B cells, switch to sIgA to execute specific humoral immune response.ConclusionOral DNA vaccine parceled with liposomes can induce specific mucosal immune response. The vaccine may be firstly intaked by M cells, then transferred to dendritic cells, thereby presenting to T and B cells to induce specific mucosal immune response.