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DNA vaccination advanced microbiology course Prof: Rula darwish Why did we choose it? Because it’s represent the most recent approach to produce vaccine against a variety of diseases , and it shows a brilliant result in veterinary field, and many studies focused on the possibility to improve DNA vaccine for diseases that infect human and have no way to prevent until now Many people even in medical field are still doesn’t know this technique introduction One of the most astonishing and important applications in the field of immunology in the last century was the invention and development of the vaccines. It was a significant leap forward in the prevention of infectious diseases that saved the lives of millions of people. Definition DNA vaccination is a third generation novel technique which includes introducing genetically engineered DNA to produce immune reaction so as to immunize the population against certain disease[10]. DNA vaccines contain the nucleotides encoding an antigenic portion of the virus such as the viral core region or envelope region. The DNA is taken up into the host cell, translated, and the protein product expressed History Experiments outlining the transfer of DNA into cells of living animals were reported as early as 1950 In 1983, Paoletti and Panicali developed multi- vaccine by inserting gene from namely Herpes simplex virus, hepatitis B and influenza into smallpox vaccine. In 1992, scientists Tang and Johnson, demonstrated that the delivery of human growth hormone into mice , resulted in developing antibodies against the human growth hormone. In 2016, there are many trial to develop a vaccine for Zika virus Vector design DNA vaccines elicit the best immune response when highly active expression vectors are used Expression plasmids contain two unites: 1. the antigen expression unit composed of promoter sequences AND antigenencoding and polyadenylation sequences (termination signal) 2. production unit composed of bacterial sequences necessary for plasmid amplification and selection a strong viral promoter to drive the in vivo transcription and translation of the gene of interest such as SV40 promoter, Rous Sarcoma Virus (RSV) promoter The most recent one is cytomegalovirus (CMV). Intron A may sometimes be included to improve mRNA stability and hence increase protein expression transcription-stop sequence such as bovine growth hormone or rabbit betaglobulin [1],[4] To construct a vector, they should treat vectors and copied genes with “restriction enzymes” which are agents that cut DNA sequences at known locations After construction, plasmids transferred to bacteria and are allowed to multiply Then when the bacterial colony grow sufficiently, the plasmids purified and used as vaccine Mechanism of action A plasmid vector that expresses the viral protein is injected into the skin or muscle of the host. After plasmid is uptaken. Translation and transcription is started then it processed into small antigenic peptides by proteases. They enter the lumen of the endoplasmic reticulum (E.R.) . In the E.R., peptides bind to MHC class I molecules and they presented on the cell surface . Subsequent CD8+ cytotoxic T lymphocytes (CTL) are stimulated and they evoke cell-mediated immunity. CTLs inhibit viruses through both cytolysis of infected cells and non-cytolysis mechanisms such as cytokine production. Also they can be presented by the MHC class II pathway by Antigen presenting cells which will elicit helper T cells (CD4+) responses. These CD4+ cells are able to recognize the peptides formed from exogenous proteins that were endocytosed by APC. Then they fragmented and uptaked by MHC class II molecules, B cells are stimulates the antibodies start to form . Delivery methods the type of immune responses induced by plasmid immunisation is significantly affected by (i) the mode and site of gene delivery, (ii) the dose of plasmid and (iii) the administration of booster injections and the interval between immunisations Saline injection most commonly used (IM) in skeletal muscle, or intradermally (ID), in extracellular spaces. assisted by electroporation; by temporarily damaging muscle fibres with myotoxins or by using hypertonic solutions of saline or sucrose. can be affected by factors including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the host Advantages I. No special delivery mechanism II. Permanent or semi-permanent expression III. pDNA spreads rapidly throughout the body Disadvantages I. Inefficient site for uptake due to morphology of muscle tissue II. Relatively large amounts of DNA used III. Th1 response may not be the response required Gene gun delivery plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant ED (abdominal skin); vaginal mucosa; surgically exposed muscle Advantages I. DNA bombarded directly into cells II. Small amounts DNA Disadvantages I. Th2 response may not be the response required II. Requires inert particles as carrier Liposome-mediated delivery Advantages I. High levels of immune response can be generated II. Can increase transfection of intravenously delivered pDNA III. Intravenously delivered liposome-DNA complexes can potentially transfect all tissues IV. Intranasally delivered liposome-DNA complexes can result in expression in distal mucosa as well as nasal muscosa and the generation of IgA antibodies Disadvantages : I. Toxicity II. Ineffectiveness in serum III. Risk of disease or immune reactions Advantages of DNA vaccines The ability to induce both cellular and humoral immune responses. highly specific and the expressed immunizing antigen is subjected to the same glycosylation and post-translational modifications as natural viral infection therapeutic potential for ongoing chronic viral infections. Obviates need for peptide synthesis, expression and purification of recombinant proteins and use of toxic adjuvants Long-term persistence of immunogen Rapid and large-scale production are available at costs considerably lower than traditional vaccines, and they are also very temperature stable making storage and transport much easier. Disadvantages May activation of oncogenes occure eliciting anti-DNA antibodies Possibility of tolerance to the antigen (protein) produced Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides) Clinical use Until now, no DNA vaccine is approved to use at human scale Most of its use is in veterinary scale (to protect horses from West Nile virus) June 2015 one human DNA vaccine had been approved for human use, the single-dose Japanese encephalitis vaccine It show a promising future Applications and clinical studies DNA vaccination and Zika virus What is Zika virus ? Why it’s important for us to develop a vaccine against it ? What are the latest results about the development of a vaccine? HPV VACCINE What is HPV? Is vaccine useful in treatment or prevention of HPV &it’s complication ? In Japan, Prevention of cervical cancer has been unsuccessful, because of low rates of cancer screening and vaccination this study indicates that HPV vaccination is associated with a reduction in the incidence of cervical abnormalities. Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases 1 . DNA toxin vaccine for cancer therapy To produce this kind of vaccine the relevant tumor antigens and their related MHC Class I and MHC Class II epitopes must be determined. 2 . DNA toxin vaccine for autoimmune diseases multiple sclerosis (MS). autoimmune encephalomyelitis (EAE) 3. Concluding remarks and future perspectives In cancer therapy, bacterial DNA toxin vaccines proved to have less sideeffects and more effective compared to conventional cancer therapy Done by: Mokhlesa Rimawi , Mays Adnan , Marwa Abdulla , Esraa Freijat , Amal AlJammal .