Syeda Hira 08-arid-1152 Ph.D (Biochemistry) DNA VACCINES Syeda Hira 08-arid-1152 Ph.D (Biochemistry)

Contents Introduction History Difference Production of DNA vaccines Method of Delivery Mode of Action Advantages and Disadvantages Future Perspective Conclusion

Introduction Small circular piece of bacterial DNA. Genetically Engineered to produce one or two specific protein from microorganism. The DNA Sequence code for antigenic protein of pathogen. As this DNA inserted into cells it is translated to form antigenic protein. As this protein is foreign to cells , so immune response raised against this protein. In this way ,DNA vaccine provide immunity against that pathogen.

History In 1990, University of Wisconsin, Jon Wolff found that injection of DNA plasmids produce a protein response in mice Since then concept of DNA vaccines started

Difference between DNA vaccine and Traditional vaccine Traditional medicine Uses weak or killed form of an infectious agent Provide humoral immunity DNA vaccines Uses only the DNA from the infectious organism Provide both humoral and cell mediated immunity

Preparation of DNA Vaccine Viral gene Recombinant DNA Technology Expression plasmid Plasmid with foreign gene

Transform into bacterial cell Plasmid DNA Bacterial cell

Plasmid DNA Purified vaccine

DNA Vaccines: Optimization strategies Plasmid optimization Gene optimization Formulation adjuvant Immune plasmid adjuvant

Plasmid Optimization Plasmid vectors for use in vaccination contain Promoter Enhancer Antigen-encoding Polyadenylation sequences Antibiotic resistance site

Gene Optimization Selection Requirements High GC content Species-specific codon utilization Consensus immunogens Nuclear localization sequences

Formulation Adjuvants Microsphere/nanoparticle Liposomes Polymers

Immune Plasmid Adjuvants Cytokine Chemokine Co stimulatory molecules Heat shock proteins

Methods of Delivery Intramuscular There are two methods immunizations both directly transfected dendritic cells and macrophages can present antigen i.m. immunizations are largely independent of DNA expression in the muscle target intramuscular deliveries of DNA tend to raise type 1 T-cell help for intracellular and plasma membrane antigens but type 2 T-cell help for secreted antigens.

Continue… Gene Gun Method Covering gold micro particles with recombinant DNA Shoot them by gas pressure normally helium gene gun immunizations directly transfected dendritic cells present antigens Gene gun immunizations depend on antigen expression at the skin target  Gene gun immunizations tend to raise type 2 T-cell help for both cell-associated and secreted antigens. 

The “gene gun” The Helios Gene Gun is a new way for in vivo transformation of cells or organisms (i.e. gene therapy and genetic immunization (DNA vaccination)). This gun uses Biolistic ® particle bombardment where DNA- or RNA-coated gold particles are loaded into the gun and you pull the trigger. A low pressure helium pulse delivers the coated gold particles into virtually any target cell or tissue. The particles carry the DNA so that you do not have to remove cells from tissue in order to transform the cells.

Mechanism of Action The DNA vaccine works in two pathways. ENDOGENOUS Antigenic Protein is presented by cell in which it is produced. EXOGENOUS Antigenic Protein is formed in one cell but presented by different cell.

ENDOGENOUS PATHWAY Nucleus Plasmid DNA MHC-I mRNA Antigenic Peptides Antigenic Protein

T- Helper Cell Multiply Memory T cells

EXOGENOUS PATHWAY Antigenic Protein come outside

Phagocytosed Antigen Presenting Cell Antigenic Peptides Memory Antibodies T- Helper Cell Cytokines Plasma B-Cell MHC-II Activated B-Cell Memory B-Cell

WHEN VIRUS ENTER IN THE BODY Memory T-Cell Viral Protein Antibodies

Advantages Elicit both humoral and cell mediated immunity Plasmid vectors can be constructed and produced quickly  and the coding sequence can be manipulated in many ways. Focused on antigen of interest Stable for storage Not require refrigeration Another important advantage of genetic vaccines is their therapeutic potential for ongoing chronic viral infections

Disadvantages Prolong immunostimulation may lead to chronic inflammation Limited to protein Antigen only.DNA can be used to raise immune responses against pathogenic proteins, certain microbes have outer capsids that are made up of polysaccharides.  This limits the extent of the usage of DNA vaccines because they cannot substitute for polysaccharide-based subunit vaccines

Safety Issues Genetic toxicity Integration of DNA vaccine into host Genome may result in Chromosome instability, turn ON Oncogenes ,Turn OFF tumor Suppressor Genes Over expression of DNA vaccine may cause acute or chronic inflammation and normal tissues destruction Generation of autoimmune diseases Resistance to antibiotics

Future Prospects Plasmid with multiple genes provide immunity against many diseases in one booster DNA Vaccines against infectious diseases such as AIDS, rabies, Malaria can be available

Conclusion Simple ,safe and cheap alternative method for generation of humoral and cell mediated immunity DNA Vaccines are in their early phase DNA vaccines are going to be Vaccines of next generation

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