ministry of higher education and scientific research university of baghdad

[vaccines and vaccination and DNA vaccination]

Prepared by Meyasa mothna &Samara kadhim Supervised By PROF.DR.AMINA AL-THWANI

2012

[ genetic

engineering and biotechnology institute for post graduate studies

]

We need vaccine for Our heath

Why?

vaccines
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters. Vaccines can be prophylactic (example: to prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen), or therapeutic (e.g. vaccines against cancer are also being investigated; see cancer vaccine) The terms vaccination and vaccine derive from the work of Edward Jenner who, over 200 years ago, showed that inoculating people with material from skin lesions caused by cowpox (L. vaccinus, of cows) protected them from the highly contagious and frequently fatal disease smallpox

Kinds of vacines
1. Killed whole organisms In this relatively crude approach, the vaccine is made from the entire organism, killed to make it harmless. The typhoid and cholera vaccines are examples. 2. Attenuated organisms Here, the organism has been cultured so as to reduce its pathogenicity, but still retain some of the antigens of the virulent form. The Bacillus Calmette-Gurin (BCG) is a weakened version of the bacterium that causes tuberculosis in cows. BCG is used as a vaccine against tuberculosis in many European countries but is rarely used in the U.S. 3. Toxoids In some diseases, diphtheria and tetanus are notorious examples, it is not the growth of the bacterium that is dangerous, but the protein toxin that is liberated by it. Treating the toxin with, for example, formaldehyde, denatures the protein so that it is no longer dangerous, but retains some epitopes on the molecule that will elicit protective antibodies. 4. Surface molecules Antibodies are most likely to be protective if they bind to the surface of the invading pathogen triggering its destruction. Several vaccines employ purified surface molecules:

Influenza vaccine contains purified hemagglutinins from the viruses currently in circulation around the world. The gene encoding a protein expressed on the surface of the hepatitis B virus, called hepatitis B surface antigen or HBsAg, can now be expressed in E. coli cells and provides the material for an effective vaccine. Hepatitis B

infection is strongly associated with the development of liver cancer. Here then is a vaccine against a cancer. The genes encoding the capsid proteins of 4 strains of human papilloma virus (HPV) can be expressed in yeast and the resulting recombinant proteins are incorporated in a vaccine (Gardasil). Because infection with some of these strains of HPV can lead to cervical cancer [Link], here is another vaccine against cancer. Some 80 different strains of Streptococcus pneumoniae cause pneumonia in humans. They differ in the chemistry of the polysaccharide capsule that surrounds them (and makes it difficult for phagocytes to engulf them by endocytosis [View]). One current vaccine consists of tiny amounts of the purified capsular polysaccharides of the 23 most common and/or dangerous strains.

5. Inactivated virus Like killed bacterial vaccines, these vaccines contain whole virus particles that have been treated (again, often with formaldehyde) so that they cannot infect the host's cells but still retain some unaltered epitopes. The Salk vaccine for polio (IPV) is an example. 6. Attenuated virus In these vaccines, the virus can still infect but has been so weakened that it is no longer dangerous. The measles, mumps, and rubella ("German measles") vaccines are examples. The Sabin oral polio vaccine (OPV) is another example. It has advantages over the Salk vaccine in that eparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters. Vaccines can be prophylactic (example: to prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen), or therapeutic (e.g. vaccines against cancer are also being investigated; see cancer vaccine).

Vaccination
Vaccination is the administration of antigenic material (a vaccine) to stimulate the immune system of an individual to develop adaptive immunity to a disease. Vaccines can prevent or ameliorate the effects of infection by many pathogens. The efficacy of vaccination has been widely studied and verified; for example, the influenza vaccine, the HPV vaccine, and the chicken pox vaccine among others. In general, vaccination is considered to be the most effective method of preventing infectious diseases. The active agent of a vaccine may be intact but inactivated (non-infective) or attenuated (with reduced infectivity) forms of the causative pathogens, or purified components of the pathogen that have been found to be highly immunogenic (e.g., the outer coat proteins of a virus). Toxoids are produced for the immunization against toxin-based diseases, such as the modification of tetanospasmin toxin of tetanus to remove its toxic effect but retain its immunogenic effect.

DNA Vaccines

DNA vaccine is DNA sequence used as a vaccine.

This 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. A new up-and-coming vaccine as explained in The Journal of Immune Based Therapies and The Journal of Experimental Medicine is called DNA Vaccines or Genetic Vaccines. Genetic immunization with naked DNA has been shown to induce cellular immune response with high efficiency (Akbari, 1999). Although not yet approved by the FDA for human use, human clinical testing has started and Genetic immunization has been approved for animal use. It is prevalent among equine vaccinations. DNA vaccines were first observed at the University of Wisconsin almost 20 years ago. Malone and Felgner at Vical Incorporated, and Wolff and colleagues at the University of Wisconsin, demonstrated that mRNA and closed loops of plasmids injected into muscle tissue could be taken up by cells at the administration site resulting in the expression of proteins not normally made by the host cell. (Moss,2009). In simpler terms, small loops of DNA in the vaccine invade body cells and incorporate themselves into the cells nuceli. Once in, the cells read the instructions and produce the genes protein.

DNA Vaccines are made using a technique called polymerase chain reaction (PCR). The first step is to make copies of a specific gene. Primers are used to find the gene and copy the sequence of its DNA. Vectors are then used because they are able to enter cells and instruct the cell on how to create proteins based on the vectors DNA code. Vectors are capable of selfreplicating within a bacterial host as long as the host is in an environment conducive to growth. The final vaccine should include only the vectors; this process requires a separation of the vector and the bacteria they are in. A detergent is added which ruptures the cell walls of the bacteria and releases the DNA within. A purifier is used to separate the altered vectors. After production of the vaccine, experiments are conducted to test for safety of the subject and how well it works.

In 1993, Merck Research Laboratories, Dr. Margaret Liu found that intramuscular injection of DNA from influenzae virus into mice produced complete immune response Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:17451749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA. In 1996, trials involving T-cell lymphoma, influenzae & herpes simplex virus were started The first clinical trials using injections of DNA to stimulate an immune response against a foreign protein began for HIV in 1995. Four other clinical trials using DNA vaccines against influenza, herpes simplex virus, T-cell lymphoma, and an additional trial for HIV were started in 1996. The technique that is being tested in humans involves the direct injection of plasmids - (aditional DNA in bacteria) that contain genes for proteins to be produced by the organism being targeted for immunity. Once injected into the host's muscle tissue, the DNA is taken up by host cells, which then start expressing the foreign protein. The protein serves as an antigen that stimulates an immune responses and protective immunological memory.

Table 1 DNA vaccines Vs Traditional vaccines

DNA vaccines

Traditional vaccines

Uses only the DNA from infectious organisms. Avoid the risk of using actual infectious organism. Provide both Humoral & Cell mediated immunity Refrigeration is not required

Uses weakened or killed form of infectious organism. Create possible risk of the vaccine being fatal. Provide primarily Humoral immunity Usually requires Refrigeration.

METHODS OF DELIVERY
DNA vaccines have been introduced into animal tissues by a number of different methods. These delivery methods are briefly reviewed in Table 2, with the advantages and disadvantages of the most commonly used methods summarized In page 21. The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. A schematic outline of the construction of a DNA vaccine plasmid and its subsequent delivery by these two methods into a host is illustrated at Scientific American. Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation. by temporarily damaging muscle fibres with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose. Immune responses to this method of delivery can be affected by many factors, including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected. Gene gun delivery, the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold ortungsten microparticles into the target cells, using compressed helium as an accelerant. Alternative delivery methods have included aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, and topical administration of pDNA to the eye and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors The method of delivery determines the dose of DNA required to raise an effective immune response. Saline injections require variable amounts of DNA, from 10 g-1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than intramuscular saline injection to raise an effective immune response. Generally, 0.2 g 20 g are required, although quantities as low as 16 ng have been reported. These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates. Saline injections

require more DNA because the DNA is delivered to the extracellular spaces of the target tissue (normally muscle), where it has to overcome physical barriers (such as the basal lamina and large amounts ofconnective tissue, to mention a few) before it is taken up by the cells, while gene gun deliveries bombard DNA directly into the cells, resulting in less wastage. Another approach to DNA vaccination is expression library immunization (ELI). Using this technique, potentially all the genes from a pathogen can be delivered at one time, which may be useful for pathogens which are difficult to attenuate or culture. ELI can be used to identify which of the pathogens genes induce a protective response. This has been tested with Mycoplasmapulmonis, a murine lung pathogen with a relatively small genome, and it was found that even partial expression libraries can induce protection from subsequent challenge. Table 2

Advantages and disadvantages of commonly used DNA vaccine delivery methods Method of Delivery

Advantage

Disadvantage Inefficient site for uptake due to morphology of muscle tissue Relatively large amounts of DNA used

Intramuscular or Intradermal injection

No special delivery mechanism Permanent or semipermanent expression

pDNA spreads rapidly throughout the body

Th1 response may not be the response required

Gene Gun

DNA bombarded directly into cells

Th2 response may not be the response required

Small amounts DNA

Requires inert particles as carrier

No particles required

Jet injection

DNA can be delivered to cells mm to cm below skin surface

Significant shearing of DNA after high-pressure expulsion 10-fold lower expression, and lower immune response

Requires large amounts of DNA (up to 300 g)

Liposomemediated delivery

High levels of immune response can be generated Can increase transfection of intravenously delivered pDNA Intravenously delivered liposome-DNA complexes can potentially transfect all tissues

Toxicity Ineffectiveness in serum

Intranasally delivered liposome-DNA complexes can result in expression in distal mucosa as well as nasal muscosa and the generation of IgA antibodies

Risk of disease or immune reactions

HOW DNA VACCINE WORKS BY 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

Mechanisms: A plasmid vector that expresses the protein of interest (e.g. viral protein) under the control of an appropriate promoter is injected into the skin or muscle of the the host. After uptake of the plasmid, the protein is produced endogenously and intracellularly processed into small antigenic peptides by the host proteases. The peptides then enter the lumen of the endoplasmic reticulum (E.R.) by membrane-associated transporters. In the E.R., peptides bind to MHC class I molecules. These peptides are presented on the cell surface in the context of the MHC class I. Subsequent CD8+ cytotoxic T cells (CTL) are stimulated and they evoke cell-mediated immunity. CTLs inhibit viruses through both cytolysis of infected cells and noncytolysis mechanisms such as cytokine production (Encke et al, 1999). The foreign protein can also be presented by the MHC class II pathway by APCs which elicit helper T cells (CD4+) responses. These CD4+ cells are able to recognize the peptides formed from exogenous proteins that were endocytosed or phagocytosed by APC, then degraded to peptide fragments and loaded onto MHC class II molecules. Depending on the the type of CD4+ cell that binds to the complex, B cells are stimulated and antibody

production is stimulated. This is the same manner in which traditional vaccines work (Schirmbeck et al., 2001).

ADVANTAGES

Elicit both Humoral & cell mediated immunity Focused on Antigen of interest Long term immunity Refrigeration is not required Stable for storage

DISADVANTAGES Limited to protein immunogen only Extended immuno stimulation leads to chronic inflammation Some antigen require processing which sometime does not occur

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

References
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Alarcon JB, Waine GW, McManus DP (1999). "DNA vaccines: technology and application as anti-parasite and anti-microbial agents". Adv. Parasitol. 42: 343 410. DOI:10.1016/S0065-308X(08)60152-9. PMID 10050276 Robinson HL, Pertmer TM (2000). "DNA vaccines for viral infections: basic studies and applications". Adv. Virus Res. 55: 174. DOI:10.1016/S00653527(00)55001-5. PMID 11050940. Sedegah, M.; Hedstrom, R.; Hobart, P.; Hoffman, S.L. (1994). "Protection against Malaria by Immunization with Plasmid DNA Encoding Circumsporozoite Protein". Proceedings of the National Academy of Sciences of the United States of America 91 (21): 9866 9870.DOI:10.1073/pnas.91.21.9866. JSTOR 2365723. PMC 44918. PMID 793790 7. Retrieved 2007-11-21. Lewis, P.J.; Babiuk, L.A. (1999). "DNA Vaccines: A Review". Advances in Virus Research (Academic Press) 54: 12988. DOI:10.1016/S00653527(08)60367-X.ISBN 978-0-12-039854-6. PMID 10547676. Retrieved 2007-1121. Daheshia, M.; Kuklin, N; Kanangat, S; Manickan, E; Rouse, BT (August 15, 1997)."Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10". The Journal of Immunology 159 (4): 19451952. PMID 9257860

Chen, Y.; Webster, R.G.; Woodland, D.L. (March 1, 1998). "Induction of CD8+ T Cell Responses to Dominant and Subdominant Epitopes and Protective Immunity to Sendai Virus Infection by DNA Vaccination 1". The Journal of Immunology 160 (5): 24252432. Sizemore DR, Branstrom AA, Sadoff JC (October 1995). "Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization". Science 270 (5234): 299302.DOI:10.1126/science.270.5234.299. PMID 7569980 Barry, M.A.; Lai, W.C.; Johnston, S.A.; Others, (1995). "Protection against mycoplasma infection using expression-library immunization". Nature 377 (6550): 632635 Fynan, E.F.; Webster, R.G.; Fuller, D.H.; Haynes, J.R.; Santoro, J.C.; Robinson, H.L. (1993)."DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations". Proc Natl Acad Sci USA 90 (24): 11478 82. DOI:10.1073/pnas.90.24.11478.PMC 48007. PMID 8265577. Retrieved 200711-21. Schirmbeck, R. and Jorg Reimann. April 2001. Revealing the Potential of DNAbased Vaccination: Lessons Learned from the Hepatitis B Virus Surface Antigen. Biol. Chem., 382:543-552. Other references
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