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DNA VACCINES



History of Vaccination:

Since the time of the Ancient Greeks, it has been recognized that people who have recovered from plague, smallpox , yellow fever , and various other infectious diseases rarely contact the diseases again.

The first scientific attempts at artificial immunizations were made in the last 18th century by Edward Jenner {1749-1823} . Jenner investigated the basis for the widespread belief of the pesants that anyone who had vacccina {cowpox} never contracted smallpox .smallpox was often fatal 10 to 40% of the vicims died and those who recovered had disfiguring pockmarks . Yet most English milkmaids ,who were readily infected with cowpox , had clear skin because cowpox was a relatively mild infection that left no scars. It was on May 14 , 1796 , that Jenner extracted the contents of a pustule from the arm of a cowpox – infected milkmaid . Sarah Nelmes , and infected it to the arm of Eight year old James Phipps . Further work on immunization was carried out by Louis Pastuer {1822-1895} . Pastuer discovered that if cultures of chicken cholera bacteria were  allowed to age for two to three months the bacteria  produced only a mild attack of cholera when inoculated into chickens . Somehow the old cultures had become less pathogenic {attenuated} for the chickens . He then found that fresh cultures of the bacteria failed to produce cholera in chickens that had been previously inoculated with old , attenunated cultures . To honor Jenner ‘s work with cowpox, Pastuer gave the name vaccine to any preparation of a weakend pathogen that was used { as was Jenner’s “vaccine virus”} to immunize against infectious disease.

INTRODUCTION:

Vaccines And Immunization:

A vaccine is a preparation of one or more microbial antigens used to induce protective immunity . it may consist of killed microorganisms , living , weakened microorganisms {attenuated vaccine} inactivated bacterial toxins {toxoids} ,purified cellular subunits , recombinant vectors {e.g. modified polio vaccines } or

DNA . Immunization is the result achieved by the successful delivery of vaccines ,it stimulates immunity . vaccination attempts to induce antibodies and activated T cells to protect a host from future infection . Many epidemics have been stayed mass prophylactic immunization . Vaccines have eradicated smallpox , pushed polio to the brink of extinction , and spared countless individuals from hepatitis A and B , influenza , measles , rotavirus , disease ,tetanus , typhoid , and dangerous other diseases. Vaccinomics , the application of genomics and bioinformatics to vaccine development , is bringing a fresh approach to the Herculean problem of making vaccines against various microorganisms and parasites .To promote a more efficient immune response , antigens in vaccines can be mix with Adjuvant which enhances the rate and degree of Immunization . Adjuvants can be any nontoxic material that prolongs antigen interaction with immune cells , assists in the Antigen presenting cells{APC} processing of antigens , or otherwise non-specifically stimulates the immune response to the antigens . several types of adjuvants can be used .Oil in water emulsions {Freund’s incomplete adjuvant} aluminium hydroxide salts{alum}, beeswax, and various combinations of bacteria {live or killed} are used in vaccine adjuvants . In most cases , the adjutant materials trap the antigen , thereby promoting a sustained release as APCs digest and degrade the preparation . In other cases, the adjuvant activates APCs so that antigen recogination , processing and presentation are more efficient .

The modern era of vaccines and immunization began in 1798 with Jenner’s use of smallpox as a vaccine against smallpox and in 1881 with Louis Pastuer ‘s anthrax vaccine . vaccination is still one of the most Cost –effective weapons for prevention of microbial disease. Vaccination of most children should begin at about 2 months . Before that age, they are protected by passive natural immunity from maternal antibodies . further vaccination of teens and adults depends on their relative risk for infectious disease .the role of vaccination as a protective therapy, it saves lives .

DNA VACCINE:

DNA vaccination is for protecting an organism against disease by injecting it with Genetically Engineered DNA to produce an immunological response . Nucleic acid vaccines are still experimental and have been applied to number of viral , bacterial and parasitic models of disease as well as to several tumor models . DNA vaccines have a number of advantages over conventional vaccines , including the ability to induce wider range of immune responses type . Vaccines are among the greatest achievements of modern medicine- in industrial nations , they have eliminated naturally-occuring cases of smallpox , nearly eliminated polio , while other diseases , such as , typhus , rotavirus , hepatitis A and B and others are well controlled. Conventional vaccines , however cover a small number of diseases and infections that lack effective vaccines kill millon of people every year , with AIDS , Hepatitis C and malaria being particularly common .

First generation vaccines are whole – organism vaccines , are able to induce Killer Tcell {Tc or CTL
} responses and antibody immunity . However there is a small risk that attenuated forms of a pathogens can revert to dangerous form and may still able to cause disease in immunocompromised people {such as those with AIDS} . While killed vaccines donot have this risk , they cannot generate specific Killer T cell responses and may not work at all for some diseases. In order to minimize these risk so called Second generation vaccines were developed . These are subunit vaccines consists of Protien antigens{such as tetanus or diphtheria toxoid} or recombinant protein components{such as hepatitis B surface antigen} . these two are able to generate TH and antibody responses, but not killer cell responses .

DNA are Third generation vaccines and hence made up of a small , circular piece of bacterial DNA called Plasmid that has been genetically engineered to produce one or two specific proteins Antigens from microorganism . the vaccine DNA is injected into the cells of body , where the “inner machinery” of the host cell “reads” the DNA and converts it into pathogenic proteins . Because these proteins are recognised as foreign . they are processed by host cells and displayed on their surface to alert the immune system , which then triggers a range of immune responses . These DNA vaccines developed from “failed” gene therapy experiments . the first demonstration of plasmid-induced immune response was when mice inoculated with a plasmid expressing Human Growth Hormone elicited antibodies instead of altering growth.

The use of DNA vaccines for neonatal/ early life :

DNA vaccines could represent a major advancement in the development of novel antigen-delivery systems to be used in early life, although the demonstration of their efficacy and safety in human adults must be awaited before any prediction on the future of such vaccines in early life can be made. Their capacity to efficiently induce adult like neonatal T helper (Th)1-type and cytolytic T lymphocyte (CTL) responses to encoded antigens in several animal species could be particularly beneficial in view of the preferential Th2-polarization and weak CTL activity frequently observed at this young age. In contrast, their frequent failure to induce early life vaccine antibody responses above those elicited by live attenuated/adjuvanted protein vaccines, remains a significant limitation. This feature calls for much more complex vaccine strategies (i.e., primeboost approaches), that may not prove feasible in countries that most need novel DNA vaccines. Similarly, the expectation that the in vivo antigen production would allow DNA vaccines to readily escape from the inhibitory influence of maternal antibodies has not been confirmed, despite the reported success in a few experimental settings. Notably, considerable heterogeneity in the quantity and quality of early life T and B cell responses to DNA immunization has been observed. These are reviewed here, underlining that a better understanding of the respective roles of the routes and methods of administration, the doses and types of antigen(s) and constructs, the co administration of antigens or immunomodulators, the ages of recipients and the titers of pre-existing maternal antibodies is clearly needed. DNA immunization models in various animal species should thus be used to progressively identify the essential parameters and characteristics of the novel DNA vaccine candidates which may be considered for use in early life.

CURRENT USE OF DNA VACCINES :

Thus far, few experimental trials have evoked a response sufficiently strong enough to protect against disease, and the usefulness of the technique, while tantalizing, remains to be conclusively proven in human trials. However, in June 2006 positive results were announced for a bird flu DNA vaccine and a veterinary DNA vaccine to protect horses from West Nile virus has been approved. In August 2007, a preliminary study in DNA vaccination against multiple sclerosis was reported as being effective.

ADVANTAGES AND

DISADVANTAGES

Advantages And Disadvantages Of Nucleic Acid-Based Immunization

Advantages

Disadvantages

  • Subunit vaccination with no risk for infection
  • Antigen presentation by both MHC class I and class II molecules
  • Able to polarise T-cell help toward type 1 or type 2
  • Immune response focused only on antigen of interest
  • Ease of development and production
  • Stability of vaccine for storage and shipping
  • Cost-effectiveness
  • Obviates need for peptide synthesis, expression and purification of recombinant proteins and the use of toxic adjuvants
  • Long-term persistence of immunogen
  • In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications
  • Limited to protein immunogens
  • Potential for atypical processing of bacterial and par
  • asite proteins

PLASMID VECTORS FOR USE IN VACCINATION:

Vector Design:

DNA vaccines elicit the best immune response when highly active expression vectors are used. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest. Intron A may sometimes be included to improve mRNA stability and hence increase protein expression. Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.Because the plasmid is the “vehicle” from which the immunogen is expressed, optimising vector design for maximal protein expression is essential. One way of enhancing protein expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT contents than the species being immunized, so altering the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.Another consideration is the choice of promoter. The SV40 promoter was conventionally used until research showed that vectors driven by the Rous Sarcoma Virus (RSV) promoter had much higher expression rates. More recently, expression rates have been further increased by the use of the cytomegalovirus (CMV) immediate early promoter. Inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev increased envelope expression. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. Additional modifications to improve expression rates have included the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences.

Vaccine Insert design:

Immunogens can be targeted to various cellular compartments in order to improve antibody or cytotoxic T-cell responses. Secreted or plasma membrane-bound antigens are more effective at inducing antibody responses than cytosolic antigens, while cytotoxic T-cell responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the major histocompatibility complex (MHC) class I pathway. This is usually accomplished by the addition of N-terminal ubiquitin signals.

Delivery methods :

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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 or tungsten microparticles into the target cells, using compressed helium as an accelerant.

Immune response raised by DNA vaccines : Helper T-Cell responses :

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Antigen presentation stimulates T cells to become either “cytotoxic” CD8+ cells or “helper” CD4+ cells. Cytotoxic cells directly attack other cells carrying certain foreign or abnormal molecules on their surfaces. Helper T cells, or Th cells, coordinate immune responses by communicating with other cells. In most cases, T cells only recognize an antigen if it is carried on the surface of a cell by one of the body’s own MHC, or major histocompatibility complex, molecules .DNA immunization is able to raise a range of TH responses, including lymphoproliferation and the generation of a variety of cytokine profiles. A major advantage of DNA vaccines is the ease with which they can be manipulated to bias the type of T-cell help towards a TH1 or TH2 response. Each type of response has distinctive patterns of lymphokine and chemokine expression, specific types of immunoglobulins expressed, patterns of lymphocyte trafficking, and types of innate immune responses generated.

Mechanistic basis for DNA raised immune responses:

DNA Uptake Mechanism

When DNA uptake and subsequent expression was first demonstrated in vivo in muscle cells, it was thought that these cells were unique in this ability because of their extensive network of T-tubules. Using electron microscopy, it was proposed that DNA uptake was facilitated by caveolae (or, non-clathrin coated pits). However, subsequent research revealed that other cells (such as keratinocytes, fibroblasts and epithelial Langerhans cells) could also internalise DNA. This phenomenon has not been the subject of much research, so the actual mechanism of DNA uptake is not known.Two theories are currently popular – that in vivo uptake of DNA occurs non-specifically, in a method similar to phago- or pinocytosis, or through specific receptors. These might include a 30kDa surface receptor, or macrophage scavenger receptors. The 30kDa surface receptor binds very specifically to 4500-bp genomic DNA fragments (which are then internalised) and is found on professional APCs and T-cells. Macrophage scavenger receptors bind to a variety of macromolecules, including polyribonucleotides, and are thus also candidates for DNA uptake.Receptor mediated DNA uptake could be facilitated by the presence of polyguanylate sequences.

Antigen presentation by bone marrow-derived cells :

A dendritic cell: Studies using chimeric mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialised B-cells called professional antigen presenting cells (APC) Iwasaki et al., 1997). After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node to present antigen. After IM and ID injections, dendritic cells have also been found to present antigen in the draining lymph node and transfected macrophages have been found in the peripheral blood.

Role of the target site :

IM and ID delivery of DNA initiate immune responses differently. In the skin, keratinocytes, fibroblasts and Langerhans cells take up and express antigen, and are responsible for inducing a primary antibody response. Transfected Langerhans cells migrate out of the skin (within 12 hours) to the draining lymph node where they prime secondary B- and T-cell responses. In skeletal muscle, on the other hand, striated muscle cells are most frequently transfected, but seem to be unimportant in mounting an immune response. Instead, IM inoculated DNA “washes” into the draining lymph node within minutes, where distal dendritic cells are transfected and then initiate an immune response. Transfected myocytes seem to act as a “reservoir” of antigen for trafficking professional APCs .

Inteferons:

Both helper and cytotoxic T-cells can control viral infections by secreting interferons. Cytotoxic T cells usually kill virally infected cells. However, they can also be stimulated to secrete antiviral cytokines such as INF-γ and TNF-α, which don’t kill the cell but place severe limitations on viral infection by down-regulating the expression of viral components. DNA vaccinations can thus be used to curb viral infections by non-destructive IFN-mediated control. This has been demonstrated for the hepatitis B virus.IFN-γ is also critically important in controlling malaria infections, and should be taken into consideration when developing anti-malarial DNA vaccines.

Alphavirus vector:

Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency. The gene encoding the antigen of interest is inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact. The Sindbis virus and Semliki Forest virus have been used to build recombinant alphavirus replicons. Unlike conventional DNA vaccinations, however, alphavirus vectors kill transfected cells, and are only transiently expressed. Also, alphavirus replicase genes are expressed in addition to the vaccine insert.

REVIEW OF LITERATURE:

A vaccine is a preparation of one or more microbial antigens used to induce protective immunity . it may consist of killed microorganisms , living , weakened ,microorganisms {attenuated vaccine} , inactivated bacterial toxins {toxoids}, purified cellular subunits, recombinant vectors { e.g. modified polio vaccine } or DNA. Immunization is the result achived by successful delivery of vaccines , it stimulates immunity . vaccination attempts to induce antibiotics and activated T cells to protect a host from infection . vaccines have eradicated smallpox, pushed polio to brink of extinction , and spared countless individuals form influenza, hepatitis A and B , measles , rotavirus disease , tetanus , typhus , and other dangerous diseases .

Many of the current vaccines re used for humans that are effective against viral and bacterial diseases consist of whole microorganisms that are either inactivated { killed } or attenuated {live but avirulent } .These are known as WHOLE CELL VACCINE .

Few experimental trials have evoked a response sufficiently strong enough to protect against disease, and the usefulness of the technique, while tantalizing, remains to be conclusively proven in human trials. However, in June 2006 positive results were announced for a bird flu DNA vaccine and a veterinary DNA vaccine to protect horses from West Nile virus has been approved. In August 2007, a preliminary study in DNA vaccination against multiple sclerosis was reported as being effective.

Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency. The gene encoding the antigen of interest is inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact.

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