| Help build the future of Wikipedia and its sister projects! Read a letter from Jimmy Wales and Michael Snow. | [Hide] [Help us with translations! ] | DNA vaccine What is antisense technology? Antisense refers to opposing the normal order (“sense”) of the code in DNA. The DNA (deoxyribonucleic acid) in genes directs cells to assemble the proteins which comprise living creatures. The order of bases in DNA corresponds to the ordering of amino acids to form the proteins. To produce protein, the DNA of the genes in cells is first transcribed into a very similar molecule called RNA.
RNA can move out of the cell’s nucleus, where the genes have to stay. In the surrounding cytoplasm, proteins are put together according to the RNA’s sequence of bases, matching the DNA instructions. Antisense molecules prevent the protein assembly machinery from seeing the genetic instructions on how to order the amino acids. If scientists make a molecule that complements the sequence of bases in the RNA, it will stick to the RNA. The antisense molecule, bound to RNA, will prevent the RNA from making protein.
Just as two complementary pieces of Velcro stick together, hiding their loops, the antisense molecules bind to RNA and hide its instructions. Thus, antisense stops the synthesis of the protein coded for by the targeted RNA. In effect antisense has turned off the specific gene, or DNA, that was coding for that protein The three-dimensional structure of the original ribozyme, the self-splicing intron of Tetrahymena (13). Green and blue ribbons indicate the path of the RNA backbone in the two major domains of the RNA, and the red star marks the active site. The making of a DNA vaccine. Nucleic acid vaccines are still experimental, and have been applied to a number of viral, bacterial and parasitic models of disease, as well as to several tumour models. DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types. Vaccines are among the greatest achievements of modern medicine – in industrial nations, they have eliminated naturally-occurring cases of smallpox, and nearly eliminated polio, while other diseases, such as typhus, rotavirus, hepatitis A and B and others are well controlled. 1] Conventional vaccines, however, only cover a small number of diseases, and infections that lack effective vaccines kill millions of people every year, with AIDS, hepatitis C and malaria being particularly common. First generation vaccines are whole-organism vaccines – either live and weakened, or killed forms. [2] Live, attenuated vaccines, such as smallpox and polio vaccines, are able to induce killer T-cell (TC or CTL) responses, helper T-cell (TH) responses and antibody immunity.
However, there is a small risk that attenuated forms of a pathogen can revert to a dangerous form, and may still be able to cause disease in immunocompromised people (such as those with AIDS). While killed vaccines do not have this risk, they cannot generate specific killer T cell responses, and may not work at all for some diseases. [2] In order to minimise these risks, so-called second generation vaccines were developed. These are subunit vaccines, consisting of defined protein antigens (such as tetanus or diphtheria toxoid) or recombinant protein components (such as the hepatitis B surface antigen).
These, too, are able to generate TH and antibody responses, but not killer T cell responses. DNA vaccines are third generation vaccines, and are made up of a small, circular piece of bacterial DNA (called a plasmid) that has been genetically engineered to produce one or two specific proteins (antigens) from a micro-organism. The vaccine DNA is injected into the cells of the body, where the “inner machinery” of the host cells “reads” the DNA and converts it into pathogenic proteins.
Because these proteins are recognised as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers a range of immune responses. [1][2] These DNA vaccines developed from “failed” gene therapy experiments. The first demonstration of a plasmid-induced immune response was when mice inoculated with a plasmid expressing human growth hormone elicited antibodies instead of altering growth. [3] Contents[hide] * 1 Current use * 2 Advantages and disadvantages of DNA vaccines * 3 Plasmid vectors for use in vaccination * 3. 1 Vector design * 3. Vaccine insert design * 4 Delivery methods * 5 Immune response raised by DNA vaccines * 5. 1 Helper T-Cell responses * 5. 1. 1 Raising of different types of T-cell help * 5. 1. 2 Mechanistic basis for different types of T-Cell help * 5. 1. 3 Practical uses of polarised T-Cell help * 5. 2 Cytotoxic T-cell responses * 5. 3 Humoral (antibody) response * 5. 3. 1 Kinetics of antibody response * 6 Mechanistic basis for DNA raised immune responses * 6. 1 DNA Uptake Mechanism * 6. 2 Antigen presentation by bone marrow-derived cells * 6. 3 Role of the target site * 6. Maintenance of immune response * 6. 5 Interferons * 7 Modulation of the immune response * 7. 1 Cytokine modulation * 7. 2 Immunostimulatory CpG motifs * 7. 3 Alternative boosts * 7. 4 Additional methods of enhancing DNA-Raised immune responses * 7. 4. 1 Formulations of DNA * 7. 4. 2 Alphavirus vectors * 8 See also * 9 References * 10 External links| [edit] Current use 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 [4] and a veterinary DNA vaccine to protect horses from West Nile virus has been approved. [5][6] In August 2007, a preliminary study in DNA vaccination against multiple sclerosis was reported as being effective. [7] [edit] Advantages and disadvantages of DNA vaccines Table 1. Advantages And Disadvantages Of Nucleic Acid-Based Immunization| Advantages| Disadvantages| Subunit vaccination with no risk for infection[1] * Antigen presentation by both MHC class I and class II molecules[1] * Able to polarise T-cell help toward type 1 or type 2[1] * Immune response focused only on antigen of interest * Ease of development and production[1] * 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[8] * Long-term persistence of immunogen[2] * In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications[2]| * Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides) * Risk of affecting genes controlling cell growth * Possibility of inducing antibody production against DNA * Possibility of tolerance to the antigen (protein) produced * Potential for atypical processing of bacterial and parasite proteins[1]| [edit] Plasmid vectors for use in vaccination [edit] 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. [9] Intron A may sometimes be included to improve mRNA stability and hence increase protein expression. [10] Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences. [1][2][11] Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein. [12] Because the plasmid is the “vehicle” from which the immunogen is expressed, optimising vector design for maximal protein expression is essential. 12] 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. [13] 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. [2] 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. [14] 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. [2] An example of DNA vaccine plasmid is pVAC, it uses SV40 promoter. [edit] 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. [1] This is usually accomplished by the addition of N-terminal ubiquitin signals. [15][16] The conformation of the protein can also have an effect on antibody responses, with “ordered” structures (like viral particles) being more effective than unordered structures. [17] Strings of minigenes (or MHC class I epitopes) from different pathogens are able to raise cytotoxic T-cell responses to a number of pathogens, especially if a TH epitope is also included. [1] [edit] Delivery methods DNA vaccine and Gene therapy techniques are similar. 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 summarised in Table 3. 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. [18] 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[19]; by temporarily damaging muscle fibres with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose. 2] Immune responses to this method of delivery can be affected by many factors, including needle type,[8] needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected. [2] 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. [2][12] Alternative delivery methods have included aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa,[12] and topical administration of pDNA to the eye[20] and vaginal mucosa. 12] Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations,[1] biodegradable microspheres,[21][12] attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa,[22] and recombinant adenovirus vectors. [12] 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. [23] Generally, 0. 2 ? g – 20 ? g are required, although quantities as low as 16 ng have been reported. 2] These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates. [1] 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 of connective 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”. [1][2] 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. 2] ELI can be used to identify which of the pathogen’s genes induce a protective response. This has been tested with Mycoplasma pulmonis, a murine lung pathogen with a relatively small genome, and it was found that even partial expression libraries can induce protection from subsequent challenge. [24] Table 2. Summary of Plasmid DNA delivery methods| Method of Delivery| Formulation of DNA| Target Tissue| Amount of DNA| Parenteral| Injection (hypodermic needle)| Aqueous solution in saline| IM (skeletal); ID; (IV, subcutaneous and intraperitoneal with variable success)| Large amounts (approximately 100-200 ? g)| | Gene Gun| DNA-coated gold beads| ED (abdominal skin); vaginal mucosa; surgically xposed muscle and other organs| Small amounts (as little as 16 ng)| | Pneumatic (Jet) Injection| Aqueous solution| ED| Very high (as much as 300 ? g)| Topical application| Aqueous solution| Ocular; intravaginal| Small amounts (up to 100 ? g)| Cytofectin-mediated| Liposomes (cationic); microspheres; recombinant adenovirus vectors; attenuated Shigella vector; aerosolised cationic lipid formulations| IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes| Variable| Table 3. Advantages and disadvantages of commonly used DNA vaccine delivery methods| Method of Delivery| Advantage| Disadvantage|
Intramuscular or Intradermal injection| * No special delivery mechanism * Permanent or semi-permanent expression * pDNA spreads rapidly throughout the body| * Inefficient site for uptake due to morphology of muscle tissue * Relatively large amounts of DNA used * Th1 response may not be the response required| Gene Gun| * DNA bombarded directly into cells * Small amounts DNA| * Th2 response may not be the response required * Requires inert particles as carrier| Jet injection| * No particles required * 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 ? )| Liposome-mediated 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 * Intranasally delivered liposome-DNA complexes can result in expression in distal mucosa as well as nasal muscosa and the generation of IgA antibodies| * Toxicity * Ineffectiveness in serum * Risk of disease or immune reactions| [edit] Immune response raised by DNA vaccines [edit] Helper T-Cell responses 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. 25] 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. [edit] Raising of different types of T-cell help The type of T-cell help raised is influenced by the method of delivery and the type of immunogen expressed, as well as the targeting of different lymphoid compartments. [2][26] Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses. [25][26] This is true for intracellular and plasma membrane-bound antigens, but not for secreted antigens, which seem to generate TH2 responses, regardless of the method of delivery. 27] Generally the type of T-cell help raised is stable over time, and does not change when challenged or after subsequent immunizations which would normally have raised the opposite type of response in a naive animal. [25][26] However, Mor et al.. (1995)[9] immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mouse malarial parasite Plasmodium yoelii (PyCSP) and found that the initial TH2 response changed, after boosting, to a TH1 response. [edit] Mechanistic basis for different types of T-Cell help It is not understood how these different methods of DNA immunization, or the forms of antigen expressed, raise different profiles of T-cell help.
It was thought that the relatively large amounts of DNA used in IM injection were responsible for the induction of TH1 responses. However, evidence has shown no differences in TH type due to dose. [25] It has been postulated that the type of T-cell help raised is determined by the differentiated state of antigen presenting cells. Dendritic cells can differentiate to secrete IL-12 (which supports TH1 cell development) or IL-4 (which supports TH2 responses). [28] pDNA injected by needle is endocytosed into the dendritic cell, which is then stimulated to differentiate for TH1 cytokine production,[29] while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation. [edit] Practical uses of polarised T-Cell help
This polarisation in T-cell help is useful in influencing allergic responses and autoimmune diseases. In autoimmune diseases, the goal would be to shift the self-destructive TH1 response (with its associated cytotoxic T cell activity) to a non-destructive TH2 response. This has been successfully applied in predisease priming for the desired type of response in preclinical models[1] and somewhat successful in shifting the response for an already established disease. [30] [edit] Cytotoxic T-cell responses One of the greatest advantages of DNA vaccines is that they are able to induce cytotoxic T lymphocytes (CTL) without the inherent risk associated with live vaccines.
CTL responses can be raised against immunodominant and immunorecessive CTL epitopes,[31] as well as subdominant CTL epitopes,[21] in a manner which appears to mimic natural infection. This may prove to be a useful tool in assessing CTL epitopes of an antigen, and their role in providing immunity. Cytotoxic T-cells recognise small peptides (8-10 amino acids) complexed to MHC class I molecules (Restifo et al. , 1995). These peptides are derived from endogenous cytosolic proteins which are degraded and delivered to the nascent MHC class I molecule within the endoplasmic reticulum (ER). [32] Targeting gene products directly to the ER (by the addition of an amino-terminal insertion sequence) should thus enhance CTL responses.
This has been successfully demonstrated using recombinant vaccinia viruses expressing influenza proteins,[32] but the principle should be applicable to DNA vaccines too. Targeting antigens for intracellular degradation (and thus entry into the MHC class I pathway) by the addition of ubiquitin signal sequences, or mutation of other signal sequences, has also been shown to be effective at increasing CTL responses. [16] CTL responses can also be enhanced by co-inoculation with co-stimulatory molecules such as B7-1 or B7-2 for DNA vaccines against influenza nucleoprotein,[31][33] or GM-CSF for DNA vaccines against the murine malaria model P. yoelii. 34] Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 and TCA3 have also been shown to increase CTL activity against HIV-1 and influenza nucleoprotein antigens. [33][35] [edit] Humoral (antibody) response Schematic diagram of an antibody and antigens Antibody responses elicited by DNA vaccinations are influenced by a number of variables, including type of antigen encoded; location of expressed antigen (i. e. intracellular vs. secreted); number, frequency and dose of immunizations; site and method of antigen delivery, to name a few. [edit] Kinetics of antibody response Humoral responses after a single DNA injection can be much longer-lived than after a single injection with a recombinant protein.
Antibody responses against hepatitis B virus (HBV) envelope protein (HBsAg) have been sustained for up to 74 weeks without boost, while life-long maintenance of protective response to influenza haemagglutinin has been demonstrated in mice after gene gun delivery. [36] Antibody-secreting cells migrate to the bone marrow and spleen for long-term antibody production, and are generally localised there after one year. [36] Comparisons of antibody responses generated by natural (viral) infection, immunization with recombinant protein and immunization with pDNA are summarised in Table 4. DNA-raised antibody responses rise much more slowly than when natural infection or recombinant protein immunization occurs.
It can take as long as 12 weeks to reach peak titres in mice, although boosting can increase the rate of antibody production. This slow response is probably due to the low levels of antigen expressed over several weeks, which supports both primary and secondary phases of antibody response. Table 4. Comparison of T-Dependent Antibody Responses raise by DNA Immunizations, Protein Inoculations and Viral Infections| | Method of Immunization| | DNA Vaccine| Recombinant protein| Natural Infection| Amount of inducing antigen| Ng| ? g| ? (ng-? g)| Duration of Ag presentation| several weeks| < 1 week| several weeks| Kinetics of Ab response| slow rise| rapid rise| rapid rise|
Number of inoculations to obtain high avidity IgG and migration of ASC to bone marrow| One| two| One| Ab isotype (murine models)| C’-dependent or C’-independent| C’-dependent| C’-independent| Additionally, the titres of specific antibodies raised by DNA vaccination are lower than those obtained after vaccination with a recombinant protein. However, DNA immunization-induced antibodies show greater affinity to native epitopes than recombinant protein-induced antibodies. In other words, DNA immunization induces a qualitatively superior response. Antibody can be induced after just one vaccination with DNA, whereas recombinant protein vaccinations generally require a boost.
As mentioned previously, DNA immunization can be used to bias the TH profile of the immune response, and thus the antibody isotype, which is not possible with either natural infection or recombinant protein immunization. Antibody responses generated by DNA are useful not just in vaccination but as a preparative tool, too. For example, polyclonal and monoclonal antibodies can be generated for use as reagents. [edit] Mechanistic basis for DNA raised immune responses [edit] DNA Uptake Mechanism When DNA uptake and subsequent expression was first demonstrated in vivo in muscle cells,[37] 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). [38] However, subsequent research revealed that other cells (such as keratinocytes, fibroblasts and epithelial Langerhans cells) could also internalize DNA. [30][39] 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,[12] or through specific receptors. [40] 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. [40][41] Receptor mediated DNA uptake could be facilitated by the presence of polyguanylate sequences. Further research into this mechanism might seem pointless, considering that gene gun delivery systems, cationic liposome packaging, and other delivery methods bypass this entry method, but understanding it might be useful in reducing costs (e. g. y reducing the requirement for cytofectins), which will be important in the food animals industry. [edit] 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)[33][42] Iwasaki et al. , 1997). After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node to present antigen. [1] After IM and ID injections, dendritic cells have also been found to present antigen in the draining lymph node[39] and transfected macrophages have been found in the peripheral blood. 43] Besides direct transfection of dendritic cells or macrophages, cross priming is also known to occur following IM, ID and gene gun deliveries of DNA. Cross priming occurs when a bone marrow-derived cell presents peptides from proteins synthesised in another cell in the context of MHC class 1. This can prime cytotoxic T-cell responses and seems to be important for a full primary immune response. [1][44] [edit] 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. [12][37][44] [edit] Maintenance of immune response DNA vaccination generates an effective immune memory via the display of antigen-antibody complexes on follicular dendritic cells (FDC), which are potent B-cell stimulators.
T-cells can be stimulated by similar, germinal centre dendritic cells. FDC are able to generate an immune memory because antibodies production “overlaps” long-term expression of antigen, allowing antigen-antibody immunocomplexes to form and be displayed by FDC. [1] [edit] Interferons 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-? and TNF-? , which don’t kill the cell but place severe limitations on viral infection by down-regulating the expression of viral components. 45] 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. [46] IFN-? is also critically important in controlling malaria infections,[47] and should be taken into consideration when developing anti-malarial DNA vaccines. [edit] Modulation of the immune response [edit] Cytokine modulation For a vaccine to be effective, it must induce an appropriate immune response for a given pathogen, and the ability of DNA vaccines to polarise T-cell help towards TH1 or TH2 profiles, and generate CTL and/or antibody when required, is a great advantage in this regard.
This can be accomplished by modifications to the form of antigen expressed (i. e. intracellular vs. secreted), the method and route of delivery, and the dose of DNA delivered. [25][26][48][49][50] However, it can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i. e. cytokines, lymphokines or co-stimulatory molecules. These “genetic adjuvants” can be administered a number of ways: * as a mixture of 2 separate plasmids, one encoding the immunogen and the other encoding the cytokine; * as a single bi- or polycistronic vector, separated by spacer regions; or * as a plasmid-encoded chimera, or fusion protein.
In general, co-administration of pro-inflammatory agents (such as various interleukins, tumor necrosis factor, and GM-CSF) plus TH2 inducing cytokines increase antibody responses, whereas pro-inflammatory agents and TH1 inducing cytokines decrease humoral responses and increase cytotoxic responses (which is more important in viral protection, for example). Co-stimulatory molecules like B7-1, B7-2 and CD40L are also sometimes used. This concept has been successfully applied in topical administration of pDNA encoding IL-10. [20] Plasmid encoded B7-1 (a ligand on APCs) has successfully enhanced the immune response in anti-tumour models, and mixing plasmids encoding GM-CSF and the circumsporozoite protein of P. yoelii (PyCSP) has enhanced protection against subsequent challenge (whereas plasmid-encoded PyCSP alone did not).
It was proposed that GM-CSF may cause dendritic cells to present antigen more efficiently, and enhance IL-2 production and TH cell activation, thus driving the increased immune response. [34] This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, and later boosting with a recombinant poxvirus expressing PyCSP. [51] However, co-injection of plasmids encoding GM-CSF (or IFN-? , or IL-2) and a fusion protein of P. chabaudi merozoite surface protein 1 (C-terminus)-hepatitis B virus surface protein (PcMSP1-HBs) actually abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone. 17] The advantages of using genetic adjuvants are their low cost and simplicity of administration, as well as avoidance of unstable recombinant cytokines and potentially toxic, “conventional” adjuvants (such as alum, calcium phosphate, monophosphoryl lipid A, cholera toxin, cationic and mannan-coated liposomes, QS21, carboxymethylcellulose and ubenimix). [1][12] However, the potential toxicity of prolonged cytokine expression has not been established, and in many commercially important animal species, cytokine genes still need to be identified and isolated. In addition, various plasmid encoded cytokines modulate the immune system differently according to the time of delivery. For example, some cytokine plasmid DNAs are best delivered after the immunogen pDNA, because pre- or co-delivery can actually decrease specific responses, and increase non-specific responses. [52] [edit] Immunostimulatory CpG motifs Plasmid DNA itself appears to have an adjuvant effect on the immune system. 1][2] Bacterially derived DNA has been found to trigger innate immune defence mechanisms, the activation of dendritic cells, and the production of TH1 cytokines. [29][53] This is due to recognition of certain CpG dinucleotide sequences which are immunostimulatory. [49][54] CpG stimulatory (CpG-S) sequences occur twenty times more frequently in bacterially derived DNA than in eukaryotes. This is because eukaryotes exhibit “CpG suppression” – i. e. CpG dinucleotide pairs occur much less frequently than expected. Additionally, CpG-S sequences are hypomethylated. This occurs frequently in bacterial DNA, while CpG motifs occurring in eukaryotes are all methylated at the cytosine nucleotide.
In contrast, nucleotide sequences which inhibit the activation of an immune response (termed CpG neutralising, or CpG-N) are over represented in eukaryotic genomes. [55] The optimal immunostimulatory sequence has been found to be an unmethylated CpG dinucleotide flanked by two 5’ purines and two 3’ pyrimidines. [49][53] Additionally, flanking regions outside this immunostimulatory hexamer must be guanine-rich to ensure binding and uptake into target cells. The innate system works synergistically with the adaptive immune system to mount a response against the DNA encoded protein. CpG-S sequences induce polyclonal B-cell activation and the upregulation of cytokine expression and secretion. 56] Stimulated macrophages secrete IL-12, IL-18, TNF-? , IFN-? , IFN-? and IFN-? , while stimulated B-cells secrete IL-6 and some IL-12. [12][56][57] Manipulation of CpG-S and CpG-N sequences in the plasmid backbone of DNA vaccines can ensure the success of the immune response to the encoded antigen, and drive the immune response toward a TH1 phenotype. This is useful if a pathogen requires a TH response for protection. CpG-S sequences have also been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have also demonstrated the stimulation of polyclonal B-cell expansion.
However, the mechanism behind this may be more complicated than simple methylation – hypomethylated murine DNA has not been found to mount an immune response. Most of the evidence for the existence of immunostimulatory CpG sequences comes from murine studies. Clearly, extrapolation of this data to other species should be done with caution – different species may require different flanking sequences, as binding specificities of scavenger receptors differ between species. Additionally, species such as ruminants may be insensitive to immunostimulatory sequences due to the large gastrointestinal load they exhibit. Further research may be useful in the optimisation of DNA vaccination, especially in the food animal industry. edit] Alternative boosts DNA-primed immune responses can be boosted by the administration of recombinant protein or recombinant poxviruses. “Prime-boost” strategies with recombinant protein have successfully increased both neutralising antibody titre, and antibody avidity and persistence, for weak immunogens, such as HIV-1 envelope protein. [1][58] Recombinant virus boosts have been shown to be very efficient at boosting DNA-primed CTL responses. Priming with DNA focuses the immune response on the required immunogen, while boosting with the recombinant virus provides a larger amount of expressed antigen, leading to a large increase in specific CTL responses.
Prime-boost strategies have been successful in inducing protection against malarial challenge in a number of studies. Primed mice with plasmid DNA encoding Plasmodium yoelii circumsporozoite surface protein (PyCSP), then boosted with a recombinant vaccinia virus expressing the same protein had significantly higher levels of antibody, CTL activity and IFN-? , and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone. [59] This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus. [51] An effective prime-boost strategy for the simian malarial model P. knowlesi has also been demonstrated. 60] Rhesus monkeys were primed with a multicomponent, multistage DNA vaccine encoding two liver-stage antigens – the circumsporozoite surface protein (PkCSP) and sporozoite surface protein 2 (PkSSP2) – and two blood stage antigens – the apical merozoite surface protein 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42). They were then boosted with a recombinant canarypox virus encoding all four antigens (ALVAC-4). Immunized monkeys developed antibodies against sporozoites and infected erythrocytes, and IFN-? -secreting T-cell responses against peptides from PkCSP. Partial protection against sporozoite challenge was achieved, and mean parasitemia was significantly reduced, compared to control monkeys.
These models, while not ideal for extrapolation to P. falciparum in humans, will be important in pre-clinical trials. [edit] Additional methods of enhancing DNA-Raised immune responses [edit] Formulations of DNA The efficiency of DNA immunization can be improved by stabilising DNA against degradation, and increasing the efficiency of delivery of DNA into antigen presenting cells. [1] This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated with cetyltrimethylammonium bromide) with DNA. Such DNA-coated microparticles can be as effective at raising CTL as recombinant vaccinia viruses, especially when mixed with alum.
Particles 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells. [1] [edit] Alphavirus vectors Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency. [1] 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.
It is not clear how alphavirus replicons raise an immune response, but it is thought that this may be due to the high levels of protein expressed by this vector, replicon-induced cytokine responses, or replicon-induced apoptosis leading to enhanced antigen uptake by dendritic cells. 1. . The Journal of Immunology 164 (11): 5905–5912. PMID 10820272. http://www. jimmunol. org/cgi/content/abstract/164/11/5905. Retrieved 2007-11-21. 2. ^ Barouch, D. H. ; Santra, S. ; Steenbeke, T. D. ; Zheng, X. X. ; Perry, H. C. ; Davies, M. E. ; Freed, D. C. ; Craiu, A. ; Strom, T. B. ; Shiver, J. W. ; Others, (August 15, 1998). “Augmentation and Suppression of Immune Responses to an HIV-1 DNA Vaccine by Plasmid Cytokine/Ig Administration 1”. The Journal of Immunology 161 (4): 1875–1882. PMID 9712056. http://www. jimmunol. org/cgi/content/abstract/161/4/1875. Retrieved 2007-11-21. 3. ^ a b Krieg, A. M. ; Yi, A. K. ; Matson, S. Waldschmidt, T. J. ; Bishop, G. A. ; Teasdale, R. ; Koretzky, G. A. ; Klinman, D. M. (1995). “CpG motifs in bacterial DNA trigger direct B-cell activation”. Nature 374 (6522): 546–549. doi:10. 1038/374546a0. PMID 7700380. 4. ^ Klinman, D. M. ; Yamshchikov, G; Ishigatsubo, Y (April 15, 1997). “Contribution of CpG motifs to the immunogenicity of DNA vaccines” * PowderMed pdf report * DyNAVacS, an Integrative Tool for Optimised DNA Vaccine Design from the Institute of Genomics and Integrative Biology. * Hooper JW, Thompson E, Wilhelmsen C, et al. (May 2004). “Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox”. J. Virol. 78 (9): 4433–43. oi:10. 1128/JVI. 78. 9. 4433-4443. 2004. PMID 15078924. PMC 387704. http://jvi. asm. org/cgi/pmidlookup? view=long&pmid=15078924. [hide] v • d • eArtificial induction of immunity / Immunization: Vaccines, Vaccination, and Inoculation (J07)| | | Development| List of vaccine ingredients · Adjuvants · Mathematical modelling · Timeline · TrialsClasses: Inactivated vaccine · Live vector vaccine (Attenuated vaccine, Heterologous vaccine) · Toxoid · Subunit/component/Virus-like particle · Conjugate vaccine · DNA vaccination| | | Administration| Global: GAVI Alliance · Policy · Schedule · Vaccine injury USA: ACIP · VAERS · VSD · Vaccine court| | |
Vaccines| Bacterial| Anthrax · Brucellosis · Cholera# · Diphtheria# · Hib# · Meningococcus# (NmVac4-A/C/Y/W-135, NmVac4-A/C/Y/W-135 – DT, MeNZB) · Pertussis# · Plague · Pneumococcal# (PPSV, PCV) · Tetanus# · Tuberculosis (BCG)# · Typhoid# (Ty21a, ViCPS) · Typhuscombination: DTwP/DTaP| | | Viral| Adenovirus · Tick-borne encephalitis · Japanese encephalitis# · Flu# (Pandemrix, LAIV, H1N1) · HAV# · HBV# · HPV (Gardasil, Cervarix) · Measles# · Mumps# (Mumpsvax) · Polio# (Salk, Sabin) · Rabies# · Rotavirus# · Rubella# · Smallpox (Dryvax) · Varicella (chicken pox)# · Yellow fever# combination: MMR · MMRVresearch: Cytomegalovirus · Epstein-Barr · HIV · Hepatitis C| | | Protozoan| Malaria · Trypanosomiasis| | | Helminthiasis| Schistosomiasis · Hookworm| | |
Other| TA-CD · NicVAX · Cancer vaccines (ALVAC-CEA vaccine)| | | | Controversy| General · MMR · NCVIA · Pox party · Simpsonwood · Thiomersal| | | See also| List of vaccine topics · Epidemiology · Eradication of infectious diseases| | | #WHO-EM. ‡Withdrawn from market. CLINICAL TRIALS: †Phase III. §Never to phase III M: BAC| bact (clas)| gr+f/gr+a(t)/gr-p/gr-o| drug(J1p, w, n, m, vacc)| | M: VIR| virs (prot)| cutn/syst (hppv, hiva, infl, zoon), epon| drugJ(dnaa, rnaa, rtva, vacc)| | | | Retrieved from “http://en. wikipedia. org/wiki/DNA_vaccination” Categories: DNA | Vaccination Personal tools * New features * Log in / create account Namespaces * Article * Discussion Variants Views Read * Edit * View history Search DNA Vaccine Outlook By Sean Henahan, Access Excellence WASHINGTON, D. C. (12/1//97) DNA vaccine technology is showing increasing promise in the treatment of human diseases, and should offer immunizations that are both safer and cheaper than conventional vaccines, according to a new consensus report released by the American Academy of Microbiology. The report, “The Scientific Future of DNA for Immunization” is based on a colloquium of 25 international experts in microbiology, infectious diseases and immunology convened in 1996. It suggests that DNA vaccination may revolutionize the practice of human immunization. Recent results obtained from DNA-vaccine testing in animal models suggest that this new technology may revolutionize the vaccination of humans,” says Harriet Robinson of Emory University, co-author of the report. “Already we have been able to induce immune responses against diarrhea-causing viruses, malarial parasites and tuberculosis. ” Conventional vaccines have prevented many millions of cases of killer diseases such as small-pox and polio. But some pathogens, such as malaria, have proven to be a considerable challenge to vaccine developers. It is in such cases that DNA vaccines may prove useful. Indeed, a promising DNA vaccine candidate has been developed for malaria.
DNA vaccines are also currently being developed for over 15 other human illnesses including AIDS, herpes, tuberculosis and rotavirus, a common cause of childhood diarrhea. Traditional vaccination methods use either a weakened or killed version of the disease-causing organism or a component of the organism, such as inactivated toxins or proteins. These component vaccines can either be purified from the organism itself or genetically engineered. The injection or oral administration of these nondisease-causing mimics mobilizes the immune system to protect the host from the disease. “Since the first vaccine was developed for smallpox in 1789, the widespread use of vaccines has resulted in the global eradication of that disease,” says Dr. Robinson. We have also eliminated polio and measles from the United States and drastically reduced the incidence of diptheria, tetanus, whooping cough, mumps and rubella. Nonetheless, infectious diseases remain major killers, despite worldwide improvements in sanitation and vaccination. ” DNA vaccination differs from traditional vaccines in that just the DNA coding for a specific component of a disease-causing organism is injected into the body. The DNA can be administered either in a saline solution injected through a hypodermic needle or on DNA-coated gold beads propelled into the body using gene guns. The actual production of the immunizing protein takes place in the vaccinated host.
This eliminates any risk of infection associated with some live and attenuated virus vaccines. The report lists a number of other advantages DNA vaccines have over classic vaccine methods: * DNA vaccination provides long-lived immune responses, unlike many component vaccines that require multiple innoculations to maintain immunity. * Vaccines for multiple diseases can all be given in a single inoculation. Currently, in the United States, the full course of childhood immunizations requires 18 visits to the doctor or clinic. * All DNA vaccines can be produced using similar techniques. The ability to use generic production methods greatly simplifies the vaccine development and production process. * They are extremely stable.
Unlike many conventional vaccines that must be held at a constant temperature, DNA vaccines can be stored under a vast array of conditions either dried or in a solution. This eliminates the need for the “cold chain” — the series of refrigerators required to maintain a vaccine during distribution. This will greatly improve the ability to deliver vaccines to remote areas in developing countries. * Candidate vaccines can be recovered from diseased tissue. Microbial DNA can be isolated from the tissue of an infected animal, purified, amplified and screened for vaccine candidates. “It is remarkable that DNA vaccines have come so far since 1992 but their real contribution is yet to come,” says Stephen Johnston of the University of Texas Southwest Medical Center, a member of the colloquium steering committee. In the next few years we will have sequenced the genomes of most if not all of the worlds pathogens. DNA vaccines probably offer the best way to translate all that sequence information into useful vaccines. The marriage of genomics and DNA vaccines may revolutionize vaccinology as applied to infectious diseases and cancer. ” DNA vaccination may have its own limitations. The most obvious is that it is limited to developing immune responses against only the protein components of pathogens. The question of which vector to use also remains controversial. Moreover, some microbes have an outer shell made of polymerized sugars, known as polysaccharides.
DNA vaccines cannot substitute for the more traditional polysaccharide-based vaccines, such as the pneumococcal vaccine for bacterial pneumonia. Antisense DNA Schematic showing how antisense DNA strands can interfere with protein translation. Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes. Some regions within a double strand of DNA code for genes, which are usually instructions specifying the order of amino acids in a protein along with regulatory sequences, splicing sites, noncoding introns, and other complicating details. For a cell to use this information, one strand of the DNA serves as a template for the synthesis of a complementary strand of RNA.
The template DNA strand is called the transcribed strand with antisense sequence and the mRNA transcript is said to be sense sequence (the complement of antisense). Because the DNA is double-stranded, the strand complementary to the antisense sequence is called non-transcribed strand and has the same sense sequence as the mRNA transcript (though T bases in DNA are substituted with U bases in RNA). DNA strand 1: sense strand DNA strand 2: antisense strand (copied to)> RNA strand (sense) Many forms of antisense have been developed and can be broadly categorized into enzyme-dependent antisense or steric blocking antisense. Enzyme-dependent antisense includes forms dependent on RNase H activity to degrade target RNA, including single-stranded DNA, RNA, and phosphorothioate antisense. The R1 plasmid hok/sok system is an example of mRNA antisense regulation process, through enzymatic degradation of the resulting RNA duplex. Double stranded RNA acts as enzyme-dependent antisense through the RNAi/siRNA pathway, involving target mRNA recognition through sense-antisense strand pairing followed by target mRNA degradation by the RNA-induced silencing complex (RISC). Steric blocking antisense (RNase-H-independent antisense)[1] interferes with gene expression or other mRNA-dependent cellular processes by binding to a target sequence of mRNA and getting in the way of other processes.
Steric blocking antisense includes 2′-O alkyl (usually in chimeras with RNase-H dependent antisense), peptide nucleic acid (PNA), locked nucleic acid (LNA), and Morpholino antisense. Antisense nucleic acid molecules have been used experimentally to bind to mRNA and prevent expression of specific genes. Antisense therapies are also in development; in the USA, the Food and Drug Administration (FDA) has approved a phosphorothioate antisense oligo, fomivirsen (Vitravene), for human therapeutic use. Cells can produce antisense RNA molecules naturally, which interact with complementary mRNA molecules and inhibit their expression. Bottom of Form Distribution Patterns of Three Dominant Tuna Species in the Indian Ocean
We analyzed fishery data provided by Taiwanese fishermen between 1967 and 1996 to investigate monthly and spatial distribution, abundance patterns, and environmental characteristics of three dominant tuna species (bigeye tuna, Thunnus obesus; yellowfin tuna, T. albacares; and albacore, T. alalunga) inhabiting the Indian Ocean using a GIS approach. Environmental preferences of these species were characterized by the monthly composites of sea surface temperature (SST) measured from AVHRR images and chlorophyll concentration derived from the Coastal Zone Color Scanner (CZCS) instrument flown aboard the Nimbus-7 satellite. Results indicate that these species show distinct distribution patterns and minor monthly variations. The peak abundance regions exhibit unique environment conditions. SST mean and range values for the three species occurred in the peak abundance regions are different.
Chlorophyll concentration index indicates that the yellowfin tuna occur in regions with higher values than the other two species. the use of discriminant function analysis to predict four abundance classes on the three species’ monthly distribution has overall prediction accuracy range from 62. 4 to 76. 1% for albacore, 52. 6-68. 0% for bigeye tuna and 58. 6-70. 9% for yellowfin tuna. With the help of remote sensing data and analyses conducted in a GIS environment, our results show some promise on predicting marine species’ abundance pattern in the Indian Ocean. Introduction Tunas are among the largest, most specialized and commercially important of all fishes (Collette and Nauen, 1983).
Belonging to the genus Thunnus of the family Scombridae, they are found in temperature and tropical oceans around the world and account for a major proportion of the world fishery products. Biologically, tuna species have complex life history. They have streamlined bodies and vary extensively in size, color and fin length. Tunas are unique among fishes because they possess body temperature several degrees higher than the ambient waters and have high metabolic rates that enable them to exhibit extraordinary growth patterns. Tunas are fast swimmer and capable of traveling more than 48 km/h (Collette and Nauen, 1983). They are migratory and have few predators.
Tunas are in great demand throughout the world market due to their excellent meat quality (Chang and Lin, 1995; FAO, 1997). The tuna industry has been a successful program in the past. However, problems on the fishing stock status have occurred due to the recently increased intensity. There is an urgency to conserve the tuna resources due to the awareness of decreasing tuna stocks (Fonteneau, 1995, 1997; Hsu, 1994, 1998). Although the data for the principal market tuna species have been accumulated for a long time, uncertainties exist in the basic distribution of these species. There have been few studies on large-scale distribution of tuna species (Hsu, 1994).
General distribution description indicates that the tunas are widely distributed among the three major oceans, though they never extend to the polar region (Collette and Nauen, 1983; Kikawa and Ferraro, 1966; Laevastu and Rossa, 1962). Information on where and when tunas occur can be critical for resource management and practical for fishery vessels. Despite the abundant literatures on the tuna resources in the Indian Ocean, there appears to have a lack on the study of distribution pattern of these tunas (Lee, 1990, 1995; Kikawa and Ferraro, 1966; Lee and Liu, 1995; Petit et al. , 1995; Wu and Chen, 1994; Yeh et al. , 1995 and see Hsu, 1994 for review).
To better conserve and maintain a sustainable yield of these species, there is a need to understand large-scale patterns of tuna stocks in space and time. Recently large-scale data obtained by remote sensing technique have become available to the scientific community through the Internet. Data, such as sea surface temperature (SST) and chlorophyll concentration, can be downloaded from NASA’s web site or a CD-ROM can be requested. With the help of a GIS, this distribution mechanism has greatly enabled scientific researches to incorporate large volume of important data in large-scale analyses. In this paper, we documented the distribution patterns of three dominant tuna species, i. e. , albacore (Thunnus alalunga), bigeye tuna (T. obesus) and yellowfin tuna (T. lbacares), in the Indian Ocean based on the catch data recorded by Taiwanese vessels between 1967 and 1996 using a GIS-approach and investigated the characteristics of high abundance (yield) regions with SST and chlorophyll concentration. Finally, we applied a discriminant function analysis to predict monthly distribution pattern using these variables. Methods The Indian Ocean, having about 20% of the global tuna production, is the second largest proportion of principal tuna market in the world (FAO, 1997). Japan, Taiwan and Korea are the major fishing countries in the Indian Ocean. In recent years, tuna fisheries are growing in this ocean, partly due to the catching of small tunas by the non-traditional tuna catching countries, such as Franch, Ivory Coast, Spain, and etc (FAO, 1997). Tuna database
Based on fishery statistics, Taiwan is the second largest tuna fishing countries in the world (FAO, 1997). Fishery data in the Indian Ocean are recorded by Taiwanese tuna longline fishing vessels between 1967 and 1996. The data set represent the best available information exist to date on the tuna resources found in the Indian Ocean because of its extensive cover in terms of both space and time. These data were compiled by the Oversea Fisheries Development Council (OFDC) of the Republic of China and include monthly summary of number of catch, total weight, and number of hooks of several tuna species in specific geographic locations. All catch data were georeferenced in a latitude and longitude system (Figure 1).
Each grid cell is 5-degree x 5-degree in size. Based on the total catch data, we identify three dominant species: albacore, bigeye tuna and yellowfin tuna. The total production of these three species comprises of more than 75% of all fishery products. Yellowfin and bigeye tunas are the second and third important commercial species of tuna, respectively, while the production of albacore over the past 30 years has been fluctuating (FAO, 1997). Despite an ever-increasing knowledge of immune mechanisms and protective antigens, Jenner’s approach of immunizing with a live, attenuated pathogen appeared to remain the most practical and perhaps most effective form of vaccine.
In 1992, an entirely new vaccine strategy was reported by Tang et al. , in which experimental animals were immunized by a naked DNA encoding a polypeptide antigen. These animals developed both humoral and cellular immune responses against that antigen [1]. This strategy, termed ‘‘genetic immunization’’ or ‘‘genetic vaccine’’ has been tested and modified by many investigators because it has several obvious advantages over more traditional vaccines that employ attenuated pathogens, purified proteins, or synthetic peptides in combination with adjuvant. First, genetic vaccines are considered to be safer because there is no risk of reversion into virulence.
Moreover, one should be able to: (1) manipulate DNA more easily, (2) isolate DNA directly from infectious pathogens or tumor cells, and (3) produce and store DNA vaccines in a more time- and cost-effective manner. Genetic immunization effectively induces both cellular and humoral immune responses and the resulting immune responses appear to last for a relatively long period without repeated boost immunizations. Thus, one obvious utility of this technology is for the initiation of protective immunity against infectious pathogens. In many experimental animals, from mice to non-human primates, specific immune responses and/or protection have been induced against a wide panel of infectious microbes, including viruses, bacteria, and parasites (see the recent review articles [2–6] for comprehensive lists of the target microbes and references).
Most recently, genetic vaccines against human immunodeficiency virus-1 (HIV-1) have been tested for the safety and immunological outcome in asymptomatic human patients with HIV-1 infection [7, 8]. Genetic vaccines have also been used to initiate protective immunity against tumor cells. In this regard, B cell lymphomas have provided an excellent model; plasmid DNA encoding variable fragments of the ‘‘idiotypic’’ immunoglobulins have been used to induce idiotype-specific immune responses [9, 10]. Prophylactic efficacy against solid tumors has been observed for DNA vaccines that encode other experimental or naturally occurring tumor-associated antigens [11–15].
Although originally developed to initiate immune responses, genetic vaccines can be used to prevent the diseases that are caused by excessive or abnormal immune reactions. For example, the onset of experimental autoimmune encephalomyelitis (EAE) has been prevented by a DNA vaccine encoding a variable region of T cell receptor expressed by pathogenic T cells [16] by DNA vaccine encoding a T cell epitope of myelin basic protein (MBP), a pathogenic antigen in EAE [17]. Moreover, DNA vaccines have also been used to prevent the onset of experimentally induced allergic responses characterized by IgE production and airway hypersensitivity [18, 19]. In summary, genetic vaccines are otentially applicable to a wide variety of diseases, including infectious diseases, cancers, autoimmune diseases, and allergic diseases. Plasmid DNA can be introduced directly into hosts either by needle injection into muscle or skin or by particle bombardment into skin with a special device called a ‘‘gene gun. ’’ Expression of the introduced plasmids and their products has Abbreviations: DC, dendritic cells; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; LC, Langerhans cells; CTL, cytotoxic T lymphocytes; GFP, green fluorescence protein; GM-CSF, granulocytemacrophage colony-stimulating factor; CMV, cytomegalovirus; IFN-g, interferon- g; TNF-a, tumor necrosis factor a; OVA, ovalbumin. Correspondence: Dr.
Akira Takashima, University of Texas Southwest Medical Center, 5323 Harry Hines Blvd. , Dallas, TX 75235-9069. Received February 10, 1999; revised March 16, 1999; accepted March 16, 1999. 350 Journal of Leukocyte Biology Volume 66, A | Mammalian Expression System| The production of proteins in mammalian cells is an important tool in numerous scientific and commercial areas. For example, the proteins expressed in and purified from mammalian cell system are routinely needed for life science research and development. In the field of biomedicine, proteins for human therapy, vaccination or diagnostic applications are typically produced in mammalian cells.
Gene cloning, protein engineering, biochemical and biophysical charact erization of proteins also require the use of gene expression in mammalian cells. Other applications in widespread use involve screening of libraries of chemical compounds in drug discovery, and the development of cell-based biosensors. | | Usage of Protein Produced in Mammalian Expression System| The proteins produced in mammalian system have the best structural and functional features that are usually most close to their cognate native form and can satisfy the following application needs or utility: Transgene expression Biochemical analyses Assay standards Functional studies of the protein (in vitro and ex vivo)
Structural studies, including protein crystallization, protein structure and NMR Protein-protein interaction experiments Enzyme kinetics Immunogen for antibodies development Proteomic and phenomics study Drug target discovery and validation Cell line development, drug screening, and in vitro model system Animal studies, including in vivo functional and ADME, PK/TK and safety studies Physiology and pathology studies Diagnostic application Therapeutic application Prophylactic (vaccine) development Protein engineering and mutagenesis studies)| Overview of Mammalian Expression Technology| Within this technology area, many aspects contribute a successful protein production in mammalian cell system.
Generally, the following lines of consideration are helpful in making a given protein expression project productive: Transgene bioinformatics analysis and genetic engineering Transgene modification and codon optimization (see expanded points below) Expression vector selection and development (see expanded description below) Host cell selection, adaptation, reengineering, and development Optimization of plasmid backbone and expression cassette for gene expression Methods for DNA introduction into mammalian cells Lipid reagents for DNA transfer into mammalian cells Reporter genes for monitoring gene expression in mammalian cells Gene targeting techniques for efficient protein production Gene transfer and amplification in mammalian cells Viral-based vectors for gene expression in mammalian cells Adenovirus Adeno-associated virus Baculovirus Epstein-Barr virus Herpes simplex virus Lentiviruses Poliovirus Retroviruses Semliki Forest virus Simian virus 40 Sindbis virus Vaccinia virus
Co-transfer of multiple plasmids/viruses to introduce several genes Matrix-attachment regions and protein production Chromatin insulators, position effects, and locus control regions Posttranslational processing, transport and secretion of proteins Pathways of mammalian protein glycosylation Metabolic engineering of mammalian cells for higher protein yield Translational regulation in mammalian cells Pathways of mammalian messenger RNA degradation Pathways of mammalian protein degradation Intracellular targeting of antibodies in mammalian cells Inducible gene expression in mammalian cells Inducible gene expression in mammalian cells Protein production in transgenic animals
Protein purification in mammalian cell culture| Consideration of Transgene Sequence Modification and Codon Optimization| The following points can be considered for preparing transgene to conduct protein expression and functional studies: Codon optimization and G/C content adaptation Inhibition of internal splicing and premature polyadenylation Prevention of creation of stable RNA secondary Introduction/avoidance of immunomodulatory CpG motifs Avoidance of direct DNA repeats and thereby recombination events Dom
