DNA vaccine - Knowledge

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immunostimulatory. 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 methylated at the cytosine nucleotide. In contrast, nucleotide sequences that inhibit the activation of an immune response (termed CpG neutralising, or CpG-N) are over represented in eukaryotic genomes. The optimal immunostimulatory sequence is an unmethylated CpG dinucleotide flanked by two 5’

116:. DNA vaccines contain DNA that codes for specific antigens from a pathogen. The DNA is injected into the body and taken up by cells, whose normal metabolic processes synthesize proteins based on the genetic code in the plasmid that they have taken up. Because these proteins contain regions of amino acid sequences that are characteristic of bacteria or viruses, they are recognized as foreign and when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers immune responses. Alternatively, the DNA may be encapsulated in protein to facilitate cell entry. If this 1671:). However, the potential toxicity of prolonged cytokine expression is not established. In many commercially important animal species, cytokine genes have not been identified and isolated. In addition, various plasmid-encoded cytokines modulate the immune system differently according to the delivery time. For example, some cytokine plasmid DNAs are best delivered after immunogen pDNA, because pre- or co-delivery can decrease specific responses and increase non-specific responses. 28: 1011: 1795:. Unlike conventional DNA vaccinations alphavirus vectors kill transfected cells and are only transiently expressed. Alphavirus replicase genes are expressed in addition to the vaccine insert. It is not clear how alphavirus replicons raise an immune response, but it 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. 1742:(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-γ-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 1517:

lymph node where they prime secondary B- and T-cell responses. In skeletal muscle, striated muscle cells are most frequently transfected, but seem to be unimportant in 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.

651: 5700: 1477: 456: 1627:(PyCSP) enhanced protection against subsequent challenge (whereas plasmid-encoded PyCSP alone did not). It was proposed that GM-CSF caused dendritic cells to present antigen more efficiently and enhance IL-2 production and TH cell activation, thus driving the increased immune response. This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, followed by boosting with a recombinant 1221: 1708:

been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have demonstrated the stimulation of polyclonal B-cell expansion. The mechanism behind this may be more complicated than simple methylation – hypomethylated murine DNA has not been found to mount an immune response.

1556:, which do not kill the cell, but limit viral infection by down-regulating the expression of viral components. DNA vaccinations can be used to curb viral infections by non-destructive IFN-mediated control. This was demonstrated for hepatitis B. IFN-γ is critically important in controlling malaria infections and is a consideration for anti-malarial DNA vaccines. 396:

inverted and tandem repeats, which are conspicuous in many commercially available cloning and expression vectors. Therefore, the reduction or complete elimination of extraneous noncoding backbone sequences would pointedly reduce the propensity for such events to take place and consequently the overall plasmid's recombinogenic potential.

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envelope protein. 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.

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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 as a preparative tool. For example, polyclonal and monoclonal antibodies can be generated for use as reagents.

1539:(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. 984:

The delivery method determines the dose required to raise an effective immune response. Saline injections require variable amounts of DNA, from 10 μg to 1 mg, whereas gene gun deliveries require 100 to 1000 times less. Generally, 0.2 μg – 20 μg are required, although quantities as

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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-γ, and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone. This can be further

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IM and ID DNA delivery initiate immune responses differently. In the skin, keratinocytes, fibroblasts and Langerhans cells take up and express antigens and are responsible for inducing a primary antibody response. Transfected Langerhans cells migrate out of the skin (within 12 hours) to the draining

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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. As many as

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The type of T-cell help raised is influenced by the delivery method and the type of immunogen expressed, as well as the targeting of different lymphoid compartments. Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses. This

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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

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epitopes than recombinant protein-induced antibodies. In other words, DNA immunization induces a qualitatively superior response. Antibodies can be induced after one vaccination with DNA, whereas recombinant protein vaccinations generally require a boost. DNA immunization can be used to bias the TH

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How these different methods operate, the forms of antigen expressed, and the different profiles of T-cell help is not understood. It was thought that the relatively large amounts of DNA used in IM injection were responsible for the induction of TH1 responses. However, evidence shows no dose-related

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Most of the evidence for immunostimulatory CpG sequences comes from murine studies. Extrapolation of this data to other species requires caution – individual species may require different flanking sequences, as binding specificities of scavenger receptors vary across species. Additionally, species

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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

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An effective vaccine must induce an appropriate immune response for a given pathogen. DNA vaccines can polarise T-cell help towards TH1 or TH2 profiles and generate CTL and/or antibody when required. This can be accomplished by modifications to the form of antigen expressed (i.e. intracellular vs.

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Besides direct transfection of dendritic cells or macrophages, cross priming occurs following IM, ID and gene gun DNA deliveries. 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

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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

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Once the plasmid inserts itself into the transfected cell nucleus, it codes for a peptide string of a foreign antigen. On its surface the cell displays the foreign antigen with both histocompatibility complex (MHC) classes I and class II molecules. The antigen-presenting cell then travels to the

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DNA vaccine expressing HBV small and middle envelope protein was injected into adults with chronic hepatitis. The vaccine resulted in specific interferon gamma cell production. Also specific T-cells for middle envelop proteins antigens were developed. The immune response of the patients was not

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Structural instability phenomena are of particular concern for plasmid manufacture, DNA vaccination and gene therapy. Accessory regions pertaining to the plasmid backbone may engage in a wide range of structural instability phenomena. Well-known catalysts of genetic instability include direct,

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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

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Plasmid DNA itself appears to have an adjuvant effect on the immune system. Bacterially derived DNA can trigger innate immune defence mechanisms, the activation of dendritic cells and the production of TH1 cytokines. This is due to recognition of certain CpG dinucleotide sequences that are

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immunization (ELI). Using this technique, potentially all the genes from a pathogen can be delivered at one time, which may be useful for pathogens that are difficult to attenuate or culture. ELI can be used to identify which genes induce a protective response. This has been tested with

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use. In some cases, protection from disease in animals has been obtained, in others not. Research is ongoing over the approach for viral, bacterial and parasitic diseases in humans, as well as for cancers. In August 2021, Indian authorities gave emergency approval to

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began tests of a different DNA vaccine against Zika in Miami. The NIH vaccine is injected into the upper arm under high pressure. Manufacturing the vaccines in volume remained unsolved as of August 2016. Clinical trials for DNA vaccines to prevent HIV are underway.

1604:, 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 (more important in viral protection). Co-stimulatory molecules such as 1699:

The innate system works 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. Stimulated macrophages secrete IL-12,

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Antibody responses elicited by DNA vaccinations are influenced by multiple variables, including antigen type; antigen location (i.e. intracellular vs. secreted); number, frequency and immunization dose; site and method of antigen delivery.

1767:) with DNA. Such DNA-coated microparticles can be as effective at raising CTL as recombinant viruses, especially when mixed with alum. Particles 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells. 2716:

Böhm W, Kuhröber A, Paier T, Mertens T, Reimann J, Schirmbeck R (June 1996). "DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection".

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As of 2021 no DNA vaccines have been approved for human use in the United States. Few experimental trials have evoked a response strong enough to protect against disease and the technique's usefulness remains to be proven in humans.

3194:"Genetic immunization of BALB/c mice with a plasmid bearing the gene coding for a hybrid merozoite surface protein 1-hepatitis B virus surface protein fusion protects mice against lethal Plasmodium chabaudi chabaudi PC1 infection" 1051:

Generally the type of T-cell help raised is stable over time, and does not change when challenged or after subsequent immunizations that would normally have raised the opposite type of response in a naïve specimen. However, Mor

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The 30kDa surface receptor binds specifically to 4500-bp 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

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Wolff JA, Dowty ME, Jiao S, Repetto G, Berg RK, Ludtke JJ, et al. (December 1992). "Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle".

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have 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

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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

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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

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White LO, Gibb E, Newham HC, Richardson MD, Warren RC (July 1979). "Comparison of the growth of virulent and attenuated strains of Candida albicans in the kidneys of normal and cortison-treated mice by chitin assay".

2213:"Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D" 507:. Immune responses to this method can be affected by factors including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the recipient. 1257:

12 weeks may be required to reach peak titres in mice, although boosting can decrease the interval. This response is probably due to the low levels of antigen expressed over several weeks, which supports both

1468:, and other delivery methods bypass this entry method, but understanding it may be useful in reducing costs (e.g. by reducing the requirement for cytofectins), which could be important in animal husbandry. 337: – 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 1115:. In autoimmune diseases, the goal is 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 159:

demonstrated that direct injection of mice with plasmid DNA encoding a flu antigen protected the animals against subsequent experimental infection with influenza virus. In 2016 a DNA vaccine for the

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Huebener N, Fest S, Strandsby A, Michalsky E, Preissner R, Zeng Y, et al. (July 2008). "A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity".

2669:"Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites" 1027:

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 has distinctive patterns of

73:. DNA vaccines have theoretical advantages over conventional vaccines, including the "ability to induce a wider range of types of immune response". Several DNA vaccines have been tested for 989:. 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 3099:"Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization" 1184:

proteins, but the principle should also be applicable to DNA vaccines. Targeting antigens for intracellular degradation (and thus entry into the MHC class I pathway) by the addition of

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Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et al. (March 1993). "Heterologous protection against influenza by injection of DNA encoding a viral protein".

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of the protein can also affect antibody responses. "Ordered" structures (such as viral particles) are more effective than unordered structures. Strings of minigenes (or MHC class I

3864:"Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens" 3278: 1570:

secreted), the method and route of delivery or the dose. It can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i.e. cytokines,

5110:"Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus" 5058:"Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein" 1504:

to present antigens. After IM and ID injections, dendritic cells present antigen in the draining lymph node and transfected macrophages have been found in the peripheral blood.

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tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences. An example of DNA vaccine plasmid is pVAC, which uses SV40

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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.

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DNA immunization is also being investigated as a means of developing antivenom sera. DNA immunization can be used as a technology platform for monoclonal antibody induction.

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enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus. An effective prime-boost strategy for the

3970:"Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA" 1623:. Plasmid encoding B7-1 (a ligand on APCs) successfully enhanced the immune response in tumour models. Mixing plasmids encoding GM-CSF and the circumsporozoite protein of 1048:

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.

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1 (C-terminus)-hepatitis B virus surface protein (PcMSP1-HBs) abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone.

1212:. Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 and TCA3 were shown to increase CTL activity against HIV-1 and influenza nucleoprotein antigens. 5005:

Sato Y, Roman M, Tighe H, Lee D, Corr M, Nguyen MD, et al. (July 1996). "Immunostimulatory DNA sequences necessary for effective intradermal gene immunization".

1990: 4084:"Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization" 330:

vectors (with multiple genes of interest) are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.

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A veterinary DNA vaccine to protect horses from West Nile virus has been approved. Another West Nile virus vaccine has been tested successfully on American robins.

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Humoral responses after a single DNA injection can be much longer-lived than after a single injection with a recombinant protein. Antibody responses against

372:(RSV) promoter had much higher expression rates. More recently, expression and immunogenicity have been further increased in model systems by the use of the 5201:

Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, et al. (April 1995). "CpG motifs in bacterial DNA trigger direct B-cell activation".

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Mancini-Bourgine M, Fontaine H, Bréchot C, Pol S, Michel ML (May 2006). "Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers".

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Prime-boost strategies have been successful in inducing protection against malarial challenge in a number of studies. Primed mice with plasmid DNA encoding

2400:"A West Nile virus DNA vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase I clinical trial" 1241:

virus (HBV) envelope protein (HBsAg) have been sustained for up to 74 weeks without boost, while lifelong maintenance of protective response to influenza

3047:"DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction" 2926:

Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA (September 2009). "Structural instability of plasmid biopharmaceuticals: challenges and implications".

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

4504:"Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope" 4912:"IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model" 1759:

The efficiency of DNA immunization can be improved by stabilising DNA against degradation, and increasing the efficiency of delivery of DNA into

4703:"Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells" 1526: 5966: 4291:"Enhancement of cell-mediated immunity against HIV-1 induced by coinnoculation of plasmid-encoded HIV-1 antigen with plasmid expressing IL-12" 3815:"Role of different lymphoid tissues in the initiation and maintenance of DNA-raised antibody responses to the influenza virus H1 glycoprotein" 3397:"Induction of CD8+ T cell responses to dominant and subdominant epitopes and protective immunity to Sendai virus infection by DNA vaccination" 5855: 1168:

molecules. These peptides are derived from cytosolic proteins that are degraded and delivered to the nascent MHC class I molecule within the

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DNA vaccines have been introduced into animal tissues by multiple methods. In 1999, the two most popular approaches were injection of DNA in

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Lode HN, Huebener N, Zeng Y, Fest S, Weixler S, Gaedicke G (December 2004). "DNA minigene vaccination for adjuvant neuroblastoma therapy".

4961:"Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity" 2620:"Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA" 5648:"Multistage multiantigen heterologous prime boost vaccine for Plasmodium knowlesi malaria provides partial protection in rhesus macaques" 2568:"Intranasal administration of plasmid DNA nanoparticles yields successful transfection and expression of a reporter protein in rat brain" 4456:

Anderson RG, Kamen BA, Rothberg KG, Lacey SW (January 1992). "Potocytosis: sequestration and transport of small molecules by caveolae".

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inner core and poly(beta-amino ester) outer coat function synergistically to increase efficiency by addressing barriers associated with

2887:"Novel engineered HIV-1 East African Clade-A gp160 plasmid construct induces strong humoral and cell-mediated immune responses in vivo" 985:

low as 16 ng have been reported. These quantities vary by species. Mice for example, require approximately 10 times less DNA than

997:) before it is taken up by the cells, while gene gun deliveries drive/force DNA directly into the cells, resulting in less "wastage". 831:

IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes

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from a pathogen, or attenuated viruses which stimulate an immune response in the vaccinated organism. DNA vaccines are members of the

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Oliveira PH, Mairhofer J (September 2013). "Marker-free plasmids for biotechnological applications - implications and perspectives".

4242:"A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increases protection conferred by a malaria DNA vaccine" 1488:

mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialised

557:, and topical administration of pDNA to the eye and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic 1763:. This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated with 6398: 249:

expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications

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Alarcon JB, Waine GW, McManus DP (1999). "DNA Vaccines: Technology and Application as Anti-parasite and Anti-microbial Agents".

1530: 6016: 4022:"Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization" 265:

Potential when using nasal spray administration of plasmid DNA nanoparticles to transfect non-target cells, such as brain cells

5365:"CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma" 405:

lymph nodes and presents the antigen peptide and costimulatory molecule signalling to T-cell, initiating the immune response.

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Table 4. Comparison of T-dependent antibody responses raise by DNA immunisations, protein inoculations and viral infections

447:) from different pathogens raise cytotoxic T-cell responses to some pathogens, especially if a TH epitope is also included. 6403: 413:

Immunogens can be targeted to various cellular compartments to improve antibody or cytotoxic T-cell responses. Secreted or

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protein is included in the DNA, the resulting vaccine can combine the potency of a live vaccine without reversion risks.

5798: 4556:"DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA" 6845: 6157: 5790: 6257: 2143: 2059: 698: 609:

escape and intracellular cargo concentration. Tested in mice, the hybrid vector was found to induce immune response.

69:(s) against which an immune response is sought, so the cells directly produce the antigen, thus causing a protective 4343:"Long-term maintenance of B cell immunity to influenza virus hemagglutinin in mice following DNA-based immunization" 680: 6938: 6886: 6824: 5799: 1643:

The advantages of genetic adjuvants are their low cost and simple administration, as well as avoidance of unstable

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Obviates need for peptide synthesis, expression and purification of recombinant proteins and use of toxic adjuvants

2838:"Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage" 6445: 6413: 6408: 6242: 5424:"IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides" 5151:"Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration" 4853:"DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state" 1631:

expressing PyCSP. However, co-injection of plasmids encoding GM-CSF (or IFN-γ, or IL-2) and a fusion protein of

1099:(IL-12) production, while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation. 6923: 6503: 6113: 676: 1712:

such as ruminants may be insensitive to immunostimulatory sequences due to their large gastrointestinal load.

380:. Additional modifications to improve expression rates include the insertion of enhancer sequences, synthetic 6301: 6011: 5530:"Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination" 164: 128: 5589:"Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine" 3345:"Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10" 1172:(ER). Targeting gene products directly to the ER (by the addition of an ER insertion signal sequence at the 4605:"The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes" 672: 259:

Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides)

4192:"Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines" 2774: 425:

responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the

1553: 6423: 5865: 5848: 3759:"Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization" 1536: 4804:"Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice" 1535:

DNA vaccination generates an effective immune memory via the display of antigen-antibody complexes on

6473: 1153:. This may prove to be a useful tool in assessing CTL epitopes and their role in providing immunity. 1146: 1142: 756: 4754: 2822: 1399:

cells, these cells were thought to be unique because of their extensive network of T-tubules. Using

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differences in TH type. The type of T-cell help raised is determined by the differentiated state of

6428: 6393: 6388: 6383: 1493: 1141:(CTL) without the inherent risk associated with live vaccines. CTL responses can be raised against 1076: 661: 291: 5056:

Weiss R, Leitner WW, Scheiblhofer S, Chen D, Bernhaupt A, Mostböck S, et al. (October 2000).

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Jones CH, Ravikrishnan A, Chen M, Reddinger R, Kamal Ahmadi M, Rane S, et al. (August 2014).

108:, because they contain a genetic information (DNA or RNA) that codes for the cellular production ( 6874: 6807: 6788: 6584: 6556: 6518: 6513: 6493: 6021: 5528:

Letvin NL, Montefiori DC, Yasutomi Y, Perry HC, Davies ME, Lekutis C, et al. (August 1997).

1760: 1664: 1637: 1056:. (1995) immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mouse 740: 665: 602: 5725:

Hooper JW, Thompson E, Wilhelmsen C, Zimmerman M, Ichou MA, Steffen SE, et al. (May 2004).

4755:"Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations" 4240:

Weiss WR, Ishii KJ, Hedstrom RC, Sedegah M, Ichino M, Barnhart K, et al. (September 1998).

1407:(or, non-clathrin coated pits). However, subsequent research revealed that other cells (such as 1245:

was demonstrated in mice after gene gun delivery. Antibody-secreting cells (ASC) migrate to the

1191:, or mutation of other signal sequences, was shown to be effective at increasing CTL responses. 6928: 6855: 6784: 5783: 2501:"Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein" 1088: 1036: 377: 168: 70: 59: 5709: 5149:

Barouch DH, Santra S, Steenbeke TD, Zheng XX, Perry HC, Davies ME, et al. (August 1998).

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Muthumani K, Zhang D, Dayes NS, Hwang DS, Calarota SA, Choo AY, et al. (September 2003).

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DNA vaccines elicit the best immune response when high-expression vectors are used. These are

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and are thus candidates for DNA uptake. Receptor-mediated DNA uptake could be facilitated by

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vectors for oral administration to the intestinal mucosa and recombinant adenovirus vectors.

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Because the plasmid – carrying relatively small genetic code up to about 200 K

295: 5473:"B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation" 4136:

Restifo NP, Bacík I, Irvine KR, Yewdell JW, McCabe BJ, Anderson RW, et al. (May 1995).

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Leitner WW, Seguin MC, Ballou WR, Seitz JP, Schultz AM, Sheehy MJ, Lyon JA (December 1997).

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US Patent 4722848 - Method for immunizing animals with synthetically modified vaccinia virus

1066:(PyCSP) and found that the initial TH2 response changed, after boosting, to a TH1 response. 6733: 6674: 6465: 6108: 5986: 5937: 5904: 5838: 5600: 5541: 5484: 5471:

Barwick BG, Scharer CD, Martinez RJ, Price MJ, Wein AN, Haines RR, et al. (May 2018).

5376: 5314: 5210: 5014: 4864: 4702: 4603:

Bennet RM, Hefeneider SH, Bakke A, Merritt M, Smith CA, Mourich D, Heinrich MC (May 1988).

4465: 4191: 4033: 3923: 3702: 3645: 3586: 3508: 3453: 3254: 2668: 2618:

Mor G, Klinman DM, Shapiro S, Hagiwara E, Sedegah M, Norman JA, et al. (August 1995).

2512: 2288: 2224: 1785: 1776: 1644: 1119: 440: 144: 109: 5253: 5150: 5108:

Sedegah M, Weiss W, Sacci JB, Charoenvit Y, Hedstrom R, Gowda K, et al. (June 2000).

4803: 4604: 4290: 4241: 4137: 3969: 3758: 3691:"DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations" 3396: 3344: 2619: 1176:) should thus enhance CTL responses. This was successfully demonstrated using recombinant 167:. The study was planned to involve up to 120 subjects aged between 18 and 35. Separately, 8: 6656: 6622: 6508: 6331: 6274: 6269: 6220: 6051: 5919: 5891: 5843: 5646:

Rogers WO, Baird JK, Kumar A, Tine JA, Weiss W, Aguiar JC, et al. (September 2001).

5423: 4959:

Cardoso AI, Blixenkrone-Moller M, Fayolle J, Liu M, Buckland R, Wild TF (November 1996).

4911: 1921: 1792: 1764: 1633: 1400: 1112: 752: 748: 287: 74: 6326: 6071: 5705: 5604: 5545: 5488: 5380: 5318: 5301:

Krieg AM, Wu T, Weeratna R, Efler SM, Love-Homan L, Yang L, et al. (October 1998).

5214: 5018: 4868: 4469: 4037: 3927: 3706: 3649: 3590: 3551: 3512: 3457: 3299:

Widera G, Austin M, Rabussay D, Goldbeck C, Barnett SW, Chen M, et al. (May 2000).

3258: 2516: 2292: 2228: 268:

Cross-contamination when manufacturing different types of live vaccines in same facility

6689: 6523: 6264: 6230: 6215: 6091: 6076: 6061: 5981: 5899: 5505: 5472: 5453: 5283: 5234: 5180: 5038: 4941: 4833: 4784: 4732: 4678: 4653: 4634: 4528: 4503: 4320: 4271: 4219: 4162: 3999: 3947: 3911: 3788: 3671: 3609: 3574: 3532: 3477: 3426: 3374: 3171: 3123: 3098: 3022: 2997: 2810: 2698: 2649: 2597: 2530: 2473: 2448: 2424: 2399: 2372: 2345: 2193: 1964: 1939: 1893: 1868: 1704:, TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL-12. 1485: 1465: 618: 389: 369: 361: 315: 303: 5751: 5726: 5082: 5057: 4580: 4555: 4108: 4083: 3888: 3863: 3839: 3814: 3266: 3218: 3193: 3071: 3046: 2903: 2886: 2862: 2837: 2784: 2566:

Harmon BT, Aly AE, Padegimas L, Sesenoglu-Laird O, Cooper MJ, Waszczak BL (May 2014).

2247: 2212: 2135: 2051: 1194:

CTL responses can be enhanced by co-inoculation with co-stimulatory molecules such as

6933: 6812: 6779: 6612: 6453: 6343: 6237: 6098: 5923: 5886: 5776: 5756: 5677: 5672: 5647: 5628: 5623: 5588: 5569: 5564: 5529: 5510: 5445: 5404: 5399: 5364: 5342: 5337: 5303:"Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs" 5302: 5275: 5226: 5172: 5131: 5087: 5073: 5030: 4982: 4933: 4892: 4887: 4852: 4825: 4776: 4724: 4683: 4626: 4585: 4533: 4481: 4438: 4399: 4364: 4312: 4289:

Tsuji T, Hamajima K, Fukushima J, Xin KQ, Ishii N, Aoki I, et al. (April 1997).

4263: 4211: 4167: 4113: 4061: 4056: 4021: 3991: 3939: 3893: 3862:

Sällberg M, Townsend K, Chen M, O'Dea J, Banks T, Jolly DJ, et al. (July 1997).

3844: 3830: 3780: 3730: 3725: 3690: 3689:

Fynan EF, Webster RG, Fuller DH, Haynes JR, Santoro JC, Robinson HL (December 1993).

3663: 3614: 3524: 3469: 3418: 3366: 3322: 3270: 3223: 3209: 3163: 3128: 3076: 3062: 3027: 2978: 2943: 2908: 2867: 2798: 2788: 2734: 2730: 2690: 2641: 2589: 2548: 2543: 2500: 2478: 2429: 2377: 2304: 2252: 2185: 2149: 2139: 2065: 2055: 1969: 1898: 1842: 1652: 1582:

mixture of 2 plasmids, one encoding the immunogen and the other encoding the cytokine

1208: 1062: 994: 637:. Even partial expression libraries can induce protection from subsequent challenge. 464: 299: 180: 83: 6758: 5742: 5663: 5587:

Sedegah M, Jones TR, Kaur M, Hedstrom R, Hobart P, Tine JA, Hoffman SL (June 1998).

5457: 5287: 5184: 4945: 4837: 4788: 4736: 4638: 4324: 4275: 4223: 4099: 4003: 3879: 3792: 3536: 3481: 3378: 3175: 2853: 2702: 2653: 2197: 952:

Intravenously delivered liposome-DNA complexes can potentially transfect all tissues

841:

Table 3. Advantages and disadvantages of commonly used DNA vaccine delivery methods

179:

In August 2021, Indian authorities gave emergency approval to ZyCoV-D. Developed by

55:

sequence into the cells of an organism as a mechanism to induce an immune response.

6738: 6723: 6579: 6440: 6353: 5746: 5738: 5667: 5659: 5618: 5608: 5559: 5549: 5500: 5492: 5435: 5394: 5384: 5332: 5322: 5265: 5238: 5218: 5162: 5121: 5077: 5069: 5042: 5022: 4972: 4923: 4882: 4872: 4851:

Mancini M, Hadchouel M, Davis HL, Whalen RG, Tiollais P, Michel ML (October 1996).

4815: 4766: 4714: 4673: 4665: 4616: 4575: 4567: 4523: 4515: 4473: 4430: 4391: 4354: 4302: 4253: 4203: 4157: 4149: 4103: 4095: 4051: 4041: 3981: 3951: 3931: 3883: 3875: 3834: 3826: 3770: 3720: 3710: 3675: 3653: 3604: 3594: 3575:"Hybrid biosynthetic gene therapy vector development and dual engineering capacity" 3516: 3461: 3430: 3408: 3356: 3312: 3262: 3213: 3205: 3155: 3118: 3110: 3066: 3058: 3017: 3009: 2970: 2935: 2898: 2857: 2849: 2780: 2726: 2680: 2631: 2601: 2579: 2538: 2520: 2468: 2460: 2419: 2411: 2395: 2367: 2357: 2296: 2242: 2232: 2177: 2131: 2047: 1959: 1951: 1888: 1880: 1549: 1419: 1138: 422: 136: 6279: 4395: 3159: 2974: 2939: 518:

delivery ballistically accelerates plasmid DNA (pDNA) that has been absorbed onto

471:

delivery. Several other techniques have been documented in the intervening years.

368:

promoter was conventionally used until research showed that vectors driven by the

139:

into vaccines that may be able to prevent other diseases. They altered the DNA of

6743: 6541: 6348: 6306: 6197: 6177: 6147: 6130: 6056: 6046: 5976: 5915: 5911: 5860: 5126: 5109: 5026: 4771: 4719: 4207: 3520: 2685: 2449:"DNA vaccination of American robins (Turdus migratorius) against West Nile virus" 1497: 1450: 1438: 1080: 1032: 488: 480: 414: 373: 311: 132: 105: 5270: 5167: 4820: 4621: 4307: 4258: 4153: 3986: 3775: 3413: 3361: 3317: 3300: 2636: 1253:

for long-term antibody production, and generally localise there after one year.

1129:

and is somewhat successful in shifting the response for an established disease.

487:(ID), delivering DNA to extracellular spaces. This can be assisted either 1) by 310:

stability and hence increase protein expression. Plasmids also include a strong

6880: 6607: 6498: 6458: 6338: 6164: 6103: 5593: