DNA vaccine
A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.[1][2]
DNA vaccines work by injecting
History
Conventional vaccines contain either specific
In 1983,
In August 2021, Indian authorities gave emergency approval to ZyCoV-D. Developed by
Applications
As of 2021[update] 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.
A veterinary DNA vaccine to protect horses from West Nile virus has been approved.[14] Another West Nile virus vaccine has been tested successfully on American robins.[15]
DNA immunization is also being investigated as a means of developing antivenom sera.[1] DNA immunization can be used as a technology platform for monoclonal antibody induction.[2]
Advantages
- No risk for infections[7]
- Antigen presentation by both MHC class I and class II molecules[7]
- Polarise T-cell response toward type 1 or type 2[7]
- Immune response focused on the antigen of interest
- Ease of development and production[7]
- Stability for storage and shipping
- Cost-effectiveness
- Obviates need for peptide synthesis, expression and purification of recombinant proteins and use of toxic adjuvants[16]
- Long-term persistence of immunogen[6]
- In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications[6]
Disadvantages
- Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides)
- Potential for atypical processing of bacterial and parasite proteins[7]
- Potential when using nasal spray administration of plasmid DNA nanoparticles to transfect non-target cells, such as brain cells[17]
- Cross-contamination when manufacturing different types of live vaccines in same facility
Plasmid vectors
Vector design
DNA vaccines elicit the best immune response when high-expression vectors are used. These are
Because the plasmid – carrying relatively small genetic code up to about 200 K
Another consideration is the choice of
Structural instability phenomena are of particular concern for plasmid manufacture, DNA vaccination and gene therapy.[24] 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, 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.[25]
Mechanism of plasmids
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 lymph nodes and presents the antigen peptide and costimulatory molecule signalling to T-cell, initiating the immune response.[26]
Vaccine insert design
Immunogens can be targeted to various cellular compartments to improve antibody or cytotoxic T-cell responses. Secreted or
The
Delivery
DNA vaccines have been introduced into animal tissues by multiple methods. In 1999, the two most popular approaches were injection of DNA in saline: by using a standard hypodermic needle, or by using a gene gun delivery.[31] Several other techniques have been documented in the intervening years.
Saline injection
Injection in saline is normally conducted intramuscularly (IM) in
Gene gun
Gene gun delivery ballistically accelerates plasmid DNA (pDNA) that has been absorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant.[6][21]
Mucosal surface delivery
Alternatives included
Polymer vehicle
A hybrid vehicle composed of bacteria cell and synthetic
ELI immunization
Another approach to DNA vaccination is
Helpful tabular comparison
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 μg) |
Gene gun | DNA-coated gold beads | ED (abdominal skin); vaginal mucosa; surgically exposed muscle and other organs | Small amounts (as little as 16 ng) | |
Pneumatic (jet) injection | Aqueous solution | ED | Very high (as much as 300 μg) | |
Topical application | Aqueous solution | Ocular; intravaginal | Small amounts (up to 100 μg) | |
Cytofectin-mediated[jargon] | formulations | IM; IV (to transfect tissues systemically); intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes | variable |
Method of delivery | Advantage | Disadvantage |
---|---|---|
Intramuscular or Intradermal injection |
|
|
Gene gun |
|
|
Jet injection |
|
|
Liposome-mediated delivery |
|
|
Dosage
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.
Immune response
Helper T cell responses
DNA immunization can raise multiple TH responses, including lymphoproliferation and the generation of a variety of
Other types of T-cell help
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.[6][42] Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses.[41][42] 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.[43]
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.[41][42] However, Mor et al.. (1995)[18] 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.
Basis for different types of T-cell help
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 differences in TH type. while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation.
Practical uses of polarised T-cell help
Polarisation in T-cell help is useful in influencing
Cytotoxic T-cell responses
One of the 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,[47] as well as subdominant CTL epitopes,[34][jargon] in a manner that appears to mimic natural infection. This may prove to be a useful tool in assessing CTL epitopes and their role in providing immunity.
Cytotoxic T-cells recognise small
CTL responses can be enhanced by co-inoculation with co-stimulatory molecules such as
Humoral (antibody) response
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.
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
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 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 primary and secondary phases of antibody response.[clarification needed] 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 robust enough to control HBV infection[53]
Method of Immunization | |||
---|---|---|---|
DNA vaccine | Recombinant protein | Natural infection | |
Amount of inducing antigen | ng | μg | ? (ng-μg) |
Duration of antigen presentation | several weeks | < 1 week | several weeks |
Kinetics of antibody response | slow rise | rapid rise | rapid rise |
Number of inoculations to obtain high 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. 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 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.[citation needed]
Mechanistic basis for DNA-raised immune responses
DNA uptake mechanism
When DNA uptake and subsequent expression was first demonstrated in vivo in
The mechanism of DNA uptake is not known.Two theories dominate – that in vivo uptake of DNA occurs non-specifically, in a method similar to
Antigen presentation by bone marrow-derived cells
Studies using
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 T-cell responses and seems to be important for a full primary immune response.[7][61]
Target site role
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 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.[21][54][61]
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.[7]
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
Immune response modulation
Cytokine modulation
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. secreted), the method and route of delivery or the dose.
- mixture of 2 plasmids, one encoding the immunogen and the other encoding the cytokine
- single bi- or polycistronic vector, separated by spacer regions
- plasmid-encoded chimera, or fusion protein
In general, co-administration of pro-inflammatory agents (such as various
This concept was applied in topical administration of pDNA encoding IL-10.[33] 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 P. yoelii (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.[50] This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, followed by boosting with a recombinant poxvirus expressing PyCSP.[68] However, co-injection of plasmids encoding GM-CSF (or IFN-γ, or IL-2) and a fusion protein of P. chabaudi merozoite surface protein 1 (C-terminus)-hepatitis B virus surface protein (PcMSP1-HBs) abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone.[30]
The advantages of genetic adjuvants are their low cost and simple administration, as well as avoidance of unstable
Immunostimulatory CpG motifs
Plasmid DNA itself appears to have an adjuvant effect on the immune system.[6][7] Bacterially derived DNA can trigger innate immune defence mechanisms, the activation of dendritic cells and the production of TH1 cytokines.[45][70] This is due to recognition of certain CpG dinucleotide sequences that are immunostimulatory.[66][71] 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.[72] The optimal immunostimulatory sequence is an unmethylated CpG dinucleotide flanked by two 5’ purines and two 3’ pyrimidines.[66][70] Additionally, flanking regions outside this immunostimulatory hexamer must be guanine-rich to ensure binding and uptake into target cells.
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.[73] Stimulated macrophages secrete IL-12, IL-18, TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL-12.[21][73][74]
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 demonstrated the stimulation of polyclonal B-cell expansion.[75] 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 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 such as ruminants may be insensitive to immunostimulatory sequences due to their large gastrointestinal load.
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.[7][76] 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-γ, and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone.
Enhancing immune responses
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.[7] 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 viruses, especially when mixed with alum. Particles 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells.[7]
Alphavirus vectors
Recombinant alphavirus-based vectors have been used to improve DNA vaccination efficiency.[7] 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 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.
See also
- Vector DNA
- HIV vaccine
- Gene therapy
- mRNA vaccine
References
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Further reading
- Hooper JW, Thompson E, Wilhelmsen C, Zimmerman M, Ichou MA, Steffen SE, et al. (May 2004). "Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox". Journal of Virology. 78 (9): 4433–4443. PMID 15078924.