DNA vaccine

ADNA vaccineis a type ofvaccinethattransfectsa specificantigen-codingDNAsequence into the cells of an organism as a mechanism to induce an immune response.^[1]^[2]

DNA vaccines work by injectinggenetically engineered plasmidcontaining the DNA sequence encoding theantigen(s) against which an immune response is sought, so the cells directly produce the antigen, thus causing a protectiveimmunological response.^[3]DNA vaccines have theoretical advantages over conventional vaccines, including the "ability to induce a wider range of types of immune response".^[4]Several DNA vaccines have been tested forveterinaryuse.^[3]In some cases, protection from disease in animals has been obtained, in others not.^[3]Research is ongoing over the approach for viral, bacterial and parasitic diseases in humans, as well as for cancers.^[4]In August 2021, Indian authorities gave emergency approval toZyCoV-D.Developed byCadila Healthcare,it is the first DNA vaccine approved for humans.^[5]

History

Conventional vaccines contain either specificantigensfrom a pathogen, or attenuated viruses which stimulate an immune response in the vaccinated organism. DNA vaccines are members of thegenetic vaccines,because they contain a genetic information (DNA or RNA) that codes for the cellular production (protein biosynthesis) of anantigen.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.^[6]^[7]Alternatively, the DNA may be encapsulated in protein to facilitate cell entry. If thiscapsidprotein is included in the DNA, the resulting vaccine can combine the potency of a live vaccine without reversion risks.^{[citation needed]}

In 1983,Enzo Paolettiand Dennis Panicali at theNew York Department of Healthdevised a strategy to producerecombinant DNAvaccines by using genetic engineering to transform ordinarysmallpox vaccineinto vaccines that may be able to prevent other diseases.^[8]They altered the DNA ofcowpoxvirus by inserting a gene from other viruses (namelyHerpes simplex virus,hepatitis Bandinfluenza).^[9]^[10]In 1993, Jeffrey Ulmer and co-workers atMerck Research Laboratoriesdemonstrated that direct injection of mice with plasmid DNA encoding a flu antigen protected the animals against subsequent experimental infection with influenza virus.^[11]In 2016 a DNA vaccine for theZika virusbegan testing in humans at theNational Institutes of Health.The study was planned to involve up to 120 subjects aged between 18 and 35. Separately,Inovio PharmaceuticalsandGeneOne Life Sciencebegan 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.^[12]Clinical trials for DNA vaccines to prevent HIV are underway.^[13]

In August 2021, Indian authorities gave emergency approval to ZyCoV-D. Developed byCadila Healthcare,it is the first DNA vaccine againstCOVID-19.^[5]

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 bothMHC class Iandclass IImolecules^[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 vivoexpression 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 areplasmidsthat usually consist of a strong viralpromoterto drive the in vivotranscriptionandtranslationof the gene (orcomplementary DNA) of interest.^[18]Intron Amay sometimes be included to improvemRNAstability and hence increase protein expression.^[19]Plasmids also include a strongpolyadenylation/transcriptionaltermination signal,such as bovinegrowth hormoneor rabbitbeta-globulinpolyadenylation sequences.^[6]^[7]^[20]Polycistronicvectors (with multiple genes of interest) are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.^[21]

Because the plasmid – carrying relatively small genetic code up to about 200 Kbp– is the "vehicle" from which the immunogen is expressed, optimising vector design for maximal protein expression is essential.^[21]One way of enhancing protein expression is by optimising thecodonusage of pathogenic mRNAs foreukaryoticcells. Pathogens often have differentAT-contentsthan the target species, so altering thegene sequenceof the immunogen to reflect thecodonsmore commonly used in the target species may improve its expression.^[22]

Another consideration is the choice ofpromoter.TheSV40promoter was conventionally used until research showed that vectors driven by theRous Sarcoma Virus(RSV) promoter had much higher expression rates.^[6]More recently, expression and immunogenicity have been further increased in model systems by the use of thecytomegalovirus(CMV) immediate early promoter, and a retroviralcis-acting transcriptional element.^[23]Additional modifications to improve expression rates include the insertion of enhancer sequences, syntheticintrons,adenovirustripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences.^[6]An example of DNA vaccine plasmid is pVAC, which uses SV40promoter.

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 orplasma membrane-bound antigens are more effective at inducing antibody responses thancytosolicantigens, whilecytotoxic T-cellresponses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into themajor histocompatibility complex(MHC) class I pathway.^[7]This is usually accomplished by the addition ofN-terminal ubiquitinsignals.^[27]^[28]^[29]

Theconformationof the protein can also affect antibody responses. "Ordered" structures (such as viral particles) are more effective than unordered structures.^[30]Strings of minigenes (or MHC class Iepitopes) from different pathogens raise cytotoxic T-cell responses to some pathogens, especially if a TH epitope is also included.^[7]

Delivery

DNA vaccines have been introduced into animal tissues by multiple methods. In 1999, the two most popular approaches were injection of DNA insaline:by using a standard hypodermic needle, or by using agene gundelivery.^[31]Several other techniques have been documented in the intervening years.

Saline injection

Injection in saline is normally conducted intramuscularly (IM) inskeletal muscle,orintradermally(ID), delivering DNA to extracellular spaces. This can be assisted either 1) byelectroporation;^[32]2) by temporarily damaging muscle fibres withmyotoxinssuch asbupivacaine;or 3) by usinghypertonicsolutions of saline orsucrose.^[6]Immune responses to this method can be affected by factors including needle type,^[16]needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the recipient.^[6]

Gene gun

Gene gundelivery ballistically accelerates plasmid DNA (pDNA) that has been absorbed ontogoldortungstenmicroparticles into the target cells, using compressedheliumas an accelerant.^[6]^[21]

Mucosal surface delivery

Alternatives includedaerosolinstillation of naked DNA onmucosalsurfaces, such as thenasalandlung mucosa,^[21]and topical administration of pDNA to the eye^[33]and vaginal mucosa.^[21]Mucosal surface delivery has also been achieved using cationicliposome-DNA preparations,^[7]biodegradablemicrospheres,^[34]^[21]attenuatedSalmonalla,^[35]ShigellaorListeriavectors for oral administration to the intestinal mucosa^[36]and recombinant adenovirus vectors.^[21]

Polymer vehicle

A hybrid vehicle composed of bacteria cell and syntheticpolymershas been employed for DNA vaccine delivery. AnE. coliinner core and poly(beta-amino ester) outer coat function synergistically to increase efficiency by addressing barriers associated withantigen-presenting cellgene delivery which include cellular uptake and internalization,phagosomalescape and intracellular cargo concentration.^[jargon]Tested in mice, the hybrid vector was found to induce immune response.^[37]^[38]

ELI immunization

Another approach to DNA vaccination isexpression libraryimmunization (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.^[6]ELI can be used to identify which genes induce a protective response. This has been tested withMycoplasmapulmonis,amurinelung pathogen with a relatively smallgenome.Even partial expression libraries can induce protection from subsequent challenge.^[39]

Helpful tabular comparison

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,subcutaneousandintraperitonealwith 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]		Liposomes(cationic); microspheres; recombinant adenovirus vectors; attenuatedShigellavector; aerosolised cationiclipidformulations	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 μg)
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 orimmune reactions^{[clarification needed]}

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.^[40]Generally, 0.2 μg – 20 μg are required, although quantities as low as 16 ng have been reported.^[6]These quantities vary by species. Mice for example, require approximately 10 times less DNA thanprimates.^[7]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 thebasal laminaand large amounts ofconnective tissue) before it is taken up by the cells, while gene gun deliveries drive/force DNA directly into the cells, resulting in less "wastage".^[6]^[7]

Immune response

Helper T cell responses

DNA immunization can raise multiple T_Hresponses, including lymphoproliferation and the generation of a variety ofcytokineprofiles. 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.^[41]Each type has distinctive patterns oflymphokineand chemokine expression, specific types ofimmunoglobulins,patterns of lymphocyte trafficking and types ofinnate immune responses.

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, Moret al..(1995)^[18]immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mousemalarialparasitePlasmodium 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.^[41]The type of T-cell help raised is determined by the differentiated state ofantigen presenting cells.Dendritic cellscan differentiate to secreteIL-12(which supports TH1 cell development) orIL-4(which supports TH2 responses).^[44]pDNA injected by needle isendocytosedinto the dendritic cell, which is then stimulated to differentiate for TH1cytokine(IL-12) production,^[45]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 influencingallergicresponses andautoimmune diseases.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 inpredisease primingfor the desired type of response inpreclinical models^[7]and is somewhat successful in shifting the response for an established disease.^[46]

Cytotoxic T-cell responses

One of the advantages of DNA vaccines is that they are able to inducecytotoxic T lymphocytes(CTL) without the inherent risk associated with live vaccines. CTL responses can be raised againstimmunodominantand immunorecessive CTL epitopes,^[47]as well assubdominant CTL epitopes,^[34]^[jargon]in a manner that appears to mimic naturalinfection.This may prove to be a useful tool in assessing CTL epitopes and their role in providing immunity.

Cytotoxic T-cells recognise smallpeptides(8-10amino acids) complexed toMHC class Imolecules.^[48]These peptides are derived from cytosolic proteins that are degraded and delivered to the nascent MHC class I molecule within theendoplasmic reticulum(ER).^[48]Targeting gene products directly to the ER (by the addition of an ER insertion signal sequence at theN-terminus) should thus enhance CTL responses. This was successfully demonstrated using recombinantvacciniaviruses expressinginfluenzaproteins,^[48]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 ofubiquitin signal sequences,or mutation of other signal sequences, was shown to be effective at increasing CTL responses.^[28]

CTL responses can be enhanced by co-inoculation with co-stimulatory molecules such asB7-1orB7-2for DNA vaccines against influenza nucleoprotein,^[47]^[49]orGM-CSFfor DNA vaccines against the murine malaria modelP. yoelii.^[50]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.^[49]^[51]

Humoral (antibody) response

Schematic diagram of an antibody and antigens

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 againsthepatitis Bvirus (HBV) envelope protein (HBsAg) have been sustained for up to 74 weeks without boost, while lifelong maintenance of protective response to influenzahaemagglutininwas demonstrated in mice after gene gun delivery.^[52]Antibody-secreting cells (ASC) migrate to thebone marrowandspleenfor long-term antibody production, and generally localise there after one year.^[52]

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 bothprimary 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]

Table 4. Comparison of T-dependent antibody responses raise by DNA immunisations, protein inoculations and viral infections
	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 highavidity IgGand migration ofASCto 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 tonativeepitopes 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 demonstratedin vivoinmusclecells,^[54]these cells were thought to be unique because of their extensive network of T-tubules. Usingelectron microscopy,it was proposed that DNA uptake was facilitated bycaveolae(or, non-clathrin coated pits).^[55]However, subsequent research revealed that other cells (such askeratinocytes,fibroblastsandepithelial Langerhans cells) could also internalize DNA.^[46]^[56]The mechanism of DNA uptake is not known.

Two theories dominate – thatin vivouptake of DNA occurs non-specifically, in a method similar tophago- orpinocytosis,^[21]or through specific receptors.^[57]These might include a 30kDa surfacereceptor,ormacrophagescavenger receptors.^{[clarification needed]}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 polyribonucleotidesand are thus candidates for DNA uptake.^[57]^[58]Receptor-mediated DNA uptake could be facilitated bythe presence ofpolyguanylate sequences.^{[clarification needed]}^{[citation needed]}Gene gundelivery systems,cationic liposome packaging,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.

Antigen presentation by bone marrow-derived cells

Studies usingchimericmice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialisedB-cellscalled professionalantigen presenting cells(APC).^[49]^[59]After gene gun inoculation to the skin, transfectedLangerhans cellsmigrate to the draininglymph nodeto present antigens.^[7]After IM and ID injections, dendritic cells present antigen in the draining lymph node^[56]and transfected macrophages have been found in the peripheral blood.^[60]

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 onfollicular 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 asIFN-γandTNF-α,which do not kill the cell, but limit viral infection by down-regulating the expression of viral components.^[62]DNA vaccinations can be used to curb viral infections by non-destructive IFN-mediated control. This was demonstrated for hepatitis B.^[63]IFN-γ is critically important in controlling malaria infections^[64]and is a consideration for anti-malarial DNA vaccines.

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.^[41]^[42]^[65]^[66]^[67]It can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i.e. cytokines,lymphokinesor co-stimulatory molecules. These "geneticadjuvants"can be administered as a:

mixture of 2 plasmids, one encoding the immunogen and the other encoding the cytokine
single bi- or polycistronic vector, separated by spacer regions
plasmid-encodedchimera,or fusion protein

In general, co-administration of pro-inflammatory agents (such as variousinterleukins,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 (more important in viral protection). Co-stimulatory molecules such asB7-1,B7-2andCD40Lare sometimes used.

This concept was applied in topical administration of pDNA encodingIL-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 ofP. 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 recombinantpoxvirusexpressing PyCSP.^[68]However, co-injection of plasmids encoding GM-CSF (or IFN-γ, or IL-2) and a fusion protein ofP. chabaudimerozoite surface protein1 (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 unstablerecombinant cytokinesand potentially toxic, "conventional" adjuvants (such asalum,calcium phosphate,monophosphoryl lipid A,choleratoxin, cationic and mannan-coated liposomes,QS21,carboxymethyl celluloseandubenimix).^[7]^[21]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.^[69]

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’purinesand two 3’pyrimidines.^[66]^[70]Additionally, flanking regions outside this immunostimulatoryhexamermust beguanine-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 encodingPlasmodium yoeliicircumsporozoite 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.^[77]This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus.^[68]An effective prime-boost strategy for thesimianmalarial modelP. knowlesihas also been demonstrated.^[78]Rhesus monkeyswere 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-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 toP. falciparumin humans, will be important in pre-clinical trials.

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 intoantigen-presenting cells.^[7]This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated withcetyltrimethylammonium 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-basedvectorshave 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. TheSindbis virusandSemliki Forest virushave been used to build recombinantalphavirus 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.

References

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