Editing DNA vaccine (section)

== 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.<ref name=Feltquate1997 /><ref name=References1996 /><ref name=Cardoso1996>{{cite journal | vauthors = Cardoso AI, Blixenkrone-Moller M, Fayolle J, Liu M, Buckland R, Wild TF | title = Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity | journal = Virology | volume = 225 | issue = 2 | pages = 293–299 | date = November 1996 | pmid = 8918915 | doi = 10.1006/viro.1996.0603 | doi-access = free }}</ref><ref name=Sato1996>{{cite journal | vauthors = Sato Y, Roman M, Tighe H, Lee D, Corr M, Nguyen MD, Silverman GJ, Lotz M, Carson DA, Raz E | display-authors = 6 | title = Immunostimulatory DNA sequences necessary for effective intradermal gene immunization | journal = Science | volume = 273 | issue = 5273 | pages = 352–354 | date = July 1996 | pmid = 8662521 | doi = 10.1126/science.273.5273.352 | s2cid = 9333197 | bibcode = 1996Sci...273..352S }}</ref><ref name=Weiss2000>{{cite journal | vauthors = Weiss R, Leitner WW, Scheiblhofer S, Chen D, Bernhaupt A, Mostböck S, Thalhamer J, Lyon JA | display-authors = 6 | title = Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein | journal = Infection and Immunity | volume = 68 | issue = 10 | pages = 5914–5919 | date = October 2000 | pmid = 10992502 | pmc = 101554 | doi = 10.1128/IAI.68.10.5914-5919.2000 }}</ref> It can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i.e. cytokines, [[lymphokine]]s or co-stimulatory molecules. These "genetic [[adjuvants]]" 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-encoded [[Fusion protein|chimera]], or fusion protein
In general, co-administration of pro-inflammatory agents (such as various [[interleukins]], [[tumor necrosis factor]], and GM-CSF) plus TH2-inducing cytokines increase antibody responses, whereas pro-inflammatory agents and TH1-inducing cytokines decrease humoral responses and increase cytotoxic responses (more important in viral protection). Co-stimulatory molecules such as [[CD80|B7-1]], [[CD86|B7-2]] and [[CD154|CD40L]] are sometimes used.

This concept was applied in topical administration of pDNA encoding [[Interleukin 10|IL-10]].<ref name=Daheshia1997 /> 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.<ref name=Weiss1998 /> This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, followed by boosting with a recombinant [[Poxviridae|poxvirus]] expressing PyCSP.<ref name=Sedegah2000>{{cite journal | vauthors = Sedegah M, Weiss W, Sacci JB, Charoenvit Y, Hedstrom R, Gowda K, Majam VF, Tine J, Kumar S, Hobart P, Hoffman SL | display-authors = 6 | title = Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus | journal = Journal of Immunology | volume = 164 | issue = 11 | pages = 5905–5912 | date = June 2000 | pmid = 10820272 | doi = 10.4049/jimmunol.164.11.5905 | doi-access = free }}</ref> However, co-injection of plasmids encoding GM-CSF (or IFN-γ, or IL-2) and a fusion protein of ''[[Plasmodium chabaudi|P. chabaudi]]'' [[Apicomplexan life cycle|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.<ref name=Wunderlich2000 />

The advantages of genetic adjuvants are their low cost and simple administration, as well as avoidance of unstable [[recombinant cytokine]]s and potentially toxic, "conventional" adjuvants (such as [[alum]], [[calcium phosphate]], monophosphoryl lipid A, [[cholera]] toxin, cationic and mannan-coated liposomes, [[QS21]], [[carboxymethyl cellulose]] and [[ubenimex]]).<ref name=Robinson2000 /><ref name=Lewis1999 /> 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.<ref name=Barouch1998>{{cite journal | vauthors = Barouch DH, Santra S, Steenbeke TD, Zheng XX, Perry HC, Davies ME, Freed DC, Craiu A, Strom TB, Shiver JW, Letvin NL | display-authors = 6 | title = Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration | journal = Journal of Immunology | volume = 161 | issue = 4 | pages = 1875–1882 | date = August 1998 | doi = 10.4049/jimmunol.161.4.1875 | pmid = 9712056 | s2cid = 36488254 | url = http://www.jimmunol.org/cgi/content/abstract/161/4/1875 | doi-access = free }}</ref>

=== Immunostimulatory CpG motifs ===

Plasmid DNA itself appears to have an adjuvant effect on the immune system.<ref name=Alarcon1999 /><ref name=Robinson2000 /> Bacterially derived DNA can trigger innate immune defence mechanisms, the activation of dendritic cells and the production of TH1 cytokines.<ref name=Jakob1998 /><ref name=Krieg1995>{{cite journal | vauthors = Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM | display-authors = 6 | title = CpG motifs in bacterial DNA trigger direct B-cell activation | journal = Nature | volume = 374 | issue = 6522 | pages = 546–549 | date = April 1995 | pmid = 7700380 | doi = 10.1038/374546a0 | s2cid = 4261304 | bibcode = 1995Natur.374..546K }}</ref> This is due to recognition of certain CpG dinucleotide sequences that are immunostimulatory.<ref name=Sato1996 /><ref name=Klinman1997>{{cite journal | vauthors = Klinman DM, Yamshchikov G, Ishigatsubo Y | title = Contribution of CpG motifs to the immunogenicity of DNA vaccines | journal = Journal of Immunology | volume = 158 | issue = 8 | pages = 3635–3639 | date = April 1997 | doi = 10.4049/jimmunol.158.8.3635 | pmid = 9103425 | s2cid = 41861994 | url = http://www.jimmunol.org/cgi/content/abstract/158/8/3635 | doi-access = free }}</ref> 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.<ref name=Krieg1998>{{cite journal | vauthors = Krieg AM, Wu T, Weeratna R, Efler SM, Love-Homan L, Yang L, Yi AK, Short D, Davis HL | display-authors = 6 | title = Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 95 | issue = 21 | pages = 12631–12636 | date = October 1998 | pmid = 9770537 | pmc = 22882 | doi = 10.1073/pnas.95.21.12631 | doi-access = free | bibcode = 1998PNAS...9512631K }}</ref> The optimal immunostimulatory sequence is an unmethylated CpG dinucleotide flanked by two 5’ [[purine]]s and two 3’ [[pyrimidine]]s.<ref name=Sato1996 /><ref name=Krieg1995 /> Additionally, flanking regions outside this immunostimulatory [[Oligomer|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.<ref name=Klinsman1996>{{cite journal | vauthors = Klinman DM, Yi AK, Beaucage SL, Conover J, Krieg AM | title = CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 7 | pages = 2879–2883 | date = April 1996 | pmid = 8610135 | pmc = 39727 | doi = 10.1073/pnas.93.7.2879 | doi-access = free | bibcode = 1996PNAS...93.2879K }}</ref> Stimulated macrophages secrete IL-12, [[IL18BP|IL-18]], TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL-12.<ref name=Lewis1999 /><ref name=Klinsman1996 /><ref name=Yi1996>{{cite journal | vauthors = Yi AK, Chace JH, Cowdery JS, Krieg AM | title = IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides | journal = Journal of Immunology | volume = 156 | issue = 2 | pages = 558–564 | date = January 1996 | doi = 10.4049/jimmunol.156.2.558 | pmid = 8543806 | s2cid = 42145608 | url = http://www.jimmunol.org/cgi/content/abstract/156/2/558 | doi-access = free }}</ref>

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.<ref>{{cite journal | vauthors = Barwick BG, Scharer CD, Martinez RJ, Price MJ, Wein AN, Haines RR, Bally AP, Kohlmeier JE, Boss JM | display-authors = 6 | title = B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation | journal = Nature Communications | volume = 9 | issue = 1 | pages = 1900 | date = May 2018 | pmid = 29765016 | pmc = 5953949 | doi = 10.1038/s41467-018-04234-4 | bibcode = 2018NatCo...9.1900B }}</ref> 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.<ref name=Robinson2000 /><ref name=Letvin1997>{{cite journal | vauthors = Letvin NL, Montefiori DC, Yasutomi Y, Perry HC, Davies ME, Lekutis C, Alroy M, Freed DC, Lord CI, Handt LK, Liu MA, Shiver JW | display-authors = 6 | title = Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 94 | issue = 17 | pages = 9378–9383 | date = August 1997 | pmid = 9256490 | pmc = 23198 | doi = 10.1073/pnas.94.17.9378 | doi-access = free | bibcode = 1997PNAS...94.9378L }}</ref> 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.<ref name=Sedegah1998>{{cite journal | vauthors = Sedegah M, Jones TR, Kaur M, Hedstrom R, Hobart P, Tine JA, Hoffman SL | title = Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 95 | issue = 13 | pages = 7648–7653 | date = June 1998 | pmid = 9636204 | pmc = 22711 | doi = 10.1073/pnas.95.13.7648 | doi-access = free | bibcode = 1998PNAS...95.7648S }}</ref> This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus.<ref name=Sedegah2000 /> An effective prime-boost strategy for the [[simian]] malarial model ''P. knowlesi'' has also been demonstrated.<ref name=Rogers2001>{{cite journal | vauthors = Rogers WO, Baird JK, Kumar A, Tine JA, Weiss W, Aguiar JC, Gowda K, Gwadz R, Kumar S, Gold M, Hoffman SL | display-authors = 6 | title = Multistage multiantigen heterologous prime boost vaccine for Plasmodium knowlesi malaria provides partial protection in rhesus macaques | journal = Infection and Immunity | volume = 69 | issue = 9 | pages = 5565–5572 | date = September 2001 | pmid = 11500430 | pmc = 98670 | doi = 10.1128/IAI.69.9.5565-5572.2001 }}</ref> [[Rhesus monkeys]] were primed with a multicomponent, multistage DNA vaccine encoding two liver-stage antigens – the circumsporozoite surface protein (PkCSP) and sporozoite surface protein 2 (PkSSP2) – and two blood stage antigens – the apical merozoite surface protein 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42). They were then boosted with a recombinant canarypox virus encoding all four antigens (ALVAC-4). Immunized monkeys developed antibodies against sporozoites and infected erythrocytes, and IFN-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 ''P. falciparum'' in 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 into [[antigen-presenting cell]]s.<ref name=Robinson2000 /> This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-co-glycolide) formulated with [[Cetrimonium bromide|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&nbsp;nm in diameter appear to be most efficient for uptake by antigen presenting cells.<ref name=Robinson2000 />

==== Alphavirus vectors ====

Recombinant alphavirus-based [[Viral vector vaccine|vectors]] have been used to improve DNA vaccination efficiency.<ref name=Robinson2000 /> 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]] [[Replicon (genetics)|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.