Editing DNA vaccine (section)

== Plasmid vectors ==

=== Vector design ===

DNA vaccines elicit the best immune response when high-expression vectors are used. These are [[plasmids]] that usually consist of a strong viral [[Promoter (genetics)|promoter]] to drive the in vivo [[Transcription (genetics)|transcription]] and [[Translation (biology)|translation]] of the gene (or [[complementary DNA]]) of interest.<ref name=Mor1995>{{cite journal | vauthors = Mor G, Klinman DM, Shapiro S, Hagiwara E, Sedegah M, Norman JA, Hoffman SL, Steinberg AD | display-authors = 6 | title = Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA | journal = Journal of Immunology | volume = 155 | issue = 4 | pages = 2039–2046 | date = August 1995 | doi = 10.4049/jimmunol.155.4.2039 | pmid = 7636255 | s2cid = 37290980 | url = http://www.jimmunol.org/cgi/content/abstract/155/4/2039 | doi-access = free }}</ref> [[Interferon alfa-2b|Intron A]] may sometimes be included to improve [[mRNA]] stability and hence increase protein expression.<ref name=Leitner1997>{{cite journal | vauthors = Leitner WW, Seguin MC, Ballou WR, Seitz JP, Schultz AM, Sheehy MJ, Lyon JA | title = Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites | journal = Journal of Immunology | volume = 159 | issue = 12 | pages = 6112–6119 | date = December 1997 | doi = 10.4049/jimmunol.159.12.6112 | pmid = 9550412 | s2cid = 37685499 | url = http://www.jimmunol.org/cgi/content/abstract/159/12/6112 | doi-access = free }}</ref> Plasmids also include a strong [[polyadenylation]]/transcriptional [[termination signal]], such as bovine [[growth hormone]] or rabbit [[beta-globulin]] polyadenylation sequences.<ref name=Alarcon1999 /><ref name=Robinson2000 /><ref name="Böhm1996">{{cite journal | vauthors = Böhm W, Kuhröber A, Paier T, Mertens T, Reimann J, Schirmbeck R | title = DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection | journal = Journal of Immunological Methods | volume = 193 | issue = 1 | pages = 29–40 | date = June 1996 | pmid = 8690928 | doi = 10.1016/0022-1759(96)00035-X }}</ref> [[Cistron|Polycistronic]] vectors (with multiple genes of interest) are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.<ref name=Lewis1999>{{cite journal | vauthors = Lewis PJ, Babiuk LA | title = DNA vaccines: a review | journal = Advances in Virus Research | volume = 54 | pages = 129–88 | year = 1999 | pmid = 10547676 | doi = 10.1016/S0065-3527(08)60367-X | url = https://books.google.com/books?id=lrMc3G9nIpkC&q=lewis&pg=PA129 | publisher = Academic Press | isbn = 978-0-12-039854-6 }}</ref>

Because the plasmid{{snd}}carrying relatively small genetic code up to about 200 K[[Base pair|bp]]{{snd}}is the "vehicle" from which the immunogen is expressed, optimising vector design for maximal protein expression is essential.<ref name=Lewis1999 /> One way of enhancing protein expression is by optimising the [[codon]] usage of pathogenic mRNAs for [[eukaryotic]] cells. Pathogens often have different [[GC-content|AT-contents]] than the target species, so altering the [[gene sequence]] of the immunogen to reflect the [[genetic code|codon]]s more commonly used in the target species may improve its expression.<ref name=Andre1998>{{cite journal | vauthors = André S, Seed B, Eberle J, Schraut W, Bültmann A, Haas J | title = Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage | journal = Journal of Virology | volume = 72 | issue = 2 | pages = 1497–1503 | date = February 1998 | pmid = 9445053 | pmc = 124631 | doi = 10.1128/JVI.72.2.1497-1503.1998 }}</ref>

Another consideration is the choice of [[promoter (biology)|promoter]]. The [[SV40]] promoter was conventionally used until research showed that vectors driven by the [[Rous Sarcoma Virus]] (RSV) promoter had much higher expression rates.<ref name=Alarcon1999 /> More recently, expression and immunogenicity have been further increased in model systems by the use of the [[cytomegalovirus]] (CMV) immediate early promoter, and a retroviral [[Cis-regulatory element|cis-acting transcriptional element]].<ref name=Muthumani1998>{{cite journal | vauthors = Muthumani K, Zhang D, Dayes NS, Hwang DS, Calarota SA, Choo AY, Boyer JD, Weiner DB | display-authors = 6 | title = Novel engineered HIV-1 East African Clade-A gp160 plasmid construct induces strong humoral and cell-mediated immune responses in vivo | journal = Virology | volume = 314 | issue = 1 | pages = 134–146 | date = September 2003 | pmid = 14517067 | doi = 10.1016/S0042-6822(03)00459-8 | doi-access = free }}</ref> Additional modifications to improve expression rates include the insertion of enhancer sequences, synthetic [[introns]], [[adenovirus]] tripartite leader (TPL) sequences and modifications to the polyadenylation and transcriptional termination sequences.<ref name=Alarcon1999 /> An example of DNA vaccine plasmid is pVAC, which uses SV40 [[promoter (biology)|promoter]].

Structural instability phenomena are of particular concern for plasmid manufacture, DNA vaccination and gene therapy.<ref>{{cite journal | vauthors = Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA | title = Structural instability of plasmid biopharmaceuticals: challenges and implications | journal = Trends in Biotechnology | volume = 27 | issue = 9 | pages = 503–511 | date = September 2009 | pmid = 19656584 | doi = 10.1016/j.tibtech.2009.06.004 }}</ref> 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.<ref>{{cite journal | vauthors = Oliveira PH, Mairhofer J | title = Marker-free plasmids for biotechnological applications - implications and perspectives | journal = Trends in Biotechnology | volume = 31 | issue = 9 | pages = 539–547 | date = September 2013 | pmid = 23830144 | doi = 10.1016/j.tibtech.2013.06.001 }}</ref>

=== 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.<ref>{{cite journal | vauthors = Kutzler MA, Weiner DB | title = DNA vaccines: ready for prime time? | journal = Nature Reviews. Genetics | volume = 9 | issue = 10 | pages = 776–788 | date = October 2008 | pmid = 18781156 | pmc = 4317294 | doi = 10.1038/nrg2432 }}</ref>

=== Vaccine insert design ===

Immunogens can be targeted to various cellular compartments to improve antibody or cytotoxic T-cell responses. Secreted or [[plasma membrane]]-bound antigens are more effective at inducing antibody responses than [[cytosolic]] antigens, while [[cytotoxic T-cell]] responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the [[major histocompatibility complex]] (MHC) class I pathway.<ref name=Robinson2000 /> This is usually accomplished by the addition of [[N-terminal]] [[ubiquitin]] signals.<ref name=Rodriguez1997>{{cite journal | vauthors = Rodriguez F, Zhang J, Whitton JL | title = DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction | journal = Journal of Virology | volume = 71 | issue = 11 | pages = 8497–8503 | date = November 1997 | pmid = 9343207 | pmc = 192313 | doi = 10.1128/JVI.71.11.8497-8503.1997 }}</ref><ref name=Tobery1997>{{cite journal | vauthors = Tobery TW, Siliciano RF | title = 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 | journal = The Journal of Experimental Medicine | volume = 185 | issue = 5 | pages = 909–920 | date = March 1997 | pmid = 9120397 | pmc = 2196169 | doi = 10.1084/jem.185.5.909 }}</ref><ref>{{cite journal | vauthors = Huebener N, Fest S, Strandsby A, Michalsky E, Preissner R, Zeng Y, Gaedicke G, Lode HN | display-authors = 6 | title = A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity | journal = Molecular Cancer Therapeutics | volume = 7 | issue = 7 | pages = 2241–2251 | date = July 2008 | pmid = 18645033 | doi = 10.1158/1535-7163.MCT-08-0109 | doi-access =  | s2cid = 35652424 | author-link8 = Lode HN, Fest S, Strandsby A, Michalsky E, Preissner R, Zeng Y, Gaedicke G, Lode HN }}</ref>

The [[protein conformation|conformation]] of the protein can also affect antibody responses. "Ordered" structures (such as viral particles) are more effective than unordered structures.<ref name=Wunderlich2000>{{cite journal | vauthors = Wunderlich G, Moura IC, del Portillo HA | title = 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 | journal = Infection and Immunity | volume = 68 | issue = 10 | pages = 5839–5845 | date = October 2000 | pmid = 10992493 | pmc = 101545 | doi = 10.1128/IAI.68.10.5839-5845.2000 }}</ref> Strings of minigenes (or MHC class I [[epitopes]]) from different pathogens raise cytotoxic T-cell responses to some pathogens, especially if a TH epitope is also included.<ref name=Robinson2000 />