Editing DNA vaccine (section)

== Mechanistic basis for DNA-raised immune responses ==

=== DNA uptake mechanism ===

When DNA uptake and subsequent expression was first demonstrated ''in vivo'' in [[muscle]] cells,<ref name=Wolff1992>{{cite journal | vauthors = Wolff JA, Dowty ME, Jiao S, Repetto G, Berg RK, Ludtke JJ, Williams P, Slautterback DB | display-authors = 6 | title = Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle | journal = Journal of Cell Science | volume = 103 | issue = 4 | pages = 1249–1259 | date = December 1992 | pmid = 1487500 | doi = 10.1242/jcs.103.4.1249 | series = 103 }}</ref> these cells were thought to be unique because of their extensive network of T-tubules. Using [[electron microscopy]], it was proposed that DNA uptake was facilitated by [[caveolae]] (or, non-clathrin coated pits).<ref name=Anderson1992>{{cite journal | vauthors = Anderson RG, Kamen BA, Rothberg KG, Lacey SW | title = Potocytosis: sequestration and transport of small molecules by caveolae | journal = Science | volume = 255 | issue = 5043 | pages = 410–411 | date = January 1992 | pmid = 1310359 | doi = 10.1126/science.1310359 | bibcode = 1992Sci...255..410A }}</ref> However, subsequent research revealed that other cells (such as [[keratinocytes]], [[fibroblasts]] and [[epithelial]] [[Langerhans cells]]) could also internalize DNA.<ref name=Raz1996/><ref name=Casares1997>{{cite journal | vauthors = Casares S, Inaba K, Brumeanu TD, Steinman RM, Bona CA | title = Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope | journal = The Journal of Experimental Medicine | volume = 186 | issue = 9 | pages = 1481–1486 | date = November 1997 | pmid = 9348305 | pmc = 2199124 | doi = 10.1084/jem.186.9.1481 }}</ref> 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 [[phagocytosis|phago]]- or [[pinocytosis]],<ref name=Lewis1999 /> or through specific receptors.<ref name=Bennett1985>{{cite journal | vauthors = Bennett RM, Gabor GT, Merritt MM | title = DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA | journal = The Journal of Clinical Investigation | volume = 76 | issue = 6 | pages = 2182–2190 | date = December 1985 | pmid = 3001145 | pmc = 424340 | doi = 10.1172/JCI112226 }}</ref> These might include a 30kDa surface [[immune receptor|receptor]], or {{Clarify | text = [[macrophage]] scavenger receptors. | date = November 2021 | reason = Unclear if 'scavanger' is a modifer for macrophage, or receptor, i.e. does scavenger meant to indicate that macrophage is a scavanger, or meant to indicate specific types of macrophage's receptors?}} 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 poly[[ribonucleotides]] and are thus candidates for DNA uptake.<ref name=Bennett1985 /><ref name=Bennet1988>{{cite journal | vauthors = Bennet RM, Hefeneider SH, Bakke A, Merritt M, Smith CA, Mourich D, Heinrich MC | title = The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes | journal = Journal of Immunology | volume = 140 | issue = 9 | pages = 2937–2942 | date = May 1988 | doi = 10.4049/jimmunol.140.9.2937 | pmid = 2452195 | s2cid = 22923379 | url = http://www.jimmunol.org/cgi/content/abstract/140/9/2937 | doi-access = free }}</ref> Receptor-mediated DNA uptake could be facilitated by {{Clarify | text = the presence of [[guanine|polyguanylate sequences]]. | date = October 2021 | reason = Where are the said polyguanylate sequences?  Presumably DNA.}}{{Citation needed | date = October 2021}} [[Gene gun]] delivery systems, [[Transfection#Lipofection|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 ===

[[Image:Dendritic cell.JPG|thumb|right|A dendritic cell]]

Studies using [[Chimera (genetics)|chimeric]] mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialised [[B-cells]] called professional [[antigen presenting cells]] (APC).<ref name=Iwasaki1997 /><ref name=Corr1996>{{cite journal | vauthors = Corr M, Lee DJ, Carson DA, Tighe H | title = Gene vaccination with naked plasmid DNA: mechanism of CTL priming | journal = The Journal of Experimental Medicine | volume = 184 | issue = 4 | pages = 1555–1560 | date = October 1996 | pmid = 8879229 | pmc = 2192808 | doi = 10.1084/jem.184.4.1555 }}</ref> After gene gun inoculation to the skin, transfected [[Langerhans cell]]s migrate to the draining [[lymph node]] to present antigens.<ref name=Robinson2000 /> After IM and ID injections, dendritic cells present antigen in the draining lymph node<ref name=Casares1997 /> and transfected macrophages have been found in the peripheral blood.<ref name=Chattergoon1998>{{cite journal | vauthors = Chattergoon MA, Robinson TM, Boyer JD, Weiner DB | title = Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells | journal = Journal of Immunology | volume = 160 | issue = 12 | pages = 5707–5718 | date = June 1998 | doi = 10.4049/jimmunol.160.12.5707 | pmid = 9637479 | s2cid = 33499198 | url = http://www.jimmunol.org/cgi/content/abstract/160/12/5707 | doi-access = free }}</ref>

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.<ref name=Robinson2000 /><ref name=Torres1997>{{cite journal | vauthors = Torres CA, Iwasaki A, Barber BH, Robinson HL | title = Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations | journal = Journal of Immunology | volume = 158 | issue = 10 | pages = 4529–4532 | date = May 1997 | doi = 10.4049/jimmunol.158.10.4529 | pmid = 9144463 | s2cid = 45069087 | url = http://www.jimmunol.org/cgi/pmidlookup?view=long&pmid=9144463 | doi-access = free }}</ref>

=== 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.<ref name=Lewis1999 /><ref name=Wolff1992 /><ref name=Torres1997 />

=== Maintenance of immune response ===
{{see also|Follicular dendritic cells#Antigen capturing, memory B-cell support|Follicular dendritic cells#Interaction with B-cells}}
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.<ref name=Robinson2000 />

=== 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 [[Interferon gamma|IFN-γ]] and [[Tumor necrosis factor alpha|TNF-α]], which do not kill the cell, but limit viral infection by down-regulating the expression of viral components.<ref name=Franco1997>{{cite journal | vauthors = Franco A, Guidotti LG, Hobbs MV, Pasquetto V, Chisari FV | title = Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice | journal = Journal of Immunology | volume = 159 | issue = 4 | pages = 2001–2008 | date = August 1997 | doi = 10.4049/jimmunol.159.4.2001 | pmid = 9257867 | s2cid = 20528634 | url = http://www.jimmunol.org/cgi/content/abstract/159/4/2001 | doi-access = free }}</ref> DNA vaccinations can be used to curb viral infections by non-destructive IFN-mediated control. This was demonstrated for hepatitis B.<ref name=Mancini1996>{{cite journal | vauthors = Mancini M, Hadchouel M, Davis HL, Whalen RG, Tiollais P, Michel ML | title = DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 22 | pages = 12496–12501 | date = October 1996 | pmid = 8901610 | pmc = 38020 | doi = 10.1073/pnas.93.22.12496 | doi-access = free | bibcode = 1996PNAS...9312496M }}</ref> IFN-γ is critically important in controlling malaria infections<ref name=Doolan1999>{{cite journal | vauthors = Doolan DL, Hoffman SL | title = IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model | journal = Journal of Immunology | volume = 163 | issue = 2 | pages = 884–892 | date = July 1999 | doi = 10.4049/jimmunol.163.2.884 | pmid = 10395683 | s2cid = 41651105 | url = http://www.jimmunol.org/cgi/content/abstract/163/2/884 | doi-access = free }}</ref> and is a consideration for anti-malarial DNA vaccines.