DNA Vaccines

In rainbow trout, up-regulation may prime immune cells such as nai ¨ve T lymphocytes fol- of MHC class II expression is found at the site of pDNA ad-lowing antigen presentation [5].. Dis

Fish & Shellfish Immunology (2008) 25,1e18

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/fsi

REVIEW

What happens to the DNA vaccine in fish?

A review of current knowledge

*

Department of Marine Biotechnology, The Norwegian College of Fishery Science,

University of Tromsø, N-9037 Tromsø, Norway

Received 12 November 2007; revised 11 March 2008; accepted 12 March 2008

Available online 19 March 2008

KEYWORDS Abstract The primary function of DNA vaccines, a bacterial plasmid DNA containing a con-DNA vaccine; struct for a given protective antigen, is to establish specific and long-lasting protective immu-Fish; nity against diseases where conventional vaccines fail to induce protection It is acknowledged Tissue distribution; that less effort has been made to study the fate, in terms of cellular uptake, persistence and

degradation, of DNA vaccines after in vivo administration However, during the last year some Plasmid DNA receptors

papers have given new insights into the fate of DNA vaccines in fish By comparing the newly acquired information in fish with similar knowledge from studies in mammals, similarities with regard to transport, blood clearance, cellular uptake and degradation of DNA vaccines have been found But the amount of DNA vaccine redistributed from the administration site after intramuscular administration seems to differ between fish and mammals This review presents up-to-date and in-depth knowledge concerning the fate of DNA vaccines with emphasis on tis-sue distribution, cellular uptake and uptake mechanism(s) before finally describing the intra-cellular hurdles that DNA vaccines need to overcome in order to produce their gene product

ª 2008 Elsevier Ltd All rights reserved

Plasmid DNA used in gene delivery studies normally contains promoter- and enhancer sequences, the gene of

A plasmid DNA (pDNA) is often used as a vehicle for gene

interest, a poly-adenylation sequence, transcriptional delivery to mammals and fish The pDNA is a circular

termination sequence, antibiotic resistance gene and origin molecule consisting of double-stranded deoxyribonucleic

of replication To express the gene of interest, pDNA is acid (DNA, not different from chromosomal DNA), usually

transcribed and mRNA is translated to protein by the cell’s own apparatus This opens up the applicability of pDNA in two important areas: gene therapy and DNA vaccination

* Corresponding author Tel.: þ47 77 6460 22; fax: þ4777 64 6020

The definition of gene therapy and DNA vaccines is not

** Corresponding author Tel.: þ47 776444 82; fax: þ47 77 64 6020

consistent in the literature Often, gene therapy also E-mail addresses:tom.tonheim@nfh.uit.no(T.C Tonheim),roy

encompasses the use of DNA vaccines The Norwegian

dalmo@nfh.uit.no(R.A Dalmo)

1050-4648/$ - see front matter ª 2008 Elsevier Ltd All rights reserved

doi:10.1016/j.fsi.2008.03.007

2 T.C Tonheim et al.

Biotechnology Advisory Board ( www.bion.no ) defines gene lymphocytes [29] In fish, the different subsets of T cells therapy in animals as the intentional transfer of genetic are not well characterised, but CD8 [23,30e32] and CD4 material to somatic cells for purposes other than in uenc- [33e35] homologues are reported Genes of MHC class IIb ing the immune system [1] Gene therapy is often aimed [36e38] and MHC class I gene [39] are found, and genes

to achieve a long-lasting physiologically matched expres- involved in MHC class I [40] and class II [41] loading path-sion of the gene, without activating the immune system ways are described in fish Only three classes of

immuno-In contrast, DNA vaccination is defined as the intentional globulins; IgM [42e46] , IgD [47e54] and IgZ/IgT [55,56]

transfer of genetic material to somatic cells for the are reported in fish, but there are no indications that this purposes of in uencing the immune system [1] For DNA repertoire of immunoglobulins are functionally retarded vaccination, a short-term expression is sufficient for evok- in any waydbesides the absence of, e.g., IgG, IgA and IgE

Immune responses are harder to investigate in fish

Working mechanism of DNA vaccines compared to mammals, due to lack of tools (e.g cell

lines, gene sequences, markers and antibodies) However, The immune response after DNA vaccination is primarily a recent publication shows that both innate and adaptive launched by antigen presenting cells (APC), e.g dendritic cell-mediated immune responses involving natural killer cells (DC)[2] Professional APC such as macrophages and DC (NK) cells and cytotoxic T lymphocytes (CTLs), respec-have been shown to contain pDNA after i.m administration tively, are triggered after a viral hemorrhagic septicaemia

[3,4] Following on, the APC at the site of administration virus (VHSV) infection [9] In rainbow trout, up-regulation may prime immune cells such as nai ¨ve T lymphocytes fol- of MHC class II expression is found at the site of pDNA ad-lowing antigen presentation [5] APC containing cytosolic ministration [57], and expression of these genes is indica-encoding pDNA may transcribe and translate the transgene tive of the recruitment of activated immunocompetent and thereby produce immunogenic proteins, mimicking an cells (e.g B cells and macrophages) [58] The CpG sites infection of an intracellular (cytosolic) pathogen and allow are regions of DNA where a cytosine nucleotide occurs

presentation of the foreign antigen peptide by major histo- next to a guanine nucleotide and is linked by a phospho-compatibility complex (MHC) class I on the surface of the diester bond These CpG sequences (CpG motifs) are nor-APC Antigen presenting cells can also take up soluble anti- mally highly methylated in mammals, but bacterial and gens released from another transgene-producing cell (e.g viral DNA possess a lower methylation frequency As

a myocyte), process it and present the peptide on MHC class such, CpG sequences are often unmethylated in a pDNA

II molecules at the cell surface The T-cell receptor (TCR) due to its bacterial origin The vertebrate immune system may recognise these peptides presented on the MHC class recognises unmethylated CpG-motifs as ‘‘foreign’’ and

I and MHC class II, which stimulate a CD 8 þ T-cell (cytotoxic ‘‘danger’’ signal Cells and animals treated with synthetic

CpG oligodeoxynucleotides (ODN) and pDNA may produce

T cell) and a CD4 þ T-cell (helper T cell) response,

respec-cytokines that may induce macrophage activation, B-cell tively In addition, APC can also take up antigen-laden

proliferation and immunoglobulin secretion [59,60] The apoptotic bodies of transfected myocytes (or other cells)

and present the relevant peptides on MHCs ( Fig 1) CpG DNA can also directly activate monocytes, macro-Under certain circumstances, exogenously derived phages and dendritic cells to secrete Th1-like cytokines peptides may be presented on both MHC I and II This [60e62] to induce a cellular immune response, which is cross-priming stems from the observation that professional the desired response for combating intracellular patho-APC could present antigen or peptides from the classical gens Leukocyte activation by CpG DNA is not mediated exogenous pathway via the MHC class I pathway of antigen through a cell surface receptor but depends on uptake presentation [6] In contrast to the TCR restricted recogni- of the DNA [59] into lysosomal compartments harbouring tion of MHC class I/II, the B lymphocyte, the precursor of specific receptors, the so-called Toll-like receptor 9

the antibody secreting cells, may directly recognise native (TLR9), for recognition [63,64] TLR9 is expressed in den-antigen through their B-cell receptor [5,6] dritic cells [63], macrophages [63], B cells [65] and liver One of the unique features of DNA vaccines is their endothelial cells [66] TLRs function as innate pattern rec-ability to stimulate both cellular (including cytotoxicity) ognition receptors (PRRs) and induce certain intracellular and humoral immune responses [2,7e9] (Fig 1) The cellu- signalling cascades that result in activation and transcrip-lar immune response is represented, for simplicity, by the tion of pro-in ammatory genes [67e69] A ligand for TLR9 activated Th1 cells that may secrete pro-in ammatory is CpG-containing pDNA and CpG ODNs The CpG induced cytokines, and CD8 þ T cells that may kill cells presenting immune responses seem to occur without apparent ad-the transgene The adaptive humoral immune response is, verse effects [70] Thus, it may be beneficial for a plasmid however, represented by activation of B lymphocytes and vector to also include CpG motifs to increase and direct

Several findings suggest that the fish immune system same immune stimulatory activities, and the effects resembles the mammalian immune system; homologies are depend on the ODN sequence, length, concentration and found between mammalian and fish MHC class I (reviewed the recipient species [71] As TLR9 has been proposed to

bind to CpG ODN, there are indications that also

CpG-in[10]), TCR[11e22] and the TCR co-receptor CD8 [23]

sug-containing pDNA bind to TLR9 [72,73] However, it is not gesting similarities in the antigen presentation process The

clear whether the immunostimulatory CpG sequences in lymphocytes detected in fish are equivalent to the

mamma-lian T and B cells [24e28] However, the teleost (bony fish) the backbone of pDNA behave similarly to that predicted

B cells possess phagocytic activity, unlike the mammalian B from studies of synthetic CpG ODN [74]

DNA vaccine in fish 3

Figure 1 A general and simplified overview of the adaptive immune response DNA vaccines that have been taken up by, e.g., muscle cells and processed may be completely degraded or may be expressed by the muscle cells The transfected cells produce and release the plasmid encoding antigens that will be subjected to uptake in APC (antigen presenting cells) APC present peptides, being the breakdown products of the antigens, on MHC I and II to CD8 þ and CD4cells following uptake of endogenously and exog- þ enously derived antigens, respectively Activation of nai ¨ve CD4cells will initiate cellular and humoral adaptive responses such as þ activation of B cells to secrete antibodies specific to this antigen, whereas binding of the T-cell receptor of CD8 þ cells to MHC I

presented peptides, with aid of cytokines produced by Th1 cells, will facilitate a cytotoxic T-cell response eliminating, e.g., virus-infected cells producing and expressing the virus antigen One feature of the adaptive immune response is the generation

of memory cells that need short time to be activated during a later infection with the same pathogen CD4, cluster of differentiation

4 co-receptor; CD8, cluster of differentiation 8 co-receptor; MHC-I, major histocompatibility complex class I protein; MHC-II, major histocompatibility complex class II protein; IgM, immunoglobulin M; IgD, immunoglobulin D; TCR, T-cell receptor; Th1, T

helper 1 Adapted from Online publication in Veterinary Sciences Tomorrow ( www.vetscite.org/publish/articles/000020/index.html ).

The TLR9 gene is found in fish [75,76] and is expressed in immune responses similar to that of mammals, such as several epithelial and lymphoid tissues [77] , but it remains activation of macrophages [78e81] , proliferation of

leuko-to be elucidated whether pDNA binds leuko-to fish TLR9 cytes [79,82,83]and stimulation of cytokine expression

However, CpG ODN administered to fish, exert various[80,84,85]

4 T.C Tonheim et al.

Advantages of DNA vaccines being tested clinically, rarely integrate [95,110,111] and

have a low probability of creating a tumorigenic event

[112] However, attempts to enhance vaccine efficacy by

A huge advantage of DNA vaccination is that pDNA, like live

modifying the vector, co-administration of vaccine with

or attenuated viruses, effectively induces both humoral and

agents that increase cellular uptake or altering the site or cell-mediated immune responses [86] Immune responses to

method of delivery, may increase the potential for integra-antigen present in soluble form, such as for recombinant

tion [105,113] Homologous recombination could occur protein, generally induce mainly antibody responses In

ad-when the foreign DNA possesses a sequence that is very sim-dition, DNA vaccines overcome the safety concerns

com-ilar to a sequence of the genomic DNA and the two se-pared to live attenuated vaccines where reversion to

quences ‘‘cross over’’, although random insertion is virulent forms may occur Another benefit compared to

con-a more frequent event [114] It should be emphasised that ventional vaccination strategies is that pDNA possesses

in-such downside effects have never been found in fish Envi-trinsic immunostimulatory capacity, due to the presence

ronmental release of pDNA may occur by several ways,

of CpG motifs [87] Furthermore, it is possible to construct

e.g leakage of DNA vaccine from the administration site

af-a vector encoding severaf-al af-antigens to be given in af-a single af-

ad-ter vaccination (mainly in fish); by consumption of pDNA res-ministration, and thus create a vaccine against multiple

dis-idues in the meat of DNA vaccinated animals; and by spills or eases From a practical point of view, the DNA vaccine is

waste of DNA vaccine from the production process After relatively inexpensive and easy to produce, and can be

man-vaccination pDNA may find its way to the intestine [115]

ufactured using identical production processes DNA

vac-where pDNA may be taken up by intestinal bacteria or se-cines are very stable as dried powder or in solution, unlike

creted by the faeces Antibiotic resistance genes may then conventional vaccines that often need to be stored under

be spread to various bacterial populations in the intestine, proper conditions (e.g cold environments) [58,88,89]

soil or water Several other advantages and disadvantages DNA vaccines administered without any additional

adju-are listed in the review by Lorenzen and LaPatra [116] vants create much less direct tissue damage or/and in

am-matory responses compared to conventional oil-adjuvanted

DNA vaccine trials in fish

vaccines [90e94] , and with no systemic toxicity [95] Per-haps the most important use of DNA vaccine technology is

DNA vaccinations against a wide range of pathogens have the possibility to create vaccines for targeted diseases

been investigated in various fish species ( Table 1) Consid-where traditional vaccines have scored sub-optimally

erable efforts have been made to develop conventional Limitation and downside effects of DNA vaccines vaccines or subunit vaccines for many of these fish

patho-gens with limited success [117e119] The IHNV and VHSV Eukaryotic transcription and production of plasmid DNA are known to cause high mortalities of cultured salmonid

encoding a non-glycosylated protein is highly feasible, but species such as Pacific salmon (Oncorhynchus sp.), Atlantic not the transcription and production of carbohydrates and salmon and rainbow trout in North America and Europe

highly glycosylated proteins This suggests that DNA vacci- Therefore, much effort has been concentrated around de-nation cannot be a substitute for the more traditional veloping DNA vaccines against these viral diseases Several polysaccharide containing vaccines in evoking immune re- parameters have been investigated to optimise the efficacy sponses against microbes that have an outer membrane of the DNA vaccines against IHNV and VHSV, such as dose made of, e.g., lipopolysaccharides [89] Following on, the and fish size [120e123] and optimal administration route pDNA contains CpG moieties that may act as an adjuvant [124e126] In short, i.m administration seems to be the

[59,96,97], andthere is a possibility that the combined adju- optimal route of administration for these DNA vaccines (

Ta-vant activity with the presence of DNA may induce antibody ble 1) and it appears beneficial to vaccinate the fish when responses to the DNA itself, but usually it does not [98e100] they are small, since larger fish require a higher dose of Such a process may be detrimental to the host, as exempli- vaccine [122,127] Early protection involves short-lived, fied by occurrence of glomerulonephritis in mice [101] non-specific, anti-viral defence mechanisms, like induction Whether there exists an antibody response to DNA in fish is of interferons and Mx proteins [126,128e130] , that may be not known An additional safety concern associated with important in protection against heterologous viruses (cross-the use of DNA vaccines after i.m administration is that my- protection) [129,131] However, the specific protection may ocytes, taking up the pDNA and expressing the encoded an- last up to 2 years after i.m administration [92]

tigen, may become targets for antigen-specific CTLs [102] The glycoprotein (G-protein) of both IHNV and VHSV This would result in the development of myositis [132,133] is known as a strong inducer of neutralising and Tolerance seems to be a problem in mainly neonatal protective antibodies Intramuscular administration of individuals, where an immature immune system could pDNA encoding the VHSV G-protein [134,135] or the IHNV recognise the DNA vaccine encoded protein as a self protein G-protein [92,136] gives high levels of protection in rainbow

[103e105] However, during neonatal DNA vaccination, tol- trout However, other rhabdoviral proteins give limited or erance is the exception rather than the rule [106] The sus- no protection [134e138] In addition, i.m administered ceptibility to tolerance induction was shown to wane within pDNA encoding IHNV G-protein also protects Atlantic

1 week after birth in mice and to increase with increasing salmon against IHNV infection [139] Finally, in 2005 a po-pDNA dose[107] Although likely not to occur, integration tential prophylactic strategy against IHNV infection was

in-of pDNA into the chromosomal DNA could lead to mutations, troduced: The IHNV DNA vaccine (Apex-IHN ) developed by genomic instability and abnormalities [105,108,109] Avail- Aqua Health Ltd (an affiliate of Novartis), was cleared for able evidence indicates that genetic vaccines, currently marketing by the Canadian Food Inspection Agency

DNA vaccine in fish 5

Table 1 An overview of studies performed with DNA vaccines encoding viral or bacterial antigens in fish Pathogena Pathogen gene in vaccine b Host Administration Protection References

routec

Nucleocapsid protein (VHSV) Rainbow trout i.m Yes [135]

Nucleocapsid protein (VHSV) Rainbow trout i.m No [138]

Aeromonas Omp38 gene, Omp48 gene Spotted sand bass i.m Partial [150]

veronii

Piscirickettsia Full expression library Coho salmon i.m Partial [153]

salmonis Renibacterium Aeromonas salmonicida DNA Rainbow trout i.m No [154]

anguillarum Atlantic salmon (Salmo salar), Asian seabass (Lates calcarifer), channel catfish (Ictalurus punctatus), chinook salmon (Oncorhynchus tshawytscha), coho salmon (O kisutch), common carp (Cyprinus carpio), hybrid striped bass (Morone saxatilis M chrysops), Japanese ounder (Paralichthys olivaceus), rainbow trout (Oncorhynchus mykiss), red seabream (Pagrus major), sockeye salmon (Oncorhynchus nerka), spotted sand bass (Paralabrax maculatofasciatus), turbot (Scophthalmus maximus)

a Pathogens: AHNV, Atlantic halibut nodavirus; CCV, channel catfish virus; HIRRV, hirame rhabdovirus; IHNV, infectious haematopoietic necrosis virus; IPNV, infectious pancreatic necrosis virus; ISAV, infectious salmon anemia virus; LCDV, lymphocystis disease virus; RSIV, red seabream iridovirus; SVCV, spring viraemia of carp virus; VHSV, viral haemorrhagic septicaemia virus

b Pathogen genes: G, glycoprotein; e.g SHRV-G, snakehead rhabdovirus glycoprotein

c Administration routes: i.m., intramuscular; i.p., intraperitoneal; i.b., intrabuccal; scarification, scarification of the skin

Distribution of plasmid DNA pDNA by endonucleases at the administration site, and

(iv) distribution of pDNA by blood, cells and lymph to various tissues

Mammals

Bureau and co-workers have suggested that as soon as pDNA is administered, it is proportionally partitioned When pDNA (DNA vaccine) is i.m administered to mam- between at least two compartments While a major part

mals, several events other than the intended immune

of the pDNA is cleared and degraded, a very small amount response are initiated: (i) uptake of pDNA by cells at the

of the administered pDNA is located in another compart-administration site, (ii) pDNA remaining extracellularly

ment where it is protected from endogenous nucleases located at the administration site, (iii) degradation of

6 T.C Tonheim et al.

[156] This compartment could be the extracellular matrix circulation [166,168,169] Plasmid DNA is detected in blood

as suggested by Bureau et al., but also the transverse tu- and several tissues after i.v administration, but is mainly bules (T-tubules) of the myocytes as suggested by others cleared from the circulation by the liver (scavenger tissue)

[157,158] In addition to myocytes [156,159], the mononu- in mice and rats [168e172] In fact the mouse liver is able clear cells [159] take up pDNA at the administration site af- to clear as much as 25 mg pDNA during a single pass [172] ter i.m administration However, shortly after i.m A rapid clearance of supercoiled pDNA from the circulation administration the majority of the pDNA is present in the in- is observed [173], with a serum half-life varying between terstitial space between myocytes at the administration 1e20 min [165,174e177] , depending on the pDNA topoform site, where pDNA is subjected to degradation [156,159] and analytical method used Plasmid DNA fragments

Degradation of pDNA at the administration site of mice is [178,179] in the blood with a molecular weight of 30 kDa observed as soon as 5 min after i.m administration [156], (1 bp z 0.6 kDa) or less may be eliminated from the circu-and nucleases degraded 95e99% of the pDNA after 90 min lation by glomerular filtration [180] The tissue distribution

[160,161] Despite this, supercoiled pDNA is detected by of pDNA after i.v administration has been compared and Southern blot analysis up to approximately 90 min after was found to be similar to that of i.m administration in i.m administration [161] Plasmid DNA is also redistributed mice [181]

from the administration site to various tissues after i.m

ad-Fish

ministration However, the majority of the pDNA remains at the administration site [111,162,163] Despite this, pDNA fragments can be detected by PCR in blood and tissues Until recently, not much was known concerning the fate of for several weeks [111,162,163] However, the pDNA copy the pDNA molecule with regard to tissue distribution, number in various tissues after i.m administration varies degradation and persistence in fish after i.m administra-between mammalian species [111,163] Blood [163], im- tion However, based upon recently published data, it was mune cells [4,159] and lymph [159,164] have all been sug- possible to create a simplified illustration ( Fig 2) showing gested to contribute to pDNA distribution after i.m that pDNA is distributed to four different body

Plasmid DNA entering the mammalian circulation is The literature suggests that pDNA enters the extracel-mainly distributed to tissues by the blood plasma after lular milieu of the administration site where it can be i.v administration [165] The transport itself may be dam- degraded, taken up by cells and/or redistributed ( Fig 2) aging to the pDNA since the plasma/serum of several spe- Local degradation of pDNA at the administration site after cies contains nucleases [160,166,167] which play an i.m administration has been observed in both Atlantic important role in the initial breakdown of pDNA in the salmon [182] and Atlantic cod [183]; however, the causal

Figure 2 Plasmid DNA may be distributed to four different compartments in fish after intramuscular administration At the administration site, some pDNA may be locally degraded and transferred to the urinary excretion system, and a fraction of the pDNA may be transferred to the circulatory system and distributed to other organs and tissues Plasmid DNA may also be subjected

to degradation by blood nucleases From extracellular compartments, pDNA will be taken up by, e.g., scavenger receptor possess-ing cells where a further breakdown may occur At the injection site, pDNA may be degraded intracellularly or be redistributed to other organs and tissues following cell migration The arrow size does not re ect the amount of pDNA transported to or from the various body compartments SEC, scavenger endothelial cells

DNA vaccine in fish 7

mechanisms yet remain to be determined In addition, the particle/apoptotic cell/pathogen involves engagement of

rate of pDNA degradation remains unknown in fish, thus pathogen-associated molecular patterns (PAMPs) by pattern making it difficult to assess the importance of the local deg- recognition receptors (PRRs) on the phagocyte [192] The radation, and this may depend upon the immunogenicity of membrane fold around the target is sealed off into a large

the transgene protein Plasmid DNA (pDNAvhsg/ihng) has vacuole, known as a phagosome The phagosome then

been observed intracellularly in myocytes up to 45 days af- usually fuses with a lysosome where the contents of the

ter i.m administration in rainbow trout [57], while pDNAluc phagolysosome are digested by hydrolytic enzymes

expresses luciferase at the administration site of Atlantic [190,193] Pinocytosis refers to the constitutive formation

salmon for at least 535 days [184] In contrast to mammals, of vesicles containing extracellular uid and

macromole-substantial amounts of the administered radiolabelled cules, specifically or non-specifically, bound to the plasma pDNA ( I- uorescein-pDNA) in Atlantic salmon12 5 [182] and membrane The term endocytosis is often used

synony-Atlantic cod [183] are re-distributed from the administra- mously with pinocytosis Pinocytosis is further divided into tion site after i.m administration From the administration uid phase endocytosis of bulk solutes (macropinocytosis)

site the pDNA is transported in the blood plasma, which was and a mediated endocytosis During

receptor-shown to degrade pDNA in both Atlantic salmon and Atlantic mediated endocytosis the macromolecules are

concen-cod (in vitro) [182,183] Nevertheless, pDNA was able to trated on the cell membrane by receptors before

reach other organs (such as kidney, spleen and gills) intact internalisation due to their receptor binding

after i.m administration, indicating that pDNA could avoid

Cells taking up DNA

local degradation at the administration site and in the

blood plasma [182,183] Following on, pDNA is cleared

pref-erentially from the circulation by Atlantic salmon kidney Using various analytical methods (polymerase chain

reac-(scavenger tissue) [182] after both i.m and i.v administra- tion (PCR), uorescent in situ hybridisation (FISH), isotope tion Similar observations were made in Atlantic cod heart and uorescence labelling of pDNA with subsequent analy-(scavenger tissues) [183] Also, following i.v and oral ad- sis), it is possible to detect DNA/DNA fragments in a wide ministration, DNA fragments (1017e151 bp) were present array of mammalian cell types, such as: cytotoxic T cells

in the kidney of Atlantic salmon [185,186] Uptake of [194], B cells [194,195], macrophages [171,194,196] , other pDNA by the kidney of Atlantic salmon and Atlantic cod leukocytes [197], dendritic cells [196], Kupffer cells [170], heart is specific and is inhibited by formaldehyde treated liver scavenger endothelial cells [170], hepatocytes [198], serum albumin, a known scavenger receptor ligand keratinocytes [199], myocytes [156,159] and microglia cells

[182,183] This suggests that scavenger-like receptors are [200] In fish, DNA has so far only been detected in rainbow involved in clearance of pDNA from the circulation in Atlan- trout myocytes [57], head kidney macrophages of Atlantic tic salmon and Atlantic cod salmon [201] and Atlantic cod [183], and in scavenger

endo-In fish, tissue distribution of pDNA or DNA fragments has thelial cells of Atlantic cod heart [183] and Atlantic salmon also been evaluated after intraperitoneal (i.p.) [187], gene kidney [182] However, the mechanisms for cellular uptake gun [188] and oral administration [185] PCR detected pDNA of DNA are not fully understood.

fragments at the administration site after i.p administra- On the basis of competitive inhibition studies, initial

tion and in blood and muscle tissue after gene gun adminis- studies suggested separate receptors for binding of oligonu-tration in rainbow trout In addition, DNA fragments were cleotides and larger DNA [197] However, several competi-detected in the kidney, liver, blood, pyloric region, mid tive inhibition experiments have provided evidence for

and distal intestine after oral administration in Atlantic a common saturable uptake pathway [202e204] To simplify salmon The wide spread tissue distribution of the pDNA matters, the next sections will mainly focus on mammalian does not seem to cause long term tissue damage since a re- and fish cells and their mechanisms used to take up pDNA. cent study showed lack of histopathological damage up to Cell lines will receive less attention in the following sections.

2 years after DNA vaccination against IHNV in rainbow trout

The thin-layered endothelium covering the walls of the blood circulatory system consists of different

subpopula-Uptake of plasmid DNA

tions of cells, which are structurally and functionally specialised depending on their anatomical location The

Endocytosis sinusoids of the mammalian liver lobules are lined by

fenestrated endothelial cells [205], with Kupffer cells (fixed Endocytosis is a common name for the various mechanisms macrophages), T lymphocytes, monocytes and natural killer used by all mammalian cells for internalisation of uids, cells, all attached to the luminal side of the endothelial

macromolecules and particles (reviewed in [189]) Depend- lining [205,206] On these liver endothelial cells (LEC),

ing on the material internalised and the mechanism numerous coated pits, vesicles and large amounts of other

involved, endocytosis can be divided into two main types: organelles associated with endocytosis have been detected

(i) phagocytosis (‘‘cell eating’’) and (ii) pinocytosis (‘‘cell [205] The liver sinusoidal endothelial cells represent a gen-drinking’’) [190] Phagocytosis is a receptor and actin eral vertebrate non-phagocytic scavenger endothelial cell

dependent process by which cells ingest large particles (SEC) system with an extensive capacity to endocytose

(>0.5 mm) [191], such as bacteria and senescent cells and degrade soluble physiological and foreign macromolec-This occurs normally in professional phagocytes (such as ular waste substances/molecules from the circulation by

granulocytes and macrophages) The recognition of the receptor-mediated endocytosis [207,208] In rat, liver

8 T.C Tonheim et al.

scavenger endothelial cells (LSEC) rather than the Kupffer In teleosts, the kidney is the major haematopoietic cells are responsible for the highest uptake and degradation tissue [232], and is considered to contain the largest

reser-of pDNA[170] The scavenger receptors on LSEC are also voir of macrophages Fish macrophages are also located in known to clear a high number of other molecules from several other tissues such as spleen, thymus, intestine the circulation [209e213] , including phosphorothioate anti- and mesentery [233] However, fish seem to lack the resi-sense oligodeoxynucleotides [214] and CpG ODN [66] dent sinusoidal macrophages in their livers [234] The mac-Scavenger receptors appear to be an ancient protein rophages of fish are not as well studied as their mammalian family that may play a role in host defence as a PRR and in counterpart, although they seem to carry out much of the antigen presentation (reviewed in [215,216]) The class of same functions [235] (see above), e.g phagocytosis of bac-scavenger receptors involved in uptake of DNA is not well teria [236e238] and antigen presentation [239] The recep-studied, although studies performed on LSEC [170] and tors involved in the phagocytic process are generally poorly brain microvessel endothelial cells [217], show uptake of described in fish However, an in vitro study of rainbow pDNA via a specific mechanism resembling scavenger recep- trout head kidney macrophages suggests that scavenger tors However, scavenger receptor class A is unlikely to be receptors are involved in phagocytic processes [240].On responsible for the uptake of pDNA, as shown in scavenger the other hand, rainbow trout macrophages seem to be of

receptor class A knockout mice [171] minor importance in the blood clearance of soluble scaven-Recent studies have shown that kidney sinusoidal endo- ger receptor ligands [241,242] A recent report showed that thelial cells of Atlantic salmon are similar to the LSEC found Atlantic salmon head kidney macrophages are able to take

in mammalian liver [207,218] However, in Atlantic cod the up CpG ODN in vitro[201], but the mechanism(s) behind the SEC are found as endocardial cells lining the luminal side of uptake remains unknown Recently published papers

the heart [207,219] A recent study showed that SEC of showed, however, that adherent anterior kidney leukocytes Atlantic cod were involved in specific uptake and degrada- from Atlantic salmon did not take up rhodamine labelled tion of pDNA [183] The study suggested additionally that pDNA (in vitro)[182], while adherent anterior kidney leuko-cod scavenger-like receptors were involved in this process cytes from Atlantic cod were able to endocytose the same Despite uptake of pDNA, no transgene expression was labelled pDNA (in vitro) [183] This difference in pDNA observed in Atlantic cod heart SEC (in vitro) [183] uptake could be explained by species differences

Macrophages arise from a common precursor in the bone Dendritic cells (DCs) are immune cells and form a part of marrow of mammals, circulate in the blood as monocytes the mammalian immune system DCs start out as immature and migrate into tissues to transform into various types of cells, but become activated into mature DCs once they tissue macrophages (e.g microglia, Kupffer and sinus come in contact with a pathogen, with or without growth histiocytes) Their distribution in the body is widespread factors and cytokines Immature DCs phagocytose, e.g

and they participate in a number of physiological and pathogens or apoptotic cells, degrade proteins and present pathological processes [220] The microglia is a type of glial upon maturation those fragments at their cell surface using cell that act as the immune cell of the central nervous sys- MHC molecules During maturation, DCs travel to lymph tem and is phagocytic [221] The Kupffer cells constitute nodes where they act as antigen presenting cells [243,244] 80e90% of the total fixed macrophages in the body [222], Immature DCs express various kinds of receptors essential and they generally reside within the lumen of the liver sinu- for endocytosis, such as the mannose receptor, Fc-g recep-soids, adherent to the endothelial lining, partially obscur- tor and scavenger receptors [245e247] In a murine DC cell ing the vascular channel [223,224] Sinus histiocytes are line (DC2.4), pDNA is taken up via a specific mechanism macrophages located in the spleen and lymph nodes The resembling scavenger receptors and subsequently degraded macrophages are multifunctional cells with phagocytic [248] In addition, it has been suggested that scavenger re-and secretory properties, re-and play an important role in ceptor class A was not involved in pDNA uptake by DC2.4 host resistance against infections, by killing and digesting cells, since significant expression of scavenger receptor invading micro-organisms [225] In addition, macrophages class A has not been reported in DCs [249,250] are professional antigen presenting cells Phagocytosis is DC or dendritic-like cells with their cytochemical signa-mediated by specific receptors, such as the scavenger tures and function have not yet been characterised in fish

receptors, mannose receptor [226,227], and various recep-tors recognising complement facrecep-tors and the Fc-g receptor Hepatocytes

[228,229] Some of these receptors also mediate effective Hepatocytes are the chief functional cells of the liver endocytosis of soluble ligands Earlier reports demon- parenchymal cells and perform an astonishing number of strated that soluble ligands like pDNA or fragmented DNA, metabolic, endocrine and secretory functions Hepatocytes are taken up by cultured macrophages [230], microglia cells constitute roughly 80% of the mass of the liver Uptake of

[200] and Kupffer cells [170] via a specific mechanism pDNA is observed in vitro in primary hepatocytes [198], but resembling scavenger receptors However, scavenger the exact mechanisms remain unknown Uptake of pDNA in receptor class A, that recognise a wide variety of anionic the hepatocytes in vivo occurs after hydrodynamic i.v ad-macromolecules is not responsible for pDNA uptake [171] ministration (rapid administration of a large administration Another receptor, Mac-1, has been suggested to mediate volume) [251] By using normal delivery during i.v adminis-uptake of oligonucleotides Mac-1 represents a heparin- tration (low administration volume and normal pressure) binding integrin located on the surface of leukocytes, the pDNA is removed mainly by the non-parenchymal cells monocytes, macrophages and natural killer cells [231] [170,252] The uptake mechanism after hydrodynamic

DNA vaccine in fish 9

administration remains unknown, and is widely discussed the early to the late endosomes is unknown However, it has (reviewed in [198,253]) However, results suggest that been suggested that early endosomes are simply

trans-pDNA is driven into the hepatocyte by a high vascular pres- formed into late endosomes The delivery of late endosomal sure after hydrodynamic administration [251] contents to lysosomes is thought to occur by the fusion of

No pDNA has been detected in hepatocytes of fish after late endosomes with pre-existing lysosomes Macromole-i.v or i.m administration cules endocytosed either by the receptor-mediated or

phagocytic pathway, are ultimately digested in the late

en-Keratinocytes dosomes or lysosomes to small molecules (<1 kDa) The

deg-radation products produced from this process may be Keratinocytes are the major cell type of the mammalian

transported to the cytoplasm [264] Plasmid DNA, like other epidermis and originate in the basal layer from the division

endocytosed molecules, travels after internalisation

of keratinocyte stem cells Epidermal keratinocytes are

through an endosomal compartment before reaching lyso-able to take up and express pDNA upon intradermal

somes where it is degraded [269] However, if transgene ex-administration [199,254].Anin vitro study shows that the

pression is to occur, pDNA needs to escape full degradation pDNA uptake in keratinocytes occurs through different

in the lysosomes How and exactly where pDNA escapes is pathways, but mainly by bulk-phase endocytosis

(macropi-poorly understood In the cytoplasm, the mesh like struc-nocytosis) [255]

ture of the cytoskeleton restricts diffusion of pDNA to the

No pDNA is detected in skin cells of fish after i.v or i.m

nucleus, thereby increasing the residence time of pDNA in administration On the other hand, green uorescent

the cytoplasm [270e273] This favours metabolic degrada-protein (GFP) expression is detected in epithelial cells of

tion of pDNA by, e.g., cytosolic nucleases [274,275] How-zebra fish skin after gene gun administration of pDNA [256]

ever, recent results published suggest that pDNA utilises the microtubule network and its associated motor proteins

Myocytes

to traffic through the cytoplasm to the nucleus [276] Mammalian myocytes (muscle cells) can take up and express

The nuclear envelope is the ultimate obstacle to the pDNA in vitro and after i.m administration [157,257,258]

nuclear entry of pDNA Entry of proteins into the nucleus Speculations and arguments regarding the mechanism(s)

has been suggested to occur through nuclear pore complex used by the myocytes to take up pDNA are many, although

(NPC), forming channels across the nuclear envelope [277] the exact mechanism(s) is not yet identified Several

mech-Molecules smaller than approximately 40 kDa may diffuse anisms are suggested to be involved in pDNA uptake: (i)

dif-passively through the NPC, whereas proteins >60 kDa are fusion of pDNA through membrane disruptions induced by

taken up by energy dependent transport Active nuclear ac-direct i.m administration [198], (ii) entry of pDNA through

cumulation requires the presence of nuclear localisation se-small transient membrane pores caused by high velocity

quence (NLS) [278] The NLS provokes considerable administration of pDNA and increased pressure [198], (iii)

conformational change in the NPC, leading to opening of entry through the T-tubular system of myofibres [157,158]

the NPC channel [279] The opening is large enough to pro-and (iv) receptor-mediated endocytosis [198,259]

vide a plausible explanation for the ability of the NPC to Plasmid DNA has been detected in rainbow trout

translocate substrates as large as 25e50 MDa [280] Accu-myocytes [57] which are able to express the transgene

mulating evidence indicates that the transport mechanisms

[260e262] However, the uptake mechanism(s) has not

for pDNA and polypeptides (with molecular masses larger been examined in detail and remains unknown

than 60 kDa) have common characteristics [281,282] In ad-dition, nuclear uptake of pDNA has been suggested to occur

Fate of plasmid DNA after cellular uptake

during cell division when the nuclear envelope disassembles

[283e285]

Transport of pDNA to the nucleus In mammals, the SECs of the liver are responsible for the

majority of the uptake and degradation of pDNA from the Plasmid DNA encounters many hurdles before transgene circulation [170] However, degradation of pDNA has not expression may occur It is estimated that at approximately been detected at a cellular level in fish

10 copies of pDNA are required per cell (HeLa cells) in the4

cytosolic compartment to ensure transcription of a trans- Immune stimulation in the lysosomal compartment

gene [263]

In general, receptors and their ligands are brought into

Once inside the lysosomal compartments, the CpG se-the cell via a vesicle after receptor-mediated endocytosis

quence (on e.g the pDNA or CpG ODN) could bind to Toll-This vesicle fuses with early endosomes and at this point

like receptor 9 (TLR9) that initiates signal transduction many receptors and ligands dissociate from each other upon

[63] The signal transduction may result in inducing a cellu-exposure to the slightly acidic pH in the early endosomes

lar immune response, as previously mentioned

The membrane-bound components (e.g the receptors) are

efficiently recycled to the cell surface for another round of

Expression of transgene(s) delivery Meanwhile, the bulk of the volume (containing the

released ligands) is destined for degradation in late

endo-somes or lysoendo-somes [264] Vesicles formed during macropi- Once inside the nucleus, pDNA can utilise the cell’s own

nocytosis can also fuse with the lysosomes, recycle machinery to transcribe the transgene(s), thereby initiating

content to the cell surface, or as in most cases, they acidify the production of the transgene encoded protein(s)

Pep-and shrink [265e268] How the ligands are transported from tides from these proteins may be presented on the cell

10 T.C Tonheim et al.

surface by the MHC class I [286], that may result in a CTL integration of DNA can also occur after oral administration, response as described earlier In addition, excretion/ as observed in mice by Schubbert and co-workers [194] escape of the transgenic protein produced in the cell could Integration of pDNA has been studied in goldfish after result in stimulation of a humoral immune response Pro- i.m administration, however, no integration was detected duction of protein after i.m administration of pDNA was by using Southern blot analysis [297] In a study using DNA first described in mouse muscle by Wolff and co-workers vaccinated Atlantic salmon, it was determined that the

in 1990 by using the luciferase reporter gene [258] Later potential risk associated with integration at the administra-they showed that the luciferase was detectable at the tion site was w43 fold less than that associated with spon-administration site for more than 19 months after i.m ad- taneous mutation rate [298]

ministration [257], even though the half-life in muscle tis-sue of both luciferase transcript and protein was less than Conclusion

24 h [258] Myocytes in mice are also known to produce Cry11Bb protein for more than 2 years after i.m

adminis-Continuous development of vaccination strategies of fish is tration of a pDNA construct [287] Fish are also able to

pro-a necessity to secure future growth of the pro-aqupro-aculture duce proteins after i.m administration of pDNA constructs

industry, contributing to world food supplies DNA vaccines and reporter gene products are detectable up to 2 years

have shown promising results as an effective strategy to after i.m administration [91,134,182e184,288e291]

combat viral diseases in fish The main advantage of this technology is that DNA vaccines induces both innate and Replication of plasmid DNA adaptive immune responses, and have proven to be

effi-cient against viral pathogens where strategies for pro-The behaviour of pDNA in the nucleus during cell division is tection still need to be optimised Additional benefits are:

not fully understood However, it has been observed [292] (i) the possibility of protection against multiple diseases as

that microinjected and nuclearly localised pDNA is found it is possible to construct a vector encoding several pro-almost exclusively within the nuclei of the daughter cells tective antigens given in a single injection, (ii) avoidance of after mitosis and is apportioned between the daughter safety concerns related to live attenuated vaccines which nuclei with a normal, Gaussian distribution can revert to virulent forms, and (iii) animal welfare issues The DNA adenine methylase-dependent (DAM) adenosine such as adhesion of internal organs and tissues which may methylation is widely used to assess pDNA replication in be due to polyvalent oil-adjuvanted vaccines However, the vivo in mammals Replicative synthesis of pDNA in bacteria significance of pDNA degradation compared to efficacy of can be proposed by the appearance of molecules harbour- transgene production as well as the possibility of pDNA ing the DAM methylation This DAM methylation is, how- replication in cells and integration into fish genome still ever, absent if pDNA is replicated in mammalian cells need to be focused upon.

[293,294] Plasmid DNA from bacteria contains a methylated adenosine within the GATC recognition site for the two References restriction enzymes: DpnI and MboI These two restriction

enzymes are used to assess the methylation of the pDNA,

[1] Foss GS Regulation of DNA vaccines and gene therapy on since they cleave the cognate recognition sequence, but

animals The Norwegian Biotechnology Advisory Board, vary in their sensitivity to methylation At GATC sites,

http://www.bion.no/publikasjoner/regulation_of_DNA_vac

MboI cleaves only completely unmethylated sites [295],

cines.pdf2003 [accessed 5 October 2007]

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[2] Restifo NP, Ying H, Hwang L, Leitner WW The promise of performed in mammals show that the pDNA methylation

nucleic acid vaccines Gene Therapy 2000;7:89e92

pattern remained as found in its bacterial form in the [3] Casares S, Inaba K, Brumeanu T-D, Steinman RM, Bona CA. muscle tissue, up to 19 months after i.m administration, Antigen presentation by dendritic cells after immunization showing that pDNA did not replicate in muscle tissue with DNA encoding a major histocompatibility complex class

[257,258,294] Similar results were observed in mammalian II-restricted viral epitope Journal of Experimental Medicine

1997;186:1481e6

liver and spleen up to 6 months [294] A study in Atlantic

[4] Chattergoon MA, Robinson TM, Boyer JD, Weiner DB Specific salmon also show that the pDNA methylation pattern

immune induction following DNA-based immunization through remains in its bacterial form in the kidney, liver, spleen

in vivo transfection and activation of macrophages/antigen-and muscle for at least 350 days macrophages/antigen-and at the administration

presenting cells Journal of Immunology 1998;160:5707e18 site for at least 535 days after i.m administration [184]

[5] Banchereau J, Steinman RM Dendritic cells and the control

of immunity Nature 1998;392:245e52

immunodeficiency virus type 1 in the past decade Clinical Microbiology Reviews 2004;17:370e89

Integration of pDNA in the host genome is a theoretical

[7] Weiner DB, Kennedy RC Genetic vaccines Scientific Ameri-possibility when pDNA enters the cell nucleus Following

can 1999;281:50e7

i.m administration, the pDNA that persists is extra

chro-[8] Whitton JL, Rodriguez F, Zhang J, Hassett DE DNA immuniza-mosomal [257,294], and integration into host DNA has been tion: mechanistic studies Vaccine 1999;17:1612e9.

postulated to be negligible [110,111,114] On the other [9] Utke K, Bergmann S, Lorenzen N, Kollner B, Ototake M, hand, new technology can increase the frequency of inte- Fischer U Cell-mediated cytotoxicity in rainbow trout, Onco-gration, as integration is detected after electroporation, rhynchus mykiss, infected with viral haemorrhagic septicae-but not after i.m administration of pDNA [113] In addition, mia virus Fish and Shellfish Immunology 2007;22:182e96.