DNA Vaccines for the Induction of Immune Responses in Mucosal Tissues

Chapter 67

DNA Vaccines for the Induction of Immune Responses in Mucosal Tissues Milan Raska School of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA; Palacky University Olomouc, Olomouc, Czech Republic

Jaroslav Turanek Veterinary Research Institute, Brno, Czech Republic

Chapter Outline Introduction1307 Brief History of DNA Vaccines 1308 Viral and Bacterial Infection Models 1308 DNA Vaccines Encode for Antigens Synthesized by Transduced Cells 1308 Structure and Principal Components of DNA Vaccine 1308 Uptake of DNA Vaccines Into the Nucleus for Antigen Expression1308 Comparison of DNA Vaccine with RNA Vaccine and Recombinant Virus Vectors 1315 Minicircles as a Minimized DNA Vaccine Form 1316 The Immune Response to DNA Vaccines 1316 Cross-presentation1316 Inherent Adjuvanticity of DNA Vaccines 1316 DNA Vaccines for Induction of Glycan-Specific Antibodies 1317 Safety of DNA Vaccines 1317 Approaches Increasing DNA Vaccine Efficacy 1317 Increasing the Efficacy of Target Cell Transduction 1317

INTRODUCTION DNA vaccines present a modern approach to the induction of cellular and humoral immune responses specific to protein or glycoprotein antigens expressed in host cells from antigen-encoding plasmid DNA administered by various routes. Because the antigen is produced by the host cells, DNA vaccination can mimic viral infection or immunization with live attenuated virus in terms of antigen expression, posttranslational modification (disulphide bridge formation, N- and O-glycosylation, proteolytic cleavage, folding, etc.), further intracytoplasmic antigen processing, major histocompatibility complex (MHC) processing and presentation, and the mechanism of host immune response stimulation. In contrast to other vaccination approaches, DNA vaccines offer rapid Mucosal Immunology. http://dx.doi.org/10.1016/B978-0-12-415847-4.00067-7 Copyright © 2015 Elsevier Inc. All rights reserved.

Physical Transduction Approaches 1317 Chemical Delivery Systems 1318 Biological Delivery Systems for DNA Vaccines 1322 Codon-Usage Optimization 1323 Enhancing Immunogenicity of DNA Vaccines with Molecular Adjuvants 1323 Targeting Innate Immune Receptors 1323 Usage of Cytokines Codelivered with DNA Vaccine 1324 Usage of Chemokines to Activate Mucosal Immune Response 1324 Targeting of DNA Vaccine to M Cells and DCs 1325 Targeting to M Cells 1325 Targeting to DC 1325 Oral DNA Vaccination 1325 Conclusion1326 Acknowledgment1326 References1326

vaccine construction based on reverse vaccinology, fast and efficacious purification, high stability, and well-documented absence of severe side effects. The level of antigen protein synthesis in the cells transduced with DNA vaccine represents one of the most important challenges for human application because, in contrast to small rodents, in which DNA vaccines are highly efficacious, in humans the quantity of antigen produced after DNA vaccination is low and the elicited immune response is generally poor and insufficient to ensure protection. Therefore, since initial DNA vaccination experiments performed in rodents in the early 1990s (Ulmer et al., 1993, 1994), intensive efforts have been made to enhance DNA vaccine efficacy and understand the molecular biological and immunological mechanisms underlying the antigen-specific immune response. 1307

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BRIEF HISTORY OF DNA VACCINES Initial DNA vaccination experiments were based on propelling DNA-coated gold microprojectiles into the skin using a handheld biolistic system (Williams et al., 1991), an approach later described as intradermal (i.d.) gene gun DNA immunization. Soon afterward, intramuscular (i.m.) immunization by unmodified naked plasmid DNA administered in buffered saline was found to stimulate immune responses of comparable intensity (Ulmer et al., 1993). The first well-known DNA immunization experiment consisted of three consecutive i.m. doses of 2 × 50 μg of DNA plasmid encoding influenza A virus nucleoprotein (NP) given to BALB/c mice. This induced both NP-specific antibody and NP-specific cytotoxic T-lymphocyte (CTL) responses (Ulmer et al., 1993), clearly demonstrating that DNA vaccine is capable of eliciting an efficacious MHC-I–restricted immune response similar to natural viral infection or immunization with attenuated viruses. Subsequent experiments demonstrated that a strong immune response could be induced in mice by i.m. immunization with a solution of naked DNA vaccine encoding influenza virus hemagglutinin (HA) or NP in doses <100 ng DNA. The mice were protected against challenge with LD90 of homologous influenza virus (Ulmer et al., 1994). Using the same construct, vaccination of nonhuman primates with 10 μg of DNA induced high-titer HA-specific neutralizing antibodies (Ulmer et al., 1994). DNA vaccines are advantageous over peptide immunization, which also induces strong MHC-I–restricted CTL responses, because they generally encode long polypeptides that can be processed and presented on various MHC-I molecules in a normal panmictic population. In contrast, peptide immunization efficacy is restricted according to the MHC haplotype of the vaccinated subject (Chaise et al., 2008).

Viral and Bacterial Infection Models DNA immunization subsequently proved to be protective against several viral, bacterial, parasitic, and even fungal agents in various animal infection models (Tables 1 and 2).

DNA VACCINES ENCODE FOR ANTIGENS SYNTHESIZED BY TRANSDUCED CELLS Structure and Principal Components of DNA Vaccine A DNA vaccine usually consists of circular double-stranded plasmid DNA coding for the vaccine antigen, under the control of a strong mammalian promoter and polyadenylation signal, with additional sequences necessary for plasmid construction, characterization, and amplification. The most frequently used promoter is derived from human cytomegalovirus (CMV) immediate/early

promoter (Boshart et al., 1985); other promoters are listed in Table 3. Research has focused on the identification of promoters effective after i.m. immunization because most DNA vaccines are administered by this route, which induces both systemic and mucosal immune responses. In humans, however, this widely used route is poorly effective for antigen expression and subsequent stimulation of immune response. Therefore, several muscle-effective promoters were identified (Table 3). Among all these, the CMV promoter generally exhibits the strongest activity in most cells and tissues (Zheng and Baum, 2005) and therefore can serve as a universal promoter for DNA vaccines. However, in therapeutic vaccine settings, the cytokine milieu or the drugs used could dramatically suppress CMV activity, as demonstrated for IFN-γ, which caused inhibition of transgene expression in most cell lines tested in vitro. Nevertheless, the same treatment led to amplification of MHC-I promoter-driven transgene expression in all cell lines (Harms et al., 1999). Therefore, for mucosal DNA vaccines, inflammatory conditions in the targeted tissue should be taken into account during optimal promoter selection. In addition to the promoter, termination/polyadenylation signals should be included. SV40 or bovine growth hormone 3′-untranslated region (BGH 3′-UTR) sequences are used in most DNA vaccines (Pfarr et al., 1986). Promoter and terminator/polyadenylation sequences are absolutely required in DNA vaccines. However, for manipulation in a bacterial host (Escherichia coli) the plasmid must also contain a bacterial origin of replication usually derived from bacterial ColE1 and pBR322 plasmids (ColE1 and pMB1 replicons) and an antibiotic resistance cassette allowing stabilization and amplification within E. coli cells during replication. The most common antibioticresistance genes used are aminoglycoside phosphotransferase, aminoglycoside acetyltransferase, or aminoglycoside nucleotidyl transferase, which inactivate gentamicin and kanamycin, β-lactamase, chloramphenicol acetyltransferase, and TetA efflux pump that provides resistance to tetracycline (Ausubel, 2002). The most frequently used DNA vaccination plasmids are derived from pUC plasmids containing a modified pMB1 replicon mutated to render them high copy-number, thereby allowing amplification in E. coli at 500–700 copies per cell (Bolivar et al., 1977; Bolivar, 1978). To increase plasmid yield during production, novel runaway R1 replicon-derived plasmids based on previously constructed pCP40 plasmid were designed (Remaut et al., 1983). Such a plasmid yields up to 19 mg of plasmid DNA per gram of dry cell weight (Bower and Prather, 2012).

Uptake of DNA Vaccines Into the Nucleus for Antigen Expression One important factor limiting the efficacy of a DNA vaccine is the transport of a large DNA molecule (3000–5000 bp or

TABLE 1  Examples of Viral Infection Models Exhibiting Protectivity of DNA Immunization Antigen Form

Host Organism

Vaccination Schedule: Dose, Form, Route

Priming Boost Strategy

Influenza H1N1

HA, NP

Mouse

3 × 100 ng, DNA, i.m.

Avian influenza H5N2

H5N1 HA

Chicken

H5N1

H5N1 HA, NA H1N1 HA NA

HBV

Challenge Dose

Note

Reference

All naked DNA

Intranasal 1 × LD90 of H1N1 (A/PR/8/34)

i.m. DNA dose <0.1 μg

Ulmer et al. (1994)

1 × 100 μg, DNA, i.m.

Single naked DNA electroporation

Oronasal 106 × ELD50 H5N2

Electroporation enhances efficacy

Ogunremi et al. (2013)

Mouse

2 × 10 or 2.5 μg, DNA, i.n.

All PEI-DNA

Intranasal 102–104 × LD50 muH5N1

PEI–DNA intranasal

Torrieri-Dramard et al. (2011)

HBsAg

Newborn chimpanzee

3 × 1 mg, DNA, i.m.

All naked DNA

Intravenous 100 × CID50

Prince et al. (1997)

HSV-1

Immediate early protein ICP27

BALB/c mice

2 × 90 μg, DNA, i.m.

All naked DNA

In scarified skin 10 × ID50

Manickan et al. (1995)

HSV-2

HSV-1 glycoprotein D

Swiss Webster mice

3 × 200 μg, DNA, i.m.

All naked DNA

Intravaginal 6.103 × PFU ∼lethal dose

Bourne et al. (1996)

HIV-1

gp160, rev, gag/pol

Chimpanzee

P* 3 × 100 μg, 160 DNA, i.m. +3 × 100 μg, 160, gag/pol DNA, i.m. B* 2 × 1 mg, 160, gag/pol DNA, i.m.

All naked DNA

Not specified 250 × CID50

First protective DNA vaccine in chimpanzee

Boyer et al. (1997)

SIV

– rAd5 env/rev, gag, nef – protein gp120

Cynomolgus macaques

P*1 × 5.108 PFU, rAd5, i.n. + oral 1 × 5.108 PFU, rAd5, intratracheal B* 2 × 100 μg, protein, i.m.

P* rAd5 B* gp120 protein or peptomer

Intrarectal 10 × MID SIVmac251

rAd5 prime protein boost strategy

Patterson et al. (2004)

SIV

– DNA SIV (mne) gag, pol, vif, vpr, rev, env, nef – protein gp160, gag/pol

Macaca fascicularis

P* 4 × 1.5 mg, DNA, i.d. + i.m. B* 2 × 250 μg, protein, i.m.

P* naked DNA B* protein

Intrarectal 20×MID SIV (mne)

DNA-induced in vivo VLP formation

Mossman et al. (2004)

Continued

Vaccines for the Induction of Immune Responses in Mucosal Tissues Chapter | 67  1309

Infection

Infection

Antigen Form

SHIV

– DNA – SIV239 gag, pol, vif, vpr, rev, env, nef – rMVA expressing SIV gag, pol, and HIV env genes

Host Organism

Vaccination Schedule: Dose, Form, Route

Priming Boost Strategy

Challenge Dose

Note

Reference

Rhesus macaques

P* 3 × 1 mg, DNA, i.n. +3 × 5 mg, DNA, i.m. +3 × 0.5 mg IL-12, DNA, i.m. B* 1 × 109 PFU, rMVA gag/pol, i.m. + 1 × 109 PFU, rMVA env, i.n.

P* naked DNA B* rMVA

Intrarectal 10 × AID SHIV 89.6P

DNA induced in vivo VLP formation IL-12 DNA

Manrique et al. (2008)

SHIV

Both vectors gag, pol, env

Rhesus macaques

P* 1 × 5.106 PFU, rVSV, i.n. B* 1 × 108 PFU, rMVA, i.m.

P* rVSV B* rMVA

Intravenous 30 × MID50 SHIV 89.6P

Monitoring 5 years after challenge

Schell et al. (2009)

IBV

Nucleocapsid protein

Chicken

2 × 150 μg, DNA, i.m.

All naked DNA

Nasal-ocular 100 × IED50

Bicistronic DNA plasmid coexpressing IL-2

Tang et al. (2008)

IBDV

VP2

Chicken

3 × 400 μg, DNA, i.m.

All naked DNA

Oral 2.103.8 × EID50/ml

Chang et al. (2003)

P*, priming; B*, boosting; CID, chimpanzee infectious dose; PFU, plaque-forming unite; MID, monkey infectious doses; rMVA, recombinant modified vaccinia virus Ankara; rVSV, recombinant vesicular stomatitis virus; rAd5, recombinant adenovirus five; IBV, infectious bronchitis virus; infectious bursal disease virus (IBDV); EID, embryo infective dose; muH5N1, mouse-adapted H5N1.

1310  SECTION | D  Mucosal Vaccines

TABLE 1  Examples of Viral Infection Models Exhibiting Protectivity of DNA Immunization—cont’d

TABLE 2  Examples of Bacterial Infection Models Exhibiting Protectivity of DNA Immunization Antigen Form

Host Organism

Vaccination Schedule: Dose, Form, Route

Priming Boost Strategy

Mycobacterium tuberculosis

Mtb39

Mouse

3 × 100 μg, DNA, i.m.

All naked DNA

Aerosol 100 × CFU of M. tuberculosis Erdman

M. tuberculosis

Rv1818PE

Mouse

3 × 100 μg, DNA, i.m.

All naked DNA

Aerogenic 500 × CFU of M. tuberculosis Erdman

PGRS domain of Rv1818 inhibits MHCI presentation

Delogu and Brennan (2001)

M. tuberculosis

ESAT-6 MPT64 MPT63 MPT83 KatG Ag85B MTB8.4 MTB39A MTB12 Rv1818c

Mouse

3 × 200 μg, total DNA, i.m.

All naked DNA each dose consists of 10 different DNA plasmids encoding ubiquitinated ags or TPA-conjugated ags

Aerogenic 50×, 500×, 300× CFU of M. tuberculosis Erdman

– Enhanced protectivity of multiple antigen DNA vaccines – Ubiqutin- or TPAconjugation enhancing cross-presentation – Protective as BCG

Delogu et al. (2002)

M. tuberculosis

Mtb72

Mouse Guinea pig

(a) 3 × 100 μg, DNA, i.m. for mouse (b) 3 × 200 μg, DNA, i.m. for guinea pig

All naked DNA

Aerosol 20–100 × CFU Of M. tuberculosis H37Rv (low dose)

Protection equivalent to BCG

Skeiky et al. (2004)

M. tuberculosis

HtpX

Mouse

(a) 2 × 108 CFU, E. coli, i.n. Or (b) 2 × 100 μg, DNA, i.m.

Naked DNA E. coli (dap−)vectored DNA

Aerogenic 200 × CFU of M. tuberculosis Erdman

Oral E. coli-vectored DNA effective as naked DNA i.m.

Brun et al. (2008)

Chlamydia trachomatis

MOMP

Pig

P* 500 μg, DNA OmpA + 100 μg, DNA LTa + 100 μg, DNA LTb + 250 μg, DNA GMCSF, all i.n. + i.vag.inj

All naked DNA

Intravaginal 108 × TCID50 of C. trachomatis serovar E

– Naked DNA administered i.n. and i.vag. inj. is protective in pigs – E. coli labile toxin adjuvant

Schautteet et al. (2012)

Chlamydophila pneumoniae

MOMP Omp2

Mouse

P* 2 × 200 μg, DNA, i.m. B* 1 × 106 IU, rSFV, s.c.

P* naked DNA B* rSFV-vectored DNA

Intranasal 106 × IFU of C. pneumoniae EBs

Naked DNA followed by rSFV exhibits protection

Penttila et al. (2000) and Penttila et al. (2004)

Infection

Challenge Dose

Note

Reference Dillon et al. (1999)

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Continued

Vaccination Schedule: Dose, Form, Route

Infection

Antigen Form

Host Organism

C. psittaci

MOMP A

Turkey

P* (a) 1 × 100 μg, DNA, i.m. +1 × 100 μg, DNA, i.n. (b) 1 × 100 μg, DNA, aerosol B* identical to P* (a) or (b)

Mycoplasma pneumoniae

p1C

Mouse

Helicobacter pylori

hspA hspB

Haemophilus influenzae Enterotoxic E. coli

Priming Boost Strategy

Challenge Dose

Note

Reference

All naked DNA i.n. dropwise administration Aerosol cirrus nebulizer

Aerosol 104 × TCID50 of C. psittaci strain 84/55 (high dose)

Naked DNA effective after mucosal administration

Vanrompay et al. (2001)

(a) 3 × 50 μg, DNA, i.m. Or (b) 3 × 50 μg, DNA, i.n.

All naked DNA

Airway 2.107 × CFU of M. pneumoniae strain M129

i.n. Administration of naked DNA induces protection

Zhu et al. (2012)

Mouse

1 × 10 μg, hspA DNA, + 10 μg, hspB DNA, both i. cutaneous

All naked DNA

Orogastric 108 × cells of H. pylori SS1.

Reduced gastritis after challenge

Todoroki et al., (2000)

P6 outer membrane

Mouse

3 × 100 μg, DNA, i.n. + Matrix-M adjuvant

All naked DNA

Intranasal 108 × CFU nontypeable H. influenzae

Effective nasal immunization with naked DNA

Kodama et al. (2011)

– DNA CfaB – rB. Subtilis expressing CfaB

Mouse

P* 2 × 100 μg, DNA, i.m. B* 2 × 1010, rB. subtilis vegetative cells or spores, intragastric

P* naked DNA B* rB. subtilis expressing CfaB

Stomach 20 × LD50 2-day-old pups

CfaB-specific mucosal IgA transmitted by milk confers protection

Luiz et al. (2008)

P*, priming; B*, boosting; Ag85B, secreted fibronectin-binding protein antigen; PstS-3, 40 kDa lipoprotein phosphatase transporter; Rv1818PE, protein transcript of the PE_PGRS (proline-glutamate) family of M. tuberculosis genes without Gly–Ala repeats extending to the C terminus of the PE_PGRS gene; MTB39, PPE (proline-proline-glutamate) family of M. tuberculosis genes member; hsp70, cytoplasmic heat shock protein 70 kDa; MOMP, major outer membrane protein encoded by OmpA gene; Npt1Cp, ADP/ATP translocase; LLO, listeriolysin; Mtb72F, 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N); TPA, tissue plasminogen activator; LTA, subunit A of the thermolabile E. coli enterotoxin; IFU, inclusion forming unit; rSFV, recombinant Semliki Forest virus; P1C, C′ terminal region of P1 adhesin protein M. pneumoniae.

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TABLE 2  Examples of Bacterial Infection Models Exhibiting Protectivity of DNA Immunization—cont’d

TABLE 3  Promoters Tested for DNA Vaccine Construction Promoter

Description, Promoted Gene

Effective in Tissue

Note

References

Human cytomegalovirus (CMV) immediate/early promoter

All tissues From mammals to fish

Strongest and most universal promoter IFN-gamma inhibits activity in colon cells Mitogen-stimulated T cells supernatant inhibits activity in colon cells

Boshart et al. (1985) and Harms et al. (1999)

rAQP5

Rat aquaporin-5

Rat submandibular glands

Tested in rat submandibular glands infected with Ad vectors encoding EGFP Tested using plasmid encoding luciferase administered by retrograde ductal instillation

Zheng and Baum (2005)

Kall

Kallikrein

Moderate in rat submandibular glands Specific to ductal cells of submandibular gland Liver Kidney

Tested in rat submandibular glands infected with Ad vectors encoding EGFP Tested using plasmid encoding luciferase administered by retrograde ductal instillation

Zheng and Baum (2005)

EF1α

Mammalian elongation factor 1α

Rat submandibular glands Long-term expression in lung

Tested in Ad-infected submandibular glands Tested with retrograde ductal instilled plasmid Persistent expression of gene in mouse lung, whereas CMV, SRV, SV40 promoted expression fell <10% within 2 weeks

Garmory et al. (2003) and Zheng and Baum (2005)

K18

Cytokeratin 18

Moderate in submandibular glands Epithelia of internal organs lung, liver, kidney, intestine

Specific to airway epithelia, not active in fibroblasts, lung endothelia

Chow et al. (1997)

K19

Cytokeratin 19

Mammal epithelial cells Lung airways epithelia Submandibular glands Esophageal and pancreas cancer cells

Tested in rat submandibular glands as one of strongest promoters

Zheng and Baum (2005) and Brembeck and Rustgi (2000)

AMY

Amylase

Salivary gland

Moderate activity in comparison to CMV

Ting et al. (1992)

Fascin-1

Mammalian actinbundling protein

DC

Successfully tested for gene gun delivery in allergy models in mice

Sudowe et al. (2006)

CD11 cS

700-bp fragment of CD11c promoter

DC

Successfully tested in therapeutic settings in mice

Ni et al. (2009)

UbC

Human ubiquitin gene

Muscle Long-term expression in lung

Persistent expression of gene in mouse lung. CMV, SRV, SV40 promoted gene expression fell < 10% within 2 weeks

Gill et al. (2001) and Jathoul et al. (2004)

CYP24A1

1,25-Dihydroxyvitamin D3 24-hydroxylase

Keratinocytes Intestinal epithelium cells

Inducible by 1,25-vitamin D3(OH)2 (Vitamin D3)

Itai et al. (2001) and Lechner et al. (2007)

BL3-6prmtr

Promoter/enhancer for MHC-I

Many tissues

IFN-γ enhances activity in colon cells Mitogen-stimulated T cells supernatant enhances activity in colon cells

Harms et al. (1999)

RSV

Rous sarcoma virus long terminal repeat

Many tissues Strongest promoter in rat submandibular glands

10-fold higher activity in rat submandibular glands compared with CMV promoter

Gorman et al. (1982) and Zheng and Baum (2005)

SV40

Simian virus 40 early promoter

Many tissues

IFN-gamma inhibits activity in colon cells Mitogen-stimulated T cells supernatant inhibits activity in colon cells

Moreau et al. (1981) and Harms et al. (1999)

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CMV

1314  SECTION | D  Mucosal Vaccines

1800–3000 kDa) from the extracellular space to the cell nucleus, where transcription of vaccine-encoding genes takes place to generate mRNA. After its translocation to the cytosol, translation into polypeptide and co- and posttranslational modifications fully rely on the cellular translational machinery. The lipid bilayers of cellular membranes represent an almost insurmountable barrier for polyanionic molecules such as DNA. It is estimated that after i.m. injection of naked DNA, approximately 90% never reaches the cytosol (Babiuk et al., 2003). Alternatively, DNA is endocytosed but mostly degraded with minimal release of intact plasmid to the cytosol. Thereafter, the nuclear membrane represents a second barrier. This contains nuclear pore complexes (NPC) consisting of several proteins allowing spontaneous transport of macromolecules <9 nm in diameter (60 kDa). DNA vaccine plasmids are above this limit (Cartier and Reszka, 2002). In vitro experiments indicate that <1% of plasmid DNA molecules present in the cytoplasm reach the nucleus (Labat-Moleur et al., 1996). By comparing the transfection efficacy of a DNA plasmid encoding HSV thymidine kinase administered by glass micropipette into cytoplasm or nucleus, it was estimated that <0.1% of cytoplasmically administered DNA is transported into the nucleus for efficient

FIGURE 1  Pathways involved in the transport of DNA from outside the cell to the nucleus. Transport of DNA into cells is initiated predominantly by endocytosis. In the case of DNA lipoplexes (blue arrows) that are formed as spherical unilamellar, spherical multilamellar, or inverted hexagonal phases, and DNA polyplexes (green arrows), endocytosis is initiated through cell surface proteoglycans or chondroitan sulfates. Fusion of DNA lipoplex phospholipid membrane with cytoplasmic membrane leads to release of the DNA close to the cytoplasmic membrane, which is associated with substantial DNA degradation (red arrows). The same is expected for naked DNA. Once DNA complexes are inside the endosome, two distinct pathways take place: DNA lipoplexes are released from endosome by lipid– lipid fusion of the membranes, leading to release of naked DNA into the perinuclear cytosolic space (L1), or intact DNA lipoplexes are released into the perinuclear cytosolic space by destabilization of the endosome membrane (L2). Thereafter, lipoplexes fuse with lipid membranes of ER, Golgi, or nucleus, leading to the release of naked DNA into cytosol or ER lumen. Release of DNA into the ER lumen is expected to facilitate the transport to the nucleus through poorly described mechanisms. DNA polyplexes are released from endosomes or after formation of lysosomes by their disruption resulting from osmotic lysis caused by the active uptake of H+ and Cl− ions and water, a situation described for polyplexes containing protonatable amines such as PEI. Lysosome and endosome disruption could be triggered by the conjugation of polyplexes with the membrane-lytic cationic peptides. After release into the perinuclear cytoplasmic space, dissociation of DNA polyplexes takes place but the mechanism is not well characterized. Naked DNA molecules located close to the nuclear membrane pass through nuclear pore complexes at a frequency of 10−3. (See color plate section.)

expression (Capecchi, 1980). DNA delivery systems based on cationic liposomes and cationic polymers increase the transfection efficacy by several mechanisms (Elouahabi and Ruysschaert, 2005), as schematically depicted in Figure 1. In contrast, active transport through the NPC can be highly effective for large DNA-containing macromolecules, as demonstrated for several DNA viruses that transport their genome through the NPC (Whittaker et al., 2000). Because the barrier function of the nuclear membrane is abolished during cell division, administration of DNA vaccines into tissue undergoing active replication leads to a dramatic increase in antigen production and an intense immune response. A striking example of such an approach is represented by hydrodynamic (HD) DNA vaccination of mice by rapid tail-vein administration in a large volume, 1 ml/10 g body weight. This leads to increased pressure and transient membrane damage in the liver owing to temporary and reversible right heart failure, and a series of reparative processes associated with increased hepatocyte division (Budker et al., 2006; Sebestyen et al., 2006). Hydrodynamic immunization of mice with DNA encoding HIV-1 gp120 induced high and long-lasting humoral and cellular immune responses, which exceeded responses induced by i.m. DNA

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vaccination by several orders of magnitude. When HD DNA immunization was combined with a subsequent mucosal (e.g., intranasal (i.n.)) protein gp120 boost, high levels of specific IgA and IgG antibodies were induced in the genital tract secretions (Raska et al., 2008). Once the DNA vaccine reaches the cell nucleus, transcription of mRNA is initiated, followed by antigen translation on cytosolic or endoplasmic reticulum (ER) ribosomes. Antigen processing inside the ER is associated either with MHC-I presentation or antigen transport into the Golgi complex, where several posttranslational modifications take place such as N- and O-glycosylation, disulphide bridge formation, cleavage at specific sites, folding, and eventually secretion out of the cell. In the case of protein synthesis on cytosolic ribosomes, the antigen is targeted to MHC-I presentation. DNA vaccination thus resembles antigen processing similar to viral infection or during tumor development, ensuring similar co- and posttranslational modifications. This intracellular synthesis of antigen is a major advantage of DNA vaccination, leading to antigen-presentation on MHC-I molecules and elicitation of antigen-specific CD8+ T cell responses.

Comparison of DNA Vaccine with RNA Vaccine and Recombinant Virus Vectors The main principle of DNA vaccination consists of the expression of antigen encoded by the plasmid. In addition, the principle of antigen expression after administration of antigen-encoding nucleic acid includes RNA vaccines as well as recombinant virus vaccines. The main difference between DNA and RNA vaccines and virus-vectored vaccines is the complexity of the delivery system, which in the case of virus-vectored vaccines (recombinant attenuated virus expressing vaccine antigen), as the most complex system, is the consequence of the natural evolution of the parent virus. Therefore, recombinant virus vaccines or vectors represent a wellproven system for the delivery of genomic nucleic acid to the cytoplasm or nucleus of target cells for effective antigen expression. The amount of plasmid DNA necessary to induce immune responses is generally many orders of magnitude higher than the amount of DNA within viral vectors. Recombinant virus vectors, often DNA viruses, are based on replication-defective adenoviruses confirmed to be safe, live, oral vaccines against adenoviral respiratory disease in military recruits, including types 4 and 7 (Hung et al., 1990). Type 5 adenovirus (rAd5) (Yoshimura et al., 1993) is currently used in several HIV-1 (Burwitz et al., 2011; Koblin et al., 2011; Schooley et al., 2010), influenza (Toro et al., 2011; Vemula and Mittal, 2010), hepatitis C (Desjardins et al., 2009), and other vaccines. Other frequently used viral vectors are derived from poxviruses, including vaccinia derivatives

NYVAC (New York Vaccinia Virus), MVA (Modified Vaccinia Virus Ankara), and canarypox-based ALVAC (Panicali and Paoletti, 1982; Paoletti, 1996; Tartaglia et al., 1992), which have been tested in several HIV-1 (Aboud et al., 2010; Goepfert et al., 2011; Gorse et al., 2012; Gudmundsdotter et al., 2009; Hanke et al., 1999; Hansen et al., 1992; Hel et al., 2002; Lai et al., 2011; Liu et al., 2012; Moise et al., 2011; Perreau et al., 2011; Ramanathan et al., 2009; Richmond et al., 1997; Winstone et al., 2009), influenza (Breathnach et al., 2004; Degano et al., 1999; Smith et al., 1983), and hepatitis C (Deng et al., 2009) models among others. The main disadvantage of viral vectors consists of their inherent immunogenicity, which leads to their recognition by the immune system and inhibition of their capacity to generate the target antigen. Such immunity could be preexisting owing to prior infection with wild-type virus or result from vaccination with a related virus or similar vector. The solutions are to develop viral vectors not commonly circulating in the immunized population and to avoid repeated immunization with the same vector (Ulmer et al., 2012). Although repeated immunization with the same recombinant virus vaccine does not boost antigen-specific T cell responses, the antibody response is enhanced (Kannanganat et al., 2010). To boost both antibody and T cell responses, a frequent approach uses DNA vaccine priming followed by recombinant virus boosting. In this case, the effect of DNA priming must be determined after boosting with recombinant virus because, although DNA priming alone might not elicit detectable immunity, it should contribute substantially to an increased T cell response (De Rosa et al., 2011). RNA-based vaccines were introduced at the same time as DNA vaccines in 1990, when Wolff et al. demonstrated that i.m. injection of RNA or DNA vectors coding for chloramphenicol acetyltransferase, luciferase, or β-galactosidase resulted in production of respective reporter protein (Wolff et al., 1990). Subsequent experiments in mice confirmed the ability of a liposome-formulated RNA vaccine to induce NPspecific CTL responses (Martinon et al., 1993). In general, RNA vaccines exhibit a similar spectrum of applications and similar dose effectiveness as DNA vaccines (Carralot et al., 2004; Pascolo, 2004). RNA vaccines are usually prepared by in vitro transcription of mRNA from linearized plasmid DNA containing a bacteriophage promoter (T7, SP6, or T3) followed by poly A extension (Lorenzi et al., 2010). RNA vaccines can also be derived from subgenomic replicons generated from Semliki Forest virus, Sindbis virus, poliovirus, tick-borne encephalitis virus, and others (Ulmer et al., 2012). In contrast to DNA vaccines, RNA vaccines are not delivered to the nucleus because cytoplasm is the main site for the translation of mRNA, which is transient and decays rapidly, thereby better mimicking antigen production during natural infection (Ulmer et al., 2012).

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Minicircles as a Minimized DNA Vaccine Form To eliminate plasmid sequences unnecessary for antigen expression in cells and enhance the transduction efficacy of DNA vaccines, strategies for preparation of minimal nucleic acid vectors (MNAV) or minicircles were developed (Weide et al., 2008). Minimal nucleic acid vectors are generated by intramolecular (cis-) recombination from a parental plasmid mediated by the activity of an inducible integrase that recognizes short sequences of DNA (att) inserted adjacent to an antigen-encoding expression cassette. An example of such a system is based on arabinose inducible PhiC31 integrase and E. coli strain ZYCY10P3S2T (Kay et al., 2010). Use of MNAV is in accordance with regulations for the manipulation of genetically modified organisms.

THE IMMUNE RESPONSE TO DNA VACCINES Cross-presentation For the induction of antigen-specific CTLs, only dendritic cells (DCs) and macrophages are capable of activating naive CD8+ T cells that can interact with antigen-derived peptide fragments presented on MHC-I molecules. Therefore, efficacious activation of the immune system requires targeting the DNA vaccine to DCs. Delivery of DNA vaccines to DCs by coupling them with ligands recognized by specific DC surface receptors significantly increases the magnitude of the elicited immune response (Condon et al., 1996). However, in most experiments, the DNA vaccine was not targeted to DC but instead administered to tissues such as muscle or epithelium formed predominantly of cells that can express the DNA-encoded antigen on MHC-I but not initiate the immune response (Corr et al., 1996; Iwasaki et al., 1997). A similar situation arises during the initiation of CTL responses toward cancers or viruses because DCs or macrophages are not affected by either process. In 1976, it was found that some exogenously delivered antigens can be recognized by CD8+ T cells, of which CTL cells are the best known (Bevan, 1976). Viral and tumor antigens are well known to be recognized by CTL (Heath and Carbone, 2001; Kurts et al., 2010). The responsible mechanism was designated as cross-priming or cross-presentation (Bevan, 1976), and DCs, especially immature DCs, are the most active cross-presenting cells. DNA vaccine-encoded antigen destined for cross-presentation by DC is produced by the transduced cell, released either alone or in association with cellular debris, and phagocytosed or pinocytosed by DCs that partially digest the engulfed antigens, leaving proteolytic fragments of appropriate size for further processing and presentation on MHC-I. Immature DC can retain nearly neutral pH for few hours (Savina et al., 2006) owing

to moderate activity of NOX2 and limited recruitment of the cytosolic subunits of the V-ATPase to endosomes and lysosomes (Rada and Leto, 2008; Savina and Amigorena, 2007). Approaches employing cross-presentation to enhance DNA vaccine efficacy could be based on altering antigen stability, as reported for lymphocytic choriomeningitis virus nucleoprotein (LCMVNP), which was targeted for degradation by N-terminal fusion to the ubiquitin-like modifier (FAT10) leading to increased cross-presentation (Schliehe et al., 2012). Alternative approaches include fusing the DNA vaccine-encoded antigen with soluble ligands for DC surface receptors that initiate crosspresentation, such as HIV-1 gag antigen fused to human programmed death-1 (PD1) receptors PD-L1 (CD274), HPV type 16 E7 antigen fused to Mycobacterium tuberculosis heat shock protein 70 kDa (hsp70) or to human hsp60, and Listeria monocytogenes immunodominant peptide antigens LLO91–99 and p60217–225 fused to murine heat shock protein (gp96) (Chen et al., 2000; Huang et al., 2007; Rapp and Kaufmann, 2004; Zhou et al., 2013). Finally, DC cross-presentation potency can be enhanced through coadministration of various stimulatory molecules such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, CD40 ligands, hsp60, and gp96 (Binder and Srivastava, 2004; Demine and Walden, 2005; Kol et al., 2000; Singh-Jasuja et al., 2001). The effectiveness of such strategies needs to be tested individually for each antigen because the nature of the antigen can substantially affect the immune response, especially with CD8+ T cells.

Inherent Adjuvanticity of DNA Vaccines Because a DNA vaccine is able to induce antigen-specific immune responses after naked plasmid administration, it may be concluded that the DNA plasmid or the route of its administration exhibits inherent adjuvanticity depending on double-stranded (ds) DNA, which interacts with intracellular pattern-recognition receptors (PRR) such as Toll-like receptor (TLR)-9 and stimulator of interferon genes (STING) (Coban et al., 2011; Ishikawa et al., 2009). TLR-9 is stimulated by CpG motifs (cytosinephosphate-guanosine dinucleotide flanked by two 5′ purines and two 3′ pyrimidines in mice), which are undermethylated when the plasmids are amplified in a bacterial host (Garmory et al., 2003). CpG induces APC to secrete Th1type cytokines IL-12 and IFN-α/β, which activate CD8+ T cells and NK cells (Klinman, 2003). Nevertheless, TLR-9–/– mice exhibit comparable levels of antigen-specific antibodies (total immunoglobulin (Ig)G, IgG1, and IgG2a), CTL, and IFN-γ after immunization with DNA vaccine encoding glycoprotein D protein from bovine herpesvirus type 1 as their wild-type TLR-9+/+ counterparts (Babiuk et al., 2004).

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Although some recent publications suggest that TLR-9 signaling is critical for elicitation of CD8+ T cell responses, it has been reported that the adjuvanticity of DNA vaccines relies on an additional mechanism identified as the B form right-handed helical structure (B-DNA) of double-stranded circular DNA plasmids. B-DNA activates STING/TANKbinding kinase 1 (TBK1)-dependent signaling independent of TLR, and induces type-I IFNs that stimulate CD4+ and CD8+ T cells and B cells (Coban et al., 2011; Fitzgerald et al., 2003; Ishii et al., 2008).

DNA Vaccines for Induction of GlycanSpecific Antibodies Induction of polysaccharide-specific antibodies is important for protection against pyogenic microorganisms, and it seems a promising strategy to prevent HIV-1. Such antibodies can be induced by DNA vaccines encoding peptides that mimic polysaccharide epitopes, thus changing the typical T-independent response to a T-dependent one with all of its advantages (long-lasting memory, high affinity, and IgG2a antibodies in mice (Beninati et al., 2006)). Antibodies against selected serotypes of Neisseria meningitidis and Streptococcus pneumoniae capsular polysaccharides and Brucella melitensis lipopolysaccharide have been experimentally induced by DNA immunization (Beninati et al., 2006, 2009; Lesinski and Westerink, 2001; Prinz et al., 2003). Identification of peptide sequences mimicking those polysaccharide epitopes could be achieved by anti-idiotype antibody technology or by using phage display.

SAFETY OF DNA VACCINES Although clinical trials using DNA vaccines against HIV-1 or HPV proved high overall safety (Guimaraes-Walker et al., 2008; Henken et al., 2012; Koblin et al., 2011), concerns focus predominantly on DNA-induced mutagenesis, spreading of bacterial resistance genes, and DNA-induced autoimmunity. The possibility of genomic integration of plasmid DNA within immunized host cells was exhaustively tested without convincing evidence of efficient integration (Wolff et al., 1992). The risk of integration is up to three orders lower than that of naturally occurring mutations (Ledwith et al., 2000a,b; Nichols et al., 1995; Sheets et al., 2006; Wang et al., 2004b). Nevertheless, the presence of DNA after i.m. injection of naked plasmid DNA in experimental mice was confirmed in muscle cell lysate up to 19 months later (Wolff et al., 1992). In the case of DNA encapsulated in liposomes, stability in experimental calves was almost two orders of magnitude higher than that of naked DNA (Orsag et al., 2008). Spreading of antibiotic resistance genes from DNA vaccine plasmids to neighboring bacteria is of high importance in mucosal tissues where a high bacterial load is typical. Genes for resistance to

clinically important antibiotics such as ampicillin are therefore not acceptable for human trials, and kanamycin resistance is preferred. In addition, minimized DNA vaccine application would be highly desirable. The last safety question deals with potential induction of an anti-DNA immune response in an autoimmune reaction. Studies in nonhuman primates as well as results from clinical trials did not provide evidence for induction of autoimmunity (Tavel et al., 2007; Wloch et al., 2008), although some studies indicated that DNA vaccination can activate autoreactive B cells to secrete IgG anti-DNA autoantibodies. However, the magnitude and duration appears to be insufficient to cause disease in normal animals or accelerate disease in autoimmuneprone mice. Based on these findings, the 2007 Food and Drug Administration (FDA) Guidance document concluded that sponsors no longer need to perform preclinical studies to specifically assess the effect of vaccination on autoimmunity (Klinman et al., 2010).

APPROACHES INCREASING DNA VACCINE EFFICACY Increasing the Efficacy of Target Cell Transduction Physical Transduction Approaches Efforts to enhance the efficacy of DNA vaccines led to the development of modified administration methods using physical or chemical/biological principles for DNAmolecule delivery to target cells. Chemical modifications include the development of complexes formed between cationic polymers and negatively charged DNA. Biological delivery systems based on recombinant viruses or bacteria increase the efficacy of targeting the DNA vaccine for expression in target cells or particular mucosal surfaces, respectively. While mucosal (buccal, sublingual, i.n., orogastric, or rectal) delivery of naked DNA is inefficient, various chemical and biological delivery systems show promise for mucosal DNA vaccines. Furthermore, some forms of systemically administered DNA vaccines can enhance mucosal immune responses owing to either the site targeted (e.g., the liver after hydrodynamic administration) or to the adjuvant effect of codelivered immunomodulatory molecules, such as mucosa-targeting chemokines. Gene Gun Gene-gun delivery, a handheld form of biolistic system used for DNA vaccination since the 1990s (Williams et al., 1991), consists of propelling DNA-coated colloidal gold microprojectiles toward the tissue surface. Gas-accelerated particles penetrate into deeper layers based on adjustment of the discharge pressure. Because the particles penetrate into the cytosol and cell nucleus, the efficacy of DNA transduction

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is substantially higher than that of other approaches and therefore substantially reduced doses of DNA plasmid are required (0.1 μg/dose for gene-gun delivery compared with 10–100 μg typically used i.m. in mice). In contrast to the Th1 immune response elicited after i.m. administration, gene-gun DNA immunization in mice preferentially induces Th2 responses associated with higher production of IL-4 and dominance of IgG1 antibodies (Gurunathan et al., 2000). The Th1/Th2 dominance and intensity of CD8+ T cell responses could be modified by the construction of the vaccine antigen. Compared with needle i.m. or biojection i.d., gene-gun i.d. application of DNA vaccine encoding HPV-16 E7 antigen fused with M. tuberculosis hsp70 generated the highest number of E7-specific CD8+ T cells (Trimble et al., 2003). Similarly, gene-gun immunization of mice with DNA coding for the V3 loop of HIV-1 Env antigen fused with hepatitis B virus midsized HBsAg (pre-S2 + S) as adjuvant stimulated V3-specific antibodies as well as CTL responses that remained stable for several weeks (Fomsgaard et al., 1998). Gene-gun immunization by the oral mucosa using DNA encoding HA from influenza A/ WSN/33 (H1N1) was effective in a hamster model (Loehr et al., 2000). Intravulval mucosal gene gun immunization of cattle with DNA encoding bovine herpesvirus-1 glycoprotein B induced a strong cellular immune response and primed humoral immune responses. Glycoprotein B was expressed early throughout the mucosa, and Langerhans cells were widely distributed in the mucosal epithelium (Wang et al., 2003). High-Pressure Delivery Biojector Needle-free jet injection is another DNA delivery approach that has been investigated extensively as a method of i.d. immunization of laboratory animals such as mice, pigs, rabbits, dogs, and monkeys (Cui et al., 2003; Mumper and Cui, 2003) and was approved by the FDA for i.d., i.m., and subcutaneous applications. This is based on the administration of a DNA vaccine in buffered saline using a device consisting of an injector and a disposable syringe that can be set to deliver DNA to distinct layers, the epidermis, dermis, or subcutaneous muscles, depending on the speed of the ejected solution. After biojection, the volume of tissue reached by the DNA is higher than after needle administration. Needle-free, i.m. jet injection of a DNA vaccine was tested in several clinical trials, including HVTN204, which consisted in the administration of HIV-1 antigens EnvA-, EnvB-, EnvC-, gagB-, polB-, and nefB-encoding DNA vaccines, followed by boosting with rAd5 containing matching inserts. This elicited both cellular and humoral immune responses (Beckett et al., 2011; Churchyard et al., 2011). Buccal mucosal jet injection was tested as booster immunization after i.m. priming with DNA vaccines coding for nef, rev, and tat regulatory proteins in HIV-1–infected individuals. The plasmids were detected in buccal biopsies. The

vaccinated side was infiltrated by T cells, granulocytes, and DCs, and a high concentration of IL-2 was detected in the mucosal transudate for 4 months. Nevertheless, only negligible antigen-specific humoral and T cell responses were induced (Lundholm et al., 2002).

Chemical Delivery Systems Free DNA or RNA is a negatively charged high-molecular hydrophilic substance. Its polyanionic character results from phosphate groups and the size of DNA results in low penetration across target cell membranes and poor transduction of target cells. Therefore, various chemical systems for complexing DNA were developed to facilitate the transport of DNA across target cell membranes and improve the stability of DNA in extracellular environments, such as serum or mucosal tissues in which DNases or low pH prevail, and intracellularly where low pH occurs in endosomes and lysosomes (Elouahabi and Ruysschaert, 2005). Individual DNA-complexing systems differ in effectiveness as a result of the folding of DNA into compact particles that resist the extracellular environment, transport of DNA complexes across cell membrane by engulfment, the release of intact DNA into the cytoplasm or entry into the cell nucleus. The trapping of DNA in polymer particles (polyplexes) or in liposomes (lipoplexes) by the targeted cells is the result of endocytosis of polyplexes or lipoplexes induced by their binding to cell surface proteoglycans or chondroitan sulfate, or it could be a result of the overall small positive charge of the complex and the negative charge of most cell surfaces owing to negatively charged membrane proteoglycans and especially sialic acid (Elouahabi and Ruysschaert, 2005; Boussif et al., 1995; Pires et al., 1999) (Figure 1). Subsequent release of intact DNA into the cytoplasm occurs by several mechanisms. The complexes are endocytosed and the endosomal vesicles transport DNA in lipoplexes to the perinuclear space, where DNA is released (Dowty et al., 1995) from endosomes by lipid–lipid fusion of the membranes, or intact DNA lipoplexes are released into the cytosol by destabilization of the endosome membrane followed by fusion with organelles such as ER, allowing DNA release (Elouahabi and Ruysschaert, 2005) (Figure 1). In the case of DNA polyplexes that do not contain a hydrophobic moiety, the endosomes are osmotically lysed owing to the active uptake of H+ and Cl– ions and water, as typically occurs with polycations such as polyethyleneimines (PEI) that contain amine groups or dendrimers that act as a “proton sponge.” Furthermore, the DNA can be released from endosomes by poorly defined mechanisms (Akinc et al., 2005; Boussif et al., 1995). Endosomal escape has been described for many delivery systems such as fusogenic lipids, pH-sensitive or cationic liposomes, lysosomatropic agents such as chloroquine, fusogenic peptides such as hemagglutinin, amphipathic peptides, cationic polymers (PEI), dendrimers, and subunits of toxins such as diphtheria toxin and Pseudomonas exotoxin

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(Wang et al., 2011). Once the DNA is in the cytosol, transport to the nucleus takes place as for naked DNA. Poly(dl-lactide-co-glycolide) Intragastric administration of poly(dl-lactide-co-glycolide) (PLG)-encapsulated plasmid DNA encoding HIV-1 Env results in transduced expression of the Env glycoprotein in the intestinal epithelium, and oral administration induces specific systemic as well as mucosal (rectal) responses (Kaneko et al., 2000). PLG-encapsulated plasmid DNA encoding rotavirus capsid protein was used for oral immunization, and induced protection against challenge with homologous rotavirus (Chen et al., 1998). Furthermore, oral immunization with PLG-DNA encoding a known CTL epitope of vesicular stomatitis virus (VSV) induced specific CTL responses (Hedley et al., 1998). Polyethylenimines Polyethylenimines (PEIs) are the second most frequently used polymer for the preparation of DNA polyplexes. Their strong positive charge given by the high nitrogen content in the backbone of the molecule leads to undesirable side effects. Therefore, various chemical modifications of PEI were tested, such as the N-acylation of grafted 25 kDa PEI (Aravindan et al., 2009), or dodecylation of 2 kDa PEI allowing five times higher transfection efficiency than the grafted 25 kDa PEI (Thomas and Klibanov, 2002). Preparation of linear PEI (Zou et al., 2000) with complete removal of N-acyl residues allowed 10,000 times higher transfection efficiency in vivo and 1500 times higher specificity for lung tissue after systemic administration in experimental mice (Thomas et al., 2005). Hydrolysis of poly(2-ethyl-2-oxazoline) in linear PEI yielding pure polycations, especially PEI87 (87 kDa) and PEI217 (217 kDa), allowed preparation of DNA polyplexes with six times higher transfection efficacy than N-deacylated 25 kDa PEI with a higher condensation of DNA and better pH-buffering capacity (Thomas et al., 2005). Further reported modifications consisted of grafting linear PEI in polyethylene glycol (PEG) (described as PEGylation) (Tang et al., 2003), folatePEG-folate (Benns et al., 2002), or dextran (Tseng and Jong, 2003). I.n. delivery of DNA/PEI complexes improved by 1000-fold the efficiency of gene transfer in the respiratory tract. I.n. administration of DNA/PEI vaccine encoding HA from influenza A H5N1 or H1N1 viruses induced high levels of HA-specific IgA detected in bronchoalveolar lavages and the serum (Torrieri-Dramard et al., 2011). PEGylated Poly (2-Dimethylamino) Ethyl Methacrylate Poly (2-(dimethylamino) ethyl methacrylate) (PDMAEMA), like PEI, is a synthetic polycation in which tertiary amine groups can be protonated at physiological pH. DNA can be

condensed and protected by the interaction between positively charged PDMAEMA and negatively charged DNA (van de Wetering et al., 1999). PEGylation dramatically decreased the PDMAEMA cytotoxicity even at N/P ratios above 30. PEGylated PDMAEMA DNA vaccine encoding HIV gag given i.n. to experimental mice significantly improved the priming effect (Qiao et al., 2010). Dendrimers and Dendrosomes Dendrimers are globular, nanoscaled, highly branched, and reactive three-dimensional polymers, with all bonds emanating from a central core. Highly controllable molecular weight and structure, large numbers of readily accessible terminal functional groups, and the possibility of encapsulating guest molecules in internal cavities give dendrimers a distinct edge over other polymers for the delivery of drugs (Liu and Frechet, 1999). Owing to their nanoscopic size, dendrimers can evade the reticuloendothelial system of the body and are extremely important for intracellular drug delivery. Inherent cationic charge and spherical shape associated with amine-terminated dendrimers make them highly potent in the delivery of genes and immunogens (Tiera et al., 2011). Cationic dendrimers possess a finite positive charge that is responsible for formation of a stable complex with DNA and subsequent transfection (Zeng and Zimmerman, 1997). Dendrimers are reported to possess hemolytic toxicity and cytotoxicity owing to their polycationic nature. The low biocompatibility of cationic dendrimers was improved by dendrosomes, which are novel, vesicular, spherical, supramolecular entities containing dendrimer/DNA complexes entrapped in a lipophilic shell. Dendrosomes possess negligible hemolytic toxicity, higher transfection efficiency and better in vivo acceptability (Dutta et al., 2010; Sarbolouki et al., 2000). In dendrosomes, vesicular layers largely protect the dendrimer/DNA complexes and lipid layers control the release of the dendrimer/DNA complex. Larger dendrosomes remain at the site of injection as a depot, which is degraded by tissue phospholipases, whereas smaller ones deliver to and efficiently transfect APC in the draining lymph nodes. Comparison of the effect of parenteral (i.m.) injection and mucosal (aerosol) DNA vaccination of turkeys demonstrated the potency of the DNA vaccination by aerosol delivery. Turkeys immunized with PEI dendrimer/DNA plasmid encoding the major outer-membrane protein from Chlamydophila psittaci by aerosol showed a level of protection against challenge that was comparable with turkeys immunized by i.m. route. Formulation of plasmid DNA in dendriplexes protects DNA against degradation during nebulization and improves transfection of antigen-presenting cells (Verminnen et al., 2010). Compatibility of dendrimers with nanoscale building blocks such as DNA makes them attractive for the development of DNA vaccine self-assembly formulations for

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mucosal application. However, there is a need to better understand the behavior of these systems in vivo, in particular their adsorption and interaction with biological surfaces and their uptake mechanisms. Complex multifunctional microparticles based on a biocompatible copolymer with polyamidoamine (PAMAM) conjugated to the surface are one possible DNA vaccine formulation for mucosal application, in which dendrimer toxicity is diminished. Such particles provide a significantly higher transgene expression than unmodified PLG microparticles (Zhang et al., 2007). Chitosan Chitosan is the common name of a linear, random copolymer of β-(1-4)-linked d-glucosamine and N-acetyl-Dglucosamine whose molecular structure comprises a backbone linked through glycosidic bonds. The basic amine groups of this polysaccharide are protonated and thus positively charged in most physiological fluids. Chitosan can be considered mostly hydrophilic but the proportion of acetylated monomers and their distribution in the chains have a critical effect on its solubility and conformation in aqueous media. Because of these molecular features, chitosan in solution exhibits pH-dependent behavior and interesting biopharmaceutical properties, such as mucoadhesiveness and the ability to open epithelial tight junctions, which make it appropriate for oral or i.n. DNA delivery. The presence of mucus on mucosal epithelia increases the residence time of mucoadhesive particles at the cell surfaces. Chitosan can be produced under GMP conditions from both animal and fungal sources. Chitosan from animal origin (crustaceans) has been mainly used as a nanocarrier for vaccines and drugs (Agnihotri et al., 2004; Lai et al., 2009; van der Lubben et al., 2001a,b). However, batch-to-batch variability is high, resulting in variable physicochemical properties, and its crustacean origin could also be associated with allergic reactions. Hence, an alternative chitosan produced, e.g., from edible Agaricus bisporus mushrooms, could be more suitable for pharmaceutical applications, especially because it is available with batch-to-batch reproducibility well-controlled for characteristics such as molecular weight and degree of acetylation. Such chitosans of pharmaceutical grade are commercially available with a narrow range of molecular weight and degree of acetylation (e.g., from Kitozyme, Belgium). Non–animal-derived chitosan produced by mushrooms under GMP conditions has been used and characterized as a DNA carrier in an in vitro model of the human follicle-associated epithelium with M-like cells (Plapied et al., 2010). Chitosan–DNA complexes made in saline or acetic acid solution partially protect DNA from nuclease degradation (MacLaughlin et al., 1998; Richardson et al., 1999). This improves delivery of DNA to APC by efficient trafficking through local lymphoid tissues and uptake by DC (Zhao et al., 2011). Thus, chitosan nanoparticles are

a suitable vehicle for oral vaccine delivery (Chadwick et al., 2010) and are capable of transfecting mammalian cells because of their ability to overcome the two steps of cellular transport, cell uptake and release from endosomes, because chitosan acts as a proton sponge (Ishii et al., 2001). Chitosan–DNA nanoparticles may be formed by ionic gelation, a mild process based on complexation between charged macromolecules (Agnihotri et al., 2004). There are at least 15 clinical trials under way listing chitosan-based systems as an intervention (http://clinicaltrials.gov), which are expected to provide critical safety information in humans in the near future (Aspden et al., 1997). In mice, DNA vaccines have been delivered orally using a variety of chitosan-based particles (Laddy and Weiner, 2006). Previous studies have reported the feasibility of gene transfer into fish by encapsulating the DNA into chitosan and incorporating into feed (Chen et al., 1998). Oral delivery of chitosan–DNA nanoparticles in mice revealed a high level of gene expression in the epithelial cells of both stomach and intestine. Because no effect on cell viability was observed and owing to its high transfection efficacy, chitosan is seen as one of the best candidates for gene delivery (Boyoglu et al., 2009; Li et al., 2009). To improve the efficacy of mucosal vaccines and prolong drug delivery, chitosan nanoparticles can be chemically modified or used together with a potent molecular adjuvant (Alves and Mano, 2008; Baudner et al., 2003). Protection against infection by Campylobacter jejuni has been demonstrated by an i.n.-administered chitosan-based DNA vaccine that enhances mucus absorption and results in transgenic DNA expression in the chicken nasopharynx. Chickens immunized with chitosan–DNA nanoparticles, containing a gene for the major structural protein, FlaA, produced significantly increased levels of serum anti-C. jejuni IgG and intestinal IgA antibody (Huang et al., 2010). Chitosan can also be used to functionalize and surfacemodify various micro- and nanoparticles such as liposomes to improve their targeting, biocompatibility, and bioadhesiveness (Channarong et al., 2011). Liposomes Owing to their biodegradability and capacity to protect and transport DNA plasmids, liposomes (phospholipid bilayer vesicles) represent an almost ideal carrier system for the preparation of synthetic vaccines. Liposomal carriers can be applied by invasive (e.g., i.m., s.c., i.d.) as well as noninvasive (transdermal and mucosal) routes. Liposomes are versatile and robust delivery systems for the induction of antibody and T- lymphocyte responses to associated subunit antigens. In the past 15 years, liposome vaccine technology has matured and several vaccines containing liposomebased adjuvants have been approved for human use or have reached late stages of clinical evaluation.

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The beneficial effect of liposomal carriers consists of the simultaneous action of several factors during transport from the site of application to the APC, and binding to and uptake by the APC. The mechanism of action includes protection and stabilization of plasmid DNA, enhanced uptake by APC by passive or active targeting, and controlled release of DNA into cytoplasm. The safety of liposomal carriers is of great importance for all administration routes, especially for mucosal applications (Watson et al., 2012). Cationic liposomes consist of basic lipids with a positively charged head group (usually tertiary or quaternary ammonium groups or polyamines) and a hydrophobic domain (comprising alkyl chains or cholesterol). The ability of positively charged liposomes to complex spontaneously with negatively charged DNA had been extensively used for in vivo nonviral gene delivery (Liu et al., 1995, 1997). Shortly after the description of immunostimulatory properties of bacterial DNA, several groups reported that cationic liposomes markedly enhanced the immunogenicity of bacterial DNA and therefore could serve as an adjuvant. Such activity can be explained by the fact that cationic liposomes can activate DCs directly (Cui et al., 2005; Foged et al., 2004; Korsholm et al., 2007), likely because the occurrence of a positive charge close to the cell surface is recognized as a danger signal, or once inside the cells they trigger cascades naturally activated by endogenous cationic compounds. They induce the expression of proapoptotic and proinflammatory mediators; and activate several kinases and proteases; induce inflammasome and NF-κB–dependent pathways, production of reactive oxygen species, and endogenous danger signals, and expression of costimulatory and maturation markers on DC (Lonez et al., 2012). Most liposome–DNA composites can also be readily lyophilized, thereby greatly increasing their shelf life and stability (Anchordoquy et al., 2001). Liposomal formulations based on molecular adjuvants derived from lipophilic analogs of muramyl dipeptide norAbuMDP (in which muramic acid was substituted with normuramic acid and l-alanine with l-2-aminobutyric acid) and norAbuGMDP (where GMDP stands for N-acetylglucosaminylmuramyl dipeptide) with new cationic lipids presents a system for delivery of DNA vaccines (Korvasova et al., 2012). Liposomal formulations of DNA vaccines were tested in several microbial infection models. For example, a DNA vaccine coding for mycobacterial hsp65 and human IL-12 formulated in hemagglutinating virus of Japan (HVJ)liposomes (DNAhsp65+IL-12/HVJ-L), when administered i.m. to experimental mice and guinea pigs, exhibited protection against intratracheal M. tuberculosis instillation. Furthermore, heterologous immunization of cynomolgus monkeys by i.d. BCG priming and i.m. DNAhsp65+IL-12/ HVJ-L boost provided excellent protection using the same model of tuberculosis as determined by survival (100%), erythrocyte sedimentation rate, body weight, chest X-ray

findings, and immune responses. In contrast, immunization with BCG alone conferred only 33% survival (Okada et al., 2007). An oral liposome-formulated vaccine based on DNA plasmid encoding M. tuberculosis Ag85A antigen induced an antigen-specific cellular and humoral mucosal immune response in mice (Wang et al., 2010). New hepatitis B vaccines are urgently needed to overcome the problems associated with immunization of immunocompromised people, and more important, for use in chronic patients, because hepatitis B virus infection is a major global health concern. A DNA vaccine encoding HBsAg (S) protein in glycol-chitosan-coated liposomes applied i.n. to BALB/c female mice elicited humoral mucosal and cellular immune responses that were significantly pronounced compared with naked DNA (Khatri et al., 2008). In the case of influenza, Vaxfectin (a novel proprietary cationic lipid-based formulation developed by Vical) and GAP-DLRIE/DOPE used a lipoplex formulation with DNAencoding influenza NP. After retrograde instillation into rat salivary glands, these DNA vaccines induced cytotoxic and humoral NP-specific responses. The humoral response was generally (but not significantly) enhanced by a second immunization. Surprisingly, no IgA specific antibodies were found in saliva (Sankar et al., 2002). I.n. immunization of BALB/c mice with DNA plasmid encoding influenza A/PR/8/34 (H1N1) HA formulated in lipoplexes stimulated both IgG and IgA humoral responses and increased IgA titers in BAL fluid, whereas immunization with naked DNA had no effect. I.m. immunization with both naked and liposome-encapsulated pCI-HA10 induced serum IgG but had no effect on IgA levels in either the serum or BAL fluid. Both i.n. and i.m. administration of HA-encoding DNA vaccines (naked or liposome-encapsulated) induced T cell proliferative responses. Thus, i.n. administration of liposome-encapsulated HA-encoding DNA was effective at stimulating mucosal, cellular, and humoral immune responses, and mice immunized i.n. were protected against lethal challenge with influenza virus (Wang et al., 2004a). I.n. administration of DOPE-DcCholesterol liposomal DNA vaccine encoding full-length HIV-1 rev plus IL-12 and granulocyte macrophage–colony-stimulating factor induced good levels of rev-specific mucosal IgA and CTL (Okada et al., 1997). Virosomes Virosomes are a special class of proteoliposomes prepared predominantly from the reconstituted influenza virus membranes (including membrane proteins HA and neuraminidase) supplemented with phosphatidylcholine (Daemen et al., 2005). These vesicles benefit greatly from the inherent delivery properties (efficient cell binding, internalization, and cytosolic release) and immunogenicity of the influenza virus (Almeida et al., 1975). Influenza virosomes

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are produced by the detergent removal of viral membrane proteins and lipids (Stegmann et al., 1987). Various viruses can be used for the production of virosomes, which can target different cell types (for review, see Daemen et al., 2005). Virosomes fuse either with the plasma membrane at a neutral pH or, after receptor-mediated endocytosis, with the endosomal membrane at an acidic pH (Schoen et al., 1999). The unique targeting features of spike proteins have been exploited in the design of DNA virosomes after the supplementation of virosomes with cationic lipids such as dioleoyloxypropyl-trimethylammonium methyl sulfate (DOTAP) and dioleoyldimethylammonium (Schoen et al., 1999; Waelti and Gluck, 1998). Mice primed i.n. with influenza DOTAP-supplemented cationic virosomes plus E. coli heat-labile toxin and boosted by i.n. administration of DNA virosomes encoding the mumps virus HA and fusion protein generated mumps virus HA- and fusion protein-specific serum IgG2a antibodies, IL-10–producing antigen-specific T cells, and a low but detectable IgA response in the bronchoalveolar lavages (Cusi et al., 2000). When the booster was performed with naked DNA vaccine, a slightly lower IgG response but no IgA response was detected. A recent study in mice immunized with DNA encoding influenza A virus (H1N1) NP adsorbed on the surface of cationic influenza virosomes demonstrated full protection against challenge with the homologous strain and induced significant protection against intra-subtypic challenge with a drift virus. Cationic influenza virosomes can transfect APC as effectively as commercial transfection reagents and trigger the expression of NFκB/AP-1. T cell production of IFN-γ and granzyme B was induced by DNA vaccine adsorbed on virosomes at 5 μg DNA, whereas induction of similar responses by naked DNA vaccine required 50 μg (Kheiri et al., 2012). The adjuvant property of influenza virosomes is related to the strong capacity of virosomes to induce maturation of DCs and trigger the secretion of cytokines such as TNF-α, IFN-γ, and IL-12 (Gluck et al., 2004; Moser et al., 2003). Lipid Emulsions Submicron emulsions based on MF59 (Novartis) is the first oil-in-water vaccine adjuvant to be commercialized in combination with seasonal influenza virus vaccine (Ott et al., 2002). MF59 has been shown to be safe and well tolerated in a number of clinical trials (Briones et al., 2001). When MF59 supplemented with cationic lipid DOTAP to bind DNA plasmid encoding HIV-1 gag was used for i.m. vaccination of rabbits, a significant increase in gag-specific serum antibodies was demonstrated compared with animals immunized with naked DNA (Ott et al., 2002). The delivery of DNA vaccines to airway mucosa might be an ideal method for mucosal immunization. I.n. administration of DNA cationic lipid emulsion vaccine encoding HBsAg to mice induced HBsAg expression in both

the nasal tissue and the lungs and, compared with naked DNA, induced higher levels of HBsAg-specific antibodies in serum and mucosal fluids, HBsAg-specific CTL in the spleen and cervical and iliac lymph nodes, and higher antigen-specific DTH (Kim et al., 2006).

Biological Delivery Systems for DNA Vaccines Bacterial Vectors for Mucosal Administration of DNA Vaccines Numerous experimental studies demonstrate the use of attenuated or commensal bacteria for targeted delivery of DNA vaccines into tissues of the mucosal immune system, eliciting mucosal or systemic immune response toward viral, bacterial, and parasitic pathogens and tumor antigens (Chinchilla et al., 2007; Chou et al., 2006; Darji et al., 2000; Guo et al., 2003; Pasetti et al., 2003; Shata et al., 2001; see also Chapter 65). The most commonly used bacterial vectors for DNA vaccines are derived from pathogens such as Salmonella enterica serovars Typhi and Typhimurium, Shigella flexneri, Shigella sonnei, L. monocytogenes, or commensal bacteria, especially Lactobacillus sp. (Bermudez-Humaran et al., 2011; Dietrich et al., 2001, 2003; Kochi et al., 2003; Lewis, 2007; Li et al., 2007). Bacterial Ghosts Bacterial ghosts (BGs) are empty bacterial envelopes of gram-negative bacteria produced by controlled expression of bacteriophage ΦX174 gene E inducing the formation of transmembrane tunnel structures in living bacteria. Tunnels allow an expulsion of all the cytoplasmic contents, whereas the inner and outer membrane structures including membrane-bound components are preserved and remain intact (Witte et al., 1992). BGs possess all the bacterial bioadhesive surface properties in their original state but do not represent a threat of infection. BGs have been developed as a novel potent carrier and adjuvant system for the delivery of DNA vaccines (Lubitz et al., 2009; Paukner et al., 2006; Tabrizi et al., 2004). Safety-profile studies concerning BGs showed no cytotoxic or genotoxic impact on various histological types of human cells after mutual co-incubation, and that endotoxicity does not limit the use of BGs as a candidate vaccine (Mader et al., 1997). In contrast to the live bacterial vectors, BGs do not represent a risk of reversion to fully pathogenic forms (Medina and Guzman, 2001; Mengesha et al., 2007). The preparation of BGs based on controlled expression of the lysis gene E was successful in a wide range of nonpathogenic, pathogenic, and probiotic gram-negative bacteria including E. coli, Shigella spp., and Neisseria spp. The self-loading, nonliving, bacterial DNA-delivery vector was developed and is based on the immobilization of plasmid DNA in the envelope complex of the plasmid-replicating

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bacterial host (self-immobilizing plasmid) owing to a specific protein–DNA interaction (Mayrhofer et al., 2005). Bacterial cell envelopes retained in BGs contain immune-stimulating pathogen-associated molecular patterns such as LPS, lipid A, and peptidoglycan, and therefore have the potential to enhance the immune response to the same extent as traditionally used adjuvants (Szostak et al., 1993). BGs elicit both humoral and cellular immune responses in animals and have been proposed as an alternative to conventionally inactivated vaccines (Mayr et al., 2005). BGs have an excellent DNA-loading capacity, varying from 4000 to 6000 plasmid copies per one BG (Kudela et al., 2005). Nevertheless, quantitative analysis showed that a relatively low DNA concentration (50 plasmids/BG) is sufficient for efficient DNA delivery to the target cells. BGs loaded with plasmid DNA are efficiently internalized and phagocytosed by APCs, especially macrophages and tumor cells (melanoma), leading to transfection efficiencies ranging from 52% to 82% (Ebensen et al., 2004; Kudela et al., 2008; Paukner et al., 2005). Cross-presentation of Ag delivered to DCs by BGs was reported. Here, the presence of bacterial LPS on the outer membrane of BGs enhances the maturation of DCs and affects endosomal acidification (Trombetta et al., 2003; Trombetta and Mellman, 2005). Several characteristics of BGs support their potential as vaccine-delivery systems (Table 4). Studies reporting BG usage for DNA vaccination are rare. BGs prepared from S. Typhi Ty21a were used as a DNA-vaccine delivery system and were explored experimentally as HIV gp140 DNA vaccine. BGs loaded with DNA vaccine were readily taken up by murine macrophage RAW264.7 cells, and gp140 was efficiently expressed in these cells in vitro. The peripheral and intestinal mucosal

TABLE 4  Main Characteristics of Bacterial Ghosts for DNA Delivery Advantages

Limitations

Nonliving carriers

Presence of lipopolysaccharides

Carriers for DNA, antigens, drugs

Inherited antigenicity of carrier

Strong adjuvant properties Good recognition and uptake by antigen-presenting cells Loading capacity and efficacy for DNA are high Targeting properties for different tissues Relatively easy biotechnology production

anti-gp120 antibody responses in mice vaccinated s.c. with BG–DNA vaccine were significantly higher than those in mice immunized with naked DNA vaccine. The enhancement of antibody responses was associated with BGinduced production of IL-10 through the TLR-4 pathway. Ty21a BG-based delivery system was shown to enhance the ability of a DNA vaccine to elicit peripheral and mucosal antibody immune responses, presumably through the upregulation of the Th2 immune response via the TLR-4 and possibly TLR-5 pathways (Wen et al., 2012).

Codon-Usage Optimization An approach to enhance antigen production after DNA immunization consists of the optimization of codons mirroring the frequency of tRNA present within the vector and host cells, to enhance the efficacy of antigen expression during DNA immunization (Andre et al., 1998; Nagata et al., 1999). Codon optimization can be simply solved by the substitution of wild-type codons with those from highly expressed human genes either by site-directed mutagenesis or by production of synthetic genes.

Enhancing Immunogenicity of DNA Vaccines with Molecular Adjuvants DNA vaccination was originally described as an approach to inducing intense systemic Th1 type responses in mice, but overall the response in humans was weak. To enhance the intensity of the immune response with optimal antibody isotypes, and CD4+ or CD8+ T cells in systemic and mucosal compartments, several molecular protein-based adjuvants were tested. They were coadministered at the site of DNA vaccine application in the form of recombinant protein encoded by the DNA plasmid. For efficient crosspresentation, covalent linkage of DNA-encoded antigen to PD1, adenylate cyclase toxoid from Bordetella pertussis, hsp proteins (hsp60, hsp70, gp96), or Fab fragments recognizing TLR-2, MR, or DEC205 may be advantageous (Masin et al., 2004; Saveanu et al., 2009; Weimershaus et al., 2012; Zhou et al., 2013).

Targeting Innate Immune Receptors Immunization of mice with dual promoter DNA plasmids encoding influenza HA or HPV16 F7 antigen together with TLR adaptor molecules such as Toll/IL-1 receptor domaincontaining adaptor inducing IFN-β (TRIF) or myeloid differentiation factor 88 (MyD88) can potentiate the effects of DNA vaccines. Incorporation of MyD88 enhanced antigen-specific humoral immune responses, whereas TRIF enhanced cellular immune responses to both HA or HPV16 F7 (Takeshita et al., 2006). I.d. immunization of mice with plasmid encoding HPV16 capsid protein L1 plus a plasmid

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encoding cholera toxin B subunit enhanced L1-specific IgA in cervical secretions and fecal extracts. Furthermore enhanced production of Th1 cytokines IL-2 and IFN-γ, and improved CD8+ T cell cytotoxicity of splenocytes was detected (Sanchez et al., 2004).

Usage of Cytokines Codelivered with DNA Vaccine Several cytokines have been tested for enhancement of DNA vaccine efficacy. Generally these efficiently enhance either cellular Th1 or Th2 or humoral responses to DNA vaccine-encoded antigens at distinct anatomical localities. I.n. immunization with lipoplex/DNA vaccine coding for HIV-1 gp160 and rev antigens coadministered with IL-12 or GM-CSF DNA plasmids induced high levels of HIV-specific CTLs and increased DTH in contrast to lipoplex/DNA vaccine alone, which induced antigen-binding or neutralizing IgA antibodies in feces, vaginal fluid, and serum, indicating a Th2 response. Thus, both IL-12 and GM-CSF exhibited modulatory activity during mucosal DNA immunization (Okada et al., 1997). In a murine model of allergen-induced airway hyperresponsiveness (AHR) induced by ovalbumin (OVA), vaccination with DNA encoding OVA alone prevented AHR development but did not affect already established AHR. In contrast, vaccination with DNA encoding OVA fused to IL-18 not only prevented AHR but also reverted established AHR and reduced allergen-specific IL-4 and increased allergen-specific IFN-γ production (Maecker et al., 2001).

Usage of Chemokines to Activate Mucosal Immune Response DNA immunization through various mucosal routes is generally poorly effective but systemic DNA immunization is at least partially relevant for induction of mucosal immune responses owing to the migration of mucosaassociated T and B lymphocytes. Coadministration of systemically administered DNA vaccines with chemokines involved in mucosal homing allows targeting of responses to mucosal tissues. Among the chemokines involved in the induction of immune responses are the homeostatic chemokines, CCL19 and CCL21, which are secreted through high endothelial venules (HEV) of peripheral lymph nodes and Peyer patches. Both are recognized by the cellular receptor, CCR7, expressed on membranes of naive and central memory T cells, naive B cells, and myeloid DC (Martin-Fontecha et al., 2008). More specific for mucosal tissues is homeostatic CCL25 (TECK) expressed and secreted by small intestine and also by thymic cortical and medullary epithelial cells (Wilkinson et al., 1999). CCL25 is involved in homing of memory and activated α4β7+ T and B cells, DC, and macrophages

to small intestinal lamina propria, and of immature pre-T cells to the thymus. These cells recognize CCL25 uniquely through the cell surface receptor CCR9 (Mora and von Andrian, 2008; Olson and Ley, 2002; Wendland et al., 2007). Recently, two DNA vaccines encoding either HIV-1 gag or influenza A neuraminidase were administered i.m. to experimental mice together with DNA plasmid encoding CCL25. In both cases, immunization led to induction of specific immune responses at distal sites including the lung and mesenteric lymph nodes. Application of CCL25 induced infiltration of cognate chemokine receptor CCR9+/CD11c+ DC cells to the site of immunization. CCL25-adjuvanted immunization enhanced IFN-γ secretion by antigen-specific CD8+ and CD4+ T cells and elevated HIV-1-specific IgG and IgA responses in peripheral blood and secondary lymphoid organs and at mucosal sites (Kathuria et al., 2012). Migration of immune cells to the colonic lamina propria, bronchial, nasal, breast, and salivary gland mucosae, and to a lesser extent other mucosal sites, is orchestrated by the chemokine CCL28 (MEC), which is expressed locally by most inflamed columnar epithelial cells and constitutively in the colon. CCL28 is recognized by receptor CCR10, expressed on α4β1+ T and surface IgA-positive B cells (Hutnick et al., 2012; Kraynyak et al., 2009a,b; Mora and von Andrian, 2008). Another ligand for CCR10 is chemokine CCL27 (CTACK), originally described as expressed by epidermal basal keratinocytes. CCL27 has an important role in the homing of memory T lymphocytes to inflamed skin but CCL27 also activates skin Langerhans cells and IgA-secreting B cells (Homey et al., 2002; Kunkel et al., 2003; Morales et al., 1999), leading to a similar B cells homing profile as expression of CCL28. Because CCL27 appears to have an important role in recruiting lymphocytes and causing an inflammatory response in the lung, several studies used coadministration of CCL27 in an effort to stimulate protective lung immunity after systemic DNA vaccination. Intramuscular immunization of mice with DNA plasmids encoding influenza An HA (pPR8HA), CCL27, and CCL28 elicited antibody production in the gut-associated lymphoid tissue and, importantly, in lung (Kutzler et al., 2010). Immunization of mice with HIV-1 gag DNA vaccine together with a CCL27-encoding plasmid enhanced specific immune responses at both systemic and mucosal sites. Furthermore, electroporationenhanced i.m. immunization of rhesus macaques with SIV gag-, Pol-, or Env-encoding DNA vaccines together with CCL27-expressing plasmid induced significant antigen-specific IFN-γ secretion, CD8+ T-cell proliferation in peripheral blood, and CD4+ T cells in the bronchiolar lavage. In contrast to the murine experiment, codelivery of HIV-1 gag DNA and CCL27 to macaques was associated with greater antigen-specific IgA only in fecal samples, not in the periphery (Kraynyak et al., 2010).

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Furthermore, mucosally administered DNA vaccines showed enhanced efficacy when immune cell immigration was activated by the coadministration of chemokines (or their genes) CCL9 (MIP1γ) and CCL20 (MIP3α). These chemokines are normally expressed and secreted by epithelial cell adjacent to mucosal lymphatic follicles, where they attract DC and lymphocytes expressing CCR1 and CCR6, respectively (Cook et al., 2000; Iwasaki and Kelsall, 2000; Zhao et al., 2003).

TARGETING OF DNA VACCINE TO M CELLS AND DCs Targeting to M Cells The difficulty in developing oral vaccines resides mainly in their poor accessibility to mucosal DC owing to dilution or degradation, continuous clearing of mucosal surfaces by peristaltic movement and production of mucus, the physical barrier formed by the mucus and the glycocalyx, and oral tolerance mechanisms that downregulate cell-mediated and humoral immune responses. Thus, efforts to develop oral vaccines focused on delivering the antigen in a form that maximizes the chances of its uptake by M cells of Peyer patches for initiation of the immune response.

Targeting to DC Cationic liposomes or other cationic delivery systems can target various cells of mucosal epithelia. DNA vaccine coding for M. tuberculosis Ag85A encapsulated in cationic liposomes (Lipofectamine 2000) is promising as a vaccine delivery system to evoke intestinal immune responses because oral administration of Ag85A DNA lipoplexes leads to Ag85 expression in the epithelium, M cells, DCs, and Peyer patches of the small intestine. Oral vaccination with Ag85A DNA lipoplexes induced antigen-specific production of IL-2 and IFN-γ and antigen-specific cytotoxicity of intraepithelial T cells from small intestine. This is important because intraepithelial lymphocytes of the small intestine have a key role in regulating the immune response to eliminate M. tuberculosis (Wang et al., 2010). Because different DC populations elicit particular types of immune responses, targeting of DNA vaccines to DC should be carefully designed according to the type of immune response desired. For example, buccal administration of plasmid DNA in mice generates robust local and systemic CD8+ T cell responses. In contrast, targeting the DNA vaccine to the anterior labial mucosa was less effective in inducing CD8+ T cells, and the most efficient antigen presentation was mediated by buccal-derived CD11c+ DCs that are capable of antigen cross-presentation. Interestingly, although Langerhans cells are indispensable for CD8+ T cell induction after buccal immunization, direct DC targeting

of DNA vaccines seems to be effective only via interstitial DCs or interstitial CD103+ langerin+ DCs, because Langerhans cells do not directly present antigens to CD8 T cells (Nudel et al., 2011). This observation highlights the need for deeper understanding of mucosal immunity to design mucosally delivered DNA vaccines effectively.

Oral DNA Vaccination As discussed, several mucosal routes for DNA vaccination were tested, including i.n., bronchial, orogastric, buccal, sublingual, vaginal, and rectal, with various efficacies and distinct types of elicited humoral and cell-mediated immune responses in systemic and various mucosal compartments. Some responses are localized to mucosal compartments, whereas other strategies induced both mucosal and systemic responses. To induce both systemic and mucosal antibodies and cell-mediated immunity by DNA vaccination, currently the heterologous prime-boost strategy is preferred, especially systemic priming immunization followed by mucosal boosting. Combination of parenteral and mucosal administration of antigen is required because parenteral vaccines are notoriously inefficient for stimulating primary immune responses in mucosal tissues but highly effective for the induction of systemic responses and avoiding mucosal tolerance. Of all tested mucosal sites, oral or i.n. delivery of DNA vaccines is an attractive route of administration because of its simplicity. Of these, buccal and especially sublingual vaccination offer potential advantages. Sublingual vaccination represents a route that effectively stimulates both systemic and mucosal antibody- and cell-mediated immune responses. The buccal administration of plasmid DNA in mice generates robust local and systemic CD8+ responses. In contrast to the i.n. route, sublingual does not result in rare but severe toxic effects caused by the penetration of antigen and adjuvants across olfactory epithelium (Azizi et al., 2010). Furthermore, the sublingual mucosa may be a useful delivery site because it harbors a dense lattice of DC, which are mobilized by mucosal adjuvants and then migrate to the proximal draining submaxillary and superficial cervical lymph nodes. Furthermore, sublingual immunization induces antigen-specific immune responses in the female reproductive tract, in addition to the respiratory tract and oral and nasal cavities (Cuburu et al., 2007). New delivery systems for sublingual and buccal vaccination are needed to prolong vaccine exposure on the mucosal surface and to keep a sufficient concentration of vaccine construct against removal by salivary flow and mechanical movement of the tongue. Future functional mucoadhesive systems offer new solutions for the construction of modern recombinant vaccines inducing both mucosal and systemic immunity. The 40-kDa outer membrane protein of Porphyromonas gingivalis (40k-OMP) sublingually administered

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with a cDNA vector plasmid encoding Flt3 ligand (pFL) was shown to elicit a protective immune response with significantly induced serum IgG and IgA, as well as salivary IgA, antibody titers. It was concluded that sublingual administration of 40k-OMP with pFL acts as an effective and safe mucosal vaccine against oral P. gingivalis infection and may be a useful tool in the prevention of chronic periodontitis (Zhang et al., 2009). In a recent study, DNA vaccine encoding influenza A virus (A/Aichi/2/1968(H3N2)) HA complexed with PEI was administered sublingually three times, followed by a recombinant HA protein boost by the same route. In subsequent i.n. challenge with live murine-adapted X-31 (H3N2) influenza virus, statistically significant protection was confirmed (Mann et al., 2013).

CONCLUSION Deoxyribonucleic acid vaccines represent an effective tool for the induction of antigen-specific immune responses in mucosal tissues in experimental animals. The number of clinical trials registered on ClinicalTrials.gov in fall 2013 claiming DNA vaccination reached a total 191, of which 63 were open and active. Nevertheless, only a few studies focused on DNA vaccine application related to mucosally transmitted infections such as HIV or influenza. Although clinical studies confirm the safety of DNA administration, the protective or therapeutic effects of DNA vaccines on mucosal or mucosally transmitted infections have not been convincingly demonstrated. Further modification of application routes as well as use of appropriate adjuvants and delivery systems identified in animal experiments are required in the future development of DNA vaccination strategies.

ACKNOWLEDGMENT This work was supported by European Social Fund grants CZ.1.07/2.3.00/20.0164, GAP304/10/1951, and GAP503/12/G147 from the Czech Science Foundation.

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1328  SECTION | D  Mucosal Vaccines

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