delivery vehicles genetic immunization: bacteria as dna …...genetic immunization: bacteria as dna...

Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=khvi20

Human Vaccines

ISSN: 1554-8600 (Print) 1554-8619 (Online) Journal homepage: https://www.tandfonline.com/loi/khvi19

Genetic immunization: Bacteria as DNA vaccinedelivery vehicles

Pablo Daniel Becker, Miriam Noerder & Carlos Alberto Guzmán

To cite this article: Pablo Daniel Becker, Miriam Noerder & Carlos Alberto Guzmán (2008) Geneticimmunization: Bacteria as DNA vaccine delivery vehicles, Human Vaccines, 4:3, 189-202, DOI:10.4161/hv.4.3.6314

To link to this article: https://doi.org/10.4161/hv.4.3.6314

Published online: 01 May 2008.

Submit your article to this journal

Article views: 326

Citing articles: 19 View citing articles

https://www.tandfonline.com/action/journalInformation?journalCode=khvi20

https://www.tandfonline.com/loi/khvi19

https://www.tandfonline.com/action/showCitFormats?doi=10.4161/hv.4.3.6314

https://doi.org/10.4161/hv.4.3.6314

https://www.tandfonline.com/action/authorSubmission?journalCode=khvi20&show=instructions

https://www.tandfonline.com/action/authorSubmission?journalCode=khvi20&show=instructions

https://www.tandfonline.com/doi/citedby/10.4161/hv.4.3.6314#tabModule

https://www.tandfonline.com/doi/citedby/10.4161/hv.4.3.6314#tabModule

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.several reporter genes.4 This and the subsequent proof‑of‑principle provided by Ulmer for the promotion of protective immunity against flu in 1993,5 triggers a literal explosion in the field of nucleic acid vaccination with literally thousands of patents being filed in a few years.

The DNA vaccination strategy is associated with many advan‑tages. The biosynthetic machinery of the host’s cells is responsible for the production of the antigen. Therefore, optimal glycosylation and folding is warranted. In addition, production and purification of individual antigens is not necessary.4 Furthermore, since cultiva‑tion is not required, vaccines can be developed against pathogens for which non appropriate cultivation techniques exist. Interestingly, the use of this approach may help to overcome maternal immu‑nity, thereby facilitating the development of vaccines for newborns. Furthermore, the stability of DNA renders nucleic acid vaccines particularly robust in terms of cold‑chain requirements. Finally, DNA vaccines exert immune stimulatory activities, due to the presence of pathogen associated molecular patterns (PAMPs), which are recognized by receptors from cells of the innate immune system (i.e., Toll‑like receptors; TLR).

By manipulating the genes coding for selected antigens (e.g., ubiquitination), it is possible to further enhance their delivery into the MHC class I‑restricted pathway.6,7 An important issue to be taken into consideration is which cell subset is responsible for gene expression. By naked DNA vaccination, in which the plasmid is usually given intramuscularly, two different cell types are transfected, namely somatic cells (e.g., myocytes) and professional antigen presenting cells (APCs). However, injected DNA is degraded by extracellular nucleases, making necessary the use of high amounts of DNA to ensure expression.8,9

The studies performed in 1980 by Walter Schaffner demon‑strating that cloned genes can be directly transferred from bacteria to mammalian cell provided the rational for using bacteria as a DNA delivery system.10 In fact, three independent groups exploited the ability of different intracellular Gram‑negative bacteria to transfer plasmids encoding eukaryotic expression systems to infected cells.11‑13 Later, it was also demonstrated that Gram‑positive bacteria, such as self‑destructing Listeria monocytogenes is able to deliver plasmid DNA.14

The delivery by live attenuated bacteria has several advantages over the normal naked DNA. There is no need for the purification of plasmid DNA, but rather a simple fermentation. The bacterial carrier

The so‑called DNA vaccination represents one of the most notable tools under development in the field of vaccinology. The concept of administering the gene coding for any given protective antigen and make responsible vaccinee’s own cells to produce the protein appeals as too simple to be true. Indeed, the implementa‑tion of this approach for mass vaccination should overcome several bottlenecks, such as need of high dosages and poor immunoge‑nicity. In this context, the use of live attenuated bacteria as delivery system for plasmid DNA has emerged as a promising alternative to overcome many of those pitfalls. In addition, this approach is not only amenable for mucosal administration, but allows to specifi‑cally target professional antigen presenting cells. This results in their transfection, as well as in their activation and maturation, due to their built‑in adjuvant properties resulting from the stimulation of pattern recognition receptors. This chapter discusses the specific features that should be taken into consideration when designing a plasmid vector, current candidate bacterial carriers for DNA delivery and main safety issues.

Introduction

In the early 1990s, DNA vaccination has emerged as a promising strategy to induce major histocompatibility complex (MHC) class I‑restricted immune responses, in particular CD8+ T‑cell mediated cytotoxicity. However, seminal work suggesting that this would be feasible approach was carried out much earlier. In 1960 Ito demonstrated that papilloma was induced by injecting nucleic acid of the Shope rabbit papilloma virus.1 In 1962, studies performed by Atanasiu demonstrated that polyoma virus DNA from infected cells injected into Hamsters lead to anti‑polyoma antibodies and tumors.2 Later, in 1979, Fried showed that polyoma virus DNA delivered by plasmids or phages was able to transform murine fibroblasts.3 However, the real potential of these observations went through unnoticed, until the 1990, when serendipity brought Wolff to observe in controls of transfection studies using cationic lipids that naked nucleic acid transfer in muscles also led to the expression of

*Correspondence to: Carlos A. Guzmán; Department of Vaccinology; Helmholtz Centre for Infection Research; Inhoffenstraße 7, D‑38124 Braunschweig, Germany; Email: carlos.guzman@helmholtz‑hzi.de

Submitted: 11/14/07; Accepted: 11/14/07

Previously published online as a Human Vaccines E‑publication: www.landesbioscience.com/journals/vaccines/article/6314

Review

Genetic immunizationBacteria as DnA vaccine delivery vehicles

Pablo Daniel Becker, Miriam noerder, Carlos Alberto Guzmán*

Department of Vaccinology; Helmholtz Centre for Infection Research; Braunschweig, Germany

Key words: bacterial vectors, DNA vaccine, bactofection, delivery system, live attenuated bacterial carriers

[Human Vaccines 4:3, 189‑202; May/June 2008]; ©2008 Landes Bioscience

www.landesbioscience.com Human Vaccines 189

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

Genetic immunization: Bacteria as DNA vaccine delivery vehicles

190 Human Vaccines 2008; Vol. 4 Issue 3

can specifically target and transfer main APCs, such as dendritic cells (DCs).15,16 Furthermore, after bacterial uptake by DCs,17 they act as natural adjuvants due to the presence of PAMPs that modulate early innate immune responses, thereby promoting robust and long‑lasting adaptive responses.18,19

Most carriers tested up to now as bactofection vectors are human intestinal pathogens (e.g., Salmonella spp., Shigella spp., Listeria spp., Escherichia coli). This is particularly advantageous due to their ability to infect human colonic mucosa following oral administration. After mucosal delivery of the carrier, both mucosal and systemic immune responses can be stimulated.20 Besides, epithelial cells in the gut or hepatocytes could serve as antigen sources for cross‑priming after infection.

Plasmid Design

Promoters and enhancers. Expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a func‑tional eukaryotic promoter. Viral promoters are very often used with this purpose, providing greater gene expression in vivo than other eukaryotic promoters. In particular, the cytomegalovirus (CMV) immediate early enhancer promoter (PCMV) has been shown to direct the highest levels of transgene expression in eukaryotic tissues when compared with other promoters. Other frequently used viral promoters are: (1) the early promoter from SV40 (PSV40), (2) the polyhedrin promoter from baculovirus (PPH), (3) the thymidine kinase promoter from Herpes simplex virus (HSV)‑1 (PTK), (4) the 5´LTR promoter from human immunodeficiency virus (HIV; PLTR) and (5) the promoter of the Rous sarcoma virus (PRSV). Since eukary‑otic and bacterial promoters have different consensus sequences, it is regarded as unlikely that promoter sequences functional in eukaryotic cells would be able to direct efficient transcription initia‑tion in bacteria.21,22 However, it was demonstrated that not only eukaryotic promoters but any type of eukaryotic DNA has a high probability to initiate transcription after transfer into bacteria. In fact, PCMV and PSV40 carry structural features required by the bacte‑rial RNA polymerase to initiate transcription,23 such as sequences with homology to the bacterial ‑10 and ‑35 promoter consensus motifs which induce expression as strong as the promoter from the bacterial TEM‑β‑lactamase gene.23 Therefore, expression of a foreign gene by a heterologous promoter must be taken into consideration when defining safety, especially when toxic products are expressed. Additionally, bacterial expression can be silenced by insertion of a transcription terminator, deletion or site‑direct mutagenesis of the bacterial ribosome binding site downstream from the promoter, in such a way that only eukaryotic cells would be able to express the gene of interest. Translation from the prokaryotic machinery can also be avoided by adding introns, thereby ensuring that expression is only possible through a eukaryotic system.

It is also important to consider that some widely used virally‑derived promoters, such as PCMV and PSV40, may not be suitable for some applications, since treatment or endogenous induction of interferon (IFN)‑γ or tumor necrosis factor (TNF)‑α may silence them, thereby inhibiting transgene expression from DNA vaccines containing these promoters.24,25 Thus, other promoter systems are currently under investigation, such as tissue specific, mammalian, synthetic and hybrid (e.g., hybrid CMV enhancer—nonviral promoter) promoters.26‑31

Polyadenylation, kozak sequence and codon usage. Another regulatory element is the polyadenylation (polyA) sequence necessary for transcriptional termination, stability, processing and translation of eukaryotic mRNA. Since the rate of transcription initiation is generally high due to the use of strong promoters/enhancers, the rate of transcriptional termination may become rate‑limiting.32 In addition, the efficiency of primary RNA transcripts processing an polyA is known to vary between the polyA sequences of different genes. Thus, the polyA sequence should be selected or, alterna‑tively, a second enhancer could be added downstream of the polyA sequence to increase expression levels.33 In addition to the polyA sequence, most eukaryotic genes possess sequences flanking the AUG initiator codon, the so‑called “kozak” consensus sequences. They are responsible for mRNA recognition by eukaryotic ribosomes, thereby affecting expression levels.34,35

It is important to consider that mammalian codon usage differs from microorganisms. Many groups have demonstrated that altering the codon use pattern may provide substantial increases in expres‑sion levels and improvements in antigenicity.36‑39 Several studies have showed that optimization of the codon usage of bacterial, parasitic and viral sequences to that of highly expressed mammalian genes significantly increased both the gene‑expression levels and the immunogenicity of the DNA vaccine.40‑42 Thus, it is useful to examine the sequence looking for clusters of rare codons, as they may retard ribosomes during translation due to exhaustion of rare tRNAs, promoting ribosomal frame‑shifting and premature truncation of the protein.43 A remarkable example of codon optimization is the case of the gene gag of HIV, its optimization resulted in a mutational inactivation of an inhibitory sequences (AU‑rich sequences respon‑sible for the dependence of gag expression on the viral Rev protein) allowing the expression of gag in a Rev‑independent manner.37,42,44

Customizing antigen coding sequence to vaccination needs.In addition to codon optimization, antigen coding genes may be modified to produce a protein that is secreted, membrane bound, cytosolic or associated with an organelle. Therefore, by altering the cellular localization it can be influenced the immune response. A protein that is naturally secreted can be modified to enhance the rate of secretion by replacement of its signal sequence with one of a highly expressed protein. A common signal used with this purpose is the tissue‑type plasminogen activator (t‑PA).45‑48 The t‑PA signal sequence of 27 amino acids can be fused to the N‑terminus of an antigen, however, the addition of a secretion signal does not warrant its secretion. Some proteins have additional motifs, such as nuclear localization signals or membrane anchors, which should be modified or deleted to achieve efficient secretion. Therefore, the modification of proteins to optimize their secretion is a good strategy to increase antibody production in vaccinated subjects.49 Likewise, if the signal sequence is removed, a naturally secreted protein can be retained in the cytoplasm.50

Targeting of gene products for degradation also represents a useful modification. Enhanced degradation reduces protein levels, however, the number of peptides available for presentation by antigen presenting cells in the context of MHC class I molecules is considerably increased. Thus, targeting to proteasomes or endo‑somes using ubiquitin or LAMP‑1 fusions38,51 is an useful strategy when induction of cytotoxic T‑lymphocytes (CTL) is required.52,53 It is also important to consider that some genes from pathogens

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



encode products which are immunosuppressive or possess potentially harmful properties. Thus, biological inactivation may be essential. For example, the reverse transcriptase and the Nef proteins of HIV exhibit enzymatic54 and immunosuppressive55 activities, whereas the human papillomavirus E7 protein represents an example of transforming activity.56

Often, it is necessary to deliver more than one gene to trigger a protective immune response (e.g., two antigens or one antigen and one cytokine). This can be achieved by the use of multiple plasmids or the generation of a bi‑ or multicistronic expression system by using an internal ribosome entry site (IRES) to initiate translation of the second gene.57 The use of polyepitopes represents a valid alterna‑tive to the expression of whole genes. Thus, a single plasmid can contain multiple expression cassettes or a large number of known epitopes linked together to be expressed as a single polypeptide with optimized flanking sequences to ensure proteosomal degradation.

Stimulatory motifs in the backbone plasmid and genetic adjuvants. Bacterial DNA, but not eukaryotic DNA, stimulates the immune system when administered to mammals. This effect is mediated through unmethylated phosphodiester linked cytosine and guanine (CpG) motifs flanked by species‑specific bases58‑60 which act through TLR‑961 to induce a series of immune stimulatory cytokines. This in turn leads to activation of B‑cells, monocytes, macrophages, DCs and natural killer cells enhancing both nonspe‑cific and antigen‑specific responses.62 There are different types of CpG that carry varying amounts of immune stimulatory motifs. They activate different types of immune cells and induce the produc‑tion of different cytokines and chemokines.63,64 On the other hand, it was demonstrated that polyG or GC‑rich encoded on oligode‑oxynucleotides suppress the immunostimulatory effect of bacterial CpG.65,66 Thus, it seems to be advantageous to define the backbone’s CpG motifs and to understand exactly their functional activity in the host cell. Species‑specific CpG motifs should be taken into consid‑eration. Undefined CpG motifs and bacterial DNA sequences that may have suppressive or undesirable immunomodulatory activities should be avoided.

Another attractive pathogen motif is the double‑stranded RNA (dsRNA) molecules associated with or derived from viral infec‑tion which are absent in normal eukaryotic cells. Such dsRNA molecules are recognized by TLR‑3 resulting in the production of IFNβ, inflammatory cytokines [TNFα, interleukine (IL)‑6 and IL‑12] and the maturation of DCs, which help to shape the overall immune response.67‑69 Furthermore, dsRNA can directly activate IRF‑3, which in turn results in a transcriptional activation of IFNα and IFNβ promoters and the IFNα/β‑responsive genes.70 It is also well established that dsRNA can activate dsRNA‑dependent protein kinase and oligoadenylate synthetase function to shut down protein translation and induce cell apoptosis.18,71‑73 Thus, plasmid‑derived hairpin RNA molecules with various stem lengths were recently evaluated. Short‑stem (40 bp) RNA molecules showed slight effect on cell apoptosis and reporter gene expression level, but were able to stimulate significantly enhanced antigen‑specific cellular immune responses. In contrast, DNA vaccines encoding long‑stem (750 bp) RNA induce vigorous cell apoptosis, but no significant improvement in cell‑mediated immune responses in immunized mice.74 These results showed that the delivered plasmid and resulting transcript are not only gene encoding sequences, but they are valuable tools for

immunoregulation. In addition, DNA plasmids can be designed to contain interference RNA (RNAi). This approach can be exploited to silence genes that might affect the overall efficiency of the stimulated immune response and/or divert from optimal response pattern.

Recently, a novel strategy to deliver genetic material into eukary‑otic cells using bacteria was described. Instead of delivering DNA, a self‑destructing L. monocytogenes strain is used to release transla‑tion‑competent mRNA directly into the cytosol.75 The simplicity of this elegant approach is based on the genetic fusion at the 5’‑end of the mRNA to an IRESEMCV element. Thus, eukaryotic translation is achieved in 5’cap‑independent manner by the infected host.76 This system may circumvent the bottleneck of limited access to the nuclear compartment of nondividing cells. Furthermore, it was demonstrated that when mRNA is delivered, translation starts earlier than when DNA plasmid is delivered.75

Genes encoding cytokines, chemokines and other molecules have been investigated for their capacity to improve the immune responses elicited against a plasmid encoded‑antigen (see Table 1). Thus, the integration of potential molecules with immunomodulatory proper‑ties into the plasmid represents a “no cost” strategy for DNA vaccine optimization.

Plasmid stabilization. During bacterial growth it is crucial to maintain plasmid among daughter cells. The well‑studied recombina‑tion site cer from the E. coli ColE1 plasmid allows multimers resolve to monomers by the XerCD recombinase.77 A promoter within cer initiates synthesis of a short transcript (Rcd) in multimer‑containing cells. The Rcd checkpoint hypothesis proposes that Rcd delays cell division until multimer resolution is complete. Thus, inclusion of the cer sequence in the plasmid backbone may assist in plasmid maintenance.78

Besides the origin of replication, antibiotic‑resistance genes are part of the standard plasmid. Antibiotic‑resistance genes are widely used as selection marker to increase plasmid stability and bacterial selection per se. However, the use of antibiotics may keep the selec‑tive pressure in vitro during biotechnological process, but high copy number plasmids are often unstable in vivo. Furthermore, there are other problems associated with antibiotic‑selection markers, namely the risk of horizontal gene transfer of the antibiotic‑resistance cassettes harbored by the plasmids and the alteration of the gene‑expression profile in mammalian cells.66,79 Thus, several strategies have emerged to achieve plasmid stabilization avoiding the use of antibiotics.

Biological stabilization systems have been developed based on active or passive approaches. Passive systems rely on metabolic auxotrophies or other gene defects, such as DNA precursor, amino acid and cell wall biosynthetic pathways, which are supplemented with the intact gene. An illustrative example of a passive contain‑ment system is the use of asd mutant strains. The asd gene coding for aspartate‑β‑semialdehyde dehydrogenase is involved in the biosyn‑thesis of diaminopimelic acid (DAP). Since DAP is not prevalent in the animal host and is a key constituent of peptidoglycan in the Gram‑negative and Gram‑positive bacterial cell wall, essentially 100% of the surviving avirulent bacteria (e.g., Salmonella spp. asd mutant) recovered from an immunized animal host still contain the recombinant plasmid and express the foreign antigen.80‑83 It has been shown that an attenuated S. enterica serovar Typhi asd mutant is safe in humans.84,85 Nevertheless, it is important to keep in mind bacterial physiology when vaccine strains with chromosomal gene

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



mutations are complemented in trans by a multicopy plasmid (pBR or pUC ori) containing the entire gene with its promoter. Using once more the example of the asd gene, when a high copy number vector (e.g., pUC‑like) coding for the asd gene is used, the Asd protein is produced in 200–300 fold excess than necessary,82 which in turns result in a slightly increased generation time and a 10‑fold increase of LD50 in mice when compared to the same strain complemented with a low‑copy (pSC101 ori) or intermediate copy (p15A ori) number plasmid.82 Thus, when designing the plasmid, it should be consid‑ered the protein level necessary to complement the system to obtain a more balanced stabilization of the expression plasmid.

Other examples of metabolic auxotrophies or gene defects are: (1) the thymidylate synthase (thyA), an essential gene involved in de novo synthesis of the DNA precursor thymidine monophosphate,86‑91 (2) the adenylosuccinate lyase (purB) which catalyzes an essential step

in the de novo synthesis of adenosine monophosphate involved in the purine biosynthetic pathway,92,93 (3) the glutamine synthetase (glnA) required for glutamine synthesis, which serves together with glutamate as primary nitrogen source in bacterial metabolism,94 (4) the tryptophanyl‑tRNA synthetase (trpS)50 and (5) the translation initiation factor 1 (infA), a small intracellular protein essential for cell viability.95

On the other hand, active stabilization provides control through the conditional production of a toxic compound whose expression is tightly controlled by an environmental factor or suppressed by internal elements. Toxin‑antitoxin systems, also known as postsegre‑gational cell killing, ensure plasmid maintenance during replication by conferring an advantage on plasmid‑retaining cells due to reduced competitiveness of their plasmid‑free counterparts. The hok‑sok system from the E. coli plasmid R1 is one of best‑characterized

Table 1 The use of live attenuated bacteria as delivery systems for DNA vaccines and therapeutic/modulatory genes

Therapeutic/ Modulatory Bacteria (strain) Gene Antigen Animal model Mouse strain Ref

S. typhimurium Human ‑ B‑cell lymphoma BALB/c 161 ΔaroA (SL5000) CD40L S. typhimurium VEGFR‑2 ‑ Melanoma, colon BALB/c and 162 ΔaroA (SL7207) (FLK‑1) and lung carcinoma C57Bl/6 S. typhimurium IFn‑γ ‑ Immuno‑ IFn‑γ–/– BALB/c 121 ΔaroA (SL7207) compromised S. typhimurium GM‑CSF Cruzipain Trypanosoma cruzi C3H/Hen 120 ΔaroA (SL7207) infection S. typhimurium IL‑10 gD and gB HSV‑2 infection BALB/c 113 ΔaroA (SL7207) from HSV S. typhimurium Antigen gp10025‑35 Melanoma C57Bl/6J, 163 ΔaroA (SL7207) fused to and TRP‑2181‑188 C57Bl/6J‑Cd8a ubiquitin epitopes tm1 Mak and C57Bl/6‑Cd4 tm1 Mak S. typhimurium PRE Tyrosine neuroblastoma A/J 164 ΔaroA (SL7207) hydroxylase S. typhimurium CD40L CEA Colon CEA‑transgenic 165 ΔaroA (SL7207) adenocarcinomas C57Bl/6J S. typhimurium CCL21 Survivin Lung cancer C57Bl/6J 166 ΔaroA, Δdam (Re88) S. typhimurium IL‑4 and IL‑18 ‑ Echinococus BALB/c 118 (SL3261) granulosus infection S. choleraesuis murine PrT gD from PsRV Pseudorabies infection BALB/c 167 S. choleraesuis Porcine PrT ‑ S. choleraesius BALB/cJ 168 infection and C3H/HeJ S. choleraesuis Thrombos‑ ‑ Melanoma C57Bl/6 169 pondin‑1 L. monocytogenes IL‑12 ‑ Leishmania BALB/c and 170 (EGD) major infection C57Bl/6 S. typhimurium IL‑2 ureB Helicobacter pylori C57Bl/6 171 ΔaroA (SL7207) infection E. coli TG‑1 PrT gD from PsRV Pseudorabies infection BALB/c 172

CEA, carcinoembryonic antigen; gD, glycoprotein D; HSV-2, herpes simplex virus type 2; PRE, posttranscriptional regulatory elements; PrT, prothymosin α; PsRV, pseudorabies virus; TRP, tyrosine-related protein; VEGFR, vascular endothelial growth factor receptor.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



toxin‑antitoxin systems. In this two‑component system, hok encodes the lethal pore forming Hok protein, whose translation is blocked by binding of the sok transcript. However, sok mRNA is highly susceptible to degradation by nucleases and its protective intracellular concentration must be maintained at a critical level by constitutive transcription from a resident plasmid carrying the hok‑sok locus.96

Safety concerns related with bacterial delivered DNA. Beside the aforementioned aspects, regulatory agencies strongly recom‑mend total avoidance of antibiotic‑resistance genes and removal of all unnecessary backbone DNA in plasmids used for vaccination or gene therapy. As for naked DNA, one of the major biosafety concerns for bacterial delivered DNA is the potential risk of chro‑mosomal integration, particularly at potentially dangerous locations, such as oncogenes or regulatory regions. Integration of plasmid sequences into the host genome could occur, since in vitro transfec‑tion with plasmid DNA almost always results in integration of the plasmid, even without selection. It is clear that live bacterial vectors (LBV)‑delivered plasmids enter the host cell nucleus. Thus, not surprisingly, plasmid integration into the genome has been found after LBV‑mediated vaccine delivery under in vitro conditions in several studies.11,14,97 However, these findings have not yet been extended to in vivo studies. Chromosomal integration after intra‑muscular vaccination with naked DNA in mice has been found to be negligible.98 In any case, careful design of the plasmid vectors is needed to minimize the risk of integration. The nucleotide sequence of the entire plasmid should be known and a careful analysis for homologies to the human genome should be performed. From a regulatory standpoint, analysis of the integration potential of a new plasmid must be carried out before any clinical trial, particularly when the backbone vector has not been tested before.

Other issues related to vaccination with DNA are the spread to and long‑term persistence of the plasmid in multiple tissues (biodis‑tribution) and the potential induction of tolerance to the immunizing antigen. Adequate guidelines and regulations concerning these issues have been published by regulatory agencies.99,100 In addition the duration of expression of the antigen, as well as the use of costimula‑tory molecules, such as cytokines and CpG DNA motifs, must be carefully evaluated since they pose further risks.101

Live Attenuated Bacterial Carriers

Currently, only three live attenuated bacterial vaccines are licensed for human use, S. enterica serovar Typhi Ty21a strain against typhoid fever, Vibrio cholera CVD 103‑HgR strain against cholera and Mycobacterium bovis bacillus Calmette‑Guerin (BCG) against tuberculosis. In addition, there are several promising live attenuated bacterial strains which have been tested in clinical trials and can potentially be exploited as delivery system for DNA vaccines (Table 2). From the point of view of DNA vaccination they should be classified in extracellular pathogens (e.g., E. coli and Yersinia spp.), intraphagosomal pathogens (e.g., Salmonella spp.) and intracytosolic pathogens (e.g., L. monocytogenes and Shigella spp.) (Fig. 1).

Gene transfer from bacteria to the eukaryotic cell is the most important parameter for the success of the DNA vaccination process. Thus, many bacteria used as DNA vectors were engineered or attenuated in such a way that they lyse upon cellular entry; such as Shigella flexneri13 and invasive E. coli,102 as a result of impaired cell wall synthesis, or L. monocytogenes,14,103 due to the production of a

phage lysine (Fig. 1). For example, a DAP auxotroph of E. coli K12, which undergoes lysis upon entry into the cytosol, is able to transfect a higher percentage of eukaryotic cells than its autotrophic coun‑terpart.102 Nevertheless, lysis of the carrier does not seem to be an absolute requirement for plasmid delivery, since immunization with S. flexneri ΔicsA, which is impaired in cell‑to‑cell spread and does not lyse, still induced a protective immune response.104

Once the plasmid is released from the carrier into the cytosol, it has to enter the nuclear compartment for transcription. Thus, an important obstacle for efficient gene transfer may be the import of plasmid DNA into the nucleus. It has been shown that nondividing cells are only hardly, if at all, transfectable with cationic lipids.105‑107 This indicates that in the case of naked plasmid DNA the nuclear membrane represents a major barrier to efficient transfer to the nucleus. However, many reports showed that using live attenuated bacteria is possible to achieve efficient transfer of plasmid to the nucleus in non dividing cells.12,16,108 It was speculated that, as in many DNA viruses, retroviruses75 and Agrobacterium tumefaciens,109 the genetic material is not merely naked, but associated with mole‑cules that prevent degradation and may even facilitate its transfer to the nucleus.110

In 2005 it was licensed the first DNA vaccine for veterinary use. This vaccine, which protects horses from the West Nile encephalitis virus, has reached the market after only a few years of research. This demonstrated that the development of nucleic acids‑based formula‑tions is much faster than antigen‑based vaccines. There are other DNA vaccines licensed for veterinary use and clinical trials (www.clinicaltrials.gov) are running in humans at the moment (e.g., influ‑enza DNA vaccine from PowerMed). Surprisingly, despite the large number of preclinical studies using attenuated bacteria as DNA delivery vectors, no clinical trials have been performed in humans. In the next section, we describe the most promising bacterial vectors under preclinical evaluation, which are expected to be translated in phase I studies in the near future.

Salmonella spp. Strains

Preclinical studies. The S. enterica serovar Typhimurium purine auxotrophic strain 22‑11 was assessed as vector for the oral delivery of a DNA vaccine encoding the major outer membrane protein of the respiratory pathogen Chlamydia trachomatis. This vaccine led to partial protection against pulmonary challenge with C. trachomatis in a murine model, demonstrating that plasmid delivery through the gut could elicit immune responses able to confer protection at a distant mucosal surface, namely the lung.111 Bacterial DNA delivery against viral diseases has also been assessed (Table 3). For example, infection with HSV‑2 can be controlled by strong T‑cell responses in the genital mucosa. Oral immunization with S. enterica serovar Typhimurium ΔaroA carrying DNA plasmids encoding the HSV‑2 glycoproteins D (gD) or B (gB) resulted in strong systemic and mucosal (vaginal) T‑cell responses, including vaginal memory T‑cells and conferred protection against a vaginal challenge with HSV, demonstrating clear superiority to intramuscular injection of the same plasmid.112,113 Furthermore, when a plasmid coding for the hepatitis B surface antigen (HBsAg) was delivered using S. enterica ΔaroA by oral route, it induced, as naked plasmid DNA, stronger CTL response than the classical alum‑adjuvanted recombi‑nant HBsAg protein, which induces mainly antibody production.114

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



These results suggest that live attenuated bacteria are a promising delivery system for therapeutic DNA vaccines against hepatitis B infection. S. enterica ΔaroA was also evaluated as delivery system of a DNA vaccine against the hepatitis C virus (HCV) encoding the NS3 antigen. This vaccination protocol resulted in the induction of strong CD8+ T‑cell responses and protection against a challenge with a recombinant HCV‑NS3 vaccinia virus.115

Most of the studies done so far in the area of tumor DNA vaccine delivery were performed in mice using Salmonella spp. as carrier

(Table 3). Paglia et al.16 were the first to demonstrate that DNA delivery using S. enterica ΔaroA as a carrier is able to induce effective cellular and humoral immune responses against a highly aggressive fibrosarcoma expressing β‑galactosidase as operational tumor associ‑ated antigen. Interestingly, this report demonstrated that by using this approach it is possible to achieve a direct targeting and transfec‑tion of DCs. The natural adjuvant effect of live attenuated bacteria was also demonstrated by the fact that the S. enterica ΔaroA vector was able per se to induce partial protection, thereby highlighting the

Table 2 Live attenuated bacterial strains used as vaccines in humans

Immunization Bacteria (strain) Attenuating mutation Route Safety Immune responses Reference

S. flexneri 2a (CVD1204) ΔguaBA Oral + Humoral 173

S. flexneri 2a (CVD1208(S)) ΔguaBA, Δset, Δsen Oral +++ Humoral and cellular 173,174

S. flexneri 2a (CVD1207) ΔguaBA, ΔvirG, Δset, Δsen Oral +++ Humoral and cellular 142

S. flexneri Y (SFL124) ΔaroD Oral +++ Humoral 175‑177

S. flexneri 2a (SFL1070) ΔaroD Oral ++ Humoral 178

S. flexneri 2a (CVD1203) ΔaroA, ΔvirG Oral + Humoral (weak) 141

S. flexneri 2a (SC602) ΔvirG, ΔiucA Oral ++ Humoral 139,140

S. flexneri (WRSS1) ΔvirG Oral ++ Humoral and cellular 179,180

S. typhi (Ty21a) ΔgalE, ΔrpoS, ΔilvD Oral +++ Humoral and cellular 181

S. typhi CDC10‑80 (PBCC211) ΔaroA, ΔaroD Oral + Humoral (weak) 182

S. typhi CDC10‑80 (PBCC222) ΔaroA, ΔaroD, ΔhtrA Oral + Humoral (weak) 182

S. typhi Ty2 (Ty800) ΔphoP, ΔphoQ Oral +++ Humoral 183

S. typhi Ty2 (Ty445) ΔphoP, ΔphoQ, ΔaroA Oral +++ Humoral (weak) 184

S. typhi Ty2 (ZH9) ΔaroC, ΔssaV Oral +++ Humoral 185

S. typhi Ty2 (M01ZH09) ΔaroC, ΔssaV, ΔSPI‑2 Oral +++ Humoral and cellular 186,187

S. typhi ISP1820 (CVD906) ΔaroC, ΔaroD Oral + Humoral 188

S. typhi Ty2 (CVD908) ΔaroC, ΔaroD Oral ++ Humoral 189,190

S. typhi Ty2 (CVD908‑htrA) ΔaroC, ΔaroD, ΔhtrA Oral +++ Humoral and cellular 135,190,191

S. typhi Ty2 (CVD909) ΔaroC, ΔaroD, ΔhtrAa Oral +++ Humoral and cellular 190,192

S. typhi Ty2 (X3927) Δcya, Δcrp Oral + Humoral 188

S. typhi Ty2 (X4073) Δcya, Δcrp, Δctd Oral ++ Humoral 85

S. typhi Ty2 (X4632) Δcya, Δcrp, Δctd, Δasd (comple‑mented with plasmid bearing

asd gene)

Oral ++ Humoralb 85

S. typhimurium TML (WT05) ΔaroC, ΔssaV Oral +++ Humoral (variable) 185

S. typhimurium (LH1160) ΔphoP, ΔphoQ, ΔpurB Oral ++ Humoral 93

S. typhimurium (VnP20009) YS72 (ΔpurI, Δxyl), ΔmsbB i.v. ++ Humoral 193

L. monocytogenes (LH1169) ΔactA, ΔplcB Oral ++ Humoral (weak) and cellular 144

F. tularensis (nDBR101) i.d. ++ nD 194,195

V. cholerae O1 El Tor Inaba (CVD103-HgR)

ΔctxA, ΔhlyA Oral +++ Humoral (also tested in children) 181,196

V. cholerae O1 El Tor Ogawa (638) ΔctxΔ hapA::celA Oral +++ Humoral 197

V. cholerae O1 El Tor Inaba (Peru‑15) ΔctxΔ recA::ctxB Oral +++ Humoral (weak in children, strong in adults)

198,199

M. bovis BCG i.d. ++ Cellular (variable) 200‑202

apromoter PtviA in Salmonella enterica serovar Typhi vaccine CVD908-htr was replaced with the constitutive promoter Ptac; bno detectable immune response against the recombinant expressed fusion protein HBc-preS;

BCG, Bacillus Calmette-Guérin; i.d., intradermal; i.p., intraperitoneal; i.v., intravenous; ND, not determined; + poorly tolerated; ++ modestly tolerated; +++ well tolerated. Licensed vaccines are shown in bold.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



import role played by the activation of the innate immune system in the overall anti‑tumor response.

As mentioned above, an important issue to take into consider‑ation is the DNA release from bacteria to the cytosol. To improve release several approaches have been pursued, such as the expression of the lambda bacteriophage S and R genes under the control of an arabinose inducible promoter.116 Another approach that could be exploited for this purpose is a recently described salicylate‑dependent regulatory control circuit, which would enable tightly‑regulated in vivo expression.117 Using this system, the live bacterial carrier could express an autolysin after induction using aspirin at therapeutic concentrations.117

On the other hand, S. enterica ΔaroA has been widely used in different experimental systems to deliver cytokines encoding genes in the context of tumor therapy, such as IL‑12, IL‑4, IL‑18 and gran‑ulocyte‑macrophage colony‑stimulating factor (GM‑CSF).118,119 This resulted in cytokine expression and partial protection against unrelated tumors.118,119 The same bacterial vector was used to deliver soluble mediators able to modulate immune response against codelivered antigens. For example, immunization with a S. enterica ΔaroA carrying a vector coding for cruzipain (Cz) from Trypanosoma cruzi alone or in combination with a strain harboring a plasmid coding for GM‑CSF resulted in the stimulation of immune responses able to control T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.120 Another application of Salmonella‑mediated bactofection was in the context of gene therapy, in order to restore a genetic deficiency at systemic level. Paglia et al demonstrated that oral administration of a S. enterica ΔaroA bearing a plasmid encoding IFNγ was able to restore the expression of IFNγ in macrophages from IFNγ –/– mice, thereby restoring the resistance of these immunocompromised animals against bacterial infections.121

Other promising attenuated derivatives of S. enterica serovar Typhimurium are those carrying the sopB mutantion. The SopB protein is encode in the Salmonella pathogenicity island (SPI)‑5 and is secreted and translocated into the cytosol through the SPI‑1 encoded type III secretion system. SopB has an inositol phosphate phosphatase activity which affects cytoskeletal rearrangement, host cell invasion and chloride homeostasis.122 The inactivation of SopB reduced inflammatory responses and fluid secretion into the intestinal lumen.123 However, they have the same tissue distribu‑tion and promote similar chemokine/cytokine profiles as wild‑type strains.124 Recent studies have demonstrated that SopB is responsible for immune escape mechanisms which can be subverted to optimize carrier performance. In fact, SopB derivatives are less toxic for DCs, which are more efficient in their capacity to process and present antigens expressed by the carrier.122

Attenuated salmonella strains tested in humans. S. enterica serovar Typhi Ty21a strain is a licensed oral vaccine against typhoid fever, which was isolated after chemical and UV mutagenesis and screened for a deficiency in the galE‑encoded enzyme uridine diphosphate (UDP)‑galactose‑4‑epimerase.125 Further untargeted mutations have later been detected in the vaccine strain (e.g., defi‑ciency in the production of the acidic Vi capsule polysaccharide and in the biosynthesis of the amino acids isoleucine and valine due to the ilvD locus mutation).126 It also harbors mutations at the rpoS locus, encoding the stationary phase‑specific sigma factor S.127,128 It is genetically stable and extremely safe.126,129 The attenuated Ty21a strain, accordingly to the directive 2001/18/EC, is not considered as

a genetically modified organism due to the methodology used for its generation. Nevertheless, it has an excellent biosafety profile, both for humans and the environment. The shedding by human volun‑teers was shown to be minimal or inexistent130,131 and the mutated rpoS locus affect bacterial resistance to environmental stresses.127,132 Ty21a, as well as the widely use attenuated S. enterica serovar Typhimurium aroA strain SL7207133 die within the phagosomal compartment of phagocytes, where they release their plasmid cargo. However, it is still unclear by which mechanism the plasmid DNA reaches the cytosol and/or the nucleus in order to be transcribed.

The S. enterica serovar Typhi CVD 908‑htrA strain is a derivative of the strain Ty2 in which aroC and aroD (involved in the biosyn‑thetic pathway to aromatic metabolites) were deleted, as well as gene htrA (involved in the cellular response to stress).134 The presence of three attenuating mutations minimizes the risk of reversion to full‑blown wild‑type virulence. In clinical trials, CVD 908‑htrA was strongly immunogenic and in contrast with the parental strain CVD 908 (Ty2 aroC aroD), CVD 908‑htrA was not detectable in the blood of vaccinees.85,135 Although the duration of excretion was short, the relatively high numbers of organisms excreted per gram of stool warranted microcosm studies. The results suggested that it is unlikely that cells released in water or soil survive for a long period of time.126 This strain represents a promising DNA carrier for human studies.

Shigella Strains

Preclinical studies. Shigella spp. entry seems to be restricted in the gut mucosa to M cell‑rich sites overlying Peyer’s patches.136 Following transcytosis across M cells, Shigella may invade macrophages, DCs and colonic mucosal cells. In contrast to Salmonella spp., Shigella spp. rapidly escape from endocytic vacuoles to the cytosol, where it may undergo lysis, thereby releasing the DNA cargo (Fig. 1). S. flexneri 2a ΔaroA ΔiscA was employed to deliver a DNA vaccine encoding gp120 of HIV‑1.104 Intranasal immunization of mice elic‑ited strong CD8+ T‑cell responses comparable to those induced by intramuscular injection of the plasmid or intraperitoneal immuniza‑tion with a recombinant vaccinia‑env vector. The immune responses mediated by the Shigella‑based DNA vaccine were protective against a vaccinia‑env challenge in a mouse model (Table 3).104

Attenuated Shigella strains tested in humans. The S. flexneri 2a SC602 strain is the vaccine candidate developed at the Institut Pasteur, by deleting the plasmid‑encoded icsA/virG virulence gene and the chromosomal iucA‑iut siderophore aerobactin locus involved in iron uptake by the bacterial cells.137 This strain is substantially attenuated and it has been tested in several clinical studies.138‑140 The obtained results demonstrated that this strain is able to evoke protection against the most severe symptoms of shigellosis. From a regulatory point of view, the residual reactogenicity of the strain at a 4‑log dose may preclude routine use in humans, since there are concerns regarding potential spread to household and workplace contacts. In addition, there are no published data focused on envi‑ronmental risk assessment.

Several other attenuated S. flexneri vaccine strains have been generated at the Center for Vaccine Development in Baltimore USA (e.g., CVD 1204, CVD 1207, CVD 1208) by deletion of the chromosomal guaBA locus. Testing performed in human volunteers showed that the CVD 1208 strain, which harbors also a deletion in the plasmid‑encoded sen and set toxin genes is the most promising

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



one.138,141,142 This strain was well‑tolerated and triggered strong immune responses, self limited fever was only observed at the higher dosages.138

Listeria Monocytogenes Strains

L. monocytogenes is the most used Gram‑positive carrier for the delivery of DNA vaccines. This intracellular pathogen is capable of invading a broad spectrum of host cell types through the intestinal mucosal surface, including macrophages, DCs, hepatocytes, intestinal epithelial cells or the endothelium of the blood‑brain barrier.143 Like Shigella spp., Listeria multiply within the cytosol of host cells and disseminate through tissues by intracellular spread. Dietrich et al14 created a L. monocytogenes Δ2 mutant harboring deletions of the genes mpl, actA and plcB. This mutant is still able to invade eukaryotic cells entering the cytosol, but has lost its ability to spread intracellularly and from cell‑to‑cell, exhibiting a high degree of attenuation in animal models14 and humans.144 Further modifications, such as the engineering for an active lysis of intracellular bacteria upon escape from the phagosome, further facilitate DNA delivery.14 Recently, a L. monocytogenes attenuated mutant, which is defective in actA and plcB, was evaluated in a Phase I clinical trials for safety and

immunogenicity. Some volunteers had abnormal liver functions, which were temporally associated with the inoculation. Therefore, further studies will be needed to evaluate this particular strain, before it can be widely implemented as conventional carrier and/or delivery system for DNA vaccines.

Preclinical studies. Vaccination with a self‑destructing attenuated L. monocytogenes Δ2 strain carrying eukaryotic expression plasmids encoding antigen 85 complex (Ag85A and Ag85B) and MPB/MPT51 (mycobacterial proteins secreted by M. bovis BCG/M. tuber‑culosis) molecules induced M. tuberculosis‑specific cellular immunity (e.g., foot pad reactions, proliferative responses and IFNγ produc‑tion), which conferred comparable levels of protection to those evoked by the Mycobacterium bovis BCG vaccine strain (Table 3).145

Administration of a recombinant Listeria vaccine strain called LM‑LLO‑E7, which secrete the HPV‑16 E7 protein fused to a nonhemolytic form of LLO, resulted in complete regression of tumors in a well‑characterized mouse model of cervical cancer (TC‑1 tumor cell line). Even when the strategy worked, Listeria cannot secrete highly hydrophobic proteins, presumably due to their diffi‑culty in translocating across the membrane or cell wall. Therefore, a Listeria‑based DNA vaccine which contains the actA‑ply118 suicide

Figure 1. Schematic representation of plasmid delivery by bacterial carriers. (I) Salmonella spp. and other intraphagosomal pathogens usually promote their own uptake into phagocytic cells. Salmonella are able to survive and deliver their DnA cargo to the nucleus. Whether this plasmid DnA is released into the cytoplasm or directly to the nucleus, as well as the underlying molecular mechanisms involved in such a process still remain to be elucidated. Attenuated strains with mutations in genes of metabolic pathways usually lyse within phagosomes, thereby improving plasmid release. (II) After transcytosis through M cells of the intestine Listeria spp. and Shigella spp. (intracytosolic pathogens) are able to invade APCs and other cellular subtypes present at the mucosa, escaping to the cytosol. (IIa) A common strategy to optimize delivery is to provide the strain with self‑destructing capacities. Thus, after escape bacteria induce their own lysis, leading to plasmid release. Despite the fact that the plasmid might be efficiently released into the cytosol, the transport into the nucleus is not as efficient as in Salmonella. A hypothesis is that the plasmid released by Listeria to the cytosol does not contain the appropriate proteins to (e.g., histone‑like) to allow an efficient transportation, however, there is still not direct evidence for this. On the other hand, it might be also possible that Salmonella resides in a particular compartment, which has preferential access to the nucleus. (IIb) There is an engineered L. monocytogenes that has a T7 RnA polymerase under the control of a cytosolic promoter, therefore, when bacteria invade the cytoplasm, not only induce their own lysis but also produce mRnA that will be translated directly in the cytosol, avoiding the transfer of the genetic material to the nucleus. The translated proteins can be directed to MHC class I restricted antigen presentation pathway in order to activate CD8+ T‑cell. Alternatively, it can be released by the cell through lysis or by adding secretory signals to the protein. This results in protein access to the MHC class II‑restricted antigen presentation pathway of bystander APCs, which in turn leads to stimulation of antibody production and CD4+ T helper responses.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



Table 3 Preclinical studies using live attenuated bacteria as delivery system for nucleic acid vaccine

Attenuating Antigen Immunization Immune responses Bacteria (strain) mutation (pathogen) Animal model route Cellular Ab Protectiona Ref

S. flexneri 2a (CVD1203) ΔaroA, ΔiscA gp120 (HIV‑1) BALB/c mouse i.m. + nD +b 104

S. flexneri 2a (CVD1204) ΔguaBA Tetanus toxin frag. C Guinea pig i.n. nD + – 203

S. flexneri 2a (CVD1208) ΔguaBA, Δset, Δsen HA (measles virus) Cotton rat i.n. + + + 204

S. flexneri 2a (15D) Δasd gp120 (HIV‑1) BALB/c mouse i.n. + + nD 205

S. flexneri 2a (15D) Δasd Fusion protein, HA, nP (measles virus)

BALB/c, BALB/c‑scid and IFn‑γ–/– C57Bl/6

mice

i.n. + + nD 160

S. flexneri 2a (SA100) rfbF::Tn5 gag (HIV‑1) CB6F1 mouse i.n. + nD nD 206

S. typhi Ty21a ΔgalE, ΔrpoS, ΔilvD gp120 (HIV‑1) BALB/c mouse i.n. + + nD 205

S. typhi Ty21a ΔgalE, ΔrpoS, ΔilvD nP (measles virus) BALB/c mouse i.p. + + nD 160

S. typhi (CVD915) ΔguaBA Tetanus toxin frag. C BALB/c mouse i.n. + + nD 207

S. typhi (CVD908‑htrA) ΔaroC, ΔaroD, ΔhtrA HA (measles virus) Cotton rat i.n. + + + 204

S. typhimurium (SL7207) ΔaroA gp120 (HIV‑1) BALB/c mouse i.n. + + nD 205

S. typhimurium (SL7207) ΔaroA env (HIV‑1) BALB/c mouse i.g. + nD nD 208

S. typhimurium (SL7207) ΔaroA ActA, listeriolysin (L. monocytogenes)

BALB/c mouse oral + + + 12

S. typhimurium (SL7207) ΔaroA HBsAg (HBV) BALB/c mouse oral + + nD 108, 114

S. typhimurium (SL7207) ΔaroA nS3 region (HCV) AAD mousec oral + nD +d 115

S. typhimurium (SL7207) ΔaroA Mp1p (P. marneffei) BALB/c mouse oral + + +/– 209

S. typhimurium (SL7207) ΔaroA cruzipain (T. cruzi) C3H/Hen mouse oral + + + 120

S. typhimurium (SL7207) ΔaroA ureB (H. pylori) C57Bl/6 mouse oral nD nD + 171

S. typhimurium (SL7207) ΔaroA H. pylori neutrophil acti‑vating protein (H. pylori)

C57Bl/6 mouse oral nD + nD 210

S. typhimurium (SL7207) ΔaroA MDR‑1 C57Bl/6 and BALB/c mice

oral + nD +e 211

S. typhimurium (SL7207) ΔaroA gp100 C57Bl/6 mouse oral + nD –f 212

S. typhimurium (SL7207) ΔaroA gp100 and TRP‑2g C57Bl/6J and C57Bl/6J scid/scid

mice

oral + nD +h 213

S. typhimurium (SL7207) ΔaroA tyrosine hydroxylase A/J mouse oral + nD +i 214

S. typhimurium (SL7207) ΔaroA β‑galactosidase BALB/c and Scid mice oral + + +/–j 215

S. typhimurium (SL7207) ΔaroA β‑galactosidase BALB/c mouse oral + + +k 16

S. typhimurium (SL7207) ΔaroA human gp100 C57Bl/6 mouse oral + nD +l 216

S. typhimurium (RE88) ΔaroA, Δdam endoglin BALB/c mouse i.g. + nD +m 217

S. typhimurium (22‑11) ΔrepA MOMP (C. trachomatis) BALB/c mouse oral nD nD +/– 111

S. choleraesuis glycoprotein D (Pseudorabies virus)

BALB/c mouse oral + + + 167

E. coli (TG‑1) glycoprotein D (Pseudorabies virus)

BALB/cBYJ mouse i.m + nD + 172

L. acidophilus (SW1) VP1 gene (Foot‑and‑mouth disease virus)

BALB/c mouse i.m., i.p., i.n.,

oral

+ + nD 218

L. monocytogenes Δ2 ΔactA, Δmpl and ΔplcB

Ag85a, Ag85b and MPB/MPT51 (M. tuberculosis)

C57Bl/6 and BALB/c mice

i.p. + nD + 145

M. smegmatis rephigh gp120 (HIV‑1) BALB/c mouse i.p. + nD nD 219

Continued on page 198

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



release system was tested in the same tumor mouse experimental model. The results demonstrated that the system was able to promote the stimulation of E7‑specifc CD8+ effector T‑cells that home to and penetrate the tumor, resulting in significant reduction of tumor growth, but it was not sufficient to induce tumor regression.146

On the other hand, L. monocytogenes can be used to deliver mRNA encoding antigens directly into the cytosol of host cells because of its intracellular lifestyle (Fig. 1). In fact, vaccination of mice with self‑destructing L. monocytogenes carriers delivering mRNA that encodes a nonsecreted form of ovalbumin (OVA) resulted in a significant OVA‑specific CD8+ T‑cell response.76

Vibrio Cholera Strains

Vibrio cholera is an extracellular pathogen, therefore, no studies have been performed until now to evaluate V. cholera as DNA delivery system. However, due to the availability of attenuated strains for human use, including the actual licensed attenuated vaccine, it is reasonable to conceive that as it was done with E. coli, V. cholera can be engineered to invade specific eukaryotic cells and deliver DNA plasmid.116

Vibrio cholerae CVD 103‑HgR. The classical Inaba recombinant cholera vaccine strain CVD 103‑HgR was constructed at the Center for Vaccine Development in Baltimore, MD, USA.147,148 Since 1994, it is the first and only licensed live oral vaccine prepared by recombinant DNA techniques. Therefore, it has served as a standard for the registration of genetically modified organism‑based vaccines. The CVD 103‑HgR was constructed by deletion of 94% of the ctxA active subunit gene of the cholera toxin operon and the deletion of the haemolysin gene (hlyA), wherein it is inserted the mercury resistance operon from the NR1 plasmid of S. flexneri.149 This vaccine strain is extremely safe and protective efficacy up to 100% against homologous challenge was observed after challenge in human volunteers.150 The strain is genetically stable during production and during transit through the gut of vaccinees. On the other hand, less than 200 CVD103‑HgR live bacteria are shed per gram faeces by 20‑30% of vaccinees from non‑endemic countries (maximum of 7 days), which is remarkable lower in comparison with a wild‑type strain (>107 vibrios per gram faeces).150,151 The vaccine strain dies within 20 days under various environmental conditions.149

Vibrio cholerae Peru‑15. The O1 El Tor Inaba vaccine strain was constructed by deletion of the entire cholera toxin locus (CTXΦ) and subsequent integration of the ctxB gene within recA locus‑ This disables the recombinational capability of the vaccine while

preserving expression of the CT‑B subunit. The final strain was also selected for motility deficiency and a nonfilamentous phenotype.152 In clinical trials this vaccine strain was well tolerated, promoting 100% protection against moderate to severe diarrhea following homologous challenge.153 However, Peru‑15 was excreted at higher levels and for longer periods of time than CVD 103‑HgR.154

Other Bacterial Vectors

An attenuated Yersinia enterocolitica strain was tested as delivery system for a DNA vaccine against Brucella abortus. Preliminary studies have shown that animals immunized with a Yersinia strain expressing the lipopolysaccharide of the serotype O:9 (cross reacting with the Brucella) showed partial protection against challenge, but not control animals immunized with a strain expressing the serotype O:3. Although anti‑lipopolysaccharide antibodies play an important role in protective immunity against Brucella, it is also known that a Th1 response seems to be critical to eliminate intracellular residing pathogens. This brought to the notion that the Yersinia strains O:9 and O:3 could be used as DNA delivery vectors for plasmids coding for the B. abortus antigens bacterioferritin and P39 (Table 3). Both prototypes induced a strong Th1 response with IFN‑γ produc‑tion. However, the best protection was achieved using Yersinia O:9 harboring the plasmid encoding for P39.155 This confirms that both cellular and humoral responses are necessary for achieving optimal protection against this pathogen. Attenuated Yersinia pseudotubercu‑losis strains have also been used to deliver DNA in vitro and in vivo. However, due to safety reasons, it will probably be more difficult to implement Yersinia strains vaccine delivery vehicles.155,156

Recently, a Lactobacillus acidophilus SW1 strain harboring a vector encoding the structural protein VP1 of the foot‑and‑mouth disease virus (FMDV) was used as DNA delivery vector (Table 3). The strongest antibody responses against VP1 were observed after intramuscular administration, followed by those of animals receiving the strain by intraperitoneal, intranasal and oral routes. In addition, immunization with L. acidophilus was able to generate moderate cellular immune responses. The results clearly demonstrate that the use of Lactobacillus as a carrier is a promising approach for DNA vaccination.

Other bacteria that have been used as DNA delivery systems are Bifidobacterium longum,157 which is a nonpathogen bacterium found in the lower small intestine of humans and Agrobacterium tumefaciens, a soil pathogen that elicit neoplastic growths on host plant species.158 In nature, Agrobacterium introduces several oncogenic genes into

Attenuating Antigen Immunization Immune responses Bacteria (strain) mutation (pathogen) Animal model route Cellular Ab Protectiona Ref

Y. enterocolitica O:9 BFR (B. abortus) P39 (B. abortus)

BALB/c mouse i.g. ++

+/‑ +

++

155

Ab, antibodies; BFR, bacterioferritin; HA, hemagglutinin; i.g., intragastric; i.m., intramuscular; i.n., intranasal; i.p., intraperitoneal; i.v., intravenous; MDR-1, multidrug resistance protein (colon/lung carcinoma); MOMP, major outer membrane protein; Mp1p, mannoprotein from Penicillium marneffei; ND, not determined; NP, nucleoprotein; TRP, tyrosinase related protein. aunless otherwise stated protection is defined as survival after challenge with the wild type organism; bchallenge with vaccinia-env virus; ctransgenic mouse expressing the alpha 1 and 2 domains of human HLA-A2.1 and the alpha 3 domain of murine H-2Dd; dchallenge with vaccinia-NS3 virus; echallenge with either MDR-1 expressing CT-26 colon carcinoma cells or MDR-1 expressing Lewis lung carcinoma cells; fchallenge with the melanoma cell line B16F1; ggenes fused to a murine ubiquitin gene; hchallenge with the melanoma cell line B16G3.26; ichallenge with NXS2 tumor cells; jchallenge with murine renal carcinoma line transfected with the lacZ gene; kchallenge with the cell line F1.A11 transduced with the lacZ gene; lchallenge with B16-hgp100 tumor cells; mchallenge with the murine D2F2 breast cancer cell line.

Table 3 Preclinical studies using live attenuated bacteria as delivery system for nucleic acid vaccine (continued)

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



the host plant, leading to formation of tumors, being widely used in the laboratory for plant genetic engineering.158,159 Agrobacterium infection requires the presence of two genetic components located on the bacterial tumor‑inducing (Ti) plasmid: the transferred DNA (T‑DNA), which is introduced into the plant cell genome and the virulence region encoding most components of the protein apparatus for T‑DNA transfer. Kunik et al demonstrated that Agrobacterium is also able to introduce T‑DNA into mammalian cells. Therefore, it would be possible to deliver DNA for noninvasive bacteria into intact mammalian cells.158

Conclusions and Outlook

The development of DNA vaccines is considerably faster than their subunit counterpart since major bottlenecks, such as difficul‑ties in antigen expression and purification, lack of posttranslational modifications, poor stability and batch‑to‑batch variability are solved. Thus, it is expected that due to their cost‑ and time‑efficient development, they will be rapidly transferred into the clinical devel‑opment pipeline. In fact, the first clinical trials of DNA vaccines are now paving the road for their transfer into the field. However, there are still major drawbacks associated with the use of DNA vaccines, such as the need of extremely high DNA dosages and poor immunogenicity. Therefore, considerable efforts are being invested in the establishment of efficient alternatives for the delivery of DNA vaccines. The use of live attenuated bacterial as carries for DNA vaccines is a very promising approach. There are several benefits over other systems, like no need for plasmid purification, built‑in immune stimulatory properties and amenability for mucosal admin‑istration. Furthermore, many of the bacterial vectors coming into consideration as carriers for DNA vaccines allow specific targeting of plasmids to APCs. Albeit pre‑existing immunity to the live carrier may affect the efficacy DNA delivery, it has been shown that not necessarily this has important detrimental effects.160 However, before live bacterial vector‑mediated DNA delivery reaches clinical trials, some roadblocks should be overcome. It is essential to elucidate the underlying molecular events leading to DNA transfer from bacteria to the cytosol and to the nucleus, which will probably differ between different carries. This is indeed a prerequisite to establish strategies to maximize DNA release. In fact, most of the attempts for vector improvement were based on empiric approaches up to now. This can in turn explain the high variability observed between studies performed using different experimental settings, as well as the fact that already planned clinical trials have been consistently delayed. It would be also of interest to develop bacterial carriers able to target specific cellular subsets in vivo, in order to fine‑tune the elic‑ited responses. In conclusion, bactofection appeals as a prominent strategy in the field of DNA vaccination, which in the coming years should prove its worth in clinical trials.

References 1. Ito Y. A tumor‑producing factor extracted by phenol from papillomatous tissue (Shope) of

cottontail rabbits. Virology 1960; 12:596‑601. 2. Atanasiu P, Orth G, Dragonas P. Delayed specific antitumoral resistance in the hamster

immunized shortly after birth with the polyoma virus. C R Hebd Seances Acad Sci 1962; 254:2250‑2.

3. Fried M, Klein B, Murray K et al. Infectivity in mouse fibroblasts of polyoma DNA inte‑grated into plasmid pBR322 or lambdoid phage DNA. Nature 1979; 279:811‑6.

4. Wolff J, Malone R, Williams P et al. Direct gene transfer into mouse muscle in vivo. Science 1990; 247:1465‑8.

5. Ulmer JB, Donnelly JJ, Parker SE et al. Heterologous protection against influenza by injec‑tion of DNA encoding a viral protein. Science 1993; 259:1745‑9.

6. Lelouard H, Ferrand V, Marguet D et al. Dendritic cell aggresome‑like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins. J Cell Biol 2004; 164:667‑75.

7. Lelouard H, Gatti E, Cappello F et al. Transient aggregation of ubiquitinated proteins dur‑ing dendritic cell maturation. Nature 2002; 417:177‑82.

8. Kawabata K, Takakura Y, Hashida M. The fate of plasmid DNA after intravenous injec‑tion in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res 1995; 12:825‑30.

9. Lew D, Parker SE, Latimer T et al. Cancer gene therapy using plasmid DNA: pharmacoki‑netic study of DNA following injection in mice. Hum Gene Ther 1995; 6:553‑64.

10. Schaffner W. Direct transfer of cloned genes from bacteria to mammalian cells. Proc Natl Acad Sci USA 1980; 77:2163‑7.

11. Courvalin P, Goussard S, Grillot‑Courvalin C. Gene transfer from bacteria to mammalian cells. C R Acad Sci III 1995; 318:1207‑12.

12. Darji A, Guzman CA, Gerstel B et al. Oral somatic transgene vaccination using attenuated S. typhimurium Cell 12 1997; 91:765‑75.

13. Sizemore DR, Branstrom AA, Sadoff JC. Attenuated Shigella as a DNA delivery vehicle for DNA‑mediated immunization. Science 1995; 270:299‑302.

14. Dietrich G, Bubert A, Gentschev I et al. Delivery of antigen‑encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat Biotechnol 1998; 16:181‑5.

15. Levine MM, Sztein MB. Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol 2004; 5:460‑4.

16. Paglia P, Medina E, Arioli I et al. Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 1998; 92:3172‑6.

17. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines. Cell 2001; 106:255‑8.

18. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20:197‑216.

19. Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat Immunol 2004; 5:971‑4.

20. Walker RI. New strategies for using mucosal vaccination to achieve more effective immuni‑zation. Vaccine 1994; 12:387‑400.

21. Bertolla F, Simonet P. Horizontal gene transfers in the environment: natural transformation as a putative process for gene transfers between transgenic plants and microorganisms. Res Microbiol 1999; 150:375‑84.

22. Nielsen K, Bones A, Smalla K et al. Horizontal gene transfer from transgenic plants to ter‑restrial bacteria—a rare event? FEMS Microbiol Rev 1998; 22:79‑103.

23. Lewin A, Mayer M, Chusainow J et al. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli. BMC Biotechnol 2005; 5:19.

24. Harms J, Oliveira S, Splitter G. Regulation of transgene expression in genetic immuniza‑tion. Braz J Med Biol Res 1999; 32:155‑62.

25. Qin L, Ding Y, Pahud D et al. Promoter attenuation in gene therapy: interferon‑gamma and tumor necrosis factor‑alpha inhibit transgene expression. Hum Gene Ther 1997; 8:2019‑29.

26. Loirat D, Li Z, Mancini M et al. Muscle‑specific expression of hepatitis B surface antigen: no effect on DNA‑raised immune responses. Virology 1999; 260:74‑83.

27. Bojak A, Hammer D, Wolf H et al. Muscle specific versus ubiquitous expression of Gag based HIV‑1 DNA vaccines: a comparative analysis. Vaccine 2002; 20:1975‑9.

28. Cazeaux N, Bennasser Y, Vidal PL et al. Comparative study of immune responses induced after immunization with plasmids encoding the HIV‑1 Nef protein under the control of the CMV‑IE or the muscle‑specific desmin promoter. Vaccine 2002; 20:3322‑31.

29. Vanniasinkam T, Reddy ST, Ertl HC. DNA immunization using a nonviral promoter. Virology 2006; 344:412‑20.

30. Tornoe J, Kusk P, Johansen TE et al. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 2002; 297:21‑32.

31. Garg S, Oran AE, Hon H et al. The hybrid cytomegalovirus enhancer/chicken beta‑actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines. J Immunol 2004; 173:550‑8.

32. Proudfoot N. How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem Sci 1989; 14:105‑10.

33. Xu Z‑L, Mizuguchi H, Ishii‑Watabe A et al. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 2001; 272:149‑56.

34. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987; 196:947‑50.

35. Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in posi‑tion +4 but is not generally affected by the nucleotides in positins +5 and +6. EMBO J 1997; 16:2482‑92.

36. Andre S, Seed B, Eberle J et al. Increased Immune Response Elicited by DNA Vaccination with a Synthetic gp120 Sequence with Optimized Codon Usage. J Virol 1998; 72:1497‑503.

37. zur Megede J, Chen MC, Doe B et al. Increased expression and immunogenic‑ity of sequence‑modified human immunodeficiency virus type 1 gag gene. J Virol 2000; 74:2628‑35.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



38. Liu WJ, Zhao K‑N, Gao FG et al. Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy. Vaccine 2001; 20:862‑9.

39. Grosjean H, Fliers W. Preferential codon usage in prokaryotic genes: the optimal codon‑ anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 1982; 18:299‑309.

40. Ko HJ, Ko SY, Kim YJ et al. Optimization of codon usage enhances the immunogenicity of a DNA vaccine encoding mycobacterial antigen Ag85B. Infect Immun 2005; 73:5666‑74.

41. Narum DL, Kumar S, Rogers WO et al. Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice. Infect Immun 2001; 69:7250‑3.

42. Deml L, Bojak A, Steck S et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol 2001; 75:10991‑1001.

43. Kane JF. Effects of rare codon clusters on high‑level expression of heterologous proteins in Escherichia coli. Current Opinion in Biotechnology 1995; 6:494‑500.

44. Schwartz S, Campbell M, Nasioulas G et al. Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev‑independent gag expres‑sion. J Virol 1992; 66:7176‑82.

45. Li Z, Howard A, Kelley C et al. Immunogenicity of DNA vaccines expressing tuberculo‑sis proteins fused to tissue plasminogen activator signal sequences. Infect Immun 1999; 67:4780‑6.

46. Leitner WW, Seguin MC, Ballou WR et al. Immune responses induced by intramuscu‑lar or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites. J Immunol 1997; 159:6112‑9.

47. Burke RL, Pachl C, Q uiroga M et al. The functional domains of coagulation factor VIII:C. J Biol Chem 1986; 261:12574‑8.

48. Chapman BS, Thayer RM, Vincent KA et al. Effect of intron A from human cytomegalovi‑rus (Towne) immediate‑early gene on heterologous expression in mammalian cells. Nucleic Acids Res 1991; 19:3979‑86.

49. Haddad D, Liljeqvist S, Stahl S et al. Comparative study of DNA‑based immunization vectors: effect of secretion signals on the antibody responses in mice. FEMS Immunol Med Microbiol 1997; 18:193‑202.

50. Ashok MS, Rangarajan PN. Protective efficacy of a plasmid DNA encoding Japanese encephalitis virus envelope protein fused to tissue plasminogen activator signal sequences: studies in a murine intracerebral virus challenge model. Vaccine 2002; 20:1563‑70.

51. Cheng W‑F, Hung C‑F, Hsu K‑F et al. Enhancement of Sindbis Virus Self‑Replicating RNA Vaccine Potency by Targeting Antigen to Endosomal/Lysosomal Compartments. Human Gene Therapy 2001; 12:235‑52.

52. Wu Y, Kipps TJ. Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome‑ dependent degradation are highly efficient inducers of cytolytic T‑lymphocytes. J Immunol 1997; 159:6037‑43.

53. Rodriguez F, Zhang J, Whitton JL. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T‑lymphocyte induction and antiviral protection but abrogates antibody induction. J Virol 1997; 71:8497‑503.

54. Isaguliants MG, Petrakova NN, Zuber B et al. DNA‑encoding enzymatically active HIV‑1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV‑1 challenge. Intervirology 2000; 43:288‑93.

55. Liang X, Fu TM, Xie H et al. Development of HIV‑1 Nef vaccine components: immuno‑genicity study of Nef mutants lacking myristoylation and dileucine motif in mice. Vaccine 2002; 20:3413‑21.

56. Smahel M, Sima P, Ludvikova V et al. Modified HPV16 E7 Genes as DNA Vaccine against E7‑Containing Oncogenic Cells. Virology 2001; 281:231‑8.

57. Ertl PF, Thomsen LL. Technical issues in construction of nucleic acid vaccines. Methods Nucleic Acid Vaccines 2003; 31:199‑206.

58. Krieg AM, Yi AK, Matson S et al. CpG motifs in bacterial DNA trigger direct B‑cell activa‑tion. Nature 1995; 374:546‑9.

59. Bauer S, Kirschning CJ, Hacker H et al. Human TLR9 confers responsiveness to bacte‑rial DNA via species‑specific CpG motif recognition. Proc Natl Acad Sci USA 2001; 98:9237‑42.

60. Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B‑cells. J Immunol 2000; 164:944‑53.

61. Hemmi H, Takeuchi O, Kawai T et al. A Toll‑like receptor recognizes bacterial DNA. Nature 2000; 408:740‑5.

62. Krieg AM, Yi A‑K, Hartmann G. Mechanisms and therapeutic applications of immune stimulatory CpG DNA. Pharmacology & Therapeutics 1999; 84:113‑20.

63. Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol 2004; 4:249‑58.

64. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20:709‑60.

65. Yamada H, Gursel I, Takeshita F et al. Effect of suppressive DNA on CpG‑induced immune activation. J Immunol 2002; 169:5590‑4.

66. Krieg AM, Wu T, Weeratna R et al. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci USA 1998; 95:12631‑6.

67. Matsumoto M, Funami K, Oshiumi H et al. Toll‑like receptor 3: a link between toll‑like receptor, interferon and viruses. Microbiol Immunol 2004; 48:147‑54.

68. Cella M, Salio M, Sakakibara Y et al. Maturation, activation and protection of dendritic cells induced by double‑stranded RNA. J Exp Med 1999; 189:821‑9.

69. Karpala AJ, Doran TJ, Bean AG. Immune responses to dsRNA: implications for gene silenc‑ing technologies. Immunol Cell Biol 2005; 83:211‑6.

70. Yoneyama M, Suhara W, Fukuhara Y et al. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF‑3 and CBP/p300. EMBO J 1998; 17:1087‑95.

71. Gil J, Esteban M. Induction of apoptosis by the dsRNA‑dependent protein kinase (PKR): mechanism of action. Apoptosis 2000; 5:107‑14.

72. Bergmann‑Leitner ES, Leitner WW. Danger, death and DNA vaccines. Microbes Infect 2004; 6:319‑27.

73. Castelli J, Wood KA, Youle RJ. The 2‑5A system in viral infection and apoptosis. Biomed Pharmacother 1998; 52:386‑90.

74. Li D, Liu Y, Zhang Y et al. Adjuvant effects of plasmid‑generated hairpin RNA molecules on DNA vaccination. Vaccine 2007; 25:6992‑7000.

75. Schoen C, Stritzker J, Goebel W et al. Bacteria as DNA vaccine carriers for genetic immu‑nization. Int J Med Microbiol 2004; 294:319‑35.

76. Loeffler DI, Schoen CU, Goebel W et al. Comparison of different live vaccine strategies in vivo for delivery of protein antigen or antigen‑encoding DNA and mRNA by virulence‑at‑tenuated Listeria monocytogenes. Infect Immun 2006; 74:3946‑57.

77. Chatwin HM, Summers DK. Monomer‑dimer control of the ColE1 Pcer promoter. Microbiology 2001; 147:3071‑81.

78. Chant EL, Summers DK. Indole signalling contributes to the stable maintenance of Escherichia coli multicopy plasmids. Mol Microbiol 2007; 63:35‑43.

79. Hartikka J, Sawdey M, Cornefert‑Jensen F et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 1996; 7:1205‑17.

80. Curtiss R 3rd, Galan JE, Nakayama K et al. Stabilization of recombinant avirulent vaccine strains in vivo. Res Microbiol 1990; 141:797‑805.

81. Zhang T, Stanley SL Jr. Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine‑rich Entamoeba histolytica protein induces an antiame‑bic immune response and protects gerbils from amebic liver abscess. Infect Immun 1996; 64:1526‑31.

82. Kang HY, Srinivasan J, Curtiss R 3rd. Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine. Infect Immun 2002; 70:1739‑49.

83. Wyszynska A, Raczko A, Lis M et al. Oral immunization of chickens with aviru‑lent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild‑type Campylobacter. Vaccine 2004; 22:1379‑89.

84. Nardelli‑Haefliger D, Kraehenbuhl JP, Curtiss R 3rd et al. Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain. Infect Immun 1996; 64:5219‑24.

85. Tacket CO, Kelly SM, Schodel F et al. Safety and immunogenicity in humans of an attenu‑ated Salmonella typhi vaccine vector strain expressing plasmid‑encoded hepatitis B antigens stabilized by the Asd‑balanced lethal vector system. Infect Immun 1997; 65:3381‑5.

86. Morona R, Yeadon J, Considine A et al. Construction of plasmid vectors with a non‑an‑tibiotic selection system based on the Escherichia coli thyA+ gene: application to cholera vaccine development. Gene1991; 107:139‑44.

87. Tacket CO, Forrest B, Morona R et al. Safety, immunogenicity and efficacy against cholera challenge in humans of a typhoid‑cholera hybrid vaccine derived from Salmonella typhi Ty21a. Infect Immun 1990; 58:1620‑7.

88. Attridge SR, Dearlove C, Beyer L et al. Characterization and immunogenicity of EX880, a Salmonella typhi Ty21a‑based clone which produces Vibrio cholerae O antigen. Infect Immun 1991; 59:2279‑84.

89. Bumann D, Metzger WG, Mansouri E et al. Safety and immunogenicity of live recombi‑nant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers. Vaccine 2001; 20:845‑52.

90. Liang W, Wang S, Yu F et al. Construction and evaluation of a safe, live, oral Vibrio cholerae vaccine candidate, IEM108. Infect Immun 2003; 71:5498‑504.

91. Fu X, Xu JG. Development of a chromosome‑plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker. Microbiol Immunol 2000; 44:551‑6.

92. DiPetrillo MD, Tibbetts T, Kleanthous H et al. Safety and immunogenicity of phoP/phoQ‑deleted Salmonella typhi expressing Helicobacter pylori urease in adult volunteers. Vaccine 1999; 18:449‑59.

93. Angelakopoulos H, Hohmann EL. Pilot study of phoP/phoQ‑deleted Salmonella enterica serovar typhimurium expressing Helicobacter pylori urease in adult volunteers. Infect Immun 2000; 68:2135‑41.

94. Ryan ET, Crean TI, Kochi SK et al. Development of a DeltaglnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae. Infect Immun 2000; 68:221‑6.

95. Hagg P, de Pohl JW, Abdulkarim F et al. A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli. J Biotechnol 2004; 111:17‑30.

96. Gerdes K, Gultyaev AP, Franch T et al. Antisense RNA‑regulated programmed cell death. Annu Rev Genet 1997; 31:1‑31.

97. Hense M, Domann E, Krusch S et al. Eukaryotic expression plasmid transfer from the intra‑cellular bacterium Listeria monocytogenes to host cells. Cell Microbiol 2001; 3:599‑609.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



98. Ledwith BJ, Manam S, Troilo PJ et al. Plasmid DNA vaccines: assay for integration into host genomic DNA. Dev Biol (Basel) 2000; 104:33‑43.

99. Cichutek K. DNA vaccines: development, standardization and regulation. Intervirology 2000; 43:331‑8.

100. Spreng S, Viret JF. Plasmid maintenance systems suitable for GMO‑based bacterial vaccines. Vaccine 2005; 23:2060‑5.

101. Robertson JS, Griffiths E. Assuring the quality, safety and efficacy of DNA vaccines. Methods Mol Med 2006; 127:363‑74.

102. Grillot‑Courvalin C, Goussard S, Huetz F et al. Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 16:862‑6.

103. Pilgrim S, Stritzker J, Schoen C et al. Bactofection of mammalian cells by Listeria monocy‑togenes: improvement and mechanism of DNA delivery. Gene Ther 2003; 10:2036‑45.

104. Shata MT, Hone DM. Vaccination with a Shigella DNA vaccine vector induces antigen‑spe‑cific CD8(+) T‑cells and antiviral protective immunity. J Virol 2001; 75:9665‑70.

105. Escriou V, Carriere M, Bussone F et al. Critical assessment of the nuclear import of plasmid during cationic lipid‑mediated gene transfer. J Gene Med 2001; 3:179‑87.

106. Fasbender A, Zabner J, Zeiher BG et al. A low rate of cell proliferation and reduced DNA uptake limit cationic lipid‑mediated gene transfer to primary cultures of ciliated human airway epithelia. Gene Ther 1997; 41173‑80.

107. Mortimer I, Tam P, MacLachlan I et al. Cationic lipid‑mediated transfection of cells in culture requires mitotic activity. Gene Ther 1999; 6:403‑11.

108. Zheng B, Woo PC, Ng M et al. A crucial role of macrophages in the immune responses to oral DNA vaccination against hepatitis B virus in a murine model. Vaccine 2001; 20:140‑7.

109. Zupan JR, Citovsky V, Zambryski P. Agrobacterium VirE2 protein mediates nuclear uptake of single‑stranded DNA in plant cells. Proc Natl Acad Sci USA 1996; 93:2392‑7.

110. Weiss S, Chakraborty T. Transfer of eukaryotic expression plasmids to mammalian host cells by bacterial carriers. Curr Opin Biotechnol 2001; 12:467‑72.

111. Brunham RC, Zhang D. Transgene as vaccine for chlamydia. Am Heart J 1999; 138:S519‑22.

112. Flo J, Tisminetzky S, Baralle F. Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus‑2 induced disease in mice. Vaccine 2001; 19:1772‑82.

113. Elias F, Flo J. Modulation of the immune response mediated by oral transgene administra‑tion of IL‑10. Cell Immunol 2002; 216:73‑81.

114. Woo PC, Wong LP, Zheng BJ et al. Unique immunogenicity of hepatitis B virus DNA vaccine presented by live‑attenuated Salmonella typhimurium. Vaccine 2001; 19:2945‑54.

115. Wedemeyer H, Gagneten S, Davis A et al. Oral immunization with HCV‑NS3‑transformed Salmonella: induction of HCV‑specific CTL in a transgenic mouse model. Gastroenterology 2001; 121:1158‑66.

116. Jain V, Mekalanos JJ. Use of lambda phage S and R gene products in an inducible lysis sys‑tem for Vibrio cholerae‑ and Salmonella enterica serovar typhimurium‑based DNA vaccine delivery systems. Infect Immun 2000; 68:986‑9.

117. Royo JL, Becker PD, Camacho EM et al. In vivo gene regulation in Salmonella spp. by a salicylate‑ dependent control circuit. Nat Methods 2007; 4:937‑42.

118. Rosenkranz CD, Chiara D, Agorio C et al. Towards new immunotherapies: targeting recombinant cytokines to the immune system using live attenuated Salmonella. Vaccine 2003; 21:798‑801.

119. Yuhua L, Kunyuan G, Hui C et al. Oral cytokine gene therapy against murine tumor using attenuated Salmonella typhimurium. Int J Cancer 2001; 94:438‑443.

120. Cazorla SI, Becker PD, Frank FM et al. Oral vaccination with Salmonella as cruzi‑pain‑DNA delivery system confers protective immunity against Trypanosoma cruzi. Infect Immun 2008;76:324‑33

121. Paglia P, Terrazzini N, Schulze K et al. In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Ther 2000; 7:1725‑30.

122. Link C, Ebensen T, Standner L et al. An SopB‑mediated immune escape mechanism of Salmonella enterica can be subverted to optimize the performance of live attenuated vaccine carrier strains. Microbes Infect 2006; 8:2262‑9.

123. Zhang S, Santos RL, Tsolis RM et al. The Salmonella enterica Serotype Typhimurium Effector Proteins SipA, SopA, SopB, SopD and SopE2 Act in Concert To Induce Diarrhea in Calves Infect Immun 2002; 70:3843‑55.

124. Reis BP, Zhang S, Tsolis RM et al. The attenuated sopB mutant of Salmonella enterica serovar Typhimurium has the same tissue distribution and host chemokine response as the wild type in bovine Peyer’s patches. Veterinary Microbiology 2003; 97:269‑77.

125. Germanier R, Fuer E. Isolation and characterization of Gal E mutant Ty 21a of Salmonella typhi: a candidate strain for a live, oral typhoid vaccine. J Infect Dis 1975; 131:553‑8.

126. Favre D, Viret JF. Biosafety evaluation of recombinant live oral bacterial vaccines in the context of European regulation. Vaccine 2006; 24:3856‑64.

127. Robbe‑Saule V, Coynault C, Norel F. The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses. FEMS Microbiol Lett 1995; 126:171‑6.

128. Coynault C, Robbe‑Saule V, Norel F. Virulence and vaccine potential of Salmonella typh‑imurium mutants deficient in the expression of the RpoS (sigma S) regulon. Mol Microbiol 1996; 22:149‑60.

129. Gentschev I, Spreng S, Sieber H et al. Vivotif—a ‘magic shield’ for protection against typhoid fever and delivery of heterologous antigens. Chemotherapy 2007; 53:177‑80.

130. Gilman RH, Hornick RB, Woodard WE et al. Evaluation of a UDP‑glucose‑4‑epimeraseless mutant of Salmonella typhi as a liver oral vaccine. J Infect Dis 1977; 136:717‑23.

131. Wahdan MH, Serie C, Cerisier Y et al. A controlled field trial of live Salmonella typhi strain Ty 21a oral vaccine against typhoid: three‑year results. J Infect Dis 1982; 145:292‑5.

132. Rozen Y, Belkin S. Survival of enteric bacteria in seawater. FEMS Microbiol Rev 2001; 25:513‑29.

133. Hoiseth SK, Stocker BA. Aromatic‑dependent Salmonella typhimurium are nonvirulent and effective as live vaccines. Nature 1981; 291:238‑9.

134. Levine MM, Galen J, Barry E et al. Attenuated Salmonella as live oral vaccines against typhoid fever and as live vectors. J Biotechnol 1996; 44:193‑6.

135. Tacket CO, Sztein MB, Wasserman SS et al. Phase 2 clinical trial of attenuated Salmonella enterica serovar typhi oral live vector vaccine CVD 908‑htrA in US volunteers. Infect Immun 2000; 68:1196‑201.

136. Philpott DJ, Edgeworth JD, Sansonetti PJ. The pathogenesis of Shigella flexneri infec‑tion: lessons from in vitro and in vivo studies. Philos Trans R Soc Lond B Biol Sci 2000; 355:575‑86.

137. Barzu S, Fontaine A, Sansonetti P et al. Induction of a local anti‑IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier. Infect Immun 1996; 64:1190‑6.

138. Riddle MS, Sanders JW, Putnam SD et al. Incidence, etiology and impact of diarrhea among long‑term travelers (US military and similar populations): a systematic review. Am J Trop Med Hyg 2006; 74:891‑900.

139. Katz DE, Coster TS, Wolf MK et al. Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers. Infect Immun 2004; 72:923‑30.

140. Coster TS, Hoge CW, VanDeVerg LL et al. Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602. Infect Immun 1999; 67:3437‑43.

141. Kotloff KL, Noriega F, Losonsky GA et al. Safety, immunogenicity and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by dele‑tions in aroA and virG. Infect Immun 1996; 64:4542‑8.

142. Kotloff KL, Noriega FR, Samandari T et al. Shigella flexneri 2a strain CVD 1207, with specific deletions in virG, sen, set and guaBA, is highly attenuated in humans. Infect Immun 2000; 68:1034‑9.

143. Vazquez‑Boland JA, Kuhn M, Berche P et al. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 2001; 14:584‑640.

144. Angelakopoulos H, Loock K, Sisul DM et al. Safety and shedding of an attenuated strain of Listeria monocytogenes with a deletion of actA/plcB in adult volunteers: a dose escalation study of oral inoculation. Infect Immun 2002; 70:3592‑601.

145. Miki K, Nagata T, Tanaka T et al. Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self‑destructing Listeria monocy‑togenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51. Infect Immun 2004; 72:2014‑21.

146. Souders NC, Verch T, Paterson Y. In vivo bactofection: listeria can function as a DNA‑cancer vaccine. DNA Cell Biol 2006; 25:142‑51.

147. Levine MM, Kaper JB, Herrington D et al. Safety, immunogenicity and efficacy of recom‑binant live oral cholera vaccines, CVD 103 and CVD 103‑HgR. Lancet 1988; 2:467‑70.

148. Ketley JM, Michalski J, Galen J et al. Construction of genetically marked Vibrio cholerae O1 vaccine strains. FEMS Microbiol Lett 1993; 111:15‑21.

149. Viret JF, Dietrich G, Favre D. Biosafety aspects of the recombinant live oral Vibrio cholerae vaccine strain CVD 103‑HgR. Vaccine 2004; 22:2457‑69.

150. Tacket CO, Cohen MB, Wasserman SS et al. Randomized, double‑blind, placebo‑con‑trolled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine CVD 103‑HgR in preventing cholera following challenge with Vibrio cholerae O1 El tor inaba three months after vaccination. Infect Immun 1999; 67:6341‑5.

151. Cryz SJ Jr, Kaper J, Tacket C et al. Vibrio cholerae CVD103‑HgR live oral attenuated vac‑cine: construction, safety, immunogenicity, excretion and nontarget effects. Dev Biol Stand 1995; 84:237‑44.

152. Kenner JR, Coster TS, Taylor DN et al. Peru‑15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172:1126‑9.

153. Cohen MB, Giannella RA, Bean J et al. Randomized, controlled human challenge study of the safety, immunogenicity and protective efficacy of a single dose of Peru‑15, a live attenu‑ated oral cholera vaccine. Infect Immun 2002; 70:1965‑70.

154. Killeen K, Spriggs D, Mekalanos J. Bacterial mucosal vaccines: Vibrio cholerae as a live attenuated vaccine/vector paradigm. Curr Top Microbiol Immunol 1999; 236:237‑54.

155. Al‑Mariri A, Tibor A, Lestrate P et al. Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen. Infect Immun 2002; 70:1915‑23.

156. Dietrich G, Spreng S, Favre D et al. Live attenuated bacteria as vectors to deliver plasmid DNA vaccines. Curr Opin Mol Ther 2003; 5:10‑9.

157. Yazawa K, Fujimori M, Nakamura T et al. Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors. Breast Cancer Res Treat 2001; 66:165‑70.

158. Kunik T, Tzfira T, Kapulnik Y et al. Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 2001; 98:1871‑6.

159. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 1984; 12:8711‑21.

160. Fennelly GJ, Khan SA, Abadi MA et al. Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J Immunol 1999; 162:1603‑10.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.



161. Urashima M, Suzuki H, Yuza Y et al. An oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium. Blood 2000; 95:1258‑63.

162. Niethammer AG, Xiang R, Becker JC et al. A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 2002; 8:1369‑75.

163. Niethammer AG, Xiang R, Ruehlmann JM et al. Targeted interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine mela‑noma. Cancer Res 2001; 61:6178‑84.

164. Pertl U, Wodrich H, Ruehlmann JM et al. Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma. Blood 2003; 101:649‑54.

165. Xiang R, Primus FJ, Ruehlmann JM et al. A dual‑function DNA vaccine encoding carci‑noembryonic antigen and CD40 ligand trimer induces T‑cell‑mediated protective immu‑nity against colon cancer in carcinoembryonic antigen‑transgenic mice. J Immunol 2001; 167:4560‑5.

166. Xiang R, Mizutani N, Luo Y et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res 2005; 65:553‑61.

167. Shiau AL, Chen YL, Liao CY et al. Prothymosin alpha enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis. Vaccine 2001; 19:3947‑56.

168. Shiau AL, Chen CC, Yo YT et al. Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin alpha. Vaccine 2005; 23:5563‑71.

169. Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella cholerae‑suis carrying thrombospondin‑1 gene leads to tumor‑specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther 2005; 12:175‑84.

170. Shen H, Kanoh M, Liu F et al. Modulation of the immune system by Listeria mono‑cytogenes‑mediated gene transfer into mammalian cells. Microbiol Immunol 2004; 48:329‑37.

171. Xu C, Li ZS, Du YQ et al. Construction of recombinant attenuated Salmonella typh‑imurium DNA vaccine expressing H pylori ureB and IL‑2. World J Gastroenterol 2007; 13:939‑944.

172. Shiau AL, Chu CY, Su WC et al. Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses. Vaccine 2001; 19:3277‑84.

173. Kotloff KL, Pasetti MF, Barry EM et al. Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208. J Infect Dis 2004; 190:1745‑54.

174. Kotloff KL, Simon JK, Pasetti MF et al. Safety and Immunogenicity of CVD 1208S, a Live, Oral DeltaguaBA Deltasen Deltaset Shigella flexneri 2a Vaccine Grown on Animal‑Free Media. Hum Vaccin 2007; 3: 268‑75.

175. Li A, Pal T, Forsum U, Lindberg AA. Safety and immunogenicity of the live oral aux‑otrophic Shigella flexneri SFL124 in volunteers. Vaccine 1992; 10:395‑404.

176. Li A, Cam PD, Islam D et al. Immune responses in Vietnamese children after a single dose of the auxotrophic, live Shigella flexneri Y vaccine strain SFL124. J Infect 1994; 28:11‑23.

177. Li A, Karnell A, Huan PT et al. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers. Vaccine 1993; 11:180‑9.

178. Karnell A, Li A, Zhao CR et al. Safety and immunogenicity study of the auxotrophic Shigella flexneri 2a vaccine SFL1070 with a deleted aroD gene in adult Swedish volunteers. Vaccine 1995; 13:88‑99.

179. Orr N, Katz DE, Atsmon J et al. Community‑based safety, immunogenicity and transmis‑sibility study of the Shigella sonnei WRSS1 vaccine in Israeli volunteers. Infect Immun 2005; 73:8027‑32.

180. Kotloff KL, Taylor DN, Sztein MB et al. Phase I evaluation of delta virG Shigella son‑nei live, attenuated, oral vaccine strain WRSS1 in healthy adults. Infect Immun 2002; 70:2016‑21.

181. Dietrich G, Griot‑Wenk M, Metcalfe IC et al. Experience with registered mucosal vaccines. Vaccine 2003; 21:678‑83.

182. Dilts DA, Riesenfeld‑Orn I, Fulginiti JP et al. Phase I clinical trials of aroA aroD and aroA aroD htrA attenuated S. typhi vaccines; effect of formulation on safety and immunogenicity. Vaccine 2000; 18:1473‑84.

183. Hohmann EL, Oletta CA, Killeen KP et al. phoP/phoQ‑deleted Salmonella typhi (Ty800) is a safe and immunogenic single‑dose typhoid fever vaccine in volunteers. J Infect Dis 1996; 173:1408‑14.

184. Hohmann EL, Oletta CA, Miller SI. Evaluation of a phoP/phoQ‑deleted, aroA‑deleted live oral Salmonella typhi vaccine strain in human volunteers. Vaccine 1996; 14:19‑24.

185. Hindle Z, Chatfield SN, Phillimore J et al. Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secre‑tion system (ssaV) mutations by immunization of healthy volunteers. Infect Immun 2002; 70:3457‑67.

186. Kirkpatrick BD, Tenney KM, Larsson CJ et al. The novel oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in 2 vaccine presentations. J Infect Dis 2005; 192:360‑6.

187. Kirkpatrick BD, McKenzie R, O’Neill JP et al. Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC‑ssaV‑) M01ZH09, with a defined mutation in the Salmonella pathogenic‑ity island 2, as a live, oral typhoid vaccine in human volunteers. Vaccine 2006; 24:116‑23.

188. Tacket CO, Hone DM, Curtiss R 3rd et al. Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers. Infect Immun 1992; 60:536‑41.

189. Tacket CO, Hone DM, Losonsky GA et al. Clinical acceptability and immunogenicity of CVD 908 Salmonella typhi vaccine strain. Vaccine 1992; 10:443‑6.

190. Tacket CO, Levine MM. CVD 908, CVD 908‑htrA and CVD 909 live oral typhoid vac‑cines: a logical progression. Clin Infect Dis 2007; 45:S20‑3.

191ß. Salerno‑Goncalves R, Wyant TL, Pasetti MF et al. Concomitant induction of CD4+ and CD8+ T‑cell responses in volunteers immunized with Salmonella enterica serovar typhi strain CVD 908‑htrA. J Immunol 2003; 170:2734‑41.

192. Wahid R, Salerno‑Goncalves R Tacket CO et al. Cell‑mediated immune responses in humans after immunization with one or two doses of oral live attenuated typhoid vaccine CVD 909. Vaccine 2007; 25:1416‑25.

193. Toso JF, Gill VJ, Hwu P et al. Phase I study of the intravenous administration of attenu‑ated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 2002; 20:142‑52.

194. Fuller CL, Brittingham KC, Porter MW et al. Transcriptome analysis of human immune responses following live vaccine strain (LVS) Francisella tularensis vaccination. Mol Immunol 2007; 44:3173‑84.

195. Hepburn MJ, Purcell BK, Lawler JV et al. Live vaccine strain Francisella tularensis is detect‑able at the inoculation site but not in blood after vaccination against tularemia. Clin Infect Dis 2006; 43:711‑6.

196. Suharyono, Simanjuntak C, Witham N et al. Safety and immunogenicity of single‑dose live oral cholera vaccine CVD 103‑HgR in 5‑9‑year‑old Indonesian children. Lancet 1992; 340:689‑94.

197. Garcia L, Jidy MD, Garcia H et al. The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers. Infect Immun 2005; 73:3018‑24.

198. Qadri F, Chowdhury MI, Faruque SM et al. Peru‑15, a live attenuated oral cholera vaccine, is safe and immunogenic in Bangladeshi toddlers and infants. Vaccine 2007; 25:231‑8.

199. Sack DA, Sack RB, Shimko J et al. Evaluation of Peru‑15, a new live oral vaccine for chol‑era, in volunteers. J Infect Dis 1997; 176:201‑5.

200. Kamath AT, Fruth U, Brennan MJ et al. New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development. Vaccine 2005; 23:3753‑61.

201. Colditz GA, Brewer TF, Berkey CS et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta‑analysis of the published literature. JAMA 1994; 271:698‑702.

202. Calmette A PH. Protective inoculation against tuberculosis with BCG. Am Rev Tuberc 1929; 19:567‑72.

203. Anderson RJ, Pasetti MF, Sztein MB et al. DeltaguaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin. Vaccine 2000; 18:2193‑202.

204. Pasetti MF, Barry EM, Losonsky G et al. Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemag‑glutinin, inducing specific immune responses and protection in cotton rats. J Virol 2003; 77:5209‑17.

205. Vecino WH, Morin PM, Agha R et al. Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vac‑cination for T‑cells against HIV. Immunol Lett 2002; 82:197‑204.

206. Xu F, Hong M, Ulmer JB. Immunogenicity of an HIV‑1 gag DNA vaccine carried by attenuated Shigella. Vaccine 2003; 21:644‑8.

207. Pasetti MF, Anderson RJ, Noriega FR et al. Attenuated deltaguaBA Salmonella typhi vac‑cine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin Immunol 1999; 92:76‑89.

208. Shata MT, Reitz MS Jr, DeVico AL et al. Mucosal and systemic HIV‑1 Env‑specific CD8(+) T‑cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector. Vaccine 2001; 20:623‑9.

209. Wong LP, Woo PC, Wu AY et al. DNA immunization using a secreted cell wall antigen Mp1p is protective against Penicillium marneffei infection. Vaccine 2002; 20:2878‑86.

210. Sun B, Li ZS, Tu ZX et al. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity. World J Gastroenterol 2006; 12:7042‑6.

211. Niethammer AG, Wodrich H, Loeffler M et al. Multidrug resistance‑1 (MDR‑1): a new target for T‑cell‑based immunotherapy. FASEB J 2005; 19:158‑9.

212. Weth R, Christ O, Stevanovic S et al. Gene delivery by attenuated Salmonella typhimurium: comparing the efficacy of helper versus cytotoxic T‑cell priming in tumor vaccination. Cancer Gene Ther 2001; 8:599‑611.

213. Xiang R, Lode HN, Chao TH et al. An autologous oral DNA vaccine protects against murine melanoma. Proc Natl Acad Sci USA 2000; 97:5492‑7.

214. Lode HN, Pertl U, Xiang R et al. Tyrosine hydroxylase‑based DNA‑vaccination is effective against murine neuroblastoma. Med Pediatr Oncol 2000; 35:641‑6.

215. Zoller M, Christ O. Prophylactic tumor vaccination: comparison of effector mechanisms initiated by protein versus DNA vaccination. J Immunol 2001; 166:3440‑50.

216. Cochlovius B, Stassar MJ, Schreurs MW et al. Oral DNA vaccination: antigen uptake and presentation by dendritic cells elicits protective immunity. Immunol Lett 2002; 80:89‑96.

217. Lee SH, Mizutani N, Mizutani M et al. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol Immunother 2006; 55:1565‑74.

218. Li YG, Tian FL, Gao FS et al. Immune responses generated by Lactobacillus as a carrier in DNA immunization against foot‑and‑mouth disease virus. Vaccine 2007; 25:902‑11.

219. Mo Y, Q uanquin NM, Vecino WH et al. Genetic alteration of Mycobacterium smegmatis to improve mycobacterium‑mediated transfer of plasmid DNA into mammalian cells and DNA immunization. Infect Immun 2007; 75:4804‑16.

delivery vehicles genetic immunization: bacteria as dna …...genetic immunization: bacteria as dna...

Documents