Bacteria as DNA vaccine carriers for genetic immunization

ARTICLE IN PRESS

International Journal of Medical Microbiology 294 (2004) 319–335 www.elsevier.de/ijmm

REVIEW

Bacteria as DNA vaccine carriers for genetic immunization Christoph Schoen, Jochen Stritzker, Werner Goebel, Sabine Pilgrim* Department of Microbiology, Biocenter of the University, D-97074 Wurzburg, Germany . Received 26 February 2004; accepted 7 March 2004

Abstract Genetic immunization with plasmid DNA vaccines has proven to be a promising tool in conferring protective immunity in various experimental animal models of infectious diseases or tumors. Recent research focuses on the use of bacteria, in particular enteroinvasive species, as effective carriers for DNA vaccines. Attenuated strains of Shigella flexneri, Salmonella spp., Yersinia enterocolitica or Listeria monocytogenes have shown to be attractive candidates to target DNA vaccines to immunological inductive sites at mucosal surfaces. This review summarizes recent progress in bacteria-mediated delivery of plasmid DNA vaccines in the field of infectious diseases and cancer. r 2004 Elsevier GmbH. All rights reserved. Keywords: Intracellular bacteria; DNA vaccines; Plasmids; Gene transfer

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Immunological aspects of bacterial DNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Bacteria used as DNA vaccine carrier strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Cellular mechanisms underlying bacteria-mediated plasmid transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Invasion of host cells and escape of the bacterial carrier from the phagosome . . . . . . . . . . . . . . . . . . . . . . . . . 323 Release of plasmid DNA from the bacterial carrier and its transfer into the nucleus . . . . . . . . . . . . . . . . . . . . . 324 Advantages and applications of bacteria-mediated delivery of DNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . 325 Vaccination against infectious diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Cancer vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

*Corresponding author. Tel.: +49-931-888-4413; fax: +49-931-888-4402. E-mail addresses: [email protected] (S. Pilgrim). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.03.001

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Introduction DNA vaccination represents one of the recent advancements of vaccine technology and has proven to confer protective immunity in animal models against a series of infectious agents including human immunodeficiency virus (HIV) (Boyer et al., 1997; Boyer et al., 1996), influenza virus (Ulmer et al., 1993), herpes simplex virus (McClements et al., 1996), Plasmodium spp. (Schneider et al., 1998), Mycobacterium tuberculosis (Huygen et al., 1996), or Borrelia burgdorferi (Luke et al., 1997). An attractive advantage of DNA vaccines from an immunological perspective is the fact that they have the potential to generate both neutralizing antibodies as well as cell-mediated immunity wherein the latter correlates well with protection against intracellular pathogens and tumors. Typical DNA vaccines are predominantly plasmids of bacterial origin composed of well-defined genetic elements for (1) plasmid replication, (2) selection and (3) mammalian expression of peptide antigens by the host cells of the vaccinees. Many companies offer basal DNA vaccine plasmids (Ertl and Thomsen, 2003) that possess multiple cloning sites to insert the coding sequence of protective antigens or epitopes derived from pathogens (viral, bacterial or protozoan) or tumors between a strong viral promoter (often CMVIE) and a suitable polyadenylation site. One of the most important aspects of a successful DNA vaccination strategy seems to be the route of delivery, and thus the application technique used to target plasmid DNA to host cells is crucial for antigen expression and priming of the immune response. A series of application routes for naked plasmid DNA have been investigated to induce either systemic and/or mucosal immune responses for over 10 years. This includes intramuscular needle injection (Ulmer et al., 1993; Wolff et al., 1990), application by bombardment using DNA-coated gold particles (Tang et al., 1992), electroporation following needle injection (Widera et al., 2000), jet injection (Furth et al., 1992), or topical exposure to mucosal sites. Most application methods were shown to induce more or less strong protective immune responses in a series of experimental animal models but were generally less successful in trials with humans (reviewed in Donnelly et al., 2003; Liu, 2003). This problem was mainly associated with the fact that high doses of plasmid DNA were necessary to induce a sufficient cell-mediated immunity (CMI) (reviewed in Donnelly et al., 2003). Therefore, much research focuses on the question how to optimize immune responses by optimization of application routes and delivery methods of the plasmid. It is generally assumed that targeting of plasmid DNA specifically to antigen-presenting cells (APC) can improve immune responses following DNA vaccination

(reviewed in Donnelly et al., 2003; Kirman and Seder, 2003). Thus, efforts are being made to complex plasmid DNA with or pack it into bite-sized particles which can be phagocytosed by APC. A number of laboratories exploit the ability to use chemically designed molecules to deliver DNA vaccines to mucosal sites (reviewed in Hobson et al., 2003). On the other hand, the use of viruses such as adenoviruses (Benihoud et al., 1999), retroviruses (Hu and Pathak, 2000) or vaccinia virus (Moorthy et al., 2003) for the transfer of plasmid DNA to a mammalian host cell is also well established and has already made its way to clinics for gene therapeutic interventions (Somia and Verma, 2000) or vaccine trials (Moorthy et al., 2003). More recently, some bacterial species were found to transfer vaccine plasmids across phylogenetic borders to mammalian host cells thereby also serving as continuous and effective producers of plasmid DNA. Gene transfer from bacteria to mammalian cells was first observed in vitro already over 20 years ago by Walter Schaffner (1980) when tandem copies of the SV40 virus genome carried by laboratory strains of E. coli were transferred into co-cultered mammalian cells. Fifteen years later, Sizemore and others (Courvalin et al., 1995; Sizemore et al., 1995) developed an improved transfer system using invasive Shigella to transfer plasmid DNA, which readily gains access to the cytosol of infected cells. In this review, we will summarize recent reports concerning principles of bacterial DNA delivery and applications of bacterial carriers for DNA vaccination.

Immunological aspects of bacterial DNA vaccines In contrast to conventional parenteral immunization, mucosal delivery of vaccines has the capability to stimulate both mucosal and systemic immune responses (Walker, 1994). With a few exceptions, most of the bacterial carrier strains for the delivery of vaccine plasmids utilized in vivo are intestinal pathogens of humans and some other mammalia. Using human enteric bacteria is particularly advantageous because of their ability to infect human colonic mucosa following oral administration. After crossing the intestinal mucosal barrier – mainly via M cells – a large fraction of bacterial carriers are taken up by APC, either in local lymphoid tissues like Peyer’s patches; or after systemic spread, in spleen, liver and lymph nodes. It has been shown that in addition to macrophages and other phagocytic cells, dendritic cells (DC) play a central role in uptake of and defense against enteric pathogens. These specialized and most efficient APC are located at the major portals of microbial entry and have evolved to monitor the environment, detect pathogens and trigger

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T cell activation by potent presentation of antigenic epitopes via MHC class I and II pathways (Banchereau and Steinman, 1998). In the gut, DC reside as immature cells in Peyer’s patches. It has been proposed that these cells are even able to open tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria, thereby monitoring the contents of the intestinal lumen (Rescigno et al., 2001). Therefore, enteric bacteria are highly qualified to deliver DNA vaccines to DC (Paglia et al., 1998) which in turn are considered the major target cells for further processing of the antigen. For direct priming of cell-mediated immune responses and the generation of specific CD8+ T (killer) cells as effectors, it is desirable that DNA vaccines specifically gain access to the biosynthetic MHC class I processing pathway of DC. After DC have taken up bacteria by phagocytosis (Mellman and Steinman, 2001), they undergo a maturation process to become efficient APC in which the recognition of products of microbial or viral pathogens, the so-called pathogen-associated molecular patterns, by pattern-recognition receptors on the surface of DCs are involved (reviewed in Medzhitov, 2001). After ingestion, bacteria transfer plasmid DNA into the cytosol of the DC (for details see below), which in turn is expressed by the cell. Consequently, the antigen is recognized as endogenous and loaded on the MHC class I molecules. The generation of a mixed immune response, comprising the induction of humoral and CMI effectors like specific CD8+ and CD4+ T cells after DNA vaccination using bacterial carriers – a phenomenon that is well established and known as cross-priming (Gurunathan et al., 2000; Sheikh and Morrow, 2003) – has been well elucidated (Darji et al., 1997; Fennelly et al., 1999; Shiau et al., 2001a). Firstly, the antigen encoded on the vaccine plasmid is produced by infected somatic or antigen-presenting cells. The antigenic protein or peptides thereof originating from the infected cell are then captured by another APC for further processing and presentation on MHC class I and II molecules to T cells. Therefore, infection of target cells other than DC by carrier bacteria could play a role of yet unknown importance in the induction and/or for the magnitude of immune responses against bacterial DNA vaccines. For example, epithelial cells in the gut or hepatocytes could serve as antigen resources for cross-priming after infection with Shigella or Listeria, respectively. Another model proposes the transfer of the vaccine plasmid DNA from one APC to another (Shata et al., 2000). Here, APC infected by bacteria undergo infectioninduced apoptosis and the membrane blebs containing the vaccine plasmids are then taken up by further APC which finally express and present the antigen. Taken together, these infected cells are not necessarily the ones which express or present the antigen.

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Bacteria used as DNA vaccine carrier strains With respect to their localization during the infective process, enteropathogenic bacteria employed as DNA vaccine carriers can be subdivided into extracellular pathogens, such as some E. coli strains or Yersinia spp., intraphagosomal pathogens like Salmonella spp., and intracytosolic pathogens, Listeria monocytogenes, Shigella spp. being well-known examples (Table 1). Yersinia enterocolitica, a member of the Enterobacteriaceae, multiplies extracellularly in abdominal lymphoid tissues after crossing the intestinal barrier (Cornelis, 2002). This survival strategy is mediated by virulence plasmid pYV encoding a series of virulence genes some of which mediate resistance to phagocytosis. Al-Mariri et al. (2002) used virulence plasmid-cured strains of Y. enterocolitica O:3 and O:9, which could thus be phagocytosed by APC, to deliver DNA vaccines encoding different Brucella abortus antigens in vivo. A characteristic feature of S. enterica serovar typhimurium (S. typhimurium) and serovar typhi (S. typhi) is that they can circumvent host defense mechanisms. They induce their own uptake into phagocytes and avoid killing mechanisms of phagocytes following internalization (Finlay and Brumell, 2000) which results in rapid multiplication within the membrane-bound phagosomal compartment. Due to an aroA mutation, the attenuated strain SL7207 of S. typhimurium (Hoiseth and Stocker, 1981) used in most of the studies with bacterial DNA vaccines employing Salmonella spp., is unable to produce aromatic amino acids necessary for its replication in vivo. S. typhi Ty21a, which has been used extensively in humans as an oral typhoid vaccine, harbors mutations that inactivate galE, leading to galactose-induced cell lysis and interference with the production of Vi polysaccharide (Germanier and Fuerer, 1975). Consequently, once they reach the lymphoid tissue after passing through M cells of intestinal mucosa into the Peyers patches, both strains die within the phagosomal compartments of macrophages and other phagocytes where they liberate copies of the DNA vaccine plasmids. Studies on primates infected with virulent Shigella spp. suggest that their initial entry in the gut mucosa is also restricted to M cell-rich sites overlying Peyer’s patches (Philpott et al., 2000). Following transcytosis across M cells, Shigella may invade macrophages, DCs and the colonic mucosal cells from below. Unlike Salmonella spp., Shigella spp. rapidly escape from endocytic vacuoles to the cytosol of the host cell where they may undergo lysis and thus be more efficient than Salmonella spp. for the transfer DNA vaccines to the host cell nucleus. Shigella flexneri Dasd 15D harbors an asd deletion mutation that interferes with the biosynthesis of diaminopimelic acid (DAP), an essential cell wall constituent. Strain 15D retains invasiveness for

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Table 1. Bacterial strains used as DNA vaccine carriers in animal models Attenuation

Phenotype

Reference

E. coli K12

TG1 DH10b

— —

(Laboratory strain) (Laboratory strain)

Shiau et al. (2001b) Cicin-Sain et al. (2003)

Y. enterocolitica

O:3, O:9

pYV

General virulence loss

Al-Mariri et al. (2002)

S. typhimurium

SL7207 RE88

aroA aroA dam aroA aroA ?

Defect Defect Defect Defect Defect Defect

Darji et al. (1997) Luo et al. (2003)

Ty21a

aroC, aroD htrA galE

CVD915

guaBA

Defect in aromatic amino acid metabolism Inability to survive and/or replicate in host tissues (htrA) Galactose-induced cell lysis, defect in production of Vi polysaccharide Defect in synthesis of guanine nucleotides

Pasetti et al. (1999)

?

Virulence attenuated

Shiau et al. (2001a)

aroA icsA guaBA guaBA set, sen asd rfbF

Defect in aromatic amino acid metabolism Loss of intercellular motility Defect in synthesis of guanine nucleotides Defect in synthesis of guanine nucleotides Loss of enterotoxins 1 and 2 Defect in diaminopimelic acid production Defect in LPS O-antigen synthesis

Shata and Hone (2001)

mpl, actA, plcB

Loss of intercellular motility

SL3261 SL7237 22–11 S. typhi

CVD 908-htrA

S. choleraesuis S. flexneri 2a

CVD1203 CVD1204 CVD1208 15D

L. monocytogenes

D2

in in in in in in

aromatic amino acid metabolism aromatic amino acid metabolism DNA adenine methylase synthesis (dam) aromatic amino acid metabolism aromatic amino acid metabolism purine metabolism

Guo et al. (2003) Darji et al. (2000) Brunham and Zhang (1999) Pasetti et al. (2003) Fennelly et al. (1999)

Anderson et al. (2000) Pasetti et al. (2003) Sizemore et al. (1997) Xu et al. (2003) Miki et al. (2004)

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Strain

C. Schoen et al. / International Journal of Medical Microbiology 294 (2004) 319–335

Carrier

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mammalian cells, yet lyses rapidly in the absence of DAP supplementation in vivo (Sizemore et al., 1995, 1997). In contrast to the Gram-negative carrier strains mentioned above, L. monocytogenes is thus far the only Gram-positive carrier applied to bacterial DNA vaccines, thus avoiding the potential toxic effects of the lipopolysaccharides of Gram-negative bacteria. This intracellular pathogen is capable of invading a broad spectrum of host cell types through the intestinal mucosal surface including macrophages, hepatocytes, intestinal epithelial cells or the endothelium of the blood–brain barrier (Vazquez-Boland et al., 2001). Like Shigella spp., Listeria multiply within the cytosol of the host cell and disseminate through tissues by intercellular spread. Dietrich et al. (1998) created an L. monocytogenes D2 mutant harboring deletions of the genes mpl, actA and plcB. This mutant is still able to invade APC and other host cells and to enter the cytosol but is unable to move from cell to cell generally leading to high virulence attenuation in animal models (Dietrich et al., 1998) and humans (Angelakopoulos et al., 2002). Two bacterial species used as DNA vaccine carriers that are not enteropathogens of humans are Bifidobacterium longum (Yazawa et al., 2001), which is a

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domestic, non-pathogenic bacterium found in the lower small intestine of humans, and Agrobacterium tumefaciens, a soil pathogen that elicits neoplastic growths on the host plant species (Kunik et al., 2001).

Cellular mechanisms underlying bacteriamediated plasmid transfer The finding of over 100 genes of putatively prokaryotic origin in the human genome (Lander et al., 2001), although cast with some doubt, strengthens the hypothesis that a transfer of genetic information between prokaryotes and mammalian eukaryotes has already happened many times in the evolutionary past. Surprisingly, at present little is known at the cell biology level of the precise events whereby the delivery of, e.g., a vaccine plasmid to mammalian cells by bacterial carriers results in an antigen-specific immune response. In general, this process may be subdivided at the cellular level into several distinct steps, some of them involving the transfer of plasmid DNA-carrying bacteria or of plasmid molecules themselves across compartmental barriers (Fig. 1).

Invasion of host cells and escape of the bacterial carrier from the phagosome

Fig. 1. Cellular mechanisms of bacteria-mediated vaccine plasmid transfer to mammalian cells. (1) Invasion of the host cell by bacterial carrier strains. (2a) Phagosomal escape of Shigella spp. or L. monocytogenes; (3a) release of plasmid DNA into the cytosol of the host cell. Alternatively, (2b) release of plasmid DNA from Salmonella carrier strains into the phagosome and (3b) consecutively from the phagosome via phagosomal leakage into the cytosol. (4) Import of plasmid DNA into the nucleus, (5) expression of plasmid-encoded antigens and (6) processing of the antigen and presentation of epitopes to T cells. (7) Alternatively, post-translationally modified antigens or epitopes thereof can be expelled from the cell. For more details see text.

Although bacterial DNA delivery to a eukaryotic host cell can in principle be achieved at a low rate using noninvasive laboratory strains of E. coli (Schaffner, 1980), the importance of effective invasion of the host cell for effective transformation of mammalian cells has been confirmed by several groups. Grillot-Courvalin et al. (1998) demonstrated that E. coli made invasive by cloning the invasion gene inv from Y. pseudotuberculosis were able to transfer DNA much more efficiently than a non-invasive strain. In fact, Cicin-Sain et al. (2003) observed that such an E. coli carrier strain was able to efficiently transfer even an entire bacterial artificial chromosome containing the complete MCMV genome to host cells in vitro, whereas almost no such transfer occurred without the plasmid-encoded invasion gene. Accordingly, L. monocytogenes impaired in their ability to invade non-phagocytic host cells due to a chromosomal deletion of the genes encoding internalin A and InlB showed a markedly reduced transfer efficacy in HEp-2 and PtK2 cells in vitro (Hense et al., 2001). Escape of bacteria or plasmid DNA from the primary vacuole further improves gene transfer efficacy. For example, co-production of the thiol-activated poreforming listeriolysin O from L. monocytogenes together with the Y. pseudotuberculosis invasin by recombinant E. coli carrier strains further increased the DNA transfer

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efficiency in vitro (Grillot-Courvalin et al., 1998). Similarly, Catic et al. (1999) demonstrated that delivery of plasmid DNA using Salmonella as carrier strain could be augmented by co-infection with a second recombinant Salmonella strain producing a pore-forming hemolysin from E. coli. Alternatively, Salmonella carrier strains engineered to secrete listeriolysin O themselves and thus made capable to escape from the phagosome led to a more efficient gene transfer in vitro (GrillotCourvalin et al., 2002). If a mutant Listeria carrier strain unable to escape from the phagosome due to deletions of the genes encoding listeriolysin O as well as phospholipases A and B was used almost no plasmid DNA transfer could be observed, again pointing out the importance of efficient phagosomal escape for an efficient transfer of DNA to the mammalian host. As mentioned above, even without escape from the phagolysosomal compartment, Salmonella carrier strains were able to transfer plasmids effectively to mammalian host cells in vivo. However, while Darji et al. (1997) and Zheng et al. (2001) reported on efficient transfer of plasmid DNA to primary macrophages using S. typhimurium DaroA also in vitro, Grillot-Courvalin et al. (2002) could only observe moderately efficient gene transfer to COS-1 cells but not to any other cell line tested including peritoneal macrophages. Therefore, there still remains a somehow enigmatic difference between the in vitro and the in vivo results obtained with S. typhimurium DaroA. Altogether, these observations may be consistent with the hypothesis that the Salmonella carrier strains are lysed within the phagolysosome, resulting in intraphagosomal release with subsequent transfer of plasmid DNA into the cytosol. This kind of leakage between these two compartments has already been proposed for transfer of certain protein antigens from the phagosome to cytosol in primary macrophages and DC (Ackerman et al., 2003; Guermonprez et al., 2003; Houde et al., 2003; KovacsovicsBankowski and Rock, 1995).

Release of plasmid DNA from the bacterial carrier and its transfer into the nucleus Most bacteria used as DNA delivery carriers were engineered to lyse upon entry of the host cell due to impaired cell wall synthesis as shown for S. flexneri (Sizemore et al., 1995) and invasive E. coli (GrillotCourvalin et al., 1998), or due to the production of a phage lysin for L. monocytogenes (Dietrich et al., 1998). The importance of efficient means for the release of plasmid DNA from bacteria into the cytosolic compartment was demonstrated by Grillot-Courvalin et al. (1998) who showed that a DAP auxotroph E. coli K12 strain which undergoes rapid lysis upon entry into the

cytosol led to a higher percentage of transgene-expressing cells than its autotrophic counterpart. Pilgrim et al. (2003) similarly demonstrated that self-destructing L. monocytogenes are much better DNA carriers than their non-destructing counterparts. In the same vein, Jain and Mekalanos (2000) showed that regulated expression of phage lambda lysis genes R and S caused dramatic lysis of V. cholerae as well as S. typhimurium cells with concomitant release of plasmid DNA in the surrounding medium. Strains equipped with the lysis genes led to a 66-fold increase in the release of plasmid DNA in the case of Salmonella and to 15-fold increase in the case of V. cholerae. On the other hand, lysis of the carrier strain may not be an absolute requirement for the release of DNA vaccine plasmid to the host cell since immunization with a DNA vaccine in S. flexneri DicsA which is impaired in cell-to-cell spread but does not lyse effectively induced protective immune responses against a model antigen (Shata and Hone, 2001) in vivo. Xu et al. (2003) supported this notion by demonstrating that other cell spread-defective mutants of S. flexneri nonetheless were able to deliver vaccine plasmid DNA effectively in vivo. Once released to the cytosol, plasmid DNA would be subjected to degradation by host cell nucleases (Pollard et al., 2001), resulting in metabolic instability and short half-life of the plasmid in the cytosol (Lechardeur et al., 1999). Furthermore, when a V. cholerae double-knockout strain lacking genes encoding extracellular DNases (dns and xds) was used, Jain and Mekalanos (2000) obtained 240 times more plasmid released into the surrounding culture broth after induction compared to the parental strain, thus demonstrating the detrimental effects of bacterial extracellular DNases for the recovery of released plasmid DNA. Although they did not report on any cell culture experiments, one might nonetheless speculate that the level of extracellular DNases expressed by the carrier strain itself may also have a great impact on the efficiency of DNA transfer to the eukaryotic host. Finally, the plasmid DNA once released from the carrier has to enter the nuclear compartment for transcription. Nuclear expression of the genetic information encoded by the vaccine plasmid was confirmed also in vivo by introducing an intron into the coding sequence of the reporter gene GFP. Darji et al. (1997) and Flo et al. (2001) showed that expression of the plasmid was indeed due to the transcription of the protein by an eukaryotic nuclear process and not following expression of the protein by the carrier bacteria itself. Therefore, besides inefficient release and detrimental effects of cytosolic as well as bacterial DNases, the import of plasmid DNA from the cytosol into the nucleus may constitute an important obstacle for efficient gene transfer. For example, it has been shown that non-dividing cells are only hardly if at all

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transfectable with cationic lipids (Escriou et al., 2001; Fasbender et al., 1997; Mortimer et al., 1999), indicating that the nuclear membrane constitutes a major barrier to efficient transfer of naked plasmid DNA residing in the cytosol. On the other hand, Grillot-Courvalin et al. (1998) pointed out that they were able to transfect X-ray-irradiated and therefore non-dividing HeLa and COS-1 cells, indicating that bacteria were able to transfer DNA also into non-dividing cells. Using attenuated S. typhimurium SL7207 as carrier strain, several groups reported that they were also able to efficiently transfect primary murine macrophages, indicating again that transfer of plasmid DNA to a nondividing cell had taken place (Darji et al., 1997; Paglia et al., 1998, 2000; Zheng et al., 2001). Since many DNA viruses (Nakanishi et al., 1996; Nelson et al., 2000; Trotman et al., 2001) as well as retroviruses (Gallay et al., 1996; Heinzinger et al., 1994) and notably also A. tumefaciens (Zupan et al., 1996) evolved elaborate mechanisms to transfer their genetic material into the nucleus of their host cells, one might speculate that plasmid DNA is not released merely as a naked DNA molecule from the carrier strain but probably in a complex with, e.g., small basic histone-like proteins (Hayat and Mancarella, 1995; Sandman et al., 1998) that may protect it from degradation in the cytosol and facilitate its nuclear import (Weiss and Chakraborty, 2002). In summary, these observations are in line with the hypothesis that carrier bacteria have to invade target cells, escape from the phagocytic vacuole and lyse as a result of either metabolic attenuation (auxotrophy), an inducible autolytic mechanism or antibiotic treatment (Hense et al., 2001). Plasmids are then released and reach the nucleus of the host cell, leading to expression of the encoded antigen. Alternatively, as in the case of intraphagosomal Salmonella spp., the plasmids have to be released from the phagocytic compartment into the cytosol after lysis of the Salmonella carrier strain via leakage from host cell phagosomes. In contrast to plasmid DNA transfer mediated by A. tumefaciens, these processes do not depend upon any special transfer machinery provided by the bacterial carrier.

Advantages and applications of bacteriamediated delivery of DNA vaccines The use of bacteria as carriers for DNA vaccines has several advantages compared to naked DNA vaccination, vaccination with viral carriers or vaccination with purified or carrier-based protein antigens, respectively. In contrast to bacteria-mediated delivery of protein antigens, bacteria-mediated delivery of DNA vaccines allows for expression of post-translationally modified

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antigens and therefore for the presentation of even conformationally restricted epitopes (Devico et al., 2002; Fouts et al., 2003). Compared to most of the viruses used as DNA vaccine carriers, bacterial carrier strains are easy to manufacture and allow for the maintenance of plasmids with a high cloning capacity. Stable replication of vaccine plasmids by different bacterial carrier species can be further ensured by introducing bacterial genes essential for survival or virulence within the host into the vaccine plasmids thereby circumventing the need to co-administrate plasmid selection markers (Galen et al., 1990, 1999; Pilgrim et al., 2003). In addition to the existence of a great number of different virulence attenuated strains, effective antibiotics are at hand in the case of an infection of an hitherto unrecognized immunocompromised vaccinee with the carrier strain, an important advantage over viral delivery systems. In contrast to immunization with naked plasmid DNA, no further plasmid amplification and purification steps are needed, thereby reducing cost and labor extensively. Most carrier bacteria allow for mucosal immunization via the oral route and show a natural tropism for inductive sites of the immune system and provide danger signals which lead to a more efficient activation of the immune system as compared to immunization with naked DNA. Besides its application to gene therapy approaches (Krusch et al., 2002; Montosi et al., 2000; Paglia et al., 2000), bacteria-mediated expression plasmid transfer has mainly been applied to genetic vaccination of model animals against infectious diseases (Table 2) and model tumors (Table 3) as will be discussed below.

Vaccination against infectious diseases Bacteria-mediated delivery of vaccine plasmids to mammalian hosts has successfully been used for the vaccination of model animals against a variety of infectious diseases of both bacterial and viral origin, particularly in models requiring cell-mediated immunity (Table 2). These include, amongst others, medically important examples such as infection with HIV-1, measles virus (MV), hepatitis B virus (HBV) and M. tuberculosis. As a large number of HIV transmissions are through human mucosal routes, the induction of high-level antiviral protection against sexually transmitted HIV-1 will only be achieved if the priming immunogen is targeted to mucosal lymphoid tissue with HIV-1-specific effector CD8+ T cells playing a central role in the control of HIV-1 replication in infected individuals. Therefore, Shata and Hone (2001) used attenuated S. flexneri 2a DaroA DicsA as carrier strain for the mucosal delivery of a vaccine plasmid encoding HIV-1

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Table 2. Vaccination of animal models against infectious diseases using bacteria-mediated delivery of DNA vaccine plasmids Pathogen

Model animal

Bacterial carrier strain

Ab

Protection

S. flexneri 2 CVD1203 S. flexneri 2a 15D, S. typhi Ty21a and S. typhimurium SL7207 S. typhimurium SL7207 S. flexneri 2 rfbFHTn5 S. typhimurium SL7207 S. typhimurium SL7207 S. flexneri 2a 15D and S. typhi Ty21a

+ +

— (+)

ND +

+a ND

Shata and Hone (2001) Vecino et al. (2002)

+ + + + +

— ND + ND +

ND ND + ND +

ND ND ND +c ND

Shata et al. (2001) Xu et al. (2003) Woo et al. (2001), Zheng et al. (2001) Wedemeyer et al. (2001) Fennelly et al. (1999)

S. flexneri 2a CVD 1208 and S. typhi CVD 908-htrA S. typhimurium SL7207 E. coli DH10b E. coli TG-1 S. choleraesuis

ND

+

+

+

Pasetti et al. (2003)

ND ND + +

+ ND + +

(+) + + +

+d + + +

Flo et al. (2001) Cicin-Sain et al. (2003) Shiau et al. (2001b) Shiau et al. (2001a, b)

S. typhimurium SL7207 L. monocytogenes D2

+ ND

+ +

+ ND

+ +

Darji et al. (1997, 2000) Miki et al. (2004)

ND

ND

ND

+

Brunham and Zhang (1999) Al-Mariri et al. (2002)

ND ND

ND +

+ +

ND

Anderson et al. (2000) Pasetti et al. (1999)

ND

+

+

+

Wong et al. (2002)

Env Gag HbsAg NS3 region Fusion protein, hemagglutinin, nucleoprotein Hemagglutinin

BALB/c mouse CB6F1 mouse Mouse AAD mouseb BALB/c mouse

Cotton rat

Herpes simplex virus-2 Cytomegalovirus Pseudorabies virus

Glycoproteins D and B MCMV-BAC Glycoproteins D and B

BALB/c mouse IFN-gR0/0 mouse BALB/c mouse

Bacteria Listeria monocytogenes Mycobacterium tuberculosis Chlamydia trachomatis Brucella abortus

ActA and LLO Ag85A, Ag85B, MPB/MPT51 MOMPe BFRf and P39

BALB/c mouse C57BL/6 and BALB/c mice BALB/c mouse BALB/c mouse

Clostridium tetani

Tetanus toxin fragment C

Guinea pig BALB/c mouse

S. typhimurium 22-11 Y. enterocolitica O:3 and O:9 S. flexneri 2a CVD 1204 S. typhi CVD 915

BALB/c mouse

S. typhimurium SL7207

a

Mp1ph

Protection against challenge with vaccinia-env virus. Transgenic mouse strain expressing the alpha 1 and 2 domains of human HLA-A2.1 and the alpha 3 domain of murine H-2Dd. c Protection against challenge with vaccinia-NS3 virus. d Protection against intravaginal chellenge with wild-type virus. e Major outer membrane protein. f Bacterioferritin. g No passive protection of animals against tetanus toxin challenge using serum samples from immunized mice. h Secreted cell wall antigen of P. marneffei. b

g

ARTICLE IN PRESS

CD4+

BALB/c mouse BALB/c mouse

Parasites Penicillium marneffei

Reference

CD8+ gp120 gp120

Hepatitis B virus Hepatitis C virus Measles virus

Immune response

C. Schoen et al. / International Journal of Medical Microbiology 294 (2004) 319–335

Viruses HIV-1

Antigen

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gp120. They showed that intranasal vaccination of mice with even a single dose of the Shigella gp120 DNA vaccine afforded significant protection against challenge with a recombinant vaccinia virus–env vector. In addition, they demonstrated that the primary CD8+ T cell response to gp120 following vaccination with the Shigella gp120 DNA vaccine was comparable to the response after vaccination with two commonly used vaccine modalities, a vaccinia–env vector and a naked gp120 DNA vaccine. They further devised two primeboost strategies, one based on priming by the Shigellabased DNA vaccine followed by a vaccinia–env boost, the other based on a Shigella-based DNA vaccine prime followed by a boost with S. typhimurium SL7207 expressing and secreting gp120. Both strategies led to an enhanced gp120-specific CD8+ T cell response when compared to a single application of one of the vaccine modalities. In a consecutive study, Shata et al. (2001) could demonstrate that intragastric inoculation of mice with S. typhimurium SL7207 carrying a HIV-1 Env DNA vaccine elicited Env-specific CD8+ T cell responses in both the systemic as well as mucosal lymphoid tissues and thus being advantageous over a parenterally administered HIV-1 DNA vaccine plasmid. Vecino et al. (2002) addressed the question of how the persistence of a carrier strain as well as the route of inoculation influences the immune responses against the plasmid-encoded antigen. They could show that in a murine intranasal immunization model, the transiently persistent S. flexneri 2a strain 15D harboring DNA vaccines induced HIV-specific IFN-g-producing CD8+ T cells among splenocytes more efficiently than either the longer persisting S. typhimurium strain SL7207 or the transiently persistent S. typhi Ty21a strain. In addition, they also found that the route of administration, i.e., intramuscular versus intranasal, had a great impact on the magnitude of mucosal and systemic antigen-specific IgA and IgG responses, with intranasally applied Shigella 15D DNA vaccines generating higher levels of HIV-specific IgA in vaginal washings than a intramuscularly injected purified DNA vaccine. Xu et al. (2003) used a cell spread-defective attenuated strain of S. flexneri 2a with a mutation in the rfbF gene leading to the synthesis of an altered O-antigen for the transfer of a vaccine plasmid encoding HIV-1 Gag. This recombinant strain was effective in inducing local and systemic immune responses after intranasal immunization of mice similar to that seen after intramuscular injection with naked DNA. They furthermore could observe a strong boosting effect in mice primed with naked vaccine DNA. Since residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current live vaccine in infants younger than 9 months of age, measles still remains the highest cause of infant and young child mortality for a vaccine-pre-

327

ventable disease in the world (Murray and Lopez, 1997; Strebel et al., 2003). As DNA vaccines are particularly suitable for priming the immune system of young animals even in the presence of low titers of maternally derived circulating antibodies, Fennelly et al. (1999) tested in a murine model the feasibility of bacteriamediated DNA vaccination against MV. They showed that the Dasd S. flexneri 15D strain harboring a DNA MV vaccine plasmid encoding different MV antigens induced a vigorous MV-specific Th1-type and a somewhat weaker Th2-type response among splenocytes from mice immunized intranasally as well as low levels of IgG and IgA in the serum. They further could demonstrate that priming for MV-specific CTL responses was even possible in mice that had a prior infection with a wild-type Shigella of the same serotype. As cotton rats are in contrast to mice extremely susceptible to pulmonary infection with MV and thus can be used to assess the efficacy of MV vaccines, Pasetti et al. (2003) tested whether the cotton rat model could be adapted to test the ability of attenuated S. flexneri 2a and S. typhi to serve as live carriers to deliver measles DNA vaccines for the priming of MV-specific immune responses. Using attenuated S. typhi CVD 908-htr and S. flexneri 2a CVD 1208 for the mucosal delivery of vaccine plasmids encoding MV hemagglutinin (H) to cotton rats, they showed that animals immunized intranasally developed MV plaque reduction neutralizing antibodies as well as proliferative responses against MV antigens. Moreover, cotton rats that received three doses of a S. flexneri 2a CVD 1208 DNA vaccine were protected against an intranasal challenge with wild-type MV as evidenced by diminished viral load in lung. Chronic HBV infection affects about 350 million people (Alter, 2003; Poovorawan et al., 2002) and is one of the leading causes of hepatocellular carcinoma worldwide (El-Serag, 2002). Since it is attributed to T cell immune tolerance to the virus, Woo et al. (2001) and Zheng et al. (2001) evaluated in a mouse model the feasibility of S. typhimurium delivering plasmid-encoded hepatitis B surface antigen (HbsAg) in eliciting effective cellular immune responses. Whereas immunization with a conventional HbsAg protein vaccine was characterized by a strong IgG1 antibody (Th 2) response accompanied by weak Th 1 and CTL responses, and in contrast to also a vigorous Th 2 and moderate CTL response after intramuscular immunization with naked DNA, the same DNA vaccine delivered by Salmonella mucosally via the oral route resulted in an only weak antibody production dominated by the IgG2 subclass but a vigorous CTL response. They concluded that the relatively absent humoral but strong cellular response could make this vaccine a potential candidate as a therapeutic vaccine for chronic HBV carriers as well as for so-called nonresponders who do not develop an antibody response to the conventional HBsAg vaccine. Furthermore, by

328

Tumor model/cell line

Vaccine antigen

gp10025–33

B78D14

TRP-225–33 gp10025–33

B16F1

TRP-225 gp100

Fibrosarcoma F1.A11

Immune response

Reference

CD8+

CD4+

DC

Ab

Protection

Genetic fusion of ubiquitin to antigen peptideminigenes

+

ND

ND

ND

+

Xiang et al. (2000)

Targeting of IL-2 to tumor tissues by coupling to a tumor-specific antibody

+

+

+

ND

+

Niethammer et al. (2001b)

Targeting of the antigen to the MHCII presentation pathway of APC w/o systemic IL-2 treatment

+

+

ND

ND

+

Weth et al. (2001)

+

ND

ND

+

+

Paglia et al. (1998)

33

Model antigen b-Gal

Breast cancer D2F2

Fos-related antigen 1

Genetic fusion of the tumor antigen to polyubiquitin. Cotransformation of the bacterial carrier with a second plasmid encoding secretory IL-18

+

+

+

ND

+

Luo et al. (2003)

Gastric cancer EAC

MG7-Ag

Genetic fusion of the antigen with helper T cell epitope PADRE

—

ND

ND

+

+

Guo et al. (2003)

Tyrosine hydroxylase

Genetic fusion of computational predicted MHCI-epitope-minigenes to ubiquitin Boosts with IL-2 targeted to tumor tissue by coupling to a tumor-specific antibody Enhanced DNA vaccine encoding the WPRE sequence

+

+

ND

ND

+

Lode et al. (2000)

+

ND

ND

ND

+

Pertl et al. (2003)

Neuroblastoma NXS2

ARTICLE IN PRESS

Melanoma B16G3.26

Additional immunomodulator(s)

C. Schoen et al. / International Journal of Medical Microbiology 294 (2004) 319–335

Table 3. Vaccination of mice against tumors using bacteria-mediated delivery of DNA vaccine plasmids

Colon carcinoma MC38

CEA CEA and CD40L

+

ND

+

ND

+

Xiang et al. (2001b)

+

ND

+

ND

+

Xiang et al. (2001a)

Boosts with IL-2 fused to a tumor-specific antibody

+

ND

+

ND

+

Niethammer et al., (2001a)

+

+

ND

ND

+

Zoller and Christ (2001)

CEA

Renal cell carcinoma RENCA

Model antigen lacZ

Lymphoma A20

No antigen

CD40L expression in the intestinal immune system

ND

ND

ND

ND

+

Urashima et al. (2000)

No antigen

Expression of plasmid-encoded cytokines (hIL-12, hGM-CSF, mIL-12, mGM-CSF) Targeting of proliferating endothelial cells in the tumor vasculature

+

ND

ND

ND

+

Yuhua et al. (2001)

ND

ND

+

Niethammer et al. (2002)

Various 4T1 and Lewis lung tumor cells Melanoma, colon carcinoma, lung carcinoma

FLK-1

+

ARTICLE IN PRESS

Lung carcinoma Lewis lung tumor cells

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Boosts with a antibody specific for a second tumor antigen coupled to IL-2 Dual-function DNA vaccine. Boosts with IL2 coupled to tumor-specific antibody

329

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co-administration of ampicillin, Woo et al. (2000) were able to enhance the humoral immune response in mice against HbsAg using S. typhi strain Ty21a as oral DNA vaccine carrier. With over 8 million new cases and 2 million deaths each year and the appearance of multi-drug-resistant M. tuberculosis strains, tuberculosis still remains an urgent public health problem worldwide (Dye et al., 1999). Since the protective efficacy of the currently used M. bovis BCG vaccine strain ranges from 0% to 85% in different controlled studies (Colditz et al., 1994), there is a great need for an improved vaccine. DNA vaccines have been shown to be one of the most promising new approaches to this end (Huygen et al., 1996). Miki et al. (2004) reported on the induction of specific protective cellular immunity against M. tuberculosis employing vaccination with recombinant attenuated L. monocytogenes strains. C57BL/6 mice immunized intraperitoneally with the attenuated self-destructing L. monocytogenes D2 strain carrying plasmids for eukaryotic expression of M. tuberculosis Ag85 complex (Ag85AA and Ag85B) and MPB/MPT51 molecules showed a specific type 1 cellular immune response. Furthermore, BALB/c mice immunized intravenously with these recombinant strains mounted protective cellular immunity against intravenous challenge with M. tuberculosis H37Rv comparable to that evoked by the conventional live BCG vaccine strain.

Cancer vaccines Cancer remains one of the major health problems with increasing numbers of people affected worldwide (Stewart and Kleihues, 2003). One of the major problems for all cancer vaccines is to overcome peripheral T cell tolerance against tumor self-antigens (Radvanyi, 2004). Since attenuated bacteria provide a danger signal resulting in enhanced stimulation of the innate immune system (Haux, 2001), they may be well suited for the delivery of vaccine plasmids encoding tumor self-antigens. Even more, it could also be shown that many bacteria specifically target tumor tissues in vivo, which may thus allow even for the selective delivery of vaccine plasmids into tumor cells (Yu et al., 2004). Most of the work being published thus far made use of attenuated S. typhimurium strains as carrier for a variety of differently engineered DNA vaccine plasmids for therapeutic vaccination of mice against model tumors (Table 3). For example, in 2000 first promising attempts to vaccinate against self-antigens of tumor tissues using SL7207 as DNA-delivery vehicle were reported by Lode et al. (2000) and Xiang et al. (2000). To enhance MHC I-dependent antigen presenta-

tion, MHC I-derived peptide-epitope-minigenes were genetically fused with ubiquitin. This resulted in breaking of self-tolerance against self-antigens which were present on melanoma or stage 4 neuroblastoma cells, respectively. Activation of antigen-specific cytotoxic T cells as well as secretion of IFN-g was demonstrated. Injection of IL-2 further enhanced protection against a murine melanoma cell line after oral vaccination with a Salmonella strain carrying a vaccine plasmid encoding the tumor antigen gp100 (Weth et al., 2001). Alternatively, injection of tumor-specific antibodies coupled to functional IL-2 in addition to oral vaccination with plasmid-encoded tumor antigens also significantly enhanced protection against different tumors like colon carcinoma (Xiang et al., 2001b), lung carcinoma (Niethammer et al., 2001a) and melanoma (Niethammer et al., 2001b). It could be demonstrated that coadministration of IL-2 in either ways resulted in upregulation of specific activation markers on antigenspecific DCs, CTL and CD4+ T-lymphocytes. Additional CD40-ligand homotrimer coexpression in a vaccine against a murine colon carcinoma (Xiang et al., 2001a) further increased efficacy of the immune response in combination with antibody-coupled IL-2 treatment and resulted in full protection of mice against a subcutaneous challenge with colon carcinoma cells. Alternatively, complete protection against hepatic metastases in a murine neuroblastoma model could be achieved by introducing a postranscriptional acting RNA element of the woodchuck hepatitis virus (WPRE) into the vaccine plasmid (Pertl et al., 2003) which resulted in increased expression of the encoded tumor antigen. Cotransformation of a second plasmid encoding the gene for murine secretory IL-18 resulted in a stronger antitumor effect in a murine breast cancer model than vaccination with a Salmonella strain carrying only the tumor antigen plasmid (Luo et al., 2003). Upregulation of activation markers on T cells, natural killer cells and DC was asserted as well as suppression of tumor angiogenesis due to the expression of IL-18. In contrast to most approaches which were directed against some specific tumor antigens, Urashima et al. (2000) and Yuhua et al. (2001) tried to protect mice against different tumors by expression of immunomodulatory molecules. Since CD40L has been shown to have a growth-suppressive effect on malignant B cells in vivo (Dilloo et al., 1997; French et al., 1999), Urashima et al. (2000) induced CD40L expression in the intestinal immune system by oral immunization of mice with a Salmonella strain carrying a plasmid containing the CD40L gene (ST40L). Up to 90% of mice survived a challenge with A20 B lymphoma cells with simultaneous oral administration of ST40L. Alternatively, to induce IL-12 and/or GM-CSF expression also known to have

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profound antitumor effects in animal models, Yuhua et al. (2001) orally immunized mice with Salmonella strains carrying plasmids encoding IL-12 and/or GM-CSF which resulted in a high degree of protection against challenge with 4T1-mammary carcinoma as well as Lewis lung tumor cells, respectively. Using a murine renal carcinoma cell line transfected . with the lacZ gene as model tumor antigen, Zoller and Christ (2001) could demonstrate that vaccination with Salmonella carrying a vaccine plasmid followed by an intravenous transfer of antigen-derived peptide-loaded DC proved to be the optimal strategy compared to other vaccination strategies such as immunization with antigen-derived peptide-loaded DCs or with naked DNA vaccine plasmids, pointing out the strength of DNA vaccination by the oral route. This finding was further substantiated by Weth et al. (2001) in a murine melanoma model which could show that oral vaccination with Salmonella is the most convenient transfer regime being further improved if the antigen is preferentially aimed at the activation of T-helper cells by genetic fusion of the antigen gene to the invariant chain cDNA resulting in presentation via the MHC II pathway. To circumvent the problem of targeting genetically unstable tumor cells, an interesting novel approach did not target antigens expressed by tumor cells but was directed against stable, proliferating endothelial cells in the tumor vasculature and thereby inhibiting angioneogenesis (Niethammer et al., 2002). The authors could show protection against lung and colon carcinoma as well as against melanoma by oral immunization of mice with a Salmonella strain carrying a plasmid containing the vascular-endothelial growth factor receptor 2 (FLK-1) gene. Apart from slight delay in wound healing, no adverse side effects such as impairment of fertility, neuromuscular performance or hematopoiesis could be detected, demonstrating the specificity of this approach.

Conclusions Before genetic immunization employing bacteriamediated transfer of vaccine plasmids can be assessed in clinical trials, biosafety issues have to be addressed such as characterization of the carrier strain with regard to the introduced attenuations, the recombinant product(s) expressed by the plasmid and integration assessment in appropriate animal models. Furthermore, it is likely that different bacterial DNA delivery systems will each require their own additional genetic engineering to optimize at the same time plasmid transfer efficiency and antigen immunogenicity as well as to minimize carrier strain reactogenicity and to enforce biosafety. But

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the results outlined above, especially the generation of cellular as well as humoral protective immune responses against a variety of infectious agents or tumors in animal model systems, are highly encouraging and suggest further development of this system is desirable. Therefore, the next logical step would be to obtain the proofof-principle in a clinical setting using a bacterial carrier strain of known safety and immunogenicity with an appropriate DNA vaccine construct.

Acknowledgements This work was supported by grants from the Bayerische Forschungsstiftung (Forimmune) and the DFG (Go168/27-1). C. Schoen gratefully acknowledges a fellowship from the BMBF (IZKF Wurzburg, . 01KS9603). The authors thank M. Kuhn and B. Joseph for critical reading of the manuscript.

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