Delivery of DNA vaccines by attenuated intracellular bacteria

TRENDS I M M U N O L O G Y TO D AY

12 Giorgi, J.V., Hausner, M.A. and Hultin, L.E. (1999) Immunol. Lett. 66, 105–110 13 Westby, M., Manca, F. and Dalgleish, A.G. (1996) Immunol. Today 17, 120–126 14 Rosenberg, E.S., Billingsley, J.M., Caliendo, A.M. et al. (1997) Science 278, 1447–1450 15 Rosenberg, E.S. and Walker, B.D. (1999) Immunol. Lett. 66, 89–93 16 Matloubian, M., Concepcion, R.J. and Ahmed, R. (1994) J. Virol. 68, 8056–8063 17 von Herrath, M.G., Yokoyama, M., Dockter, J., Oldstone, M.B. and Whitton, J.L. (1996) J. Virol. 70, 1072–1079 18 Heeney, J., Bogers, W., Buijs, L. et al. (1996) Immunol. Lett. 51, 45–52 19 Leandersson, A.C., Bratt, G., Hinkula, J. et al. (1998) AIDS 12, 157–166

20 Patterson, S., Robinson, S.P., English, N.R.

(1999) Immunol. Lett. 66, 151–157

and Knight, S.C. (1999) Immunol. Lett. 66,

28 Lehner, T., Wang, Y., Cranage, M. et al. (1996)

111–116

Nat. Med. 2, 767–775

21 Albert, M.L., Sauter, B. and Bhardwaj, N.

29 Stebbings, R., Stott, J., Almond, N. et al.

(1998) Nature 392, 86–89

(1998) AIDS Res. Hum. Retroviruses 14,

22 Albert, M.L., Pearce, S.F.A., Francisco, L.M.

1187–1198

et al. (1998) J. Exp. Med. 188, 1359–1368

30 Bogers, W.M., Dubbes, R., ten Haaft, P. et al.

23 Parren, P.W., Mondor, I., Naniche, D. et al.

(1997) Virology 236, 110–117

(1998) J. Virol. 72, 3512–3519

31 Haigwood, N.L., Pierce, C.C., Robertson,

24 Cameron, P.U., Freudenthal, P.S., Barker, J.M.

M.N. et al. (1999) Immunol. Lett. 66, 183–188

et al. (1992) Science 257, 383–387

32 Calarota, S., Bratt, G., Nordlund, S. et al.

25 Zolla-Pazner, S., Xu, S., Burda, S. et al. (1998)

(1998) Lancet 351, 1320–1325

J. Infect. Dis. 178, 1502–1506

33 Heeney, J.L., Teeuwsen, V.J.P., van Gils, M.

26 Barker, E., Bossart, K.N. and Levy, J.A.

et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,

(1998) Proc. Natl. Acad. Sci. U. S. A. 95,

10803–10808

1725–1729

34 Li, T.S., Tubiana, R., Katlama, C. et al. (1998)

27 Lochner, C.P., Blackbourn, D.J. and Levy, J.A.

Lancet 351, 1682–1686

Delivery of DNA vaccines by attenuated intracellular bacteria Guido Dietrich, Ivaylo Gentschev, Jürgen Hess, Jeffrey B. Ulmer, Stefan H.E. Kaufmann and Werner Goebel Professional antigen-presenting cells (APCs) play a key role in the

I

n principle, DNA vaccines consist of bacterial plasmids that code for antigens under the control of strong eukaryotic promoters1. These vaccines can either be injected as an aqueous solution by an intramuscular route or coated onto gold particles and introduced into the dermis via a gene gun. Both routes of application elicit strong cellular and humoral immune responses against a variety of pathogens, tumor antigens and allergens. Recent work has demonstrated that professional antigen-presenting cells (APCs), which migrate to lymphoid organs after vaccination by either route, are essential for priming immune responses1–3, either through direct transfection of APC (Ref. 4), crosspriming2,3, or both. Delivery of DNA to APCs can be accomplished by attenuated intracellular bacteria. After phagocytosis by APCs, the bacteria PII: S0167-5699(98)01431-5

host cell phagosome or cytosol. The released DNA can enter the nucleus, resulting in expression of encoded antigens and subsequent induction of humoral and cellular immune responses.

induction of immune responses evoked by vaccination with plasmid DNA. Use of attenuated intracellular bacteria as delivery vehicles has the potential to efficiently target DNA vaccines to

Gram-negative bacteria as DNAvaccine vehicles

professional APCs.

survive inside these cells either by modifying the phagosomal compartment, e.g. by arresting the phagosomal continuum at distinct stages, or by egress from the phagosome into the host cell cytosol5. Most intracellular bacteria can be transformed easily with DNA vaccine vectors. Accordingly, attenuated mutants, which undergo lysis inside APCs, can carry these plasmids into the

Attenuated mutant strains of the invasive bacteria Shigella flexneri and Salmonella typhimurium have been used for the delivery of eukaryotic expression vectors6–8 (Fig. 1). These strains were attenuated by deletion of genes involved in the production of metabolites essential for cell wall synthesis. Therefore, these auxotrophic bacteria infect mammalian cells and then lyse due to the lack of these metabolites. S. flexneri can be transformed with eukaryotic expression vectors containing 0167-5699/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.

J U N E Vo l . 2 0

1 9 9 9 No.6

251

TRENDS I M M U N O L O G Y T O D AY

MHC class I

6

3a

2 ? 1

4

5

APC Nucleus

3b

3d

3c

Fig. 1. DNA vaccine delivery to an APC by attenuated intracellular bacteria. (1) Salmonellae, shigellae or listeriae carrying DNA vaccine vectors (red) adhere to an APC. (2) Bacteria are phagocytosed by the APC. (3a) Salmonellae lyse inside the phagosome, releasing the plasmid molecules into this compartment, from where they enter the cytoplasm via unknown mechanisms. (3b) Shigellae and listeriae lyse the phagosomal membrane, escape into the cytosol (3c) and release the plasmid DNA directly into the cytoplasm upon bacterial desintegration (3d). (4) The plasmid DNA enters the nucleus. (5) Plasmid-encoded antigens are expressed by the APC, processed and presented in the context of MHC class I and possibly also MHC class II (either directly or by crosspriming) molecules (6). Abbreviations: APC, antigen-presenting cell; MHC, major histocompatibility complex.

origins of replication that are functional in Escherichia coli. Because S. flexneri enters the host cell cytosol after phagocytosis, the bacteria can deliver the plasmids directly to this intracellular compartment. Plasmidencoded antigens are subsequently expressed by the host cell, as has been shown in vitro for several cell types6. An invasive phenotype of the bacteria is necessary for successful plasmid delivery. S. flexneri has also been used in mice for plasmid delivery by intranasal application of the bacteria. This immunization protocol led to strong cellular and humoral immune responses against the model antigen b-galactosidase7. Unlike S. flexneri, S. typhimurium remains in the phagosomal compartment of the host cell5. Nevertheless, attenuated strains of S. typhimurium can deliver eukaryotic expression vectors into macrophages and splenic dendritic cells in vivo with high efficiency, leading to strong expression of plasmidencoded reporter genes8,9. Attenuated S. typhimurium were also used for in vivo delivery of a DNA vaccine vector encoding listeriolysin8, which is a protective T-cell

J U N E 252

antigen of the intracellular bacterium Listeria monocytogenes and is responsible for lysis of the phagosomal membrane of infected host cells10. Vaccination of mice via the oral route induced CD81 and CD41 T-cell responses as well as high antibody titers to this antigen. The phenotype of the T helper (Th)-cell responses was Th1-like with high levels of interferon g (IFN-g) and no detectable interleukin 4 (IL-4). The DNA vaccine-induced immune response protected mice against a lethal challenge with L. monocytogenes and could be boosted by oral application of the S. typhimurium–DNA vaccine construct. It is not yet known how the plasmid DNA is transported from the phagosomal compartment into the host cell nucleus, but infection with Salmonella possibly causes leakage of the phagolysosome. In support of this hypothesis, infection of primary macrophages with S. typhimurium results in the presentation of phagosomal proteins in the context of major histocompatibility complex (MHC) class I molecules10. Alternatively, there might be a specific and as yet unidentified transport process for the translocation of the

1 9 9 9 Vo l . 2 0

No.6

plasmid vectors across the phagosomal membrane. Interestingly, Salmonella failed to deliver plasmid DNA in cell lines6,8, suggesting that functional DNA delivery is more efficient in mature APCs. S. flexneri and S. typhimurium are both well suited for delivery of macromolecules into mammalian cells due to their invasive properties. Using a different approach, a cell wall mutant of a noninvasive and apathogenic bacterium, the genetically welldefined laboratory strain E. coli K12 was equipped with genes conferring invasiveness11,12. This strain was transformed with the 200 kb virulence plasmid of S. flexneri, which contains the genes for invasion, as well as for intra- and intercellular motility11. Alternatively, the invasin gene inv of Yersinia pseudotuberculosis and the hly gene of L. monocytogenes, coding for listeriolysin, were introduced into E. coli (Ref. 12). Both strains delivered eukaryotic expression vectors directly to the host cell cytosol with high efficiency in vitro11,12, but have not yet been used for vaccination.

DNA delivery by Gram-positive bacteria Gram-negative DNA vaccine carriers have the disadvantage of containing abundant amounts of toxic lipopolysaccharides (LPS), which might interfere with the synthesis of plasmid-encoded antigens by host cells. To avoid this, Gram-positive bacteria have been used as DNA vaccine carriers. A good candidate is L. monocytogenes, which is found in high numbers in the cytosol of splenic APCs shortly after infection through the intestinal mucosa. L. monocytogenes has been attenuated by deletion of the genes responsible for intracellular mobility and cell-to-cell spreading after bacterial escape from the host cell phagosome13. Direct delivery of plasmid vectors to the host cell cytosol was achieved by subsequent autolysis of listeriae due to a listeria-specific phage lysin produced upon bacterial egress from the phagosome13. Delivery of eukaryotic expression vectors by these attenuated suicide L. monocytogenes and subsequent expression of plasmidencoded reporter genes have been demonstrated in a murine macrophage cell line. Additionally, delivery of ovalbumin

TRENDS I M M U N O L O G Y TO D AY

expression vectors to primary murine macrophages leads to efficient presentation of the major ovalbumin epitope in the context of MHC class I molecules13 (Fig. 1).

Potential risks Although the invasive nature of the employed bacterial carriers is advantageous for efficient DNA vaccine delivery, it also carries a potential risk for the host. Reversion to virulence is always a concern for attenuated pathogens. Another safety consideration at least for Gram-negative bacteria is the toxic effect of the LPS. However, attenuated Salmonella strains, which are used as vaccines in human and veterinary medicine, have been shown to be safe. An important issue concerning safety of DNA vaccines in general is their potential for integration into the host cell genome, which might promote oncogenesis. Whereas immunization with naked DNA has been found not to lead to genomic integration1, delivery of eukaryotic expression vectors by invasive E. coli (Ref. 11) and attenuated L. monocytogenes (Ref. 13) resulted in chromosomal integration in vitro. These findings might be due to the high plasmid copy numbers that are delivered to single cells by the bacterial carriers. The potential of integration using these vectors in vivo is not yet known. A mitigating factor of integration might be that cells presenting foreign antigens (derived from the DNA vaccine or the bacterial carriers) can be destroyed by cytotoxic T lymphocytes.

Concluding remarks Delivery of DNA vaccines by bacterial vectors has several advantages over naked DNA. First, the application of DNA vaccines by intramuscular injection or gene-gun inoculation transfects only very limited numbers of professional APCs, whereas delivery of DNA vaccines by appropriate bacterial carriers could target plasmid vectors specifically to APCs in immunologically relevant organs. In addition to macrophages, which seem to be the preferred host cells of salmonellae and listeriae5, it should be advantageous to target bacterial carriers to dendritic cells, which are best equipped for

antigen presentation when compared with mononuclear phagocytes14. After oral immunization of mice with S. typhimurium harboring a eukaryotic expression vector encoding green fluorescent protein, fluorescent dendritic cells were demonstrated, suggesting delivery of plasmid DNA to these cells9. Second, bacterial components such as LPS from Gram-negative bacteria and lipoteichoic acids from Gram-positive bacteria, as well as unmethylated sequence motifs in the bacterial DNA, could serve as adjuvants, thus potentiating the immunogenicity of DNA vaccines. Finally, bacterial carriers also have the advantage of being applicable via the oral route8, leading to transfection of cells of the gut-associated lymphoid tissue, which should promote efficient mucosal immune responses. Several approaches exist to enhance DNA delivery by intracellular bacterial carriers. The delivery by auxotrophic cell wall synthesis mutants relies on passive disintegration of the bacteria due to the lack of essential metabolites, however, active lysis of the carriers (e.g. by the expression of a phage lysin) could promote plasmid transfer. Whereas S. flexneri and L. monocytogenes transfer plasmid molecules directly to the host cell cytosol, the efficiency of plasmid delivery by bacterial carriers, which are usually confined to the phagosome, can be substantially enhanced by bacterial expression of an enzyme with phagosomal escape function, such as listeriolysin12. Therefore, Salmonella strains that enter the cytosol of infected host cells due to secretion of active listeriolysin10 are interesting candidates for DNA vaccine delivery. Despite the numerous advantages, the risk factors associated with the delivery of DNA vaccines by attenuated intracellular bacteria must be addressed carefully. These include safety of the bacterial carriers and possible genomic integration of the plasmid DNA. Furthermore, the broad applicability of these systems to a wide range of antigens and animal models awaits further investigation before application in humans can be envisaged. However, if these issues can be resolved, delivery of DNA vaccines by attenuated intracellular bacteria has the potential to combine the advantages of live vaccine vectors with those of naked DNA vaccines.

We thank M. Dietrich, B. Knapp and S. Spreng for critical reading of the manuscript.

Guido Dietrich ([email protected]. com) is at Preclinical Research Vaccines, ChironBehring, D-35006 Marburg, Germany; Ivaylo Gentschev and Werner Goebel are at the Dept of Microbiology, University of Würzburg, D97074 Würzburg, Germany; Jürgen Hess and Stefan Kaufmann are at the Max Planck Institute for Infection Biology, D-10117 Berlin, Germany; Jeffrey Ulmer is at Chiron Technologies, Chiron Corporation, Emeryville, CA, USA. References 1 Donnelly, J.J., Ulmer, J.B., Shiver, J.W. et al. (1997) Annu. Rev. Immunol. 15, 617–648 2 Ulmer, J.B., Deck, R.R., DeWitt, C.M. et al. (1996) Immunology 89, 56–67 3 Selby, M., Walker, C.M. and Ulmer, J.B. Exp. Opin. Invest. Drugs (in press) 4 Condon, C., Watkins, S.C., Celluzzi, C.M. et al. (1996) Nat. Med. 2, 1122–1128 5 Kaufmann, S.H.E. (1998) in Fundamental Immunology (Paul, W.E., ed.), pp. 1335–1371, Lippincott-Raven 6 Sizemore, D.R., Branstrom, A.A. and Sadoff, J.C. (1995) Science 270, 299–302 7 Sizemore, D.R., Branstrom, A.A. and Sadoff, J.C. (1997) Vaccine 15, 804–807 8 Darji, A., Guzman, C.A., Gerstel, B. et al. (1997) Cell 91, 765–775 9 Paglia, P., Medina, E., Arioli, I. et al. (1998) Blood 92, 3172–3176 10 Hess, J., Gentschev, I., Miko, D. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1458–1463 11 Courvalin, P., Goussard, S. and Grillot-Courvalin, C. (1995) C. R. Acad. Sci. 318, 1207–1202 12 Grillot-Courvalin, C., Goussard, S., Huetz, F. et al. (1998) Nat. Biotechnol. 16, 862–866 13 Dietrich, G., Bubert, A., Gentschev, I. et al. (1998) Nat. Biotechnol. 16, 181–185 14 Bancherau, J. and Steinman, R. (1998) Nature 392, 245–252

Trends If you would like to contribute to the Trends section of Immunology Today then please contact our Editorial Office at the following numbers: Fax 144 1223 464430; e-mail [email protected]

J U N E Vo l . 2 0

1 9 9 9 No.6

253