Tailoring host immune responses to Listeria by manipulation of ...

FEMS Immunology and Medical Microbiology 35 (2003) 243^253

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Tailoring host immune responses to Listeria by manipulation of virulence genes ^ the interface between innate and acquired immunity Christian Peters

b

a;b

, Eugen Domann c , Abdelhak Darbouche c , Trinad Chakraborty c , Martin E.A. Mielke b;d;

a Aventis Behring, Clinical Research and Development, 1020 First Avenue, King of Prussia, PA 19406, USA Institut fu«r Infektionsmedizin, Medizinische Mikrobiologie und Infektionsimmunologie, Universita«tsklinikum Benjamin Franklin, Freie Universita«t Berlin, Hindenburgdamm 27, 12203 Berlin, Germany c Institute for Medical Microbiology, Justus-Liebig University of Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany d Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany

Received 31 July 2002; accepted 2 October 2002 First published online 20 January 2003

Abstract Although attenuated strains of microbial pathogens have triggered vaccine development from its origin, the role of virulence factors in determining host immunity has remained largely unexplored. Using the murine listeriosis model, we investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes. We intentionally deleted specific genes of Listeria monocytogenes, including those encoding the positive regulatory factor (prfA), hemolysin (hly), the actin nucleator (actA), and phospholipase B (plcB). The resulting strains showed decisive differences in their immunogenic properties. In particular, we identified a double-deletion mutant that retained Listeria’s profound ability to induce protective CD8þ T cells, but that is strongly attenuated and exhibits a significantly reduced ability to induce CD4þ T cell-mediated inflammation. We conclude that this mutant, L. monocytogenes vactAvplcB, is at present the most promising mutant for a bacterial vaccine vector and is able to safely induce potent CD8þ T cell-mediated immunity. 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Vaccination ; Listeria; Virulence gene; Protective CD8þ T cell; Immunity

1. Introduction The Gram-positive facultative intracellular bacterium Listeria monocytogenes has been used for decades for the induction and analysis of T cell-mediated immunity [1^5]. These studies have led to extensive and detailed information on the host response to infection, and have triggered strong interest in bacterial virulence factors that allow entry into the host, support intracellular survival and facilitate dissemination of these bacteria during infection [6,7]. Studies of cell biology revealed that invading bacteria use

* Corresponding author. Tel. : +49 (30) 4547 2233 ; Fax : +49 (30) 4547 2612. E-mail addresses : [email protected] (C. Peters), [email protected] (M.E.A. Mielke).

active mechanisms to escape from the encapsulating phagosome, and that they can replicate in the cytoplasm of both professional phagocytes and parenchymal cells, such as hepatocytes. These studies have also explained the cellular basis of Listeria’s potent intrinsic capacity to induce major histocompatibility complex (MHC) class I-restricted protective CD8þ T cells [3,8,9]. This in turn resulted in the current interest in developing L. monocytogenes as a viable bacterial T cell vaccine vector as an alternative to Salmonella, which mainly induces CD4þ T cell responses [10]. Indeed, recombinant L. monocytogenes expressing foreign antigens has already been shown to be a highly e¡ective vector for the induction of speci¢c T cells active in the prevention of tumor growth and for protection against viral infections [11^21]. Recent studies showing that antivector immunity does not prevent the development of a primary cytotoxic T-lymphocyte re-

0928-8244 / 03 / $22.00 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-8244(02)00469-8

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C. Peters et al. / FEMS Immunology and Medical Microbiology 35 (2003) 243^253

sponse to a foreign antigen [22] have underlined its suitability. For increased safety, however, a more appropriate vaccine vector would be a well-characterized mutant that is reliably attenuated in virulence but that still retains the favorable immunological, i.e. CD8þ T cell-inducing properties of the parental strain. In addition, the accompanying CD4þ T cell response mediating delayed type hypersensitivity (DTH) and granulomatous in£ammation [8,23] should be downmodulated in order to prevent in£ammatory tissue destruction. Knowledge about the speci¢c effects of bacterial virulence genes in inducing and modifying the immune response would allow the rational tailoring of the vector, but is currently incomplete. To this end, we sought to identify Listeria strains that combine low virulence with the ability to induce potent T cell-mediated speci¢c protection but a reduced degree of DTH. We systematically examined the immunological properties of isogenic Listeria mutants lacking single or multiple virulence determinants : those with speci¢c deletions within the hemolysin (hly), phospholipase (plcB), and actin nucleator (actA) genes of L. monocytogenes. In addition, we examined a recombinant derivative of the naturally nonvirulent strain of Listeria innocua complemented with L. monocytogenes-derived virulence factors. Of these, only the double deletion mutant L. monocytogenes vactAvplcB elicited the desired immune response and is at present the most promising candidate for a Listeria-based bacterial vaccine vector.

2. Materials and methods 2.1. Mice Female C57BL/6 mice of 9^12 weeks of age were used for all experiments. They were raised in our own breeding facilities under speci¢ed pathogen-free conditions and were serologically screened to demonstrate absence of common viral pathogens. 2.2. Bacteria L. monocytogenes EGD, serovar 1/2a, parental strain for deletion mutants. This virulent mouse-adapted experimental strain is known to possess the following virulence factors: phosphatidylinositol-speci¢c phospholipase C (PlcA), hemolysin (Hly), metalloprotease (Mpl), actin-binding protein (ActA), phosphatidylcholine-speci¢c phospholipase C (PlcB), and internalins A and B. These virulence factors mediate entry into various cell lines, escape from the phagolysosome, intracellular motility, and the spread of bacteria in infected tissue [6,7,24]. L. monocytogenes EGD vactA2, serotype 1/2a. Isogenic deletion mutant lacking the actA gene as described previously [25].

L. monocytogenes EGD vactAv vplcB, serotype 1/2a. Isogenic deletion mutant lacking the actA gene and the plcB gene. In order to generate the vactAvplcB double mutant strain an 1193-bp-long PCR fragment was ampli¢ed using oligonucleotides 240 5P-CGGATCCAGAATTTAGTTCCGCAGTGG-3P (BamHI) and 7752 5P-ATCTTCGCTAGCGGCCGCTGCAAATATTATGTC-3P (NotI) and a second PCR fragment using oligonucleotides 7753 5P-AAAGGATACAGCGGCCGCTACTGGAGCTAGAC-3P (NotI) and 7751 5P-ATAACGGAATTCTAATGGTCACTG-3P (EcoRI). Both fragments were digested with endonuclease NotI and used in a ligation reaction. An entire PCR fragment of 2203 bp, lacking actA and plcB genes, was ampli¢ed using oligonucleotides 240 and 7751, digested with restriction enzymes BamHI and EcoRI and cloned into the BamHI and EcoRI restriction sites of the pAUL-A plasmid. Transformation of this pAULvactAvplcB plasmid into L. monocytogenes EGD and generation of chromosomal in-frame deletion of the actA and plcB genes was performed as described recently [26]. Finally, the ¢rst 30 amino acid residues of ActA and the last 25 amino acid residues of PlcB remained. The chromosomal deletion was con¢rmed by nucleotide sequencing and immuno blotting (data not shown). The prepared supernatant £uids of the wild-type strain EGD and the mutant vactAvplcB were developed with polyclonal antibodies directed against ActA and monoclonal antibody O85 which is speci¢c for PlcB. L. monocytogenes EGD vhly2 plus prfA. Isogenic deletion mutant lacking the hemolysin gene but containing additional copies of the prfA gene on plasmid pERL-3, leading to enhanced expression of all remaining virulence factors [27,28]. L. innocua transformed with the hly and prfA genes. Complementation of the avirulent species L. innocua with plasmid pERL-3 harboring the hly and prfA genes [28,29]. All deletions were con¢rmed by Western blot analysis as lack of expression of the respective protein (data not shown). All strains of bacteria were mouse-passaged prior to their use in order to stabilize their behavior in vivo [5]. Bacteria were grown from spleen homogenates in trypticase soy broth, harvested in exponential phase, dispensed in 0.5 ml aliquots, and frozen at 370‡C until needed. An inoculum of bacteria, depending on the type of mutant used, was prepared for i.v. injection by thawing an aliquot and appropriately diluting it in phosphate bu¡ered saline (PBS). 2.3. Bacterial antigens Two types of soluble antigen from L. monocytogenes were used. 1. Somatic soluble antigen was prepared by culturing L. monocytogenes in tryptic soy broth for 18 h, washing

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it in PBS, and subsequently subjecting it to ultrasonication. 1 g (wet weight) of bacterial cells was suspended in 10 ml of PBS and sonicated ¢ve times for 1 min each time (87.5% output, degree 7 on a soni¢er model S-125; Branson Sonic Power Co., Danbury, CT, USA) on ice. The sonicated suspension was centrifuged at 39 000Ug for 50 min, and the supernatant was ¢lter sterilized (pore size, 0.45 Wm) and stored at 320‡C at a dilution of 1:100 in PBS [5]. 2. Released soluble antigens from L. monocytogenes EGD were obtained through preparation of a culture ¢ltrate. For this, 500 ml of cell culture medium RPMI 1640 was inoculated with viable L. monocytogenes EGD and incubated for 16^24 h at 37‡C in 5% CO2 . The suspension was centrifuged at 1500Ug for 30 min, the supernatant passed through a sterile ¢lter (pore size 0.22 Wm), and the ¢ltrate stored at 320‡C. Optimal lymphocyte stimulation was achieved with these antigens at protein concentrations between 10 and 100 ng ml31 .

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by an intravenous injection of viable bacteria in a volume of 0.2 ml of PBS. Bacterial growth in spleens and livers was determined by plating 10-fold serial dilutions of the organ homogenates on tryptic soy agar. The detection limit of this procedure was 102 colony forming units (CFU) per organ. Colonies were counted after 24 h of incubation at 37‡C. For secondary infection, mice were injected with 106 listeriae on day 28 after primary infection [30]. 2.7. Histology For investigation of spleen and liver histomorphology mice were killed and spleen as well as one liver lobe were removed. Organs were ¢xed in 4% formalin/PBS, embedded in para⁄n, sectioned (3^4 Wm) and stained with hematoxylin and eosin according to standard procedures. 2.8. Reverse transcriptase (RT)-PCR-assisted ampli¢cation of host cell mRNA

2.4. Monoclonal antibodies (mAbs) mAbs were obtained from ascitic £uid from pristaneprimed nude mice injected i.p. with the relevant hybridoma cell line. The hybridoma lines 2.43 (anti-Lyt-2, rat IgG2b, and GK 1.5 [anti-L3T4, rat IgG2b]) were obtained from the American Type Culture Collection, Rockville, MD, USA (Tumor Immunology Bank [TIB] 107, 210, and 207). The mAbs designated 23-7 (unrelated speci¢city, rat IgG) was kindly provided by T. Diamantstein, Department of Immunology, Freie Universita«t Berlin, Berlin, Germany, and served as a control for nonspeci¢c e¡ects of ascitic £uid. Ascitic £uid was cleared and delipidated by centrifugation. The concentration of antibodies was determined by an enzyme-linked immunosorbent assay with speci¢city for rat IgG and calculated with a commercially available standard of rat IgG2b (catalog no. 1330, Becton Dickinson, Paramus, NJ, USA) as a reference. Dilutions containing 500 Wg of mAbs per ml in PBS were stored at 370‡C until use. All experiments were performed with portions of the same batch of the respective mAb preparation [5,30]. 2.5. Preparation of culture supernatants, SDS^PAGE, and immunoblotting Culture supernatants from the Listeria strains were prepared, separated by SDS^PAGE, and blotted onto nylon membranes as described previously. The corresponding immunoblots were developed with ActA- and PlcB-speci¢c polyclonal and mAbs, respectively [31,32]. 2.6. Experimental infection and determination of bacterial load in infected organs Primary infection with L. monocytogenes was performed

Ampli¢cation of cytokine mRNA has been done as described by Ehlers et al. [33,34]. Spleens of killed animals were removed in toto and homogenized in 5 ml lysis bu¡er, consisting of 4 M guanidine thiocyanate (Merck, Darmstadt, Germany), 1 M sodium citrate (pH 7, Sigma, Deisenhofen, Germany), 0,5% N-lauroylsarcosine (Sigma), and 1% 2-mercapto-ethanol (Sigma). Triplicate samples of a 1:5 dilution of this homogenate were frozen at 370‡C until total RNA preparation was performed using a phenol-chloroform extraction standard procedure. RNA precipitates were pelleted at 4‡C, washed once with 75% ethanol in diethylpyrocarbonate-treated distilled water (DEPC-dH2 O) and repelleted at 12 000Ug for 15 min. Vacuum-dried pellets were resuspended in 10 Wl DEPCdH2 O containing 0.2 Wg oligo-dT (12^18 mer, Sigma) and incubated at 65‡C for 10 min. After cooling on ice, the mixture was incubated with 10 Wl of RT-mixture (4 Wl 5U ¢rst strand bu¡er, 2 Wl 0,1 M dithiothreitol, 1Wl Superscript II RT, all from Gibco), 2 Wl 0.01 M dNTPs (cont. dATP, dCTP, dGTP, dTTP, Roth, Germany) and 1 Wl RNasin (40 U Wl31 , Promega) for 90 min at 42‡C. Tubes were then heated to 95‡C for 10 min, cooled on ice and 180 Wl of dH2 O was added to 20 Wl reaction mixture. Samples were stored at 4‡C or 320‡C until further use, respectively. RT reactions were always performed simultaneously for samples to be directly compared. Cytokine speci¢c 5P and 3P primer pairs complementary to sequences in di¡erent exons or spanned exon^exon junctions and therefore mRNA/cDNA speci¢c were designed (TIB MOLBIOL). 5 Wl of cDNA (representing 1/ 2000th of total spleen homogenate) was ampli¢ed in 0.5 ml GeneAmp reaction tubes (Perkin-Elmer, Norwalk, CT, USA) in the presence of 5 Wl primers at a ¢nal concentration of 50 nM, 0.5 U of Taq polymerase (InViTek, Berlin),

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and 2.5 Wl PCR bu¡er containing 160 mM (NH4 )2 SO4; 500 mM Tris^HCl (pH 8.0 at 25‡C), 0.1% Tween 20, 1.25 Wl 50 mM MgCl2 (kit from InViTek) and 0.5 Wl 10 mM dNTP in a ¢nal volume of 25 Wl. The reaction mixture was topped with a drop of light mineral oil and PCR was performed in a DNA thermal cycler (Perkin-Elmer) for 25 cycles: 30 s denaturation at 95‡C, 60 s annealing at 60‡C and 1 min extension at 72‡C. The reaction product was visualized by subjecting 20 Wl of the reaction mix to electrophoresis at 120 V for 60 min in 2% agarose in 0.5UTBE bu¡er with 1 Wg ml31 ethidium bromide; 1 Wg DNA-ladder (100 bp, Gibco-Life Technologies) was run in parallel as molecular mass markers. PCR-assisted mRNA ampli¢cation was repeated at least twice for at least two separately prepared cDNA samples ; data shown are representative of at least three di¡erent experiments yielding identical results. Samples run without RT showed no amplicon band (data not shown). L2 -microglobulin : 5P-TGACCGGCTTGTATGCTATC-3P; 5P-TGACCGGCTTGTATGCTATC-3P IL-1L L : 5P-CCCATACTTTAGGAAGACACGGATT-3P; 5P-TCATGGGATGATGATGATAACCTGCT-3P IL-2: 5P-TGATGGACCTACAGGAGCTCCTGAG-3P; 5P-GAGTCAAATCCAGAACATGCCGCAG-3P IL-2R: 5P-TCCTGGAGCAGCAACTGCCAGTGCA-3P; 5P-CTTATACTCCATTGTGAGCACAAATG-3P IL-3: 5P-GACCCTCTCTGAGGAATAAG-3P; 5P-CTCCAGATCGTTAAGGTGGA-3P IL-4: 5P-AGTAATCCATTTGCATGATGCTCTT-3P; 5P-ACAAAAATCACTTGAGAGAGATCAT-3P IL-6: 5P-CTGGTGACAACCACGGCCTTCCCTA-3P; 5P-ATGCTTAGGCATAACGCACTAGGTT-3P IL-10: 5P-ACCTGGTAGAAGTGATGCCCCAGGC-3P; 5P-CTATGCAGTTGATGAAGATGTCAAA-3P IL-12 P40: 5P-CCTCAGAAGCTAACCATCTCCT-3P; 5P-CAGCCATGAGCACGTGAACCGT-3P IL-15R: 5P-ATCTATTGAGCATGCTGACA-3P; 5P-GTCATTGGTACTGTTTCCAT-3P TNF-K K : 5P-ACACCCATTCCCTTCACAGAGCAAT-3P; 5P-AGCCCACGTCGTAGCAAACCACCAA-3P IFN-QQ : 5P-ATCAGCAGCGACTCCTTTTCCGCTT-3P; 5P-GAAAGCCTAGAAAGTCTGAATAACT-3P GM-CSF: 5P-GAAGAGGTAGAAGTCGTCTCTA-3P; 5P-GCTGCTTATGAAATCCGCATAGG-3P 2.9. DTH response to somatic listerial antigen For determination of DTH responsiveness, 50 Wl of somatic soluble L. monocytogenes EGD antigen diluted 1:100 in sterile PBS (¢nal protein concentration : 60 ng ml31 ) was injected into the left hind footpads of mice. Twenty-four hours later, thickness of the left and right footpads of individual mice were measured with dial-gauge calipers (Kro«plin, Schlu«chtern, Germany). DTH-induced footpad swelling was calculated by subtracting the mean di¡erences between left and right footpad thickness of in-

jected naive control mice from those of immune mice [5]. Non-speci¢c footpad swelling never exceeded 0.2 mm. 2.10. Stimulation of spleen cells in vitro for cytokine production Spleens of naive mice and of those immunized 7 or 28 days earlier were removed and used to prepare in vitro cell cultures. Single-cell suspensions were generated by injecting the spleens with PBS and dissociating the tissue mechanically. After spontaneous sedimentation of cell clusters, the single-cell suspension was ¢ltered through a 100Wm metal sieve and washed three times in culture medium RPMI 1640. Erythrocytes were lysed by osmotic shock. The cell count in trypan blue demonstrated over 95% viable cells. Cells were incubated on the basis of spleen equivalents (1U108 cells/5 ml) in 25 cm2 cell culture £asks in RPMI 1640, supplemented with 2 mM glutamine, 5U105 35 M 2-mercaptoethanol, and 5% fetal calf serum, as well as antibiotics (50 U ml31 penicillin, 50 Wg ml31 streptomycin). Lymphokine secretion was stimulated by released soluble antigens of L. monocytogenes EGD (concentration 10^100 ng). At the end of incubation, the cells were harvested and centrifuged in order to produce cellfree culture supernatant. The supernatant was passed through a 0.45 Wm ¢lter, fractionated, and stored at 370‡C until it was thawed only once before use in the various cytokine assays. 2.11. Quanti¢cation of cytokines in serum and cell culture supernatants Interferon-Q (IFN-Q) was detected in serum and the supernatants of splenocytes by using an ELISA system: 96-well ELISA plates (Nunc-Immuno Module, Maxisorp F16 ; Nunc, Wiesbaden, Germany) were coated with a rat anti-mouse monoclonal antibody to IFN-Q (Clone R46A2; ELISA Capture-Antibody ; 1 Wg ml31 diluted in 0.1 ml NaHCO3 , pH 8.2; 100 Wl per well) and incubated overnight at 4‡C. Plates were washed twice with PBS/ Tween (0.5 ml Tween 20 l31 PBS, ELISA washing bu¡er). Addition of bovine serum albumin (3% BSA in 200 Wl PBS; for 2 h at room temperature or at 4‡C overnight) prevented unspeci¢c binding. Plates were washed again twice with PBS/Tween and incubated with 100 Wg per well of either recombinant mouse IFN-Q (0.15^10 ng ml31 ) or splenocyte supernatants at 4‡C overnight. After thorough washing (four times, PBS/Tween) a detection antibody to IFN-Q (biotinylated rat anti-mouse mAb IFN-Q clone XMG 1.2; 1 Wg ml31 ) was added for 45 min at 37‡C in humidi¢ed air. Again plates were washed (six times, PBS/Tween). Binding of the rat antibody was detected by streptavidin horseradish peroxidase (1 Wg ml31 , 100 Wl well31 ; Amersham Life Science, Amersham/Buchler, Braunschweig, Germany) incubation for 30 min at 37‡C followed by eight washing steps. Substrate solution

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Fig. 1. Course of primary infection with L. monocytogenes EGD and Listeria mutants. Mice were infected with 103 CFU of L. monocytogenes EGD and with 107 CFU of the various Listeria mutants. On days 1, 2, 3, 5, 8 and 10 after infection, the numbers of CFU in the spleens and the livers of three animals per group were determined. Experiments were repeated three times. One representative experiment is shown. Standard deviation is not displayed for reasons of clarity. *P 9 0.05 (EGD vactA2 vs. EGD vactAvplcB or EGD vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA vs. EGD vactA2 and EGD vactAvplcB).

was 3,3P,5,5P-tetramethylbenzidine dihydrochloride in 0.05 M phosphate-citrate bu¡er (TMB tablets, 1 mg tablet31 , Sigma, Deisenhofen, Germany). 1 TMB-tablet was diluted in 10 ml bu¡er (cont. 25.7 ml of 0.2 M Na2 HPO4 , Merck, Darmstadt, Germany, and 0.1 M citric acid, Sigma, Deisenhofen, Germany) in 50 ml triple-distilled water, pH 5.0 and 2 Wl 30% hydrogen peroxide solution (Merck) added just before use. 50 Wl H2 SO4 per well (Merck) stopped the reaction. Optical densities of the end products were compared with a corresponding standard curve for IFN-Q concentrations to determine the units ml31 in supernatants. Detection limit was 0.15 ng ml31 . 2.12. In vivo depletion of T cell subsets In vivo depletion of T cell subsets was carried out by i.p. administration of 500 Wg puri¢ed mAb (GK 1.5 = antiL3T4 and/or 2.43 = anti-Lyt-2 as well as an irrelevant control mAb) 3 days prior to primary or secondary infection, as described previously [5,30]. This protocol consistently resulted in the reduction of the targeted subsets to 6 1.0%, using £uorescence-activated cell sorting analysis of total spleen cells. 2.13. Statistics Data were statistically analyzed with the non-parametric Mann^Whitney U-test. P values lower than or equal to

Fig. 2. Detection of cytokine-speci¢c mRNA in the spleen 3 days after infection with L. monocytogenes and the various Listeria mutants. Mice were infected with 103 CFU of L. monocytogenes EGD or 107 CFU of the various Listeria mutants. Organs were removed 3 days after infection, homogenized in guanidine thiocyanate bu¡er and total RNA was used for RT-PCR to evaluate the expression of cytokine mRNA. Polaroid photographs of ethidium-bromide-stained agarose gels are shown. PCR-assisted mRNA ampli¢cation was repeated three times for at least two separately prepared cDNA samples; data shown are representative of at least three di¡erent experiments yielding identical results.

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d2 d3

d2 d3

d2 d3

d2 d3

d2 d3

Fig. 3. IFN-Q concentration in the serum of animals infected with Listeria strains. Mice were infected with 103 CFU of L. monocytogenes EGD or 107 CFU of the various Listeria mutants. On day 3, animals were killed and blood was obtained by heart puncture. Serum concentration of IFN-Q was determined by ELISA. Mean values of mice used in the experiment presented in Fig. 1 are shown.

0.05 were considered to indicate statistically signi¢cant di¡erences.

3. Results 3.1. Growth kinetics of L. monocytogenes EGD wild-type strain and various Listeria mutants in murine spleen and liver The ¢rst and crucial step in the in vivo characterization of the various mutants was the determination of their growth kinetics in spleen and liver. After the intravenous injection of various sublethal doses of each of the Listeria mutants (1U104 ^1U107 CFU per mouse) into separate groups of mice, the numbers of bacteria in the spleen and liver of infected mice were determined on days 1, 2, 3, 5, 8 and 10 after infection. The third day after infection is the last day prior to, and the day that is the most critical for, the induction of the T cell response [33,43]. For all subsequent analyses, therefore, an inoculum of the various strains was aspired that resulted in a bacterial load at least as high as the one present on day 3 of the typical sublethal experimental infection (1U103 CFU per mouse) with the virulent wild-type strain of L. monocytogenes EGD (Fig. 1). This, however, could not be achieved even with high inocula of strains EGD vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA. The growth kinetics of the wild-type strain of L. monocytogenes EGD di¡ered from that of all mutants. Even though the inoculum was smaller, the number of wildtype bacteria increased between day 1 and day 3, while for all mutants the number of bacteria decreased between day 1 and day 3. On day 3, the number of wild-type bacteria peaked at the same level as the bacterial load observed for the mutants EGD vactA2 and EGD vactAvplcB on this day. After day 5, the number of viable

wild-type bacteria declined. In all groups bacteria were completely cleared from the spleen and liver by day 10 of the infection. Strain EGD vactAvplcB was cleared from the spleen more rapidly than EGD vactA2 after day 3 of infection. In the liver, however, both mutants persisted at a constant level for 5 days, after which they began to be eliminated with kinetics that paralleled those of the wild-type strain. In contrast, regardless of the dose of infection, the mutants EGD vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA were rapidly cleared from both organs from the outset. On day 3 p.i., bacterial load of the latter had already fallen to levels of approximately 103 ^104 CFU in liver and spleen even after infection with 107 viable bacteria per mouse. 3.2. Spleen morphology on day 3 after infection Because the spleen is the lymphatic organ where the immune response is initiated following an i.v. inoculum, we conducted a histomorphological analysis of this organ on day 3 after infection. Infection with the various bacterial strains resulted in marked di¡erences in the intensity of leukocyte in¢ltration of the white pulp in the spleen (data not shown). The wild-type of L. monocytogenes induced the most vigorous in¢ltration, whereas EGD vactA2 showed reduced, and EGD vactAvplcB only subtle leukocytic in¢ltration of the white pulp. Infection with the mutants EGD vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA did not result in histomorphological changes on day 3 after infection. 3.3. Cytokines present on day 3 after infection The Listeria-induced cytokine pattern was investigated by means of PCR-assisted mRNA-ampli¢cation in organ homogenates from infected animals. In the spleen, mRNA encoding IL-6, IL-10, IL-12 and IFN-Q could be detected only after infection with the wild-type strain of L. monocytogenes and the vactA2 and vactAvplcB mutants. Infec-

8 days p.i

42 days p.i

Fig. 4. Listeria-induced IFN-Q production by spleen cells 8 and 42 days after infection (p.i.). Mice were infected with 103 CFU of L. monocytogenes EGD or with 107 CFU of the various Listeria mutants. On days 8 and 42 after infection, mice were killed and spleens removed. Single cell suspensions were stimulated in vitro with secreted soluble Listeria antigen to produce IFN-Q. After 48 h, culture supernatants were tested for presence of IFN-Q by ELISA.

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the observations obtained in vitro were con¢rmed in vivo by antigen-elicited skin responses showing corresponding results (Fig. 5). 3.5. Expression of acquired immunity in normal and T cell subset-depleted mice

Fig. 5. DTH response to listerial antigen after infection with various Listeria mutants on day 8. Mice were infected with 103 CFU of L. monocytogenes EGD or 107 CFU of the various Listeria mutants. Eight days after infection, DTH was triggered through injection of soluble somatic listerial antigen. Twenty-four hours later, speci¢c skin response was determined. Experiments were repeated twice. The mean value U S.D. of ¢ve animals of a representative experiment is shown. *P 9 0.05 (EGD vactA2 vs. EGD vactAvplcB).

tion with L. monocytogenes EGD vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA did not give rise to mRNA encoding these cytokines. In addition, only L. monocytogenes induced detectable amounts of mRNA for GM-CSF (granulocyte macrophage colony stimulating factor). Neither in the liver (data not shown) nor in the spleen of any of the animals was mRNA speci¢c for IL-2, IL-3 or IL-4 detected on day 3 after infection Fig. 2. To complement the PCR-assisted cytokine analysis, we performed a quantitative analysis of Listeria-induced IFNQ production. Infection with the wild-type strain and with the vactA2 and vactAvplcB mutants resulted in detectable but varying levels of IFN-Q in the serum of infected animals. The wild-type strain induced the highest serum levels of IFN-Q, followed by the vactA2 mutant. The double deletion mutant vactAvplcB induced the lowest levels of IFN-Q (Fig. 3). No IFN-Q was detected in sera on day 3 after infection with the vhly2 plus pERL-prfA mutant of L. monocytogenes and the L. innocua plus pERL-hly/prfA strain.

Currently, the only way to demonstrate the induction of protective CD8þ T cells in listeriosis is by secondary infection of T cell subset-depleted mice. Therefore, we immunized groups of mice with the mutants under study, and then challenged them with a secondary infection of 100ULD50 of the wild-type strain. Sixty hours after the challenge, we measured bacterial loads in organs. Immunization with the vactA2 or vactAvplcB mutant conferred antibacterial protection as potent as that observed in animals immunized with the wild-type strain (Fig. 6). The immunization with the vhly2 plus pERL-prfA strain of L. monocytogenes or L. innocua plus pERL-hly/prfA did not result in any protection so that these animals succumbed to the challenge infection. In successfully immunized animals, depletion of CD4þ T cells did not impair

3.4. Listeria-induced IFN-Q production of spleen cells in vitro and DTH-response in vivo Antigen-induced CD4þ T cell-derived IFN-Q production of spleen cells was measured as an in vitro correlate of DTH to determine whether immunization with the various Listeria mutants may lead to quantitative di¡erences in the proin£ammatory T cell response. Day 8 after infection was chosen for the analysis of the primary immune e¡ector phase, and day 42 after infection was chosen to test memory e¡ector functions. Spleen cells from mice immunized with the L. monocytogenes vactAvplcB mutant produced signi¢cantly lower levels of IFN-Q when compared with the results after immunization with the wild-type strain or vactA2 mutant. The EGD vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA strains failed to prime T cells for the production of IFN-Q (Fig. 4). Most notably,

Fig. 6. E¡ect of T cell subset depletion on the elimination of bacteria from spleens and livers in the course of a secondary infection with L. monocytogenes EGD. Mice were immunized with 103 CFU of L. monocytogenes EGD or with 107 CFU of the various Listeria mutants, respectively. Three days prior to re-infection (28 days p.i.), the animals were treated with 500 Wg of CD4-speci¢c mAb or a mixture of 500 Wg CD4- and 500 Wg CD8-speci¢c mAb. The animals were killed 60 h after re-infection and spleen and liver were removed to determine the number of CFU in the organs. The experiments were repeated three times. Mean value U S.D. of three animals each of a representative experiment is shown. *P 9 0.05 (KCD4/8 vs. KCD4). ‘2’, all of the mice expired.

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the animals’ ability to eliminate the bacteria, while depletion of both CD8þ and CD4þ T cells severely impaired acquired immunity.

4. Discussion In this study we show that selective virulence gene depletion can generate mutant bacteria of the genus Listeria that retain the marked protective immunogenic potential of the wild-type strain but are attenuated in virulence as well as in their capacity to induce CD4þ T cell-mediated in£ammation. Four Listeria deletion and/or complemented mutants in addition to the wild-type strain were assessed: L. monocytogenes EGD vactA2, vactAvplcB and vhly2 plus pERL-prfA, and L. innocua plus pERL-hly/ prfA. The Listeria mutant vhly2 plus pERL-prfA lacks the gene for hemolysin, which is essential for Listeria’s escape from the phagolysosome [6,35]. Its additional copies of the positive regulation factor A (prfA) on plasmid pERL-3 result in overexpression of all other PrfA-regulated virulence factors [6,28,36]. Because hemolysin is known to be critical for the induction of T cell-mediated immunity [37^ 39], we selected this mutant to determine whether increased production of the remaining virulence factors regulated by PrfA can compensate for the absence of hemolysin [40]. In contrast, the recombinant L. innocua plus pERL-hly/prfA is an avirulent species supplemented with genes for hemolysin and prfA on plasmid pERL-3 [29]. This mutant was chosen to determine whether expression of hemolysin alone, in a bacterium otherwise free of known virulence factors, could su⁄ce to induce T cellmediated immunity. The mutant EGD vactA2 is identical to the wild-type, except that it lacks the entire actA gene, crucial for actin polymerization, and thus it lacks Listeria’s intracellular actin-based motility [25]. The double deletion mutant EGD vactAvplcB lacks both the actA gene and the gene for phosphatidylcholine-speci¢c phospholipase (plcB) from the lecithinase operon, which impairs mechanisms by which Listeria escapes from host cell-derived vacuoles [41]. These mutants were chosen because they lack genes associated with discrete steps in the infectious intracellular lifestyle of Listeria following escape from the endosomal compartment, namely intracellular motility and cell-to-cell spread but still can produce hemolysin. We selected our investigation parameters to correspond to critical steps of the host response during (i) the preimmune phase, (ii) the primary immune e¡ector phase, and (iii) the memory immune e¡ector phase of the infection [4]. Day 3 of a Listeria infection marks the end of the pre-immune phase, i.e. it is the last day before the expansion of speci¢c T cells in this model [33,42]. The presence of viable bacteria on this day has been shown to be critical for the successful induction of T cell-mediated immunity

[43]. On day 3, therefore, we investigated (i) bacterial load, (ii) histomorphology, and (iii) the pattern of cytokine mRNA expression in the spleen and the liver as well as serum levels of IFN-Q. Day 8 corresponds to the primary immune e¡ector phase, that is, acquired resistance. On this day, we measured DTH to soluble antigen in vivo as well as T cell-derived IFN-Q production by spleen cells, known to be an in vitro correlate of DTH reaction and a measure of CD4þ T cell activity [5]. Day 42 p.i. was selected to investigate acquired immunity using the parameters mentioned above. In addition, we measured bacterial load in spleens and livers in secondarily infected but T cell subsetdepleted mice in order to determine the contribution of CD4þ and CD8þ T cells to protective immunity [8]. Bacterial growth kinetics in vivo indicate not only the virulence of a bacterium but are also associated with the induction of T cell-mediated host responses [44]. Experiments that temporally abrogated Listeria infection with antibiotics [43] have revealed that the persistence and number of viable microorganisms are important parameters for e⁄cient induction of T cell-mediated immunity. Accordingly, the growth kinetics of the various investigated mutants in the spleen can be divided into two groups. While the vactA2 and vactAvplcB mutants were present in substantial numbers for at least 3 days, the strains vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA were rapidly eliminated from day 1 onwards even when high inocula were used for infection/vaccination. Within the ¢rst group, di¡erences ¢rst appeared on day 5, when the mutant vactAvplcB was present in lower numbers in the spleens of infected animals. In the liver, both vactA2 and vactAvplcB mutants persisted for at least 5 days before the total number of bacteria declined. As in the spleen, the bacterial numbers in the livers of the mutants vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA began to fall immediately after infection. The di¡erences in bacterial eradication between the vactA2 and vactAvplcB mutants suggest that PlcB may be involved in Listeria’s resistance against the T cell-induced bactericidal mechanisms in professional phagocytes, the major host cells in the spleen, whereas its role for survival may be negligible in the liver, where less welldefended (permissive) parenchymal cells are also infected [45]. Studies of the histomorphology of infected spleens have shown that the wild-type strain and the mutants vactA2 and vactAvplcB induce monocytic in¢ltrations of the white pulp. The degree of in¢ltration on day 3 p.i., however, was shown to be most intensive with the wild-type strain of L. monocytogenes, intermediate with the mutant EGD vactA2, and barely detectable after infection with the double deletion mutant vactAvplcB in spite of similar bacterial load. The mutants vhly2 plus pERL-prfA and L. innocua plus pERL-hly/prfA did not induce any detectable changes in spleen morphology. The intensity of the histomorphological alterations in the spleen paralleled the

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level of Listeria-induced DTH responses. No such correlation was observed in the liver, where the wild-type strain, vactA2, and vactAvplcB mutants all produced signi¢cant in£ammatory lesions. PCR-assisted analysis of the cytokine mRNA patterns on day 3 p.i. showed that only vaccination with mutants with the capacity to induce T cell-mediated immunity, i.e. the wild-type strain and the vactA2 and vactAvplcB mutants, resulted in appreciable levels of type 1 cytokine transcripts like IL-12 and IFN-Q as well as the IL-2 and IL-15 receptors. This ¢nding was also supported by quantitative di¡erences in the amount of IFN-Q in sera from mice on day 3 of infection. In addition, the levels of IFN-Q in the supernatants of spleen cell cultures from immunized mice followed the same pattern. In accordance with data presented by Yang et al. [46], IFN-Q turned out to be a most helpful in vivo and in vitro parameter predicting the strength of the proin£ammatory, CD4þ T cell-mediated immune response to the bacteria. Only the wild-type strain and the mutants EGD vactA2 and vactAvplcB that displayed the ability to induce T cell-mediated immunity induced detectable levels of IFN-Q in sera and in spleen cell supernatants of infected animals. Recent advances in techniques to investigate the speci¢c T cell response, in particular of the CD8þ subpopulation with peptide loaded tetrameric MHC, intracellular cytokine staining and the use of transgenic animals [53^ 57,62,63] have provided tremendous insight as to how the speci¢c immune response is mounted. It is the elegant work by Pamer’s group that also revealed, that priming of CD8þ T cells by heat-killed bacteria must be di¡erentiated from di¡erentiation into e¡ector cells [54]. Therefore, the most reliable approach to assess the complex ability of T cells to protect animals against a lethal dose of microorganisms remains the demonstration of the in vivo function of T cells by the e¡ect of T cell subset depletion (CD4þ and CD4þ plus CD8þ ) in previously immunized and subsequently challenged animals [5]. In contrast to animals immunized with mutants unable to induce a DTH response, all animals immunized with L. monocytogenes EGD, the vactA2 or vactAvplcB mutants were protected against 100ULD50 of virulent Listeria, despite signi¢cant di¡erences in their DTH responses. T cell subset depletion revealed that this protection was CD4þ T cellindependent, but that it was severely impaired when both CD4þ and CD8þ T cells were depleted. As a result and in accordance with the literature [6,35,38,39,47^49], hemolysin has been shown as absolutely necessary not only for survival of the bacteria but also for the induction of both DTH and protection mediating T cells. All other virulence factors, even if over-expressed, cannot compensate for the absence of hemolysin as may be suspected from the work of Marquis and coworkers [50]. Most noteworthy, the ability to produce hemolysin is not su⁄cient to convert L. innocua into a protection inducing bacterial vector.

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The expression of plcB has been shown to increase resistance to the bactericidal mechanisms of macrophages, which are the major host cells in the spleen [50,52]. Consequently, its absence may lead to earlier eradication from and less vigorous stimulation of the infected macrophages resulting in the reduced production of T cell co-stimulatory molecules. In the liver, in contrast, cell-to-cell spread between permissive (MHC class I-expressing) hepatocytes may occur even in the absence of ActA and PlcB, e.g. via the release of bacteria from degrading parenchymal cells. This may explain the similarities between strains vactA and vactA/vplcB with respect to growth kinetics in the liver and the ability to induce protective CD8þ T cells. The delineation of its immunological properties shows that the doubly attenuated L. monocytogenes vactA/vplcB retains the favorable immunological properties of the parental strain and ful¢lls our posited criteria for a suitable live bacterial T cell vaccine vector. Recently a comparable Listeria mutant has been investigated in an initial clinical safety study as a prototype L. monocytogenes-based vector in adults. The results demonstrate the safety of this attenuated L. monocytogenes strain when studied in healthy adult volunteers [51]. In addition, recent reports suggest that L. monocytogenes as a live vaccine vector can induce T cell-mediated immune responses in models of antiviral immunity and tumor regression [58^61]. In conclusion, this mutant appears to be the Listeria vector of choice because of its immunological properties and its safety pro¢le.

Acknowledgements The work presented herein was made possible by grants from the German^Israeli Foundation for Scienti¢c Research and Development (GIF No. I-314-169) to M.M. and T.C. and the Deutsche Forschungsgemeinschaft to E.D. (SFB 535). The authors thank U. Ru«schendorf and A. Finke for expert technical help.

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