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Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Correspondence
Fred R. Frankel
frankelf{at}mail.med.upenn.edu
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pamer, 2004).
The merit of L. monocytogenes as a potential vaccine vector results from the close interface of its unique life cycle and the cellular immune system of its host. The micro-organism can enter phagocytic cells through Fc receptors or type I macrophage scavenger receptors (Dunne et al., 1994; Suzuki et al., 1997) and can invade non-phagocytic cells using the bacterial surface proteins InlA and InlB (Gaillard et al., 1991, 1996). Although the majority of engulfed organisms are killed in the phagosomal compartment of phagocytes (de Chastellier & Berche, 1994), a small fraction of the organisms escape that vacuole by means of the virulence factors listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC) (Portnoy et al., 1988; Smith et al., 1995) and colonize the host cell cytosol. Proteins secreted by the organism in this milieu can be accessed directly by the MHC class I pathway of antigen processing and presentation (Ada, 1990; Braciale et al., 1987). As a result, mice infected with a sublethal dose of wild-type bacteria develop long-lasting protective immunity, mediated predominantly by CD8+ T-cells, with little production of antibody (Finelli et al., 1999; Kaufmann, 1993; Pamer, 2004). Since the natural route of infection by L. monocytogenes is by ingestion of contaminated foods, these infections activate the mucosal immune system (Marzo et al., 2002).
These properties of L. monocytogenes have made it attractive as a potential live vaccine vector, and recombinant strains expressing foreign antigens have successfully protected mice against infection with lymphocytic choriomeningitis virus (Goossens et al., 1995; Shen et al., 1995), Mycobacterium tuberculosis (Miki et al., 2004), papilloma virus (Jensen et al., 1997; Kadish & Einstein, 2005) and influenza virus (Ikonomidis et al., 1997) and against tumour challenge (Brockstedt et al., 2005; Bruhn et al., 2005; Gunn et al., 2001; Yoshimura et al., 2006). Nevertheless, several issues, such as anti-vector immunity and safety, need to be addressed before L. monocytogenes can be considered as a vaccine vector for human use. Unlike the situation with viral vectors, existing antilisterial immunity does not appear to diminish the therapeutic capacity of recombinant L. monocytogenes (Bouwer et al., 1999; Starks et al., 2004; Stevens et al., 2005). However, safety remains an important concern, since wild-type L. monocytogenes poses a serious risk to neonates, infants, pregnant women, the elderly and immunocompromised individuals (Gellin & Broome, 1989). An ideal vaccine strain of L. monocytogenes should be attenuated and avirulent, but still retain immunogenicity. We previously generated a conditional lethal strain, L. monocytogenes daldat (Lmdd), attenuated by virtue of deletions in two genes necessary for synthesis of D-alanine, a rare amino acid required for peptidoglycan and lipoteichoic acid formation (Thompson et al., 1998). Brief administration of D-alanine at the time of immunization was adequate to allow minimal but sufficient bacterial replication for the induction of an Lm-specific immune response in mice.
To obviate the dependence on exogenous D-alanine without significantly compromising the safety associated with the original strain, we developed several new second-generation attenuated strains of D-alanine-independent Lmdd. These strains carried either an IPTG-inducible or a recombinase-sensitive Bacillus subtilis racemase (dal) gene whose product could transiently complement the D-alanine deficiency of Lmdd (Li et al., 2005; Zhao et al., 2005). In this study we describe a different attenuation system that combines a truncated racemase gene promoter with an ssrA tag at the 3' terminus of the B. subtilis dal gene. The ssrA tag encodes a short peptide sequence at the C-terminus of the nascent racemase chain, which can stimulate its proteolysis by C-terminal-specific proteases (Gottesman et al., 1998; Karzai et al., 1999; Keiler et al., 1996). This system resulted in an attenuated strain that retained good immunogenicity, without requiring D-alanine administration.
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METHODS |
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). In order to use standardized and known quantities of bacteria, many experiments were performed with thawed cultures prepared from 3–4 h growth of 1 : 10- or 1 : 20-diluted overnight cultures. Bacteria were stored at –80 °C in BHI. E. coli strains used for molecular cloning were grown in Luria–Bertani (LB) medium at 30 °C with agitation. Antibiotics were used at the following concentrations: ampicillin, 100 µg ml–1; chloramphenicol (Cam), 10 µg ml–1; streptomycin (Sm), 50 µg ml–1. Tissue culture cells were grown in Dulbecco's modified Eagle medium (DMEM; Mediatech) supplemented with 10 % fetal bovine serum and 2 mM L-glutamine (Mediatech) at 37 °C in a 5 % CO2/air atmosphere. Plasmid pKSV7-Bsdal contains the B. subtilis racemase gene (dal) with 551 upstream base pairs.
Construction of plasmids with the B. subtilis dal gene containing various lengths of upstream region, –551, –80, –48 and –18 bp, before the racemase start codon.
The structures of plasmids are shown in Fig. 1. The B. subtilis dal gene with 551 bp upstream region was amplified from pKSV7-Bsdal using the 5' primer 5'-GCTCTAGAGCTTTGAATTTAATAAACAATTTG-3' (XbaI site underlined here and in the following) and 3' primer 5'-GCGTCTAGATTATTATGCATAATCTGGAACATCATATGGATAATTGCTTATATTTACCTGCAATAAAGG-3' (HA epitope in bold) to yield a 1772 bp product containing the long upstream region, dal coding sequence, an HA (YPYDVPDYA) tag, a downstream translation termination sequence, and XbaI sites at the 5' and 3' termini. Ligating the XbaI-digested B. subtilis dal gene fragment into the XbaI site of shuttle vector pKSV7 generated the plasmid designated pK551. A PCR product with an ssrA tag added onto the 3' end of HA on the B. subtilis dal gene was generated using the earlier 5' primer and 3' primer 5'-GCGTCTAGATTATTAGGCAGCGAAAGCTAGGTTTTGTTTTTCTTTGCCAGTTGCATAATCTGGAACATCATATGGATAATTGC-3' (ssrA italicized), and ligated into pKSV7 to form pK551S. In like manner, additional constructs in pKSV7 were pK80 (5' primer 5'-GCTCTAGACGTTAGACATCGTTTCCCTTAGC-3'), pK80S, pK48 (5' primer 5'-GGGTCTAGATTAGCATGATATGTAAATG-3'), pK48S, pK18 (5' primer 5'-GCGTCTAGAAAGCTAGGAAGTGTCGTAATG-3') and pK18S. Those constructs that carried the ssrA tag were generated using the 3' primer above that contained both the HA and ssrA sequences. Those without ssrA used the 3' primer above that expressed HA only. Constructs prepared in plasmid pAM401 were designated pA551, pA551S, pA80 and pA80S.
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In vitro growth.
For uniformity of growth, retention of plasmid and to generate equivalent starting cultures for most experiments, Lmdd constructs were routinely grown overnight in BHI+Cam+D-alanine liquid medium. One millilitre of overnight culture was added to 19 ml of BHI+/–Cam broth, adjusted to the same OD600 value, aliquoted into multiple 1 ml cultures, and grown at 30 °C. Wild-type Lm+ and Lmdd in the presence of D-alanine (200 µg ml–1) were controls. At various times, samples were taken for OD600 measurement.
Plasmid stability in vitro.
One millilitre of overnight culture was added to 19 ml BHI+/–Cam broth, and grown to OD600 0.8 at 30 °C. Bacteria were counted on BHI+/–Cam and BHI+D-alanine agar plates. The fraction of bacteria with plasmid was calculated as the percentage of those that were D-alanine-independent (growth on bhi plates) relative to the total number of viable bacteria (growth on bhi+D-alanine plates).
RNA and DNA isolation.
Total RNA from strains of L. monocytogenes was prepared after suspending bacteria in RNAprotect Bacteria Reagent (Qiagen), lysozyme lysis, proteinase K digestion, and isolation using the RNeasy mini-kit (Qiagen) according to the manufacturer's protocols. RT controls were performed to assure the complete removal of genomic DNA. For isolation of total genomic DNA plus plasmid DNA, cells were treated with lysozyme, proteinase K and RNase, and then extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol.
Real-time PCR.
cDNA was synthesized using the Superscript First-Strand Synthesis System kit according to the manufacturer's suggested protocol (Invitrogen). Two microlitres of cDNA was used in a 25 µl reaction mixture for real-time PCR amplification with Power SYBR Green PCR Master Mix (Applied Biosystems). The reactions were performed using an iQ 5 Cycler Real-Time PCR detection system (Bio-Rad). Reaction conditions were optimized and data were analysed using iQ5 Optical system software. Primers were designed to amplify a 186 bp sequence from the racemase-encoding dal gene of B. subtilis and a 182 bp sequence from the hly gene of L. monocytogenes. The dal gene forward primer was 5'-AATTGAAAGGGACCGACATC-3' and reverse primer, 5'-TTAATGGTTTCGAGCCTTCC-3'. The internal control hly gene primers were 5'-GCAAGCTAGCTCATTTCACATC-3' and 5'-ATTTCGGATAAAGCGTGGTG-3'. dal gene amplification began with 3 min at 95 °C followed by 45 cycles of 95 °C for 10 s, 59.6 °C for 30 s and 78 °C for 6 s; hly gene amplification was 95 °C for 3 min followed by 45 cycles of 95 °C for 10 s, 58.4 °C for 30 s and 75 °C for 6 s. Melt curves were performed to ensure detection of the correct product. Products were initially analysed by agarose gel electrophoresis. Threshold cycles (CT) (PCR cycles at which fluorescence first accumulates above background) were determined for each amplification. The CT values from serial dilutions of cDNA were plotted against log input of the genomic DNA copies isolated from our standard control, Lmdal– dat– Bsdal+, to generate a standard curve. Unknown samples were quantified by comparison of their CT values with that curve.
Plasmid copy number in cells of the different Listeria constructs was determined using real-time PCR to measure the number of plasmid-bearing dal genes in preparations of total genomic DNA, relative to the number of hly chromosomal genes detected in those same preparations. We assumed that a single Listeria chromosome was present in each of the cells in our cultures.
D-Alanine determination.
D-alanine pools were examined by a spectrophotometric method that coupled the oxidative deamination of D-alanine by the enzyme D-amino acid oxidase to the reduction of the resulting product, pyruvate, in the presence of lactic dehydrogenase and NADH (Bergmeyer & Grassl, 1983). Catalase was present to prevent the alternative conversion of pyruvate to acetic acid and carbon dioxide by accumulated hydrogen pyroxide. The reaction was linear from 10 µg ml–1 to 500 µg ml–1 D-alanine. Prior to assay, cultures (20–100 ml) at OD600 0.8 were concentrated to 1 ml, lysed with lysozyme and three freeze–thaw cycles, and then extracted with perchloric acid followed by neutralization with potassium bicarbonate to remove protein.
Detection of B. subtilis racemase.
Bacteria were grown from overnight cultures in BHI+Cam+D-alanine medium at 30 °C and samples were collected at various times for electrophoresis. Equivalent amounts of bacteria, based on total protein analysis (Dc protein assay; Bio-Rad), were lysed using lysozyme at 37 °C for 1 h as described elsewhere (Zhao et al., 2005). Samples were electrophoresed in SDS-PAGE gel, and transferred to Immun-Blot PVDF membrane (Bio-Rad). Anti-HA monoclonal antibody was the primary antibody (Roche), followed by sheep anti-murine IgG linked with horseradish peroxidase (Amersham Biosciences). Bands were detected with the ECL Western Blotting Analysis System kit (Amersham Biosciences).
Analysis of plaque formation on L2 fibroblasts.
Assays of plaque formation on mouse L2 fibroblast cell monolayers were performed as previously described (Sun et al., 1990), with some modification. Briefly, L2 cell monolayers were grown to confluence in six-well tissue culture plates. Approximately 1x106 bacteria from a frozen stock culture were used to infect the monolayers for 1 h in DMEM. Monolayers were washed three times with PBS, and a DMEM/0.7 % agar overlay containing 10 µg gentamicin ml–1 was added. Plates were incubated for 3 days to allow plaque formation. At day 4 the overlay was removed, and the cells were fixed in PBS/10 % paraformaldehyde for 60 min, stained with 0.1 % crystal violet/20 % ethanol for 5 min, washed and air-dried. The diameter of plaques in the monolayers was measured after 10x amplification.
Bacterial proliferation in spleen.
To examine the virulence of Lmdd constructs in mice, animals were infected intravenously (i.v.) with 0.2 LD50 of bacteria (i.e. 1.4x107 of Lmdd in the presence of D-alanine, 4x106 of Lmdd/pA80S or 2x103 of Lm+). Viable bacteria in spleen were determined at days 1, 3, 5 and 7 post-infection. The organ was homogenized in 3 ml Hanks' buffered salt solution, and splenocytes were lysed in H2O and dilutions plated on BHI+D-alanine+Sm or BHI+Sm. Since the wild-type strain from which Lmdd was derived is streptomycin-resistant, inclusion of this antibiotic allowed selection against possible contaminant organisms.
Primary T-cell responses and protection induced by Lmdd/pA80S.
Six- to eight-week-old BALB/c (H-2d) female mice were infected i.v. with 2x103 of Lm+, 1.4x107 of Lmdd +/–20 mg D-alanine, or 4x106 of Lmdd/pA80S. At 8 days after infection, spleens were homogenized as above, splenocytes were collected, red blood cells removed with ACK lysis buffer, and the washed lymphocytes stained with anti-CD8a-FITC, anti-CD11a-PE (eBiosciences) and LLO91–99-MHC I tetramers labelled with APC. IFN-
intracellular staining was performed according to methods described elsewhere (Pearce et al., 2004). Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson) and the data were analysed using FlowJo software (Tree Star Inc.). For the protection studies, mice were infected as above with 0.2 LD50 of each strain, challenged 21 days later with 2x104 wild-type L. monocytogenes, and sacrificed at day 3 postchallenge. Spleens and livers were removed, homogenized as above, and bacterial content enumerated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To obviate this requirement for an external source of the amino acid and to produce a potentially more useful vaccine vector, we generated several plasmids that carried the B. subtilis dal gene; this gene encodes racemase, one of the two D-alanine-synthesizing enzymes used by L. monocytogenes. Each plasmid carried a dal gene with a different length of upstream region: 551 bp, 80 bp, 48 bp or 18 bp. The dal genes were also modified by addition of an ssrA tag at the 3'-terminus. These dal gene sequences were inserted into two plasmids used for gene transfer in L. monocytogenes: pKSV7, a shuttle vector with a temperature-sensitive origin of replication (Smith & Youngman, 1992), and pAM401, whose replication is not temperature sensitive (Wirth et al., 1986) (Fig. 1
). As shown previously, B. subtilis racemase is able to complement the growth defect of Lmdd (Li et al., 2005; Zhao et al., 2005).
The growth in vitro of each of these strains was examined. Growth of constructs prepared in the pKSV7 vector is shown in Fig. 2(a). Those constructs with the shorter upstream regions, 48 bp and 18 bp, either grew very slowly or failed to grow at all at 30 °C in the absence of D-alanine. The two constructs with the longer upstream regions, 551 bp and 80 bp, grew well in the absence of D-alanine, although they grew more slowly than Lmdd+D-alanine during the 6 h culture. There was little effect on growth of the ssrA tag at the 3' ends of the genes. However, ssrA did affect the growth of constructs prepared with plasmid pAM401 (Fig. 2b). Both Lmdd/pA551S and Lmdd/pA80S grew somewhat more slowly than their counterparts without the 3' tag.
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, when growth of the bacteria was at 23 °C. Although the 551 bp and the 80 bp upstream regions allowed similar rates of colony growth, if either of the dal genes was tagged with ssrA, colony growth was severely delayed. After 24 h incubation, colonies of Lmdd/pA551 and Lmdd/pA80 were obvious (Fig. 3a) while those of Lmdd/pA551S or Lmdd/pA80S were barely detectable. By 96 h, the slow-growing bacteria had almost caught up with their counterparts, so that at this time there was little difference in colony size between constructs with or without the ssrA tags (Fig. 3c).
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). Conversely, the Lmdd/pA80S cultures contained mainly long rods as well as large numbers of incompletely septate bacteria.
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). Therefore we were surprised to observe that this was not the case for the pKSV7 constructs. After growth in D-alanine-free liquid media, only 21–35 % of the Lmdd harbouring the temperature-sensitive pKSV7-based plasmids formed colonies on D-alanine-free plates (compared to colony formation on D-alanine-containing plates). This was observed regardless of the temperature of growth of the cultures or the plates, between 23 °C and 37 °C. We infer that in these cultures, Lmdd that had lost their plasmids were able to grow on D-alanine released from the plasmid-containing bacteria. Because of this plasmid instability, we focused most of our efforts to devise a more useful vaccine strain on the stable pAM401-containing constructs.
Effects on transcription of the B. subtilis dal gene
In order to examine the effects on gene expression of the dal gene modifications introduced into each of the plasmids, we examined the level of dal gene transcription by real-time RT-PCR. A strain of Lmdd that contained only one integrated copy of the B. subtilis dal gene and one natural chromosomal copy of the haemolysin (hly) gene of Listeria (strain Lmdal– dat– Bsdal+) served as the control for these tests. The ratio of mRNA molecules (dal/hly) transcribed from these two genes in these bacteria was found to be 1.08 in this assay. In cells containing plasmids that express the B. subtilis dal gene, and only one chromosomal hly gene, the dal/hly ratio should be higher. The results of RT-PCR of each of the plasmid-bearing constructs are shown in Table 1. In the pKSV7 series, the dal/hly ratio for bacteria carrying the full-length upstream region, Lmdd/pK551, was over 40. Truncation of the upstream sequence from 551 bp to 80 bp had no detectable effect, while further shortening to 48 bp reduced the ratio of dal/hly transcripts to 23.5, and additional shortening to 18 bp reduced the ratio to 5.3. To determine the transcription efficiency from each plasmid in the constructs, plasmid copy number was ascertained and used to calculate mRNA copies per plasmid template (Table 1). These data show that decreasing the upstream region from 551 bp to 80 bp decreased the number of dal transcripts per plasmid template by approximately 20 %. Further truncation to 48 bp and 18 bp had still more drastic effects on transcription efficiency.
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To summarize these results, shortening the upstream region of the B. subtilis dal gene below 80 bp led to striking reductions in transcription of the gene. The truncation from 551 bp to 80 bp had a smaller effect on the total number of transcripts in the cell, but when examined on the basis of transcription per plasmid, the decrease was significant. The presence of ssrA on the 3' end of the dal gene did not appear to affect transcription, but did lead to in an increased plasmid copy number. As shown later, ssrA-tagging of the dal gene was associated with a decrease in D-alanine pool size. Plasmid number may increase in these cells in an attempt to accommodate this change in D-alanine pool size through a feedback mechanism.
Racemase expression and the effect of ssrA
Eubacteria utilize tmRNA, encoded by the ssrA gene, to allow the release of ribosomes stalled on stop-codon-deficient mRNA (Keiler et al., 2000). The mechanism generates a short peptide tag at the C-terminus of the abortive protein product which functions as a signal for enhanced proteolysis. If the ssrA sequence that was placed at the 3' end of the B. subtilis dal gene functions in this way, we would expect to find lower quantities of racemase, the product of the dal gene, in cells that harbour this modified gene. Cultures of Lmdd/pA551, Lmdd/pA551S, Lmdd/pA80 and Lmdd/pA80S were grown at 30 °C, diluted into fresh medium and sampled at several subsequent time points. Fig. 5 shows Western blots from two experiments. In both, the amount of HA-tagged racemase was significantly lower for the strains possessing the ssrA sequence at the 3' end of the dal gene. The ssrA effect appeared to be greater in the early exponential phase of growth than at later time points.
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. D-Alanine pools were found to be slightly larger in our wild-type Listeria than in Escherichia coli (Mengin-Lecreulx et al., 1982), not unexpected since the latter has a much thinner peptidoglycan layer.
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verifies that Lmdd grown in the presence of D-alanine (and then washed) contains very high levels of D-alanine, which are depleted by additional growth in the absence of the amino acid for 20 or 45 min.
Plaques produced by Lmdd constructs on L2 fibroblast monolayers
We sought to determine whether the properties of the new Lmdd constructs described thus far would affect their ability to interact with host cells, that is, to infect, multiply and spread in eukaryotic cells, or affect their virulence and ability to elicit an immune response in mice. An in vitro test, infection of monolayers of murine L2 fibroblasts, leads to growth of the micro-organism, its cell-to-cell spread and visible plaque formation (Sun et al., 1990). Fibroblast monolayers were infected with Lm+, Lmdd+/–D-alanine, Lmdd/pA551, Lmdd/pA551S, Lmdd/pA80 and Lmdd/pA80S, and the presence and diameter of plaques produced were measured. Table 3 shows that Lm+ and Lmdd+D-alanine produced comparable, large plaques, while Lmdd in the absence of D-alanine failed to produce any plaques. Lmdd/pA551 and Lmdd/pA80 produced slightly smaller than normal diameter plaques. The plaques that resulted from infection with Lmdd/pA551S and Lmdd/pA80S, which carried ssrA-tagged dal genes, were reduced in size by 14 % and 26 %, respectively, relative to Lmdd+D-alanine.
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). To assess the safety of the new constructs, the number of bacteria found in these organs of infected host animals was examined at various times after infection. Groups of mice infected i.v. with about 0.2 LD50 of bacteria (1.4x107 of Lmdd+D-alanine, 4x106 of Lmdd/pA80S or 2x103 of wild-type Lm) were examined at various time points up to 7 days post-infection for total viable bacteria in spleen. As indicated in Fig. 6, wild-type Lm multiplied in this organ, reaching a maximum number of organisms at day 3, and was then eliminated by host immune defences by day 7. In contrast, Lmdd+D-alanine and Lmdd/pA80S failed to replicate significantly and their numbers fell continuously. They were cleared from the organ in about 3 days. Thus Lmdd/pA80S had a similar low virulence in mice as the original Lmdd (+D-alanine) strain, despite the fact that the former was able to satisfy its own need for D-alanine.
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; Kathariou et al., 1987). This protein also contains the dominant CD8+ T-cell epitope of Listeria in BALB/c mice (Wipke et al., 1993). We therefore assessed the immune response of mice to Lmdd/pA80S by determining the level of activated (CD11ahi) LLO-specific CD8+ effector T-cells in spleen following immunization. As shown in the FACS analysis presented in Fig. 7(a), Lmdd/pA80S elicited a similar percentage of LLO-tetramer+ CD8+ T-cells in spleen as was induced by wild-type Lm. Most of these splenocytes also functioned as effector cells, as evidenced by their expression of intracellular IFN-, shown in Fig. 7(b). Lmdd administered to mice without a supply of D-alanine generated few antigen-specific T-cells.
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a) and livers (Fig. 8b) of mice immunized by Lmdd/p80S were similar to those after immunization with wild-type Lm and Lmdd+D-alanine, with more than a six-log10 reduction in bacterial numbers compared to the challenged naïve group. Prior immunization with Lmdd in the absence of D-alanine led to little protection (Thompson et al., 1998).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Schafer et al., 1992; Shen et al., 1995; Starks et al., 2004). However, the wild-type organism can cause listeriosis, a serious food-borne illness that poses a health threat especially for pregnant women and other immunocompromised individuals (Gellin & Broome, 1989). Live micro-organisms must be attenuated if they are to be used as human vaccine vectors but a balance between their safety and immunogenicity needs to be achieved since overattenuation can result in loss of the desired immune response.
L. monocytogenes has been attenuated in various ways. Genes altered for this purpose have included actA (Kocks et al., 1992) and actA in combination with plcB (Angelakopoulos et al., 2002; Peters et al., 2003) or with intA/B (Brockstedt et al., 2004); lipoate protein ligase LplA1 (O'Riordan et al., 2003); aro (Stritzker et al., 2004); and even psoralen-inactivated whole populations of recombinant Listeria (Brockstedt et al., 2005). Our work has focused on a strain of L. monocytogenes in which the two genes responsible for D-alanine synthesis, dal and dat, have been deleted (Thompson et al., 1998). These bacteria, Lmdd, are absolutely dependent on an exogenous supply of D-alanine for cell wall synthesis, both in vitro and in vivo. The strain was shown to effectively induce protective immunity against challenge by the wild-type organism itself or by other organisms expressing a common antigen, when infection was initiated with a small dose of D-alanine (Rayevskaya & Frankel, 2001; Thompson et al., 1998).
In order to generate a derivative of Lmdd that would obviate its need for exogenous administration of the amino acid yet still retain attenuation, in this study we cloned a B. subtilis dal gene (which can complement the deficiency of Lmdd) with different lengths of upstream region into vectors pKSV7 and pAM401 and transformed the resulting plasmids into Lmdd. We found that a minimum 80 bp promoter length was necessary to allow D-alanine-independent growth of Lmdd (Fig. 2a). The promoterless construct, Lmdd/pK18, with only a Shine–Dalgarno sequence, and Lmdd/pK48 failed to grow. The number of dal gene transcripts in cells carrying these constructs was severely reduced (Table 1). Thus, the sequence between –48 bp and –80 bp must contain important elements for the dal gene promoter. When comparing the number of dal gene transcripts generated from the two long promoter regions (551 bp and 80 bp) there was little or no difference in the total number of transcripts, although the number per plasmid copy was significantly reduced for the 80 bp construct.
A general mechanism used by many species of bacteria to rescue stalled ribosomes relies on the intervention of tmRNA encoded by the ssrA gene. This stable RNA molecule binds to ribosomes and its encoded peptide tag is cotranslationally added to the abortive polypeptide chain, targeting it for proteolysis (Karzai et al., 2000). In Gram-positive bacteria, the proteolysis appears to be mediated by ClpXP protease (Wiegert & Schumann, 2001). We sought to utilize this salvage mechanism in our constructs to destabilize accumulated racemase, by intentionally tagging the dal gene at its 3' end with the ssrA gene. This had the effect of greatly decreasing the steady-state concentration of racemase in the cells (Fig. 5), presumably by enhancing its degradation. We showed that this in turn was associated with decreased pools of free D-alanine. This evidently restricted the overall growth rate of the bacteria (Fig. 3) and limited their ability to multiply, spread and form plaques in monolayers of L2 fibroblasts (Table 3).
The goal of these studies was to generate a strain of L. monocytogenes for use as a vaccine vector with reduced virulence but strong immunogenicity. Compared with wild-type L. monocytogenes, the approximate LD50 of Lmdd/pA80S is increased by at least 3-log10 (1x104 vs approximately 2x107). While the reduced length of upstream region of the B. subtilis dal gene from 551 bp to 80 bp had a fairly small effect on total transcription of the gene (Table 1), we have observed in some preliminary experiments a fourfold higher LD50 for constructs that carried the longer upstream sequence (data not shown). A greater contribution to the attenuation of these bacteria was provided by the ssrA gene tag on the dal gene, which resulted in a greatly reduced concentration of racemase and a notable reduction of D-alanine pool size. Following infection of mice with 4x106 of Lmdd/pA80S, at day 3 there was a 4-log10 decline in number of organisms in the spleen, whereas at day 3 after infection with 103 wild-type Lm+ there was a 2-log10 increase (Fig. 6). These results attest to the attenuation of Lmdd/pA80S.
Despite early indications to the contrary (North et al., 1981), experiments that temporally abrogate Listeria infection of mice with antibiotics revealed that 24 h of infection may suffice to generate a T-cell response of normal magnitude (Mercado et al., 2000). Consistent with this, despite the short time that Lmdd/pA80S survives in animals, the strain was able to induce an effective immune response. A strong induction of LLO-specific CD8+ effector T-cells as shown by LLO-tetramer and IFN- intracellular staining was obtained (Fig. 7). Furthermore, a high level of protection against subsequent challenge by the wild-type organism was achieved (Fig. 8).
In summary, we have characterized a new vaccine system for L. monocytogenes based on Lmdd, a D-alanine-requiring mutant. The new strain Lmdd/pA80S satisfies the need for D-alanine through limited expression of a racemase gene. Immunization of mice with these bacteria resulted in an effective immune response that led to protection against subsequent challenge. Oral immunization of rhesus macaques with recombinant strains of these organisms revealed them to be a safe and effective inducer of cellular immune responses in this nonhuman primate model (R. Ruprecht and others, personal communication). Lmdd/pA80S may thus be a candidate as a live bacterial vaccine vector for oral immunization that meets the important medical need for vectors able to elicit cellular and mucosal immunity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 March 2006;
revised 7 July 2006;
accepted 17 July 2006.
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