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PATHOGENICITY AND VIRULENCE

Expression of two Listeria monocytogenes antigens (P60 and LLO) in Lactococcus lactis and examination for use as live vaccine vectors

Mohammed Bahey-El-Din, Pat G. Casey, Brendan T. Griffin, Cormac G. M. Gahan

  • 1School of Pharmacy, University College Cork, Cork, Ireland
  • 2Department of Microbiology, University College Cork, Cork, Ireland
  • 3Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Egypt
  • 4Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
  • Correspondence
    Cormac G. M. Gahan
    c.gahan{at}ucc.ie

    • Journal of Medical Microbiology 2010; 59(8):904–912 · https://doi.org/10.1099/jmm.0.018770-0

    View at publisher PubMed

    Abstract

    Listeria monocytogenes is a food-borne intracellular pathogen that mainly infects pregnant and immunocompromised individuals. The pore-forming haemolysin listeriolysin O (LLO), the main virulence factor of Listeria monocytogenes, allows bacteria to escape from the harsh environment of the phagosome to the cytoplasm of the infected cell. This leads to processing of bacterial antigens predominantly through the cytosolic MHC class I presentation pathway. We previously engineered the food-grade bacterium Lactococcus lactis to express LLO and demonstrated an LLO-specific CD8+ response upon immunization of mice with the engineered L. lactis vaccine strains. In the present work, we examined the immune response and protective efficacy of an L. lactis strain co-expressing LLO and a truncated form of the listerial P60 antigen (tP60). Oral immunization revealed no significant protection against listeriosis with L. lactis expressing LLO, tP60 or the combined LLO/tP60. In contrast, intraperitoneal vaccination induced an LLO-specific CD8+ immune response with LLO-expressing L. lactis but no significant improvement in protection was observed following vaccination with the combined LLO/tP60 expressing L. lactis strain. This may be due to the low level of tP60 expression in the LLO/tP60 strain. These results demonstrate the necessity for improved oral vaccination strategies using LLO-expressing L. lactis vaccine vectors.

    INTRODUCTION

    Listeria monocytogenes is a Gram-positive food-borne pathogen that primarily infects immunocompromised individuals. Pregnant women and newborns are particularly susceptible to listerial infection with disease culminating in miscarriage and meningitis, respectively (Meng & Doyle, 1997). Listeriolysin O (LLO) is a critical listerial virulence factor and immunodominant antigen. LLO facilitates the escape of Listeria monocytogenes from the phagosomal compartment to the cytoplasm of infected cells, a process mediated by membrane pore-forming activity (Vazquez-Boland et al., 2001). LLO-mediated bacterial translocation into the cytoplasm leads to the processing of listerial antigens through the MHC class I antigen presentation pathway. This ultimately results in the development of CD8+ cell-mediated immunity against the major immunodominant listerial antigens LLO and P60 (Pamer, 2004). P60 is a listerial protein that has important roles in cell division and in host cell invasion (Faith et al., 2007; Kuhn & Goebel, 1989; Wuenscher et al., 1993).

    The unique phagosomal permeabilizing activity of LLO means that accompanying antigens can access the cytosolic antigen presentation pathway. This feature has been exploited by several investigators to develop or enhance cell-mediated immune responses against specific antigens by co-expression of these antigens with LLO in a live vaccine vector (Huang et al., 2008; Radford et al., 2002; Tvinnereim et al., 2002).

    Lactococcus lactis is a GRAS (generally regarded as safe) food-grade bacterium that has long been utilized in the food industry, especially in fermented dairy products. During the last two decades L. lactis has been investigated as a potential vaccine delivery platform for the delivery of various antigens (Bahey-El-Din et al., 2010). L. lactis is non-pathogenic, non-invasive and non-commensal Gram-positive bacterium that lacks the pro-inflammatory LPSs associated with Gram-negative bacteria. Although L. lactis has innate immunoadjuvant activity, it elicits only a weak immune response against itself, which is an obvious advantage as a vaccine live vector (Robinson et al., 1997; Wells & Mercenier, 2008).

    We have previously demonstrated that LLO-expressing L. lactis strains are capable of eliciting an antigen-specific CD8+ immune response and protection against Listeria monocytogenes challenge in the murine infection model (Bahey-El-Din et al., 2008). The objective of the present work was to examine the protective efficacy of an L. lactis vaccine strain that dually expresses two listerial antigens: LLO and a truncated form of the P60 listerial antigen (tP60) to determine if it provides greater vaccination efficacy than either antigen expressed alone. We also hypothesized that the pore forming activity of LLO would facilitate the translocation of tP60 to the cytosol of infected cells with subsequent antigen presentation through the MHC class I pathway.

    METHODS

    Bacterial strains, plasmids and culture conditions.

    A summary of the bacterial strains and plasmids used in this study is shown in Table 1⇓. Luria–Bertani (LB) broth was used for Escherichia coli cultures, while M17 broth (Oxoid) supplemented with 0.5 % glucose (i.e. GM17) was used for Lactococcus. For Listeria monocytogenes, brain heart infusion (BHI) broth (Oxoid) was used. Technical agar (1.5 % w/v) (Merck) was added when solid media were required. Incubation temperatures were 30 °C for L. lactis, and 37 °C for Listeria monocytogenes, and E. coli. When required, ampicillin was used at a concentration of 100 μg ml−1 for E. coli, while chloramphenicol (Cm) was used at 10 μg ml−1 for E. coli and 5 μg ml−1 for L. lactis. All cell culture media and reagents were obtained from Gibco unless otherwise stated.

    Table 1.

    Bacterial strains and plasmid vectors

    Cloning of DNA encoding listerial antigens in pNZ8048-derivative plasmid vectors.

    Three plasmids were constructed for the expression of the two listerial antigens individually or in combination. Plasmid pNZPnisA : SEC-LLO was created for secreted LLO antigen, plasmid pNZPnisA : SEC-tP60 for secreted truncated P60 (tP60) antigen and plasmid pNZPnisA : SEC-LLO/tP60 for dually secreted LLO and tP60 antigens (Fig. 1⇓). Plasmid pNZPnisA : SEC-LLO containing the hly gene encoding LLO was created as described in our previous work (Bahey-El-Din et al., 2008) where LLO is secreted with the aid of the N-terminal Usp45 secretion signal upon nisin induction of the PnisA promoter. The Usp45 protein is a common lactococcal secreted protein (van Asseldonk et al., 1990) and the use of the Usp45 secretion signal (here designated SEC) has been reported to direct the secretion of heterologous proteins in L. lactis (Steidler et al., 2003; Wells et al., 1993).

    Figure image not available in archive
    Fig. 1.

    Diagrammatic representation of the three plasmids constructed in this study. PnisA, nisin-inducible promoter; RBS, ribosome-binding site; SEC, secretion signal of Usp45 protein; H, six histidine tag; T, transcription terminator. hly and iap are the genes encoding LLO and P60 antigens, respectively.

    The pQE30 plasmid (Qiagen) was used as an intermediate cloning vector to create DNA encoding His-tagged P60 antigen for subsequent cloning into pNZ8048 derivatives. The iap gene (encoding P60) of Listeria monocytogenes EGDe (accession number AL591824) minus the secretion signal was PCR amplified from chromosomal DNA using primers P60-pQE30 (forward primer) and P60-pQE30 (reverse primer) (Table 2⇓). This amplified iap gene was digested with BamHI and KpnI, and ligated using T4 DNA ligase (Roche) to a similarly digested pQE30 plasmid (Qiagen) that introduced an N-terminal six histidine tag to P60. The ligation reaction mixture was used to transform chemically competent E. coli NovaBlue (Novagen) by heat shock following the manufacturer's instructions and plated onto LB agar containing ampicillin. Colony PCR was used to identify positive colonies and plasmid (pQE30/iap) was extracted from E. coli NovaBlue using a miniprep kit (Qiagen). The integrity of the DNA sequence was confirmed by sequencing (Cogenics).

    Table 2.

    Oligonucleotide primers

    A construct composed of the SEC signal DNA [with a ribosome-binding site (RBS)] and DNA encoding a His-tagged truncated form of P60 was made using the splicing by overlap extension (SOE) technique (Horton et al., 1990). The primers used in the PCRs are shown in Table 2⇑. This construct was cloned downstream of hly in the pNZPnisA : SEC-LLO plasmid between KpnI and XbaI restriction sites (Fig. 1⇑). Briefly, the SEC signal (including its native RBS) was amplified from the L. lactis MG1363 genome using primers 37 and 4. Using pQE30/iap as a template, primers 5 and 38 were used to amplify a DNA sequence corresponding to an N-terminus His-tag plus amino acids 26–402 of the native P60 protein (i.e. tP60). The two PCR products, i.e. SEC (with RBS) and truncated iap (encoding tP60), were SOE-spliced using primers 37 and 38. The spliced PCR product (designated RBS-SEC-tP60) was sequentially digested with KpnI and XbaI, and ligated using T4 DNA ligase (Roche) to a similarly digested pNZPnisA : SEC-LLO. The ligation mix was used to transform commercial chemically competent E. coli Top10 (Invitrogen) following the manufacturer's instructions and plated onto LB agar containing Cm. After incubation at 37 °C for 24–48 h, positive colonies were detected by colony PCR. The created plasmid pNZPnisA : SEC-LLO/tP60 was extracted from E. coli Top10 using the miniprep kit (Qiagen) and the DNA sequence was confirmed by sequencing (Cogenics).

    Plasmid pNZPnisA : SEC-tP60 was created for expression of tP60 alone (Fig. 1⇑). The SEC signal was amplified (without its native RBS) from the L. lactis MG1363 genome using primers 34 and 4. Primers 5 and 38 were used to amplify DNA encoding a His-tagged tP60 from pQE30/iap plasmid as described above. These two PCR products (SEC and truncated iap) were spliced using primers 34 and 38 and the SOE technique (Horton et al., 1990) resulting in the construct SEC-tP60. After sequential digestion of both SEC-tP60 and pNZ8048 (Kuipers et al., 1998) with NcoI and XbaI, they were ligated and used to transform chemically competent E. coli Top10 (Invitrogen) following the manufacturer's instructions and plated onto LB agar containing Cm. After incubation at 37 °C for 24–48 h, positive colonies were detected by colony PCR and the DNA sequence of the created plasmid pNZPnisA : SEC-tP60 was confirmed by sequencing (Cogenics).

    All PCRs were performed using the high fidelity KOD hot start DNA polymerase (Novagen) following the manufacturer's instructions.

    Transformation of Lactococcus strains with the engineered plasmid vectors.

    L. lactis NZ9000 ΔhtrA (Lindholm et al., 2004) (kindly provided by Professor Airi Palva, Faculty of Veterinary Medicine, University of Helsinki, Finland) was used as a host for the three engineered plasmids. It should be noted that HtrA is an extracellular housekeeping protease (Poquet et al., 2000) and HtrA-deficient Lactococcus mutants have been reported to be more efficient in secreting expressed protein (Le Loir et al., 2005; Lindholm et al., 2004). Electrocompetent L. lactis ΔhtrA cells, prepared as described by Holo & Nes (1989), were transformed with the corresponding plasmid vectors using a Gene Pulser (Bio-Rad), plated onto GM17 agar containing Cm and incubated at 30 °C for 24 h. Colonies were checked by colony PCR and positive clones were preserved in glycerol stocks at −80 °C. In addition, plasmid pNZ8048 was transformed into L. lactis NZ9000 ΔhtrA to create a negative control Lactococcus strain.

    Investigation of LLO production by SDS-PAGE and Western blotting.

    Overnight cultures of the three engineered Lactococcus strains (i.e. containing pNZPnisA : SEC-LLO, pNZPnisA : SEC-tP60 or pNZPnisA : SEC-LLO/tP60) were subcultured (5 % v/v) in fresh GM17 broth and grown statically at 30 °C to an optical density at 600 nm of 0.5. At this point, nisin, used as a filter-sterilized culture supernatant of the nisin-producer strain Lactococcus NZ9700 (Kunji et al., 2003), was added at a concentration of 0.2 % (v/v). These strains were allowed to grow for a further 3 h. Culture supernatants of the induced vaccine strains were collected and secreted proteins were precipitated using cold trichloroacetic acid precipitation (15 % w/v final concentration) as described by (Piard et al., 1997).

    For Western blotting, SDS-PAGE gels were blotted onto nitrocellulose membranes (Hybond-ECL; Amersham Biosciences) and the membrane was then blocked overnight at 4 °C in 5 % (w/v) skimmed milk in Tris-buffered saline buffer (0.8 % w/v sodium chloride, 20 mM Tris/HCl, pH 7.6). Primary mouse anti-penta-His-tag antibody (Abgent) and secondary anti-mouse antibody (ECL Western blotting system) were used at 1/1000 and 1/1500 dilutions in 5 and 10 % (w/v) skimmed milk, respectively. Western blot detection was performed using the Amersham ECL western blotting system (Amersham Biosciences) according to the protocol recommended by the manufacturer.

    Assessment of LLO haemolytic activity.

    The haemolytic activity of LLO in induced culture supernatants was assessed using the method described by Kohda et al. (2002) with some modifications. Briefly, aliquots of 100 μl 0.5 % (v/v) sheep red blood cells suspended in PBS (pH 5.9) were added to twofold dilutions (in PBS pH 5.9) of dialysed culture supernatants of the cultured induced strains, to a final volume of 1 ml. A positive control (distilled water) and negative controls (PBS pH 5.9 and culture supernatant of L. lactis NZ9000 ΔhtrA(pNZ8048)) were also included. Tubes were incubated statically at 37 °C for 45 min after which they were centrifuged and the supernatants collected. Absorbance was measured at 415 nm and haemolytic activity was expressed in terms of complete haemolytic units (CHU), defined as the reciprocal of the highest dilution of supernatant showing complete haemolysis (Hess et al., 2000).

    Murine immunization protocols.

    Female BALB/c mice, 6–8 weeks of age, were used in all animal experiments. All animal procedures were reviewed and approved by the ethical assessment committee of University College Cork. Overnight cultures of Lactococcus vaccine and control strains were subcultured into fresh warm GM17 and incubated statically at 30 °C until an OD600 of 0.5 was reached, whereupon nisin (0.2 % v/v) was added and the cultures were incubated for a further 1 h. Cells were then pelleted by centrifugation, washed once with PBS and resuspended in PBS for intraperitoneal (IP) injection (107 c.f.u. per dose) or resuspended in a bicarbonate buffer (0.2 M sodium bicarbonate, 5 % casein hydrolysate, 0.5 % glucose) (Robinson et al., 2004) for oral inoculation (5×109 c.f.u. per dose). In the oral vaccination experiment we used 5 μg Cm ml−1 instead of the higher levels (10 μg ml−1) reported previously (Bahey-El-Din et al., 2008) for plasmid selection to decrease any inhibitory effect of Cm on acid tolerance in Lactococcus (Kim et al., 2002). We postulated that this may increase lactococcal survival in the gastric acidic medium upon oral vaccination. As a positive control we vaccinated mice with a murinized Listeria monocytogenes (designated Listeria monocytogenes EGDm) strain (recently created in our lab with a mutated internalin A virulence factor to allow improved murine gut invasion by the oral route) (I. R. Monk, C. Hill & C. G. M. Gahan, unpublished data). An overnight culture of this strain was diluted in the aforementioned bicarbonate buffer and inoculated orally (106 c.f.u. per dose) in the oral vaccination experiments. Groups treated with a sublethal IP Listeria monocytogenes EGDe (2×103 c.f.u. per dose) were also included as a positive control for the IP vaccination experiments. L. lactis NZ9000 ΔhtrA(pNZ8048)-treated groups (IP and oral), and PBS-injected groups and bicarbonate buffer orally treated groups, were included as negative controls in the IP and oral vaccination experiments, respectively.

    Groups were given seven doses of the vaccine or control strains by oral gavage on days 1, 2, 3, 28, 29, 30 and 37. This vaccine regimen is similar to a successful regimen utilized for L. lactis-based vaccination studies (Robinson et al., 2004). For IP vaccination, three doses were given on days 1, 15 and 30. The final dose volume per mouse was 200 μl for both oral and IP inoculations.

    Detection of LLO-specific CD8+ T cells by the enzyme-linked immunospot (ELISPOT) test.

    The ELISPOT test was used as described by Carvalho et al. (2001) to detect gamma interferon (IFN-γ)-secreting CD8+ cells specific to the H2 Kd-restricted LLO epitope LLO91–99, GYKDGNEYI and the P60 epitope P60217–225, KYGVSVQDI (Peptide Protein Research) (Vijh & Pamer, 1997). Mouse mastocytoma line P815-1-1 cells (from the European Collection of Cell Cultures) were used as antigen presenting cells (APCs). P815-1-1 cells pulsed with 10−6 M LLO91–99 or P60217–225 peptides or non-pulsed were used to stimulate splenocytes from vaccinated and control mice. Briefly, MultiScreen 96-well plates (Millipore) were first coated with anti-mouse IFN-γ antibodies (clone R4-6A2) (Biolegend), and the splenocytes along with APCs were incubated in the coated wells for 20–24 h in a tissue culture incubator (37 °C and 5 % CO2 atmosphere). The wells were subsequently washed with PBS containing 0.05 % (v/v) Tween 20 and treated with biotin anti-mouse IFN-γ antibodies (clone XMG1.2) (Biolegend) followed by avidin-horseradish peroxidase (Biolegend). Spots corresponding to epitope-specific IFN-γ-secreting cells developed upon adding the peroxidase substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Sigma). The number of spots was determined using a stereomicroscope.

    Murine challenge with Listeria monocytogenes.

    Listerial challenge was performed as previously described (Stack et al., 2005). Briefly, mice were challenged with IP injection of 200 μl containing 2×106 c.f.u. ml−1 (4×105 c.f.u. per mouse) Listeria monocytogenes EGDe. For the oral vaccination experiment, challenge was performed orally with the murinized Listeria monocytogenes EGDm (6×109 c.f.u. per mouse) on day 42. Mice were euthanized 3 days post-challenge and the listerial burden was determined from the spleens by organ homogenization, serial dilutions and plating on BHI agar plates. Plates were incubated at 37 °C for 1–2 days and Listeria counts were calculated per spleen. The limit of detection (LOD) of Listeria was 50 c.f.u. per organ.

    Interpretation of data and statistical analysis.

    Data satisfying the assumptions of normality were analysed using the Student's t-test. Data not showing normal distribution were analysed by Kruskal–Wallis one-way ANOVA by ranks with post hoc comparison using Dunn's method. In all cases, P values less than 0.05 were considered statistically significant.

    RESULTS AND DISCUSSION

    Production of LLO and tP60 by engineered lactococcal strains

    SDS-PAGE followed by Western blotting using anti-penta-His-tag antibody (Abgent) revealed efficient production of LLO and tP60 in the corresponding L. lactis NZ9000 ΔhtrA strains (Fig. 2⇓). However, it was observed that the level of tP60 production in NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) was significantly lower than its production level in NZ9000 ΔhtrA(pNZPnisA : SEC-tP60) (Fig. 2⇓). Assessment of the haemolytic activity of secreted LLO confirmed the secretion of biologically active haemolysin. Culture supernatants of NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) and L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) showed 8 CHU, whereas no detectable haemolytic activity was observed with L. lactis NZ9000 ΔhtrA(pNZ8048) or L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-tP60).

    Figure image not available in archive
    Fig. 2.

    Western blotting of culture supernatant protein precipitates following nisin induction. Mouse anti-His tag antibodies were used as primary antibodies to detect the His-tagged LLO and tP60. Lanes 1–4 represent trichloroacetic acid precipitates of the supernatants of nisin-induced cultures of NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) (lane 1), NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) (lane 2), NZ9000 ΔhtrA(pNZPnisA : SEC-tP60) (lane 3) and NZ9000 ΔhtrA(pNZ8048) (lane 4).

    Assessment of short-term immune responses

    CD8+ T lymphocytes specific for the H2 Kd-restricted LLO91–99 epitope but not the P60217–225 epitope were elicited by the vaccine strains following IP immunization.

    The ELISPOT assay was used to analyse the development of epitope-specific CD8+ cells. Mouse mastocytoma P815-1-1 cells were used as APCs as they express restricted H2 Kd MHC class I molecules. As a result, only spots due to epitope-specific CD8+ cells are produced (Carvalho et al., 2001). The short-term immune response was assessed on day 42 from the start of vaccination by the IP or oral routes. The two LLO-secreting strains, i.e. L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) and L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60), demonstrated LLO91–99-specific spots following a three-dose IP vaccination regimen (Fig. 3⇓). No P60217–225-specific spots were observed following this three-dose IP regimen with any of the tP60-secreting lactococcal vaccine strains whether in presence or absence of LLO co-expression (Fig. 3⇓). Following oral vaccination, only the positive control murinized Listeria monocytogenes EGDm strain was found to elicit LLO91–99- and P60217–225-specific spots in the ELISPOT test (data not shown).

    Figure image not available in archive
    Fig. 3.

    ELISPOT test results following a three-dose IP vaccination regimen with the lactococcal vaccine strains. Mouse groups (n=4) were vaccinated by IP injection on days 1, 15 and 30 then examined by ELISPOT on day 42. Asterisks indicate a P<0.05 as compared to the negative control groups. Error bars represent the mean±sem. White bar, P60217–225; black bars, LLO91–99.

    Protection against Listeria monocytogenes challenge following IP vaccination.

    Mice were challenged intraperitoneally with Listeria monocytogenes EGDe on day 42 after the first vaccination dose. Three days following challenge, mice were euthanized and the Listeria counts were determined in the spleens. Spleen listerial counts following the three-dose IP vaccination revealed significant protection (P<0.05) with both L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) and L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) strains when compared with negative control groups or with groups vaccinated with L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-tP60) (Fig. 4⇓). No significant difference in protection was found between L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) and L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) (Fig. 4⇓).

    Figure image not available in archive
    Fig. 4.

    Results of the challenge experiment following a three-dose IP vaccination regimen. Mouse groups (n=5) were IP-vaccinated on days 1, 15 and 30 then challenged with IP Listeria monocytogenes EGDe on day 42. Mice were euthanized 3 days later and the Listeria in the spleens counted. Asterisks indicate a P<0.05 as compared to negative control groups. Error bars represent the mean±sem. nd, Not detectable.

    In oral vaccination experiments, no significant difference was observed between the orally vaccinated groups and the negative control groups for any of the three constructed vaccine strains after challenge with oral murinized Listeria monocytogenes EGDm (data not shown). This failure to protect by the oral route may be due to the rapid killing of L. lactis in the gastrointestinal tract (GIT) that has been reported by Kimoto et al. (2003) and by Vesa et al. (2000). This shows the necessity of an improved strategy to protect L. lactis during GIT transit in the murine model for enhanced presentation of the cloned antigens to the immune system. Other factors that may play critical roles upon oral vaccination are the vaccination regimen used, bacterial inoculation doses, amount and nature of the expressed antigens in lactococci, and the particular L. lactis strain utilized. Moreover, other harsh GIT conditions, such as the presence of bile, may play a significant role in preventing the survival of L. lactis. A possible future approach to solve this problem may be the use of L. lactis harbouring BilE, the bile resistance system of Listeria monocytogenes, which allows better L. lactis survival and persistence in the murine gut (Watson et al., 2008).

    Assessment of long-term immune responses following an IP vaccination regimen

    As seen above, the three-dose IP vaccination regimen did not show any protective difference between L. lactis dually expressing LLO and tP60, and L. lactis that expressed LLO alone. Moreover, we did not detect a significant shift in the immune response against tP60 towards a cell-mediated CD8+ response or T helper type 1 (Th1) response when LLO was co-expressed in NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60). Hence, we decided to increase the frequency of IP doses to detect any weak immune response that might have been obscured due to suboptimal immunization. In addition, the immune response was assessed on day 69 rather than day 42 of vaccination to examine longer term immunity. Eight IP bacterial doses, according to a previously determined regimen from our work (Bahey-El-Din et al., 2008), were injected by the IP route on days 1, 2, 7, 14, 21, 28, 35 and 36, and the immune response was assessed on day 69 from the start of vaccination. Fig. 5⇓ summarizes the different vaccination regimens used in the present study.

    Figure image not available in archive
    Fig. 5.

    Diagrammatic representation of the different vaccination regimens used in this study. The numbered arrows represent the days when vaccine inoculations were carried out. The numbered stars at the end (right side) of the regimen diagram represent the day of vaccine challenge or ELISPOT test.

    ELISPOT results indicated significant LLO91–99-specific responses in both mice treated with NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) or NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60), but no significant difference was found between these two groups (Fig. 6⇓). A P60217–225-specific CD8+ response was detectable at low levels in only two out of five mice vaccinated with NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) but the overall P60217–225-specific spots (evaluating all mice) were statistically insignificant as compared to negative control groups (Fig. 6⇓). No P60217–225-specific spots were observed in NZ9000 ΔhtrA(pNZPnisA : SEC-tP60)-treated mice (Fig. 6⇓). These results indicate that the effect of LLO on introducing tP60 to the MHC class I pathway was limited, perhaps due to the low levels of tP60 expression in NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) (Fig. 2⇑).

    Figure image not available in archive
    Fig. 6.

    ELISPOT test results following an eight-dose IP vaccination regimen (long-term investigation). Mouse groups (n=5) were vaccinated by IP injection on days 1, 2, 7, 14, 21, 28, 35 and 36 then examined by ELISPOT on day 69. Asterisks indicate a P<0.05 as compared to negative control groups. Error bars represent the mean±sem. White bar, P60217–225; black bars, LLO91–99.

    Following IP challenge with Listeria monocytogenes EGDe on day 69, significant protection, as evidenced by splenic listerial counts, was achieved in mice treated with NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) or NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) as compared to negative control groups (Fig. 7⇓). However, no statistical difference in protection was found between these two groups. In addition, no protection was observed in mice treated with IP NZ9000 ΔhtrA(pNZPnisA : SEC-tP60). These results further confirm that tP60 expressed alone in L. lactis cannot afford protection against listeriosis and also the effect of LLO/tP60 combined expression in L. lactis does not offer a more pronounced protection than L. lactis expressing LLO alone.

    Figure image not available in archive
    Fig. 7.

    Results of the challenge experiment following an eight-dose IP vaccination regimen (long-term investigation). Mouse groups (n=5) were vaccinated by IP injection on days 1, 2, 7, 14, 21, 28, 35 and 36 then challenged with IP Listeria monocytogenes EGDe on day 69. Mice were euthanized 3 days later and the Listeria in the spleens counted. Asterisks indicate a P<0.05 as compared to negative control groups. Error bars represent the mean±sem.

    Conclusions

    Listeria monocytogenes is a food-borne pathogen that can cause miscarriage and meningitis in the pregnant and newborn, respectively. Several listerial outbreaks have been reported by Ramaswamy et al. (2007) and immunocompromised individuals are at a higher risk of infection. In a previous work, we demonstrated that LLO-expressing L. lactis vaccine vectors could elicit antigen-specific CD8+ immune responses and protection against Listeria monocytogenes challenge in the murine model (Bahey-El-Din et al., 2008). The use of the GRAS L. lactis as a live vaccine vector represents a safe and advantageous approach in current vaccine research, particularly in terms of the stimulation of mucosal immunity. In the present work, we co-expressed a truncated form of P60 antigen of Listeria monocytogenes along with LLO in an L. lactis strain and examined the vaccine potential of this construct in a murine model of listeriosis. Control L. lactis strains expressing LLO or tP60 individually were also included and examined to allow comparison between the single and dual expression vaccine candidates. Regardless of tP60 expression, we found that LLO expression by L. lactis is critical for successful vaccination against Listeria monocytogenes, most probably due to its reported immunodominance (Vijh & Pamer, 1997). Although P60 antigen is known to be immunogenic, LLO is considered more important for a protective cytotoxic CD8+ immune response, a phenomenon demonstrated by Hess and co-workers who used Salmonella typhimurium aroA− attenuated strains as live vectors for LLO and P60 antigens (Hess et al., 1996).

    It is well known that the protective immune response against listeriosis is dependent on a cytotoxic CD8+ immune response (Pamer, 2004). Since cytoplasmic access favours antigen processing through the MHC class I presentation pathway, we hypothesized that LLO activity would facilitate this process for the accompanying tP60 antigen. However, it seems that the process was limited by the relatively moderate amount of tP60 expressed in L. lactis NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60). The amount of antigen expressed in L. lactis vectors is reported to play a critical role in the resultant immune response and higher levels of antigen are usually associated with a better immune response (Wells & Mercenier, 2008). Although appreciably high anti-P60 IgG titres were observed in mice treated intraperitoneally with tP60-expressing strains, no significant difference was observed between serum anti-P60 IgG antibodies in the absence or presence of LLO co-expression (data not shown). Furthermore, examination of anti-P60 IgG1 (indicative of Th2 response) and IgG2a (indicative of Th1 response) subclasses did not show any shift towards a particular anti-P60 immune response (Th1 or Th2) when LLO was co-expressed with tP60 (data not shown). The reasons behind these results are not clear.

    Surprisingly, the positive control Listeria monocytogenes IP-treated group showed a lower number of LLO91–99-specific CD8+ T cells than those observed with NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) or NZ9000 ΔhtrA(pNZPnisA : SEC-LLO) in the long-term IP regimen (Fig. 6⇑). It is known that the pool of LLO91–99-specific CD8+ T cells shrinks in time following parenteral administration of Listeria monocytogenes (Vijh & Pamer, 1997). Indeed we observed this phenomenon in a previous study (Bahey-El-Din et al., 2008). Our observations may reflect the different rates of expansion and contraction of LLO91–99-specific T lymphocytes. To examine this, it would be necessary to follow up the kinetics of the LLO91–99-specific CD8+ response at different time intervals following a specific vaccination regimen.

    We had two objectives in mind for the current study: first, to provide a safe bivalent L. lactis vaccine vector against listeriosis; and second, to examine the immunomodulatory effects of LLO on the accompanying antigen, tP60. LLO is known to direct the immune system towards cell-mediated immunity due to its ability to permeabilize the phagosomal membrane (Darji et al., 1995; Dietrich et al., 2001). When E. coli was engineered to express both LLO and ovalbumin (OVA), as a model antigen, the engineered strain could deliver an OVA Kb-restricted epitope for MHC class I presentation and mice were protected upon challenge with an OVA-expressing melanoma cell line (Radford et al., 2002). More recently, Bacillus subtilis was genetically manipulated to express both LLO and PA (the protective antigen of Bacillus anthracis), and an enhanced Th1 immune response against PA was evident (Huang et al., 2008). Nevertheless, it is clear that the use of the advantageous GRAS L. lactis as an LLO vector represents a potentially safe alternative platform for the cytosolic antigen presentation pathway.

    Igwe and colleagues co-expressed large C-terminal portions of LLO and P60 antigens (LLO51–363 and P60130–477, respectively) in an attenuated Salmonella enterica serovar Typhimurium strain (Igwe et al., 2002). However, for cytoplasmic translocation of antigens they used the type III secretion system of Salmonella rather than the pore forming activity of LLO. Following oral vaccination in mice it was shown that the combined expression and translocation of the truncated LLO and P60 antigens offered better protection against wild-type Listeria monocytogenes challenge than either antigen alone (Igwe et al., 2002). In our study, we could not find a significant difference in efficacy between LLO-expressing L. lactis and L. lactis expressing combined LLO/tP60 whether by the IP or the oral route. The apparent discrepancy between the findings of Igwe and co-workers and our data has a number of possible explanations. Firstly, L. lactis is non-invasive and is not adapted to survive intracellularly whether in the phagosome or intracytoplasmically (Bahey-El-Din et al., 2008). In contrast Salmonella is an invasive bacterium that is able to persist in the phagosomal compartment, even when attenuated, which means a longer period of antigen production and presentation to the immune system (Carrol et al., 1979). Also, in our hands the level of tP60 expression in NZ9000 ΔhtrA(pNZPnisA : SEC-LLO/tP60) was found to be much lower than the tP60 level expressed by NZ9000 ΔhtrA(pNZPnisA : SEC- tP60) (Fig. 2⇑). Although the reason for this is not clear, it is likely to have an effect on the immunization potential of the combined LLO/tP60-expressing vaccine strain. Finally, the mechanism by which tP60 was intended to access the MHC class I presentation pathway in our study (LLO permeabilization) is different from the approach used by Igwe and co-workers (type III secretion system) (Igwe et al., 2002). Despite these findings we believe that the safety profile of L. lactis suggests that this vector may be more applicable as a delivery vehicle in humans and that further work is necessary to improve vaccine delivery using this platform.

    In brief, we successfully created L. lactis vaccine strains that secrete the listerial antigens LLO and tP60 in combination or singly. In the murine model, no protective immunity was observed upon oral vaccination whilst IP immunization could elicit protective anti-listerial immunity with LLO-producing lactococcal strains. The immunodominant LLO antigen proved to be critical for CD8+ cell-mediated immunity and protection, while tP60 alone did not elicit protection and did not significantly improve the immune outcome upon combined expression with LLO in L. lactis. It is postulated that higher tP60 expression levels may be necessary for sufficient cytoplasmic access of the antigen upon LLO permeabilization of the phagosome.

    Acknowledgments

    The authors would like to thank Ian Monk and Maire Begley for their help in this study. The authors would like also to acknowledge the funding received from the Egyptian Education and Culture Bureau (London) and the funding of the Alimentary Pharmabiotic Centre by the Science Foundation of Ireland Centres for Science, Engineering and Technology (CSET) programme.

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