Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli
Summary
The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression. Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.
Introduction
Live microbial vaccine vectors, viable at target sites of mucosal immunization, represent efficient delivery systems to facilitate immune responses concurrently at mucosal and systemic sites. 1 Observations to date have underlined the superiority of attenuated pathogenic viruses and bacteria over non-replicating antigens for the induction of mucosal immune responses. Oral subunit-vaccine approaches based upon peptides or purified recombinant proteins may therefore be deficient in this one important requisite, the induction of protective immunity in the gastrointestinal (GI) tract itself. 2–4 Oral vaccination remains safe and inexpensive whilst maintaining the potential for single-dose immunity, contributing therefore to improved compliance rates in vaccination programmes. Currently, vaccination against tetanus involves tetanus toxoid (TT) formulations and maintains poor coverage and contamination concerns that run concurrent with all needle-delivery vaccines. Tetanus toxin fragment C (TTFC) is the 50 000-molecular weight (MW) non-toxic papain cleavage product of the tetanus holotoxin and is an alternative protective immunogen that is currently utilized in several live-vector systems under development. 5 The delivery of vaccine subunits to the mucosal surfaces by a suitable live microbial vector is a rational response to the obstacles encountered by parenteral vaccines. However, potential safety and environmental considerations, particularly the immune status of the vaccine recipients in developing countries, still negates employment of the majority of mucosally delivered vector candidates such as Escherichia coli, Salmonella and Vaccinia virus. 6 Therefore, non-pathogenic, food grade or commensal bacterial vectors have begun to receive attention for their vaccine potential.5,7,8
Commensal bacteria maintain a sophisticated, non-invasive ecology with the host and although surveyed by the immune system are not necessarily susceptible to immune clearance from their ecological niches. 9 The predominance of lactobacilli in various regions of the aerodigestive tracts indicates their particular potential as live oral vaccines. Their ‘generally recognized as safe’ (GRAS) status is evident from applications in the food industry,10,11 and their capacity to enhance immune responses has been demonstrated with co-administered DxRRV rhesus-human reassortent oral rotavirus vaccine 12 and trinitrophenyl (TNP)-conjugated antigen, 13 effects probably attributable to the macrophage-activating and interferon-γ (IFN-γ)-inducing properties of the Gram-positive peptidoglycan 14 and lipoteichoic acid fractions. 15
Immune homeostasis at the mucosa, in which lactobacilli participate, is a combination of physical exclusion, immunoglobulin A (IgA) secretion and active regulation by T-cell subsets. 9 In this study we investigated the possibility of avoiding antigen-specific peripheral tolerance following oral delivery (oral tolerance) by vaccinating with TTFC in particulate form (contrasting soluble dietary antigens), to permit processing and presentation by mechanisms ordinarily contributing to immune regulation of intestinal flora. In particular, cell-surface presentation of antigen by recombinant bacteria has been reported as immunologically advantageous.16,17 However, as proteolytic environments in nasal and GI tracts are a potential basis for antigen degradation, in this report we examined whether intracellular accumulation of TTFC in lactobacilli preferentially maintains epitope integrity and consequently effective immunogenicity. Our data indicate that the Lactobacillus plantarum 256 strain is more effective in this respect than L. casei 393, and that delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression under the experimental conditions tested.
Materials and methods
Recombinant DNA techniques
Escherichia coli DH5α was used as a host strain for manipulation of the previously described pLP401 or pLP503 shuttle vectors. 18 The 1329-bp DNA coding for TTFC (kindly provided by A. Mercenier, Lille, France) was elongated at its 3′ end with XhoI and NcoI restriction sites using the polymerase chain reaction (PCR) to facilitate cloning into the pLP401 and pLP503 shuttle vectors.
Prior to transfer of the plasmids into lactobacilli by electroporation, the Tldh terminator sequence present in the shuttle vectors was removed by NotI digestion, resulting in the pLP401 or pLP503 plasmids defined in Table 1. Religation of the vectors juxtaposed the TTFC protein-encoding sequences in-frame with the codons of the translation initiation region present downstream of the regulatable amylase gene (α-amylase) or constitutive lactate dehydrogenase (ldh) gene promoter sequences present in pLP401 and pLP503, respectively. Following electroporation of competent lactobacilli, transformants were selected on erythromycin (5 µg/ml) agar plates. General molecular cloning techniques and transformation of lactobacilli were carried out essentially as described previously. 18
| Strain or plasmid | Relevant genotype or phenotype | Source/reference |
|---|---|---|
| L. plantarum | 256, Wild type | This work |
| L. casei | 393 | ATCC |
| pLP503-TTFC | p-ldh, ampr, eryr, TTFC | Pouwels et al. (1996) 7 |
| pLP401-TTFC | p α-amylase, ampr, eryr, ssAmy, Anchor, TTFC | Pouwels et al. (1996) 7 |
- amp r, ampicillin-resistance gene; anchor, anchor peptide (117 aa) encoding sequences of Lactobacillus casei;eryr, erthromycin resistance gene; p α-amylase, promoter sequence of the L. amylovorusα-amylase gene; p-ldh, promoter sequence of the L. casei ldh gene; ssAmy, sequences encoding the secretion signal (36 aa) of the α-amylase gene of L. casei; TTFC, 1329 bp of DNA encoding fragment C of tetanus toxin.
Lactobacillus strain selection
Lactobacillus spp. appropriate as host strains for transformation were identified by quantitative cultures of faecal samples obtained following inoculation of mice with single intragastric doses of 109 cells of a wide panel of rifampicin-resistant wild-type lactobacilli. L. plantarum 256 (kindly provided by P. Conway, Sydney, Australia), which persisted in the GI tract for up to 12 days, and L. casei (American Type Culture Collection [ATCC] 393, Baltimore, MD), which became undetectable within 72 hr, were selected as prototype host strains (data not shown). Following transformation, both L. casei and L. plantarum recombinant strains exhibited identical GI-tract persistence characteristics as compared to their wild-type strains (data not shown).
Gel electrophoresis and Western blotting
Recombinant L. casei and L. plantarum containing the plasmids listed in Table 1 were routinely prepared from glycerol stocks by semianaerobic overnight growth of a 1 : 50 dilution in Mann–Rogosa–Sharp (MRS) medium containing 5 µg/ml erythromycin.
Transformants with plasmids containing the constitutive ldh promoter were optimally grown at 37° in antibiotic selective lactobacillus-carrier medium (LCM) medium containing 2% (w/v) glucose. Transformants with plasmids containing the regulatable α-amylase promoter were grown by diluting an overnight culture of cells 1 : 50 in LCM medium containing 2% (w/v) mannitol. Total cell extracts were obtained from the bacteria by sonicating the cells four times on a 30-second on/30-second off cycle, using a W870 Branson sonicator, to release both cytoplasmic and cell membrane-bound proteins.
Proteins in 30 µg of total cell extracts or fractions were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% acrylamide, 400 m m Tris [pH 8·9]) run in a 25-m m Tris, 192 m m glycine buffer (pH 8·3) at 200 V for 45 min. Protein was transferred electrophoretically onto nitrocellulose using a Bio-Rad (Richmond, CA) electrophoresis unit. Immunoblots were developed using optimally diluted rabbit TTFC-specific antiserum and goat-anti-rabbit immunoglobulin G (IgG)-specific phosphatase conjugates (Nordic, Tilburg, the Netherlands).
Flow cytometric analysis of cell-surface expression of TTFC
At defined time-points, bacteria were prepared for analysis by fluorescence-activated cell sorter (FACScan; Becton-Dickinson, San Jose, CA) analysis. Cells were washed twice and resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Fifty microlitres of optimally diluted rabbit TTFC-specific antiserum was added to the cells for 1 hr. Cells were again washed twice and bound antibody was detected by a 30-min incubation with fluorescein isothiocyanate-conjugated (FITC) anti-rabbit antibody at a dilution of 1 : 1000. Cells were then washed twice prior to analysis for light scatter and fluorescence using a FACscan. A gate was set around appropriate size events, as determined by cytograms of forward and side scatter. Controls were prepared by staining wild-type L. casei 393 or L. plantarum 256 recombinants using non-immune rabbit serum or by excluding the rabbit TTFC-specific antiserum. All procedures were performed on ice with 1% BSA. For each sample, data was collected for 10 000–20 000 gated events. The fluorescence obtained from bacterial cell suspensions was represented by fluorescence histograms, and mean channel intensities were calculated.
Immunization
BALB/c or C57BL/6 mice, aged 6–8 weeks, were immunized intragastrically or intranasally with preparations of recombinant L. casei or L. plantarum that expressed TTFC. Bacteria obtained from the overnight cultures were diluted 1 : 50 in MRS or LCM medium containing 1% glucose and grown for 6 hr at 37° until an optical density (OD) at 695 nm of between 0·6 and 0·8 (mid-exponential phase) was reached. Cells were pelleted by centrifugation at 4°, washed once with PBS and appropriate concentrations of bacteria were prepared in sterile PBS.
For oral immunization, 2–5 × 109 cells were administered intragastrically in a 250-µl volume of 0·2 m NaHCO3 on three consecutive days. For intranasal immunization, 2–5 × 109 cells were administered to the nares of non-anaesthetized mice in a 20-µl volume of PBS. Control mice received identical doses of wild-type lactobacilli. Plate counts were performed with all inoculum samples to confirm the number of colony-forming units (CFU) administered to the mice.
Sample collection and enzyme-linked immunosorbent assay (ELISA)
Serum was prepared from blood samples obtained from the tail vein of preimmune mice and at subsequent 7-day intervals beginning 21 days following immunization.
To obtain brochoalveolar lavage samples, mice were killed at specific time-points and the lungs were cannulated and washed repeatedly with 0·7 ml of PBS containing 0·1% BSA, following which the collected wash volume was centrifuged at 1000 g and the supernatants stored at −80°.
Antigen-specific IgG levels were evaluated as described previously, 13 using microtitre plates coated overnight with 50 µl of a 0·16-µg/ml solution of TT (RIVM, Bilthoven, the Netherlands). Individual serum samples were titrated by serial log2 dilutions and assayed in duplicate. Bound antibody was detected by the addition of 50 µl of optimally diluted goat anti-mouse IgG-phosphatase conjugate (Nordic). Following addition of the p-nitrophenyl phosphate (PNPP) chromogen substrate [1 mg/ml in 0·1 m di-ethanolamine (DEA)/MgCl2], antibody levels were quantified by measuring plate absorbance (A), at 405-nm, 30–90 min following initiation of the reaction. End-point titres were calculated using a cut-off determined from the mean absorbance (A = 0·2) of a 1 : 10 dilution of serum obtained from preimmune mice. For evaluating antigen-specific immunoglobulin A (IgA) levels in bronchoalveolar lavage fluid, an identical procedure was performed, using an optimally diluted goat anti-mouse IgA phosphatase conjugate (Nordic).
Antigen-specific T-lymphocyte proliferation assays and enzyme-linked immunospot (ELISPOT) analysis
At 12 and 21 days following the last immunization, the spleens and cervical lymph nodes (CLN) of mice were removed aseptically. Single-cell suspensions were prepared by passage through a cell strainer (70 µm Nylon; B & D, Le Pont de Claix, France) and centrifugation at 480 g for 10 min. Viable, unfractionated cell numbers were assessed by Trypan Blue dye exclusion.
For testing antigen-specific T-lymphocyte proliferation, 19 cells were resuspended and plated at concentrations of 3 × 105 cells/spleen or 5 × 105 cells/lymph node (LN) in a final volume of 200 µl of culture medium (RPMI-1640, supplemented with 10% heat-inactivated fetal calf serum [FCS], 2 m m l-glutamine, 20 U/ml of penicillin and 20 µg/ml of streptomycin; all Gibco, Paisley, Strathclyde, UK), and 50 µm 2-mercaptoethanol (Sigma, St Louis, MO) in sterile flat-bottomed 96-well culture plates (Nunc, Roskilde, Denmark). Control wells contained medium only, and antigens were added to triplicate cultures over the indicated dose range. All cells were maintained in a humidified 5% CO2 atmosphere at 37° for 4 days. The cells were pulse-labelled with 0·6 µCi of [3H]thymidine ([3H]TdR, 5 Ci/mmol, TRA 120; Amersham, Bucks., UK) in 30-µl volumes/culture well during the last 16–18 hr before harvesting. Cells were collected using an ILACON cell harvester and deposited onto glass fibre filter discs (Whatman, Kent, UK). [3H]TdR incorporation was assessed by gas scintillation spectrometry (β-plate counter, Canberra Packard, Meriden, CT) and the results were calculated as the mean c.p.m (± SD) from triplicate cultures and expressed as a stimulation index (SI).
Quantifying the number of TT-specific antibody-secreting cells (ASC) in the spleen and CLN was undertaken according to Czerkinsky et al. 20 Microtitre plates (Maxisorp plates, Nunc) were coated overnight with 50 µl of a 0·16-µg/ml solution of TT (RIVM) or 50 µl of PBS as a control. After extensive washing and blocking of the plate with PBS containing 0·1% BSA, cells were added at concentrations of 1 × 106 and 2 × 105 cells/spleen or 5 × 105 and 1 × 105 cells/LN into a final volume of 50 µl culture medium and incubated for 4 hr at 37° in a humidified 5% CO2 atmosphere. The plates were rinsed and incubated for 20 min with ice-cold PBS containing 10 m m EDTA to remove the cells, and washed again with PBS containing 0·05% Tween-20 and then with PBS containing 0·5% BSA. Bound antibody was detected by the addition of 50 µl of optimally diluted rabbit anti-mouse immunoglobulin phosphatase-conjugate (DAKO, Glostrup, Denmark) overnight at 4°. Plates were washed extensively and incubated with 1 mg/ml of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) in amino methyl propanol (AMP) buffer containing 1% low-melting-temperature agarose. The plates were inverted over a light source and the number of blue dots was scored macroscopically.
Results
Expression of recombinant TTFC in lactobacilli
Expression of cytoplasmic or cell wall-bound TTFC by L. plantarum and L. casei transformants was demonstrated by collection of cells in mid-exponential phase followed by sonication to disrupt the bacteria. Proteins were separated by SDS–PAGE and immunoblots developed using TTFC-specific rabbit antiserum. As shown in Fig. 1(a), lactobacilli containing pLP503-TTFC expressed only the intracellular 50 000-MW TTFC polypeptide. L. plantarum containing the vector pLP401-TTFC expressed a surface-anchored 75 000-MW polypeptide corresponding to the 50 000-MW TTFC fused to an anchor sequence of 25 000 MW, at a level higher than previously reported 15 for L. casei pLP401-TTFC. Exposition of TTFC on the cell-wall of L. plantarum and L. casei through fusion to the anchor sequence was confirmed by FACS analysis ( Fig. 1b).
(a) Expression of tetanus toxin fragment C (TTFC) from Lactobacillus casei and L. plantarum transformants. pLP401-TTFC transformants (surface-anchored expression) were grown in LCM medium (+ 2% mannitol) and pLP503-TTFC transformants (intracellular expression) were grown in MRS (both supplemented with 5 µg/ml erythromycin), at 37° to an optical density (OD) at 695 nm of 0·6, pelleted and disrupted by sonication. Thirty micrograms of total protein was analysed on a 10% sodium dodecyl sulphate–polyacrylamide gel, and separated proteins were transferred electrophoretically to nitrocellulose. The TTFC was visualized using rabbit anti-TTFC (1 : 500 dilution) and a phosphatase/p-nitrophenyl phosphate (PNPP) chromogen combination. Lane 1, 0·5 ng of purified TTFC; lane 2, L. plantarum pLP503-TTFC; lane 3, L. plantarum pLP401-TTFC; lane 4, L. plantarum 256; lane 5, L. casei pLP503-TTFC; lane 6, L. casei 393; lane 7, molecular weight markers. (b) Immunofluorescence analysis of recombinant L. plantarum pLP401-TTFC (black shading) and L. casei pLP401-TTFC (grey shading) expressing TTFC as a surface-anchored product. Lactobacilli were gated on the basis of forward and side scatter and stained with rabbit TTFC-specific antiserum diluted 1 : 500. Bound antibody was detected with optimally diluted fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (IgG). Fluorescence levels from cells collected at an OD 695 nm of 0·6 were analysed using a fluorescence-activated cell sorter and are shown in histogram form, presented in relation to levels of fluorescence obtained with non-recombinant lactobacilli (no shading). Ten-thousand cells were analysed in each experiment.
TTFC-specific antibody responses after intranasal immunization with recombinant lactobacilli
BALB/c and C57BL/6 mice receiving three intranasal doses of L. plantarum or L. casei (expressing intracellular TTFC) on days 1–3 were strongly primed for a secondary response to TTFC following booster intranasal administrations on days 28–30. The titre of the TTFC-specific response in BALB/c mice following immunization with recombinant L. plantarum was higher (although not significantly) than recombinant L. casei ( Fig. 2a), with IgG detectable as rapidly as day 28, rising to titres of 103·5 and 102·9 by day 49, respectively.
Tetanus toxin fragment C (TTFC)-specific immunoglobulin G (IgG) levels following immunization of groups of mice with live recombinant lactobacilli. Serum was collected from preimmune mice and at 7-day intervals, beginning on day 7. TTFC-specific serum IgG levels in individual or pooled serum was measured by enzyme-linked immunosorbent assay (ELISA) in microtitre plates coated overnight at 4° with 0·16 µg/ml of tetanus toxoid in phosphate-buffered saline (PBS). Bound antibody was detected by the addition of anti-mouse alkaline phosphatase (AP) conjugate and p-nitrophenyl phosphate (PNPP) substrate. The absorbance (A) 405 nm values of each well were measured at 90 min. End-point titres were determined using a cut-off value calculated as the mean A + 2 SD (≈ 0·2) of preimmune sera diluted 1 : 10. (a) Three BALB/c mice were immunized intranasally with three doses of 5 × 109Lactobacillus plantarum pLP503-TTFC (●) or with three doses of 5 × 109L. casei pLP503-TTFC () in 20 µl of PBS on days 1–3. Identical booster immunizations were administered on days 28–30. (b) Sixteen C57BL/6 mice were immunized with three doses of 5 × 109L. plantarum pLP503-TTFC intranasally in 20 µl of PBS (●) or orally in 200 µl of NaHCO3 () on days 1–3. Identical booster immunizations were administered on days 28–30. (c) Three C57BL/6 mice were immunized with 5 × 109L. plantarum pLP503-TTFC intranasally in 20 µl of PBS on days 1 and 28 (▪) or with three doses of L. plantarum pLP401-TTFC intranasally in 20 µl of PBS on days 1–3 followed by a booster with either 5 × 109L. plantarum pLP503-TTFC on days 28–30 (), or L. plantarum pLP401-TTFC on days 28–30 and 49–51 (;), intranasally in 20 µl of PBS.
C57BL/6 mice demonstrated mean end-point titres higher (although not significantly) than BALB/c mice (see Fig. 2b) and therefore were selected for further analysis by intranasal immunization using a single-dose priming and booster schedule with the L. plantarum transformants that expressed TTFC intracellularly ( Fig. 2c). In comparison to the results presented in Fig. 2(c) obtained in BALB/c mice, higher mean titres of serum IgG (104·2) were induced in C57BL/6 by day 49. However, regardless of whether a triple- or single-dose priming and boosting immunization schedule was used, C57BL/6 mice demonstrated serum IgG end-point titres that were not significantly different.
In contrast, in order to induce TTFC-specific serum IgG in C57BL/6 mice immunized intranasally with L. plantarum expressing TTFC at the cell surface, it was necessary to prime mice on days 1–3 and subsequently boost at least twice (on days 28–30 and 49–51) before antigen-specific responses were measurable ( Fig. 2c). Interestingly, however, mice primed (as described above) with the L. plantarum pLP401-TTFC did induce TTFC-specific serum IgG (with more rapid kinetics than responses obtained from naive mice) when boosted on days 28–30 using the L. plantarum pLP503-TTFC transformant ( Fig. 2c). This observation, intriguingly, implies that antigen-specific sensitization of the mice has in fact occurred following administration of L. plantarum transformed with pLP401-TTFC, although maturation or measurability of the immune response is, for numerous reasons, prevented from occurring. In surprising contrast, however, L. casei expressing TTFC on the cell surface failed to demonstrate any priming effect in either BALB/c or C57BL/6 strains as did the L. plantarum transformant itself administered on days, 1, 28 and 56 as single doses (data not shown).
In addition, as shown in Fig. 3(a), 3(b), C57BL/6 mice receiving three doses (on days 1–3) of L. plantarum expressing TTFC were primed mucosally for a TT-specific IgA response in bronchoalveolar lavage fluids measured 12 and 21 days following booster intranasal administration on days 28–30. Bronchoalveolar lavages obtained from mice immunized intranasally with wild-type L. plantarum 256 demonstrated no reactivity with TT coated onto microtitre plates either on day 12 or day 21, respectively (data not shown).
Induction of tetanus-toxoid specific immunoglobulin A (IgA) antibodies in bronchoalveolar lavage (BAL) fluids after intranasal immunization of mice with Lactobacillus plantarum-pLP503-tetanus toxin fragment C (TTFC). C57BL/6 mice were immunized on days 1–3 with 5 × 109L. plantarum pLP503-TTFC, followed by an identical booster immunization on days 28–30, either intranasally (a) and (b) or orally (c) and (d). On day 12 (a) and (c), and day 21 (b) and (d), after the last boost four animals per group were killed and BAL were obtained by flushing the lung, through a cannula, with 0·7 ml of phosphate-buffered saline (PBS) containing 0·1% bovine serum albumin (BSA). TT-specific IgA in these samples was measured with enzyme-linked immunosorbent assay (ELISA) in microtitre plates coated overnight at 4° with 0·16 µg/ml of TT in PBS. Bound antibody was detected by the addition of anti-mouse alkaline phosphatase (AP) conjugate and p-nitrophenyl phosphate (PNPP) substrate. The absorbance (A) 405 nm values of each well were measured after overnight incubation at 4°.
TTFC-specific antibody responses after oral immunization with recombinant lactobacilli
In a comparative study of intranasal versus oral immunization, C57BL/6 mice received three doses of L. plantarum expressing intracellular TTFC on days 1–3, followed by booster administrations on days 28–30. Following oral immunization, induction of TTFC-specific serum IgG responses occurred in nine of the 16 mice tested ( Fig. 2b). After intranasal immunization, all 16 mice responded with high TTFC-specific end-point titres, with a mean titre of 104·3 within 7 days of the booster, remaining high for 3 weeks. The mean end-point titres of the oral responders was 101·6 at day 7 and reached a peak of 102·4 at 14 days after boosting. In contrast to the intranasal group, in orally immunized mice no TT-specific IgA response could be measured in bronchoalveolar lavages on either day 12 or 21, respectively ( Fig. 3c, 3d).
In an additional study, mice were orally primed on days 1, 2 and 3 and boosted 2, 3 or 4 weeks later. Irrespective of the timing of the boost, a TTFC-specific IgG response was observed within 7 days of the booster inoculation (data not shown).
In contrast, BALB/c or C57BL/6 mice immunized orally with the L. casei transformants (expressing TTFC either intracellularly or surface-anchored), or L. plantarum expressing TTFC on the cell surface, failed to induce detectable TTFC-specific serum IgG responses at all equivalent time-points examined (data not shown). Mice receiving wild-type lactobacilli or irrelevant vectors demonstrated no TTFC-specific serum IgG responses at any time-point (data not shown).
TT-specific ASC and T-cell responses in spleens and CLN
C57BL/6 mice were immunized either orally or intranasally on days 1–3 with either 5 × 109L. plantarum pLP503-TTFC transformants or the wild-type L. plantarum 256 as a control. This was followed by an identical booster immunization on days 28–30. At 12 or 21 days following the last boost, mice were killed and spleens and CLN cell suspensions were prepared.
In Table 2, the number of TT-specific ASC, determined by ELISPOT, present in spleen or CLN are shown. Following intranasal immunization with L plantarum transformants, high numbers of TT-specific ASC were found in spleens and CLN of intranasally immunized mice, at 12 days after the last boost, and these numbers had decreased by day 21. These data suggest that antigen-specific sensitization of the mice had occurred locally at the CLN level, as well as systemically in the spleen. In the orally immunized group only two out of eight spleen cell suspensions tested contained TT-specific ASC, whereas no ASC was found in the CLN.
| Groups * | Day 12 | Day 21 | ||
|---|---|---|---|---|
| Spleens | CLN | Spleens | CLN | |
| Oral L. plantarum 256 | 0 † | 0 | 0 | 0 |
| Oral L. plantarum TTFC | 0 | 0 | 0·4 (0–2) | 0 |
| Intranasal L. plantarum 256 | 0 | 0 | 0 | 0 |
| Intranasal L. plantarum TTFC | 48 (7–107) | 195 (156–233) | 16 (4–31) | 13 (5–27) |
- *Sixteen C57Bl/6 mice per group were immunized intranasally on days 1–3 or orally with 5 × 10 9 Lactobacillus plantarum (either L. plantarum 256 or L. plantarum pLP503-TTFC transformants), followed by an identical booster immunization on days 28–30. Twelve or 21 days after the last boost, eight animals per group were killed and cell suspensions were prepared from spleens and cervical lymph nodes (CLN; pooled per two animals). The amount of TT-specific immunoglobulin-producing cells were determined using ELISPOT.
- †The data, presented as ASC per 10 6 cells, represent the mean (range) of eight spleen samples or four CLN samples, measured in triplicate wells.
- TTFC, 1329 bp of DNA encoding fragment C of tetanus toxin.
In Fig. 4, the antigen-specific T-cell responses of spleen or CLN are presented. In mice immunized intranasally with the L. plantarum pLP503-TTFC transformants, proliferation was measurable following restimulation with TTFC, TT or TT peptide P30, suggesting that intranasal immunization induced specific immunity initially via local LN activation. Antigen-specific T-cell responses of spleen and CLN were also obtained in BALB/c mice (data not shown). No antigen-specific proliferation was observed in cells obtained from mice immunized orally or identically to wild-type L. plantarum ( Fig. 4).
Induction of antigen-specific T cells by intranasal immunization of mice with Lactbacillus plantarum pLP503 tetanus toxin fragment C (TTFC). C57BL/6 mice were immunized on days 1–3 intranasally or orally with 5 × 109L. plantarum (either L. plantarum 256 or L. plantarum pLP503-TTFC transformants), followed by an identical booster immunization on days 28–30. Twelve (a) and (c) or 21 (b) and (d) days after the last boost, eight animals per group were killed and spleen (a) and (b) and cervical lymph node (CLN) (pooled per two animals) (c) and (d) cell suspensions were prepared. The cells were examined for [3H]thymidine incorporation following in vitro incubation of 3 × 105 spleen cells or 5 × 105 CLN cells per well for 72 hr with TTFC, TT, TT peptide P30 or medium alone. [3H]Thymidine was added to the cultures for the final 18 hr of incubation. Results are expressed as the stimulation index (SI) calculated from the mean counts per minute (c.p.m.) of triplicate test cultures of cells divided by the mean c.p.m. of cultures receiving buffer alone. The background values of cultures receiving buffer alone varied as follows: (a) 220–760 c.p.m.; (b) 150–240 c.p.m.; (c) 1000–4000 c.p.m.; and (d) 150–560 c.p.m.
Discussion
The purpose of this study was to evaluate whether the heterologous gene-expression capabilities recently developed for Lactobacillus spp. had provided a tool for appropriate antigenic profiling and effective antigen delivery to mucosal surfaces. In the present work, the pLP401/pLP503 plasmids uniquely comprised fully homologous Lactobacillus gene-expression elements that enabled directed (surface or intra-cellular) and regulatable expression of TTFC at levels and efficiencies not previously attainable in Lactobacillus.4,21 In addition, the L. plantarum pLP503-TTFC that expressed TTFC intracellularly was demonstrated to be highly immunogenic following intranasal delivery, priming on days 1–3 and boosting on days 28–30. Immunogenicity was shown at the systemic level by high TT-specific IgG serum titres, as well as at the mucosal level showing TT-specific IgA in the BAL fluids. In addition, TT-specific ASC and antigen-specific T-cell proliferation were demonstrated at the systemic level in spleens as well as locally in CLN. Following oral delivery of L. plantarum pLP503-TTFC, low to moderate titres of TT-specific serum IgG was measured.
The results reported in this study confirm the efficiency of the nasal route for immunization, and imply that the L. casei and L. plantarum are sampled efficiently by the M-like cells found in the nasal tracts 22 and are capable of inducing substantial levels of immunoglobulins in serum. The variation between the recombinant strains in regard to the levels of TTFC expression, sustainable levels of non-degraded TTFC and persistence in the GI tract, may have pivotal influences on the potency of the recombinant vaccine and may account for the often surprising observations distinguishing the L. plantarum and L. casei strains.
In contrast to our expectations, this present report demonstrates that the L. plantarum pLP401-TTFC recombinant strains, expressing relatively low levels of surface-exposed TTFC, are immunogenic following intranasal administration, although it was necessary to prime mice (on days 1–3) and subsequently boost twice (on days 28–30 and 49–51) before antigen-specific responses were measurable. Although surface expression of TTFC would enable direct binding by immunoglobulins present on the surface of B cells, potentially augmenting immunogenicity, this surface antigen expression may be particularly susceptible to low pH, bile acid or proteolytic environments encountered by vaccine vectors following mucosal immunization. Interestingly, however, mice primed, as described above, with the L. plantarum pLP401-TTFC did induce TTFC-specific serum IgG (with more rapid kinetics than responses obtained from naive mice), when boosted as early as days 28–30 using the L. plantarum pLP503-TTFC transformant instead. This observation, intriguingly, implies that antigen-specific sensitization of the mice has in fact occurred following administration of L. plantarum transformed with pLP401-TTFC, although enhancement, maturation or measurability of the immune response is, for numerous reasons, prevented. This would suggest that high levels of ‘stable’ surface expression may not be as critical as accumulated intracellular antigen levels, in order to minimize antigen degradation and ensure that sufficient antigen remains available to immune inductive sites.
The present study provides a significant endorsement for Lactobacillus-based vaccines by extending the observations of immunogenicity following nasal immunization to demonstrate, for the first time, that oral immunization of C57BL/6 mice with 5 × 109L. plantarum expressing TTFC intracellularly induces TTFC-specific serum IgG responses. In contrast, BALB/c or C57BL/6 mice immunized orally with the L. casei trans-formants (expressing TTFC either intracellularly or surface-anchored), or L. plantarum expressing TTFC on the cell surface failed to induce detectable TTFC-specific serum IgG responses.
L. casei and L. plantarum were selected for study owing to their different durations of persistence in the GI tract. It is interesting to speculate that the differences in persistence do explain the differences found in immunogenicity. However, in the preliminary studies reported here we are obliged to assume only that the recombinant strains function as particulate antigen-delivery vehicles, and to be cautious not to claim which of the multitude of additional factors may combine to influence the immunogenicity of the lactobacilli. This caution is especially pertinent as the dose of cells delivered (109) represents an ecologically extreme quantity that overwhelms the homeostatic levels established in the normal murine gut, making assessment of the impact of viable versus non-viable cells difficult to ascertain. In this regard, Lactococcus lactis has proved to be a potent delivery vehicle for TTFC, in spite of its non-viable and therefore non-replicating status on entry into the intestinal tract. The absolute levels of the TTFC expressed by the lactobacilli may particularly impact immunogenicity, and the capacity of the lactobacilli to immunomodulate more precisely may well be contingent on attributes that are present only in live bacterial populations.
The antigen-delivery vehicles described in this work emphasize the need to carefully define host–vector combinations and to evaluate the impact that cell viability, cell numbers, adhesion to the mucosa and the mechanism of triggering of the immune system has on the immunogenicity of the recombinant lactobacilli. This first demonstration of TTFC immunogenicity following delivery of recombinant lactobacilli by the oral route has endorsed the continued development of safe Lactobacillus-based oral neonatal vaccines to combat, for example, the current 400 000 deaths, recorded annually, that are caused by tetanus. 23 Accelerating the kinetics of the response and increasing the specific immune responses at systemic and mucosal levels by defining optimal host–vector combinations, will provide a sound basis to analyse protective efficacy against lethal toxin challenge as well as an expanded application of this technology to a catalogue of additional pathogens that manifest their pathology through the mucosal surfaces.
Acknowledgments
We appreciate the technical contributions of Claudia Antonissen. This study was supported in part by an EC-Biotech (B104-CT-0542) award. We thank Dr P. Conway, University of New South Wales (Sydney, Australia) for kindly providing L. plantarum 256 and Dr Annick Mercenier (Institute Pasteur de Lille, France) for providing the gene encoding TTFC.
