Mucosal Immune Responses and Protection against Tetanus Toxin after Intranasal Immunization with RecombinantLactobacillus plantarum

Bacterial inocula.Two recombinant L. plantarumNCIMB8826 TTFC-producing strains were used in this study. They were obtained by electroporation with a plasmid carrying the TTFC-encoding gene under the control of a constitutive (p_ldh) or inducible (p_nisA) promoter, pMEC4 (erythromycin resistance) or pMEC46 (chloramphenicol resistance), respectively (24; Chagnaud et al., unpublished). A control strain harboring the nonexpressing plasmid pTG2247 (chloramphenicol resistance) was used as a negative control (7). Bacteria were grown in MRS broth (Difco, Detroit, Mich.) containing the appropriate antibiotic, erythromycin or chloramphenicol, at a final concentration of 5 or 10 μg per ml, respective. An overnight culture was used to inoculate fresh medium at a 1:20 dilution, and cells were grown to the exponential phase (optical density at 660 nm [OD₆₆₀] of ≈2). For strain 8826(pMEC46), induction was performed by adding 20 ng of nisin (Sigma, St. Louis, Mo.) per ml 1 h after dilution and further incubation at 37°C for 5 h (24). The cells were then washed twice with sterile phosphate-buffered saline (PBS) and adjusted to 10¹¹ CFU per ml. For chemical inactivation of bacteria, suspensions of 5 × 10⁹ CFU per ml in MRS medium were treated with 200 μg of mitomycin C (MitC; Sigma) per ml for 90 mn at 37°C. The treated cells were washed twice in sterile PBS and resuspended at 10¹¹CFU per ml for immunization. An aliquot of serial dilutions was plated on selective MRS agar in order to calculate the efficiency of the treatment.

Heat-killed bacteria were prepared for T-cell in vitro stimulation by heating a cell suspension of wild-type L. plantarumNCIMB8826 (10⁹ CFU per ml in PBS) for 1 h at 70°C. The absence of viable strains was checked by plating on MRS agar.

Immunoblotting assay.Ten microliters (10⁹ CFU) of each inoculum was resuspended in 1 ml of 10 mM Tris (pH 8) buffer containing lysozyme (1 mg/ml; Sigma). After 30 min of incubation at 37°C, the cells were washed once in 10 mM Tris-HCl (pH 8) and lysed by addition of 100 μl of lysis buffer (10% glycerol, 2% Sodium dodecyl sulfate, 375 mM Tris-HCl [pH 7.6]). An aliquot (10 μl) of each cell extract was submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 10% gel as described by Laemmli (12). After transfer to nitrocellulose membranes by electroblotting, the specific proteins were detected with a rabbit anti-TTFC polyclonal serum (kindly provided by Innogenetics N.V., Ghent, Belgium) and revealed by alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG; Promega, Madison, Wis.).

Immunizations.Eight-week-old female C57BL/6 N Crl BR mice (Charles River Laboratories, St. Aubin-les-Elbeuf, France) were immunized intranasally. Initially, groups of eight mice were anesthetized by intraperitoneal injection of 200 μl of a 5% sodium pentobarbital solution (Sanofi, Libourne, France) or 150 μl of a cocktail (as described in reference 2) containing 20% Imalgene 1000 (Merial, Lyon, France), 0.5 mg of Valium (Roche, Neuilly-sur-Seine, France), and 62.5 μg of atropine (Aguettant Laboratory, Lyon, France) per ml. Ten microliters (10⁹ CFU) of the bacterial suspension was then instilled into one nostril of each mouse. Control groups received 10 μl of buffer alone (PBS) or 10 μg of purified recombinant TTFC (Boehringer Mannheim, Mannheim, Germany) mixed with 1 μg of cholera toxin B subunit (CTB) as the mucosal adjuvant (Sigma). Antigens were given at 3-week intervals by one (single dose) or two consecutive (double dose at 24-h interval) intranasal administrations. Ten days after each administration (priming and two boosts), serum samples were collected and stored at −20°C until use. Bronchoalveolar lavage fluids (BALF) were obtained 10 days after the final boost by three consecutive lavages of the lungs with a total volume of 400 μl of PBS containing complete protease inhibitor cocktail (Boehringer Mannheim). After 10 min of centrifugation at 4,000 × g (at 4°C), the supernatants were collected and stored at −20°C until analysis.

ELISA for detection of antigen-specific serum or mucosal antibody responses.Purified antigen (TTFC; Boehringer Mannheim) was coated overnight at 4°C on enzyme-linked immunosorbent assay (ELISA) plates (Immulon I; Dynatech, McLean, Va.) at 200 ng per 100 μl in 0.1 M carbonate-bicarbonate buffer (pH 9.5). After 1 h of saturation with PBS containing 3% bovine serum albumin (BSA; Sigma), samples were tested using twofold serial dilutions from a 1:50 dilution (for primary antisera) or a 1:2 dilution (for BALF) in PBS containing 1% BSA. After overnight incubation at 4°C, the plates were washed three times in PBS containing 0.1% Tween, and secondary anti-mouse biotinylated conjugate was applied in PBS containing 1% BSA and 0.1% Tween 20 at a 1:20,000 or 1:2,000 dilution for anti-mouse IgG (and IgG subtypes) or anti-mouse IgA (Southern Biotechnology Associates, Inc., Birmingham, Ala.), respectively. After 1 h of incubation at room temperature, horseradish peroxidase-conjugated streptavidine (Amersham, Buckinghamshire, United Kingdom) was applied at a 1:2,000 dilution in PBS containing 0.1% Tween for 30 min. After intensive washes (eight times), 1 mg of o-phenylenediamine substrate (Sigma) per ml in 0.2 M Na₂HPO₄–0.1 M citrate buffer (pH 5.5) containing 0.2% H₂O₂ was added and incubated for 30 min at 37°C. The reaction was stopped by addition of 50 μl of 2 N HCl, and the OD₄₉₀ was measured with an Elx800GUV automated microplate reader (Bio-Tek Instruments, Inc., Vinooski, Vt.). Endpoint titers were calculated as the reciprocal of the dilution producing the same OD₄₉₀ as 3 times the background, using the KC4 program (Kineticalc for Windows; Bio-Tek Instruments).

As the concentration of total IgA in BALF is not constant, the amount of TTFC-specific IgA was normalized to the total IgA concentration in each sample. Total IgA levels were determined by ELISA, as described above, with microplates coated with 100 μl of anti-mouse IgA α chain specific (Sigma) at 5 μg per ml. The concentration of each sample was calculated from a standard curve of mouse myeloma IgA_k (Sigma) with twofold serial dilutions from 0.1 μg per ml. Endpoint titers were calculated as described above for IgG. Results are expressed as specific activity calculated by dividing the endpoint titer by the level of total IgA concentration for each serum.

Statistical analysis.The results are expressed as the mean ± standard error of the mean (SEM). Statistical significance was evaluated by Mann-Whitney U test. Differences were considered significant at P < 0.05 .

TTFC-specific T-cell response.Cervical lymph nodes (CLN) were aseptically removed (10 days after the last boost) and pooled for each group of animals; single-cell suspensions were prepared by mechanical dissociation using a homogenizer tube and passage through a nylon cell strainer (Becton Dickinson, Franklin Lakes, N.J.). After two washes, the cells were resuspended in complete medium RPMI 1640 (Gibco-BRL, Paisley, Scotland) containing 10% fetal calf serum (Boehringer Mannheim), 2 mM glutamine, 8 μg of gentamicin/ml, and 0.05 mM 2-mercaptoethanol. The cells were cultured at 37°C at a density of 2.5 × 10⁶ cells per ml, in the presence or absence of purified TTFC (different final concentrations) or heat-killed L. plantarum (10⁷ CFU/ml), for 4 days. To measure antigen-specific T-cell proliferative responses, [methyl-³H]thymidine (2.5 μCi/ml) was added to the culture 16 h before harvesting. The stimulation index (experimental/control) was determined in duplicate cultures.

Direct protection assay: TT challenge of mice.Immunized and control mice were challenged 13 days after the last boost (day 54) by subcutaneous inoculation of a standard challenge solution of TT. The number of 50% lethal doses (LD₅₀) administered in the experiment was concomitantly determined by injection of dilutions of the challenge solution in three groups of three age-matched naive mice and by calculation of the LD₅₀ by the method of Reed and Muench (27). The animals were observed each day. Mice showing no signs of paralysis 96 h after the challenge were considered fully protected; those surviving with signs were considered partially protected. Unprotected mice died at the latest on the second day after the challenge.

Indirect protection assay: determination of TT neutralizing antibodies in mouse sera.The toxin neutralization test was performed on the pooled sera of each group of immunized and control mice according to the specifications of the European Pharmacopoeia, with slight modifications. In brief, mixtures containing serial twofold dilutions of a pool and a definite amount of TT (L+/4,000 level) were injected subcutaneously in two OF1 mice (obtained from the animal facility, Institut Pasteur de Bruxelles) per mixture. A series of dilutions of a standard antitoxin solution mixed with the same toxin amount was included in each experiment. The neutralizing antibody content is expressed, in international units (IU) per milliliter, as a range that includes the actual value. Due to the small volumes of sera that were available, the detection limit was 0.0025 IU per ml of pooled sera. The protective level of tetanus antitoxin is usually considered to be 0.01 IU/ml (16).