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

Abstract

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.

Figure 1

(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.

Figure 2

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.

Figure 3

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°.

Figure 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.