A foreign protein incorporated on the Tip of T3 pili in Lactococcus lactis elicits systemic and mucosal immunity

Abstract

The use of Lactococcus lactis to deliver a chosen antigen to the mucosal surface has been shown to elicit an immune response in mice and is a possible method of vaccination in humans. The recent discovery on Gram-positive bacteria of pili that are covalently attached to the bacterial surface and the elucidation of the residues linking the major and minor subunits of such pili suggests that the presentation of an antigen on the tip of pili external to the surface of L. lactis might constitute a successful vaccine strategy. As a proof of principle, we have fused a foreign protein (the Escherichia coli maltose-binding protein) to the C-terminal region of the native tip protein (Cpa) of the T3 pilus derived from Streptococcus pyogenes and expressed this fusion protein (MBP*) in L. lactis. We find that MBP* is incorporated into pili in this foreign host, as shown by Western blot analyses of cell wall proteins and by immunogold electron microscopy. Furthermore, since the MBP* on these pili retains its native biological activity, it appears to retain its native structure. Mucosal immunization of mice with this L. lactis strain expressing pilus-linked MBP* results in production of both a systemic and a mucosal response (IgG and IgA antibodies) against the MBP antigen. We suggest that this type of mucosal vaccine delivery system, which we term UPTOP (for unhindered presentation on tips of pili), may provide an inexpensive and stable alternative to current mechanisms of immunization for many serious human pathogens.

FIG. 1.

Regions of the FCT-3 pilus cluster encoded by plasmids used in the present study. The position of the HA epitope tag in Cpa is indicated by a triangle. The region encoding MBP, inserted between the 5′ and 3′ regions of cpa, is shown as a rectangle (not to scale). The location of the His₆ tag is shown as a square. Expression of the pilus gene cluster regions in pJRS9550, pJRS9565, and pJRS9566 are under the control of the strong constitutive P23 promoter of pJRS9508 (1).

FIG. 2.

MBP* is surface exposed and incorporated into HMW T3 pilus polymers in L. lactis. (A and B) Whole-cell dot blots of MG1363/pJRS9565 (blot 1) and MG1363/pJRS9566 (blot 2) analyzed with anti-MBP (A) and anti-T3 (B). (C and D) Western blots of cell wall extracts (lanes 1 to 4) and trichloroacetic acid-precipitated supernatants (lanes 5 to 8) boiled in SDS and reacted with monoclonal anti-MBP antibody (C) or polyclonal anti-T3 antiserum (D). Lanes: 1, 2, 5, and 6, MG1363/pJRS9565 (MBP*); 3 and 7, MG1363/pJRS9566 (−SrtC2); 4 and 8, MG1363/pJRS9545 (empty vector). The location of the MBP* monomer and the MBP*-T3 heterodimer are indicated by arrows to the right of the figure. The sizes of molecular mass standards (in kilodaltons) are indicated to the left of each blot.

FIG. 3.

Immunogold EM localization of MPB* in T3 pili. Negative-stain transmission EM images of whole bacteria. MG1363/pJRS9545 (vector control; panel A), MG1363/pJRS9550 (T3 pili with HA-tagged Cpa; panels B, E, F, and G), and MG1363/pJRS9565 (T3 pili with MPB* fusion; panels C, D, H, and I). In panels A to C, the bacteria were incubated with anti-T3 antiserum, followed by a gold-conjugated secondary antibody. Pili and gold labeling are absent for the vector control strain (A), but present for the strains expressing HA-tagged Cpa (B) or MBP* (C). In panels D to F, the bacteria were incubated with anti-HA antiserum, followed by a gold-conjugated secondary antibody. In panels G to I, the bacteria were incubated with anti-MBP antiserum, followed by a gold-conjugated secondary antibody. The gold particles specific for HA-tagged Cpa or MPB* can be seen at the tips of the pilus fibers (arrows in panels E, F, H, and I). Similar labeling is absent for the control reactions (D and G). Scale bar, 500 nm.

FIG. 4.

MBP* incorporated into T3 pili in L. lactis retains activity. Whole-cell lysates of MG1363/pJRS9565 (MBP*) (lanes 1 to 3), MG1363/pJRS9566 (−SrtC2) (lanes 4 to 6), and MG1363/pJRS9550 (wild-type pilus cluster without MBP) (lanes 7 to 9) were purified with amylose resin and analyzed with monoclonal anti-MBP antibody (A) or purified anti-T3 antibody (B). Molecular masses (in kilodaltons) are indicated to the left of the figure. The locations of MBP* and the MBP*-T3 heterodimer are shown to the right of the figure. Fractions are indicated as follows: C, crude lysate; FT, flowthrough; and E, eluate.

FIG. 5.

MBP-specific antibody response in mucosal secretions. MBP-specific IgA in lung lavage samples collected 10 days after the last immunization (day 39). The IgA response was determined in undiluted samples from individual mice by ELISA performed in quadruplicate. Each data point represents the average response in an individual animal, and horizontal lines represent the median response. Samples were taken from naive mice (▴, n = 2) or mice inoculated i.n. with MG1363/pJRS9545 (empty vector; •, n = 8) or MG1363/pJRS9565 (MBP*; ⧫, n = 10). The statistical significance between the experimental MG1363/pJRS9565 and the control group (MG1363/pJRS9545) determined by using the Student unpaired t test was P < 0.001.

FIG. 6.

MBP-specific antibody response in serum. MBP-specific IgG in serum samples of experiment described in Fig. 5. The IgG response was determined in samples (diluted 1:50) from individual mice by ELISA performed in quadruplicate. Each data point represents the average response in an individual animal, and horizontal lines represent the median response. Samples were taken from naive mice (▴, n = 2) or mice inoculated i.n. with MG1363/pJRS9545 (empty vector; •, n = 8) or MG1363/pJRS9565 (MBP*; ⧫, n = 10). The statistical significance between the experimental MG1363/pJRS9565 and the control group (MG1363/pJRS9545) determined by using the Student unpaired t test was P < 0.0001.

FIG. 7.

T3-specific antibody response in mucosal secretion. T3-specific IgA in lung lavage collected 10 days after the last immunization (day 39) from naive mice (▴, n = 2) or mice inoculated i.n. with MG1363/pJRS9545 (empty vector; ▪, n = 8) or MG1363/pJRS9565 (MBP*; ⧫, n = 10). The IgA response was determined in undiluted samples from individual mice by ELISA performed in quadruplicate. Each data point represents the average response in each mouse, and horizontal lines represent the median response. The statistical significance between the experimental MG1363/pJRS9565 and the control group (MG1363/pJRS9545) determined by using the Student unpaired t test was P < 0.0001.

FIG. 8.

T3-specific antibody response in serum. T3-specific IgG in serum samples of the experiment described in Fig. 7. The IgG response was determined in samples (1:500) from untreated mice (▪, n = 2) or mice inoculated i.n. with MG1363/pJRS9545 (empty vector; □, n = 8) or MG1363/pJRS9565 (MBP*; ░⃞, n = 10). The ELISA was performed in quadruplicate, and the bars represent the average response in each group. The standard deviation is indicated by a vertical line, and the asterisk indicates that the experimental (MG1363/pJRS9565) and the control group (MG1363/pJRS9545) are significantly different as determined by using the Student unpaired t test (P < 0.001).