Sublingual Adjuvant Delivery by a Live Attenuated Vibrio cholerae-Based Antigen Presentation Platform

Abstract

A sublingually delivered heterologous antigen presentation platform that does not depend on antigen or adjuvant purification would be of great benefit in protection against diarrheal disease. In proof-of-concept studies, we previously showed that when a fusion protein comprised of the Vibrio cholerae biofilm matrix protein RbmA and the B subunit of cholera toxin (R-CTB) is expressed from a plasmid within V. cholerae, R-CTB is sequestered in the biofilm matrix, leading to decoration of the cell surface. Sublingual delivery of live attenuated R-CTB-decorated cells results in a mucosal immune response to CTB. To improve the immune response to diarrheal antigens presented by this platform, we have engineered our live attenuated vaccine to express the mucosal adjuvant mmCT (i.e., multiply mutated CT). Here we report that delivery of this adjuvant via sublingual administration of our vaccine enhances the mucosal immune response to V. cholerae LPS and elicits a systemic and mucosal immune response to CTB. However, provision of R-CTB with mmCT selectively blunts the mucosal immune response to CTB. We propose that mmCT delivered by this live attenuated Vibrio cholerae vaccine platform may serve as a mucosal adjuvant for heterologous antigens, provided they are not too similar to mmCT.IMPORTANCE Diarrheal disease is the most common infectious disease of children in the developing world. Our goal is to develop a diarrheal antigen presentation platform based on whole Vibrio cholerae cells that does not depend on protein purification. We have previously shown the feasibility of genetically fusing antigens to the V. cholerae biofilm matrix protein RbmA for presentation on the cell surface. A mucosal adjuvant could improve immunogenicity of such a vaccine at the mucosal surface. Here we engineer a live attenuated V. cholerae vaccine to constitutively synthesize mmCT, a nontoxic form of cholera toxin. When this vaccine is delivered sublingually, in vivo-synthesized mmCT acts as both an adjuvant and antigen. This could greatly increase the magnitude and duration of the immune response elicited by codelivered heterologous antigens.

Keywords: Vibrio cholerae; cholera toxin adjuvants; live vector vaccines; mmCT; vaccine; vaccine platform.

FIG 1

Chromosomally expressed RΔ-CTB is poorly immunogenic. (A) Genotype of an O139 serotype vaccine strain in which the entire CTX phage, including the recombination sites, as well as the tcpA gene, has been deleted. Two R→A mutations (R116A and R234A) were introduced into rbmA. CTB is genetically coupled to the 3′ end of rbmA and placed under the native rbmA promoter on the chromosome (cRΔ-CTB). (B) Western analysis of CTB fused to the C-terminal end of wild-type RbmA (WT) or an RbmA protein carrying the R116A and R234A point mutations (Δ) was detected in the V. cholerae cell pellet. (C) Quantification of cell-associated CTB in femtograms per cell when expressed from a plasmid or on the chromosome. (D) Vaccination scheme for the live attenuated vaccine. Open triangles indicate vaccination. Arrows indicate blood and stool sample collection. (E) Nanograms of LPS-specific antibodies per milliliter of serum or per microgram of total IgA in stool after immunization with a live attenuated vaccine strain expressing cRΔ-CTB. (F) Nanograms of CTB-specific antibodies per milliliter of serum or per microgram of total IgA in stool after immunization with a live attenuated vaccine strain expressing cRΔ-CTB. Error bars in panel C denote standard deviation. For panel C, * indicates P ≤ 0.05 using two-tailed, unpaired Student’s t test. For panels E and F, * indicates P ≤ 0.05 and **** indicates P ≤ 0.0001 using one-way ANOVA followed by Dunnett’s test for multiple comparisons. Horizontal bars mark the mean. Each vaccination group included 10 mice.

FIG 2

Characterization of mmCT as an in vivo antigen and adjuvant. (A) Genotype of an O139 serotype vaccine strain in which the entire CTX phage, including the recombination sites, as well as the tcpA gene has been deleted. The genes encoding mmCT (a cholera toxin variant carrying multiple mutations in the A subunit of cholera toxin) are integrated in frame into the lacZ gene on the V. cholerae chromosome. A ribosome-binding site is included at the 5′ end of the sequence encoding mmCT, allowing the lacZ promoter to drive transcription and translation of mmCT. (B) Constitutive production of mmCT in LB medium does not affect bacterial fitness as measured by growth over time. (C) Western blot analysis of cell pellets and supernatants (Supt) after overnight LB broth culture of the vaccine strain noted in panel A with or without mmCT. Rabbit sera raised against CT followed by secondary anti-rabbit antibody were used for detection. (D) Quantification of mmCT in the supernatant of an overnight culture in LB and after incubation of the vaccine preparation in PBS. Vaccines were kept on ice for less than 1 h before administration. (E) Quantification of the amount of CTB delivered as R-CTB and as part of mmCT, compared to the amount of purified CTB in Dukoral vaccine. The amount of CTB from supernatant mmCT was measured after 1 h of incubation at 22°C in PBS.

FIG 3

In situ production of mmCT alone after sublingual vaccine delivery elicits a robust and long-lived immune response to CTB. (A) Genotype of vaccine strain used. Expression of genes encoding mmCT is driven by the constitutive lacZ promoter on the chromosome of the vaccine strain. (B) Vaccination scheme for the live attenuated vaccines with constitutive expression of mmCT. Open triangles indicate vaccination. Arrows indicate blood and stool sample collection. (C) Nanograms of LPS-specific antibodies per milliliter of serum or per microgram of total IgA in stool after immunization with a live attenuated vaccine strain expressing mmCT. (D) Nanograms of CTB-specific antibodies per milliliter of serum or per microgram of total IgA in stool after immunization with a live attenuated vaccine strain expressing mmCT. *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, and ****, P ≤ 0.0001, using one-way ANOVA and Dunnett’s test. Horizontal bars mark the mean. Each vaccination group included 10 mice.

FIG 4

Coexpression of the adjuvant mmCT with chromosomal RΔ-CTB attenuates the immune response to CTB. (A) Genotype of an O139 serotype vaccine strain in which the entire CTX phage, including the recombination sites, as well as the tcpA gene has been deleted. Constitutive expression of genes encoding mmCT is driven by the native, chromosomal lacZ promoter. CTB is genetically coupled to the 3′ end of rbmA carrying R116A and R234A mutations and placed under the native rbmA promoter on the chromosome (cRΔ-CTB). (B) Vaccination scheme for the live attenuated vaccine with chromosomal RΔ-CTB and constitutive expression of mmCT. Open triangles indicate vaccination. Arrows indicate blood and stool sample collection. (C and D) Nanograms of LPS-specific antibodies per milliliter of serum or micrograms per total IgA in stool (C) and nanograms of CTB-specific antibodies per milliliter of serum or per microgram of total IgA in stool (D) after immunization with a live attenuated vaccine strain expressing cRΔ-CTB and mmCT. Data were log transformed prior to calculation of statistical significance. n.s., not significant (P ≥ 0.05), *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, and ****, P ≤ 0.0001, using one-way ANOVA and Dunnett’s multiple-comparison test. Horizontal bars mark the mean. Each vaccination group included 10 mice. (E and F) Comparison of (E) vibriocidal titers and (F) LPS-specific IgG1 and IgG2a for vaccines expressing cRΔ-CTB with or without mmCT at days 56 and 180. Differences were not statistically significant.