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. Copyright 2018 Liao et al.