Comparison of a regulated delayed antigen synthesis system with in vivo-inducible promoters for antigen delivery by live attenuated Salmonella vaccines

Induction of strong immune responses against a vectored antigen in hosts immunized with live attenuated Salmonella vaccines is related in part to the amount of antigen delivered and the overall fitness of the Salmonella vector in relation to its ability to stimulate the host immune system. Constitutive high-level antigen synthesis causes a metabolic burden to the vaccine vector strain that can reduce the vaccine strain's ability to interact with host lymphoid tissues, resulting in a compromised immune response. A solution to this problem is the use of systems that regulate antigen gene expression, permitting high levels of antigen synthesis only after the vaccine strain has reached its target tissues. In vivo-inducible promoters (IVIPs) are often used to accomplish this. We recently developed an alternative strategy, a regulated delayed antigen synthesis (RDAS) system, in which the LacI-repressible P_trc promoter controls antigen gene expression by adding arabinose. In this paper, we compared the RDAS system with two commonly used IVIPs, P_ssaG and P_pagC. Three nearly identical plasmids, differing only in the promoter used to direct transcription of the pneumococcal pspA gene, P _trc, P_ssaG, or P_pagC, were constructed and introduced into isogenic Salmonella vaccine strains with or without arabinose-inducible LacI synthesis. Mice immunized with the RDAS strain developed slightly higher titers of mucosal and serum anti-PspA antibodies than P_pagC-immunized mice, while titers in mice immunized with the P _ssaG strain were 100-fold lower. Both the RDAS and P_pagC strains conferred similar levels of protection against Streptococcus pneumoniae challenge, significantly greater than those for the P_ssaG strain or controls. Thus, RDAS provides another choice for inclusion in the live vaccine design to increase immunogenicity.