Expression of TetC fusion proteins from Salmonella in a gaseous environment that models conditions found in mammalian tissues.

There is an increasing body of evidence to suggest that attenuated strains of Salmonella can be used as live multivalent vaccine delivery systems for guest antigens from other pathogens. However, the constitutive expression of guest antigens, particularly from eukaryotic pathogens, can result in segregation and loss of the recombinant expression plasmid. To maintain expression of these guest antigens, an in vivo inducible promoter can be used. This offers an attractive alternative to the use of plasmids containing genes that confer antibiotic resistance, which is routinely used in vitro but is not acceptable for clinical applications. The modified Escherichia coli nitrite reductase nirB promoter is induced under conditions of reduced oxygen tension and has been used with great success to direct the expression of guest antigens in vivo (8). An attenuated S. typhi construct harboring a plasmid containing fragment-C from tetanus toxin (TetC) of Clostridium tetani under the transcriptional control of nirB is under evaluation as a combined human typhoid-tetanus vaccine (5). The Salmonella-TetC expression system has been used to enable the expression of a variety of antigens fused to TetC. The induction of the nirB promoter has been described using a number of different industrial anaerobic induction regimes. These regimes have proved impractical for scaling down to suit primary research purposes. This report compares expression of antigens from the human malaria parasite Plasmodium falciparum with TetC as a fusion partner driven from the nirB promoter in aerobic, microaerophilic and anaerobic conditions, using a commercially available small-scale atmosphere generating system. We show that the nirB promoter is inducible under microaerophilic conditions that model those found in lymphoid organs colonized by Salmonella in vivo and so can be used as a tool to predict the efficacy of a multivalent vaccine before administration. The malarial antigens studied were the Cand N-terminal non-repeat regions of liver stage antigen-1 (LSA-1) (11). Immune responses to peptides within the N-terminal region are associated with an absence of blood stage parasitemia (1) and with protection against severe malaria (4). The codon bias of a gene can have a major impact on the expression level of the corresponding protein and hence its immunogenicity in a live bacterial vaccine. Increased expression of TetC after codon optimization may have contributed to the success of the Salmonella-TetC tetanus vaccine (5). The gene fragments of the malarial antigens used in this study were not codon-optimized for bacterial expression. The use of A+T bases in the genome of P. falciparum, at greater than 80%, is higher than any other organism whose DNA has been characterized (9). This codon bias is a novel feature of the genome of this protozoan parasite and differs significantly from Salmonella, which has an A+T content of 48%. Hence, it may be predicted that malarial antigens may be expressed at low levels in a Salmonella host. Vaccine formulations of Salmonella are grown aerobically to a stationary phase. When administered orally, the bacteria persist in the lymphoid organs, such as liver and spleen, until cleared by the host’s immune system (5). To further our research on the use of Salmonella as a vaccine delivery system for malaria antigens, we sought to determine the expression of TetC fusion proteins from a vaccine strain. Previous studies have shown that expression of the lacZ reporter gene from nirB increases significantly when Salmonella enter mammalian macrophages in vitro (2). As direct examination of fusion protein expression in vivo is not feasible, we have developed a method by which expression can be determined Benchmarks