Construction of a tetR-integrated Salmonella enterica serovar Typhi CVD908 strain that tightly controls expression of the major merozoite surface protein of Plasmodium falciparum for applications in human Vaccine production

Abstract

Attenuated Salmonella strains are an attractive live vector for delivery of a foreign antigen to the human immune system. However, the problem with this vector lies with plasmid segregation and the low level of expression of the foreign gene in vivo when constitutive expression is employed, leading to a diminished immune response. We have established inducible expressions of foreign genes in the Salmonella enterica serovar Typhi CVD908 vaccine strain using the tetracycline response regulatory promoter. To set up this system, a tetracycline repressor (tetR) was integrated into a defined Delta aroC locus of the chromosome via suicide plasmid pJG12/tetR-neo. To remove the neo gene conferring kanamycin resistance from the locus, a cre expression vector under the control of the tetracycline response promoter was transformed into the clone; expression of the Cre recombinase excised the neo gene and generated the end strain CVD908-tetR. Expression of the luciferase reporter gene in this strain is dependent on the presence of tetracycline in the medium and can be regulated up to 4,773-fold. Moreover, the tightly controlled expression of major merozoite surface protein 1 (MSP1) and parts of Plasmodium falciparum was achieved, and the product yield was increased when the inducible expression system was employed. Inoculation of bacteria harboring plasmid pZE11/MSP1(42) in mice produced the protein in liver and spleen controlled by the inducer. The persistence of the plasmid-carrying bacteria in mice was determined. Peak colonization of both liver and spleen was detected on the third day postinoculation and was followed by a decline in growth curves. After 14 days postinfection, the majority of the bacteria (>90%) recovered from the liver and spleen of the mice retained the plasmid when expression was induced; this clearly indicated that stability of the expression vector in vivo was improved by inducible expression. Establishment of the regulatory system in the vaccine strain may broaden the range of its use by enhancing plasmid stability and expression levels in vivo. Moreover, the availability of the vaccine strain inducibly expressing the entire MSP1 provides possibilities for examining its immunogenicity, particularly the cellular response in animal models.

FIG. 1.

Cre/LoxP-mediated mobilization of the tetR cassette into the ΔaroC locus of the chromosome of strain CVD908.

FIG. 2.

PCR identification of integrated CVD908-tetR-neo-loxP. Lanes: M, DNA markers; 1, CVD908 (N32/N33); 2, CVD908 (Tet2/N33); 3, CVD908 merodiploid (N32/N33); 4, CVD908 merodiploid (Tet2/N33); 5, CVD908-tetR-neo-loxP (N32/N33); 6, CVD908-tetR-neo-loxP (Tet2/N33).

FIG. 3.

PCR identification of strain CVD908-tetR. Lanes: M, DNA markers; 1, Tet1/Tet2; 2, N32/N33; 3, Tet1/N32; 4, Tet2/N33.

FIG. 4.

Detection of luciferase activity in strain CVD908-tetR. Bars: 1, RLU in the presence of aTc; 2, RLU in the absence of aTc. The luciferase activity was defined as the RLU in the strain and is represented as the number of RLU per 10² cells.

FIG. 5.

Tetracycline-controlled expression of the entire MSP1 and parts thereof in strain CVD908-tetR detected by Western blot analysis in the presence of aTc (lane 2) and in the absence of aTc (lane 1). (A) MSP1₃₁ and rabbit anti-MSP1₃₁ serum. (B) MSP1₄₂ and MAb5.2. (C) MSP1 and MAb5.2.

FIG. 6.

CFU of S. enterica serovar Typhi CVD908-tetR harboring plasmid pZE11/MSP1₄₂ in mice. (A) Spleen. (B) Liver. kan, kanamycin; amp, ampicillin.