Novel methods for expression of foreign antigens in live vector vaccines

Abstract

Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In this approach, genes encoding protective antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using low copy expression plasmids, here we pursue expression of foreign proteins from the live vector chromosome. Our strategy is designed to compensate for the inherent disadvantage of loss of gene dosage (vs. plasmid-based expression) by integrating antigen-encoding gene cassettes into multiple chromosomal sites already inactivated in an attenuated Salmonella enterica serovar Typhi vaccine candidate. We tested expression of a cassette encoding the green fluorescent protein (GFPuv) integrated separately into native guaBA, htrA or clyA chromosomal loci. Using single integrations, we show that expression levels of GFPuv are significantly affected by the site of integration, regardless of the inclusion of additional strong promoters within the incoming cassette. Using cassettes integrated into both guaBA and htrA, we observe cumulative synthesis levels from two integration sites superior to single integrations. Most importantly, we observe that GFPuv expression increases in a growth phase-dependent manner, suggesting that foreign antigen synthesis may be "tuned" to the physiology of the live vaccine. We expect this novel platform expression technology to prove invaluable in the development of a wide variety of multivalent live vector vaccines, capable of expressing multiple antigens from both chromosomal and plasmid-based expression systems within a single strain.

Keywords: Salmonella; chromosomal expression; foreign gene; live vector; vaccine.

Figure 1. Schematic depiction of the strategy for chromosomal integration of the foreign antigen cassette P_ompC-gfpuv, encoding the model fluorescent antigen GFPuv. Details of this approach are thoroughly presented in the Materials and Methods. Briefly, an osmotically controlled GFPuv-encoding cassette (tandem white circle and hatched thick arrow) was constructed and linked to an aph marker encoding resistance to kanamycin (shaded thick arrow), flanked by FRT recombination sites (black triangles). The incoming P_ompC-gfpuv-aph cassette was integrated into the live vector chromosome using the λ Red recombination system, followed by removal of the aph marker using FLP recombinase, to yield the final live vector strain bearing no genes encoding resistance to antibiotics. The bacterial chromosome is represented by 5′-proximal and 3′-terminal darkened rectangles, and the black circle labeled with a “P” represents the wildtype chromosomally encoded promoter of the deleted target open reading frame (i.e either guaBA or htrA).

Figure 2. Flow cytometry histograms of GFPuv-mediated fluorescence encoded by P_ompC-gfpuv gene cassettes integrated into either the guaBA (thick solid line), htrA (thin hatched line) or clyA (thick broken line) sites of the attenuated S. Typhi live vector vaccine candidate CVD 910, compared with the vaccine strain alone (thin dotted line). Fluorescence intensities are measured for individual bacterial cells grown under inducing conditions of 200 mM NaCl in rich medium at 37°C/250 rpm for 16 h.