Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

Abstract

Enteric fever is a major global healthcare issue caused largely by Salmonella enterica serovars Typhi and Paratyphi A. The objective of this study was to develop a novel, bivalent oral vaccine capable of protecting against both serovars. Our approach centred on genetically engineering the attenuated S. Typhi ZH9 strain, which has an excellent safety record in clinical trials, to introduce two S. Paratyphi A immunogenic elements: flagellin H:a and lipopolysaccharide (LPS) O:2. We first replaced the native S. Typhi fliC gene encoding flagellin with the highly homologous fliC gene from S. Paratyphi A using Xer-cise technology. Next, we replaced the S. Typhi rfbE gene encoding tyvelose epimerase with a spacer sequence to enable the sustained expression of O:2 LPS and prevent its conversion to O:9 through tyvelose epimerase activity. The resulting new strain, ZH9PA, incorporated these two genetic changes and exhibited comparable growth kinetics to the parental ZH9 strain. A formulation containing both ZH9 and ZH9PA strains together constitutes a new bivalent vaccine candidate that targets both S. Typhi and S. Paratyphi A antigens to address a major global healthcare gap for enteric fever prophylaxis. This vaccine is now being tested in a Phase I clinical trial (NCT04349553).

Keywords: Paratyphi A; Typhi; bacteria; enteric fever; salmonella; synthetic biology; vaccine.

Conflict of interest statement

All authors are or were employees of Prokarium Ltd. at the time of the design and execution of this study. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Figures

Figure 1

Replacing the S. Typhi (H:d) flagellin with S. Paratyphi A (H:a) flagellin. (a) The genetic engineering process to generate S. Typhi ZH9 expressing S. Paratyphi A flagellin (ZH9PF). Adapted with permission from Bloor and Cranenburgh, 2006 [23]. (b) Fluorescence microscopy with S. Typhi ZH9 and the derivative strain, ZH9PF, probed with H:d antiserum (anti-S. Typhi) or H:a antiserum (anti-S. Paratyphi A) plus Dylight 488 secondary antibodies; the left column images are phase contrast images, and the right column images are immuno-fluorescence images. Images were taken at 100× magnification. Scale bars represent 10 µm. Representative images were based on three independent experimental repeats.

Figure 2

Modifying LPS (O:9) to LPS (O:2). (a) Part of the wild-type O-antigen locus from S. Typhi ZH9 was modified using two test approaches: by deleting the majority of the rfbE cistron to generate S. Typhi ZH9PL2 or by replacing the rfbE cistron with a spacer DNA sequence to maintain the original reading frame to generate S. Typhi ZH9W. (b) Fluorescence microscopy images showing the parental S. Typhi ZH9 and derivative strains, ZH9PL2 and ZH9W, probed with anti-S. Typhi LPS (O:9) or anti-S. Paratyphi A LPS (O:2) monoclonal antibodies followed by Dylight 488 secondary antibodies; the left column images are phase contrast images and the right column images are immuno-fluorescence micrographs. Images were taken at 100× magnification. Scale bars represent 10 µm. Representative images based on three independent experimental repeats. (c) Silver-stained polyacrylamide gel of LPS extracts from the parental S. Typhi ZH9 and derivative strains, ZH9PL2 and ZH9W, indicating the short and long O-antigen chains. LPS = lipopolysaccharide; mAb = monoclonal antibody.

Figure 3

Converting flagellin and LPS in the final new strain, ZH9PA. (a) Fluorescence microscopy images showing the S. Typhi ZH9 derivative strain, ZH9PA, probed with anti-S. Typhi (H:d) or anti-S. Paratyphi A (H:a) flagellin antiserum and anti-S. Typhi (O:9) or anti-S. Paratyphi A (O:2) LPS mAbs; the left images are phase contrast images and right images are immuno-fluorescence micrographs. Images were taken at 100× magnification. Scale bars represent 10µm. Representative images based on three independent experimental repeats. (b) Western blots of membrane fractions probed with anti-S. Typhi (H:d) or anti-S. Paratyphi A (H:a) flagellin antisera using ZH9 or SPAV as positive controls, respectively. Purified flagellin proteins were also included as a positive control. (c) Dot blot probed with anti-S. Typhi and anti-S. Paratyphi A LPS mAbs. (d) Silver-stained polyacrylamide gel of LPS preparations from S. Typhi ZH9 and derivative strains, ZH9PA, indicating the short and long O-antigen chains. LPS = lipopolysaccharide; mAb = monoclonal antibody; SPAV = attenuated S. Paratyphi A.

Figure 4

Comparison of growth profiles. Bacteria were seeded into LB broth cultures at OD600 nm = 0.1 and grown for 24 h. At regular intervals, samples were taken and analysed by spectrophotometry or by titration on agar plates. Optical density (a measure of growth density) and bacterial titre were plotted for ZH9 (the parental strain) and ZH9PA (the modified strain). The late exponential growth phase (5 to 8 h) is shown in grey. (a) OD600 nm and CFU/mL measurements compared within each individual strain. (b) OD600 nm or CFU/mL measurements compared between both strains. Statistical comparisons were made using a two-way ANOVA, based on triplicate cultures in a single experiment. CFU = colony-forming units; ml = millilitres; nm = nanometres.

Figure 5

Anti-LPS IgG antibody responses following in vivo vaccination. (a) Specific IgG antibody responses against S. Typhi LPS (O:9). (b) Specific IgG antibody responses against S. Paratyphi A LPS (O:2). Antibody responses were evaluated by ELISA in Balb/c mouse serum at 35 or 42 days following subcutaneous vaccination with 108 CFU ZH9 (•), 108 CFU ZH9PA (♦) or a 1:1 mix of 0.5 × 108 CFU of ZH9 and 0.5 × 108 CFU of ZH9PA (Entervax™ basic formulation (▲)). Pre-vaccination (d0) samples were pooled across individual mice to generate the negative assay control (dotted line). Each data point represents an individual mouse, and data were pooled across three independent experiments. Mean values are represented by the horizontal bar. Statistical comparisons were made using a one-way ANOVA. ELISA = enzyme-linked immunosorbent assay; EPT = end point titre; IgG = immunoglobulin G; LPS = lipopolysaccharide; OD = optical density.