Journal: International Journal of Molecular Sciences

Article Title: Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

doi: 10.3390/ijms22063287

Figure Lengend Snippet: 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 . ( 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.

Article Snippet: LPS analysis was carried out by staining bacteria with anti- S. Typhi LPS (clone B348M; GeneTex, Insight Biotechnology, Wembley, UK) or anti- S. Paratyphi A LPS (clone 10B10G; Bio-Rad Laboratories, Hemel Hempstead, UK) monoclonal antibodies.

Techniques: Expressing, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

doi: 10.3390/ijms22063287

Figure Lengend Snippet: 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.

Article Snippet: LPS analysis was carried out by staining bacteria with anti- S. Typhi LPS (clone B348M; GeneTex, Insight Biotechnology, Wembley, UK) or anti- S. Paratyphi A LPS (clone 10B10G; Bio-Rad Laboratories, Hemel Hempstead, UK) monoclonal antibodies.

Techniques: Modification, Sequencing, Fluorescence, Microscopy, Bioprocessing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

doi: 10.3390/ijms22063287

Figure Lengend Snippet: 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.

Article Snippet: LPS analysis was carried out by staining bacteria with anti- S. Typhi LPS (clone B348M; GeneTex, Insight Biotechnology, Wembley, UK) or anti- S. Paratyphi A LPS (clone 10B10G; Bio-Rad Laboratories, Hemel Hempstead, UK) monoclonal antibodies.

Techniques: Fluorescence, Microscopy, Western Blot, Membrane, Purification, Positive Control, Dot Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

doi: 10.3390/ijms22063287

Figure Lengend Snippet: 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 10 8 CFU ZH9 (•), 10 8 CFU ZH9PA (♦) or a 1:1 mix of 0.5 × 10 8 CFU of ZH9 and 0.5 × 10 8 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.

Article Snippet: LPS analysis was carried out by staining bacteria with anti- S. Typhi LPS (clone B348M; GeneTex, Insight Biotechnology, Wembley, UK) or anti- S. Paratyphi A LPS (clone 10B10G; Bio-Rad Laboratories, Hemel Hempstead, UK) monoclonal antibodies.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Formulation, Control

Journal: International Journal of Molecular Sciences

Article Title: Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

doi: 10.3390/ijms22063287

Figure Lengend Snippet: Bacterial strains and plasmids used directly in this study.

Article Snippet: LPS analysis was carried out by staining bacteria with anti- S. Typhi LPS (clone B348M; GeneTex, Insight Biotechnology, Wembley, UK) or anti- S. Paratyphi A LPS (clone 10B10G; Bio-Rad Laboratories, Hemel Hempstead, UK) monoclonal antibodies.

Techniques: Plasmid Preparation