Oral immunization of mice with vaccine of attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit.
OBJECTIVE
To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection.


METHODS
Using genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected.


RESULTS
The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed.


CONCLUSIONS
The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.
Yang
Xing-Long
XL
Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, Shaanxi, China. foru3000@hotmail.com
Liu
Wen-Chao
WC
Yang
Wu-Wei
WW
Zhong
Dong
D
Liu
Yu-Hu
YH
Zhang
Jing-Dong
JD
Jiang
Jian-Hui
JH
Li
Shan-Shan
SS
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000008:administration & dosage* / Q000276:immunology*; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016481:Helicobacter Infections / Q000276:immunology / Q000517:prevention & control; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000097:blood; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D012486:Salmonella typhimurium / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014510:Urease / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D015431:Weight Loss
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D007074:Immunoglobulin G; D014613:Vaccines, Attenuated; D016753:Interleukin-10; D007371:Interferon-gamma; D014510:Urease