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China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (12): 1-8    DOI: 10.13523/j.cb.20191201
Expression and Immunological Properties of Multivalent Epitope Vaccine CWAE Against Helicobacter pylori
GUO Le1,3,WANG Shu-e3,HE Meng3,ZHANG Fan3,LIU Hong-peng3,**(),LIU Kun-mei2,3,**()
1 Provincial Key Laboratory of Clinical Pathogenic Microbiology, General Hospital of Ningxia Medical University, Yinchuan 750004, China
2 Breeding Base of State Key Laboratory for Craniocerebral Diseases, Ningxia Medical University, Yinchuan 750021, China
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Objective: To evaluate antigenic structure of multivalent epitope vaccine CWAE against Helicobacter pylori (H. pylori) by bioinformatics softs, obtain the purified CWAE protein after prokaryotic expression, and further identify the immunological properties of CWAE.Methods: The antigenic structure of CWAE was analyzed by bioinformatics softs. The recombinant plasmid pET28a-CWAE was obtained by using a synthetical WAE gene to replace the UE gene in pET28a-CUE plasmid. Then, the recombinant plasmid pET28a-CWAE was transformed into E. coli BL21 (DE3). After induction by IPTG, the antigen protein CWAE was purified by Ni-NTP nickel ion affinity chromatography. The adjuvant activity of CTB components in CWAE was identified by GM1-ELISA. Finally, the ability of CWAE to induce antibodies and lymphocyte immune response against H. pylori was detected by ELISA and spleen lymphocyte proliferation test.Methods: The multivalent epitope vaccine CWAE had a scientific and reasonable structure. The CWAE gene in recombinant plasmid pET28a-CWAE was consistent with the design sequence. After induction with IPTG, CWAE protein mainly exists as inclusion body. The purified CWAE was about 93.2% after purification.Results: from GM1-ELISA confirmed that CTB components in CWAE had good mucosal adjuvant activity. ELISA results confirmed that CWAE could stimulate BALB/c mice to produce anti-H. pylori antibodies. Moreover, CWAE vaccine could stimulate lymphocyte responses against various pathogenic factors from H. pylori.Conclusion: H. pylori multivalent epitope vaccine CWAE has scientific and reasonable antigen structure, and CWAE with high purity can be obtained by prokaryotic expression. Furthermore, the CWAE vaccine can stimulate specific antibodies and lymphocyte response against H. pylori. Experimental evidences for the development of multivalent epitope vaccine against H. pylori will be provided.

Key wordsHelicobacter pylori      Multivalent epitope vaccine      Specific antibody     
Received: 27 May 2019      Published: 15 January 2020
ZTFLH:  Q93  
Corresponding Authors: Hong-peng LIU,Kun-mei LIU     E-mail:;
Cite this article:

GUO Le,WANG Shu-e,HE Meng,ZHANG Fan,LIU Hong-peng,LIU Kun-mei. Expression and Immunological Properties of Multivalent Epitope Vaccine CWAE Against Helicobacter pylori. China Biotechnology, 2019, 39(12): 1-8.

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Fig. 1 Antigenic structure of CWAE vaccine and analysis of the linkers by DNAstar (a) Antigenic structure of CWAE vaccine (b) Analysis of the linkers (DPRVPSS, KK, GS and GGG) The linker (DPRVPSS, KK, GS and GGG) is marked in gray (c) Antigenicity analysis of CWAE
Fig.2 Construction and identification of recombinant plasmid pET28a-CWAE (a) The construction of recombinant plasmid pET28a-CWAE (b) PCR amplification of gene WAE (c) The pET28a-CWAE plasmids were digested with Kpn I and Xho I M:DNA marker; 1: The double restriction map (d) The pET28a-CWAE plasmids were digested with Nco I and Xho I M:DNA marker; 1: The double restriction map (e) Identification of plasmid pET28a-CWAE by gene sequencing Linker (DPRVPSS) is marked by a black line. Besides, the UreA27-53 Th epitope is marked by a red line
Fig.3 Expression and purification of antigen protein CWAE (a) Expression and purification of antigen protein CWAE M:Protein marker; 1:CWAE expression after induction for 6h; 2:CWAE expression after induction for 5h; 3:CWAE expression after induction for 4h; 4:CWAE expression after induction for 3h; 5:CWAE expression after induction for 2h (b) Purification of antigen protein CWAE M:Protein marker; 1:The inclusion body of CWAE; 2:The peak of loading protein sample; 3: The impurity protein eluted by wash buffer; 4: The purified CWAE protein washed down by elution buffer
Fig.4 Adjuvant activity of CTB component in CWAEwas identified by GM1-ELISA A GM1-ELISA was performed to evaluate the adjuvanticity of CTB component in CWAE vaccine. The CTB protein was used as a positive control. ELISA plates were coated with 1μg/well of GM1 ganglioside or BSA. The proteins CWAE or CTB were diluted from 100μg/ml to 0.78μg/ml
Fig.5 IgG and IgA antibodies against H. pylori after CWAE immunization (a) IgG antibodies against H. pylori lysates by ELISA (b) IgA antibodies against H. pylori lysates by ELISA.
Fig.6 Proliferation of splenic lymphocytes after CWAE immunization Splenic lymphocytes were separated from H. pylori-infected BALB/c mice after CWAE immunization, and were incubated with CWAE, Urease, UreA, UreB or H. pylori lysates
[1]   Parsonnet J, Friedman G D, Vandersteen D P , et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med, 1991,325(16):1127-1131.
[2]   Hunt R H, Xiao S D, Megraud F , et al. Helicobacter pylori in developing countries. J Gastrointestin Liver Dis, 2011,20(3):299-304.
[3]   Lucas B, Bumann D, Walduck A , et al. Adoptive transfer of CD4 + T cells specific for subunit A of Helicobacter pylori urease reduces H. pylori stomach colonization in mice in the absence of interleukin-4 (IL-4)/IL-13 receptor signaling . Infect Immun, 2001,69(3):1714-1721.
[4]   Vermoote M, Flahou B, Pasmans F , et al. Protective efficacy of vaccines based on the Helicobacter suis urease subunit B and gamma-glutamyl transpeptidase. Vaccine, 2013. 31(32):3250-3256.
[5]   Yamaguchi H, Osaki T, Kai M , et al.Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter pylori. Infect Immun, 2000,68(6):3448-3454.
[6]   Flach C F, Svensson N, Blomquist M , et al. A truncated form of HpaA is a promising antigen for use in a vaccine against Helicobacter pylori. Vaccine, 2011,29(6):1235-1241.
[7]   Satin B, Del Giudice G, Della Bianca V , et al. The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor. J Exp Med, 2000,191(9):1467-1476.
[8]   Liu K Y, Shi Y, Luo P , et al. Therapeutic efficacy of oral immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori CagA, VacA and UreB fusion proteins in mice model. Vaccine, 2011,29(38):6679-6685.
[9]   Moss S F, Moise L, Lee D S , et al. HelicoVax: epitope-based therapeutic Helicobacter pylori vaccination in a mouse model. Vaccine, 2011,29(11):2085-2091.
[10]   Czinn S J, Blanchard T . Vaccinating against Helicobacter pylori infection. Nat Rev Gastroenterol Hepatol, 2011,8(3):133-140.
[11]   Liu C, Luo J, Xue R Y , et al. The mucosal adjuvant effect of plant polysaccharides for induction of protective immunity against Helicobacter pylori infection. Vaccine, 2019,37(8):1053-1061.
[12]   Zeng M, Mao X H, Li J X , et al. Efficacy, safety, and immunogenicity of an oral recombinant Helicobacter pylori vaccine in children in China: a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet, 2015,386(10002):1457-1464.
[13]   Rutherford J C . The emerging role of urease as a general microbial virulence factor. PLoS Pathog, 2014,10(5):e1004062.
[14]   Sette A, Fikes J . Epitope-based vaccines: an update on epitope identification, vaccine design and delivery. Curr Opin Immunol, 2003,15(4):461-470.
[15]   Nezafat N, Eslami M, Negahdaripour M , et al. Designing an efficient multi-epitope oral vaccine against Helicobacter pylori using immunoinformatics and structural vaccinology approaches. Mol Biosyst, 2017,13(4):699-713.
[16]   Liu W, Peng Z, Liu Z , et al. High epitope density in a single recombinant protein molecule of the extracellular domain of influenza A virus M2 protein significantly enhances protective immunity. Vaccine, 2004,23(3):366-371.
[17]   Kovacs-Nolan J, Mine Y . Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice. Biochim Biophys Acta, 2006,1760(12):1884-1893.
[18]   Sun J B, Mielcarek N, Lakew M , et al. Intranasal administration of a Schistosoma mansoni glutathione S-transferase-cholera toxoid conjugate vaccine evokes antiparasitic and antipathological immunity in mice. J Immunol, 1999,163(2):1045-1052.
[19]   Kang S M, Yao Q, Guo L , et al. Mucosal immunization with virus-like particles of simian immunodeficiency virus conjugated with cholera toxin subunit B. J Virol, 2003,77(18):9823-9830.
[20]   Liljeqvist S, Stahl S, Andreoni C , et al. Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding. J Immunol Methods, 1997,210(2):125-135.
[21]   Guo L, Yin R, Xu G , et al. Immunologic properties and therapeutic efficacy of a multivalent epitope-based vaccine against four Helicobacter pylori adhesins (urease, Lpp20, HpaA, and CagL) in Mongolian gerbils. Helicobacter, 2017,22(6).
[22]   Guo L, Yin R, Liu K , et al. Immunological features and efficacy of a multi-epitope vaccine CTB-UE against H. pylori in BALB/c mice model. Appl Microbiol Biotechnol, 2014,98(8):3495-3507.
[23]   Ermak T H, Giannasca P J, Nichols R , et al. Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses. J Exp Med, 1998,188(12):2277-2288.
[24]   Romagnani S . Th1/Th2 cells. Inflamm Bowel Dis, 1999,5(4):285-294.
[25]   Yang J, Dai L X, Pan X , et al.Protection against Helicobacter pylori infection in BALB/c mice by oral administration of multi-epitope vaccine of CTB-UreI-UreB. Pathog Dis, 2015,73(5).
[26]   Chen J, Li N, She F . Helicobacter pylori outer inflammatory protein DNA vaccine-loaded bacterial ghost enhances immune protective efficacy in C57BL/6 mice. Vaccine, 2014,32(46):6054-6060.
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