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Development of a peptide ELISA to discriminate vaccine-induced immunity from natural infection of hepatitis A virus in a phase IV study

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Abstract

Hepatitis A virus (HAV) is a highly infectious agent that causes acute liver disease. The infection can trigger the production of antibodies against the structural and non-structural proteins of HAV. Nonetheless, vaccination with an HAV vaccine leads to the production of a primary antibody against the structural proteins. Because the non-structural proteins are only produced during active virus replication, there is no or very little antibody production against the non-structural proteins. However, the current commercial immunoassay cannot distinguish between antibodies produced during natural infection and those from vaccination against HAV. In our study, six immune-dominant epitopes from the non-structural proteins were designed, synthesized, linked together and cloned into pGEX-5X-1 plasmid. The recombinant protein was expressed in E. coli and purified by Ni2+-coated magnetic agarose beads. Then the purified recombinant protein was used as an ELISA antigen to detect antibodies for HAV non-structural proteins in serum samples. Seventy-seven attenuated and 89 inactivated vaccinated samples collected from our previous phase IV study of HAV vaccines were detected by peptide ELISA developed in this study. The mean OD450 value for the vaccination samples and acute infection samples were 0.529 (0.486 for the attenuated group and 0.567 for the inactivated group) and 1.187, respectively. According to the receiver operating characteristic (ROC) curve, the sensitivity and specificity of the peptide ELISA were 93.80% and 91.00%, respectively. This peptide ELISA was confirmed to discriminate vaccine-induced immunity from natural infection of HAV in a phase IV study with high sensitivity and specificity.

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Author notes

  1. Q. Sun & J. Yang

    Present address: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), 935 Jiao Ling Road, Kunming, Yunnan Province, 650118, People’s Republic of China

Authors and Affiliations

  1. Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), 935 Jiao Ling Road, Kunming, Yunnan Province, 650118, People’s Republic of China

    C. Ye, J. Luo, X. Wang, J. Xi, Y. Pan, J. Chen, X. Yang & G. Li

  2. Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming, 650118, People’s Republic of China

    C. Ye, J. Luo, X. Wang, J. Xi, Y. Pan, J. Chen, X. Yang, G. Li, Q. Sun & J. Yang

  3. Kunming Medical University, Kunming, 650500, People’s Republic of China

    C. Ye & J. Luo

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  1. C. Ye
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  2. J. Luo
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Corresponding authors

Correspondence to Q. Sun or J. Yang.

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Funding

This research was supported by the National “Twelfth Five Year” “major new drug discovery” technology major projects (grant no. 2012ZX09104–302), the Foundation of the CAMS Initiative for Innovative Medicine (CAMS-I2M) (grant no. 2016-I2M-1-19), and the Natural Science Foundation of Yunnan Province (2012FB188 and 2016FA029).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All participants were informed of the study aims, and written informed consent was received from each participant before sample collection. The study protocol was approved by the Institutional Ethics Committee (Institute of Medical Biology, Chinese Academy of Medical Sciences, and Peking Union Medical College) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Electronic supplementary material

Fig. S1

DNA sequence and the peptides of recombinant protein. The underlined sequences were endonuclease site sequences of BamH I(above) and EcoR I (below), respectively. (GIF 144 kb)

High resolution image (TIFF 39976 kb)

Fig. S2

(A) Bacterial colony PCR.Lane 1, E. coli BL21 cells without recombinant protein. Lane 2–7, E. coli BL21 cells successfully transformed with plasmids expressing recombinant protein. (B) Restriction enzyme digestion. Lane 1–2, empty vector. Lane3–4, recombinant vector. Target DNA was indicated by the arrow. (ZIP 232 kb)

High resolution image (ZIP 8790 kb)

Fig. S3

Lanes 1–3, E. coli cells transformed with empty vector. Lane 1, not induced; lane 2, induced for 4 h; lane 3, induced for 6 h; Lanes 4–6, E. coli cells transformed with recombinant plasmid. Lane 4, not induced; lane 5, induced for 4 h; lane 6: induced for 6 h. (GIF 116 kb)

High resolution image (TIFF 69804 kb)

Fig. S4

Purification of recombinant protein. Lane 1, E. coli cells transformed with empty vector induced 6 h; lane 2, total protein after sonication; lane 3, supernatant after centrifugation of inclusion bodies washed with 2 M urea. Lane 4, flow-through of NI2+ coated magnetic agarose beads; lane 5, eluate washed with 300 mM imidazole. Lane 6, eluate washed with 400 mM imidazole; lane 7, eluate washed with 500 mM imidazole. (GIF 83 kb)

High resolution image (TIFF 13077 kb)

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Ye, C., Luo, J., Wang, X. et al. Development of a peptide ELISA to discriminate vaccine-induced immunity from natural infection of hepatitis A virus in a phase IV study. Eur J Clin Microbiol Infect Dis 36, 2165–2170 (2017). https://doi.org/10.1007/s10096-017-3040-6

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  • DOI: https://doi.org/10.1007/s10096-017-3040-6

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