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. 2020 Apr 21;17(1):55.
doi: 10.1186/s12985-020-01325-x.

Disulfide isomerase ERp57 improves the stability and immunogenicity of H3N2 influenza virus hemagglutinin

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Disulfide isomerase ERp57 improves the stability and immunogenicity of H3N2 influenza virus hemagglutinin

Jialing Wu et al. Virol J. .

Abstract

Background: Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA's immunogenicity, and many efforts have been made to stabilize the expressed HA proteins.

Methods: In this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA.

Results: The percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells.

Conclusion: ERp57 could improve the stability and immunogenicity of H3N2 HA.

Keywords: ERp57; Hemagglutinin; Immunogenicity; Protein disulfide isomerase; Stability.

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Conflict of interest statement

All the authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Overexpression and characterization of PDI proteins in 293 T cells. NC denotes the normal culture of 293 T cells; PDI and ERp57 denote overexpression of PDI and ERp57 proteins respectively; PDI/HA and ERp57/HA denote co-expression of H3 HA with ERp57 and PDI proteins respectively. a Western-blot analysis of PDIprotein expression under reducing condition. b Western-blot analysis of ERp57 protein expression under reducing condition. ImageJ was used to quantify PDIs. c Indirect immunofluorescence assay of PDI protein expression. d Indirect immunofluorescence assay of ERp57 protein expression. Cells nuclei were stained with DAPI (blue). PDI/ERp57 proteins were reacted with primary antibody and FITC/Cy3-conjugated secondary antibody (green/red). Scale bars indicate 25 μm
Fig. 2
Fig. 2
Expression and characterization of HA proteins in 293 T cells. HA denotes the single expression of H3 HA proteins; PDI/HA denotes co-expression of PDI and H3 HA proteins; ERp57/HA denotes co-expression of ERp57 and H3 HA proteins. a Western-blot analysis of HA protein expression under reducing condition. ImageJ was used to test the quantity of HA proteins. b Indirect immunofluorescence assay of HA protein expression. Cells nuclei were stained with DAPI (blue), and HA proteins were reacted with primary antibody and Cy3-conjugated secondary antibody (red). Scale bars indicate 25 μm. c Flow cytometry analysis of HA protein expression
Fig. 3
Fig. 3
The percentage of the disulfide-bonded HA trimers significantly increased in the PDI-overexpressed 293 T cells. a Western-blot analysis of the disulfide-bonded HA trimers under non-reducing condition. b A statistical graph of the disulfide-bonded HA trimers analyzed by ImageJ. T denotes Trimer, D denotes Dimer, and M denotes Monomer. ** represents P < 0.01, *** represents P < 0.001
Fig. 4
Fig. 4
The percentage of the disulfide-bonded HA trimers significantly descended when the ERp57 proteins were down-expressed. a Real-time qPCR analysis of ERp57 gene expression. iERp57 denotes the expression of ERp57 genes in the ERp57 gene-knockdowned cells. ** represents P < 0.01. b Western-blot analysis of ERp57 expression under reducing condition; iERp57 denotes the expression of ERp57 proteins in the ERp57 gene-knockdowned cells. c Western-blot analysis of HA expression under reducing condition; Mock denotes the conventional 293 T cells transfected by blank plasmid; HA denotes the expression of HA proteins in the conventional 293 T cells; NC denotes the expression of HA proteins in the 293 T cells transfected by negative control siRNA which don’t interference any genes expression in the 293 T cells; iERp57 denotes the expression of HA proteins in the ERp57 gene-knockdowned cells. d Western-blot analysis of the disulfide-bonded HA trimers under non-reducing condition; iERp57 denotes the expression of HA proteins in the ERp57 gene-knockdowned cells. e A statistical graph of the disulfide-bonded HA trimers analyzed by ImageJ. *** represents P < 0.001
Fig. 5
Fig. 5
H3 HA/ERp57 elicited significantly higher HA-specific antibodies than H3 HA. H1N1, H3N2, and H9N2 influenza viruses were purified and inactivated as antigens for assay. BALB/c mice (n = 5) were immunized twice with H3 HA or H3 HA/ERp57 or PBS as control. a The HI titer against homologous (H3N2) and heterologous (H1N1 or H9N2) antigens. The collected serum samples were assayed for HI titers following the conventional HI assay. ND denotes HI antibody not detectable. b The HA-specific antibodies against homologous (H3N2) and heterologous (H1N1 or H9N2) antigens. * represents p < 0.05, ** represents p < 0.01, **** represents p < 0.0001
Fig. 6
Fig. 6
Mice immunized with H3 HA/ERp57 showed less body weight loss against heterologous (H1N1) challenges than those immunized with H3 HA. BALB/c mice (n = 5) immunized with H3 HA or H3 HA/ERp57 were infected i.n. by 3 × MLD50 H1N1 viruses 3 weeks after the second immunization. The mice were monitored daily for their survival rates and body weight for 14 days. The body weight percentage was calculated according to the formula of (retained body weight/initial body weight) × 100%. The survival rate was calculated according to the formula of (number of survival mice/number of challenged mice) × 100%. a Weight loss of immunized mice against infection of H1N1 influenza virus, ** represents p < 0.01. b Survival rates of immunized mice against infection of H1N1 influenza virus, * represents p < 0.05

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