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Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

ABSTRACT: We evaluated the antigenicity of recombinant infectious bronchitis virus (IBV) S1 protein expressed in mammalian cells. Recombinant S1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using Ni Sepharose. The purified protein was analyzed by Western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. Six weeks after immunization, anti-IBV neutralizing titer and anti-S1 ELISA titer were determined; immunized chickens then were inoculated with IBV via the trachea and ciliary activity was observed. Results showed that the recombinant S1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant S1 protein was lower than that of inactivated virus. However, anti-S1 ELISA indicated that the recombinant S1 protein induced antibodies against S1. These results suggest that the recombinant S1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. In conclusion, the neutralizing antigenicity of recombinant S1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies.

SUBMITTER: Andoh K

PROVIDER: S-EPMC7114517 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

Andoh Kiyohiko K Suenaga Kiyotaka K Sakaguchi Masashi M Yamazaki Kenichi K Honda Takashi T

Virus research 20150622

We evaluated the antigenicity of recombinant infectious bronchitis virus (IBV) S1 protein expressed in mammalian cells. Recombinant S1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using Ni Sepharose. The purified protein was analyzed by Western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. Six weeks after immunization, anti-IBV neutralizing titer and anti-S1 ELISA titer were determined; immunized ...[more]

PMID: 26113306

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Project description:The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

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Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

Publications

Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

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