Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody.

Satoko Matsunaga,Izumi Matsuo,Hiroyuki Tsukagoshi,Naoki Yamamoto,Hirokazu Kimura,Yuki Matsushima,Shiho Kawakami,Akiko Okayama,Nobuhiko Okabe,Hisashi Hirano,Akihide Ryo,Hideaki Shimizu,Ayumi Kudoh

doi:10.3389/fmicb.2014.00208

Abstract

Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

Highlights

Human parainfluenza viruses (HPIVs) are major causes of lower respiratory infections in infants, young children, the immunocompromised, the chronically ill, and the elderly (Glezen et al, 1984; Counihan et al, 2001; Weinberg et al, 2009)
His-tagged Human parainfluenza virus 3 (HPIV3)-HN protein was synthesized by this procedure in a large scale
Balb/c mice were immunized with purified full-length HPIV3-HN protein

Summary

Introduction

Human parainfluenza viruses (HPIVs) are major causes of lower respiratory infections in infants, young children, the immunocompromised, the chronically ill, and the elderly (Glezen et al, 1984; Counihan et al, 2001; Weinberg et al, 2009). HPIVs belong to the Paramyxoviridae family of medium-sized enveloped viruses and their genomes are organized on a single negative-sense strand of RNA. HPIV3 is second only to respiratory syncytial virus (RSV) as a cause of pneumonia and bronchiolitis in infants and young children. The two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), are necessary for viral entry, cell-fusion and syncytium formation (Horvath et al, 1992; Hu et al, 1992; Takimoto et al, 2002; Porotto et al, 2012)

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Conclusion