Clin Exp Vaccine Res.  2017 Jan;6(1):61-66. 10.7774/cevr.2017.6.1.61.

Preparation and immunogenicity of influenza virus-like particles using nitrocellulose membrane filtration

• Park YC
• Song JM

• KMID: 2366893
• DOI: http://doi.org/10.7774/cevr.2017.6.1.61

Abstract

PURPOSE
Nitrocellulose membrane-based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity.
MATERIALS AND METHODS
Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment.
RESULTS
Influenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus-specific antibody responses.
CONCLUSION
Our results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.

Keyword

Virus-like particle; Vaccines; Nitrocellulose membrane; Baculovirus

MeSH Terms

Animal Experimentation
Antibody Formation
Baculoviridae
Collodion*
Filtration*
Hemagglutinins
Influenza, Human*
Membranes*
Neuraminidase
Orthomyxoviridae
Sucrose
Ultracentrifugation
Vaccines
Collodion
Hemagglutinins
Neuraminidase
Sucrose
Vaccines

Figure

• 

  Fig. 1 Strategy of influenza virus like particles (VLPs) production using nitrocellulose membrane–based filtration system (NCFS). Influenza VLPs were generated by baculovirus-insect cell expression system and purified by NCFS and ultracentrifugation. HA, hemagglutinin; NA, neuraminidase.

• 

  Fig. 2 Schematic diagram of stirred cell for nitrocellulose membrane–based filtration system and ultracentrifugation. Stirred cell device with nicrocellulose membrane under N2 pressure (A) and two layered ultracentrifugation (B). VLPs, virus-like particles.

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  Fig. 3 Comparison of hemagglutinin (HA) titer during purification of influenza virus like particles in each step. HA titer after clarification (A), total HA activity after ultrafiltration (B), unit HA activity after ultracentrifugation (C). HAU, hemagglutinating units.

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  Fig. 4 Characterization of influenza virus–like particles (VLPs). (A) hemagglutinin (HA) protein and M1 were detected by inactivated H5N8 virus immunized mice serum. (B) M1 protein were detected by mouse anti-influenza M1 IgG. (C) Identification of H5N8 VLPs by transmission electron microscopy.

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  Fig. 5 H5N8 virus specific antibody response in immunized serum, virus-specific IgG response in immunized serum (A), virus-specific IgA response in immunized serum (B). IM, intramuscular; IN, intranasal.

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