A Novel Application of Virus Like Particles in the Hemagglutination Inhibition Assay

Abstract

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP's antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen.

Keywords: avian influenza; hemagglutination inhibition assay; vaccinal seed; virus like particles.

Figure 1

Gel electrophoresis of the restriction enzyme digestion screening of the combined pFastBac1 vector containing the three genes (HA, NA and M1). Lane 1 indicates the 1 Kilo base (kB) ladder (500 bp, 800 bp, 1600 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, etc.). The even No. lanes are combined pFastBac1 vectors that are undigested by restriction enzyme Tth111I and are considered the control, while the odd No. lanes are combined pFastBac1 vectors digested by restriction enzyme Tth111I. The figure shows that the colony in lane No. 3 (marked by the white arrow) is the only one that gave specific bands (5158 bp, 2211 bp, 1719 bp) with the negative control of the same colony without restriction enzyme digestion as shown in lane 2.

Figure 2

The hemagglutination assay (HA assay) of the recombinant baculovirus yielded from passage 1 (P1) using 1% of RBCS. The figure shows the HA activity of the passage 1 harvest at 2⁶ hemagglutination unit (HAU) titers, approximately. The two rows show HA titer for two different recombinant baculovirus candidates.

Figure 3

Characterization of VLP-expressed proteins using SDS-PAGE and Western blot. Figure (a) shows the VLP proteins separated using 4–12% gradient SDS-PAGE. The protein ladder used is SeeBlue^® Plus2 Pre-Stained Standard (Invitrogen). The gel shows the full-length HA protein (64 kDa) and M1 protein (30 kDa). NA protein did not appear as it was expressed less than the other influenza virus proteins. Figure (b) shows the Western blot of the specific band of the whole HA and M1.

Figure 4

Characterization of VLPs using negative stain transmission electron microscopy. The micrograph shows the spherical VLPs with the spikes of HA and NA proteins protruding from its envelope and resembling the natural virus particles. The size bar indicates the size of the VLP particles. The normal size varies from 80 to 120 nm, the natural Avian Influenza virus size.

Figure 5

Individual HI titers of the different serum antibody groups using homologous relevant vaccinal antigens (the antibodies derived from chickens vaccinated by the same antigen used in the HI assay) against the heterologous VLP antigen.

Figure 6

Individual HI titer of serum antibodies from SPF chickens vaccinated by VLPs using the homologous relevant vaccinal antigen (VLP) against heterologous the A/chicken/Egypt/18-H/2009(H5N1) antigen.

Figure 7

Alignment of the H5 HA1 amino acid sequences (excluding signal peptide) of the different vaccinal seeds used in this study, including the prepared VLPs’ seed. The HA1 includes the residues relevant to the epitopes in the predetermined major antigenic sites A–D. Single letter codes are shown for amino acids and colors indicate their major biochemical properties such as Red-acidic; Blue-basic.