Figure 1.
Construction and cellular expression of influenza virus-like particles (VLPs) in mammalian cells.
(A) Diagrams of mammalian expression vectors of influenza VLPs. Indicated are the CMV constitutive promoter (CMV), tetracycline repressor gene (TetR), polyadenylation signals (PA), CMV doxycycline (Dox)-inducible promoter (CMV-TO), chimeric intron (In), encephalomyocarditis virus internal ribosomal entry site (IRES), and influenza genes (M1 and M2, matrix 1 and 2; HA, hemagglutinin; NA, neuraminidase). (B), (C) Expression of viral genes in selected quadruple VLP producer cells. The total cell lysates were extracted from VLP producer cells without (−) or with (+) Dox induction, and then probed with respective specific antibodies against HA, NA, M1, and M2 in Western blot assays. Expression of N2 was detected by RT-PCR. Molecular masses of expressed HA, NA, M1, and M2 are indicated on the right. (D), (E) In vivo immunofluorescence staining of expressed viral proteins in VLP producer cells with antibodies against HA and NA (red) as marked and counterstained with DAPI (blue).
Figure 2.
Morphology and antigen presence determinations of purified mammalian VLPs.
(A), (B) Secreted VLPs and virus were purified by sucrose density gradient ultracentrifugation, negatively stained with 2% uranyl acetate, and observed by TEM at 100,000× magnification; pictures of respective virus strains are shown below the VLPs. (C), (D) Immunogold electron microscopy of purified VLPs. The primary antibodies used are shown below individual panels. Secondary antibody was goat anti-rabbit conjugated to 12 nm gold beads.
Figure 3.
Dynamic light scattering (DLS) measurements of particle sizes and distributions of mammalian VLPs in solution.
Average particle diameters of H3N2-VLPs (A), H3N2 virus (B), and H5N1-VLPs (C), and H5N1 virus (D) in phosphate buffer (pH 7.4), at 25°C. Two representative determinations of different batches of VLPs and virus are shown as red and green lines. The size distribution of VLP populations ranged from 70 to 200 nm with a 95% confidence interval (CI).
Figure 4.
Characterization of purified mammalian VLPs.
The total proteins of purified H3N2-VLPs and H3N2 virus (A) and H5N1-VLPs and H5N1 virus (C) were resolved by SDS-PAGE in a 7.5–17.5% gradient gel and stained with Coomassie blue. Molecular masses of protein markers are shown on the right. Each relevant band, as marked with an arrow and a number, was subjected to LC/MS/MS analysis to identify its composition. The identified viral protein bands of H3N2- and H5N1-VLPs (marked with parentheses in panels A and C) confirmed by Western blot analyses with relevant specific antibodies for H3N2-VLPs and virus (B) and for H5N1-VLPs and virus (D). The relative abundances of HA and NA, quantified by Chemigenius 2 (SYNGENE, Frederick, MD) and GeneTools (version 3.07) software, were 4∶1 for H3N2-VLPs and 3∶2 for H5N1-VLPs. The HA protein attributes to 12.8% and 18% of total proteins in H3N2- and H5N1-VLPs, respectively. (E) Assessment of HA function by hemagglutination assay. The amounts of mammalian VLPs or virus used are indicated, in a twofold serial dilution. TNE (buffer of VLPs) and PBS were included as the negative controls in the hemagglutination assays.
Table 1.
Mammalian VLPs associated viral and cellular proteins identified by mass spectrometry (LC/MS/MS).
Table 2.
Unique cellular proteins identified by LC/MS/MS with high Mascot scores in mammalian VLPs.
Figure 5.
Specific integration of cellular proteins in influenza VLPs.
Purified H5N1-VLPs were either mock-treated (Untr.) or digested overnight with typsin (Prot.) followed by purification with a 20% sucrose cushion. Mock-treated VLPs (lane 1) or typsin-treated VLPs (lane 2), 2.5 µg each, were further analyzed by Western blotting with antibodies against the indicated proteins including HA, NA, M1, M2, β-actin, tubulin, annexin A2, and clathrin (A). Molecular weight markers are marked on the left. (B) Immunogold labeling of clathrin on the surface of purified VLPs. Secreted VLPs purified from conditioned medium were immunogold labeled with antibody against clathrin, negatively stained with 2% uranyl acetate, and observed by electron microscopy (100,000× magnification).
Figure 6.
Glycosylation profiling of HA and NA in mammalian VLPs.
Mock-treated and deglycosylase-treated proteins of H5N1-VLPs were separated by SDS-PAGE, stained with Coomassie blue (A), and further analyzed by Western blotting using antibodies against H5 (B) and N1 (C). In parallel, mock-treated and deglycosylase-treated proteins of H3N2-VLPs were analyzed by Western blotting using the antibody against H3 (D). Untreated and deglycosylase-treated proteins of respective VLPs and virus are compared in panels B and D. Glycosylated HA1, HA2, NA, and H3 are denoted as HA1**, HA2**, NA**, and H3**, respectively. # represents glycosylated proteins harboring a residue moiety of complex-type glycans sensitive to PNGase F, but not Endo-H. π represents unknown posttranslational modifications on the NA of H5N1-VLPs.
Figure 7.
Humoral immune response of mammalian VLPs.
(A) Regimen of prime and boost vaccination was followed by viral challenge. IM0, IM1, and IM2 represent the mouse serum collected before immunization (day -1), and 14 days after priming, and boosting, respectively. (B) The antigen-specific IgG antibodies from the serum of each mouse group taken at IM2 were assayed against distinct antigens of H3N2-VLP, H5 protein (recombinant baculovirus expressed), and H5N1-VLP (same as the immunization antigen) by ELISA after vaccination of H5N1-VLP or inactivated whole virus (IWV). Groups of mice (n = 8–12) were either intramuscularly immunized with 2.5 µg and 10 µg dose of VLPs, or IWV, as marked. The dilution of used serum samples in the ELISA assays are labeled at the top. H3N2-VLPs containing all the host cell proteins integrated into the VLP antigen but with different subtype of HA and NA was used as negative control, whereas the baculovirus-produced H5 protein constituted the positive control. The employed amounts of coating antigens are shown. (C) ELISA of serum IgG antibodies induced by H5N1-VLPs or IWV vaccines against H5N1 virus as ELISA antigen. (D) Western blot analysis of mice serum IgG antibodies elicited by either H5N1-VLP (left panel) or IWV (right panel) vaccines. The used antigens and individual amounts are indicated at the top of panels. (E) The specific IgG isotype and IgA elicited by VLPs and IWV vaccines were assayed using H5N1-VLP as ELISA antigen. (F) HI titer of each vaccinated mouse; mean values of the same group are plotted. HI titer of 40 was set as threshold of seroprotection.
Figure 8.
Vaccine Protection against lethal-dose challenge of H5N1 virus.
At day 42, vaccinated mice were challenged intranasally with a lethal dose (100 LD50) of recombinant H5N1 (NIBRG-14) virus and monitored daily for weight loss and mortality. The percentages of survival rate and changes of body weight and temperature were recorded. (A) Survival for H5N1-VLP groups, (B) survival for IWV groups, Mice that lost greater than 30% body weight were euthanized. (C) Body weight and temperature for H5N1-VLP groups. For th groups receiving 0.3 µg and 1.5 µg antigen dose, only data of surviving mice are shown. (D) Body weight and temperature for IWV groups.