Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 May;84(9):4513-23.
doi: 10.1128/JVI.01931-09. Epub 2010 Feb 24.

Assembly and Biological and Immunological Properties of Newcastle Disease Virus-Like Particles

Affiliations
Free PMC article

Assembly and Biological and Immunological Properties of Newcastle Disease Virus-Like Particles

Lori W McGinnes et al. J Virol. .
Free PMC article

Abstract

Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.

Figures

FIG. 1.
FIG. 1.
Proteins in ND VLPs. Proteins in purified ND VLPs (VLP) and egg-grown, purified NDV (virus) were separated on polyacrylamide gels and visualized by silver staining. Lanes 1, 3, and 5 show proteins in VLPs from three different preparations. Proteins in purified virus are shown in lanes 2 (strain AV), 4 (strain B1), and 6 (strain B1). Lanes 1 to 4 show proteins electrophoresed in the absence of reducing agent (−βME). Lanes 5 and 6 show proteins electrophoresed in the presence of reducing agent (+βME). HN, hemagglutinin-neuraminidase protein, either dimer or monomer; F0, uncleaved fusion protein; NP, nucleocapsid protein; M, membrane protein; Fnr, nonreduced fusion protein (mix of uncleaved F0 and disulfide-linked F1-F2). The asterisk alongside lane 2 indicates BSA that copurifies with some preparations of virus.
FIG. 2.
FIG. 2.
Attachment activity of ND VLPs. Different amounts (1×, 0.5×, and 0.1×) of radioactively labeled ND VLPs were added to avian cell monolayers on ice and incubated for 30 min. Unbound particles were removed, and the cells washed with PBS and lysed. Viral proteins in the resulting cell extracts were electrophoresed on polyacrylamide gels and visualized by autoradiography. Lane 1, input VLPs (1×); lane 2, negative control (particles purified from supernatants of vector-transfected cells); lane 4, 1× VLPs added to cells; lane 5, 0.5× VLPs added to cells; lane 6, 0.1× VLPs added to cells. Lane 3 shows the binding of 1× VLPs to cells in the presence of anti-NDV antiserum. The relative ratios of proteins in VLPs shown in Fig. 2 appear different than those shown in Fig. 1 due to the variations in numbers of methionine and cysteine residues in the NDV proteins.
FIG. 3.
FIG. 3.
Hemagglutination activities of ND VLPs. The hemagglutination titer was determined as described in Materials and Methods. Twofold dilutions of the three preparations of ND VLPs (VLP#1, VLP#2, and VLP#3) described in Table 1 or of purified NDV (strain B1) were added, in duplicate, to wells of a microtiter plate. RBCs were added to each well. Shown is a digitally acquired photograph of the plate.
FIG. 4.
FIG. 4.
Membrane fusion activities of ND VLPs. Purified VLPs formed with NP, HN, F, and M proteins, VLPs formed with NP, HN, and M proteins (37), and VLPs formed with F, NP, and M proteins (37) were loaded with R18 as described in Materials and Methods. The VLPs were added to RBCs on ice and warmed to 37°C. The figure shows the increase in fluorescence with time after transfer to 37°C. The data were normalized for the R18 loading in each VLP as determined by fluorescence after chemical dequenching induced by the addition of Triton X-100. Shown are the averages of the results of three experiments and error bars indicating standard deviations.
FIG. 5.
FIG. 5.
ELISA titers of NDV-specific antibodies after immunization with ND VLPs or NDV. Groups of five mice were immunized with the total protein concentrations of VLPs or virus indicated at the bottom of each panel. All mice received a boost of 10 μg of total VLP or virus protein at day 26. Sera were collected at 10, 20, 37, and 49 days postimmunization. The figure shows the titers of antibodies with the use of total NDV proteins of purified NDV strain AV as the target antigen. Titer was defined as the reciprocal dilution of antibody that resulted in an OD that was 5-fold greater than that of the background. Top, titers obtained after ND VLP immunization; bottom, titers obtained after immunization with UV-inactivated NDV. Horizontal bars, mean; vertical bars, standard deviation.
FIG. 6.
FIG. 6.
Activation of T cells by ND VLPs. Activation of CD8 or CD4 T cells to secrete IFN-γ was measured by intracellular cytokine staining. Splenocytes from groups of four mice immunized with different concentrations of ND VLPs or UV-inactivated virus (shown at the bottom of each panel) were stimulated in vitro as described in Materials and Methods. The percentages of total cells that were CD8+, IFN-γ+ (A) or CD4+, IFN-γ+ (B) were determined by flow cytometry. The percentage of cells stimulated with anti-CD3 antibody is shown as a positive control. The mean and standard error of the mean, calculated using Prism Graph Pad software, are show for each set of mice.
FIG. 7.
FIG. 7.
Incorporation of HN and F proteins from NDV strain B1 into ND VLPs. Avian cells were transfected with the cDNAs encoding the NP and M proteins from strain AV, as well as various combinations of cDNAs encoding HN and F proteins from either strain AV or strain B1, as indicated at the top of the figure. Two sets of cells were pulse labeled with [35S]methionine and then chased with nonradioactive medium as described in Materials and Methods. Radioactively labeled proteins in cell lysates of one set of cells prepared at the end of the pulse labeling are shown in the left panel. VLPs harvested at the end of the nonradioactive chase in the other set of cells were purified, and the proteins present in extracts and VLPs were precipitated with a cocktail of antibodies (anti-NDV antibody, anti-HN protein antibody, anti-F protein antibodies, and anti-M protein). Proteins in the precipitate were resolved on polyacrylamide gels and detected by autoradiography.
FIG. 8.
FIG. 8.
Incorporation of peptides into ND VLPs. Avian cells were transfected with cDNAs encoding the NDV M protein, as well as various combinations of untagged and HA-tagged HN protein, untagged and FLAG-tagged F protein, and untagged and HA-tagged NP protein, as indicated below each panel. F protein cDNA containing a mutation in the cleavage site (20), as described in Results, was used in order to resolve the F protein separately from NP on polyacrylamide gels. (A) VLPs were radioactively labeled as described in Materials and Methods and the legend to Fig. 7. Proteins present in the pulse-labeled cell extracts (left panel) or in purified VLPs (right panel), harvested from supernatants of cells subjected to a radioactive pulse and a nonradioactive chase, were detected by autoradiography of polyacrylamide gels containing proteins precipitated using a polyclonal antibody cocktail. (B) Avian cells were transfected with the cDNAs indicated below the panel. VLPs were harvested at 24 h posttransfection. The figure shows the proteins in purified VLPs detected by Western blotting (WB) using an antibody cocktail (described in the legend to Fig. 7) (top panel) or anti-HA antibody (bottom panel). M, marker NDV-infected cell extract.
FIG. 9.
FIG. 9.
Construction of HN-NiV G chimeric protein. The diagram shows the locations of the cytoplasmic domain (CT), transmembrane domain (TM), and ectodomain of the NDV HN protein and the NiV G protein and the domains present in two NDV HN-NiV G chimeric proteins. Below the bars, the sequences at the junctions of the TM and ectodomains of the two wild-type proteins, as well as those of two different chimeric proteins, HN/NiVG#1 and HN/NiVG#2, are shown.
FIG. 10.
FIG. 10.
HN/NiVG expression and assembly into ND VLPs. (A) The first through the sixth lanes show the total protein immunoprecipitated from radioactively labeled extracts of cells expressing the HN protein, the NiV G protein, or one of the two chimeric proteins. HN protein was precipitated with anti-NDV antibodies (first and second lanes). The NiV G protein and the chimeric proteins were precipitated with anti-NiV antibody (third through sixth lanes). The 7th through the 12th lanes show biotinylated surface-expressed protein levels in cells transfected with the HN protein, NiV G protein, or chimeric protein cDNAs. Biotinylated proteins were sequentially precipitated with neutravidin-agarose and then anti-NDV antibodies (seventh and eighth lanes) or anti-NiV G protein antibody (9th through 12th lanes). (B) Radioactively labeled NiV G protein sequences in VLPs prepared from cells transfected with the cDNAs indicated at the bottom of the panel for each lane. Purified VLPs were lysed, and the proteins precipitated with anti-NiV G protein antibody and detected by autoradiography.

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback