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. 2008 Dec;82(23):11813-23.
doi: 10.1128/JVI.01076-08. Epub 2008 Sep 10.

Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses

Bao-Zhong Wang  1 , Fu-Shi Quan, Sang-Moo Kang, Jadranka Bozja, Ioanna Skountzou, Richard W Compans
Affiliations

Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses

Bao-Zhong Wang et al. J Virol. 2008 Dec.

Abstract

We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H(1)N(1)) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular immune responses, and the protective efficacy against homologous and heterologous live virus challenge of the resulting VLPs were tested after intramuscular administration in a mouse model. The results demonstrated that flagellin-containing VLPs elicited higher specific immunoglobulin G (IgG) responses than standard HA and M1 VLPs, indicating the adjuvant effect of flagellin. Enhanced IgG2a and IgG2b but not IgG1 responses were observed with flagellin-containing VLPs, illuminating the activation of Th1 class immunity. The adjuvant effects of flagellin were also reflected by enhanced specific cellular responses revealed by the secretion of cytokines by freshly isolated splenocyte cultures when stimulated with pools of major histocompatibility complex class I or II peptides. When immunized mice were challenged with homologous live PR8 virus, complete protection was observed for both the standard and cVLP groups. However, when a heterosubtypic A/Philippines (H(3)N(2)) virus was used for challenge, all of the standard VLP group lost at least 25% of body weight, reaching the experimental endpoint. In contrast, for the cVLP group, 67% of mice survived the challenge infection. These results reveal that cVLPs designed by incorporating flagellin as a membrane-anchored adjuvant induce enhanced cross-protective heterosubtypic immune responses. They also indicate that such cVLP vaccines are a promising new approach for protection against pandemic influenza viruses.

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Figures

FIG. 1.
FIG. 1.
Construction of membrane-bound flagellin and VLP production. (A) Schematic diagram of the membrane-anchored flagellin construct. The mellitin SP and HA TM-CT coding sequences were fused to the 5′ and 3′ ends of flagellin-encoding gene fliC, respectively, to form the full-length chimeric fliC. (B) Cellular and cell surface expression of membrane-anchored flagellin. Surface expression of the membrane-anchored flagellin was detected by cell surface biotinylation. Lanes: 1, cell lysate from cells infected with rBV expressing membrane-anchored flagellin; 2, mock rBV (rBV expressing human immunodeficiency virus Gag)-infected cells. (C) Optimization of VLP production: Sf9 cells were infected with rBVs expressing HA, M1, and flagellin at different MOI as designated at the bottom. VLPs were prepared as described in Materials and Methods. The resulting VLPs were analyzed by Western blotting. HA and M1 bands were probed with mouse anti-influenza serum. The band below M1 was variable in different VLP preparations and may represent a degradation product. Membrane-anchored flagellin (FliC) was probed with rabbit antiflagellin polyclonal antibody. (D) Electron microscopy of influenza VLPs. Influenza VLPs containing flagellin, HA, and M were negatively stained as described in Materials and Methods.
FIG. 2.
FIG. 2.
Glycosylation and TLR-5 agonist activity of membrane-anchored flagellin. (A) Ten-microgram aliquots of flagellin-containing VLPs were left untreated (lane 2) or were digested by PNGase F (lane 3) or endo-H (lane 4). Lane 1 is cell lysate from cells infected by membrane-anchored flagellin expressing rBV. Samples were resolved by Western blotting. (B) TLR-5-positive and -negative RAW264.7 cells were activated with soluble flagellin (sFliC) and flagellin-containing VLPs (FliC/HA/M1 VLPs), respectively. Standard HA/M1 VLPs were used as controls. Supernatants were collected 24 h after stimulation, and the TNF-α concentration in the supernatant was determined by ELISA as described in Materials and Methods. TLR-5-specific bioactivity was expressed by the production by TNF-α of TRL-5-positive cells, from which was subtracted that of TLR-5-negative cells stimulated by flagellin, flagellin-containing VLPs, or standard HA/M1 VLPs at the same concentration. Data represent means ± standard errors from triplicate repeats.
FIG. 3.
FIG. 3.
Serum IgG and isotype endpoint titers. Serum antibodies specific for influenza A/PR8 virus were determined. The highest serum dilution (n-fold) which gave an OD450 two times higher than that of naive mice was designated as the serum antibody endpoint titer. Representative data are the mean ± standard deviation (SD) of six mice/group and were analyzed by an unpaired t test. A two-tailed P value of <0.05 is designated as a significant difference. (A to D) Serum IgG (*, P < 0.05) (A), IgG1 (B), IgG2a (*, P < 0.05) (C), and IgG2b (*, P < 0.05) (D). sFliC, soluble flagellin.
FIG. 4.
FIG. 4.
Neutralization and HI titers against influenza A/PR8 virus, and the effect of preexisting antiflagellin immunity. (A) Neutralization activities were determined using the capacity of sera to neutralize plaque formation by influenza PR8 virus in MDCK cell cultures. Serial dilutions of sera were incubated with influenza PR8 virus (about 100 PFU) at 37°C for 1 h. A standard plaque reduction assay was performed using MDCK cells. (B) HI titers of sera were determined using the capacity of sera to inhibit virus hemagglutination of chicken red blood cells (*, P < 0.05). (C) The preexisting antiflagellin IgG titer was determined with ELISA. A group of six mice was preimmunized twice intramuscularly at a 4-week interval with 10 μg of soluble recombinant flagellin and subsequently immunized twice with 10 μg cVLPs at a 4-week interval. A six-mouse group without preimmunization was used as the control. Serum antiflagellin and anti-inactivated PR8 virus IgG titers were determined by ELISA. For flagellin-specific IgG titers, microplates were coated with 100 μl of recombinant flagellin per well at 5 μg/ml. IgG titer determinations are described in Materials and Methods. Representative data are the mean ± SD from six mice in each group. sFliC, soluble flagellin.
FIG. 5.
FIG. 5.
Serum IgG endpoint and HI titers against the heterosubtypic virus A/Philippines (H3N2). Serum IgG endpoint titer (A) and HI titer (B) were determined as described in Materials and Methods. Data depict the mean ± SD from six mice per group (*, P < 0.05). sFliC, soluble flagellin.
FIG. 6.
FIG. 6.
Cytokine secretion from immunized mouse splenocytes. Splenocytes were isolated from immunized six-mouse groups 3 weeks after the boosting immunization. Cells (1 × 106) were seeded into 96-well cell culture plates with 200 μl RPMI 1640 medium. The MHC-I- or MHC-II-specific HA peptides of A/PR8 virus were added into cell culture medium, and secreted cytokines were determined as described in Materials and Methods. Data depict the mean ± SD of six mice per group with similar results in triplicate assays (*, +, or ^, P < 0.05). (A) IL-2. (B) IFN-γ. (C) TNF-α. (D) IL-4. FliC, flagellin.
FIG. 7.
FIG. 7.
Protection from challenge with A/PR8 or A/Philippines virus. Mouse groups containing six mice were challenged with 40×LD50 of PR8 (H1N1) or A/Philippines (H3N2) virus. Mice were monitored daily for 14 days for body weight changes (A) and percentages of survival (B) after PR8 virus challenge or for body weight changes (C) and percentages of survival (D) after A/Philippines virus challenge. sFliC, soluble flagellin.
FIG. 8.
FIG. 8.
Lung viral load on day 4 postchallenge. Six mice in each group were challenged with 40×LD50 of PR8 (H1N1) or A/Philippines (H3N2) virus. Mouse lung samples were collected on day 4 postchallenge. Six lungs in each group were pooled, ground, and cleared in 6 ml of DMEM. Lung virus loads were determined using a standard plaque assay with MDCK cells. Bars represent mean virus titers ± standard errors from three independent assays (*, P < 0.05). FliC, flagellin.
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References

    1. Ali, A., R. T. Avalos, E. Ponimaskin, and D. P. Nayak. 2000. Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein. J. Virol. 748709-8719. - PMC - PubMed
    1. Ben-Yedidia, T., and R. Arnon. 1998. Effect of pre-existing carrier immunity on the efficacy of synthetic influenza vaccine. Immunol. Lett. 649-15. - PubMed
    1. Bright, R. A., D. M. Carter, S. Daniluk, F. R. Toapanta, A. Ahmad, V. Gavrilov, M. Massare, P. Pushko, N. Mytle, T. Rowe, G. Smith, and T. M. Ross. 2007. Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin. Vaccine 253871-3878. - PubMed
    1. Ciacci-Woolwine, F., I. C. Blomfield, S. H. Richardson, and S. B. Mizel. 1998. Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line. Infect. Immun. 661127-1134. - PMC - PubMed
    1. Ciacci-Woolwine, F., P. F. McDermott, and S. B. Mizel. 1999. Induction of cytokine synthesis by flagella from gram-negative bacteria may be dependent on the activation or differentiation state of human monocytes. Infect. Immun. 675176-5185. - PMC - PubMed

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