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. 2010 Nov 29;5(11):e13972.
doi: 10.1371/journal.pone.0013972.

Intranasal immunization with influenza VLPs incorporating membrane-anchored flagellin induces strong heterosubtypic protection

Bao-Zhong Wang  1 , Rui Xu, Fu-Shi Quan, Sang-Moo Kang, Li Wang, Richard W Compans
Affiliations

Intranasal immunization with influenza VLPs incorporating membrane-anchored flagellin induces strong heterosubtypic protection

Bao-Zhong Wang et al. PLoS One. .

Abstract

We demonstrated previously that the incorporation of a membrane-anchored form of flagellin into influenza virus-like particles (VLPs) improved the immunogenicity of VLPs significantly, inducing partially protective heterosubtypic immunity by intramuscular immunization. Because the efficacy of mucosal vaccination is highly dependent on an adjuvant, and is particularly effective for preventing mucosal infections such as influenza, we determined whether the membrane-anchored flagellin is an efficient adjuvant for VLP vaccines by a mucosal immunization route. We compared the adjuvant effect of membrane-anchored and soluble flagellins for immunization with influenza A/PR8 (H1N1) VLPs by the intranasal route in a mouse model. The results demonstrate that membrane-anchored flagellin is an effective adjuvant for intranasal (IN) immunization, inducing enhanced systemic and mucosal antibody responses. High cellular responses were also observed as shown by cytokine production in splenocyte cultures when stimulated with viral antigens. All mice immunized with flagellin-containing VLPs survived challenge with a high lethal dose of homologous virus as well as a high dose heterosubtypic virus challenge (40 LD(50) of A/Philippines/82, H3N2). In contrast, no protection was observed with a standard HA/M1 VLP group upon heterosubtypic challenge. Soluble flagellin exhibited a moderate adjuvant effect when co-administered with VLPs by the mucosal route, as indicated by enhanced systemic and mucosal responses and partial heterosubtypic protection. The membrane-anchored form of flagellin incorporated together with antigen into influenza VLPs is effective as an adjuvant by the mucosal route and unlike standard VLPs, immunization with such chimeric VLPs elicits protective immunity to challenge with a distantly related influenza A virus.

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Conflict of interest statement

Competing Interests: B.Z.W., S.M.K., R.W.C. and Emory University have equity interests in Zetra Biologicals, which is developing virus-like particle technology under license from Emory University. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Mucosal IgA and IgG titers against PR8 virus.
Samples were collected as described in Materials and Methods. Collected mucosal samples were cleared by a brief centrifugation (8000 rpm for 10 min), and assessed by ELISA. Microtiter plates were coated with 100 µl/well of inactivated PR8 virus (5 µg/ml) and HRP-conjugated goat anti-mouse IgA or IgG antibodies were used for detection. Antibody endpoint titers were defined as the highest serum dilution (fold) which gave an OD450 value 2-fold higher than naïve mice at the lowest dilution (200×). Representative data are the geometric mean ± standard deviation (SD) of six mice per group. Comparisons were performed using the non-matching two-way ANOVA followed by Bonferroni's post test. P<0.05(*), P<0.01(**), P<0.001(***), P>0.05(n.s.). A, Mucosal IgA titers; B, Mucosal IgG titers. Bars 1, 2, 3 and 4 presented groups of M1 only VLPs, HA/M1VLPs, HA/FliC/M1 cVLPs, and HA/M1VLPs + sFliC, respectively. FliC, flagellin; sFliC, soluble flagellin.
Figure 2
Figure 2. Serum IgG endpoint titers.
Endpoint titers of immune serum IgG specific to A/PR8 (A) or cross-reactive binding to A/Philippines (B) were determined using ELISA as described in Materials and Methods. HRP-conjugated goat anti-mouse IgG antibody was used for detection. Endpoint titers were defined as in Fig 1. M1, M1 only VLPs; HA/M1, HA/M1 VLPs; HA/F/M1, HA/flagellin (FliC, F)/M1 cVLPs; HA/M1+sF, mixture of HA/M1 VLPs plus soluble flagellin (sFliC, sF).
Figure 3
Figure 3. Endpoint titers of PR8-specific IgG subclasses.
IgG subclass endpoint titers were determined as for serum IgG endpoint titer but using HRP-conjugated goat anti-mouse IgG1(A), IgG2a (B), IgG2b (C) or IgG3 (D) for detection.
Figure 4
Figure 4. Neutralization activity against PR8.
Immune sera were diluted 2-fold stepwise and incubated with virus (100 PFU) at 37°C for 1 h. A standard plaque reduction assay was performed using MDCK cell cultures.
Figure 5
Figure 5. Hemagglutination inhibition (HI) titers against H1N1 PR8 or H3N2 Philippines virus.
A, HI titers to PR8 virus; B, HI titers to Philippines virus. HI titers of immune sera were determined as the capacity of sera to inhibit virus hemagglutination of chicken red blood cells. Representative data are the geometric mean ± S.D.
Figure 6
Figure 6. Cytokine secretion from immunized mouse splenocytes.
ELIspot assays were used to detect the proliferation of antigen-specific CD4+ secreting INF-γ (A) or IL-4 (B) cells as described in “Materials and Methods”. Splenocytes were isolated from immunized mice 4 weeks after the boosting immunization and seeded into pre-coated Multiscreen plates (Millipore). Defined peptides (MHC I or MHC II) or inactivated PR8 viruses were added to stimulate antigen-specific T cell proliferation. Cytokine secreting clones were visualized by spot staining following the manufacturer's instruction. Representative data were mean ± S.D. of spot number in 1×106 splenocytes of six mice.
Figure 7
Figure 7. Live virus challenge using A/PR8 (H1N1) or A/Philippines (H3N2) viruses.
Immunized mice were challenged with a lethal dose (40 LD50) of A/PR8 (A and B) or A/Philippines (C and D) in a volume of 25 µl by IN instillation. Mouse survival (A, C) and body weight changes (B, D) were monitored daily.
Figure 8
Figure 8. Lung viral loads on day 4 post-challenge infection with the homologous virus A/PR8 or the heterosubtypic virus A/Philippines.
Mice immunized IN with designed doses of VLPs were challenged with a lethal dose (40 LD50) of A/PR8 or A/Philippines. Six mice were included in each viral challenge group. Four days after infection, mouse lungs were collected and extracted with medium RPMI 1640. The extract was adjusted to 1 ml per lung. Virus titers in lung extracts were titrated using MDCK-based plaque assay as described in Materials and Methods. The lung virus titer is presented as pfu per lung.
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