Dual-linker gold nanoparticles as adjuvanting carriers for multivalent display of recombinant influenza hemagglutinin trimers and flagellin improve the immunological responses in vivo and in vitro

Abstract

Vaccination is the most cost-effective means of infectious disease control. Although current influenza vaccines are effective in battling closely matched strains, such vaccines have major limitations such as the requirement to produce new vaccines every season, an egg-dependent production system, long production periods, uncertainty in matching the vaccine to circulating strains, and the inability to react to new influenza pandemics resulting from genetic drift or shift. To overcome the intrinsic limitations of the conventional influenza vaccine, we have designed dual-linker gold nanoparticles (AuNPs) conjugated with both recombinant trimetric A/Aichi/2/68 (H3N2), hemagglutinin (HA) and TLR5 agonist flagellin (FliC) as a novel vaccine approach. Click chemistry and metal-chelating reactions were used to couple the two proteins. The conjugated proteins were found to possess high coupling specificity, high stability in harsh environments, high conjugation efficiency, and the ability to keep the appropriate protein conformations for immunogenicity and immunostimulation. Both AuNPs-HA/FliC and AuNPs-HA formulations induced higher levels of antibody responses than a mixture of soluble HA and FliC proteins when administered via a single intranasal immunization in mice. To further investigate the adjuvancy of these nanoparticles, in vitro experiments were conducted in both the JAWS II dendritic cell (DC) line and bone marrow-derived DC (BMDC) models. The results showed that dual-conjugated AuNPs were rapidly targeted and taken up by DCs. Consequently, DCs were induced toward maturation, as demonstrated by high levels of cytokine secretions and membrane costimulatory molecule expression. T cell proliferation was observed when splenic T cells were cocultured with AuNPs-HA/FliC-primed BMDCs. These results suggest that dual-conjugated AuNPs are effective at simultaneously displaying antigens and adjuvants in an oriented, multivalent format and can promote a strong immune response by activating DCs and T cells.

Keywords: adjuvant; co-delivery; dendritic cells; gold nanoparticle; influenza vaccine.

Figure 1

Characterization of HA and FliC protein-functionalized AuNP. Notes: (A, B) Transmission electron micrographs of AuNPs and HA/FliC-modified AuNPs. (C) UV–visible absorption spectra of bare AuNPs, dual-linker modified AuNPs, and HA/FliC-conjugated AuNPs. (D) Agarose gel electrophoresis of different amount of proteins conjugated to the same amount of AuNPs. The direction of migration is top to bottom. Lane 1, 5 μg/0.4 mg; lane 2, 30 μg/0.4 mg; lane 3, 50 μg/0.4 mg; lane 4, bare AuNPs. (E) TLR5-specific bioactivity of AuNP-HA/FliC and soluble FliC. (F) HA titers in AuNP-HA/FliC and soluble HA protein. A and B share the same scale bar. Abbreviations: AuNP, gold nanoparticle; FliC, flagellin; HA, hemagglutinin; UV, ultraviolet.

Figure 2

Systemic IgG immune responses. Notes: Mice were immunized intranasally with different AuNP formulations containing 5 μg of HA and 0.25 μg of FliC. Blood samples were collected at day 21 post vaccination. ELISA plates were coated with 4 μg/mL of A/Aichi/2/68 virus. HRP-conjugated goat anti-mouse IgG was used for detection. Significant difference: *P<0.05; ***P<0.005 (mean ± SEM, n=5). Abbreviations: AuNPs, gold nanoparticles; FliC, flagellin; HA, hemagglutinin.

Figure 3

Uptake of AuNPs-HA/FliC in vitro. Notes: (A, B) CLSM image of BMDCs incubated with Alexa 488-labeled AuNPs-HA/FliC or AuNPs-HA for 2 h. Nanoparticles were recognized as black dots in the bright field and merge images. The nucleus was stained blue with DAPI. (C) After 18 h incubation with BMDCs, cells were analyzed for CD11c⁺ and Alexa 488⁺ cell frequencies using FACS. Blue: control; green: AuNPs-HA; red: AuNPs-HA/FliC. (D) Quantitative analysis of uptake efficacy of AuNPs-HA and AuNPs-HA/FliC by BMDCs. The Y-axis is MFI of Alexa 488 after gating of CD11c⁺ cells, ns: P>0.05. (E, F) CLMS immunofluorescence images of JAWS II treated with AuNPs-HA/FliC or soluble HA/FliC for 12 h. Cells were then fixed and treated with Aichi-positive sera and DyLight™ 649 anti-mouse IgG Ab. HA was recognized by fluorescent-labeled Ab (red). Nuclei were stained by DAPI (blue). The concentrations used in treatments were 5 μg/mL of HA and 0.5 μg/mL of FliC. Abbreviations: Ab, antibody; AuNPs, gold nanoparticles; BMDCs, bone marrow-derived dendritic cells; CLSM, confocal laser scanning microscopy; DAPI, 4′,6-diamidino-2-phenylindole; FliC, flagellin; HA, hemagglutinin; MFI, mean fluorescence intensity; ns, no significance; TL, transmitted light.

Figure 4

Cytokines produced by JAWS II cells and BMDCs stimulated with different AuNP formulations or PBS. Notes: Cells were treated with soluble HA, soluble HA/FliC, AuNPs-HA, or AuNPs-HA/FliC at various HA and FliC concentrations. The concentration indicated in the figure was conjugated HA concentration. The ratio between HA and FliC was 5:1 (w/w). R848 was used as positive control (1 μg/mL). Significant difference: *P<0.05, ***P<0.005 versus control (mean ± SEM, n=2–3). Abbreviations: AuNPs, gold nanoparticles; BMDCs, bone marrow-derived dendritic cells; FliC, flagellin; HA, hemagglutinin; IL, interleukin; SEM, standard error of the mean; PBS, phosphate buffered saline.

Figure 5

Maturation of BMDCs and JAWS II cells. Notes: Costimulatory molecules CD40, CD80, CD86, and MHC class II on CD11c⁺ BDMCs and JAWS II cells treated with PBS (gray), HA/FliC (red), AuNPs-HA (green), and AuNPs-HA/FliC (blue) were compared by FACS. The MFI on CD11c⁺ gated cells were indicated. Significant difference: *P<0.05; **P<0.01 (mean ± SEM, n=2–3). Abbreviations: AuNPs, gold nanoparticles; BMDCs, bone marrow-derived dendritic cells; FACS, fluorescence activated cell sorting; FliC, flagellin; HA, hemagglutinin; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; SEM, standard error of the mean.

Figure 6

AuNPs-HA/FliC enhanced antigen presentation by BMDCs and stimulation of CD3⁺ and CD8⁺ T cells. Notes: CFSE-labeled splenic cells were cocultured with stimulated BMDCs (DC: splenic cell ratio is 1:5). Five days later, the proliferation of CFSE-positive CD3 and CD8 cells was analyzed by flow cytometry. After blocking, cells were stained with APC-conjugated anti-CD8α and PerCP-Cy5.5-conjugated anti-CD3ε. (A) Representative dot plots of CD3⁺ T cell proliferation. (B) Representative dot plots of CD8⁺ T cell proliferation. (C) Percentages of CD3 and CD8 positive. Significant difference: *P<0.05; **P<0.01 (mean ± SEM, n=2–3). Abbreviations: APC, antigen-presenting cell; AuNPs, gold nanoparticles; BMDCs, bone marrow-derived dendritic cells; CFSE, carboxyfluorescein succinimidyl ester; DC, dendritic cell; FliC, flagellin; HA, hemagglutinin; SEM, standard error of the mean; PBS, phosphate buffered saline.

Scheme 1

Design of dual-linker modified AuNPs to quantitatively conjugate HA and FliC. Abbreviations: AuNPs, gold nanoparticles; FliC, flagellin; HA, hemagglutinin; PEG, polyethylene glycol; N₃-PEG-SH, Azido PEG thiol; SH-NTA, N-[Nα,Nα-bis (carboxymethyl)-l-lysine]-12-mercaptododecanamide.