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. 2014 Feb;9(2):237-51.
doi: 10.2217/nnm.13.58. Epub 2013 Jul 5.

Gold nanoparticle-M2e conjugate coformulated with CpG induces protective immunity against influenza A virus

Wenqian Tao  1 , Katherine S Ziemer, Harvinder S Gill
Affiliations

Gold nanoparticle-M2e conjugate coformulated with CpG induces protective immunity against influenza A virus

Wenqian Tao et al. Nanomedicine (Lond). 2014 Feb.

Abstract

Aim: This study aimed to develop a novel influenza A vaccine by conjugating the highly conserved extracellular region of the matrix 2 protein (M2e) of influenza A virus to gold nanoparticles (AuNPs) and to test the vaccine in a mouse influenza challenge model.

Materials & methods: Citrate-reduced AuNPs (diameter: 12 nm) were synthesized, and characterized by transmission electron microscopy and dynamic light scattering. M2e was conjugated to AuNPs through thiol-gold interactions to form M2e-AuNP conjugates. Particle stability was confirmed by UV-visible spectra, and M2e conjugation was further characterized by x-ray photoelectron spectroscopy. Mice were immunized with M2e-AuNPs with or without CpG (cytosine-guanine rich oligonucleotide) as an adjuvant with appropriate control groups. Sera was collected and M2e-specific immunoglobulin (IgG) was measured, and immunized mice were challenged with PR8-H1N1 influenza virus.

Results: M2e-capped AuNPs could be lyophilized and stably resuspended in water. Intranasal vaccination of mice with M2e-AuNP conjugates induced M2e-specific IgG serum antibodies, which significantly increased upon addition of soluble CpG as adjuvant. Upon challenge with lethal PR8, mice vaccinated with M2e-AuNP conjugates were only partially protected, while mice that received soluble CpG as adjuvant in addition to M2e-AuNP were fully protected.

Conclusion: Overall, this study demonstrates the potential of using the M2e-AuNP conjugates with CpG as an adjuvant as a platform for developing an influenza A vaccine.

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Figures

Figure 1
Figure 1. Morphology and size distribution of gold nanoparticles.
(A) Transmission electron micrograph and (B) dynamic light scattering data showing size distribution.
Figure 2
Figure 2. Characterization of gold nanoparticles.
(A) Absorption spectrum of AuNPs before and after conjugation of M2e, and after addition of soluble CpG. Inset shows a close-up of absorption peaks. (B) Absorption spectrum of nonlyophilized AuNPs, lyophilized unconjugated AuNPs or lyophilized M2e–AuNP conjugates after resuspension in water. Inset shows a photograph of lyophilized M2e–AuNP conjugates. (C) Transmission electron micrograph of nonlyophilized M2e–AuNP conjugates. (D) Transmission electron micrograph of a resuspension of lyophilized M2e–AuNP conjugates. AuNP: Gold nanoparticle.
Figure 3
Figure 3. X-ray photoelectron spectroscopy spectra of M2e-conjugated and unconjugated gold nanoparticles.
(A) Tight scan spectra of Au 4f region. Arrows point to the high-energy shoulders. (B) Tight scan spectra of C 1s region. (C) Tight scan spectra of N 1s region. AuNP: Gold nanoparticle.
Figure 4
Figure 4. M2e-specific IgG antibody response in mouse serum.
(A) Titration curve for pooled day 21 serum. (B) Titration curve for pooled day 42 serum. (C) M2e-specific IgG antibodies in 1:200 diluted serum of individual mice of a group. OD measured at 490 nm wavelength. Each diamond in (C) represents an animal in a group (n = 3 in M2e alone group, n = 5 in M2e + CpG group and n = 8 for the remainder of the groups), the vertical column represents the mean, and error bars represent standard error of the mean. The data presented combine results of two independent experiments. **p < 0.01; ***p < 0.001. AuNP: Gold nanoparticle; OD: Optical density.
Figure 5
Figure 5. M2e-specific IgG subtypes in mouse serum.
(A) Titration curve for IgG1 in pooled day 42 serum. (B) Titration curve for IgG2a in pooled day 42 serum. (C) IgG1 and IgG2a antibodies in 1:200 diluted serum of individual mice of a group. OD measured at 490 nm wavelength. Each diamond in (C) represents an animal in a group (n = 3 in M2e alone group, n = 5 in M2e + CpG group and n = 8 for the remainder of the groups), the vertical column represents the mean, and error bars represent standard error of the mean. The data presented combine results of two independent experiments. ***p < 0.001. AuNP: Gold nanoparticle; OD: Optical density.
Figure 6
Figure 6. Cross-reactivity of mouse serum IgG (1:50 dilution) against native M2 expressed on three influenza A viruses.
(A) OD measured at 490 nm wavelength. Each diamond represents an animal in a group (n = 3 in M2e alone group, n = 5 in M2e + CpG group and n = 8 for the remainder of the groups). The vertical column represents the mean. (B) The table provides sequences of the consensus M2e used for immunization and M2e present on the surface of the three influenza A viruses used in the cross-reactivity ELISA assay (Philippines-H3N2, PR8-H1N1 and WSN-H1N1). The data presented combine results of two independent experiments. *p < 0.05; ***p < 0.001. AuNP: Gold nanoparticle; OD: Optical density.
Figure 7
Figure 7. Response of mice to a lethal dose (5 × lethal dose 50%) of influenza PR8 virus.
(A) Weight loss of mice after challenge. (B) Survival of mice after challenge. The data presented combine results of two independent experiments (n = 3 in M2e alone group, n = 5 in M2e + CpG group and n = 8 for the remainder of the groups). AuNP: Gold nanoparticle.
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