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. 2017 May:141:62-72.
doi: 10.1016/j.antiviral.2017.01.021. Epub 2017 Feb 2.

Consensus M2e peptide conjugated to gold nanoparticles confers protection against H1N1, H3N2 and H5N1 influenza A viruses

Wenqian Tao  1 , Brett L Hurst  2 , Akhilesh Kumar Shakya  1 , Md Jasim Uddin  1 , Rohan S J Ingrole  1 , Mayra Hernandez-Sanabria  3 , Ravi P Arya  3 , Lynn Bimler  3 , Silke Paust  3 , E Bart Tarbet  2 , Harvinder Singh Gill  4
Affiliations

Consensus M2e peptide conjugated to gold nanoparticles confers protection against H1N1, H3N2 and H5N1 influenza A viruses

Wenqian Tao et al. Antiviral Res. 2017 May.

Abstract

The extracellular domain of influenza A ion channel membrane matrix protein 2 (M2e) is considered to be a potential candidate to develop a universal influenza A vaccine. However poor immunogenicity of M2e presents a significant roadblock. We have developed a vaccine formulation comprising of the consensus M2e peptide conjugated to gold nanoparticles (AuNPs) with CpG as a soluble adjuvant (AuNP-M2e + sCpG). We demonstrate that intranasal delivery of AuNP-M2e + sCpG in mice induces lung B cell activation and robust serum anti-M2e immunoglobulin G (IgG) response, with stimulation of both IgG1 and IgG2a subtypes. Using Madin-Darby canine kidney (MDCK) cells infected with A/California/04/2009 (H1N1pdm) pandemic strain, or A/Victoria/3/75 (H3N2), or the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) as immunosorbants we further show that the antibodies generated are also capable of binding to the homotetrameric form of M2 expressed on infected cells. Lethal challenge of vaccinated mice with A/California/04/2009 (H1N1pdm) pandemic strain, A/Victoria/3/75 (H3N2), and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) led to 100%, 92%, and 100% protection, respectively. Overall, this study helps to lay the foundation of a potential universal influenza A vaccine.

Keywords: Adjuvants; CpG; Gold nanoparticles; Influenza vaccine; Intranasal vaccination; M2e; Universal influenza vaccine.

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Figures

Fig. 1
Fig. 1
(A) Scheme of vaccine design. M2e is conjugated to AuNPs. By keeping M2e in excess in the solution, complete surface-coverage of AuNPs with M2e is ensured at all times. Soluble CpG (sCpG) is an unmethylated CG-rich oligonucleotide found in viral and bacterial genome. sCpG, is a known TLR-9 agonist, which enhances the immune response and is used as an adjuvant in the formulation. We used CpG 1826: 5′-TCCATGACGTTCCTGACGTT-3′ with phosphorothioate linkages. sCpG stays in solution and does not attach to AuNPs. (B) Vaccination schedule. Mice (n=37) were vaccinated with AuNP-M2e+sCpG on days 0 and 21, and serum was collected on days 0, 21 and 42 for analysis. (C) M2e-specific IgG antibody response in mouse serum. M2e-specific IgG antibody in 1:1600 diluted serum of individual mice at days 0, 21 and 42. Each circle* represents an individual animal and the horizontal bar represents the mean. (D) M2e-specific IgG1 and IgG2a response in mouse serum. M2e-specific IgG1 and IgG2a antibodies in 1:1600 diluted serum of individual mice at day 42. Each circle* represents an individual animal and the horizontal bar represents the mean. Optical density (OD) of the ELISA reaction was measured at 492 nm wavelength. *: Serum of one vaccinated mouse was not available in sufficient quantity and it was thus not included in the ELISAs.
Fig. 2
Fig. 2. M2e-specific IgG and IgA antibody response in nasal wash and lung wash
(A) M2e-specific IgG antibody in 1:512 diluted nasal and lung wash of individual mice. (B) M2e-specific IgA antibodies in 1:64 diluted nasal and lung wash of individual mice. The vertical columns represent the mean and the error bars represent standard deviation. Optical density (OD) of the ELISA reaction was measured at 492 nm wavelength.
Fig. 3
Fig. 3. M2e specific T cell response in spleen, bone marrow and lung
Cytokine production after in vitro re-stimulation by using medium, AuNP, M2e peptide and ConA was analyzed by cytokine multiplex assay. Study included two groups (n=5 per group, vaccinated and naïve). (A) IL-1α, (B) IL-2, (C) IL-17, (D) TNFα and (E) RANTES in spleen; (F) IL-17 in bone marrow. Each symbol represents an animal and the horizontal bar represents the mean. (G) IL-2 in lung. Samples were prepared as duplicates.
Fig. 4
Fig. 4. B cells are increased in lungs of AuNP-M2e+sCpG vaccinated mice, and respond rapidly to in vitro restimulation with AuNP-M2e
B cell frequencies and CD86 expression was determined by flow cytometry. Hematopoietic cells were identified as CD45+ cells, and B cells as CD45+CD19+B220+CD3–cells. Study included two groups (n=5 per group, vaccinated and naïve). (A) Average frequency and standard deviation of B cells in the lungs, spleens and bone marrow (BM) of naïve vs. AuNP-M2e+sCpG vaccinated donor mice, regardless of their in vitro re-stimulation. (B) CD86 expression was determined on B cells isolated from naïve or AuNP-M2e+sCpG vaccinated donor lungs after 96 hours of media control or AuNP-M2e restimulation in vitro.
Fig. 5
Fig. 5. Immunofluorescence Assay (IFA) on influenza-infected MDCK cells
Acetone-fixed-influenza virus-infected MDCK cells were used to evaluate the ability of antibodies generated by AuNP-M2e+sCpG to bind M2 expressed on cells. Immunofluorescence assays were performed using day 0 and day 42 mouse serum at 1:50 dilution after pooling sera of vaccinated mice. Uninfected MDCK cells were used as a negative control. California-H1N1pdm: A/California/04/2009 (H1N1pdm); Victoria-H3N1: A/Victoria/3/75 (H3N2); Vietnam-H5N1: A/Vietnam/1203/2004 (H5N1).
Fig. 6
Fig. 6. Cell-based ELISA using influenza-infected MDCK cells
MDCK cells that were acetone fixed before infection or after infection with A/California/04/2009 (H1N1pdm) (California-H1N1pdm), A/Victoria/3/75 (H3N2) (Victoria-H3N2), or A/Vietnam/1203/2004(H5N1) (Vietnam-H5N1) were used as immunoabsorbants to evaluate the ability of antibodies induced by the AuNP-M2e+sCpG vaccine to bind M2 in its homotetrameric form expressed on infected cells. (A) Day 42 mouse serum was used at 1:50 dilution after pooling sera of vaccinated mice. The vertical columns represent the mean and the error bars represent standard deviation. Optical density (OD) of the ELISA reaction was measured at 492 nm wavelength. (B) Amino acid sequences of: consensus M2e, M2e used for immunization, and M2e for the three viruses used to infect MDCK cells.
Fig. 7
Fig. 7. Effectiveness of AuNP-M2e+sCpG vaccine in mice against H1N1, H3N2 and H5N1 influenza A subtypes
Mice were vaccinated with AuNP-M2e+sCpG or saline (placebo) on days 0 and 21, and challenged on day 42 with 3xLD50 of California-H1N1pdm: A/California/04/2009 (H1N1pdm); or 3xLD50 of Victoria-H3N1: A/Victoria/3/75 (H3N2); or 1xLD90 of Vietnam-H5N1: A/Vietnam/1203/2004 (H5N1). (A) Survival rate and (B) body weight after pdm-CA2009-H1N1 virus infection. n=12 in AuNP-M2e+sCpG group, n=13 in placebo group. (C)Survival rate and (D) body weight after Victoria-H3N2 virus infection. n=13 in both groups. (E)Survival rate and (F) body weight after Vietnam-H5N1 virus infection. n=12 in both groups.
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