Nanostructured glycan architecture is important in the inhibition of influenza A virus infection

Journal name:
Nature Nanotechnology
Volume:
12,
Pages:
48–54
Year published:
DOI:
doi:10.1038/nnano.2016.181
Received
Accepted
Published online

Rapid change1 and zoonotic transmission to humans2 have enhanced the virulence of the influenza A virus (IAV)3. Neutralizing antibodies fail to provide lasting protection from seasonal epidemics1, 4. Furthermore, the effectiveness of anti-influenza neuraminidase inhibitors has declined because of drug resistance5. Drugs that can block viral attachment and cell entry independent of antigenic evolution or drug resistance might address these problems. We show that multivalent 6′-sialyllactose-polyamidoamine (6SL–PAMAM) conjugates, when designed to have well-defined ligand valencies and spacings, can effectively inhibit IAV infection. Generation 4 (G4) 6SL–PAMAM conjugates with a spacing of around 3 nm between 6SL ligands (S3–G4) showed the strongest binding to a hemagglutinin trimer (dissociation constant of 1.6 × 10−7 M) and afforded the best inhibition of H1N1 infection. S3–G4 conjugates were resistant to hydrolysis by H1N1 neuraminidase. These conjugates protected 75% of mice from a lethal challenge with H1N1 and prevented weight loss in infected animals. The structure-based design of multivalent nanomaterials, involving modulation of nanoscale backbone structures and number and spacing between ligands, resulted in optimal inhibition of IAV infection. This approach may be broadly applicable for designing effective and enduring therapeutic protection against human or avian influenza viruses.

At a glance

Figures

  1. Design and synthesis of multivalent 6SL–PAMAM dendrimer conjugates.
    Figure 1: Design and synthesis of multivalent 6SL–PAMAM dendrimer conjugates.

    a, Reaction scheme showing the reductive amination of the reducing sugar, 6SL, and the primary amino groups of PAMAM dendrimers (G2–G5) using NaCNBH3. b, Structures of various 6SL–PAMAM dendrimer conjugates designed for synthesis. S represents the number of 6SL ligands and G represents generation in the S–G conjugates. The different valencies and spacings of the ligands are illustrated.

  2. The ligand number calculated for the S3–G4 6SL dendrimer conjugates provides for the detailed structure shown.
    Figure 2: The ligand number calculated for the S3–G4 6SL dendrimer conjugates provides for the detailed structure shown.

    The irregular spacing between the 6SL residues is a representative structure and illustrates the Poisson distribution in 6SL incorporation. The 1H NMR spectra below the structure are for 6SL (top), the PAMAM dendrimer (G4, middle) and the conjugate (S3–G4, bottom) and provide the basis for this calculation. The integral of the distinctive signal at 2.07 ppm, corresponding to the –CH3 of the 6SL acetyl group, was used to determine the number of 6SL ligands on each dendrimer. For the S3–G4 dendrimer conjugate, the integral value at 2.07 ppm was 61.25, which when divided by 3H from the acetyl group results in a value of 20.4, corresponding to the average number of 6SL ligands attached to one molecule of PAMAM dendrimer G4.

  3. In vitro inhibitory activity of 6SL–PAMAM dendrimer conjugates against H1N1 virus infection.
    Figure 3: In vitro inhibitory activity of 6SL–PAMAM dendrimer conjugates against H1N1 virus infection.

    a, The influence of ligand spacing on the inhibitory potency of 6SL–PAMAM dendrimers. Error bars indicate the mean ± s.e.m., n = 5. As the ligand spacing increases, IC50 decreases. b, The interaction between the 6SL–PAMAM dendrimer conjugates and HA measured by SPR. S3–G4 shows the strongest interaction with HA. c, The sialic dendrimer binding signal (in response units (RU)) to immobilized HA demonstrates that binding increases with increased ligand spacing. S3–G4 shows a nearly optimal spacing. Error bars are the mean ± s.d., n = 3. d. TEM analysis of S3–G4, the purified H1N1 virus alone, a mixture of G4 and purified H1N1, and a mixture of S3-G4 and purified H1N1. The samples were spread on a carbon-coated grid and stained with uranyl acetate before TEM analysis. White arrows point to blebs in H1N1 where S3–G4 is bound to HA. S3–G4 particles (∼7 nm) with spherical shapes (top left panel) and bleb formation on the surface of H1N1 resulting from the binding of S3–G4 (bottom right panel) were observed. Bleb formation was not observed on treatment with the G4 PAMAM dendrimer without 6SL ligands (bottom left panel). e, Time-lapse images of R18-labelled HIN1 and dual-colour-labelled viruses (both DiOC18-labelled and R18-labelled H1N1) showing virus internalization; no green and yellow viral particles appeared in the dual-labelled viruses treated with S3–G4. Scale bars, 20 µm. In the control rows, binding and internalization are shown in red and green, respectively. In S3–G4-treated samples, no colour is seen. f. Infection inhibition of S3–G4 against an H3N2 virus (A/Shandong/3/93) and different types of H1N1 virus including A/NWS/33, A/Puerto Rico/8/43 and A/California/07/2009. The viral NP protein, cell membranes and cell nuclei were labelled in green, red and blue, respectively. Scale bars, 100 µm. In the control column panels, the green and yellow colours correspond to IAV infection not observed in the S3–G4-treated column panels.

  4. In vivo inhibitory efficacy of S3–G4 against H1N1 and HA binding models.
    Figure 4: In vivo inhibitory efficacy of S3–G4 against H1N1 and HA binding models.

    a–c, S3–G4 administration reduces viral replication and inflammation in the lungs of mice infected with H1N1. Mice were administered intranasally with S3–G4, followed by H1N1 infection via the same route. The lungs were collected for the analysis of viral replication on day 3. a, After three days, mice were killed and the viral titer was measured from each mouse lung homogenate. The viral titers were determined from the TCID50 by using MDCK cells. Error bars are the mean ± s.e.m., n = 5 (Student's t-test). **P < 0.05. S3–G4 decreases the viral titer. b, Slide sections were prepared from the left lobe of each lung and stained with hematoxylin and eosin for histopathological examination (n = 5). The increased number of leukocytes (blue-stained nuclei) observed in H1N1 (without S3–G4 treatment) corresponds to inflammation. Less inflammation is seen in the treated samples. c, The production of IL-6, TNF-α, IL-10, CCL2, CXCL1 and CXCL2 in lung homogenates was measured by ELISA. Error bars are mean ± s.e.m., n = 5 (two-way ANOVA). **P < 0.05. Two cytokines, IL6 and CCL2, decrease on treatment with S3–G4. d, Kaplan–Meier plot of mice survival (n = 8 mice/group). Survival and weight loss were monitored in response to S3–G4 administered intranasally to mice 10 min before challenge with a mouse-lethal dose of the A/NWS/33 H1N1 virus. Differences in survival were evaluated by the log-rank test. S3–G4 protects 75% of mice from a lethal challenge with IAV. e, Top view of an HA trimer in a complex with 6SL (left panel, red as a stick representation, PDB accession 1HGG (ref. 28)). Black dotted lines represent the distance between the 6SL binding sites of the HA trimer. There is a discrete spacing of 4 nm in the HA trimer of IAV. f, Representation of the interactions between multivalent 6SL–PAMAM dendrimer conjugates and receptor binding sites on HA trimers. S3–G4, which has a near-optimal ligand spacing, allows the simultaneous binding of the three HAs in the IAV HA trimer, whereas S6–G4 does not.

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Author information

  1. These authors contributed equally to this work

    • Seok-Joon Kwon,
    • Dong Hee Na &
    • Jong Hwan Kwak

Affiliations

  1. Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005, 110 8th St., Troy, New York 12180, USA

    • Seok-Joon Kwon,
    • Marc Douaisi,
    • Fuming Zhang,
    • Ravi S. Kane,
    • Jonathan S. Dordick &
    • Robert J. Linhardt
  2. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea

    • Dong Hee Na &
    • Eun Ji Park
  3. School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea

    • Jong Hwan Kwak
  4. Laboratory Animal Medicine, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea

    • Jong-Hwan Park
  5. Department of Biochemistry, College of Medicine, Konyang University, Daejeon 302-718, Republic of Korea

    • Jong-Hwan Park &
    • Kyung Bok Lee
  6. College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea

    • Hana Youn &
    • Chang-Seon Song
  7. Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Biotech 4005, 110 8th St., Troy, New York 12180, USA

    • Robert J. Linhardt

Contributions

S.-J.K, D.H.N., and J.H.K. performed all of the experiments and analysed all data. M.D. performed the microneutralization assays. F.Z. performed the SPR analyses. E.J.P. synthesized the 6SL–PAMAM dendrimer conjugates. J.-H.P., H.Y. and C.-S.S. conducted the murine experiments. R.S.K. and J.S.D. provided critical feedback on the manuscript. S.-J.K and K.B.L. planned the experiments, interpreted the results, and wrote the manuscript with R.J.L. and J.S.D.

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The authors declare no competing financial interests.

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