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. 2016 Sep 20;111(6):1173-1179.
doi: 10.1016/j.bpj.2016.07.046.

Identification of HIV-1-Based Virus-like Particles by Multifrequency Atomic Force Microscopy

Irene González-Domínguez  1 , Sonia Gutiérrez-Granados  2 , Laura Cervera  2 , Francesc Gòdia  2 , Neus Domingo  3
Affiliations

Identification of HIV-1-Based Virus-like Particles by Multifrequency Atomic Force Microscopy

Irene González-Domínguez et al. Biophys J. .

Abstract

Virus-like particles (VLPs) have become a promising platform for vaccine production. VLPs are formed by structural viral proteins that inherently self-assemble when expressed in a host cell. They represent a highly immunogenic and safe vaccine platform, due to the absence of the viral genome and its high protein density. One of the most important parameters in vaccine production is the quality of the product. A related bottleneck in VLP-based products is the presence of cellular vesicles as a major contaminant in the preparations, which will require the set up of techniques allowing for specific discrimination of VLPs from host vesicular bodies. In this work novel, to our knowledge, multifrequency (MF) atomic force microscopy (AFM) has permitted full structural nanophysical characterization by its access to the virus capsid of the HIV-based VLPs. The assessment of these particles by advanced amplitude modulation-frequency modulation (AM-FM) viscoelastic mapping mode has enhanced the imaging resolution of their nanomechanical properties, opening a new window for the study of the biophysical attributes of VLPs. Finally, the identification and differentiation of HIV-based VLPs from cellular vesicles has been performed under ambient conditions, providing, to our knowledge, novel methodology for the monitoring and quality control of VLPs.

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Figures

Figure 1
Figure 1
EM analysis of HIV-1 Gag VLP preparations. (a) TEM analysis. (b) SEM analysis, from deposits on carbon-coated grids. (Straight blue arrows) HIV-1 Gag VLPs; (red dashed arrows) cellular vesicle structures. To see this figure in color, go online.
Figure 2
Figure 2
TEM and AFM imaging of an individual HIV-1 Gag VLP. (a) TEM image. (b) AFM 3D topography imaging. (c) AFM amplitude and (d) phase image of the fundamental mode. (e) Second amplitude (A2) and (f) phase (Φ2) images of bimodal AFM, corresponding to the dynamics of the first excited eigenmode. (g) First excited eigenmode frequency shift (Δf2) image of AM-FM viscoelastic mapping mode, and the corresponding calculated (h) Young’s modulus image. To see this figure in color, go online.
Figure 3
Figure 3
Individual VLP deposited on mica substrate. (a) AFM topography image of VLPs on mica substrate. (b) 3D topography image of a single VLP structure. (c) Δf2 imaging of an isolated VLP structure, as obtained by AM-FM viscoelastic mapping mode and (d) the corresponding calculated Young’s modulus distribution. To see this figure in color, go online.
Figure 4
Figure 4
“Mock” sample deposited on mica substrate. (a) AFM topography image of empty cellular vesicles deposited on mica substrate; (b) corresponding AFM phase image, showing the contrast due to the presence of vesicles. To see this figure in color, go online.
See this image and copyright information in PMC

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