Journal of Colloid and Interface Science 545 (2019) 259–268

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Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Regular Article

Probing and pressing surfaces of hepatitis C virus-like particles

Simon Collett a, Joseph Torresi b, Linda Earnest-Silveira b, Dale Christiansen b,
Aaron Elbourne a,⇑, Paul A. Ramsland a,c,d,⇑
a
School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, VIC 3000, Australia
b
Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia
c
Department of Immunology, Monash University, Melbourne, VIC 3004, Australia
d
Department of Surgery Austin Health, University of Melbourne, Heidelberg, VIC 3084, Australia

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o a b s t r a c t

Article history: Hepatitis C virus-like particles (VLPs) are being developed as a quadrivalent vaccine candidate, eliciting
Received 14 January 2019 both humoral and cellular immune responses in animal trials. Biophysical, biomechanical and biochem-
Revised 7 March 2019 ical properties are important for virus and VLP interactions with host cells and recognition by the
Accepted 9 March 2019
immune system. Atomic force microscopy (AFM) is a powerful tool for visualizing surface topographies
Available online 11 March 2019
of cells, bionanoparticles and biomolecules, and for determining biophysical and biomechanical attri-
butes such as size and elasticity. In this work, AFM was used to define morphological and nanomechan-
Keywords:
ical properties of VLPs representing four common genotypes of hepatitis C virus. Significant differences in
Atomic force microscopy
Bio-interface
size of the VLPs were observed, and particles demonstrated a wide range of elasticity. Ordered packing of
Enveloped virus-like particles the core and potentially envelope glycoproteins was observed on the surfaces of the VLPs, but detailed
Nanostructure structural characterization was hindered due to intrinsic dynamic fluctuations or AFM probe-induced
Vaccine development damage of the VLPs. All VLPs were shown to be glycosylated in a manner similar to native viral particles.
Young’s elastic modulus Together, the results presented in this study further our understanding of the nanostructure of hepatitis C
VLPs, and should influence their uptake as viable vaccine candidates.
Ó 2019 Elsevier Inc. All rights reserved.

1. Introduction

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA

⇑ Corresponding author at: School of Science, College of Science, Engineering and hepacivirus, infecting around 71 million people globally [1]. Infec-
Health, RMIT University, Melbourne, VIC 3000, Australia (P.A. Ramsland). tion causes chronic liver disease, cirrhosis, and liver cancer with
E-mail addresses: aaron.elbourne@rmit.edu.au (A. Elbourne), paul.ramsland@ high mortality rates. Currently, there is no approved vaccine
rmit.edu.au (P.A. Ramsland).

https://doi.org/10.1016/j.jcis.2019.03.022
0021-9797/Ó 2019 Elsevier Inc. All rights reserved.
260 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268
against HCV, but recently, highly effective antiviral treatments for
HCV have become available [2]. Globally, HCV is most prevalent in
the developing world and among injecting drug users, which are
groups with the most limited access to antiviral treatment. In addi-
tion, reinfection following successful treatment has been shown to
be common among at risk populations [3]. Factors including the
high cost of antiviral treatment and low diagnosis rates make a
vaccine an important addition to the approaches aimed at the
reduction of the global HCV disease burden [4].
A successful HCV vaccine needs to generate neutralizing anti-
bodies against multiple viral serotypes and genotypes [5].
Antibody-mediated immunity to HCV has been shown by passive
transfer of serum from anti-HCV positive donors [6–10] or antibod-
ies raised against recombinant E1 and E2 envelope proteins [11–
13]. Cytotoxic T lymphocyte immune responses are also important
for clearing HCV infections [14–16]. To achieve protective immu-
nity and effective clearance of acquired infections, next generation
vaccines will therefore need to elicit both humoral and cell-
mediated immune responses [17–19].
Recombinant expression of viral structural proteins in cell cul-
ture has been successfully used to produce self-assembling virus-
like particles (VLPs), which are non-infectious, but present viral
proteins in a manner faithful to the conformations found in native
viruses [20–23]. Compared to other nanoparticle formulations,
VLPs are ideally suited to present multiple and context-
dependent viral epitopes [24]. For these reasons, VLPs are ideal
vaccine candidates, as demonstrated by approved VLP-based vacci-
nes with efficacy against non-enveloped (human papilloma virus,
HPV) and enveloped (hepatitis B virus, HBV) viruses [25–27]. Pre-
clinical vaccination trials demonstrate HCV-derived VLPs elicit
immunological responses in mouse models [21,28,29]. As a strong
indicator of potential to induce protective immunity, the VLPs
were shown to elicit HCV specific humoral and cellular immune
responses [21,30,31].
Viruses and VLPs are sub-microscopic (<200 nm) particles, pre-
cluding visualization by traditional light microscopy. Enveloped
viruses typically contain a high proportion of lipids, often derived
from host cell membranes, making crystallization (and therefore
structure determination by X-ray crystallography) of intact viruses
difficult. Advances in atomic force microscopy (AFM) have enabled
interrogation of a wide variety of biological systems including sin-
gle cells, sub-microscopic particles (e.g., viruses and VLPs) and
biomacromolecules in biologically relevant aqueous environments
[32–39]. However, systematic studies of virus or VLP morphologi-
cal and physical variation of samples with major sequence differ-
ences (genotypes) have been largely neglected. To the best of our
knowledge, there are no published 3D structures of intact HCV or
HCV VLPs by X-ray crystallography, cryoEM or AFM. Most studies
have relied on negatively stained transmission electron micro-
scopy (TEM) images to derive information on particle size and
shape. A cryo-electron tomography study of HCV VLPs (genotype
1a) provided 3D isosurface reconstructions as incomplete spherical
particles with single or double membranes [21].
In this work, we utilize in situ amplitude modulated (AM)-AFM
to visualize morphological features of four genotypes of HCV VLPs
(genotypes 1a, 1b, 2a and 3a) currently being developed as a
quadrivalent vaccine candidate [21]. Furthermore, the nanome-
chanical properties of the VLPs were probed using force spec-
troscopy for each HCV genotype. The VLPs investigated were
shown to be effectively internalized by Huh7 cells, and were
shown to be glycosylated by interaction with lectins. We present
the first reported AFM topographical images and biomechanical
data for HCV VLPs. The approach reported here should be generally
applicable for characterization and development of enveloped VLPs
as next-generation vaccine candidates.
2. Materials and methods
2.1. Production of HCV VLPs
HCV VLPs were produced by recombinant expression of viral
core, E1 and E2 proteins in Huh7 cells as previously reported
[21,30]. HCV VLPs of genotypes 1a, 1b, 2a and 3a were examined
in this study.
2.2. VLP cellular uptake by flow cytometry
HCV VLPs were labelled with fluorescein isothiocyanate (FITC.
Sigma, >90%) by combining 25 mg of each VLP in 200 mL of FITC,
2 mg/mL in DMSO (Sigma, >99%), and incubating for 24 h at 4 °C,
then dialysed against PBS for 24 h at 4 °C. The labelled HCV VLPs
were then stored at
20 °C until required. Huh7 cells were grown
in Dulbecco’s modified Eagle’s medium (DMEM. Gibco, Thermo
Fisher) supplemented with 10% foetal calf serum (FCS. Gibco,
Thermo Fisher) and streptomycin 50 mg/mL at 37 °C in 5% CO2.
For cell entry assays, cells were harvested in log growth phase
(80–90% confluent), using enzyme-free cell dissociation buffer
(Gibco, Thermo Fisher) so as to preserve cell surface receptors,
and washed in 0.05% FCS, phosphate buffered saline (PBS. Sigma,
>98%) FITC labelled VLPs of each genotype at 1 mg/mL, 2 mg/mL,
5 mg/mL and 10 mg/mL were incubated with 2  105 cells in
100 mL of 0.05% FCS, PBS at 4 °C for 1 h for binding followed by
1 h incubation at 37 °C for entry. This protocol has previously been
shown to induce uptake of HCV VLPs [21]. Triplicates of each geno-
type at each concentration were prepared. Cells were then washed
in 1 mL 0.05% FCS, PBS to remove any unbound HCV VLPs. The cells
were fixed in Cytofix (BD BioSciences) for 30 min at 4 °C, and entry
of FITC-labelled HCV VLP was then determined by flow cytometry
(FACS Canto II, BD Bioscience). The presence of the CD81 marker on
cells was confirmed using an APC-conjugated mouse anti-human
antibody (BD Pharmingen) with an APC conjugated mouse IgG
isotype control (Invitrogen). Data analysis was performed using
the WEASEL 2.0 software package (Walter and Eliza Hall Institute,
Melbourne, Australia), with further analysis performed using
Graphpad Prism. Results are expressed as percentage (%) of
positive cells.
2.3. Lectin binding of HCV VLPs
Enzyme-linked immunosorbent assays (ELISAs) were per-
formed to investigate lectin binding to each of the four VLP geno-
types. The HCV VLPs (50 mL) at 5 mg/mL in 20 mM Tris (Sigma,
reagent grade), 150 mM NaCl (Sigma, >99%), 0.05% (v/v) tweenÒ20
(Sigma) (TBST) buffer were incubated in nunc flat bottomed 96-
well plates (Thermo Scientific, USA) overnight at 4 °C to allow par-
ticles to adsorb to the wells. Plates were blocked with 0.5% poly
vinyl alcohol (PVA) (Merck, >98%) in TBST, incubated with biotiny-
lated concanavalin A (ConA) (Vector Laboratories Inc, USA, 99%) at
1 mg/mL for 1 h at 37 °C, and binding was detected with PierceÒ
NeutrAvidin-HRP conjugate (Thermo Scientific, USA Lot number
PA193455) (50 mL of a 1:20,000 dilution for 30 min at 37 °C). Plates
were washed thoroughly between each step in excess TBST. Finally,
lectin binding was quantified by the addition of 2,20 -Azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate (Roche Diag-
nostics, Germany >98%), with absorbance at 405 nm measured on a
clariostar plate reader (BMG Labtech, Germany) after 20 min.
Negative controls included no VLPs and ConA incubated with its
conjugate sugars methyl-a-D-mannopyranoside (MMA. Sigma,
>98%) and methyl-a-D- glucopyranoside (MGA. Sigma, >98%). In
both cases all other steps were followed as above.
 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268
2.4. AFM measurements of HCV VLPs
A Cypher ES AFM (Oxford Instruments, Asylum Research, Santa
Barbara, CA, USA) was used to obtain the measurements presented
in this study. The instrument was operated at ambient tempera-
ture (25 °C) for all experiments. Small amplitude (AM)-AFM was
employed for all images. Biolever BL-AC40TS cantilevers (Oxford
Instruments, Asylum Research, Santa Barbara, CA, USA, nominal
spring constant kc = 0.09 N/m) were used for all measurements.
Cantilevers were calibrated prior to each experiment via the ther-
mal spectrum method and the lever sensitivity was determined
using force spectroscopy. In real space, the cantilever amplitude
was determined based on the individual tune of the cantilever,
with free amplitudes set to between 20 nm and 6 nm (free ampli-
tude). Imaging amplitudes of between 14 nm and 3 nm were rou-
tinely used by using setpoint ratios of between 0.7 and 0.5.
Imaging ratios were image- and cantilever-dependent, and were
determined during experiments to achieve the best possible
images. Imaging forces were minimized via a setpoint ratio (imag-
ing amplitude (A)/free amplitude (A0)) of >0.5, which has been rou-
tinely shown to minimize tip-sample distortions [32]. The
maximum force applied to particles via the cantilever during imag-
ing was calculated to be 0.52 nN (Fig. S1). Images were obtained at
a sampling resolution (pixel size) of between 1.9 and 5.8 nm/pixel
(scans > 1 mm), and between 0.2 and 0.6 nm/pixel (scans < 1 mm),
with rates of acquisition between 256 and 512 pixels/line.
Samples and all buffers were filtered through 0.45 mm filters
(Millipore, Australia) prior to imaging to ensure that the VLPs were
the only particulate matter being imaged. Samples were diluted to
approximately 25 ng/mL in Tris-buffered saline (TBS, Sigma, >99%)
to ensure imaging of single particles, transferred to a freshly
cleaved muscovite (mica) surface and incubated at room tempera-
ture for approximately 1 h (performed within the sealed unit of the
AFM, which minimizes evaporation of buffer, and subsequent pH
changes, and fluctuations in temperature) prior to imaging to allow
particles to adsorb to the surface. All experiments were conducted
in a droplet of approximately 100 mL of TBS. Superimposed trace
and retrace signals showed negligible differences, meaning images
obtained were an accurate representation of the surface adsorbed
material [40].
Processing of AFM data involved using a combination of the
Asylum Research software, custom MATLAB codes, and the Gwyd-
dion software package [41]. The grains analysis function of the
Gwyddion package [41] was utilized to assess size distribution of
VLP particles in the AFM data. Grains were marked by the water-
shed method, and further thresholded by height and diameter. A
nominal cut-off of 10 nm equivalent disk diameter and 5 nm
height was chosen based on the dimensions for HCV E2 (the largest
of the proteins present in the HCV VLPs), which is 5  6 nm based
on crystallographic data [42].
2.5. Determining elasticity of intact HCV VLPs
Force versus distance curves (FD curves) were obtained from
the central region of suitable particles; defined as VLPs that
appeared intact, stable to imaging, isolated from others, and of at
least 15 nm height to allow for adequate indentation while avoid-
ing surface confinement effects. After imaging particles in AM
mode, the AFM was switched to contact mode. FD curves were first
obtained on an area of bare mica next to the particle to ensure lin-
ear (non-elastic) FD curves were observed, which provided a refer-
ence for determining elastic regime of FD curves obtained from
particles and to ensure cantilevers were properly calibrated. The
spring constant (kc) of the cantilever was determined for each can-
tilever used as described above (values in the range of 0.05–0.1 N/
m were obtained for all cantilevers). The tip was then directed to
261
the central region of the particle to obtain accurate indentation
data. Upward of 100 FD curves were recorded across several parti-
cles for each genotype. Importantly, to avoid surface confinement
effects, parameters were set to ensure indentation was not more
than 20% of the total height of each particle. To ensure the force
applied was not physically disrupting the particles, particles were
imaged after FD curves were taken. In no instances were particles
observed to be damaged by the force applied. A representative
sequence of images obtained prior-to and after force indentation
of a single VLP particle is shown in Fig. S2. This reveals that the par-
ticle is undamaged during the measurement process.
Determination of the contact point between the AFM cantilever
and the VLP was performed independently for each force curve
analysed using methods previously described in the literature
[43]. Specifically, the contact point between the AFM cantilever
tip and the VLP is defined as the point at which the cantilever first
makes physical contact with the surface adsorbed particle. Follow-
ing this point, the cantilever bends in response to interactions with
the VLP, while the particle itself is indented. In a raw force curve
(z-displacement (Z) vs. cantilever deflection (d)), this point of first
contact is defined as Z0 and d0, respectively. This is observed in the
AFM force curve as an increase in force (y-axis), above the zeroed
baseline, as a function of distance (x-axis prior to zeroing the
indentation). As such, the indentation of the tip into the VLP (d)
can be calculated using;
d ¼ ðz
z0 Þ
ðd
d0 Þ ð1Þ
which can then be transformed into indentation vs. force curves
using Hooke’s law;
F ¼ Kc  d ð2Þ
The non-linear portion (elastic regime) of the resulting indenta-
tion graph was then fitted to the Hertz/Sneddon Eq. (3) to obtain a
Young’s elastic modulus (E) for each of the fitted curves. Using cus-
tom MATLAB scripts, curves were fitted to the equations:
2
F cone ¼ tanaE d2 ð3Þ
p
and;
E
E ¼ ð4Þ
1
v2
where F is loading force, d is indentation depth, a is the cone open-
ing angle, E* is the apparent elastic modulus, and v is the Poisson’s
ratio. A conal tip angle of 34.4° and a Poisson’s ratio of 0.5 was used
for processing of all force curves. All elasticity values were obtained
from curves fitted with an R2 value of 0.9 or above. Data falling
below this quality was rejected for further analysis.
2.6. Statistical analysis
Statistical analysis was performed using a combination of
Graphpad Prism, MATLAB and Python scripts, utilizing the NumPy
suite.
3. Results
3.1. Four genotypes of HCV VLPs display similar attachment and entry
into human liver cells
To examine genotypic variation in cell binding and entry of the
VLPs, cell entry assays were performed by flow cytometry using
Huh7 cells. After initial interaction between membrane lipopro-
teins and host cell scavenging receptor B1 (SR-B1), the HCV E1/
E2 dimer is known to bind CD81 as the first step of the attachment
and entry pathway [44,45]. For all genotypes of VLPs, median
262 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268

fluorescence increased in a dose-dependent manner. Percentage Table 1

VLP entry into Huh7 cells was seen to plateau around 10 mg/mL EC50 and EC90 values for cell entry assay.

VLP concentration (Fig. 1). EC90 values (obtained from % entry) Genotype EC50 (mg/mL) EC90 (mg/mL)
were seen to be highest for genotype 2a and lowest for genotype 1a 0.48 3.33
1b (Table 1). However, from the observed range of EC90 values (less 1b 0.89 2.90
than 2-fold difference) it can be concluded that the different VLPs 2a 1.26 5.04
are all capable of equivalent entry of cells. 3a 0.96 3.57

3.2. Lectin binding indicates high mannose glycosylation of HCV VLPs

similar to native HCV virions

Glycosylation of native HCV envelope glycoproteins influence

protein folding and cellular interactions [46,47]. To assess glycosy-
lation of the VLPs, ELISAs were performed. The lectin concanavalin
A (ConA), known to bind high-mannose type glycans, clearly inter-
acted with all HCV VLP genotypes (Fig. 2). Negative controls with
no VLPs showed background level absorbance, while inhibiting
the lectin with its conjugate sugars, methyl-a-D-
mannopyranoside (MMA) and methyl-a-D-glucopyranoside
(MGA), resulted in complete inhibition of binding, with absorbance
values equal to the negative controls.

Fig. 2. Binding of lectin concanavalin A (ConA) to HCV VLPs. Shown are genotypes
3.3. Probing the surfaces of HCV VLPs reveals semi-ordered viral 1a (circles), 1b (squares), 2a (upward facing triangles) and 3a (downward facing
protein arrays within a loose envelope triangles). Two negative controls (ConA incubated with the competitive binding
carbohydrates methyl-a-D-mannopyranoside (MMA) and methyl-a-D-glucopyra-
noside (MGA), blue symbols, and no VLP, red symbols) were included. Data shown
AFM imaging in tris buffered saline (TBS) was used to determine are for n = 10 technical replicates for HCV VLPs, and n = 2 for each negative control.
topographical detail of HCV VLPs of four genotypes. Fig. 3 shows Data are shown as median ± SD. (For interpretation of the references to color in this
low-resolution AM-AFM images (1 mm scan size) of the four VLP figure legend, the reader is referred to the web version of this article.)
genotypes following immersion on a freshly cleaved mica surface.
Inspection of the images reveals small particulate matter, of vari-
ous sizes, adsorbed to the underlying atomically smooth substrate.
Individual particles for each system were imaged at higher res-
olution, with scan sizes between 100 and 500 nm, to investigate
the finer structure of the adsorbed particles. Representative images
of each genotype are shown in Fig. 4, with the first image for each
genotype (top left) a higher resolution image of the particle indi-
cated by the colored box in Fig. 3. Although considerable morpho-
logical variation can be seen both within and between the
genotypes, icosahedral (pentameric or hexameric) symmetry is
discernible for some particles, similar to the structural organiza-
tion of the protein subunit constituents previously reported for
HCV VLPs, as displayed in Fig. 5.

Fig. 3. Large scale (1 mm scan size) AFM images were obtained for the 4 genotypes
of the quadrivalent HCV VLP vaccine. All images are displayed as ortho projections
as viewed at a 0° angle. White scale bars in the bottom right of each image
represent 500 nm. For ease of comparison, all images were scaled to a 50 nm z-
dimension false color scale bar.

Further, the semi-ordered or arrayed protrusions (interpreted

Fig. 1. HCV VLP binding and entry to Huh7 cells, shown as % entry as a function of
log2[VLP concentration] for genotypes (A) 1a, (B) 1b, (C) 2a and (D) 3a. Binding and
entry by VLPs was seen to reach saturation by 10 mg/mL (the second highest
concentration) for all genotypes. Data shown are for n = 3 technical replicates.
Experiments were repeated twice with the same trend observed for both exper-
iments. Data are shown as median ± SD. Data are so invariant that error bars are not
shown for most points.
as imprints in the VLP surface from the icosahedral core and the
surface displayed E1/E2 proteins) were easily disrupted by
repeated AFM scans on some particles, while others appeared
extremely stable to imaging. Fig. S3 shows a particle undamaged
by 10 consecutive scans over a period of 30 min. The nanostructure
of VLPs was also often obscured by what appear to be larger pro-
trusions and valleys across the envelope or membranes that encase
the VLPs, and closer inspection reveals smaller, less regular,
surface-adsorbed material in all images (see Fig. 3). Such features
are likely unstructured or dissociated protein and lipid components
(<10 nm diameter) from the VLPs. Notably, a heterogeneous distri-
bution of particles in terms of both size and shape is observed for
all VLPs investigated.
 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268 263

Fig. 4. Nanoscale (100–500 nm scan size) AFM images obtained for individual HCV VLPs. All images are displayed as ortho projections as viewed at a 0° angle, with the first
image for each genotype (top left) a higher resolution image of the particle indicated by the coloured box in Fig. 3. White scale bars in the bottom right of each image
represent 50 nm. For ease of comparison, all images were scaled to a 50 nm z-dimension false colour scale bar, although several of the particles displayed are over 50 nm in
height, as highlighted by the white, false-colour thresholding. Geometric features of the VLP have been highlighted with 2D projections of the proposed icosahedral symmetry
on 2 particles of genotype 1b. More particles than those presented were observed displaying such features; included here are particles displaying heterogeneous, pleomorphic
surface topologies to more accurately represent the VLP preparations in their entirety. For comparison, 2-dimensional representations of all images in Fig. 4 are provided in
Fig. S5.

Fig. 5. Diagrammatic representation of assembly of HCV virions and VLPs. (A) shows proposed assembly of HCV core protein adopting an icosahedral configuration displaying
2-fold, 3-fold and 5-fold symmetry. Envelope proteins, as multimeric arrangements of E1/E2 dimers, are thought to be distributed within the lipid membrane in low
abundance. (B) and (C) show a cross-section displaying HCV core, E1/E2 proteins, and lipid membrane. B) wild-type virions with genetic material and C) VLPs lacking genetic
material.

3.4. Size distribution analysis of HCV VLPs reveals a highly
heterogeneous population
Infectivity, and immunological responses, have been shown to
be higher for HCV virions (or VLPs) around 60 nm in diameter
[48]. Size distribution was therefore determined for height
(Fig. 6) and diameter (Fig. 7). Median heights of between 6.4 and
12.9 nm and median diameters of between 26.4 and 34.2 nm were
observed for all genotypes (Table 2). Genotype 2a was seen to have
the largest average height and diameter, while genotype 1a
264 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268

displayed the smallest. Large interquartile ranges for both height

and diameters were observed for all genotypes (Table 2).
This can be accounted for by a large number of small particles
and a small number of very large particles (Figs. 3, 4). As such,
the distributions displayed in Figs. 6 and 7 are representative of
both whole VLPs and the smaller, more abundant fragments, driv-
ing averages towards smaller values. For all genotypes, height dis-
tribution was seen to be heavily skewed toward the smaller
particles (Fig. 6), while diameter showed a roughly normal distri-
bution, but with a long tail representing a small number of larger
particles (Fig. 7). Overall, genotype 2a was observed to have the
most consistent distribution. A significant difference in diameter
(p = 7.75e
4) and height (p = 1.74e
29) was found between all
genotypes when compared using the Kruskal-Wallis test (applying
an alpha value of 0.05). When genotypes were compared individu-
ally using the Mann-Whitney U test (with Bonferonni correction
for multiple comparisons) a significant difference in diameter
was observed between genotypes 1a and 1b (p = 0.007), and 1a
and 2a (p = 0.003), and a significant difference in height was
observed between all genotypes (Table 3). Fig. 7. Diameter distributions for HCV VLP genotypes (A) 1a n = 428, (B) 1b n = 259,
Dynamic light scattering (DLS) data collected from HCV VLP (C) 2a n = 179 and (D) 3a n = 432 as measured by Gwyddion software [41] grains
analysis function using the threshold by watershed tool. All genotypes display a
(40 mg/mL in PBS) is in agreement with AFM data, showing a pop-
roughly normal distribution with a tail toward the right indicating a small
ulation of particles heterogeneous for diameter with peaks around proportion of considerably larger particles.
20 nm, 107 nm and 1070 nm diameter (with the largest peak
explained by aggregation of the sample). DLS analysis shows a
majority of particles display a diameter centered around 20 nm, Table 2
with a population of larger particles with 50–500 nm diameter. Size distribution for 4 genotypes of HCV VLPs. Median height and diameter (nm) with
the interquartile range show a broad size range for all genotypes.
Fig. S4 shows representative DLS data for genotype 1a.
Genotype 25% Percentile Median 75% Percentile
3.5. Elasticity of individual particles varies within samples of HCV VLP Height (nm)
genotypes 1a 5.6 6.4 8.8
1b 7.4 9.5 14.7
2a 9.0 12.9 16.2
To investigate particle elasticity (stiffness) as a function of 3a 5.8 6.9 10.3
genotype, Young’s elastic moduli (E) were calculated from force
Diameter (nm)
versus distance (FD) curves obtained from the central region of 1a 22.4 26.2 37.4
intact and isolated VLP particles (Fig. 8). The first challenge in con- 1b 21.6 32 46.2
version of FD curves to nanoindentation data is establishing the 2a 23.4 34.2 49.2
point where contact with the sample is made. Multiple approaches 3a 20.2 29.4 39.6

exist for determining the contact point, with each giving slightly
different results in the interpretation of force spectroscopic mea-
surements. For the systems investigated in this study, the rela- Table 3
P values derived from Mann-Whitney U test for comparison of height and diameter
for all genotypes.

Genotype 3a 2a 1b
Height
1a p = 0.024 p = 4.2e
13 p = 5.2e
23
1b p = 3.8e
14 p = 0.0162
2a p = 2.2e
09
Diameter
1a p = 0.15 p = 0.004 p = 0.007
1b p = 0.29 p = 0.69
2a p = 0.06

tively high ionic strength of the buffer solution (0.1 M NaCl,

Fig. 6. Height distributions for HCV VLP genotypes (A) 1a n = 428, (B) 1b n = 259, (C)
2a n = 179 and (D) 3a n = 432 as measured by Gwyddion software [41] grains
analysis function using the threshold by watershed tool. A varied distribution was
seen between the genotypes, but all were skewed heavily to the smaller particles.
Insets show a magnified view of data from larger height values for easy comparison.
0.02 M Tris, pH 7.4) in which the particles were imaged is known
to reduce the contribution of long-range electrostatic surface
forces to tip/particle interactions. Hence, we were able to assume
the point of contact to be defined by the point of deviation from
zero deflection (Fig. 8A). At large tip-sample separations the can-
tilever’s motion is unimpeded by the surrounding liquid environ-
ment, meaning that a net-zero force is recorded as the sample
approaches the cantilever. This is observed in the indentation
curve at distances designated as negative values (see Fig. 8A).
On contact with the VLP, the cantilever beam begins to deflect
in response to physically engaging with the sample, while
 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268
Fig. 8. Fitting of VLP indentation curves to determine Young’s elastic modulus (E).
A) Fitting protocol followed using the Hertz/Sneddon equation to convert exper-
imentally derived force versus distance (FD) curves into indentation data (blue
circles) and to fit the elastic (non-linear) region to calculated E values (orange line).
Data obtained on a hard mica surface is shown for comparison (black line). A single
representative curve obtained for genotype 1a is shown. B) Data from multiple force
curve measurements across several particles from each genotype. Y axis shows
Young’s elastic modulus (E) in MPa. Data are shown as median ± SD. Detailed
statistical data for each genotype are provided in Table 4. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
this article.)
simultaneously indenting the ‘‘soft” material. This action produces
a non-linear, elastic response, which can be observed in the repre-
sentative single indentation curve (from genotype 1a) shown in
Fig. 8A.
For comparison, a curve obtained on the hard mica surface is
superimposed over the elastic data, revealing negligible indenta-
tion. Using this methodology, the raw FD curves can be converted
to indentation data as shown in Fig. 8A. Following conversion, the
Herztian model can be used to calculate the Young’s elastic modu-
lus (E) by fitting the non-linear elastic regime of the indentation
curves as described by multiple groups for both virus [49–51]
265
and other biological entities [43,52–55]. Notably, a study by de
Pablo et al. [39] used the Hertzian model to appropriately describe
the indentation of broken viral constituents, which were signifi-
cantly softer than their intact capsid counterparts. This highlights
the validity of using the Hertz model to describe the VLPs investi-
gated in this work. Employing this methodology, FD curves were
obtained and used to calculate E values from numerous HCV VLPs
of four genotypes (Fig. 8B). A representative curve from each geno-
type, together with an image of the particle from which it was
taken, is shown in Fig. S6. Evaluation of the resulting elasticity fit-
ting reveals median E values ranging from 1.5 MPa (1a) to
11.6 MPa (2a), with a large range observed within all genotypes
(Table 4). This large variation reveals that there is no meaningful
difference in elasticity between the HCV VLP genotypes – a result
which is consistent with the heterogeneity observed for particle
sizes. Fitting of curves to a range of indentation depths (from
5 nm to 20 nm, while keeping within the 20% indentation regime
described above in methods) showed identical values (within
error) are obtained regardless of indentation depth for larger par-
ticles (Fig. S7 and Table S1).
4. Discussion
Advances in knowledge of structural and biomechanical fea-
tures of viruses and their functional molecules are leading to
increased understanding of key processes of the viral life cycle,
allowing rational design of therapeutic and preventative agents
[56]. HCV remains a public health issue despite the recent intro-
duction of effective direct acting antiviral treatments [57]. A vac-
cine would enable control and eventual eradication of the disease
[19]. VLP-based vaccines show great promise, and a quadrivalent
HCV VLP-based vaccine is currently in development [21,29,31].
We report here an AFM-based approach that permitted the
detailed morphological and nanomechanical characterization of
all genotypes of HCV VLPs in the quadrivalent candidate vaccine.
The HCV VLPs investigated in this work were all decorated with
high-mannose, N-linked glycans, likely attached to the E1 and E2
proteins, which are known to be heavily glycosylated [46]. In
native HCV, due to limited trafficking through the Golgi, E1/E2 gly-
cosylation is mostly limited to mannose-rich glycans [58], with
Man7-9GlcNAc2 species shown to predominate [59]. The native-
like glycosylation of the VLPs likely results in cellular interactions
similar to those of native HCV, as demonstrated here by similar
activities for all genotypes of HCV VLPs in cellular entry assays.
Thus, we next sought to examine the structural and biomechanical
properties of HCV VLPs as a means to characterize the quadrivalent
vaccine candidate and to further inform vaccine formulation.
From AM-AFM measurements, we were able to observe mor-
phologies and nanoscale structural organization for all four geno-
types of HCV VLPs (see Figs. 3 and 4). Considerable variation was
seen between the genotypes. However, the organization of
repeated protein subunits was observed for all genotypes, and
the expected icosahedral (pentameric or hexameric) symmetry
was evident to a greater or lesser extent in several independently
imaged VLPs (Fig. 4). Thus, some VLPs could be considered to fit
with the classification of HCV as an icosahedral virus [60], where
the core proteins are thought to assemble into triangular subunits
arranged in a repeated symmetrical fashion, displaying 2-fold, 3-
fold and 5-fold symmetries. Indeed, Yu et al. [61] reported infec-
tious HCV particles with a fairly homogenous 50 nm diameter,
and modelling reconstruction based on these data indicate the
membrane bilayer adopts a slightly polygonal shape instead of
the expected spherical shape of a lipid vesicle, revealing the under-
lying icosahedral core protein layer. Computational modeling and
size-exclusion chromatography data suggest that E1/E2 form
266 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268
Table 4
Statistical data for elasticity modulus measurements.
Genotype Minimum 25% Median 75%
Percentile Percentile
GT 1a 1.0 1.4 1.5 6.1
n = 90
GT 1b 2.0 2.8 3.6 6.9
n = 90
GT 2a 5.2 9.2 11.6 16.0
n = 90
GT 3a 0.25 5.0 7.3 10.1
n = 90
heterodimers, which were proposed as a building block for envelope
trimers that could be further assembled into hexamers in the HCV
envelope [62]. However, it remains unclear whether the dimeric
envelope protein assembly actually assumes the proposed icosahe-
dral structure. Indeed, numerous HCV VLP particles were imaged
where these features were not discernible. Collectively, our AM-
AFM topographical images demonstrate that a mix of structured,
loosely-structured, and unstructured morphologies exists within
the population of HCV VLPs for all genotypes investigated.
Congruent with these observations are reports that infectious
HCV particles appear to contain a non-icosahedral capsid with a
diameter of around 45 nm, and observations that the viral envel-
ope may not be tightly associated with the capsid [48]. Recently,
Piver et al. [63] proposed that the HCV nucleocapsid may not be
as rigid as classically observed viral nucleocapsids. Instead, the
HCV core protein may be only loosely associated with the viral
RNA, meaning that the HCV glycoprotein envelope may be much
more deformable than that of other enveloped viruses. Analo-
gously, dengue (an enveloped, icosahedral flavivirus) is known to
undergo a loosening of the envelope protein structures when
exposed to physiological temperatures [64].
Understanding size variation in virus and VLP systems is impor-
tant for ensuring vaccine viability and cell uptake. All HCV VLP
genotypes displayed a high degree of heterogeneity in particle
morphology (size and shape), which is also a feature of native
HCV. In serum from HCV-infected patients and cell culture super-
natants, infectious enveloped virions and non-infectious particles
occur [65–67]. Non-infectious particles include those composed
solely of E1/E2 and host lipoproteins [68,69], and non-enveloped
capsids [70]. Two main HCV particles predominate in Huh7 cell
cultures with diameters of approximately 45 nm (non-enveloped
capsids) and 60 nm (enveloped infectious virions). Additionally,
minor populations of large vesicular (average diameter of 85 nm)
and multi-vesicular HCV particles (average diameter of 115 nm)
were reported in the HCV/Huh7 cell culture system [48].
Young’s elastic modulus (E) is a measure of a material’s stiff-
ness, defining the relationship between applied force and deforma-
tion, and is generally measured in units of Pascals (Pa). Here, fitted
median E values for HCV VLPs ranged from 1.5 MPa (genotype 1a)
to 11.6 MPa (genotype 2a), but due to large variations within sam-
ples, elasticity can be considered equivalent between genotypes.
Elasticity reported here for HCV VLPs is relatively low compared
to values reported for other viruses or VLPs, where E values have
been reported in the 100 s of MPa to 2.8 GPa range [37,71]. How-
ever, relatively little published data exists for enveloped viruses,
which in part may be due to challenges in interpreting results of
nanoindentation studies of enveloped viruses, compared to non-
enveloped viruses, due to the complex interplay between viral
and host-cell derived molecular components, including lipids
[72]. For comparison to other biological materials, indentation
measurements have shown elasticity values in the kPa range
(1–90 kPa) for mammalian cells [73,74] and the MPa range for
bacteria, yeasts and fungi [33,43,75–78].
Maximum Mean Std. Std. Error of Lower 95% CI of Upper 95% CI of
Deviation Mean Mean Mean
11.8 2.93 3.2 0.3 2.7 4.1
37.1 5.7 5.2 0.5 4.6 6.8
30.9 13.1 5.4 0.6 12.0 14.2
18.6 7.7 4.5 0.4 6.8 8.7
A number of factors may explain the comparatively low E values
observed here for the HCV VLPs, as outlined below. Firstly, the
enveloped hepatitis B virus (HBV) was reported to have E values
of 360–370 MPa [79], markedly lower than other viruses. The
lower elasticity of HBV can be rationalized as its capsid proteins
are more loosely packed than those of other, similar viruses [80].
A similar situation may exist for HCV, where the core is thought
to be dynamically unstable and E1/E2 proteins are present at rela-
tively low densities in the envelope. Secondly, for many viruses,
packaging of genetic material encapsulated within the virus has
been shown to contribute to a more stable and robust particle
[81,82]. The absence of genetic material in the HCV VLPs investi-
gated here may well lead to lower elasticity of the VLPs than for
intact HCV virus. Thirdly, in infected cells, HCV virions are secreted
as low-density lipoprotein (LDL)-HCV complexes and are therefore
associated with host apolipoproteins A and E (ApoA and ApoE) and
are rich in triglycerides [83,84], meaning they behave essentially as
liposomes. The VLPs investigated here have previously been shown
to contain ApoC and ApoE [21]. An E value of 2 MPa was calculated
using the Hertzian model for phosphatidylcholine liposomes [55],
which is in the range we measured for the HCV VLPs. In light of
these factors, the low MPa elastic modulus values reported here
for HCV VLPs are not surprising.
5. Conclusions
A quadrivalent HCV vaccine is currently being developed and
shows promising efficacy in pre-clinical evaluations [21,29,31].
This is the first morphological and biomechanical characterization
of all four HCV VLP genotypes using AM-AFM and force
spectroscopy. All HCV VLP genotypes were seen to be biologically
functional, based on cell entry assays, and were decorated with
high-mannose type, N-linked glycans similar to native HCV.
As expected, HCV VLP preparations show a greater heterogene-
ity, and a lower elasticity, than many other reported VLP systems
[85,86]. Young’s elastic moduli were found to be in the low MPa
range similar to values expected for liposomes, which may yet
prove to be typical of some enveloped viruses. Significant differ-
ences were observed in average size between all genotypes. Some
detail of structural organization of HCV core or envelope glycopro-
teins was observable, but like beans in a bag, the semi-ordered
structures were dynamic and transient during AFM scanning.
The characterization techniques employed here are broadly
applicable to multiple enveloped and non-enveloped virus and
VLP systems, could be used for bioengineering of more uniform
and stable VLPs, and may also be of use for more detailed analysis
of biomechanical responses in the viral life-cycle. The images pre-
sented here represent an improvement on previous morphological
characterization [20,21,87,88], particularly as imaging was per-
formed in near physiological conditions, and elasticity data is the
first reported for HCV VLPs. This work provides a framework for
further AFM investigation of HCV VLPs and their behavior under
 S. Collett et al. / Journal of Colloid and Interface Science 545 (2019) 259–268
various conditions, including VLP interactions with other classi-
cally employed model AFM substrates, and in situ interactions of
VLPs with other biological material could be investigated with
nanoscale resolution. Broadly, the results described here will
impact on the VLP and the AFM imaging community as a whole.
Conflicts of interest
None.
Acknowledgments
The authors thank Dr Andrew J. Guy and Dr Madeleine F.
Dupont (RMIT University) for advice and support for computa-
tional implementation and statistical analysis. SC is supported by
a research training program stipend scholarship from the Aus-
tralian Government, Department of Education and Training. AFM
work was conducted in the MicroNano Research Facility (MNRF)
at RMIT University and the Cypher ES AFM instrument was funded
in part by grant LE170100096 from the Australian Research
Council (ARC). This work was supported by the National Health
and Medical Research Council (NHMRC) of Australia, grant number
1126379. JT is supported by an NHMRC Practitioner Fellowship,
number 106043.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jcis.2019.03.022.
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