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Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in ferrets

Authors:
  • Erasmus MC, Rotterdam, Netherlands

Abstract and Figures

Halting transmission The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein binds to host cells and initiates membrane fusion and cell infection. This stage in the virus life history is currently a target for drug inhibition. De Vries et al. designed highly stable lipoprotein fusion inhibitors complementary to a conserved repeat in the C terminus of S that integrate into host cell membranes and inhibit conformational changes in S necessary for membrane fusion. The authors tested the performance of the lipoproteins as a preexposure prophylactic in a ferret-to-ferret transmission study. Intranasal administration of the peptide 2 days before cohousing with an infected ferret for 24 hours completely protected animals in contact from infection and showed efficacy against mutant viruses. Because ferrets do not get sick from SARS-CoV-2, disease prevention could not be tested in this model. Science , this issue p. 1379
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Cite as: R. D. de Vries et al., Science
10.1126/science.abf4896 (2021).
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First release: 17 February 2021 www.sciencemag.org (Page numbers not final at time of first release) 1
Infection by SARS-CoV-2 requires membrane fusion between
the viral envelope and the host cell, at either the cell surface
or the endosomal membrane. The fusion process is mediated
by the viral transmembrane spike glycoprotein (S). Upon vi-
ral attachment or uptake, host factors trigger large-scale con-
formational rearrangements in S, including a refolding step
that leads directly to membrane fusion and viral entry (13).
Peptides corresponding to the highly conserved heptad re-
peat (HR, Fig. 1A) domain at the C terminus of the S protein
(HRC peptides, Fig. 1B) can prevent this refolding and inhibit
fusion, thereby preventing infection (48). The HRC peptides
form six-helix bundle-like assemblies with the extended in-
termediate form of the S protein trimer, disrupting the struc-
tural rearrangement of S that drives membrane fusion (4)
(see also movie S1).
Our approach in designing SARS-CoV-2 S-specific fusion
inhibitors builds on our previous work that demonstrated
that lipid conjugation of HRC-derived inhibitory peptides
markedly increases antiviral potency and in vivo half-life (9,
10). These peptides successfully inhibit human parainfluenza
virus type 3 (HPIV-3), measles virus, influenza virus, and
Nipah virus infection (9, 1113). Furthermore, the
combination of dimerization and lipopeptide integration into
cell membranes protects the respiratory tract and prevents
systemic lipopeptide dissemination (14). Lipid-conjugated
peptides administered intranasally to animals reached high
concentrations both in the upper and lower respiratory tract,
and the lipid could be designed to modulate the extent of
transit from the lung to the blood and organs (9, 14). We pro-
pose a SARS-CoV-2 specific lipopeptide as a candidate antivi-
ral for pre-exposure and early post-exposure prophylaxis for
SARS-CoV-2 transmission in humans.
We recently described a monomeric SARS-CoV-2 HRC-
lipopeptide fusion inhibitor (4) against SARS-CoV-2 with in
vitro and ex vivo efficacy superior to previously described
HRC-derived fusion inhibitory peptides (6, 7). To further im-
prove antiviral potency, we compared monomeric and di-
meric HRC-peptide derivatives (Fig. 1C). Figure 1D shows
antiviral potency in a quantitative cell-cell fusion assay of
four monomeric and two dimeric SARS-CoV-2 S-derived 36-
amino acid HRC-peptides (Fig. 1B, see also figs. S1A and S3
for structure and characterization), without or with ap-
pended cholesterol. Dimerization increased the peptide po-
tency for both non-lipidated peptides and their lipidated
Intranasal fusion inhibitory lipopeptide prevents
direct-contact SARS-CoV-2 transmission in ferrets
Rory D. de Vries1*, Katharina S. Schmitz1*, Francesca T. Bovier2,3,4*, Camilla Predella2,5, Jonathan Khao6,
Danny Noack1, Bart L. Haagmans1, Sander Herfst1, Kyle N. Stearns2,3,7, Jennifer Drew-Bear2,3, Sudipta Biswas8,
Barry Rockx1, Gaël McGill6,9, N. Valerio Dorrello2, Samuel H. Gellman10, Christopher A. Alabi8†,
Rik L. de Swart1†, Anne Moscona2,3,7,11†, Matteo Porotto2,3,4
1Department of Viroscience, Erasmus MC, Rotterdam, Netherlands. 2Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, USA. 3Center for
Host–Pathogen Interaction, Columbia University Irving Medical Center, New York, NY, USA. 4Department of Experimental Medicine, University of Campania “Luigi
Vanvitelli,” Caserta, Italy. 5Department of Biomedical Engineering, Politecnico di Milano, Milan, Italy. 6Digizyme Inc., Brookline, MA, USA. 7Department of Physiology and
Cellular Biophysics, Columbia University Irving Medical Center, New York, NY, USA. 8Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell
University, Ithaca, NY, USA. 9Center for Molecular and Cellular Dynamics, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, MA, USA. 10Department of Chemistry, University of WisconsinMadison, Madison, WI, USA. 11Department of Microbiology and Immunology, Columbia University
Irving Medical Center, New York, NY, USA.
*These authors contributed equally to this work.
Corresponding author. Email: caa238@cornell.edu (C.A.A.); r.deswart@erasmusmc.nl (R.L.d.S.);
am939@cumc.columbia.edu (A.M.); mp3509@cumc.columbia.edu (M.P.)
Containment of the COVID-19 pandemic requires reducing viral transmission. SARS-CoV-2 infection is
initiated by membrane fusion between the viral and host cell membranes, mediated by the viral spike
protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection, and
based on in vitro efficacy and in vivo biodistribution selected a dimeric form for evaluation in an animal
model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact
transmission during 24-hour co-housing with infected animals, under stringent conditions that resulted in
infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate
into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2.
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counterparts (Fig. 1D). A dimeric cholesterol-conjugated
lipopeptide based on the HPIV-3 F protein HRC domain, used
as a negative control, did not inhibit fusion at any concentra-
tion tested (black line in Fig. 1D, see fig. S1, B and C, for ad-
ditional negative controls). Among the monomeric
lipopeptides, the peptide bearing PEG24 was most potent. The
dimeric cholesterol-conjugated peptide ([SARSHRC-PEG4]2-
chol; red line in Fig. 1D) was the most potent lipopeptide
against SARS-CoV-2 among our panel. This peptide also ro-
bustly inhibited fusion mediated by the S proteins of several
emerging SARS-CoV-2 variants [including D614G (15)], the
recent variants of concern B.1.1.7 and B.1.351 (16, 17) and the
S protein of SARS-CoV and MERS-CoV (Fig. 1E). Proposed an-
choring of the dimeric lipopeptide in the host cell membrane
and interactions with the viral S protein are shown in fig. S2
and movie S1. Collectively, these data suggest that the
[SARSHRC-PEG4]2-chol lipopeptide is equipped to combat an
evolving pandemic.
For other enveloped respiratory viruses, we previously
showed that both ex vivo and in vivo dimeric lipopeptides
(administered intranasally) displayed increased retention in
the respiratory tract compared to monomeric compounds
(14). Here, we compared local and systemic biodistribution of
our most potent monomeric and dimeric lipopeptides
(SARSHRC-PEG24-chol and [SARSHRC-PEG4]2-chol) at 1, 8, and
24 hours after intranasal inoculation or subcutaneous injec-
tion in humanized K18 hACE2 mice (Fig. 2 and fig. S4). The
two lipopeptides reached a similar lung concentration at 1
hour after intranasal administration (~1 to 2 μM). At 8 and
24 hours, the dimeric [SARSHRC-PEG4]2-chol lipopeptide re-
mained at high levels in the lung with minimal entry into the
blood, but the monomeric peptide entered the circulation
and the lung concentration decreased (Fig. 2A). The dimeric
[SARSHRC-PEG4]2-chol lipopeptide was distributed through-
out the lung after intranasal administration (Fig. 2B). A cel-
lular toxicity (MTT) assay in primary HAE cells showed
minimal toxicity even after 6 days at the highest concentra-
tions tested (<20% at 100 μM), and no toxicity at its IC90 entry
inhibitory concentrations (~35 nM) (fig. S5). The longer res-
piratory tract persistence of [SARSHRC-PEG4]2-chol, in concert
with its in vitro efficacy, led us to advance this dimeric
lipopeptide.
The lead peptide, [SARSHRC-PEG4]2-chol, was assessed for
its ability to block entry of SARS-CoV-2 in VeroE6 cells or
VeroE6 cells overexpressing the protease TMPRSS2, one of
the host factors thought to facilitate viral entry at the cell
membrane (2). Whereas viral fusion in VeroE6 cells predom-
inantly occurs after endocytosis, the virus enters TMPRSS2-
overexpressing cells by fusion at the cell surface, reflecting
the entry route in airway cells (18). This difference is high-
lighted by chloroquine’s effectiveness against SARS-CoV-2 in-
fection in Vero cells but failure in TMPRSS2-expressing Vero
cells and human lung (19). The [SARSHRC-PEG4]2-chol peptide
dissolved in an aqueous buffer containing 2% dimethylsulfox-
ide (DMSO) inhibited virus entry after 8 hours with an IC50
~300 nM in VeroE6 and ~5 nM in VeroE6-TMPRSS2 cells
(Fig. 3A). To strengthen translational potential toward hu-
man use, the lipopeptide was reformulated in sucrose instead
of DMSO, resulting in equivalent in vitro potency (Fig. 3B). A
control dimeric fusion-inhibitory lipopeptide directed
against HPIV-3 blocked infection by HPIV-3, but did not in-
hibit SARS-CoV-2 infection. The in vitro efficacy data are
summarized in table S1.
Ferrets are an ideal model for assessing respiratory virus
transmission, either by direct contact or by aerosol transmis-
sion (20, 21). Mustelids are highly susceptible to infection
with SARS-CoV-2, as also illustrated by frequent COVID-19
outbreaks at mink farms. Direct contact transmission of
SARS-CoV in ferrets was demonstrated in 2003 (22), and both
direct contact and airborne transmission have been shown in
ferrets for SARS-CoV-2 (20, 23). Direct contact transmission
in the ferret model is highly reproducible (100% transmission
from donor to acceptor animals), but ferrets display limited
clinical signs. After infection via direct inoculation or trans-
mission, SARS-CoV-2 can readily be detected in and isolated
from the throat and nose, and viral replication leads to sero-
conversion.
To assess the efficacy of [SARSHRC-PEG4]2-chol in prevent-
ing SARS-CoV-2 transmission, naïve ferrets were dosed
prophylactically with the lipopeptide before being co-housed
with SARS-CoV-2 infected ferrets. In this setup, transmission
via multiple routes can theoretically occur (aerosol, orofecal,
and scratching or biting), and ferrets are continuously ex-
posed to infectious virus during the period of co-housing,
providing a stringent test for antiviral efficacy. The study de-
sign is shown in fig. S6. Three donor ferrets (gray in diagram)
were inoculated intranasally with 5 × 105 TCID50 SARS-CoV-2
on day 0. Twelve recipient ferrets housed separately were
treated by nose drops with a mock preparation (red) or
[SARSHRC-PEG4]2-chol peptide (green) 1 and 2 days post-inoc-
ulation (DPI) of the donor animals. The [SARSHRC-PEG4]2-chol
peptides for intranasal administration were dissolved to a
concentration of 6 mg/ml in an aqueous buffer containing 2%
DMSO, administering a final dose of 2.7 mg/kg to ferrets
(450 μl, equally divided over both nostrils). Peptide stocks
and working dilutions had similar IC50’s, confirming that pep-
tide-treated ferrets were dosed daily with comparable
amounts (fig. S7, A and B). Six hours after the second treat-
ment on 2 DPI, one infected donor ferret (highly positive for
SARS-CoV-2 by RT-qPCR) was co-housed with four naïve re-
cipient ferrets (two mock-treated, two peptide-treated). After
a 24-hour transmission period in three separate, negatively
pressurized HEPA-filtered ABSL3-isolator cages, co-housing
was stopped and donor, mock-treated and peptide-treated
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ferrets were housed as separate groups. Additional [SARSHRC-
PEG4]2-chol peptide treatments were given to recipient ani-
mals on 3 and 4 DPI.
The viral loads (detection of viral genomes via RT-qPCR)
for directly inoculated donor animals (gray), mock-treated re-
cipient animals (red) and lipopeptide-treated recipient ani-
mals (green) are shown in Fig. 4, A and B. All directly
inoculated donor ferrets were productively infected, as
shown by SARS-CoV-2 genome detection in throat and nose
swabs, and efficiently and reproducibly transmitted the virus
to all mock-treated acceptor ferrets (Fig. 4, A and B, red
curves). Productive SARS-CoV-2 infection was not detected in
the throat or nose of any of the peptide-treated recipient an-
imals (Fig. 4, A and B, green curves). A slight rise in viral
loads in samples collected at 3 DPI was detected (at the end
of the co-housing), confirming that peptide-treated animals
were exposed to SARS-CoV-2. In Fig. 4C the area under the
curve (AUC) shows the striking difference between the mock
treated and the peptide treated animals. No infectious virus
was isolated from lipopeptide-treated ferrets, while infectious
virus was detected in all mock-treated ferrets (Fig. 4D). Virus
isolation data correlated with genome detection (Fig. 4E).
Seroconversion occurred in donor ferrets and 6/6 mock-
treated animals by 21 DPI, but in none of the peptide-treated
recipient animals, as shown by S- and N-specific IgG enzyme-
linked immunosorbent assay (ELISA) and virus neutraliza-
tion (Fig. 4, F to H). Successful challenge infection confirmed
that in-host virus replication had been completely blocked by
the [SARSHRC-PEG4]2-chol treatment (Fig. 4I and fig. S8) and
that none of the peptide-animals were protected, whereas the
mock-treated animals (which had seroconverted) were all
protected. Collectively, these data show that intranasal
prophylactic administration of the [SARSHRC-PEG4]2-chol
peptide had protected 6/6 ferrets from transmission and pro-
ductive infection.
In light of the persistence of the dimeric lipopeptide in
the murine lung (Fig. 2 and fig. S4), we assessed the potential
for a single administration of sucrose-formulated lipopeptide
in a ferret transmission experiment two hours before co-
housing to prevent or delay infection. In this experiment, we
used a dimeric HPIV-3-specific lipopeptide as mock control
(fig. S9). Although sucrose formulation had resulted in prom-
ising results in vitro at small scale (Fig. 3B), formulation at
larger scale resulted in incomplete dissolution. As a conse-
quence, the sucrose-formulated [SARSHRC-PEG4]2-chol
lipopeptide was administered at a substantially lower con-
centration than in the experiment with the DMSO-
formulated lipopeptide (fig. S7, C and D). Nevertheless, the
SARS-CoV-2 lipopeptide provided a significant level of pro-
tection as compared to the HPIV-3 control group, and four
out of six SARS-CoV-2 lipopeptide-treated animals were pro-
tected against infection. This experiment suggests that single-
administration pre-exposure prophylaxis is promising, while
the optimal formulation and dosing regimen is an area of on-
going experimentation.
The intranasal [SARSHRC-PEG4]2-chol peptide presented in
this study is the first successful prophylaxis that prevents
SARS-CoV-2 transmission in a relevant animal model, provid-
ing complete protection during a 24-hour period of intense
direct contact. Parallel approaches to prevent transmission
that target the interaction between S and ACE2 have shown
promise in vitro [e.g., the “miniprotein” approach (24)]. The
lipopeptide described here acts on the S2 domain after shed-
ding of S1 (fig. S2 and movie S1), and is complementary to
strategies that target S1’s functions or maintain S in its pre-
fusion conformation, e.g., synthetic nanobodies (25, 26). Fu-
sion-inhibitory lipopeptides could be used for pre- and post-
exposure prophylaxis in combination with these strategies,
and in conjunction with treatments [e.g., ribonucleoside an-
alogs (27)] that reduce replication in a treated infected indi-
vidual. A combination of drugs that target different aspects
of the viral life cycle is likely ideal for this rapidly-evolving
virus. Of note, the [SARSHRC-PEG4]2-chol lipopeptide is
equally active against several emerging SARS-CoV-2 variants
including the D614G as well as the recent variants of concerns
(B.1.1.7 and B.1.351). The [SARSHRC-PEG4]2-chol peptide has a
long shelf life, does not require refrigeration and can easily
be administered, making it particularly suited to treating
hard-to-reach populations. This is key in the context of
COVID-19, which has reached every community with the bur-
den falling disproportionately on low-income and otherwise
marginalized communities. This HRC lipopeptide fusion in-
hibitor is feasible for advancement to human use and should
readily translate into a safe and effective nasal spray or inha-
lation administered fusion inhibitor for SARS-CoV-2 prophy-
laxis, supporting containment of the ongoing COVID-19
pandemic.
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ACKNOWLEDGMENTS
We thank J. S. Orange, S. G. Kernie, M. Lamers, E. Verveer, A. Mykytyn and
M. Koopmans for their contributions to this study. Funding: This work was
supported by funding from the National Institutes of Health (AI146980,
AI121349, and NS091263 to M.P., AI114736 to A.M., HHSN272201400008C to
S.H.), the Sharon Golub Fund at Columbia University Irving Medical Center
(CUIMC), the Children’s Health Innovation Nucleation Fund of the Pediatrics
Department at CUIMC and a Harrington Discovery Institute COVID-19 Award to
A.M. A.M. is the inaugural Sherie L. Morrison Professor of Immunology.
Author contributions: Conceptualization: R.D.d.V., S.H.G., C.A.A., R.L.d.S.,
A.M., M.P. Formal analysis: R.D.d.V., K.S.S., F.T.B., C.P., N.V.D., C.A.A.,
R.L.d.S., A.M., M.P. Funding acquisition: B.L.H., R.L.d.S., A.M., M.P.
Investigation: R.D.d.V., K.S.S., F.T.B., C.P., J.K., D.N., K.N.S., S.H., J.D.-B.,
S.B., G.M., N.V.D., S.H.G., C.A.A., R.L.d.S., A.M., M.P. Resources: B.L.H., B.R.,
N.V.D., C.A.A., R.L.d.S., A.M., M.P. Supervision: R.L.d.S., N.V.D., C.A.A.,
A.M., M.P. Visualization: R.D.d.V., K.S., F.T.B., J.K., G.M., N.V.D., S.H.G., C.A.A.,
A.M., M.P. Writing original draft: R.D.d.V., R.L.d.S., A.M., M.P. Writing – final
version: all co-authors provided feedback to the final draft. Competing
interests: R.D.d.V., F.T.B., R.L.d.S., A.M. and M.P. are listed as inventors of
[SARSHRC-PEG4]2-chol on a provisional patent application covering findings
reported in this manuscript. Data and materials availability: All data are
available in the manuscript or the supplementary materials. Materials are
available by MTA with the Trustees of Columbia University, NYC. This work is
licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0)
license, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. To view a copy of this
license, visit https://creativecommons.org/licenses/by/4.0/. This license does
not apply to figures/photos/artwork or other content included in the article that
is credited to a third party; obtain authorization from the rights holder before
using such material. Reagents are available from the corresponding authors
under a material agreement with Columbia University.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/cgi/content/full/science.abf4896/DC1
Materials and Methods
Figs. S1 to S10
Table S1
References (2834)
MDAR Reproducibility Checklist
Movie S1
29 October 2020; resubmitted 4 January 2021
Accepted 9 February 2021
Published online 17 February 2021
10.1126/science.abf4896
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Fig. 1. Peptide-lipid conjugates that inhibit SARS-CoV-2 spike (S)mediated fusion
.
(
A
) The functional domains of SARS-CoV-2 S protein: receptor-binding domain (RBD)
and heptad repeats (HRN and HRC) are indicated. (
B
) Sequence of the peptides derived
from the HRC domain of SARS-CoV-2 S. (
C
) Monomeric and dimeric forms of lipid
tagged SARS-CoV-2 inhibitory peptides that were assessed in cell-cell fusion assays.
(
D
) Cell-cell fusion assays with different inhibitory peptides. The percentage inhibition
is shown for six different SARS-CoV-2-specific peptides and a control HPIV-3-specific
peptide at increasing concentrations. Percent inhibition was calculated as the ratio of
the relative luminescence units in the presence of a specific concentration of inhibitor
and the relative luminescence units in the absence
of inhibitor, corrected for
background luminescence. % inhibition = 100 × [1 (luminescence at X
background)/(luminescence in absence of inhibitor background)]. The difference
between the results for [SARSHRC-PEG4]2-chol and SARSHRC-PEG4-chol lipopeptides was
statistically significant (two-way ANOVA, P < 0.0001). (
E
) Fusion inhibitory activity of
[SARSHRC-PEG4]2-chol peptide against emerging SARS-CoV-2 S variants, MERS-CoV-2
S, and SARS-CoV S. Data in (D) and (E) are means ± standard error of the mean (SEM)
from three separate experiments with the curve representing a four-parameter dose-
response model.
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Fig. 2. Biodistribution of [SARSHRC-PEG4]2-chol and
SARSHRC-PEG24 peptides after intranasal administration
to mice.
(
A
) The concentration of lipopeptides (y axis) was
measured by ELISA in lung homogenates and plasma
samples (peptide-treated n = 3 to 4, mock n =1). Median is
indicated by horizontal bar. (
B
) Lung sections of [SARSHRC-
PEG4]2-chol-treated (or vehicle-treated) mice were stained
with anti-SARS-
HRC antibody (red) confirming broad
distribution of [SARSHRC-PEG4]2-chol in the lung (8 hours
post-inoculation, 8HPI). Scale bar = 500 μm in lung tile scan,
50 μm in magnification, representative images and a full tile
scan are shown. Nuclei were counterstained with DAPI
(blue).
Fig. 3. Inhibition of infectious SARS-CoV-2 entry by [SARSHRC-PEG4]2-
chol and [HPIV-3HRC-PEG4]2-chol peptides.
(
A
and
B
) The percentage
inhibition of infection is shown on VeroE6 and VeroE6-TMPRSS2 cells
with increasing concentrations of [SARSHRC-PEG4]2-chol (red lines) and
[HPIV-3HRC-PEG4]2-chol (gray lines). DMSO-formulated (A) and sucrose-
formulated stocks (B) were tested side-by-side. Mean ± SEM of
triplicates are shown, dotted lines show 50% and 90% inhibition.
Additionally, the potency of [HPIV-3HRC-PEG4]2-chol was confirmed by
inhibition of infectious HPIV-3 entry (dotted green lines, performed on
Vero cells).
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Fig. 4. [SARSHRC-PEG4]2-chol prevents SARS-CoV-2 transmission in
vivo.
(
A
and
B
) Viral loads detected in throat (A) and nose (B) swabs by
RT-qPCR. (
C
) Comparison of the area under the curve (AUC) from
genome loads reported in B for mock- and peptide-treated sentinels.
(
D
) Viral loads detected in throat swabs by virus isolation on VeroE6.
(
E
) Correlation between viral loads in the throat as detected via RT-qPCR
and virus isolation. Presence of anti-S (
F
) or anti-N (
G
) antibodies was
determined by IgG ELISA assay. Presence of neutralizing antibodies was
determined in a virus neutralization assay (
H). Virus neutralizing
antibodies are displayed as the endpoint serum dilution factor that
blocks SARS-CoV-2 replication. Direct inoculation of peptide-treated or
mock-treated animals with SARS-CoV-2 led to productive infection in
only the previously peptide-treated animals (
I
), in the absence of S-
specific, N-specific and neutralizing antibodies. Donor animals shown in
gray, mock-treated animals in red, peptide-treated animals in green.
Symbols correspond to individual animals (defined in fig. S6). Line
graphs in (A), (B), (D), and (F) to (I) connect the median of individual
animals per time point. Mock- and peptide-
treated groups were
compared via two-way ANOVA repeated measures [(A), (B), and (F) to
(I)] or Mann-Whitney test (C).
on February 25, 2021 http://science.sciencemag.org/Downloaded from
ferrets
Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in
Gellman, Christopher A. Alabi, Rik L. de Swart, Anne Moscona and Matteo Porotto
Sander Herfst, Kyle N. Stearns, Jennifer Drew-Bear, Sudipta Biswas, Barry Rockx, Gaël McGill, N. Valerio Dorrello, Samuel H.
Rory D. de Vries, Katharina S. Schmitz, Francesca T. Bovier, Camilla Predella, Jonathan Khao, Danny Noack, Bart L. Haagmans,
published online February 17, 2021
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REFERENCES http://science.sciencemag.org/content/early/2021/02/16/science.abf4896#BIBL
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Thesis
Les virus respiratoires aéroportés sont particulièrement préoccupants du fait de la difficulté de contrôler leur transmission. Parmi ces virus, le virus du syndrome respiratoire aigu sévère 2 (SARS-CoV-2), le virus de la rougeole (VR) et les Henipavirus Nipah (NiV) et Hendra (HeV) peuvent infecter également le système nerveux central (SNC) chez l’homme et provoquent alors souvent des encéphalites létales. Par exemple, le SARS-CoV-2, responsable de la pandémie de COVID-19, entraine un syndrome respiratoire aigu sévère et des atteintes neurologiques. De son coté, et malgré un vaccin efficace, la rougeole connait une réémergence inquiétante et cause la mort de plus de 200 000 personnes par an. Le VR peut entrainer des encéphalites rougeoleuses à corps d’inclusion (MIBE) dans un contexte d’immunodéficience ou une panencéphalite sclérosante subaiguë (PESS) parfois des décennies après l’exposition au virus chez des patients immunocompétents. NiV et HeV sont des Paramyxovirus zoonotiques hautement pathogènes du genre Henipavirus. Malgré le faible nombre de cas humains recensés depuis leur émergence à la fin des années 1990, les NiV et HeV sont classés parmi les huit pathogènes prioritaires pour la recherche par l’Organisation Mondiale de la Santé en raison de leur fort potentiel pandémique. Certaines souches sont mortelles dans plus de 70% des cas en moyenne. À ce jour il n’existe pas de traitement efficace commercialisé pour traiter ces infections virales chez l’homme. De plus, les étapes précoces de l’infection du SNC par ces virus restent peu documentées car la majorité des données proviennent d’analyses réalisées post mortem. L’objectif global de cette thèse a été d’identifier des facteurs influençant l’invasion du SNC par ces virus. Le tropisme initial, la dissémination, ainsi que l’implication des glycoprotéines virales de surface et l’évolution génétique virale ont été analysées pour le SARS-CoV-2, le VR et plusieurs souches d’Henipavirus à l’échelle organique, cellulaire et moléculaire. Deux nouveaux modèles de cultures organotypiques de poumons et de tronc cérébral chez le hamster ont été développés et caractérisés. Ces modèles ex vivo sont susceptibles à l’infection par le SARS-CoV-2 et par le NiV. En revanche, un mutant hyperfusogène du VR, pourtant capable de fusionner en l’absence de récepteur connu, n’infecte que les cultures de cerveau. Ces cultures organotypiques ont permis de valider le tropisme initial du SARS-CoV-2 dans les poumons et démontré la permissivité de certains neurones dans le cerveau. Ces modèles ont également permis d’établir que l’infection par le SARS-CoV-2 induit une réponse interféron spécifique et une réponse immunitaire innée, ainsi qu’une mort cellulaire par apoptose, nécroptose et pyroptose dans ces organes. Enfin, ces cultures organotypiques ont montré leur pertinence dans la validation de l’effet d’antiviraux. L’étude de VR portant des mutations dans leur protéine de fusion observées lors d’encéphalites rougeoleuses a montré l’importance du caractère hyperfusogène de ces mutants pour se disséminer dans le SNC pourtant dépourvu de récepteurs connus. Des différences dans la machinerie de fusion de trois souches pathogènes d’Henipavirus ont aussi été identifiées et analysées.Grace aux cultures organotypiques cérébrales de hamster et de souris transgéniques plusieurs candidats antiviraux ont été testés pour bloquer la dissémination du VR sauvage et de variants neuroinvasifs, mais aussi du NiV et du SARS-CoV-2. Ces résultats donnent des perspectives nouvelles d’utilisation de ces modèles ex vivo pour étudier l’infection par des virus émergents et pour évaluer l’efficacité de traitements en amont de validation in vivo. L’étude comparative de l’infection des cultures organotypiques par ces virus respiratoires à pathogénicité variable a illustré comment la machinerie de fusion peut influencer la dissémination virale dans le cerveau.
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The virulence of avian gamma-coronavirus infectious bronchitis viruses (IBV) for the kidney has led to high mortality in dominant-genotype isolations, but the key sites of viral protein that determine kidney tropism are still not fully clear. In this study, the amino acid sequences of the S2 subunit of IBVs with opposing adaptivity to chicken embryonic kidney cells (CEKs) were aligned to identify putative sites associated with differences in viral adaptability. The S2 gene and the putative sites of the non-adapted CN strain were introduced into the CEKs-adapted SczyC30 strain to rescue seven mutants. Analysis of growth characteristics showed that the replacement of the entire S2 subunit and the L1089I substitution in the S2 subunit entirely abolished the proliferation of recombinant IBV in CEKs as well as in primary chicken oviduct epithelial cells. Pathogenicity assays also support the decisive role of this L1089 for viral nephrotropism, and this non-nephrotropic L1089I substitution significantly attenuates pathogenicity. Analysis of the putative cause of proliferation inhibition in CEKs suggests that the L1089I substitution affects neither virus attachment nor endocytosis, but instead fails to form double-membrane vesicles to initiate the viral replication and translation. Position 1089 of the IBV S2 subunit is conservative and predicted to lie in heptad repeat 2 domains. It is therefore reasonable to conclude that the L1089I substitution alters the nephrotropism of parent strain by affecting virus-cell fusion. These findings provide crucial insights into the adaptive mechanisms of IBV and have applications in the development of vaccines and drugs against IB.
Preprint
Currently the world is dealing with the third outbreak of the human-infecting coronavirus with potential lethal outcome, cause by a member of the Nidovirus family, the SARS-CoV-2. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused the last worldwide pandemic. Successful development of vaccines highly contributed to reduce the severeness of the COVID-19 disease. To establish a control over the current and newly emerging coronaviruses of epidemic concern requires development of substances able to cure severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA using antisense oligonucleotides (ASOs) and identify locked nucleic acid gapmers (LNA gapmers) potent to reduce by up to 96% the intracellular viral load in vitro . Our results strongly suggest promise of our preselected ASOs for further development as therapeutic or prophylactic anti-viral agents. One sentence summary ASOs (LNA gapmers) targeting the SARS-CoV-2 RNA genome have been effective in viral RNA (load) reduction in vitro .
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Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system.
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Vaccine protects against B1.1.7 variant The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B1.1.7 (VOC 202012/01) variant that emerged in late 2020 in the United Kingdom has many changes in the spike protein gene. Three of these are associated with enhanced infectivity and transmissibility, and there are concerns that B.1.1.7 might compromise the effectiveness of the vaccine. Muik et al. compared the neutralization efficacy of sera from 40 subjects immunized with the BioNTech-Pfizer mRNA vaccine BNT162b2 against a pseudovirus bearing the Wuhan reference strain or the lineage B.1.1.7 spike protein (see the Perspective by Altmann et al. ). Serum was derived from 40 subjects in two age groups 21 days after the booster shot. The vaccine remained effective against B.1.1.7 with a slight but significant decrease in neutralization that was more apparent in participants under 55 years of age. Thus, the vaccine provides a significant “cushion” of protection against this variant. Science , this issue p. 1152 ; see also p. 1103
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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative infection of a global pandemic that has led to more than 2 million deaths worldwide. The Moderna mRNA-1273 vaccine has demonstrated ~94% efficacy in a Phase 3 study and has been approved under Emergency Use Authorization. The emergence of SARS-CoV-2 variants with mutations in the spike protein, most recently circulating isolates from the United Kingdom (B.1.1.7) and Republic of South Africa (B.1.351), has led to lower neutralization from convalescent serum by pseudovirus neutralization (PsVN) assays and resistance to certain monoclonal antibodies. Here, using two orthogonal VSV and lentivirus PsVN assays expressing spike variants of 20E (EU1), 20A.EU2, D614G-N439, mink cluster 5, B.1.1.7, and B.1.351 variants, we assessed the neutralizing capacity of sera from human subjects or non-human primates (NHPs) that received mRNA-1273. No significant impact on neutralization against the B.1.1.7 variant was detected in either case, however reduced neutralization was measured against the mutations present in B.1.351. Geometric mean titer (GMT) of human sera from clinical trial participants in VSV PsVN assay using D614G spike was 1/1852. VSV pseudoviruses with spike containing K417N-E484K-N501Y-D614G and full B.1.351 mutations resulted in 2.7 and 6.4-fold GMT reduction, respectively, when compared to the D614G VSV pseudovirus. Importantly, the VSV PsVN GMT of these human sera to the full B.1.351 spike variant was still 1/290, with all evaluated sera able to fully neutralize. Similarly, sera from NHPs immunized with 30 or 100μg of mRNA-1273 had VSV PsVN GMTs of ~ 1/323 or 1/404, respectively, against the full B.1.351 spike variant with a ~ 5 to 10-fold reduction compared to D614G. Individual mutations that are characteristic of the B.1.1.7 and B.1.351 variants had a similar impact on neutralization when tested in VSV or in lentivirus PsVN assays. Despite the observed decreases, the GMT of VSV PsVN titers in human vaccinee sera against the B.1.351 variant remained at ~1/300. Taken together these data demonstrate reduced but still significant neutralization against the full B.1.351 variant following mRNA-1273 vaccination.
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Coronavirus entry is mediated by the spike protein which binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells, and more frequently formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.
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Nanobodies that neutralize Monoclonal antibodies that bind to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show therapeutic promise but must be produced in mammalian cells and need to be delivered intravenously. By contrast, single-domain antibodies called nanobodies can be produced in bacteria or yeast, and their stability may enable aerosol delivery. Two papers now report nanobodies that bind tightly to spike and efficiently neutralize SARS-CoV-2 in cells. Schoof et al. screened a yeast surface display of synthetic nanobodies and Xiang et al. screened anti-spike nanobodies produced by a llama. Both groups identified highly potent nanobodies that lock the spike protein in an inactive conformation. Multivalent constructs of selected nanobodies achieved even more potent neutralization. Science , this issue p. 1473 , p. 1479
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The coronavirus disease 2019 (COVID-19) pandemic is having a catastrophic impact on human health¹. Widespread community transmission has triggered stringent distancing measures with severe socio-economic consequences. Gaining control of the pandemic will depend on the interruption of transmission chains until vaccine-induced or naturally acquired protective herd immunity arises. However, approved antiviral treatments such as remdesivir and reconvalescent serum cannot be delivered orally2,3, making them poorly suitable for transmission control. We previously reported the development of an orally efficacious ribonucleoside analogue inhibitor of influenza viruses, MK-4482/EIDD-2801 (refs. 4,5), that was repurposed for use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently in phase II/III clinical trials (NCT04405570 and NCT04405739). Here, we explored the efficacy of therapeutically administered MK-4482/EIDD-2801 to mitigate SARS-CoV-2 infection and block transmission in the ferret model, given that ferrets and related members of the weasel genus transmit the virus efficiently with minimal clinical signs6–9, which resembles the spread in the human young-adult population. We demonstrate high SARS-CoV-2 burden in nasal tissues and secretions, which coincided with efficient transmission through direct contact. Therapeutic treatment of infected animals with MK-4482/EIDD-2801 twice a day significantly reduced the SARS-CoV-2 load in the upper respiratory tract and completely suppressed spread to untreated contact animals. This study identified oral MK-4482/EIDD-2801 as a promising antiviral countermeasure to break SARS-CoV-2 community transmission chains.
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SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.
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SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers ( in vitroex vivo
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Miniproteins against SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is decorated with spikes, and viral entry into cells is initiated when these spikes bind to the host angiotensin-converting enzyme 2 (ACE2) receptor. Many monoclonal antibody therapies in development target the spike proteins. Cao et al. designed small, stable proteins that bind tightly to the spike and block it from binding to ACE2. The best designs bind with very high affinity and prevent SARS-CoV-2 infection of mammalian Vero E6 cells. Cryo–electron microscopy shows that the structures of the two most potent inhibitors are nearly identical to the computational models. Unlike antibodies, the miniproteins do not require expression in mammalian cells, and their small size and high stability may allow formulation for direct delivery to the nasal or respiratory system. Science , this issue p. 426
Preprint
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Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.
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The COVID-19 pandemic, which is caused by the novel coronavirus SARS-CoV-2, has been associated with more than 470,000 fatal cases worldwide. In order to develop antiviral interventions quickly, drugs used for treatment of unrelated diseases are currently being repurposed to combat COVID-19. Chloroquine is a anti-malaria drug that is frequently employed for COVID-19 treatment since it inhibits SARS-CoV-2 spread in the kidney-derived cell line Vero1–3. Here, we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block SARS-CoV-2 infection of the TMPRSS2-positive lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not operative in lung cells and is unlikely to protect against SARS-CoV-2 spread in and between patients.