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\begin{document}

\title{Molecular definition of SARS-CoV-2 RBD mutations: receptor affinity
versus neutralization of receptor interaction}



\author[1]{Monique Vogel}%
\author[2]{Gilles Augusto}%
\author[2]{Xinyue Chang}%
\author[3]{Xuelan Liu}%
\author[3]{Daniel Speiser}%
\author[2]{Mona Mohsen}%
\author[3]{Martin Bachmann}%
\affil[1]{Inselspital University Hospital Berne Institute of Immunology}%
\affil[2]{University of Bern}%
\affil[3]{Inselspital University Hospital Bern}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Several new variants of SARS-CoV-2 have emerged since fall
2020 which have multiple mutations in the receptor binding domain (RBD)
of the spike protein. It is unclear which mutations affect receptor
affinity versus immune recognition. Methods: We produced RBD with single
mutations (E484K, K417N or N501Y) or with all three mutations combined
and tested their binding to ACE2 by biolayer interferometry (BLI). The
ability of convalescent sera to recognize RBDs and block their
interaction with ACE2 was tested as well. Results: We demonstrated that
single mutation N501Y increased binding affinity to ACE2 but did not
significantly affect its recognition by convalescent sera. In contrast,
single mutation E484K had almost no impact on the binding kinetics, but
essentially abolished recognition of RBD by convalescent sera.
Interestingly, combining mutations E484K, K417N and N501Y resulted in a
RBD with both features: enhanced receptor binding and abolished immune
recognition. Conclusion: Our data demonstrate that single mutations
either affect receptor affinity or immune recognition while triple
mutant RBDs combine both features.%
\end{abstract}%



\sloppy


\textbf{Molecular definition of SARS-CoV-2 RBD mutations: receptor
affinity versus neutralization of receptor interaction}

Monique Vogel\textsuperscript{1,2\&}, Gilles Sousa
Augusto\textsuperscript{1,2\&}, Xinyue Chang\textsuperscript{1,2},
Xuelan Liu\textsuperscript{1,3}, Daniel Speiser\textsuperscript{1,2},
Mona O. Mohsen\textsuperscript{1,2} and Martin F.
Bachmann\textsuperscript{1,2,3 *}

\textbf{Affiliations}

\textsuperscript{1} Department of Immunology, University clinic of
Rheumatology and Immunology, Inselspital,

Bern, Switzerland

\textsuperscript{2} Department of BioMedical Research, University of
Bern, Bern, Switzerland

\textsuperscript{3} International Immunology Centre, Anhui Agricultural
University, Hefei, China, Dermatology,

\textsuperscript{4} Nuffield Department of Medicine, Centre for Cellular
and Molecular Physiology (CCMP),

The Jenner Institute, University of Oxford, Oxford, UK

\textsuperscript{\&}equal contribution

\textbf{Address for Correspondence:}

Martin Bachmann, University Clinic of Rheumatology and Immunology,
University Hospital Bern, Salihaus 2, CH-3010 Bern, Switzerland. E-mail:
martin.bachmann@dbmr.unibe.ch.

\textbf{Acknowledgement}

We thank Marianne Zwicker for production of wild type and mutant RBDs.
We thank PD Dr. Alexander Eggel and Dr. Daniel Brigger for providing
biotinylated and non-biotinylated ACE2-mFc.

\textbf{Funding}

We thank Saiba AG, the Swiss National Science Foundation (SNF grant
31003A\_185114) and the International Immunology Centre, Anhui
Agricultural University, Hefei, China for the financial support.

\textbf{Conflict of Interests}

M. F. Bachmann is a board member of Saiba AG, involved in the
development of RBD-CuMV a vaccine against COVID-19. All other authors
declare no conflict of interest

\textbf{Author contributions}

M.V., G.S.A., X.C. designed, performed and interpreted most experiments;
X.L. M.M. provided serum sample; M.V., G.S.A., D.S. wrote the
manuscript, M.F.B. designed experiments and wrote the manuscript.

\textbf{Abstract}

\textbf{Background} : Several new variants of SARS-CoV-2 have emerged
since fall 2020 which have multiple mutations in the receptor binding
domain (RBD) of the spike protein. It is unclear which mutations affect
receptor affinity versus immune recognition.

\textbf{Methods:} We produced RBD with single mutations (E484K, K417N or
N501Y) or with all three mutations combined and tested their binding to
ACE2 by biolayer interferometry (BLI). The ability of convalescent sera
to recognize RBDs and block their interaction with ACE2 was tested as
well.

\textbf{Results:} We demonstrated that single mutation N501Y increased
binding affinity to ACE2 but did not significantly affect its
recognition by convalescent sera. In contrast, single mutation E484K had
almost no impact on the binding kinetics, but essentially abolished
recognition of RBD by convalescent sera. Interestingly, combining
mutations E484K, K417N and N501Y resulted in a RBD with both features:
enhanced receptor binding and abolished immune recognition.

\textbf{Conclusions:} Our data demonstrate that single mutations either
affect receptor affinity or immune recognition while triple mutant RBDs
combine both features.

\textbf{Keywords}

SARS-CoV-2, RBD, affinity, neutralization, ACE2, biolayer interferometry

\textbf{Introduction}

SARS-CoV-2 has caused a pandemic that is characterized in many countries
by several waves of infection \textsuperscript{1} \textsuperscript{2}.
While the origin of these infection waves may differ in different
regions of the world, the latest increase seen in numbers of infected
individual is apparently caused by the occurrence of mutated viral
strains \textsuperscript{3}. The most prominent mutated strains are
following variants: B.1.1.7. (N501Y, D614G), P.1 (K417N/T, E484K, N501Y)
and B.1.351 (K417N, E484K, N501Y, D614G) \textsuperscript{4}, which show
mutations in the receptor binding domain (RBD) and receptor binding
motif (RBM) of the spike (S) glycoprotein. RBD and in particular RBM are
responsible for interaction with the cellular receptor ACE2 and are the
primary target of neutralizing antibodies \textsuperscript{5} (Fig.1).
Mutant viruses may spread more efficiently because they show increased
affinity for the receptor or because they escape neutralizing antibody
responses. The importance of receptor affinity has been illustrated by
SARS-CoV-1, which showed a 4-fold lower affinity for ACE2 and also was
much less contagious and showed strongly reduced transmission compared
to SARS-CoV-2 \textsuperscript{6}.

Viruses that escape neutralization are typically called serotypes and
usually may only occur when a large proportion of individuals show
antibody-based immunity against the original strain and further spread
may only be possible by escape of neutralizing antibody
responses\textsuperscript{7}. For SARS-CoV-2, this is certainly not the
case on a global scale but certain regions of the world may show
infections rates that are compatible with serotype formation.

Here we assessed the molecular basis for antibody escape and how the RBD
mutations present in two variants of concern (B.1.1.7 and P.1) influence
the affinity to the receptor.

\textbf{Material and Methods}

\emph{Protein expression and purification}

The SARS-CoV-2 receptor-binding domain (RBD\textsubscript{WT}) and the
RBD mutant (RBD\textsubscript{K417N}, RBD\textsubscript{N501Y},
RBD\textsubscript{K417N,} RBD\textsubscript{TRIP}) were expressed using
Expi293F cells (Invitrogen, ThermoFisher Scientific, MA, USA). The genes
that encode SARS-CoV-2 RBD\textsubscript{WT} (residues Arg319-Phe541) or
RBD mutants with a C-terminal 6-His-tag was inserted into pTwist CMV
BetaGlobin WPRE Neo vector (Twist Bioscience, San Francisco, USA). The
construct plasmids were transfected into Expi293F cells using
ExpiFectatmine 293 transfection kit (Gibco, ThermoFisher Scientific, MA,
USA). The supernatant of cell culture containing the secreted RBDs was
purified using His-Trap HP column (GE Healthcare, USA). Collected
RBD\textsubscript{WT} or RBD mutated proteins were equilibrated in PBS
and kept at -20@\selectlanguage{english}C. ACE2-mFc was purchased from Sino Biological
(Eschborn, Germany). Biotinylated and non-biotinylated soluble human
ACE2 fused to mouse IgG2a Fc proteins were kindly provided by PD Dr.
Alexander Eggel (University Clinic of Rheumatology and Immunology,
Inselspital, Bern, Switzerland) who received the plasmid from Prof.
Peter Kim (Stanford University, CA, USA).

\emph{Human sera}

Human sera were obtained from 11 COVID-19 convalescent patients which
were recruited t the University Hospital of Bern, Bern,
Switzerland\textsuperscript{13}. All patients provided written informed
consent before participation in the studies.

\emph{ELISA Assay}

Corning half area 96-well plates were coated with 1 \selectlanguage{greek}µ\selectlanguage{english}g/ml
RBD\textsubscript{WT} or mutated RBDs in PBS overnight at 4\selectlanguage{ngerman}°C and then
blocked with PBS/0.15\% casein. Convalescent human sera were added,
serially diluted 1:3 and incubated on plates for one hour at room
temperature. Bound IgG antibodies were detected with goat anti-human
IgG-POX antibody (Nordic MUbio, Susteren, The Netherlands). ELISA was
developed with tetramethylbenzidine (TMB), stopped by adding equal 1 M
H\textsubscript{2}SO\textsubscript{4} solution, and read at
OD\textsubscript{450nm}.

\emph{RBDwt and RBDmut kinetics by Bio-Layer Interferometry (BLI)}

The analysis of binding kinetics of RBD\textsubscript{WT} and
RBD\textsubscript{TRIP} to ACE2-mFc was performed by Bio-Layer
Interferometry (BLI) using an Octet RED96e (Fortebio) instrument. High
precision Streptavidin (SAX, ForteBio, Fremont, CA, USA) biosensors were
saturated with 7.5 \selectlanguage{greek}µ\selectlanguage{english}g/ml biotinylated ACE2-mFc in BLI assay buffer (PBS,
0.1\% BSA, 0.02\% Tween 20) for 10 min. RBD\textsubscript{WT} and
RBD\textsubscript{TRIP} were prepared as twofold serial dilution
(typically 50, 25, 12.5, 6.25 and 3.125 nM) in BLI assay buffer plus
buffer blanks. Kinetic~values were calculated by ForteBio data analysis
software using a 1:1 binding model.

\emph{BLI-based competitive assay}

The ability of the sera of the COVID convalescent patient to compete
with ACE2 for binding to RBD\textsubscript{WT} and
RBD\textsubscript{TRIP} was tested in a sandwich format assay on the
Octet RED96e (Fortebio). Anti-penta-His (HIS1K) biosensors were loaded
for 10 min with RBD\textsubscript{WT} and RBD\textsubscript{TRIP} at a
concentration of 7.5 \selectlanguage{greek}µ\selectlanguage{english}g/ml in BLI assay buffer followed by addition of
samples (diluted 1:20 in BLI assay buffer) from convalescent human sera.
To assess whether the sera can inhibit the binding of ACE2 to
RBD\textsubscript{WT} and RBD\textsubscript{TRIP}, ACE2-mFc (50 nM) was
added to biosensor. For control two additional sensors with BLI buffer
were used, one for baseline and one without serum sample to determine
binding of ACE2-mFc alone. The results are expressed of single
individual. The response data were normalized using ForteBio data
analysis software version1.2.0.1.55.

\emph{Data and statistical analysis}

All statistical tests were performed using GraphPad PRISM 8.0 (GraphPad
Software, Inc.). ELISA data in graphs are displayed as enpoint titres
measured at a cut off 0.15 OD 450nm. Comparison between
RBD\textsubscript{WT} and mutated RBDs were analyzed by two-ways Anova
test for ELISA and paired two-tailed Student`s t-test for BLI assay.
\selectlanguage{greek}α\selectlanguage{english}=0.05 and statistical significance are displayed as p[?]0.05 (*), p[?]0.01
(**), p[?]0.005 (***), p[?]0.001 (****).

\textbf{Results}

\emph{Structural model and binding kinetics of SARS-CoV-2 RBD variants
to ACE2}

To address the questions of antibody binding strength and competition
mechanistically, we have expressed the RBD of the isolate P.1 exhibiting
RBD mutations (K417N, E484K and N501Y) (RBD\textsubscript{Trip}), two of
which are located in the RBM (E484K, N501Y) (shown in Fig.
1A)\textsuperscript{8}. In addition, we generated each of the respective
single mutants (RBD\textsubscript{K417N}, RBD\textsubscript{E484K}, and
RBD\textsubscript{N501Y}). All RBDs were purified to homogeneity and the
affinity to recombinant ACE2 was determined by Biolayer Interferometry
using Octet technology \textsuperscript{9}. The BLI assays showed that
the affinity of ACE2 for RBD\textsubscript{TRIP} (shown in Fig. 2B, 2F,
Table 1, K\textsubscript{D} = 10.3 nM) was about twice as high as for
RBD\textsubscript{WT} (shown in Fig. 2A, 2F, Table 1,
K\textsubscript{D}= 20.5 nM). Having in mind that the affinity of the of
SARS-CoV-2 for ACE2 is only 4-fold higher compared to SARS-CoV-1, this
factor of 2 is expected to be biologically significant and most likely
reflects enhanced infectivity. In contrast, the mutation at position 484
in RBD\textsubscript{484} did not affect receptor affinity (shown in
Fig. 2D, F). For comparison, the dissociation constant observed for
RBD\textsubscript{N501Y} was 3-fold lower (K\textsubscript{D} = 6.2 nM,
shown in Fig. 2C, 2F, Table 1). Interestingly, the mutation at position
417 in RBD\textsubscript{417} resulted in completely altered binding
properties (shown in Fig. 2E). RBD\textsubscript{K417N} showed much
lower association rates and plateau levels a non-monovalent pattern of
dissociation rates (shown in Fig. 2E, Table 1 K\textsubscript{D} could
not be determined in a meaningful way). Presence of aggregates was not
responsible for this effect, as purification by size exclusion
immediately before measurements did not alter the binding kinetics
observed (data not shown). However, since this mutation does not occur
on its own in viral strains, we did not further investigate this effect.

\emph{Reduced ability of convalescent sera to recognize RDB variants}

To determine whether RBD\textsubscript{WT} -specific immune sera might
have a reduced ability to bind to mutated RBDs we performed ELISA and
Biolayer Interferometry using sera from convalescent patients (shown in
Fig. 3). As expected RBD\textsubscript{WT} was well recognized by
convalescent sera in ELISA experiments. In contrast,
RBD\textsubscript{K417N} and RBD\textsubscript{N501Y} were recognized in
a slightly impaired fashion (shown in Fig. 3A). In marked contrast,
mutation at position 484 essentially abolished recognition of both
RBD\textsubscript{E484K} and RBD\textsubscript{TRIP}. Corresponding
results were obtained using Biolayer Interferometry (shown in Fig. 3B).
RBD-specific neutralizing antibodies typically block interaction of RBD
with the viral receptor ACE2. We therefore assessed whether reduced
binding of convalescent sera to RBD\textsubscript{TRIP} was paralleled
by reduced ability of these antibodies to block binding of ACE2 to the
triple mutant (shown in Fig. 3C). These experiments demonstrate that
human convalescent sera essentially failed to block binding of ACE2 to
RBD\textsubscript{TRIP,} explaining why SARS-CoV-2- induced antibodies
largely fail to neutralize the triple mutant variants.

\textbf{Discussion}

The newly emerging mutant RBDs may affect the affinity for the viral
receptor. In addition, such mutations at the virus-receptor interaction
face may alter the ability of RBD-specific antibodies - induced by
previous infection - to neutralize the mutant viruses. A previous study
showed that serum neutralization is not compromised by N501Y (found in
the strain B.1.1.7) \textsuperscript{10}. In contrast, E484K (found in
B.1.1.7 and in P.1 strains) was associated with reduced
neutralization\textsuperscript{11,12}. Interestingly, studies applying
\emph{in vitro}pressure produced similar mutations as those that
occurred naturally\textsuperscript{13}. When we investigated whether
distinct mutations may affect receptor affinity we found that N501Y
mutation enhanced affinity for the viral receptor ACE2 both as a single
mutation as well as in the triple mutation, while E484K mutation alone
did not affect the interaction with ACE2 .

In addition, such mutations at the virus-receptor interaction interface
may alter the ability of RBD-specific antibodies to recognize and
neutralize the mutant variants. A previous study showed that serum
neutralization is not compromised by N501Y (also found in the strain
B.1.1.7) \textsuperscript{10} \textsuperscript{14}. In contrast, E484K
(found B.1.1.7 and in P.1 strains) was associated with reduced
neutralization \textsuperscript{11,12}. Interestingly, studies
applying\emph{in vitro} pressure produced similar mutations as those
that occurred naturally \textsuperscript{13}. In this study we showed
that convalescent sera has reduced ability to recognize
RDB\textsubscript{TRIP} variants explaining why the mutant SARS-CoV-2
strain P.1 is more infectious \textsuperscript{15} and less susceptible
to neutralization by antibodies induced with RBD\textsubscript{WT}.

In summary, our data demonstrate that distinct mutations may affect
receptor affinity which likely affects viral infectivity versus
recognition by convalescent sera which likely affects neutralization.
These observations may shed light on the potential origin of the viral
mutants. The variant with the mutation N501Y shows enhanced affinity but
almost normal recognition by convalescent antibodies. This indicates
that this variant spread largely by increased infectivity while
recognition by antibodies of previously infected individuals was less
relevant, a phenotype consistent with the epidemiology in the UK, where
overall infection rates remain relatively low, rendering the previously
infected individuals a relatively unimportant source of viral
spread\textsuperscript{16}. In contrast, the triple mutant variant shows
enhanced infectivity and escape from antibody-recognition. It may
therefore not be a coincidence that this variant originated in Manaus, a
region in Brazil previously seen to have seroprevalence of
\textgreater{}80\%, forcing the virus to escape immunity for further
spreading \textsuperscript{17}.

\textbf{References}

1. Choudhary S, Sreenivasulu K, Mitra P, Misra S, Sharma P. Role of
Genetic Variants and Gene Expression in the Susceptibility and Severity
of COVID-19. \emph{Ann Lab Med.} 2021;41(2):129-138.

2. Bontempi E. The europe second wave of COVID-19 infection and the
Italy ''strange'' situation. \emph{Environ Res.} 2021;193:110476.

3. Bakhshandeh B, Sorboni SG, Javanmard AR, et al. Variants in ACE2;
potential influences on virus infection and COVID-19
severity.\emph{Infect Genet Evol.} 2021;90:104773.

4. Zhao S, Lou J, Cao L, et al. Quantifying the transmission advantage
associated with N501Y substitution of SARS-CoV-2 in the United Kingdom:
An early data-driven analysis. \emph{J Travel Med.} 2021.

5. Yi C, Sun X, Ye J, et al. Key residues of the receptor binding motif
in the spike protein of SARS-CoV-2 that interact with ACE2 and
neutralizing antibodies. \emph{Cell Mol Immunol.} 2020;17(6):621-630.

6. Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional
properties of SARS-CoV-2 spike protein: potential antivirus drug
development for COVID-19. \emph{Acta Pharmacol
Sin.}2020;41(9):1141-1149.

7. Bachmann MF, Zinkernagel RM. The influence of virus structure on
antibody responses and virus serotype formation. \emph{Immunol
Today.}1996;17(12):553-558.

8. Faria NR, Mellan TA, Whittaker C, et al. Genomics and epidemiology of
a novel SARS-CoV-2 lineage in Manaus, Brazil. \emph{medRxiv.} 2021.

9. Petersen RL. Strategies Using Bio-Layer Interferometry Biosensor
Technology for Vaccine Research and Development. \emph{Biosensors
(Basel).} 2017;7(4).

10. Xie X, Liu Y, Liu J, et al. Neutralization of SARS-CoV-2 spike 69/70
deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited
sera.\emph{Nat Med.} 2021.

11. Wibmer CK, Ayres F, Hermanus T, et al. SARS-CoV-2 501Y.V2 escapes
neutralization by South African COVID-19 donor plasma.
\emph{bioRxiv.}2021.

12. Greaney AJ, Starr TN, Gilchuk P, et al. Complete Mapping of
Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape
Antibody Recognition. \emph{Cell Host Microbe.} 2021;29(1):44-57 e49.

13. Andreano E, Piccini G, Licastro D, et al. SARS-CoV-2 escape in vitro
from a highly neutralizing COVID-19 convalescent plasma.
\emph{bioRxiv.}2020.

14. Rathnasinghe R, Jangra S, Cupic A, et al. The N501Y mutation in
SARS-CoV-2 spike leads to morbidity in obese and aged mice and is
neutralized by convalescent and post-vaccination human
sera.\emph{medRxiv.} 2021.

15. Corum J, Zimmer C. Coronavirus variants and mutations. \emph{The New
York Times.} March 22, 2021, 2021.

16. News B. Coronavirus infection levels continue to drop in the UK. In.
5 March ed2021.

17. Candido DS, Claro IM, de Jesus JG, et al. Evolution and epidemic
spread of SARS-CoV-2 in Brazil. \emph{Science.}2020;369(6508):1255-1260.

\textbf{Table 1} Kinetic parameters of RBD\textsubscript{WT} and mutated
RBDs calculated by BLI\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Analyte} & \textbf{K\textsubscript{D} {[}M{]}} &
\textbf{k\textsubscript{on}
{[}M\textsuperscript{-1}s\textsuperscript{-1}{]}} &
\textbf{k\textsubscript{off} {[}s\textsuperscript{-1}{]}}\tabularnewline
\midrule
\endhead
RBD\textsubscript{WT} & 20.5 x 10\textsuperscript{-9} & 1.34 x
10\textsuperscript{5} & 2.91 x 10\textsuperscript{-3}\tabularnewline
RBD\textsubscript{TRIP} & 10.3 x 10\textsuperscript{-9} & 1.69 x
10\textsuperscript{5} & 1.75 x 10\textsuperscript{-3}\tabularnewline
RBD\textsubscript{N501Y} & 6.2 x 10\textsuperscript{-9} & 1.69 x
10\textsuperscript{5} & 1.85 x 10\textsuperscript{-3}\tabularnewline
RBD\textsubscript{E484K} & 19.7 x 10\textsuperscript{-9} & 1.64 x
10\textsuperscript{5} & 3.24 x 10\textsuperscript{-3}\tabularnewline
RBD\textsubscript{K417N} & ND* & ND* & ND*\tabularnewline
\bottomrule
\end{longtable}

*ND = not determined

\textbf{Figure Legends}

\textbf{Fig. 1} Key mutations of variants B.1.351 and P1 fall on the
interface between the RBD and ACE2. (left) S monomer (purple ribbon)
bound to ACE2 ectodomain (orange surface). In detail, the positions of
residues K417, E484, and N501 (blue sticks) are highlighted. Mutations
E484K and N501K occur on the RBM segments (dark purple ribbon), while
K417N occurs on helix \selectlanguage{greek}α\selectlanguage{english}4 of RBD. From PDB files 6ACG and 6M0J.

\textbf{Fig. 2} Binding kinetics of RBD\textsubscript{WT} (A),
RBD\textsubscript{TRIP} (B), RBD\textsubscript{N501Y} (C),
RBD\textsubscript{E484K} (D), and RBD\textsubscript{K417N} (E) to hACE2.
In all assays, both association and dissociation were performed in 300
s. The resulting k\textsubscript{on} and k\textsubscript{off} values
from each condition are compared in panel (F).

\textbf{Fig. 3} Sera of COVID-19 convalescent patients bind less to
RBD\textsubscript{WT} and mutated RBDs (RBD\textsubscript{TRIP},
RBD\textsubscript{N501Y}, RBD\textsubscript{E484K}, and
RBD\textsubscript{K417N}). A) Binding of sera from 12 COVID-19
convalescent patients and one COVID negative individual to
RBD\textsubscript{WT} and mutated RBDs was determined by ELISA. Endpoint
titres (EPT) (dilution factor) individuals are shown as dots. Direct
binding of sera to RBD\textsubscript{WT} and mutated RBDs (B) and
competitive inhibition of ACE2-mFc interaction to
RBD\textsubscript{WT}and mutated RBD (C) were assessed by BLI. The same
sera (dilution 1:20) for each individual was usual. p[?]0.05 (*), p[?]0.01
(**), p[?]0.005 (***), p[?]0.001 (****).

\textbf{Figure 1}\selectlanguage{english}
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\textbf{Figure 2}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}



\textbf{Figure 3}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
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