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\begin{document}

\title{Association of Toll-like receptor (TLR) 3 \& 9 genes' polymorphism with
Hepatitis C virus-specific cell-mediated immunity outcome among Egyptian
healthcare workers}



\author[1]{Sayed Abdelwahab}%
\author[2]{Shaimaa Hamdy}%
\author[3,4]{Ahmed Osman}%
\author[5]{Zainab Zakaria}%
\author[6]{Iman Galal}%
\author[7]{Maha Sobhy}%
\author[8]{Mohamed Hashem}%
\author[9]{Walaa Allam}%
\author[10]{Mohamed Abdel-Samiee}%
\author[10]{Eman Rewisha}%
\author[10]{Imam Waked}%
\affil[1]{Minia University Faculty of Medicine}%
\affil[2]{The Egyptian Holding Company for Biological Products and Vaccines }%
\affil[3]{Cairo University}%
\affil[4]{Cairo University Faculty of Science}%
\affil[5]{Heliopolis University for Sustainable Development}%
\affil[6]{The Egyptian Holding Company for Biological Products and Vaccines (VACSERA),}%
\affil[7]{ The Egyptian Holding Company for Biological Products and Vaccines (VACSERA)}%
\affil[8]{University of Maryland School of Medicine}%
\affil[9]{Zewail City of Science and Technology - Zewail City Campus}%
\affil[10]{Menoufia University National Liver Institute}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Immune response variations could define successful resistance to
Hepatitis C Virus (HCV) infection. Toll-like receptors (TLR)-3 are
innate detectors of dsRNA viruses while bacterial and viral unmethylated
CpG motifs are recognized by TLR9. We previously reported that
TLR3.rs3775290 ``CC'' genotype was associated with HCV chronicity, while
TLR9 gene played no major role in this infection. This study identified
the role of TLR3.rs3775290 (c.1377C/T), TLR9.rs5743836 (-1237T-C) and
TLR9.rs352140 (G2848A) gene polymorphisms in predicting the outcome of
HCV-specific cell-mediated immunity (CMI) among Egyptian healthcare
workers (HCWs) and patients. We enrolled 546 subjects (409 HCWs and 137
patients) divided into four groups. Group1: 265 seronegative, aviraemic
subjects; group2: 25 seronegative, viraemic subjects; group3: 87
subjects with spontaneously resolved HCV infection; and group4: 169
chronic HCV HCWs and patients. All subjects were genotyped by
PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for the
TLR3.rs3775290, TLR9.rs5743836 and TLR9.rs352140 SNPs. We, also,
quantified HCV-specific CMI in 265 HCWs distributed among the four
groups using an interferon gamma (IFN-\selectlanguage{greek}γ\selectlanguage{english}) enzyme-linked immunospot
(ELISpot) assay in response to nine HCV genotype 4a overlapping 15mer
peptide pools covering the whole viral genome. No statistically
significant difference was found between CMI responding subjects with
different HCV states and TLR3.rs3775290 genotype or TLR9.rs352140.
However, there was a significant relationship between the outcome of the
HCV-specific CMI and the TLR9.rs5743836 genotype among the responding
subjects (p=0.005) and the chronic HCV patients (p=0.044). In
conclusion, TLR9.rs5743836 SNP; but not TLR3.rs3775290 or TLR9.rs352140
genotypes; could predict the outcome of HCV-specific CMI responses among
genotype-4-infected Egyptians.%
\end{abstract}%



\sloppy


\textbf{Association of Toll-like receptor (TLR) 3 \& 9 genes'
polymorphism with Hepatitis C virus-specific cell-mediated immunity
outcome among Egyptian healthcare workers}

\textbf{Running title} : TLR3\&9 SNPs in HCV-infected Egyptians

Sayed F. Abdelwahab\textsuperscript{1,2,3,\#,*}, Shaimaa
Hamdy\textsuperscript{1,4,\#}, Ahmed M. Osman\textsuperscript{4}, Zainab
A. Zakaria\textsuperscript{1,5}, Iman Galal\textsuperscript{1}, Maha
Sobhy\textsuperscript{1}, Mohamed Hashem\textsuperscript{1,6}, Walaa R.
Allam\textsuperscript{1,7}, Mohamed Abdel-Samiee\textsuperscript{8},
Eman Rewisha\textsuperscript{8}, Imam Waked\textsuperscript{8}

\emph{\textsuperscript{1}The Egyptian Holding Company for Biological
Products and Vaccines (VACSERA), 51 Wizaret El-Zeraa St., Agouza, Giza
22311, Egypt,\textsuperscript{2}Division of Microbiology, Department of
Pharmaceutics and Industrial Pharmacy, Taif College of Pharmacy,
Al-Haweiah, PO Box 888, Taif 21974,Kingdom of Saudi
Arabia;\textsuperscript{3}Department of Microbiology and Immunology,
Faculty of Medicine, Minia University, Minia 61511,
Egypt,\textsuperscript{4}Department of Zoology, Faculty of Science,
Cairo University, Giza 12613, Egypt, \textsuperscript{5}Biomedical
Research Laboratory, Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt, \textsuperscript{6}Department of
Epidemiology and Public Health, University of Maryland School of
Medicine, Baltimore, MD, USA, \textsuperscript{7}Centre for Genomics,
University of Science and Technology, Zewail City of Science and
Technology, Giza, Egypt,\textsuperscript{8}Department of Hepatology and
Gastroenterology, National Liver Institute, Menoufia University,
Menoufia 32511, Egypt}

\textsuperscript{\#} Contributed equally to this work

\textsuperscript{*}\emph{Corresponding Author} :

Sayed F. Abdelwahab, PhD

Professor, Department of Microbiology and Immunology, Faculty of
Medicine, Minia University, Minia 61511, Egypt and Department of
Microbiology, Taif Faculty of Pharmacy, Al-Haweiah, PO Box 888, Taif
21974, KSA

Email: icpminia@yahoo.com

Telephone:~+20-10-9000-8885; +966-54-122-1361, Fax: +20-86-234-2813

ORCID: http://orcid.org/0000-0002-9636-7485

\textbf{Key words:} CMI, Cell-mediated immunity; HCV; Healthcare
workers; SNP; TLR3; TLR9.

\emph{Abbreviations}\textbf{:} CMI, Cell-mediated immunity; HCV,
hepatitis C virus; HCWs, healthcare workers; TLR, toll-like receptor

\textbf{Abstract word count:} 249; \textbf{Text word count}(Introduction
to end of discussion): 3713



\textbf{Summary:}

Immune response variations could define successful resistance to
Hepatitis C Virus (HCV) infection. Toll-like receptors (TLR)-3 are
innate detectors of dsRNA viruses while bacterial and viral unmethylated
CpG motifs are recognized by TLR9. We previously reported that
TLR3.rs3775290 ``CC'' genotype was associated with HCV chronicity, while
TLR9 gene played no major role in this infection. This study identified
the role of TLR3.rs3775290 (c.1377C/T), TLR9.rs5743836 (-1237T-C) and
TLR9.rs352140 (G2848A) gene polymorphisms in predicting the outcome of
HCV-specific cell-mediated immunity (CMI) among Egyptian healthcare
workers (HCWs) and patients. We enrolled 546 subjects (409 HCWs and 137
patients) divided into four groups. Group1: 265 seronegative, aviraemic
subjects; group2: 25 seronegative, \emph{viraemic} subjects; group3: 87
subjects with spontaneously resolved HCV infection; and group4: 169
chronic HCV HCWs and patients. All subjects were genotyped by
PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for the
TLR3.rs3775290, TLR9.rs5743836 and TLR9.rs352140 SNPs. We, also,
quantified HCV-specific CMI in 265 HCWs distributed among the four
groups using an interferon gamma (IFN-\selectlanguage{greek}γ\selectlanguage{english}) enzyme-linked immunospot
(ELISpot) assay in response to nine HCV genotype 4a overlapping 15mer
peptide pools covering the whole viral genome. No statistically
significant difference was found between CMI responding subjects with
different HCV states and TLR3.rs3775290 genotype or TLR9.rs352140.
However, there was a significant relationship between the outcome of the
HCV-specific CMI and the TLR9.rs5743836 genotype among the responding
subjects (\emph{p} =0.005) and the chronic HCV patients (\emph{p}
=0.044). In conclusion, TLR9.rs5743836 SNP; but not TLR3.rs3775290 or
TLR9.rs352140 genotypes; could predict the outcome of HCV-specific CMI
responses among genotype-4-infected Egyptians.

\textbf{Introduction}

Immune response variations may help define successful resistance to
Hepatitis C virus (HCV) infection particularly among seronegative
healthcare workers, reviewed elsewhere {[}1{]}. Toll-like receptors
(TLR)-3 are innate detectors of dsRNA of viruses while TLR9 recognizes
bacterial and viral unmethylated CpG motifs. HCV virions bind to the
cell surface and get into the cell through receptor-mediated
endocytosis. The structure of HCV with different parts are recognized by
different TLRs. The core and non-structural (NS) proteins are vital
sequences recognized by pattern recognition receptors (PRRs), including
TLRs. They are, also, important inhibitors of TLR signalling {[}2, 3{]}.
HCV core and NS proteins are important pathogen-associated molecular
patterns (PAMPs) for TLR2, TLR3, TLR4, TLR7, 8, and 9. TLR3 is critical
for its antiviral immune actions, as it stimulates non-parenchymal liver
to regulate HCV replication through the synthesis of interferon (IFN)-\selectlanguage{greek}β\selectlanguage{english}
{[}4, 5{]}. In chronic HCV infections, TLR3 mRNA is significantly
increased in monocytes {[}6{]}. An IFN-responsive element has been
identified in the TLR3 gene promoter region and, so, it appears likely
that expression TLR3 is responsive to IFN treatment in HCV infection
{[}7{]}. Myeloid dendritic cells (mDCs) have ordinary operative TLR3 and
are capable of producing interleukin (IL)-12, IL-6, IL-10, IFN-\selectlanguage{greek}γ, \selectlanguage{english}and
tumour necrosis factor (TNF)-\selectlanguage{greek}α \selectlanguage{english}with TLR3 stimulation regardless of HCV
infection {[}8{]}.

HCV viral proteins activate TLR signalling, which plays a significant
role in viral immune clearance. However, HCV can at the same time evade
immune clearance by targeting and ruining TLR signal transduction via
numerous mechanisms. First, HCV intervenes with signalling via the
TIR-domain-containing adapter-inducing IFN\selectlanguage{greek}β/\selectlanguage{english}TANK-binding kinase
(TRIF)/TBK)1-Interferon regulatory factor 3 (IRF3) pathway. The HCV NS3
protein induces degradation of TRIF, while the NS3/4A protein prevents
IRF3 and NF\selectlanguage{greek}κ\selectlanguage{english}B activation by downgrading the amount of TRIF in serum and
by producing cleavage products with dominant-negative action {[}4, 9{]}.
NS3/4A, also, interacts directly with TBK1 to reduce the interaction
between TBK1 and IRF3 and, so inhibit IRF3 activation {[}10{]}. HCV,
also, interferes with the TLR-MyD88 (Myeloid differentiation primary
response 88) pathway through NS5A interaction with MyD88 to prevent
Interleukin-1 receptor associated kinase 1 (IRAK1) recruitment and
cytokine production in response to ligands for TLR2, TLR4, TLR7, and
TLR9 {[}11{]}.

HCV has been shown to regulate the expression of TLR9 through the
transcription factor (Elk-1), which is an essential signal integration
point between T-Cell Receptor (TCR) and CD28 in T helper 1 (Th1) cell
activation {[}12{]}. HCV, also, impedes the production of IFN-\selectlanguage{greek}α \selectlanguage{english}and
IFN-\selectlanguage{greek}β \selectlanguage{english}through TLR9 and decreases human leukocyte antigen DR (HLA-DR)
expression by pDCs, linked to diminished activation of na\selectlanguage{ngerman}ïve T cells
{[}13{]}. TLR9 signalling in mDCs is unchanged {[}8, 13{]}.
Consequently, it is clear that compartmentalization of the outcomes on
TLR function is a crucial strategy by which HCV can escape immune
surviellance yet can lead to chronic inflammatory hepatic injury and
fibrosis.

We previously showed that TLR3.rs3775290 ``CC'' genotype was associated
with HCV chronicity, while TLR9 gene played no major role in HCV
infection {[}14{]}. This study identified the role of TLR3.rs3775290
(c.1377C/T), TLR9.rs5743836 (-1237T\selectlanguage{english}-C) and TLR9.rs352140 (G2848A) single
nucleotide polymorphisms (SNP) in predicting the outcome of HCV-specific
cell-mediated immunity (CMI) among Egyptian healthcare workers (HCWs)
and patients. We show that TLR9.rs5743836 SNP; but not TLR3.rs3775290 or
TLR9.rs352140 genotypes; could predict the outcome of HCV-specific CMI
responses among genotype-4-infected Egyptians.

\textbf{Subjects and Methods}

\textbf{Study subjects}

Two hundred sixty-five HCWs were selected for the evaluation of IFN-\selectlanguage{greek}γ
\selectlanguage{english}producing T-cells by an Enzyme Linked Immuno-Spot (ELISpot) assay in
response to nine HCV genotype 4a overlapping 15mer peptide pools
covering the whole viral genome. The NLI-Institutional Review Board
(IRB) approved the study protocol, and all subjects had given informed
consent prior to enrolment. The demographic characteristics of the
participants were previously reported {[}14{]}.

\textbf{Laboratory testing}

\textbf{HCV molecular diagnosis and quantification of HCV-RNA}

HCV diagnosis was determined by using the Murex third generation
anti-HCV ELISA assay (version 4.0 USA) that utilizes antigens from the
core, NS3, NS4 and NS5 regions of the virus. All the study subjects were
tested by quantitative real-time RT-PCR (qPCR) for detection of current
HCV infection and quantification of the HCV viral load using QIAamp\selectlanguage{ngerman}®
Viral RNA purification kit (QIAGEN, USA). The amplification step was
conducted using AgPath-ID one-step assay as instructed by the
manufacturer (Applied Biosystems-Life Technologies Corporation, CA).

\textbf{HCV-genotyping and genotyping for TLR3.rs3775290 (c.1377C/T)
SNP}

PCR-RFLP was used for HCV genotyping after reverse transcription as
described {[}15{]}.

PCR-RFLP was, also, used for the identification of the TLR3.rs3775290
(c.1377 C/T) gene SNPs. PCR was conducted in a total of 25 \selectlanguage{greek}μ\selectlanguage{english}l reaction
volume with 20 pmol of each of the forward and reverse primers as
described {[}16{]}.

\textbf{Genotyping for TLR9.rs5743836 (-1237T-C) and TLR9.rs352140
(G2848A) SNPs}

The total reaction volume of TLR9.rs5743836 (-1237TTBC) PCR was 25 \selectlanguage{greek}μ\selectlanguage{english}l
with 20 pmol of each of the forward and reverse primers as described
{[}17{]}. Also, the total reaction volume of TLR9.rs352140 (G2848A) PCR
was 25 \selectlanguage{greek}μ\selectlanguage{english}l with 20 pmol of each of the forward and reverse primers as
reported {[}16{]}.

\selectlanguage{greek}\textbf{Evaluation of IFN-γ producing cells using ELISPOT assay}\selectlanguage{english}

An ELISPOT assay was used to quantify IFN-\selectlanguage{greek}γ \selectlanguage{english}synthesis by PBMC separated
from the study subjects in response to nine HCV genotype 4a isolate ED43
peptide antigen pools consisting of 15 (15mer) and overlapping by 10
amino acids. These were 600 peptides combined in nine pools
corresponding to all the HCV proteins. These synthetic peptides were
specially manufactured by Mimotopes (Australia). About 15ml of blood was
collected in EDTA vacutainer tubes (Becton Dickinson Biosciences, NJ,
USA). PBMC were separated from whole blood using Ficoll-Hypaque density
gradient centrifugation. Cellular viability was determined by trypan
blue exclusion method. Briefly, 2x10\textsuperscript{5} PBMC
(200\selectlanguage{greek}µ\selectlanguage{english}l/well) were incubated in triplicate cultures in the ELISpot plates
(Whatman Unifilter, USA) coated with anti-human IFN\selectlanguage{greek}γ \selectlanguage{english}antibody and
incubated for \textasciitilde{}16 hours with or without recombinant HCV
antigens at 3\selectlanguage{greek}μ\selectlanguage{english}g/ml of each single peptide in complete RMPI-1640 medium.
A medium containing DMSO alone and 0.1\selectlanguage{greek}μ\selectlanguage{english}g / ml of SEB (or other
polyclonal stimuli) represented the negative and positive controls,
respectively. The assay was developed at the end of the incubation
period until the spots appeared. The wells were, then, rinsed with tap
water to stop the reaction. The number of spots/well was calculated
using an automated ELISpot reader (Cellular Technology Ltd., Cleveland,
USA) as reported {[}18{]}. The mean number of spot forming cells (SFC)
in control wells were subtracted from the mean number of
peptide-stimulated wells to correct for background cytokine production
and are expressed as SFC/million PBMC. A positive antigen-specific HCV
response was considered if the SFC in the presence of antigen was at
least three times the number of SFCs in the medium control and
\textgreater{} 55 SFC / million PBMC were present, as previously
reported {[}19{]}.

\textbf{Statistical analysis}

Allele and genotype frequencies of each SNP were calculated by direct
counting. Genotype and allele frequencies between groups were compared
using the \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} test and Fisher's exact test when
appropriate. For allelic associations, p values with Yates correction
and odds ratio (OR) with 95\% confidence intervals (CI) were calculated
using Statcalc program (Epiinfo version 6.0.4, CDC, Atlanta, GA).
Pair-wise LD (D', r\textsuperscript{2}) was estimated by use of
Haploview software version 4.2. For categorical data, chi-square tests
were performed, while the student's \emph{t} -test (or Mann Whitney U
test where appropriate) was performed to compare the continuous data.
Differences in mean with a \emph{p} value \textless{}0.05 were deemed
significant. Standard errors of the means (and/or deviations) were also
calculated

\textbf{Results}

\selectlanguage{greek}\textbf{Relationship between the TLR3.rs3775290 (c.1377C/T) genotype and
the HCV-specific IFN-γ ELISpot response}\selectlanguage{english}

All subjects enrolled into the study were tested for IFN-\selectlanguage{greek}γ \selectlanguage{english}production by
PBMCs using ELISpot assay to evaluate CMI response against nine HCV
genotype 4a isolate ED43 peptide antigen pools composed of 15 (15mer)
and overlapping by 10 amino acids. Only 140 (53\%), 20 (80\%), 35 (40\%)
and 70 (41.4\%) samples had valid CMI data among the four study groups
(seronegative, aviraemic subjects; seronegative,
\emph{viraemic}subjects; spontaneously resolved subjects and chronic HCV
patients groups, respectively).

There was no association between the results of the HCV-specific immune
response and the TLR3.rs3775290 genotype among the 263 HCWs with valid
CMI responses and TLR3.rs3775290 genotyping tests (\emph{p=} 0.222).
Also, there was a significantly (\emph{p} =0.004) higher proportion of
the CC genotype (73.3\%) in responding subjects than in non-responding
seronegative, aviraemic individuals (65.1\%). In addition, there was a
differential distribution of the three TLR3.rs3775290 genotypes among
the total study participants with and without HCV-specific CMI (Table
1).

The average totals (\selectlanguage{ngerman}±SEM) of IFN-\selectlanguage{greek}γ \selectlanguage{english}responses measured in SFC per million
PBMCs in the \emph{responding} total subjects were 455\selectlanguage{ngerman}±90, 507±122, and
317±45 with the TLR3.rs3775290 genotype CC (n=37), CT (n=16) and TT
(n=6). On the other hand, among the \emph{non-responding} subjects, the
average totals (±SEM) of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC per million PBMCs in total subjects
were 29\selectlanguage{ngerman}±5, 23±5, and 49±19 with the TLR3.rs3775290 genotype CC (n=130),
CT (n=65) and TT (n=9), respectively. There was no significant
difference between the responding total subjects with different
TLR3.rs3775290 genotypes (\emph{p} =0.89; Figure 1A).

For the \emph{seronegative, aviraemic} subjects, the average totals of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
responding subjects were 404±97.7, 1058±807.5, and 249±21.9, among those
with the TLR3.rs3775290 CC genotype (n=22), CT (n=4) and TT (n=4)
genotypes, respectively. Differences were not statistically significant
(\emph{p} =0.88). On the other hand, the average totals of IFN-\selectlanguage{greek}γ
\selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}± SEM) of the non-responding subjects
were 26.7±5.7, 16.7±4, and 13.3±13.3, among those with the
TLR3.rs3775290 CC (n=71), CT (n=36) and TT (n=2) genotypes, respectively
(Figure 1B).

Regarding \emph{seronegative} , \emph{viraemic} subjects, the average
totals (±SEM) of HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs
for\emph{responding} subjects were 657\selectlanguage{ngerman}±443, and 163, among those with
the TLR3.rs3775290 genotype CC (n=3) and CT (n=1) genotypes,
respectively. There were no responding subjects with the TT genotype.
Differences were not statistically significant (\emph{p} =0.18). On the
other hand, the average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs
(\selectlanguage{ngerman}±SEM) of the non-responding subjects were 42±22, 30±11 and 8±8, among
those with the TLR3.rs3775290 CC (n=9), CT (n=5) and TT (n=2) genotypes,
respectively (Figure 1C).

For \emph{spontaneously resolved subjects,} the average totals of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of
the\emph{responding} subjects were 793±605.8, and 325±121, among those
with the TLR3.rs3775290 CC (n=2), CT (n=5) genotypes, respectively. No
TT genotype was recorded in this category. Differences were not
statistically significant (\emph{p} =0.698). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
non-responding subjects were 51±21, 15±12 and 58, among those with the
TLR3.rs3775290 CC (n=14), CT (n=12) and TT (n=1) genotypes, respectively
(Figure 1D).

For \emph{chronic HCV patients} , the average totals of HCV-specific
IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
\emph{responding}subjects were 439±214, 349 ±181, and 453±9, among those
with the TLR3.rs3775290 CC (n=10), CT (n=6) and TT (n=2) genotypes,
respectively. Differences were not statistically significant (\emph{p}
=0.228). On the other hand, the average totals of IFN-\selectlanguage{greek}γ
\selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the non-responding chronic HCV
patients were 26±8, 38±13, and 84±36, among those with the
TLR3.rs3775290 CC (n=36), CT (n=13) and TT (n=4) genotypes, respectively
(Figure 1E).

\selectlanguage{english}\textbf{Relationship between the TLR9.rs5743836 (-1237T-C) genotype and
the HCV-specific IFNγ ELISpot response}

There was no association between the results of the HCV-specific CMI
response and the TLR9.rs5743836 genotype among the 265 HCWs with valid
CMI responses and TLR9.rs5743836 genotyping tests (Table 2,\emph{p=}
0.12). However, there was a statistically significant (\emph{p} =0.019)
lower proportion of the TT genotype in responding spontaneously resolved
individuals compared to non-responding spontaneously resolved subjects
(20\% Vs 80\%, respectively, Table 2).

The average totals (\selectlanguage{ngerman}±SEM) of IFN-\selectlanguage{greek}γ \selectlanguage{english}responses measured in SFC per million
PBMCs in the responding total subjects were 624\selectlanguage{ngerman}±125, and 381±100 with
the TLR9.rs5743836 CT (n=18) and TT (n=41) genotypes, respectively.
There were no responding subjects with the CC genotype. Additionally,
among the non-responding subjects, the average totals (±SEM) of IFN-\selectlanguage{greek}γ
\selectlanguage{english}SFC per million PBMCs in total subjects were 40\selectlanguage{ngerman}±23, 40±10, and 26±4,
among those with the TLR9.rs5743836 CC (n=4), CT (n=40) and TT (n=162),
respectively. There was a significant difference between the responding
total subjects with different TLR9.rs5743836 genotypes (\emph{p} =0.005;
Figure 2A).

For the \emph{seronegative, aviraemic} subjects, the average totals of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of
the\emph{responding} subjects were 617±192, and 398±164, among those
with the TLR9.rs5743836 CT (n=10) and TT (n=20) genotypes, respectively.
There were no responding subjects with the CC genotype. Differences were
not statistically significant (\emph{p} =0.159). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
non-responding seronegative, aviraemic subjects were 32±31, 44±14, and
18±3, among those with the TLR9.rs5743836 CC (n=3), CT (n=25) and TT
(n=82) genotypes, respectively (Figure 2B).

For \emph{seronegative} , \emph{viraemic} subjects, the average totals
of HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of
the\emph{responding} subjects were 238, and 632±455, among those with
the TLR9.rs5743836 CT (n=1) and TT (n=3) genotypes, respectively. There
were no responding subjects with the CC genotype. Differences were not
statistically significant (\emph{p} =0.654). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
non-responding subjects were 65, 18±14, and 38±18, among those with the
TLR9.rs5743836 CC (n=1), CT (n=4) and TT (n=11) genotypes, respectively
(Figure 2C).

For \emph{spontaneously resolved subjects} , the average total
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±) of
the\emph{responding} subjects was 795±345, and 206±49, among those with
the TLR9.rs5743836 CT (n=3) and TT (n=4) genotypes, respectively. There
were no responding subjects with the CC genotype. Differences were not
statistically significant (\emph{p} =0.077). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}± )
of\emph{non-responding subjects} were 1, and 36±12, among those with the
TLR9.rs5743836 CT (n=1) and TT (n=27) genotypes, respectively. Also,
there were no subjects with the CC genotype among non-responding
subjects (Figure 2D).

For \emph{chronic HCV patients} , the average totals of HCV-specific
IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}± SEM) of the
\emph{responding}subjects were 610±212, and 354±156, among those with
the TLR9.rs5743836, CT (n=4) and TT (n=14) genotypes, respectively.
There were no responding subjects with the CC genotype. Differences were
statistically significant (\emph{p} =0.044). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
\emph{non-responding chronic} HCV subjects were 43±19 and 32±8, among
those with the TLR9.rs5743836 CT (n=10) and TT (n=42) genotypes,
respectively. There were no subjects with the CC genotype among the
non-responding chronic patients (Figure 2E).

\selectlanguage{greek}\textbf{Relationship between the TLR9.rs352140 (G2848A) genotype and the
HCV-specific IFN-γ ELISpot response}\selectlanguage{english}

There was no association between the results of the HCV-specific CMI
response and the TLR9.rs352140 (G2848A) genotype among the 258 HCWs with
valid CMI responses and TLR9.rs352140 genotyping tests (\emph{p=}
0.356). Also, there was no significant difference (\emph{p=} 0.299) in
the proportion of the GG genotype in responding (46.4 \%) and in the
non-responding seronegative aviraemic subjects (31.4 \%; Table 3).

The average totals (\selectlanguage{ngerman}±SEM) of IFN-\selectlanguage{greek}γ \selectlanguage{english}responses measured in SFC per million
PBMCs in the responding total subjects were 587\selectlanguage{ngerman}±176, 390±85, and 327±45,
among those with the TLR9.rs352140 GG (n=24), GA (n=23) and AA (n=10)
genotypes, respectively. On the other hand, among the non-responding
subjects, the average totals (±SEM) of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC per million PBMCs in
the total subjects were 30\selectlanguage{ngerman}±6, 31±6, and 26±7, among those with the
TLR9.rs352140 (G2848A) GG (n=66), GA (n=86) and AA (n=49) genotypes,
respectively. There was no significant difference between the responders
of the total subjects with the TLR9.rs352140 genotypes (\emph{p} =0.652;
Figure 3A).

For the \emph{seronegative, aviraemic} subjects, the average totals of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of
the\emph{responding} subjects was 672±278.8, 307±86, and 357±68, among
those with the TLR9.rs352140 GG (n=13), GA (n=10) and AA (n=5)
genotypes, respectively. Differences were not statistically significant
(\emph{p} =0.594). On the other hand, the average totals of IFN-\selectlanguage{greek}γ
\selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) among the \emph{non-responding
seronegative, aviraemic} subjects were 23±7, 31±8, and 17±5, among those
with the TLR9.rs352140 GG (n=33), GA (n=43) and AA (n=29) genotypes,
respectively (Figure 3B).

For \emph{seronegative,} \emph{viraemic} subjects, the average totals of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the
responding subjects were 214±24.2, and 853±689, among those with the
TLR9.rs352140 GG (n=2) and GA (n=2) genotypes, respectively. There were
no responding subjects with the AA genotype. Differences were not
statistically significant (\emph{p} =1.000). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) were
12±2.22, 30±10.4, and 60±47.6, among the \emph{non-responding
seronegative} , \emph{viraemic} subjects with the TLR9.rs352140 (G2848A)
GG (n=3), GA (n=9) and AA (n=4) genotypes, respectively (Figure 3C).

For \emph{spontaneously resolved subjects} , the average totals of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of
the\emph{responding} subjects were 244±48.2, and 619±301, among those
with the TLR9.rs352140 GG (n=3) and GA (n=4) genotypes, respectively.
There were no responding subjects with the AA genotype. Differences were
not statistically significant (\emph{p} =1.000). On the other hand, the
average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) were
26±14.1, 60±26.9, and 10±10, among the \emph{non-responding
spontaneously resolved} \emph{subjects} with the TLR9.rs352140 GG
(n=11), GA (n=10) and AA (n=7) genotypes, respectively (Figure 3D).

For \emph{chronic HCV patients} , the average totals of HCV-specific
IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs (\selectlanguage{ngerman}±SEM) of the responding subjects
were 698±357.2, 245±51.5, and 296±57.8, among those with the
TLR9.rs352140 GG (n=6), GA (n=7) and AA (n=5) genotypes, respectively.
Differences were not statistically significant (\emph{p} =0.775). On the
other hand, the average totals of IFN-\selectlanguage{greek}γ \selectlanguage{english}SFC/10\textsuperscript{6} PBMCs
(\selectlanguage{ngerman}±SEM) of \emph{non-responding chronic HCV subjects} were 46±13.8,
18±7.9, and 50.9±23.1, among those with the TLR9.rs352140 GG (n=19), GA
(n=24) and AA (n=9) genotypes, respectively (Figure 3E).

\textbf{Discussion}

In this study, there was no statistically significant difference between
CMI responding individuals in Egyptian HCWs with diverse HCV states with
different TLR3.rs3775290 (c.1377C/T) genotypes. Also, there was no
association between the results of the HCV-specific CMI response and the
TLR3.rs3775290 (c.1377C/T) genotype among the 263 subjects tested. This
agrees with another report {[}20{]}. TLR3's function was investigated in
four different viral models of infection in TLR3-/- mice, including
lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus
(VSV), Murine Cytomegalovirus (MCMV), and reovirus. The researchers
found that TLR3 is not always required for effective antiviral responses
to be generated, as the absence of TLR3 did not alter either viral
pathogenesis or the generation of adaptive antiviral responses to these
viruses by the host. Remarkably, intracellular transduction of poly I: C
signals begins TLR3-independent activation of an IFN response,
therefore, restricting the role of TLR3 in the IFN pathway {[}21{]}.
Other reports {[}22, 23{]} indicate that HCV may use TLR3 pathway to
escape immune scrutiny through HCV NS3/4A protease-mediated cleavage of
the TLR3 adaptor protein TRIF. Also, HCV NS3/4A impedes RIG-I, which is
a key factor in TRIF-independent signalling {[}21{]}. In addition, it
was suggested that several polymorphisms that alter TLR3 amino acids
initiate resulting protein changes and that gene expression may be
down-regulated while TLR3 activities required for proper signalling may
be reduced. {[}24{]}.~Lower TLR3 activity results in inability to
identify the invading microorganisms and consequently insufficient
immune responses. This increases the risk of infections and infectious
diseases {[}25{]}.

Our results provide strong indirect evidence that TLR9 could play a
greater role than previously expected in HCV infection. We identified an
association between the TLR9.rs5743836 polymorphism within the TLR9 gene
and the natural history of HCV infection. In this study, there was a
strong association between the results of the HCV-specific CMI response
and the TLR9.rs5743836 (-1237T\selectlanguage{english}-C) genotype among the 265 subjects with
valid CMI responses in total responders of individual HCWs
(\emph{p}=0.005). Also, there was a statistically significant difference
between responding individuals in chronic HCV patients with distinct
TLR9.rs5743836 genotypes (\emph{p} = 0.044).

We show that the TLR9-1237T allele~was significantly~associated~with
HCV-specific CMI response. In this regard, the TT genotype is
transcribed more effectively than the CC genotype as reported by others
{[}26{]} who showed that the wild-type construct stimulates higher
transcriptional activity compared to the CC allele. The data showed that
individuals with the ``favourable'' TT allele would have HCV-specific
CMI response when compared to those with the ``unfavourable'' CC allele.
Also, several reports found that the mutant allele ``T'' imparts the
immunity against the various pathogens. However, statistically
inconsiderable, the mutant allele was declared to be linked with the
depressed microbial load in Africans {[}27-29{]}. In addition, the
mutant allele T manipulate immunity against establishing the infections,
which is convincing that the TLR9 gene had bear the influence of genetic
assortment to cope with the infections {[}30{]}. Functional analyses of
the impact of TLR9 polymorphisms on basal promoter activity, on the
other hand, revealed that the rs5743836 SNP causes higher gene
expression than the wild type promoter. The findings of others {[}31{]}
could explain the increased promoter activity of TLR9.rs573836. That
study {[}31{]} showed that the variant C allele at rs5743836 establishes
a potential NF-\selectlanguage{greek}κ\selectlanguage{english}B-binding site that increases the gene's transcriptional
activity . The presence of this extra putative NF-\selectlanguage{greek}κ\selectlanguage{english}B-binding site
promotes transcription of TLR9 more effectively than the wild type
TLR9.rs5743836 T sequence in response to different stimuli. This finding
agrees with another report {[}32{]}, where TLR9 promoter SNPs were
associated with the natural course of HCV infection and exhibit higher
transcriptional activities and imply the role of the DNA sensor TLR9 in
natural immunity against HCV. Our data are analogous to other studies
{[}1, 33{]}, where the rs5743836 TT variant was reported to have a
greater promoter activity than the CC genotype. That study suggested
that this activity could lead to increased pro-inflammatory cytokine
production during malaria infection, leading to successful malaria
control and elimination {[}33{]}.

There was no association between the results of the HCV-specific immune
response and the TLR9.rs352140 (G2848A) genotype among the 258 Egyptian
HCWs with valid CMI responses and TLR9.rs352140 genotyping. There have
been no studies to date that indicate an association between
TLR9.rs352140 (G2848A) and HCV infection. The region around 2848 is the
major coding region of the TLR9 protein {[}34, 35{]} and a TLR9 2848 GA
genotype polymorphism has been reported at the transcriptional level to
reduce TLR9 expression {[}35, 36{]}. Down regulated TLR9 could reduce
the functions of the innate immune response against HCV infection. Our
results are related to another study that indicates that the TLR9 gene
may play a role in cervical carcinogenesis but has a minor (or no) role
in tumour progression where individuals with the favourable'' GG allele
were reported to be more frequent than the ``unfavourable'' AA allele
{[}37{]}. In addition, a recent meta-analysis showed that there is no
relationship between TLR9.rs352140 (G2848A) and cervical cancer
vulnerability {[}38{]}.~ Our findings suggest that the TLR9.rs352140
(G2848A) genotype does not affect the natural course of HCV infection.

In conclusion, this study shows that TLR9.rs5743836 SNP; but not
TLR3.rs3775290 or TLR9.rs352140 genotypes; could predict the outcome of
HCV-specific CMI responses among genotype-4-infected Egyptians.

\textbf{Acknowledgement} : This study was supported by the Egyptian
Science and Technology Development Fund (STDF) Contract no. 1664 to S.F.
Abdelwahab.

\textbf{Author contributions} :

Conceptualization and design of experiments: SFA, SH, AO, ZZ, ER, and
IW; Data collection, experimentation, analysis, and investigation: SH,
SFA, MA, ZZ, IG, MS, WA; Funding acquisition and resources: SFA, ER, and
IW; Writing-original draft (SH and SFA); Writing-review and editing
(SFA, AO, MS, MH, WA, MA, ER, and IW). All authors approved the final
version of the manuscript.

\emph{Compliance with Ethical Standards} :

\textbf{Conflict of Interest:} None declared.

\textbf{Data Sharing and Data Accessibility:} All data pertinent to this
article are included herein.

\textbf{Research involving human participants and/or animals} :

All procedures performed in studies involving human participants were in
accordance with the ethical standards of the institutional and/or
national research committee and with the 1964 Helsinki declaration and
its later amendments.

\textbf{Informed Consent} : Informed consent was obtained from all
enrolled study participants.

\textbf{Table (1):} Relationship between TLR3.rs3775290 (c.1377C/T) and
HCV-specific cell mediated immune (CMI) response among NLI HCWs in terms
of CMI responders frequencies.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\emph{p}\textbf{-value} & Total & \textbf{TLR3.rs3775290 genotype} &
\textbf{TLR3.rs3775290 genotype} & \textbf{TLR3.rs3775290 genotype} &
\textbf{GROUP} & \textbf{GROUP}\tabularnewline
\midrule
\endhead
& & \textbf{TT} & \textbf{CT} & \textbf{CC} & &\tabularnewline
0.004 & 30 (21.6) & 4 (13.3) & 4 (13.3) & 22 (73.3) & Responder &
\textbf{Seronegative, aviraemic}\tabularnewline
& 109 (78.4) & 2 (1.8) & 36 (33) & 71 (65.1) & Non-responder
&\tabularnewline
0.694 & 4 (20) & 0(0) & 1 (25) & 3 (75) & Responder &
\textbf{Seronegative, viraemic}\tabularnewline
& 16 (80) & 2 (12.5) & 5 (31.3) & 9 (56.3) & Non-responder
&\tabularnewline
0.425 & 7 (20.6) & 0(0) & 5 (71.4) & 2 (28.6) & Responder &
\textbf{Resolved}\tabularnewline
& 27 (79.4) & 1 (3.7) & 12 (44.4) & 14 (51.9) & Non-responder
&\tabularnewline
0.57 & 18 (25.7) & 2 (11.1) & 6 (33.3) & 10 (55.5) & Responder &
\textbf{Chronic}\tabularnewline
& 52 (74.3) & 4 (7.7) & 12 (23.1) & 36 (69.2) & Non-responder
&\tabularnewline
\textbf{0.222} & \textbf{59 (22.3)} & \textbf{6 (10.2)} & \textbf{16
(27.1)} & \textbf{37 (62.7)} & \textbf{Responder} & Total\tabularnewline
& \textbf{204 (77.7)} & \textbf{9 (4.4)} & \textbf{65 (31.9)} &
\textbf{130 (63.7)} & \textbf{Non-responder} &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table (2)} : Relationship between TLR9.rs5743836 (-1237T-C) and
HCV-specific cell mediated immune (CMI) response among NLI HCWs in terms
of CMI responders frequencies.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\emph{p}\textbf{-value} & Total & \textbf{TLR9.rs5743836 genotype} &
\textbf{TLR9.rs5743836 genotype} & \textbf{TLR9.rs5743836 genotype} &
\textbf{GROUP} & \textbf{GROUP}\tabularnewline
\midrule
\endhead
& & \textbf{TT} & \textbf{CT} & \textbf{CC} & &\tabularnewline
0.353 & 30 (21.4) & 20 (66.7) & 10 (33.3) & 0 (0) & Responder &
\textbf{Seronegative, aviraemic}\tabularnewline
& 110 (78.5) & 82 (74.5) & 25 (22.7) & 3 (2.7) & Non-responder
&\tabularnewline
0.87 & 4 (20) & 3 (75) & 1 (25) & 0 (0) & Responder &
\textbf{Seronegative, viraemic}\tabularnewline
& 16 (80) & 11 (68.8) & 4 (25) & 1 (6.3) & Non-responder
&\tabularnewline
\textbf{0.019} & 7 (20) & 4 (57.1) & 3 (42.9) & 0 (0) & Responder &
\textbf{Resolved}\tabularnewline
& 28 (80) & 27 (96.4) & 1 (3.6) & 0 (0) & Non-responder &\tabularnewline
0.74 & 18 (25.7) & 14 (77.8) & 4 (22.2) & 0 (0) & Responder &
\textbf{Chronic}\tabularnewline
& 52 (74.3) & 42 (80.8) & 10 (19.2) & 0 (0) & Non-responder
&\tabularnewline
\textbf{0.12} & \textbf{59 (22.3)} & \textbf{41 (69.5)} & \textbf{18
(30.5)} & \textbf{0 (0)} & Responder & Total\tabularnewline
& \textbf{206 (77.7)} & \textbf{162 (78.6)} & \textbf{40 (19.4)} &
\textbf{4 (1.9)} & Non-responder &\tabularnewline
\bottomrule
\end{longtable}

\par\null

\textbf{Table (3)} : Relationship between TLR9.rs352140 (G2848A) and
HCV-specific cell mediated immune (CMI) response among NLI HCWs in terms
of CMI responders frequencies.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\emph{p}\textbf{-value} & Total & \textbf{TLR9.rs352140 genotype} &
\textbf{TLR9.rs352140 genotype} & \textbf{TLR9.rs352140 genotype} &
\textbf{GROUP} & \textbf{GROUP}\tabularnewline
\midrule
\endhead
& & \textbf{AA} & \textbf{GA} & \textbf{GG} & &\tabularnewline
0.299 & 28 (21.1) & 5 (17.9) & 10 (35.7) & 13 (46.4) & Responder &
\textbf{Seronegative, aviraemic}\tabularnewline
& 105 (78.9) & 29 (27.6) & 43 (41) & 33 (31.4) & Non-responder
&\tabularnewline
0.32 & 4 (20) & 0 (0) & 2 (50) & 2 (50) & Responder &
\textbf{Seronegative, viraemic}\tabularnewline
& 16 (80) & 4 (25) & 9 (56.3) & 3 (18.8) & Non-responder
&\tabularnewline
0.299 & 7 (20) & 0 (0) & 4 (57.1) & 3 (42.9) & Responder &
\textbf{Resolved}\tabularnewline
& 28 (80) & 7 (25) & 10 (35.7) & 11 (39.3) & Non-responder
&\tabularnewline
0.627 & 18 (25.7) & 5 (27.8) & 7 (38.9) & 6 (33.3) & Responder &
\textbf{Chronic}\tabularnewline
& 52 (74.3) & 9 (17.3) & 24 (46.2) & 19 (36.5) & Non-responder
&\tabularnewline
\textbf{0.35} & \textbf{57 (22.1)} & \textbf{10 (17.5)} & \textbf{23
(40.4)} & \textbf{24 (42.1)} & \textbf{Responder} & Total\tabularnewline
& \textbf{201(77.9)} & \textbf{49 (24.4)} & \textbf{86 (42.8)} &
\textbf{66 (32.8)} & \textbf{Non-responder} &\tabularnewline
\bottomrule
\end{longtable}

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\section*{}

{\label{section}}

\textbf{Figure legends} :

\textbf{Figure (1)} : \textbf{Relation between TLR3.rs3775290
(c.1377C/T) genotype and total HCV-specific CMI response among total
subjects (A), seronegative, \emph{aviraemic} subjects (B),
seronegative\emph{viraemic} subjects (C), spontaneously resolved
subjects (D),and} \textbf{chronic HCV patients (E).} Data are shown as
scatter plots. For each subject, the total cumulative number of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}spot-forming cells (SFC) per
10\textsuperscript{6}PBMCs is shown for responding and non-responding
healthcare workers (HCWs) and patients. Error bars represent the SEM.

\textbf{Figure (2)} : \textbf{Relationship betweenTLR9.rs5743836
(-1237T-C) genotype and total HCV-specific CMI response among the total
subjects (A), seronegative \emph{aviraemic} subjects (B),
seronegative\emph{viraemic} subjects (C), spontaneously resolved
subjects (D),and} \textbf{chronic HCV patients (E).} Data are shown as
scatter plots. For each subject, the total cumulative number of
HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}spot-forming cells (SFC) per
10\textsuperscript{6}PBMCs is shown for responding and non-responding
healthcare workers (HCWs) and patients. Error bars represent the SEM.

\textbf{Figure (3): Relationship between TLR9.rs352140 (G2848A) genotype
and total HCV-specific CMI response among the total subjects (A)
seronegative, \emph{aviraemic} subjects (B) seronegative,\emph{viraemic}
subjects (C) spontaneously resolved subjects (D)} and\textbf{chronic HCV
patients (E).} Data are shown as scatter plots. For each subject, the
total cumulative number of HCV-specific IFN-\selectlanguage{greek}γ \selectlanguage{english}spot-forming cells (SFC)
per 10\textsuperscript{6} PBMCs is shown for responding and
non-responding HCWs and patients. Error bars represent the SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1:\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2:\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3:

\selectlanguage{english}
\FloatBarrier
\end{document}

