\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Memory T-cell targeted immune response in patients of liver cirrhosis
and immunocompetent recipient of ChAdOx1nCoV-19 Vaccine (Covishield)''}



\author[1]{Geeta Yadav}%
\author[2]{Himanshu Dandu}%
\author[3]{Amit Goel}%
\author[1]{Manish Kumar}%
\author[2]{Hardeep Malhotra}%
\author[3]{Harshita Katiyar}%
\author[4]{Monica Agarwal}%
\author[2]{Neeraj Kumar}%
\author[5]{Pragya Pandey}%
\author[2]{Shivani Rani}%
\affil[1]{King George's Medical University Department of Pathology}%
\affil[2]{King George's Medical University Faculty of Medical Sciences}%
\affil[3]{Sanjay Gandhi Post Graduate Institute of Medical Sciences}%
\affil[4]{King George's Medical University Department of Community Medicine}%
\affil[5]{King George's Medical University Faculty of Dental Sciences}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Despite the effectiveness of COVID-19 vaccination in reducing the
severity of the disease, the demand for booster is increasing in
vulnerable populations like elderly and immunocompromised individuals
especially with each new wave of COVID-19 in different countries. There
is limited data on the sustained immunity against COVID-19 in patients
with liver cirrhosis. The study was aimed to compare the T-cell and
humoral immune response after one year of ChAdOx1nCoV-19 Vaccine in
patients with liver cirrhosis and healthy health-care workers (HCW).
This was a prospective observational study including 36 HCW,19 liver
cirrhosis patients and 10 unvaccinated individuals. Anti-SARS-CoV-2S
antibody, neutralizing antibody and memory T-cell subsets were evaluated
by ELISA and flow cytometry respectively in all three groups after one
year of initial vaccination. Compared to HCW and unvaccinated
individuals, liver cirrhosis patients had significantly depleted
T-cells, although CD4:CD8+ T-cell ratio was normal. Significant
difference was noted in various memory subsets {[}effector memory RA (P=
0.141, P= 0.000), effector memory (P= 0.000, P= 0.00), central memory
(P= 0.000, P= 0.00), stem cell memory (P= 0.009, P= 0.08) and na\selectlanguage{ngerman}ïve (P=
0.000, P= 0.02){]} of CD4+T and CD8+T respectively. However, on post-hoc
analysis no difference was noted in the extent of memory T-cells between
cirrhotic patients and HCW. Patients with liver cirrhosis developed
comparable memory T-cells after vaccination which can evoke sustainable
immune response on reinfection. Therefore, additional vaccine doses may
not be necessary for cirrhosis patients.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Title Page~}

\textbf{Title:} Memory T-cell targeted immune response in patients of
liver cirrhosis and immunocompetent recipient of ChAdOx1nCoV-19 Vaccine
(Covishield)''

\textbf{Running Title:} T-cell immune response to ChAdOx1nCoV-19 Vaccine
(Covishield) in cirrhotic patients

\textbf{Authors Name:}

Himanshu Dandu\textsuperscript{1}, Amit Goel\textsuperscript{2}, Manish
Kumar\textsuperscript{3}, Hardeep Singh Malhotra\textsuperscript{4},
Harshita Katiyar\textsuperscript{2}, Monica Agarwal\textsuperscript{5},
Neeraj Kumar\textsuperscript{4}, Pragya Pandey\textsuperscript{6},
Shivani Rani\textsuperscript{1}, Geeta Yadav\textsuperscript{3}

\textbf{Author's Institutional affiliation:}

\textsuperscript{1}Department of Internal Medicine, King George's
Medical University, Lucknow, India, 226003

\textsuperscript{2}Department of Hepatology, Sanjay Gandhi Post-graduate
Institute of Medical Sciences, Lucknow, India, 226014

\textsuperscript{3}Department of Pathology, King George's Medical
University, Lucknow, India, 226003

\textsuperscript{4}Department of Neurology, King George's Medical
University, Lucknow, India, 226003

\textsuperscript{5}Department of Community Medicine, King George's
Medical University, Lucknow, India, 226003

\textsuperscript{6}Department of Conservative Dentistry and Endodontics,
King George's Medical University, Lucknow, India, 226003

\textbf{Corresponding Author:}

Dr. Geeta Yadav, Additional Professor, Department of Pathology, King
George's Medical University, Lucknow, India, 226003

Email address: geetayadav@kgmcindia.edu

Contact number: +919795199085

\textbf{Abstract~}

Despite the effectiveness of COVID-19 vaccination in reducing the
severity of the disease, the demand for booster is increasing in
vulnerable populations like elderly and immunocompromised individuals
especially with each new wave of COVID-19 in different countries. There
is limited data on the sustained immunity against COVID-19 in patients
with liver cirrhosis. The study was aimed to compare the T-cell and
humoral immune response after one year of ChAdOx1nCoV-19 Vaccine in
patients with liver cirrhosis and healthy health-care workers (HCW).
This was a prospective observational study including 36 HCW,19 liver
cirrhosis patients and 10 unvaccinated individuals. Anti-SARS-CoV-2S
antibody, neutralizing antibody and memory T-cell subsets were evaluated
by ELISA and flow cytometry respectively in all three groups after one
year of initial vaccination. Compared to HCW and unvaccinated
individuals, liver cirrhosis patients had significantly depleted
T-cells, although CD4:CD8+ T-cell ratio was normal. Significant
difference was noted in various memory subsets {[}effector memory RA (P=
0.141, P= 0.000), effector memory (P= 0.000, P= 0.00), central memory
(P= 0.000, P= 0.00), stem cell memory (P= 0.009, P= 0.08) and na\selectlanguage{ngerman}ïve (P=
0.000, P= 0.02){]} of CD4+T and CD8+T respectively. However, on post-hoc
analysis no difference was noted in the extent of memory T-cells between
cirrhotic patients and HCW. Patients with liver cirrhosis developed
comparable memory T-cells after vaccination which can evoke sustainable
immune response on reinfection. Therefore, additional vaccine doses may
not be necessary for cirrhosis patients.



\textbf{Introduction}

The COVID-19 pandemic has continued for more than two years infecting
half of the world population either by natural infection or by
vaccination. More than 760 million people have been infected and about
6.9 million deaths have occurred owing to SARS-CoV-2 infection as of
April 2023.\textsuperscript{1} The available vaccines in India at the
time of study were the Astra Zeneca (ChAdOx1 nCoV-19) vaccine
(Covishield, Oxford/Astra Zeneca COVID-19 AZD1222, Serum Institute of
India), a non-replicating viral vector vaccine (NRVV) and Bharat biotech
BBV152 (Covaxin {[}BBV152{]}) containing inactivated whole virus; both
of which required two doses for effective action. Covishield vaccine
uses modified viral vector platform of chimpanzee adenovirus (ChAdOx1)
that permits it to transfer spike protein of COVID-19 virus into human
cells. Study based on the in-vitro live-virus neutralization and T-cell
immune responses to the spike protein, showed that the efficacy of two
doses of Covishield vaccine against moderate-to-severe COVID-19 was
\textasciitilde{} 81.5\%.\textsuperscript{3} Multiple studies have
quoted that after natural SARS-CoV-2 infection or vaccination, vigorous
T-cells response is responsible for production of anti-spike
Neutralizing antibodies against multiple viral epitopes like spike
Anti-receptor binding domain (RBD) \& N-Terminal domain
(NTD).\textsuperscript{4-6} The mRNA and adjuvant vaccines promote
intracellular production of spike protein which is presented by antigen
presenting cells to naïve T-cells (both CD8+ and CD4+ T cells) causing
activation and differentiation of T-cells into effector cells and
different subsets of memory T-cells. These memory cells once invoked
either by natural infection or by vaccination, can persists even after
decades. The interplay of this cellular and humoral response is
essential for effective immunity.\textsuperscript{7-9}

Vaccination against COVID-19 has reduced the severity of the disease,
yet we are still facing waves of COVID-19 infection like the recent
surge in China and India.\textsuperscript{10,11} Demand for booster dose
is brought up with each wave of COVID-19, especially in vulnerable
population like elderly, cancer patients and immunocompromised
population.\textsuperscript{12} Studies have monitored the titer of
neutralizing antibody at different time interval to check for adequate
humoral immune response in healthy
individuals.\textsuperscript{13-15}Few studies have mentioned waning
effect of humoral response at six months of vaccination by measuring IgG
neutralizing antibodies (NAb) levels and proposed that reactivity \&
durability of cellular response ( CD8+ cells) can prevent severe disease
against viral variant even when they escape from Nab. Equally, T-cell
response at different time period has also been studied in healthy
individuals. \textsuperscript{4,16,17}But there is paucity of data in
immunocompromised individuals for sustained immune response especially
with regards to T-cell-based immunity. Cirrhosis is associated with
impairment of innate and adaptive immune system leading to acquired
immunodeficiency. Both B and T-cell mediated immunity is hampered in
form of B and T-cell depletion and dysfunction because of multifactorial
causes like impaired production, proliferation and increased
apoptosis.\textsuperscript{18,19} Hence, we selected patients of
cirrhosis as immunocompromised population in our study. We studied
different subset of T-cell memory cells along with antibody titer at
baseline and at one year duration in vaccinated healthy individuals and
patients with liver cirrhosis at one year to observe any difference in
immune response.



\textbf{Material and Methods:}

Study design and population:

This was a prospective longitudinal cohort study involving 36 health
care workers (HCW) and 19 cirrhosis patients at King George's Medical
University and Sanjay Gandhi Postgraduate Institute of Medical Sciences,
Lucknow. Blood samples of 10 healthy unvaccinated (and unexposed as per
history) individuals were obtained from rural area and they were
referred for vaccination at primary health care center. The normal
healthy HCW were defined as without any co-morbidity or medication.
Patients were selected for the cirrhotic group based on clinical,
biochemical and radiological finding along as well as evidence of portal
hypertension and AST-Platelet ratio index. Hepatic decompensation was
determined by the presence of ascites or hepatic encephalopathy in cases
of hepatitis B-related liver cirrhosis, or by a markedly raised serum
bilirubin level along with prolonged prothrombin time or international
normalized ratio \textgreater{}1.5. \textsuperscript{20} Venous blood
was collected from all participants before receiving the first dose (day
0) and before booster dose (day 270±14 after second dose or \selectlanguage{english}[?] one year
after first dose). The time interval between first and second dose was
three months as per government protocol at the time of vaccination.
Samples were collected in plain and EDTA vial for anti-SARS-CoV-2
antibody and flow cytometry assay respectively. All participant signed
written informed consent form and study was approved by institutional
ethical committee.

Serological assay:

Anti-receptor binding domain (RBD) or anti-Spike antibody titer and
neutralizing antibody (NAb) were measured in stored serum sample by
double-antigen sandwich enzyme-linked immunoassay (ELISA) through
Elecsys(r) Anti-SARS-CoV-2 S (Roche Diagnostics GmbH, Germany) and
SARS-CoV-2 Neutralizing Antibody competitive ELISA Kit (Invitrogen,
Thermo-Fischer) respectively. The Elecsys(r) Anti-SARS-CoV-2 S antibody
titer is expressed as U/mL and value \textgreater{}0.8 U/mL was
considered positive (quantitation limit of 0.40--250 U/mL). For
SARS-CoV-2 Neutralizing Antibody, samples with more than 20\% calculated
neutralization were considered positive.

\par\null

Flow Cytometry Assay:

The sample preparation for flow cytometric analysis was done by
stain-lyse-wash protocol. The antibodies utilized in the assay were
procured from Becton Dickinson (BD) Biosciences, San Jose, California,
USA. The fluorochrome and clones of antibody used were CD3 PerCP-Cy5.5
(SP34-2)/ CD4 PE-Cy7 (SK3)/ CD8 APC-H7 (SK1)/ CD197 APC (2-L1-A)/ CD95
BV421 (DX2)/ CD45RA BV480 (H1100)/ CD27 PE (M-T271) in the memory cell
tube. A blank tube without antibody was also run. While FACS Canto II
flow cytometer, BD Biosciences was used for sample acquisition, data
analysis was performed using FACS Diva version 8 software. At least
10,000 CD3+T cells were acquired in each case to ensure adequate number
of subset population to be studied. Based on light scatter properties,
lymphocytes were identified. From this population, CD3+ total T-cells as
well as CD4+ and CD8+ subsets were subsequently defined. On the basis of
differential expression of CD45RA and CD197 (CCR7), central memory (CM),
effector memory (EM) and effector memory RA (EMRA) subsets were
identified on both helper and cytotoxic cell. From the CD45RA+CD197+
gate, CD95+ stem cell memory and CD95- naive T-cells were further
identified. The gating strategy has been depicted in figure 1.
(Figure-1)

Statistical analysis:

Normality assessment of data was done with Shapiro Wilk test. Continuous
data was expressed as mean (with standard deviation) or median (with
range) with respect to its distribution. To compare the expression of
antibodies, Mean fluorescence intensity (MFI) was used. Non-parametric
assessments were used to compare the different groups; 2-independent
sample analysis was done using Kolmogorov-Smirnov Z test while
\textgreater{}2-independent sample analysis was done by Kruskal Wallis
ANOVA with post-hoc pairwise comparisons. All assessments were 2-tailed
and a P-value of \textless{}0.05 was taken as significant; all values
\textless{}0.001 were expressed as \textless{}0.001. Data was analyzed
with IBM SPSS package for Windows, Version 24.0, Armonk, NY.

\textbf{Results:}

\textbf{Demography and hematological parameters}

We evaluated 19 patients of cirrhotic liver disease, 36 normal health
care workers and 10 unvaccinated healthy individuals at one year
duration after initial dose of vaccination. All individuals received two
doses of ChAdOx1nCoV-19 vaccine. The demographic and laboratory findings
of study population are described in Table I. The median age of
cirrhotic patients was 55 (44-67) years with male female ratio of 2.8:1.
Underlying causes of cirrhosis were Hepatitis C virus (52.63\%),
alcoholic hepatitis (15.78\%), hepatitis B virus (10.52\%), cryptogenic/
nonalcoholic steatohepatitis (10.52\%) and other (10.52\%) respectively.
Of 19 patients, 84.21 \% had compensated and 15.78\% had non-compensated
cirrhosis with 78.94\% patients belonging to Child--Turcotte--Pugh (CTP)
class A (15; 78.94\%) and B (4; 21.05\%) cirrhosis.

\textbf{Anti-RBD antibody and neutralizing antibody at baseline and at
one year}

The baseline anti-RBD antibody (U/mL) and neutralizing antibody (\%)
titer in cirrhotic and HCW was 0.675 (0.4-10478) \& 54.7 (0.04-1403.0)
and 11.2 (-17.39-97.35) and 15.02 (-10.04-97.3) respectively. Three
patients in cirrhotic group had higher levels of anti-RBD antibody and
NAbs levels even before vaccination which could be due to asymptomatic
natural SARSCoV-2 infection. All participants in cirrhotic and HCW group
showed development of antibodies and the median titers after one year
were 2132 (11.09-42594) \& 8444 (101.6-23044) and 35.57 (-22.29-97.4)
and 94.3 (-0.8-97.6) respectively. There was no difference between
anti-RBD and neutralizing antibody titer at one year between cirrhotic
and HCW group (P=0.312, P=0.277). The unvaccinated group had
\textless{}0.4U/mL and \textless{}20\% anti-RBD antibody (U/mL) and
neutralizing antibody levels. (Figure-2)

\textbf{Memory T-cell response at one year}

Significant depletion of T-cells was noted in cirrhotic patients (5.8
{[}0.5-20.1{]}) as compared to HCW (13.1 {[}1.6-31.3{]}) and
unvaccinated individuals (12.3 {[}7.8-19.6{]}; (P=0.001 \& P=0.000)

respectively. However, the ratio of CD4+T to CD8+T cells was not altered
in any group. By analyzing expression of CD45RA and CD197 (CCR7),
subsets of central memory (CM), effector memory (EM) and effector memory
RA (EMRA) were identified in both CD4+ or CD8+ T-cell. From the
CD45RA+CD197+ gate, CD95+ stem cell memory and CD95- naive T cells were
further identified. Significant difference was noted in various memory
subsets {[}EMRA (P= 0.141, P= 0.000), EM (P= 0.000, P= 0.00), CM (P=
0.000, P= 0.00), SCM (P= 0.009, P= 0.08) and naive (P= 0.000, P=
0.02){]} of CD4+T and CD8+T in all three group respectively. (Table II)

On post-hoc analysis, directed at cirrhotic versus HCW, only T-cell
distributions were found to be statistically different in the two groups
(p\textless{}0.001; Kolmogorov-Smirnov Z = 2.119). The various memory
cells subsets in cirrhotic and HCW group did not show any difference,
though there was significantly raised in comparison to unvaccinated
individual. (Figure-3)

CM and SCM are responsible for long term maintenance of memory cells
which were absent in unvaccinated individuals as they were not
vaccinated. All HCW and 89.47\% cirrhotic patients developed
CD4+T\textsubscript{CM}. 63.88\% HCW and 47.36\% cirrhotic patients
developed CD4+T\textsubscript{SCM} . 36.11\% HCW and 36.84\% cirrhotic
patients developed CD8+T\textsubscript{CM}. 41.66\% HCW and 42.10\%
cirrhotic patients developed CD8+T\textsubscript{SCM.} There was no
difference between memory cells on T-cells between vaccinated cirrhotic
patients and vaccinated HCW.(Figure-4)

\textbf{Discussion:}

This study compares the cellular and humoral response in cirrhotic and
immunocompetent HCW after one year of receiving ChAdOx1nCoV-19 Vaccine
(Covishield). All individuals received two doses of vaccine. The study
found that both groups showed had similar seroconversion as denoted by
titers of anti RBD and neutralizing antibodies (P=0.312, P=0.277).
Though the T-cells were significantly reduced in cirrhotic patients,
CD4:CD8 T-cell ratio was normal in cirrhotic patients. Both CD4+ and
CD8+T-cell subsets developed comparable CM and SCM in cirrhotic and HCW,
which is suggestive of similar cellular immune response.

Due to the ongoing fear of developing fatal complication of SARS-CoV-2
infection, immunocompromised patients like HIV, cancer, and patients
with solid organ transplant, needs special care and
precaution.\textsuperscript{21} Lee A et al in a meta-analysis of 83
studies using mRNA, VVV \& inactivated whole virus vaccine in
immunocompromised patients (solid cancer, inflammatory diseases, HIV \&
transplant recipients) found that after first dose of vaccination
seroconversion rate was half in immunocompromised patients compared to
the immunocompetent one. Second dose of vaccine was associated with
consistently improved seroconversion in all groups with lesser magnitude
in transplant individuals. Based on the above data authors advocated for
the targeted intervention (third or booster dose) in immunocompromised
patients.\textsuperscript{22} Costiniuk et al in their study on HIV
patients, found that anti-RBD and anti S antibody levels were maintained
in 92 \% patients after 6 months and in 100\% patients after one month
of third dose and suggested timely booster administration in HIV
patients for improved immune response.\textsuperscript{23} Studies have
shown reduced seroconversion in immunocompromised
patients.\textsuperscript{24} Therefore, many European countries
recommend booster (third) dose for immunocompromised individuals; even
some countries advocate extended (forth dose) or yearly dose in
susceptible and vulnerable population with different cut-off for age,
citing the waning effect of vaccination over time.\textsuperscript{25}

Effective vaccination develops both humoral and cellular response that
can elicit adequate immune response on reinfection. Effector memory (EM)
T-cells serves as immediate effector function at frontline barriers and
recall responses are mediated by CM and SCM even in the absence of
neutralizing antibodies.\textsuperscript{26} Crucial role of T-cell
immune response in SARS-CoV-1 infection has been clearly studied in
animal models. On long term follow-up, only 8.69\% (2/23) patients of
SARS-CoV-1 patients have shown detectable levels of IgG at six years
indicating reduce humoral immunity over time whereas durable memory
T-cells were detected against SARS-CoV-1 at even \textgreater{}10 years
after infection. These findings may help us to understand the potential
cross-reactivity with existing SARS-CoV-2 and effectiveness of T-cell
memory cells.\textsuperscript{7}

Cirrhosis is associated with dysfunction of both innate and adaptive
immune system and is a state of acquired
immunodeficiency.\textsuperscript{18} Cirrhotic patients developing
COVID-19 infection seem to have a worse outcome than those
otherwise.\textsuperscript{27} Data on the duration of vaccine efficacy
towards COVID-19 in patients with cirrhosis is sparse. Literature
suggests that the underlying immune dysfunction in cirrhosis may lead to
suboptimal response to vaccination as seen with hepatitis B and
pneumococcal vaccines.\textsuperscript{28,29} However, recent studies
have shown an improved outcome, reduced hospital stay and mortality in
patients with cirrhosis undergoing COVID-19 vaccination.

Our study shows concordance in equal seroconversion in cirrhotic \&
immunocompetent HCW in developing comparable titers of anti-RBD and
neutralizing antibodies after one year of initial dose of vaccination,
however, differ in concept of booster dose requirement as persistent
cellular immune response in form of induced stem cell memory \& central
memory were intact and similar in both cirrhotic and HCW which upon
antigenic exposure have ability to mediate adequate immune response.

Several studies have revealed the disparity between B cell and T-cell
mediated immune response in recipients of kidney transplant . It has
been observed that prevalence of S-protein specific CD4+T in transplant
recipient is comparable to healthy control despite a lower humoral
response after two dose of vaccination.\textsuperscript{30}Additionally,
research has demonstrated that cytokines are produced by
S-protein-reactive T-cells on ex-vivo antigenic stimulation indicating
that they can mediate antiviral response activity and safeguard patients
against severe COVID-19, despite the lack of antibodies. Consequently,
relying solely on antibody detection to assess vaccination response may
underestimate existing antiviral protection.\textsuperscript{31}

Feng et al in their randomized control trial of vaccine efficacy study
determined a binding antibody unit of 264 or a pseudo-virus
neutralization assay titer of 26 IU/ml corresponded to 80\% efficacy
against symptomatic COVID-19 though they could not determine threshold
for asymptomatic infections in their
study.\textsuperscript{32}Therefore, more studies with a long-term
follow-up on larger population (including vulnerable and different
spectrum of immunocompromised individuals) are required to define the
reliable vaccine specific correlates of protection as in Hepatitis B.

In summary, the study found that both healthy individuals and cirrhosis
patients generate similar levels of memory T-cells after being
vaccinated, which is an indication of effective and durable immunity.
Therefore, cirrhosis patients may not need additional vaccine doses
compared to healthy individuals.

One of the strengths of the study is that it examined the detailed
cellular and humoral immune responses of cirrhotic patients with varying
degrees of liver disease severity and causes, over a one-year period
after receiving the ChAdOx1nCoV-19 vaccine. However, a limitation of the
study is the small sample size, with fewer patients in the CTP class C
category.

\textbf{Acknowledgment:}

We thank Mr. Hirendra Kumar and Devendra Kumar (Laboratory technician,
Department of Pathology, King George's Medical University) for their
effort in processing the samples.



\textbf{References:}

1. WHO Coronavirus (COVID-19) Dashboard; https://covid19/who/int/.

2. Acosta-Coley I, Cervantes-Ceballos L, Tejeda-Benitez L, et al.
Vaccines platforms and COVID-19: what you need to know. Trop Dis Travel
Med Vaccines. 2022;8(1):20

3. Thiruvengadam R, Awasthi A, Medigeshi G, et al. Effectiveness of
ChAdOx1 nCoV-19 vaccine against SARS-CoV-2 infection during the delta
(B.1.617.2) variant surge in India: a test-negative, case-control study
and a mechanistic study of post-vaccination immune responses. Lancet
Infect Dis. 2022;22(4):473-482.

4. Guo L, Wang G, Wang Y, et al. SARS-CoV-2-specific antibody and T-cell
responses 1 year after infection in people recovered from COVID-19: a
longitudinal cohort study. Lancet Microbe. 2022;3(5):e348-e356.

5. Guerrera G, Picozza M, D'Orso S, et al. BNT162b2 vaccination induces
durable SARS-CoV-2-specific T cells with a stem cell memory phenotype.
Sci Immunol. 2021;6(66):eabl5344.

6. Bertoletti A, Le Bert N, Tan AT. SARS-CoV-2-specific T cells in the
changing landscape of the COVID-19 pandemic. Immunity.
2022;55(10):1764-1778.

7. Liu WJ, Zhao M, Liu K, et al. T-cell immunity of SARS-CoV:
Implications for vaccine development against MERS-CoV. Antiviral Res.
2017;137:82-92.

8. Sallusto F, Lanzavecchia A, Araki K, Ahmed R. From vaccines to memory
and back. Immunity. 2010;33(4):451-463.

9. Golubovskaya V, Wu L. Different Subsets of T Cells, Memory, Effector
Functions, and CAR-T Immunotherapy. Cancers (Basel). 2016;8(3):36.

10. Impact of surge in China COVID-19 cases on epidemiological situation
in EU/EEA. Available from
https://www.ecdc.europa.eu/en/news-events/impact-surge-china-covid-19-cases.
Accessed January 2022.

11. Ministry of Health and Family Welfare -- GOI''. Available from
https://www.mohfw.gov.in. Accessed April 24, 2023.

12. Tartof SY, Slezak JM, Puzniak L, et al. Analysis of mRNA COVID-19
Vaccine Uptake Among Immunocompromised Individuals in a Large US Health
System. JAMA Netw Open. 2023;6(1):e2251833.

13. Furukawa K, Tjan LH, Kurahashi Y, et al. Assessment of Neutralizing
Antibody Response Against SARS-CoV-2 Variants After 2 to 3 Doses of the
BNT162b2 mRNA COVID-19 Vaccine. JAMA Netw Open. 2022;5(5):e2210780.

14. Nickel O, Rockstroh A, Wolf J, et al. Evaluation of the systemic and
mucosal immune response induced by COVID-19 and the BNT162b2 mRNA
vaccine for SARS-CoV-2. PLoS One. 2022;17(10):e0263861

15. Trombetta CM, Marchi S, Leonardi M, et al. Evaluation of antibody
response to SARS-CoV-2 variants after 2 doses of mRNA COVID-19 vaccine
in a correctional facility. Hum Vaccin Immunother. 2022;18(7):2153537.

16. Tarke A, Coelho CH, Zhang Z, et al. SARS-CoV-2 vaccination induces
immunological T cell memory able to cross-recognize variants from Alpha
to Omicron. Cell. 2022;185(5):847-859.e11.

17. Levin EG, Lustig Y, Cohen C, et al. Waning Immune Humoral Response
to BNT162b2 Covid-19 Vaccine over 6 Months. N Engl J Med.
2021;385(24):e84.

18. Albillos A, Lario M, Alvarez-Mon M. Cirrhosis-associated immune
dysfunction: distinctive features and clinical relevance. J Hepatol.
2014;61(6):1385-1396.

19. Lario M, Munoz L, Ubeda M, et al. Defective thymopoiesis and poor
peripheral homeostatic replenishment of T-helper cells cause T-cell
lymphopenia in cirrhosis. J Hepatol. 2013;59(4):723-730.

20. Goel A, Verma A, Tiwari P, et al. Serological Immune Response
Following ChAdOx1 nCoV-19 Vaccine (Covishield(r)) in Patients with Liver
Cirrhosis. Vaccines (Basel). 2022;10(11):1837.

21. Mohammed AH, Blebil A, Dujaili J, Rasool-Hassan BA. 2020. The risk
and impact of COVID-19 pandemic on immunosuppressed patients: Cancer,
HIV, and solid organ transplant recipients. \emph{AIDS Rev} .
22:151--157.

22. Lee ARYB, Wong SY, Chai LYA, et al. Efficacy of covid-19 vaccines in
immunocompromised patients: systematic review and meta-analysis. BMJ.
2022;376:e068632.

23. Costiniuk CT, Singer J, Lee T, et al. COVID-19 vaccine
immunogenicity in people with HIV. AIDS. 2023;37(1):F1-F10.

24. Reeg DB, Hofmann M, Neumann-Haefelin C, Thimme R, Luxenburger H.
SARS-CoV-2-Specific T Cell Responses in Immunocompromised Individuals
with Cancer, HIV or Solid Organ Transplants. Pathogens. 2023;12(2):244.

25. Good practice statement on the use of second booster doses for
COVID-19 vaccines 18 August 2022 Background {[}Internet{]}. Available
from: https://www.who.int/groups/strategic-advisory.\textbf{~}

26. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector
memory T cell subsets: function, generation, and maintenance. Annu Rev
Immunol. 2004;22:745-763.

27. Sharma P, Kumar A, Anikhindi S, et al. Effect of COVID-19 on
Pre-existing Liver disease: What Hepatologist Should Know?. J Clin Exp
Hepatol. 2021;11(4):484-493.

28. McCashland TM, Preheim LC, Gentry MJ. Pneumococcal vaccine response
in cirrhosis and liver transplantation. J Infect Dis.
2000;181(2):757-760.

29. Gutierrez Domingo I, Pascasio Acevedo JM, Alcalde Vargas A, et al.
Response to vaccination against hepatitis B virus with a schedule of
four 40-\selectlanguage{greek}μ\selectlanguage{english}g doses in cirrhotic patients evaluated for liver
transplantation: factors associated with a response. Transplant Proc.
2012;44(6):1499-1501.

30. Thieme CJ, Anft M, Paniskaki K, et al. The Magnitude and
Functionality of SARS-CoV-2 Reactive Cellular and Humoral Immunity in
Transplant Population Is Similar to the General Population Despite
Immunosuppression. Transplantation. 2021;105(10):2156-2164.

32. Feng S, Phillips DJ, White T, et al. Correlates of protection
against symptomatic and asymptomatic SARS-CoV-2 infection. Nat Med.
2021;27(11):2032-2040.

\textbf{~}

\section*{Table and Figure Legends}

{\label{table-and-figure-legends}}

Table I : Demographic and laboratory parameters of study population

Data are presented as the median (range), or n (\%). NASH (non-alcoholic
steatohepatitis), CTP (Child-Turcot- Pugh)

Table II: Mean Florescence intensity (MFI) of various memory T-cells in
respective study population

Mean florescence intensity is shown in median (range), EMRA (effector
memory RA), EM (effector memory), CM (central memory), SCM (stem cell
memory)

Figure-1 Flow plot showing sequential gating strategy; singlets followed
by viable cells followed by T cells (SSC vs CD3), followed by CD4+T and
CD8+T cells (CD4+ vs CD8+). From either CD4+ or CD8+ T cell gate,
central memory (CM), effector memory (EM) and effector memory RA (EMRA)
subsets were identified based on differential expression of CD45RA and
CD197 (CCR7). From the CD45RA+ CD197+ gate, CD95+ stem cell memory and
CD95 - na\selectlanguage{ngerman}ïve T cells were further identified.

Figure-2.(A) Box plot comparing the anti RBD antibodies levels at
baseline, (B) Box plot comparing the anti RBD antibodies levels after
one year of vaccination, (C) Box plot comparing the neutralizing
antibodies levels at baseline, (D) Box plot comparing the neutralizing
antibodies levels after one year of vaccination among health care
workers and cirrhotic patients.

Figure-3 Three-dimensional scatter plot of mean frequency of
distribution of memory T-cells on CD4+ \& CD8+T cells among cirrhotic,
health care workers and unvaccinated individuals

Figure-4 Flow plot showing frequency of expression of different subset
of memory cells (both CD4+ and CD8+T cells) in cirrhotic, health care
workers and unvaccinated individuals.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

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

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`Table I.docx`  available at \url{https://authorea.com/users/618791/articles/643727-memory-t-cell-targeted-immune-response-in-patients-of-liver-cirrhosis-and-immunocompetent-recipient-of-chadox1ncov-19-vaccine-covishield}

\textbf{Hosted file}

\verb`Table II.docx`  available at \url{https://authorea.com/users/618791/articles/643727-memory-t-cell-targeted-immune-response-in-patients-of-liver-cirrhosis-and-immunocompetent-recipient-of-chadox1ncov-19-vaccine-covishield}

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

