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\begin{document}

\title{Ad5-nCoV vaccination could induce HLA-E restricted CD8 + T cell
responses specific for epitopes on severe acute respiratory syndrome
coronavirus 2 spike protein}



\author[1]{yuling wang}%
\author[1]{Lu Yang}%
\author[1]{Kang Tang}%
\author[1]{Yusi Zhang}%
\author[1]{Chunmei Zhang}%
\author[1]{Yun Zhang}%
\author[1]{Boquan Jin}%
\author[1]{Yuan Zhang}%
\author[1]{Ran Zhuang}%
\author[1]{Ying Ma}%
\affil[1]{Air Force Medical University Department of Immunology}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
\textbf{Objectives:} To evaluate cellular immune responses induced by
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines
immunization in population based on HLA-E-restricted CD8
\textsuperscript{+} T cell epitopes identification. \textbf{Methods:}
HLA-E-restricted SARS-CoV-2 CD8 \textsuperscript{+} T cell nonamer
peptides were predicted with software. HLA-E-transfected K562 cells
binding assay was used to screen for high-affinity peptides. IFN-\selectlanguage{greek}γ
\selectlanguage{english}enzyme-linked immunospot assay were used to identify HLA-E-restricted
epitopes. HLA-E/epitope tetramer was employed to detect the frequencies
of epitope-specific CD8 \textsuperscript{+} T cells. \textbf{Results:}
Four CD8 \textsuperscript{+} T cell epitopes on spike protein of
SARS-CoV-2 restricted by both HLA-E*0101 and E*0103 were identified.
HLA-E-restricted epitope specific IFN-\selectlanguage{greek}γ-\selectlanguage{english}secreting CD8
\textsuperscript{+} T cell responses could be detected in individuals
vaccinated with SARS-CoV-2 vaccines. Importantly, the frequencies of
epitope-specific CD8 \textsuperscript{+} T cell in Ad5-nCoV vaccinated
individual were higher than that in individuals vaccinated with
recombinant protein or inactivated vaccines. Moreover, frequencies of
epitope-specific CD8 \textsuperscript{+} T cells could maintain for at
least 120 days after only one dose Ad5-nCoV vaccination. While
frequencies of epitope-specific CD8 \textsuperscript{+} T cells
decreased in individuals after two doses of Ad5-nCoV vaccination.
\textbf{Conclusions:} These findings may contribute to more
comprehensive evaluating protective effects of vaccines for SARS-CoV-2,
meanwhile may provide information to characterize HLA-E-restricted CD8
\textsuperscript{+} T cell immunity against SARS-CoV-2 infection.%
\end{abstract}%



\sloppy


\textbf{Ad5-nCoV vaccination could induce HLA-E restricted}
\textbf{CD8\textsuperscript{+} T cell responses specific for epitopes on
severe acute respiratory syndrome coronavirus 2 spike protein}

Yuling Wang\textsuperscript{1}, Lu Yang\textsuperscript{1}, Kang
Tang\textsuperscript{1}, Yusi Zhang\textsuperscript{1}, Chunmei
Zhang\textsuperscript{1}, Yun Zhang\textsuperscript{1}, Boquan
Jin\textsuperscript{1}, Yuan Zhang\textsuperscript{1}, Ran
Zhuang\textsuperscript{1*}, Ying Ma\textsuperscript{1*}

\textsuperscript{1} Department of Immunology, the Air Force Medical
University, Xi'an, China

\textsuperscript{\textbf{*}} \textbf{Correspondence:}

Dr. Ying Ma

E-mail: mayingying@fmmu.edu.cn

Dr. Ran Zhuang

E-mail: fmmuzhr@fmmu.edu.cn

\textbf{Abstract}

\textbf{Objectives:} To evaluate cellular immune responses induced by
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines
immunization in population based on HLA-E-restricted
CD8\textsuperscript{+} T cell epitopes identification.

\textbf{Methods:} HLA-E-restricted SARS-CoV-2 CD8\textsuperscript{+} T
cell nonamer peptides were predicted with software. HLA-E-transfected
K562 cells binding assay was used to screen for high-affinity peptides.
IFN-\selectlanguage{greek}γ \selectlanguage{english}enzyme-linked immunospot assay were used to identify
HLA-E-restricted epitopes. HLA-E/epitope tetramer was employed to detect
the frequencies of epitope-specific CD8\textsuperscript{+} T cells.

\textbf{Results:} Four CD8\textsuperscript{+} T cell epitopes on spike
protein of SARS-CoV-2 restricted by both HLA-E*0101 and E*0103 were
identified. HLA-E-restricted epitope specific IFN-\selectlanguage{greek}γ-\selectlanguage{english}secreting
CD8\textsuperscript{+} T cell responses could be detected in individuals
vaccinated with SARS-CoV-2 vaccines. Importantly, the frequencies of
epitope-specific CD8\textsuperscript{+} T cell in Ad5-nCoV vaccinated
individual were higher than that in individuals vaccinated with
recombinant protein or inactivated vaccines. Moreover, frequencies of
epitope-specific CD8\textsuperscript{+} T cells could maintain for at
least 120 days after only one dose Ad5-nCoV vaccination. While
frequencies of epitope-specific CD8\textsuperscript{+} T cells decreased
in individuals after two doses of Ad5-nCoV vaccination.

\textbf{Conclusions:} These findings may contribute to more
comprehensive evaluating protective effects of vaccines for SARS-CoV-2,
meanwhile may provide information to characterize HLA-E-restricted
CD8\textsuperscript{+} T cell immunity against SARS-CoV-2 infection.

\textbf{Keywords:}

severe acute respiratory syndrome coronavirus 2; Ad5-nCoV; vaccination;
HLA-E; CD8\textsuperscript{+} T cell response; epitope

\textbf{Introduction}

The acute infectious disease coronavirus disease 2019 (COVID-19), caused
by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
infection, is characterized by fever and upper respiratory symptoms, and
even multiple organ failure such as acute respiratory distress syndrome
{[}1{]}. COVID-19 began to appear in December 2019 and posed a huge
threat to global public health and human life. The World Health
Organization (WHO) declared that COVID-19 has become a global pandemic
in March 2020. COVID-19 spread across nearly every country. Up to now,
COVID-19 has accumulated more than 661 million confirmed cases and more
than 6.7 million deaths worldwide.

Vaccination is the most effective way to prevent SARS-CoV-2 infection.
At present, many types of vaccines against SARS-CoV-2 have been widely
vaccinated in the world and achieved certain preventive effects. Among
them, recombinant SARS-CoV-2 vaccine with adenovirus type 5 vector,
which was developed and produced in China (Ad5-nCoV, CanSinoBIO, China),
has been proved to induce high level of both humoral and cellular immune
responses {[}2-4{]}. Importantly, Ad5-nCoV has been widely vaccinated in
the population and approved by WHO as COVID-19 vaccine ''Emergency Use
List'' in May 2022 {[}5-7{]}.

SARS-CoV-2 is single positive-stranded, enveloped RNA virus that can
infect many animal species and humans. The genome containing six open
reading frames (ORFs), which encode non-structural proteins such as RNA
dependent RNA polymerase (RdRp), and four structure proteins, namely
spike protein (S), envelope protein (E), membrane protein (M) and
nucleocapsid proteins (N) {[}8{]}. The S protein could induce high level
of specific antibody. However, antibody responses against SARS-CoV-2
always could not be well maintained for a long time {[}9{]}.

It has been reported that SARS-CoV-2-specific CD8\textsuperscript{+} T
cell responses in peripheral blood of COVID-19 patients were closely
related to remission of the disease {[}10, 11{]}. In fact, SARS-CoV-2
specific CD8\textsuperscript{+} T cell responses could be detected in
acute phase of COVID-19 within 7 days after onset of symptoms, which
always reached the strongest at 14 days {[}12{]}. Studies in SARS-CoV-2
infected animal models have found that although neutralizing antibodies
could protect against viral attack, CD8\textsuperscript{+} T cell
responses could provide more important clinical protection when antibody
levels were low or reduced {[}13{]}. CD8\textsuperscript{+} T cell
epitopes of SARS-CoV-2 restricted by classical HLA-I molecules, such as
HLA-A1/ORF1a\textsubscript{1637}, HLA-A2/S\textsubscript{269},
HLA-A3/N\textsubscript{361}, HLA-A24/S\textsubscript{1208},
HLA-B7/N\textsubscript{105} and HLA-B40/N\textsubscript{322}, were used
to further study the effects and mechanisms of specific
CD8\textsuperscript{+} T cell responses in SARS-CoV-2 infection {[}14,
15{]}. Among them, NP\textsubscript{105-113} epitope restricted by
HLA-B7 has been proved to induce specific CD8\textsuperscript{+} T cell
response with strong antiviral activity {[}16{]}. Although most studies
focused on classical HLA-I restricted CD8\textsuperscript{+} T cell
epitopes and specific responses, non-classical HLA-I molecule HLA-E
restricted CD8\textsuperscript{+} T cells also play an important role
for immune protection and regulation in many viral infectious diseases
{[}17-19{]}. Notably, HLA-E is a ubiquitous HLA locus with only two
different alleles HLA-E*0101 and HLA-E*0103, both of which have a
combined frequency of over 99\% in global population. The two molecules
just differ in position 107 of the mature HLA-E protein (arginine for
HLA-E*0101 and glycine for HLA-E*0103){[}20{]}. It has been proposed to
use HLA-E restricted CD8\textsuperscript{+} T cells to treat severe
COVID-19 patients in the early stage of SARS-CoV-2 infection {[}21{]}.
Therefore, it is necessary to identify CD8\textsuperscript{+} T cell
epitopes that can overcome the polymorphism of HLA molecules. Moreover,
detection of SARS-CoV-2 epitope-specific CD8\textsuperscript{+} T cell
responses may be important for evaluation of the protective effects
induced by SARS-CoV-2 vaccines.

In this study, specific CD8\textsuperscript{+} T cell responses in
different vaccines inoculated populations, especially in Ad5-nCoV
vaccinated subjects, were detected based on identification of four
epitopes on S protein of SARS-CoV-2 restricted by both HLA-E*0101 and
E*0103. These findings may provide crucial information to evaluate the
effects of COVID-19 vaccines immunization in population according to
HLA-E-restricted SARS-CoV-2 specific CD8\textsuperscript{+} T cell
responses.

\textbf{Methods}

\textbf{Study cohort}

A total of 56 samples from 43 individuals vaccinated with SARS-CoV-2
vaccines were enrolled in this study. The ages of vaccinated subjects
were ranging between 21 and 32 years. The vaccinated donors who were
suffering from infectious diseases or autoimmune diseases, and
experiencing any inflammation or infection within a week before sampling
were excluded from this study. Subjects were divided into three groups
according to different types of vaccine they received, including
inactivated vaccine group (CoronaVac, Sinovac Biotech, China),
recombinant protein subunit vaccine group (ZF2001, Zhifei Biological,
China) and adenovirus-vectored vaccine group (Ad5-nCoV, CanSino
Biologics, China). The basic information of the enrolled volunteers is
summarized in Table 1.

\textbf{Sample collection}

Peripheral venous blood samples of SARS-CoV-2 vaccination individuals
were drawn into pyrogen-free blood collection tubes with citrate as an
anticoagulant. Peripheral blood mononuclear cells (PBMCs) were isolated
using a standard Ficoll-Hypaque (Sigma-Aldrich, MO) density gradient
centrifugation.

\textbf{Epitopes prediction}

The Immune Epitope Database and Analysis Resource (IEDB), NetMHC4.0
Server and NetMHCpan 4.1 Server are used to predict binding of peptides
to MHC molecule of known sequence using artificial neural networks
(ANNs). Specifically, nine-mer peptide as the peptide length option was
selected. HLA-E*0101 or HLA-E*0103-restricted SARS-CoV-2 nine-mer
peptide epitopes were predicted by inputting 1,273 amino acids of S
protein sequence, 75 amino acids of E protein sequence, 222 amino acids
of M protein sequence and 419 amino acids of N protein sequence of
SARS-CoV-2 prototype strain Wuhan-Hu-1 (GenBank accession number:
NC\_045512.2) into the databases, respectively.

There is only HLA-E*0101 allele option could be selected in NetMHC4.0
Server database, while both HLA-E*0101 and E*0103 alleles could be
selected in NetMHCpan 4.1 Server database under the loci or species
option with HLA-E. Both the two methods specified if a sequence is a
strong MHC binder (SB) or a weak MHC binder (WB) based on a \%Rank
score. By default, \%Rank \textless{} 0.5\% and \%Rank \textless{} 2\%
thresholds are considered for detecting as SB and WB for HLA-E molecule.

\textbf{Peptides synthesis}

The predicted SARS-CoV-2 nine-mer epitopes were synthesized at least
90\% purity as assessed by high-performance liquid chromatography and
mass spectrometry (ChinaPeptides, Shanghai, China). Peptides from
HLA-Cw*03\textsubscript{3-11} or HLA-A*02\textsubscript{3-11} leading
sequences VMAPRTLIL or VMAPRTLVL, which have been confirmed to be the
HLA-E-restricted epitopes, were synthesized and used as positive
control. The peptide melanoma antigen-encoding gene (MAGE-1) with
sequence EADPTGHSY, which couldn't bind to HLA-E molecules, was
synthesized and used as the negative control. All the peptides were
stored at 1 mM concentration at -70\selectlanguage{ngerman}°C and repeated freeze-thawing was
avoided.

\textbf{K562 cell lines transfected with HLA-E molecule}

An immortalized chronic myelogenous leukemia cell line K562 with MHC
molecules expression deficiency was obtained from ATCC (CCL-243). We
stably transfected K562 cell lines with single allele HLA-E*0103 or
HLA-E*0101 to generate mono-allelic cell lines with lentivirus
transfected methods. Stably transfected cells showed strong expression
(\textgreater{}95\%) of HLA-E*0103 or HLA-E*0101 according to flow
cytometry (FCM) analysis.

\textbf{K562/HLA-E cells binding assay}

The stable transformed K562/HLA-E*0103 or K562/HLA-E*0101 cells were
respectively incubated with 10 \selectlanguage{greek}μ\selectlanguage{english}M each indicated peptide and 1 \selectlanguage{greek}μ\selectlanguage{english}M human
\selectlanguage{greek}β\selectlanguage{english}2-microglobulin (\selectlanguage{greek}β\selectlanguage{english}2m, Sigma) in serum-free RPMI 1640 medium for 16 h at
26\selectlanguage{ngerman}°C with 5\% CO2, while K562/HLA-E cells with no peptide incubation
under the same culture conditions were used as blank control. These
cells binding with peptides were further incubated at 37°C for 2 h for
thermal stability. We detected the expression of HLA-E molecules on
surface of K562/HLA-E cells by staining with PE-labeled anti-HLA-E
monoclonal antibody (mAb) (3D12; BioLegend, USA), using ACEA NovoExpress
system (Agilent Technologies, USA). The results are presented as the
fluorescence index (FI). FI \selectlanguage{english}[?] 1 represents high-affinity peptide,
indicating that the stable combination of the peptide with HLA-E
molecules on surface of K562/HLA-E cells could increase the mean
fluorescence of the HLA-E molecules by at least one-fold.

\textbf{Isolation of CD8\textsuperscript{+} T cells with magnetic beads
kits}

The CD8\textsuperscript{+} T cells from PBMCs were isolated by magnetic
streptavidin nanobeads of Biolegend
MojoSort\textsuperscript{TM}Isolation Kits. Incubating PBMCs with biotin
antibody cocktail followed by incubation with magnetic streptavidin
nanobeads. Targeted cells were depleted by magnetically labeled fraction
using a magnetic separator, while untouched
CD3\textsuperscript{+}CD8\textsuperscript{+} cells were collected.

\textbf{K562/HLA-E cells pre-incubation with peptides}

Before incubation with peptides, K562/HLA-E*0103 cells were incubated at
26\selectlanguage{english} for 24 hours to adapt to the condition for antigen presentation in
advance. K562 cells were then incubated with peptides constructed (ul/10
cells) at 26\selectlanguage{english} for 19h for the next assay.

\emph{Ex Vivo} \selectlanguage{greek}\textbf{IFN-γ enzyme-linked immunospot assay (ELISPOT)}\selectlanguage{english}

The human IFN-\selectlanguage{greek}γ \selectlanguage{english}ELISPOT kit (DAKEWE Biotech Company, China) was used to
detect the capacity of IFN-\selectlanguage{greek}γ \selectlanguage{english}production by CD8\textsuperscript{+} T
cells stimulated with specific antigenic peptides presented by
K562/HLA-E cells as previously. The spots representing peptide-specific
IFN-\selectlanguage{greek}γ-\selectlanguage{english}producing CD8\textsuperscript{+} T cells were counted using an
automatic ELISPOT reader (Cellular Technology Limited, USA). Adjusted
spot-forming cells (SFCs) after subtracting negative values are
expressed as SFCs/10\textsuperscript{6} cells.

\textbf{Peptide/HLA-E*0103 tetramer staining}

Two peptides with high binding affinity S17 and S19 were selected for
construction of peptide/HLA-E tetramer labeled with PE by Epigen Biotech
(Nantong, China). The PBMCs of the SARS-CoV-2 vaccinated individuals
were stained with each PE-labeled peptide/HLA-E tetramer for 10 min, and
subsequently stained with FITC-labeled anti-human CD8 mAb (clone SK1,
Biolegend, US) and APC-labeled anti-human CD3 mAb (clone OKT3,
Biolegend, US). Approximately 300,000 cells gated on
CD3\textsuperscript{+} cells were captured. The gate for
CD8\textsuperscript{+} tetramer\textsuperscript{+} T cells was set up by
matching non-tetramer staining isotype control (BioLegend). Compensation
controls were checked regularly to avoid false-positive results and
individually determined for each experimental setup.

\textbf{Statistical analysis}

Statistical analysis was performed using GraphPad Prism software,
version 6 (GraphPad; La Jolla, CA, USA). The Mann--Whitney \emph{U} test
was used for parameter comparison between two subject groups. The
frequencies of epitope-specific CD8\textsuperscript{+} T cells are
presented as median with 95\% confidence interval (CI95\%) or median
with range.\emph{P} -Value (two-tailed) below 0.05 (\emph{P} [?] 0.05) was
considered to be statistically significant.

\textbf{Results}

\textbf{Thirty-six HLA-E-restricted peptides on SARS-CoV-2 were
predicted and synthesized}

HLA-E*0101 or HLA-E*0103-restricted peptides on SARS-CoV-2 structure
protein S protein, E protein, M protein and N protein were predicted.
According to the binding affinity, thirty-six predicted nonapeptides on
SARS-CoV-2 with strong binding or weak binding ability in either of the
two databases were selected. A total of 36 SARS-CoV-2-derived
HLA-E-restricted nonapeptides with the detail information including 25
peptides on S protein, one peptide on E protein, six peptides on M
protein and four peptides on N protein were listed in Table S1.

\textbf{Four nonapeptides of} \textbf{SARS-CoV-2 exhibited high binding
affinity to bothHLA-E*0103 and HLA-E*0101 molecules}

Then we used the K562/HLA-E cell binding assay to further screen the
high binding affinity SARS-CoV-2 nonapeptides to HLA-E*0103 or
HLA-E*0101 molecules, respectively. Among the 36 predicted HLA-E
restricted SARS-CoV-2 peptides, six peptides including S7, S10, S13,
S15, S19 and S25 could increase the expression of HLA-E*0101 on
K562/HLA-E cell surface characterized by fluorescence index (FI) [?] 1,
indicating a high binding affinity to HLA-E*0101 molecule (Figure 1A).
Moreover, four of the 23 predicted peptides including S7, S13, S19 and
S25 could also increase the HLA-E*0103 expression on K562/HLA-E cell
surface with FI [?] 1, suggesting a high binding affinity to HLA-A*0103
molecule as well (Figure 1B). Therefore, the four nonapeptides S7, S13,
S19 and S25 of SARS-CoV-2 exhibited high binding affinity to both
HLA-E*0103 and HLA-E*0101 molecules. However, there may be slight
difference of the binding affinity among these peptides according to the
FI value. Specifically, S19 showed higher binding affinity to HLA-E*0103
molecule, while S7 and S15 exhibited higher binding affinity to
HLA-E*0101 molecule. Detailed information for the screened peptides with
high binding affinity were summarized in Table S2.

\selectlanguage{greek}\textbf{HLA-E restricted epitopes ofSARS-CoV-2 were identified by
inducing specific IFN-γ-producing CD8\textsuperscript{+} T cell
responses}\selectlanguage{english}

We next assessed the capacity of each peptide to elicit epitope-specific
CD8\textsuperscript{+} T cell responses by detecting the secretion of
IFN-\selectlanguage{greek}γ \selectlanguage{english}\emph{in vitro} in SARS-CoV-2 vaccinated subjects. The results
showed that four peptides (S7, S13, S19, S25) could effectively elicit
epitope-specific IFN-\selectlanguage{greek}γ-\selectlanguage{english}secreting CD8\textsuperscript{+} T cell responses
in peripheral blood of vaccinated subjects (Figure 2A), indicating that
the four SARS-CoV-2 peptides could be defined as HLA-E restricted
CD8\textsuperscript{+} T cell epitopes. Specifically, the median (range)
was 108 (13-685) SFC/10\textsuperscript{6} PBMCs for peptide S7, 87
(10--516) SFC/10\textsuperscript{6} PBMCs for peptide S13, 102 (15--588)
SFC/10\textsuperscript{6} PBMCs for peptide S19 and 104 (8-591)
SFC/10\textsuperscript{6} PBMCs for peptide S25. However, there was only
11 out of 56 samples collected showed the HLA-E restricted
epitopes-specific CD8\textsuperscript{+} T cell responses. Moreover, the
frequencies of IFN-\selectlanguage{greek}γ-\selectlanguage{english}secreting CD8\textsuperscript{+} T cell responses
showed no difference among the four HLA-E restricted SARS-CoV-2 epitopes
(Figure 2B).

\textbf{The frequencies of HLA-E restricted SARS-CoV-2 epitopes-specific
CD8\textsuperscript{+} T cells could be detected in peripheral blood of
vaccinated individuals}

The SARS-CoV-2 epitopes S7 and S19 were selected to construct
HLA-E/peptide tetramer to accurately detect the frequencies of
epitope-specific CD8\textsuperscript{+} T cells in the peripheral blood
of SARS-CoV-2 vaccinated subjects. The frequencies of both epitope S7
and epitope S19-specific CD8\textsuperscript{+} T cells could be
detected (Figure 3A). The frequency of epitope S7-specific
CD8\textsuperscript{+} T cells in 56 samples ranged from 0.020\% to
1.120\% (median values: 0.11\%, CI95\%: 0.1234\%-0.2398\%), while the
frequency of epitope S19-specific CD8\textsuperscript{+} T cells ranged
from 0.020\% to 0.74\% (median value: 0.10\%, CI95\%:
0.1280\%-0.2156\%). Notably, there was no difference of the frequencies
of epitope-specific CD8\textsuperscript{+} T cells between the two
epitopes in each SARS-CoV-2 vaccination group (Figure 3B). When compared
the frequencies of epitope-specific CD8\textsuperscript{+} T cells among
three different of SARS-CoV-2 vaccines immunization groups for each
epitope, the frequencies of epitope-specific CD8\textsuperscript{+} T
cells in individuals receiving adenovirus-vectored vaccine group seemed
higher than that in inactivated vaccine group and recombinant protein
subunit vaccine group (Figure 3C).

\textbf{The frequencies of epitope-specific CD8\textsuperscript{+} T
cells were decreased in the individuals receiving two doses of
SARS-CoV-2 vaccination than only one dose}

The frequencies of epitope-specific CD8\textsuperscript{+} T cells in
individuals who received only one dose of Ad5-nCoV vaccine were further
analyzed according to the time interval between the latest vaccination
and sample collection. The results showed that the frequencies of
epitope-specific CD8\textsuperscript{+} T cells were elevated from the
time less than 30 days to the time 30 days-60 days, then decreased at
the time 60 days-90 days, and again elevated at the time 90 days-120
days, even more than 120 days. The trends of the frequencies of
epitope-specific CD8\textsuperscript{+} T cells at different time
quantum was similar between the epitope S7 and epitope S9 (Figure
4A-4B). Then we compared the frequencies of epitope-specific
CD8\textsuperscript{+} T cells in individuals who received different
doses of Ad5-nCoV vaccination. Notably, the frequencies of
epitope-specific CD8\textsuperscript{+} T cells in subjects with two
doses vaccination were lower compared with that in subjects with only
one dose vaccination (\emph{P} \textless{}0.05 for epitope S7) (Figure
4C-4D).

\textbf{Discussion}

SARS-CoV-2 vaccines inducing specific antibody production, meanwhile the
specific antiviral T cell responses may present better protective
effects for people. Therefore, exploring the vaccine-induced
SARS-CoV-2-specific CD8\textsuperscript{+} T cell responses based on
CD8\textsuperscript{+} T cell epitopes might be one of the important
approaches. In this study, we identified four nonamer epitopes on
SARS-CoV-2 restricted by HLA-E molecule, which could elicit
epitope-specific CD8\textsuperscript{+} T cell responses in SARS-CoV-2
vaccinated population. Importantly, the HLA-E-restricted SARS-CoV-2
epitope-specific CD8\textsuperscript{+} T cells showed high frequencies
in Ad5-nCoV vaccinated individuals. The frequencies of epitope-specific
CD8\textsuperscript{+} T cells were decreased in the individuals
receiving two doses of Ad5-nCoV vaccination than only one dose. These
results may greatly advance the understanding of the cellular immune
defense against SARS-CoV-2 infection, meanwhile contribute to more
comprehensive evaluating the protective effects of vaccines for
SARS-CoV-2 covering all the HLA-diverse populations.

The most ideal result after infection or vaccination is highly
protective and lasting immunity, so as to establish a high level of
immunity of the population. The large-scale inoculation of SARS-CoV-2
vaccine in population has greatly reduced the rates of infection and
severity. T cell response plays a key role in vaccine mediated
protection. It has been confirmed that vaccines that could induce
SARS-CoV-2 specific T cell response could effectively control infection,
avoid severe tissue damage, and significantly reduce hospitalization
rate and mortality. In clinical trials of SARS-CoV-2 mRNA vaccine
(BNT162b2; Modern VRC) or adenovirus vector vaccine (AdV5; ChAdOx),
SARS-CoV-2 specific CD8\textsuperscript{+} T cells could secrete high
level of IFN- \selectlanguage{greek}γ, \selectlanguage{english}which was similar to the level of specific
CD8\textsuperscript{+} T cell response in patients with COVID-19. The
inoculation of SARS-CoV-2 mRNA vaccine or adenovirus vector vaccine
could induce high frequency of CD8\textsuperscript{+} T cell response
specific to S protein with
CCR7\textsuperscript{-}CD45RA\textsuperscript{+}effect phenotype,
meanwhile could induce persistent memory CD8\textsuperscript{+} T cells
{[}3, 22-24{]}. Moreover, the strong SARS-CoV-2 specific T cell
responses could be detected in naturally infected individuals after
vaccination. Our results showed that the HLA-E restricted SARS-CoV-2
epitopes-specific CD8\textsuperscript{+} T cell responses could be
detected in the subjects vaccinated with adenovirus vector vaccine
Ad5-nCoV, further indicating that Ad5-nCoV inoculation could induce
effective T cell responses in the population.

HLA-E has been proved to present epitope to interact with \selectlanguage{greek}αβ\selectlanguage{english}TCR on
CD8\textsuperscript{+} T cells in several infectious diseases. Since
HLA-E is a ubiquitous HLA allele locus in the population, HLA-E could be
used as the first choice in more specific and effective peptide vaccine
design and research. The current research mainly focuses on the
mechanism of HLA-E mediated NK cell function in the process of
SARS-CoV-2 infection. It has been found that SARS-CoV-2 could induce
HLA-E expression on the surface of pulmonary epithelial cells, which
could bind peptides derived from S protein and interact with CD94/NKG2A
on NK cells {[}25{]}. The peptide derived from nsp13 of SARS-CoV-2
presented by HLA-E could not interact with NKG2A on NK cells, thus
mediating the killing effects of NK cells to virus-infected target cells
{[}26{]}. Therefore, it is necessary to identify HLA-E restricted
epitope of SARS-CoV-2, which would provide information for people who
suffered from COVID-19 disease or vaccinated with SARS-CoV-2 vaccine.
Based on the general rule of epitope binding motifs, nonapeptides which
could be presented by HLA-E molecule always showed mainly anchor
residues methionine (M), isoleucine (I) or leucine (L) at position 2,
and isoleucine (I) or leucine (L) at position 9. Therefore, the six
SARS-CoV-2 nonapeptides we identified with residue M/I/L at position 2
and residue L at C-terminal were conformed the general rule of
HLA-E-restricted peptides. Notably, our study found that two epitopes S7
and S19 on SARS-CoV-2 restricted by HLA-E could also be resented by
HLA-A*02 reported in other studies {[}14{]}. Considering that 86\%
sequence was similar between HLA-E and HLA-A*02 molecules, and HLA-A*02
molecules may share binding peptides with HLA-E molecule. Therefore,
this is a common phenomenon that HLA-E*01 and HLA-A*02 molecules always
could present the same epitopes such as influenza
M\textsubscript{159-167}, M\textsubscript{158-166} and Epstein-Barr
virus BZLF\textsubscript{139-147} due to conserved deep pockets
{[}27{]}. The co-presentation of antigen epitopes by both HLA-E*01 and
HLA-A*02 seems to be more important in inducing CD8\textsuperscript{+} T
cell response for HLA-E restriction epitopes.

It has been proved that receptor-binding domain (RBD) on S protein
contains a variety of conformational epitopes that could induce high
levels of antibody production {[}8{]}, especially specific antibody with
neutralizing activity. In addition, S protein also contains multiple
dominant T cell epitopes, which could induce effective T cell immune
response {[}28{]}. Therefore, S protein is the primary antigen used in
development of SARS-CoV-2 vaccines. RBD region of S protein has become
the core target for research of therapeutic neutralizing antibody and
the mechanisms for specific T cell responses. HLA-E restricted specific
CD8\textsuperscript{+} T cell epitopes identified in this study were all
from S protein of SARS-CoV-2. Although these epitopes aa269-aa277,
aa576-aa584, aa958-aa966 and aa1185-aa1193 were not locus within the RBD
region (aa329-aa521), they are still important for the potential
applications in future.

In fact, although SARS-CoV-2 non-structural protein (nsp) and open
reading frames (ORFs) such as ORF3, nsp3, nsp4 and nsp12 showed low
levels in SARS-CoV-2 infected cells, they also contain very important
CD8\textsuperscript{+} T cell epitopes. For example, ORF9b derived
epitopes could induce high-level virus specific CD8\textsuperscript{+} T
cell responses in COVID-19 patients, confirming good immunogenicity of
epitopes \emph{in vivo} {[}29{]}. Therefore, identification of HLA-E
restricted epitopes on nsp and ORFs of SARS-CoV-2 also make sense for
research the effects of CD8\textsuperscript{+} T cells in further study.

In conclusion, HLA-E-restricted SARS-CoV-2 epitope-specific
CD8\textsuperscript{+} T cell responses could be detected in Ad5-nCoV
vaccinated population. Importantly, the levels of epitope specific
CD8\textsuperscript{+} T cell responses could maintain for a long time
after only one dose vaccination. The study may be a good supplement for
evaluation of vaccination effects of Ad5-nCoV from the perspective of T
cell responses. However, HLA-E-restricted CD8\textsuperscript{+} T cell
responses induced in naturally infected individuals after vaccination
still need to be investigated in the future.

\textbf{Conflict of Interest}

The authors have no competing interests to declare.

\textbf{Funding Source}

This work was supported by the National Natural Science Foundation of
China, grant number 81871239; Natural Science Foundation of Fourth
Military Medical University, China, grant number 2020SWAQ02; Key
projects of natural science basic research plan in Shaanxi Province,
China, grant number 2023-JC-ZD-49.

\textbf{Ethical Approval statement}

The study was conducted in accordance with the Declaration of Helsinki,
and approved by the Institutional Review Board of the Fourth Military
Medical University. The research involving humans and human materials
was also approved by the Ethical Review Board of the Fourth Military
Medical University (KY20212112-C-1), and the related data were
anonymized before use.

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10.1016/j.cell.2021.05.046.\selectlanguage{english}
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