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\begin{document}

\title{Inactivated vaccine fueled adaptive immune responses to Omicron in
2-year COVID-19 convalescents}



\author[1]{William J. Liu}%
\author[1]{Maoshun Liu}%
\author[2]{Jie Zhang}%
\author[1]{Lei Li}%
\author[3]{Jinmin Tian}%
\author[1]{Mengjie Yang}%
\author[1]{Bingli Shang}%
\author[3]{Xin Wang}%
\author[3]{Min Li}%
\author[1]{Hongmei Li}%
\author[4]{Can Yue}%
\author[1]{Sijia Yao}%
\author[3]{Ying Lin}%
\author[3]{Yuanyuan Guo}%
\author[1]{Kexin Zong}%
\author[3]{Danni Zhang}%
\author[3]{Yingze Zhao}%
\author[5]{Kun Cai}%
\author[5]{Shaobo Dong}%
\author[5]{Shengping Xu}%
\author[5]{Jianbo Zhan}%
\author[5]{Yongzhong Jiang}%
\author[1]{George-Fu Gao}%
\affil[1]{Wenzhou Medical University School of Laboratory Medicine and Life Sciences}%
\affil[2]{Capital Medical University Affiliated Beijing Ditan Hospital}%
\affil[3]{Chinese Center for Disease Control and Prevention}%
\affil[4]{University of the Chinese Academy of Sciences Savaid Medical School}%
\affil[5]{Hubei Provincial Center for Disease Control and Prevention}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Over three years, humans have experienced multiple rounds of global
transmission of SARS-CoV-2 and its variants. In addition, the widely
used vaccines against SARS-CoV-2 involve multiple strategies of
development and inoculation. Thus, the acquired immunity established
among humans is complicated, and there is a lack of understanding within
a panoramic vision. Here, we provide the special characteristics of the
cellular and humoral responses in 2-year convalescents after inactivated
vaccines, in parallel to vaccinated COVID-19 na\selectlanguage{ngerman}ïve persons and
unvaccinated controls. The decreasing trends of the IgG, IgA, and NAb,
but not IgM of the convalescents were reversed by the vaccination. Both
cellular and humoral immunity in convalescents after vaccination were
higher than the vaccinated COVID-19 naïve persons. Notably, inoculation
with inactivated vaccine fueled the NAb to BA.1, BA.2, BA.4, and BA.5 in
2-year convalescents, much higher than the NAb during 6 months and 1
year after symptoms onset. And no obvious T cell escaping to the S
protein was observed in 2-year convalescents after inoculation. The
study provides insight into the complicated features of human acquired
immunity to SARS-CoV-2 and variants in the real world, indicating that
promoting vaccine inoculation is essential for achieving herd immunity
against emerging variants, especially in convalescents.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Inactivated vaccine fueled adaptive immune responses to Omicron
in 2-year COVID-19 convalescents}

Maoshun Liu\textsuperscript{1,2,\#}, Jie
Zhang\textsuperscript{3,4,5,\#}, Lei Li\textsuperscript{1,2}, Jinmin
Tian\textsuperscript{6,2}, Mengjie Yang\textsuperscript{1,2}, Bingli
Shang\textsuperscript{1,2}, Xin Wang\textsuperscript{2,7}, Min
Li\textsuperscript{2}, Hongmei Li\textsuperscript{1,2}, Can
Yue\textsuperscript{2,8}, Sijia Yao\textsuperscript{1,2}, Ying
Lin\textsuperscript{2,7}, Yuanyuan Guo\textsuperscript{2,7}, Kexin
Zong\textsuperscript{1,2}, Danni Zhang\textsuperscript{2}, Yingze
Zhao\textsuperscript{2}, Kun Cai\textsuperscript{9}, Shaobo
Dong\textsuperscript{10}, Shengping Xu\textsuperscript{10}, Jianbo
Zhan\textsuperscript{9}, Yongzhong Jiang\textsuperscript{9}, George F.
Gao\textsuperscript{6,1,2,7,8,11,12,*}, and William J.
Liu\textsuperscript{1,2,12,*}

\textsuperscript{1}Zhejiang Provincial Key Laboratory of Medical
Genetics, Key Laboratory of Laboratory Medicine, Ministry of Education,
School of Laboratory Medicine and Life Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China.

\textsuperscript{2}NHC Key Laboratory of Biosafety, National Institute
for Viral Disease Control and Prevention, Chinese Center for Disease
Control and Prevention (China CDC), Beijing 102206, China.

\textsuperscript{3}Beijing Key Laboratory of Emerging Infectious
Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital,
Capital Medical University, Beijing 100015, China.

\textsuperscript{4}Beijing Institute of Infectious Diseases, Beijing,
100015, China.

\textsuperscript{5}National Center for Infectious Diseases, Beijing
Ditan Hospital, Capital Medical University, Beijing 100015, P.R. China.

\textsuperscript{6}School of Ophthalmology \& Optometry, Wenzhou Medical
University, Wenzhou 325035, China.

\textsuperscript{7}Department of Epidemiology, School of Public Health,
Cheeloo College of Medicine, Shandong University, Shandong 250100,
China.

\textsuperscript{8}Savaid Medical School, University of Chinese Academy
of Sciences, Beijing 100049, China.

\textsuperscript{9}Hubei Provincial Center for Disease Control and
Prevention, Wuhan 430079, China.

\textsuperscript{10}Macheng Center for Disease Control and Prevention,
Huanggang 438300, China.

\textsuperscript{11}CAS Key Laboratory of Pathogen Microbiology and
Immunology, Institute of Microbiology, Chinese Academy of Sciences
(CAS), Beijing 100101, China.

\textsuperscript{12}Research Unit of Adaptive Evolution and Control of
Emerging Viruses, Chinese Academy of Medical Sciences, Beijing 102206,
China.

\# Maoshun Liu and Jie Zhang contributed equally to this work.

* \textbf{Corresponding Author:}

William J. Liu, PhD; National Institute for Viral Disease Control and
Prevention, Chinese Center for Disease Control and Prevention (China
CDC), Changping District, Beijing 102206, China. Tel.: 86-10-63510565;
Fax: 86-10-63510565; E-mail:\emph{liujun@ivdc.chinacdc.cn};

George F. Gao, DPhil; Institute of Microbiology, Chinese Academy of
Sciences (CAS), Beijing 100101, China. Tel.: 86-10-64807688; Fax:
86-10-64807882; E-mail:\emph{gaof@im.ac.cn}.

\textbf{Running Head:} Hybrid adaptive immune responses to Omicron.

\textbf{Abstract}

Over three years, humans have experienced multiple rounds of global
transmission of SARS-CoV-2 and its variants. In addition, the widely
used vaccines against SARS-CoV-2 involve multiple strategies of
development and inoculation. Thus, the acquired immunity established
among humans is complicated, and there is a lack of understanding within
a panoramic vision. Here, we provide the special characteristics of the
cellular and humoral responses in 2-year convalescents after inactivated
vaccines, in parallel to vaccinated COVID-19 na\selectlanguage{ngerman}ïve persons and
unvaccinated controls. The decreasing trends of the IgG, IgA, and NAb,
but not IgM of the convalescents were reversed by the vaccination. Both
cellular and humoral immunity in convalescents after vaccination were
higher than the vaccinated COVID-19 naïve persons. Notably, inoculation
with inactivated vaccine fueled the NAb to BA.1, BA.2, BA.4, and BA.5 in
2-year convalescents, much higher than the NAb during 6 months and 1
year after symptoms onset. And no obvious T cell escaping to the S
protein was observed in 2-year convalescents after inoculation. The
study provides insight into the complicated features of human acquired
immunity to SARS-CoV-2 and variants in the real world, indicating that
promoting vaccine inoculation is essential for achieving herd immunity
against emerging variants, especially in convalescents.

\textbf{Keywords:} Omicron; SARS-CoV-2; convalescents; inactivated
vaccine; T cell; antibody.

\textbf{Introduction}

The coronavirus disease 2019 (COVID-19) pandemic has been wreaking havoc
for nearly three years, with more than 758 million infected cases
worldwide as of March 2023 (\emph{https://covid19.who.int/}). The herd
immunity raised by the vaccination or natural infection has been
recognized as the barrier to cut off the transmission of the virus.
Thus, the immune memory characteristics of the convalescents and the
vaccinees, including cellular and humoral immunity have raised wide
concern.\textsuperscript{1-3} Cellular immune memory can last up to 1
year after the COVID-19 patient's recovery,\textsuperscript{2,3} while
for humoral immunity, neutralizing antibodies (NAb) are still detectable
16 months after symptoms onset, but the neutralizing activity of
antibodies has marked declined over time.\textsuperscript{4} But the
immune statuses of convalescents become complicated in consideration of
the application of vaccines.

In the past three years, to effectively prevent COVID-19 and achieve
herd immunity with minimal loss of life, different types of vaccines
have been developed and widely used globally, such as mRNA, inactivated,
polypeptide subunit, and adenovirus vector
vaccine.\textsuperscript{5-8}It was indicated that S-RBD IgG, NAb, and
CD4\textsuperscript{+} T cell responses waned quickly after a single
inactivated vaccine dose, and further increased after the second dose.
Virus-specific CD4\textsuperscript{+} T cell responses generated by two
doses of vaccination lasted more than 2 months.\textsuperscript{7}A
significantly reduced neutralizing activity against Omicron was observed
in the convalescent and two-dose BBIBP-CorV vaccination group, which was
elevated by a third homologous inactivated vaccine
booster.\textsuperscript{9} Studies also showed that the Omicron
breakthrough infected individuals immunized with inactivated vaccines
maintained high level of neutralization against wild type (WT) and
variants, and the enhancement effects by breakthrough infection were of
significant differences between 2-dose and 3-dose
groups.\textsuperscript{10}Prior immunization with inactivated prototype
strain vaccines substantially restrains pneumonia development, reduces
cytokine storms, and facilitates clinical recovery after virus
breakthrough.\textsuperscript{11} However, the features of the humoral
and cellular immune memory are still largely unknown, especially among
COVID-19 convalescents in the first wave during the pandemic who later
received two or three doses of inactivated vaccines.

The emergence of different severe acute respiratory syndrome coronavirus
type 2 (SARS-CoV-2) variants, notably Delta and Omicron, has led to
successive waves of pandemics, posing enormous public health
challenges.\textsuperscript{12} Studies showed that Omicron is more
transmissible than other variants.\textsuperscript{13-16} The Omicron
variant harbors 32 mutations in spike
glycoproteins,\textsuperscript{17}raising concern about the Omicron
escaping from immunity established by WT infection or vaccination.
Studies have shown that sera from COVID-19 convalescents or individuals
who were vaccinated exhibited substantially diminished neutralizing
activity against Omicron.\textsuperscript{18-23}It has been reported
that mRNA-1273, BNT162b2, Ad26.COV2.S, and NVX-CoV2373 vaccines elicit
highly conserved cellular immunity to Omicron.\textsuperscript{24-28}
The memory status of T-cell immunity and the cross-reactivity to Omicron
among inactivated vaccines were still unexplored, especially in terms of
the vaccines recovered from COVID-19.

In this study involving COVID-19 and control cohorts, we assessed the
antibody and T-cell responses to WT and Omicron strains in COVID-19
convalescents who have later inoculated with inactivated vaccines, and
healthy individuals after routine two- or three-dose vaccination. This
study will expand knowledge of the immune features and the memory
persistence in vaccinated COVID-19 convalescents.

\textbf{2. Methods}

\textbf{2.1 Sample collection}

A total of 112 participants were recruited in Macheng and Beijing,
China. Participants comprised 51 COVID-19 vaccinated convalescents
(2Y-Vac), 35 healthy vaccinated controls (HC-Vac), and 26 healthy
unvaccinated controls (HC-Unvac). 23 out of the 51 2-year convalescents
were also kept prior to vaccination during the followed up at 6 months
(6m) and 1 year (1Y) after symptoms onset (Table 1). Sera and PBMCs of
whole blood were separated and frozen immediately for subsequent
analysis (Supplementary Methods).

\textbf{2.2 SARS-CoV-2 antibodies test}

The titer of anti-SARS-CoV-2 IgG, IgM, and IgA were measured by
enzyme-linked immunosorbent assay (ELISA) (WANTAI BioPharm, China) and
magnetic chemiluminescence enzyme immunoassay (MCLIA) (Bioscience based
on indirect immunoassay, Tianjin, China) as described
previously.\textsuperscript{3} Neutralizing antibodies (NAb) against
S-RBD of WT, Delta, BA.1, BA.2, and BA.4/5 strains (Supplemental Figure
3), were measured using competitive enzyme-linked immunosorbent assay
(cELISA) (Vazyme, China) (Supplementary Methods).

\textbf{2.3 Peptide pools}

15- to 18-mer peptides with 10 amino acids of overlap were synthesized
and covered the full length of S, M, and N proteins. Meanwhile, the
peptide pools spanned WT and BA.1 RBD were also designed. PBMCs were
cultured for 9 days with S1, S2, M, and N peptide pools stimulation,
respectively (Supplementary Methods).\textsuperscript{3}

\textbf{2.4 SARS-CoV-2-specific T cell responses}

Enzyme-linked immunospot (ELISpot) assay (BD, USA) and intracellular
cytokine staining (ICS) were used to test SARS-CoV-2-specific T cell
reaction in 2-year COVID-19 convalescents and health controls, as
described in our previous study (Supplementary
Methods).\textsuperscript{3}

\textbf{2.5 Statistical analysis}

The statistical analyses were conducted with IBM SPSS Statistics 26.0. A
comparison of antibodies and T-cell reaction between groups were
analyzed using the Mann-Whitney U-test and Paired Wilcoxon Signed-Rank
Test. The difference in positive rate was performed by the Chi-square
test. The figures were done with GraphPad Prism 8.0.2. The statistical
significance of these results was defined as * p \textless{} 0.05, ** p
\textless{} 0.01, and *** p \textless{} 0.001.

\textbf{3. Results}

\textbf{3.1 SARS-CoV-2 antibody features in 2-year convalescents after
inactivated vaccines}

To increase the vaccination rate coverage of SARS-CoV-2 and construct
herd immunity, the 2-year COVID-19 convalescents in Macheng were
vaccinated in the second half of 2021. Herein, to assess
SARS-CoV-2-specific antibody features of COVID-19 convalescents, who had
recovered for 2 years after inactivated vaccines (CoronaVac or
BBIBP-CorV or WIBP-CorV), sera from participants was evaluated for the
presence of IgG, IgM, IgA, and NAb together with the vaccinated healthy
control donors in the same town (Figure 1A). We found that over 96\% of
2-year convalescents after inactivated vaccines have a positive IgG
response to WT, as presented by the double positive for ELISA and MCLIA
tests (Supplementary Table 1). No significant difference was observed in
the positive proportion of IgG between the healthy individuals (ELISA:
85.7\%, MCLIA: 94.3\%) and 2-year convalescents (96.1\% for both
methods) after the inoculation of inactivated vaccines (Supplementary
Table 1). However, IgG levels of 2-year convalescents after inactivated
vaccines were higher compared to healthy individuals with either
vaccinated (p \textless{} 0.0001) or unvaccinated (p \textless{} 0.0001,
Figure 1B, C). 2-year convalescents after vaccination exhibited IgG
titers that were 1.2-fold more than healthy individuals after
vaccination (Figure 1B, C). Certain levels of IgM could be detected in
both vaccinated 2-year convalescents and healthy individuals but with no
significant difference (31.4\% vs 28.6\% in ELISA; 23.5\% vs 20.0\% in
MCLIA) (Figure 2A, B and Supplementary Table 1).

To reveal the dynamic antibody responses within the convalescents, we
retrieved the paired sera at the 6-month and 1-year follow-up visits of
the convalescents. The 2-year convalescents had higher IgG antibodies
following vaccination in contrast with the 1-year visit, with 50\%
(9/18) of employees increased more than 1-fold (p = 0.005 for ELISA and
p = 0.004 for MCLIA), but with no difference with the 6-month visit
(Figure 1D, E). However, a significant reducing trend was observed for
IgM levels among the convalescents from 6 months to 2-year recovery,
although the convalescents were vaccinated before the 2-year visit
(Figure 2C, D). We next analyzed whether severity affected the levels of
IgG/IgM, and no differences were observed among vaccinated 2-year
convalescents with different disease severities (Figure 1F, G and Figure
2E, F).

We also tested the IgA antibody within the sera of the donors. 58.8\%
(30/51) of 2-year convalescents had positive IgA responses to SARS-CoV-2
S protein RBD, which were significantly higher compared with the
vaccinated individuals without prior infection (Figure 1H). For a
longitudinal analysis of the IgA antibodies among the convalescents, the
avidity of IgA antibodies decreased from 6-month to 1-year visits but
increased significantly during the 2-year visit in the convalescents
(Figure 1I).

To demonstrate the potential influence of the vaccination dose on the
antibody levels among the convalescents and the healthy individuals, we
compared the antibodies from healthy individuals after 3 doses and
2-year convalescents vaccinated with 2 doses. The IgG, IgA, and NAb
levels of 2-year convalescents after 2 doses of inactivated vaccines
were even higher than healthy individuals with 3 doses of vaccination
(Supplementary Figure 1A, B, E, F). No IgM differences were observed
between the two groups (Supplementary Figure 1C, D). Overall, these
results suggest that 2-year convalescents after inoculated inactivated
vaccines possibly develop and maintain stronger IgG, IgA, and NAb but
not IgM antibodies responses to the WT stain than vaccinated healthy
individuals.

\textbf{3.2 T cell reactivity to SARS-CoV-2 in 2-year convalescents
after inactivated vaccines}

We also compared the SARS-CoV-2-specific T cell responses among the
2-year convalescents and healthy controls after inactivated vaccines,
assessed by ELISpot and ICS assays (Figure 3A). In the \emph{ex
vivo}ELISpot assay based on freshly-isolated PBMCs, both two groups have
low-frequency antigen-specific T cell responses, with no obvious
difference between 2-year convalescents and healthy controls (Figure
3B). After incubating with viral S1, S2, M, and N peptides \emph{in
vitro} , the positive proportion with IFN-\selectlanguage{greek}γ \selectlanguage{english}responses in the population
showed a noteworthy increase (Figure 3C). 92.2\% (47/51) of the 2-year
convalescents had positive T-cell response memory to at least one of
these four antigens, which was markedly higher than the corresponding
proportion (54.3\%) of vaccinated healthy controls (Figure 3F and
Supplementary Table 2). Meanwhile, the altitudes of T cell responses to
M and N peptide pools, as well as S1 and S2, were greater in the 2-year
convalescents (median 500 SFU/10\textsuperscript{6}~PBMCs {[}IQR
190-1582.5{]} for M; median 485 SFU/10\textsuperscript{6}~PBMCs {[}IQR
200-1487.5{]} for N) than in the healthy controls following vaccination
(median 20 SFU/10\textsuperscript{6}~PBMCs {[}IQR 0-105{]} for M; median
20 SFU/10\textsuperscript{6} PBMCs {[}IQR 0-222.5{]} for N) (Figure 3C).
We also compared the T cell immune responses between the four peptide
pools and found that proteins M and N overlapping peptide pools-specific
T cell responses were much higher than S protein among the 2-year
convalescents after vaccination (Figure 3C). 2-year convalescents after
2 doses immunization can produce even significantly higher IFN-\selectlanguage{greek}γ
\selectlanguage{english}responses than healthy controls inoculated with the 3 doses vaccination,
which further underlined the significance of memory T-cell responses
induced by the previous infection, and boosted by the following
vaccination (Supplementary Figure 2A, B). But there were no significant
differences in T cell immune responses between the 2-year convalescents
with different severity during the acute infection (Figure 3D, E).

To further investigate the SARS-CoV-2-specific CD8\textsuperscript{+}and
CD4\textsuperscript{+} T cells in participants, we stimulated PBMCs with
peptide pools of four antigens (S1, S2, M, and N) and measured cytokine
production (IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-2, and TNF-\selectlanguage{greek}α\selectlanguage{english}) using flow cytometry (Figure 4A).
Though without statistical significance, the median ratios of
cytokine-secreting T cells against four peptide pools were higher in the
2-year convalescents than in healthy controls who had been vaccinated
(Figure 4B, C). Furthermore, specific T cells among both groups
developed stronger cellular immunity than individuals with neither
infection nor vaccination (Figure 4C).

\textbf{3.3 The Omicron variant cross-recognizing feature of ancestral
SARS-CoV-2-specific T cell and antibody}

We also explored whether the Omicron variant immune evasion exists for
the antibodies and T cell responses induced by the inactivated vaccine
inoculated 2-year convalescents. We performed a competitive ELISA assay
to further evaluate the S protein RBD-binding neutralizing antibodies
against the WT strain as well as Delta and Omicron (BA.1, BA.2, and
BA.4/5). When comparing the titers at three time points, i.e. 6 months,
1 year and 2 years, neutralizing capacities of sera showed a decreasing
trend between 6-month and 1-year cohorts against the WT as well as other
variants (Figure 5A). The inhibition rates of NAb to each strain were
markedly raised in the vaccinated 2-year convalescents compared to their
unvaccinated time points at 6 months and 1 year, with median inhibition
rates against five strains (WT, Delta, BA.1, BA.2, and BA.4/5) in the
2-year visit that were 1.3, 1.4, 4.5, 2.0, and 2.9 times those for the
1-year visit (Figure 5A). None of the individuals had detectable
neutralizing antibodies in the unvaccinated cohort (Figure 5B). In the
sera of 2-year convalescents with vaccination, the median NAb inhibition
rates against the WT, Delta, BA.1, BA.2, and BA.4/5 strains (94.5, 90.9,
39.3, 47.8, and 37.6\%), were much higher than in healthy controls with
vaccination against these strains (74.7, 54.7, 11.8, 16.7, and 14.0\%)
(Figure 5B). The median anti-RBD NAb inhibition levels against WT,
Delta, BA.1, BA.2, and BA.4/5 strains were higher by 1.3-, 1.7-, 3.3-,
2.9, 2.7-fold, respectively, in the vaccinated 2-year convalescents as
compared to the vaccinated healthy controls (Figure 5B). The Nab
inhibition against WT and other variants was directly compared within
2-year convalescents and healthy controls after vaccination (Figure 5C,
D). The positive proportions of vaccinated 2-year convalescents sera
against BA.1 (70.6\%), BA.2 (78.4\%), and BA.4/5 (82.4\%) variants were
lower than WT (94.1\%) and Delta (94.1\%) but mostly above the detection
threshold (Figure 5C and Supplementary Table 3). However, we found
statistical differences among all five strains, except for BA.1 and
BA.4/5 strains (p \textless{} 0.001, Figure 5C). For the 2-year
convalescents who had a history of vaccination, non-reduction in the
median ratio of neutralizing activity of Delta, a 2.4-fold reduction of
BA.1, a 2.0-fold reduction of BA.2, and a 2.5-fold reduction of BA.4/5
was shown, compared with the neutralization of WT strain (Figure 5C).
Even lower proportions of healthy controls after vaccinations showed
positive neutralization responses against BA.1 (34.3\%), BA.2 (37.1\%)
as well as BA.4/5 (34.3\%), and a markedly drop in comparison with WT
(85.7\%) and Delta (80.0\%) was observed (Figure 5D and Supplementary
Table 3). Notably, in the healthy controls, the cross-neutralizing
antibodies were much lower than that for 2-year convalescents after
vaccination. The drop level of NAb inhibition rates against Delta, BA.1,
BA.2, and BA.4/5 was 1.4-, 6.3-, 4.5-, and 5.3-fold respectively,
compared to WT (Figure 5D).

Comparing the geometric mean ratio of four SARS-CoV-2 variants NAb
inhibition rates normalized WT, these data indicate that 2-year
vaccinated convalescents lead to more broadly cross-neutralization of
BA.1 and BA.4/5 as compared with healthy individuals with vaccination,
but not for Delta and BA.2 (Figure 5E). We also calculated the
seropositivity rates of NAb against all four SARS-CoV-2 strains,
regarding cross-protection of neutralizing antibodies. We observed a
higher proportion of antibodies neutralized overall stains in the
convalescent participants (68.6\%) compared to the healthy controls
following vaccination (28.6\%) (p = 0.001, Figure 5F). These data show
that 2-year COVID-19 convalescents with vaccination have more tolerance
to the Omicron variant than healthy controls after vaccination, though
both groups were inoculated with the inactivated vaccine for the WT
strain.

Furthermore, we assessed the T cell responses from 2-year convalescents
and healthy controls after vaccination to WT and Omicron BA.1 peptide
pools (RBD, S1, and S2) (Figure 6A). T cell reactivities in 2-year
convalescents after inoculated inactivated vaccines were significantly
higher than in healthy controls against WT and BA.1 peptide pools (p
\textless{}0.001) (Figure 6B). Notably, we found no remarkable
difference in IFN-\selectlanguage{greek}γ \selectlanguage{english}levels between WT and BA.1 for 2-year convalescents
(median 150 SFU/10\textsuperscript{6}~PBMCs for WT S1; median 130
SFU/10\textsuperscript{6}~PBMCs for BA.1 S1) and healthy controls
following immunization (Figure 6B). Based on the profile of cytokine
expression (IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-2, TNF-\selectlanguage{greek}α\selectlanguage{english}) after stimulation with either WT or BA.1
S1/S2 antigens among the 2-year convalescents and healthy controls after
vaccination, we demonstrated that high cross-CD4\textsuperscript{+} and
CD8\textsuperscript{+} T cell responses retained against BA.1 in both
2-year convalescents and healthy controls after vaccination (Figure 6C,
D). In conclusion, the spike-specific T cell reactivity produced by
vaccination and/or previous infection is vastly preserved against
Omicron BA.1.



\textbf{4. Discussion}

Since its emergence in 2019, the SARS-CoV-2 virus has continued to
evolve into different mutant strains, posing continuous challenges to
the control of COVID-19.\textsuperscript{29,30} To achieve the goal of
herd immunity, a variety of vaccines have been introduced
worldwide.\textsuperscript{31} One of the main vaccines promoted in
China is the inactivated vaccines, which has a high inoculation coverage
among the population up to date.\textsuperscript{32} Thus, features of
the immune memory triggered by natural infections of SARS-CoV-2 have
become complicated, as several COVID-19 convalescents were vaccinated.
In this study, we mainly focused on the SARS-CoV-2-specific humoral and
cellular immunity and the ability to protect against Omicron variants
after inoculated inactivated vaccines in 2-year convalescents and
general healthy controls. Data from our study showed that IgG, IgA, and
neutralizing antibodies were significantly higher in vaccinated
convalescents than in vaccinated healthy controls. Though both groups
had lower cross antibodies to Omicron variants, the vaccinated 2-year
convalescents kept higher cross-antibody responses than the vaccinated
healthy controls. Meanwhile, the convalescents after vaccination carried
robust T cell immune memory to WT and no T cell immune escape was
observed for Omicron BA.1, and with a higher T cell immunity level than
the vaccinated healthy controls.

Our previous longitudinal study of COVID-19 convalescents demonstrated
that SARS-CoV-2-specific humoral immunity was present within 95\% of
convalescents at 1-year post-disease onset,\textsuperscript{3} which was
also concordant with other cohort
studies.\textsuperscript{1,2,33,34}Recently, it was reported that NAbs
against SARS-CoV-2 could persist for one and a half years among COVID-19
convalescents.\textsuperscript{4,35}However, most of the studies
indicated a decreasing trend of the antibody levels with time among the
convalescents.\textsuperscript{1,4}In our study, through the antibody
titers decreased from 6 months to 1 year, a sharply increasing can be
observed among the 2-year convalescents after administration with
inactivated vaccines, showing the robust antibody-inducing capacity of
the inactivated vaccines,\textsuperscript{7,36,37} especially among the
population after natural infection. The enhanced antibody level may play
a positive role in the convalescents to avoid re-infection. Thus,
vaccination should still be encouraged for the
convalescents.\textsuperscript{38}

Although the IgG antibody could be obviously reinforced by the
inactivated vaccine among the 2-year convalescents after symptoms onset,
our data revealed positive rates of IgM antibody continued the
decreasing trend even among the convalescents after vaccination,
indicating that IgG among the convalescents could be more easily
enhanced by the inactivated vaccines.\textsuperscript{39}

Previous studies showed that IgA dominates the early neutralizing
antibody response to SARS-CoV-2 and may play an important role in the
prevention of severe illness.\textsuperscript{40,41} And a higher
concentration of IgA antibody can be detected in the convalescents who
received booster vaccination of mRNA vaccine, but there is no
significant increase in booster-vaccinated COVID-19 na\selectlanguage{ngerman}ïve
people.\textsuperscript{33} Our studies indicated that the decreasing of
IgA antibodies in follow-up sera from 6 months to 1 year after symptoms
onset, and observed inactivated vaccines could also induce an increase
of IgA antibody titer at 2 years post-infection.

The persistence of T-cell immunity stimulated by natural infection may
play an important role in alleviating severe disease in the
reinfection.\textsuperscript{42} Here we found that T cell immune memory
was much higher in the 2-year convalescents after vaccination than the
vaccinated even boosted healthy controls, demonstrating that T cell
immune memory by natural infection could persist up to 2 years after
symptoms onset. Several studies show that mRNA vaccines could stimulate
a certain level of virus-specific CD4\textsuperscript{+} and
CD8\textsuperscript{+} T-cell immunity,\textsuperscript{8,43,44} while
T-cell immunity induced by inactivated vaccines is far from being
well-defined. However, unlike inactivated vaccines, mRNA vaccines are
designed primarily to focus on the S protein, the cellular immunity
stimulated by this vaccine only targets the S
protein.\textsuperscript{8} Our data showed that inactivated vaccines
could stimulate T cell immunity against structural proteins S, M, and N
in COVID-19 naïve people. Nevertheless, when evaluating the inactivated
vaccine-related T cell responses, the potential baseline cross-T cell
immunity against M or N proteins of common cold coronaviruses would be
considered.\textsuperscript{3}

Despite extensive mutations-related neutralization escape against
Omicron could be observed, the overall T cell responses induced by
vaccination or infection could cross-recognize the
variants.\textsuperscript{28} We verified that the neutralizing ability
of the antibodies to Delta, Omicron BA.1, BA.2, and BA.4/5 decreased
among vaccinated 2-year convalescents and healthy controls. However, the
cross-neutralizing ability against the variants among the convalescents
was significantly stronger than that of the vaccinated healthy controls.
This may indicate a special cross-reactive antibody-inducing feature by
natural infection and the necessity for vaccination among the
convalescents. Previous studies determined that cellular immunity
elicited by the mRNA vaccine could cross-recognize the
Omicron.\textsuperscript{26,28,45} We herein showed that the cellular
immunity stimulated by inactivated vaccine also has a cross-response to
Omicron. As it turns out, the vaccination of inactivated vaccine is
beneficial to the population against the WT and its variants, and it is
an effective method to realize herd immunity.

Our current study has several limitations. First, the number of
subjects' PBMCs available for the ICS test is limited, thus the
statistical analysis has difficulties showing the differences between
groups in the ICS test. However, our main conclusions are based on the
statistical differences within the ELISpot assays and antibody tests. We
also lacked PBMCs from unvaccinated healthy controls to estimate T cell
responses and it was impossible to recruit additional volunteers since
herd immunity had been established in China. Second, we did not perform
PCR testing on the healthy controls to exclude asymptomatic infection,
but this was supported by our earlier epidemiological
analysis.\textsuperscript{46} Third, we use the ELISA kit with the
antigen S-RBD as a surrogate for live virus neutralization assay, the
detection of the neutralizing ability may be inaccurate because other
regions could also be neutralizing. However, previous studies suggested
that using RBD as an immunogen might be a better strategy for eliciting
a robust NAb response compared with full-length S
protein,\textsuperscript{47} and the ELISA results were highly
responsive to neutralizing antibody levels.\textsuperscript{48}

In summary, we provide the special characteristics of the cellular and
humoral immune responses in 2-year convalescents after the inoculation
of inactivated vaccines. The decreasing trends of the IgG, IgA, and
neutralizing antibodies, but not IgM of the convalescents were reversed
by the vaccination. And both cross-reactive cellular and humoral
immunity to Omicron variants in convalescents after vaccination were
higher than in the vaccinated healthy controls. Thus, promoting vaccine
inoculation is an essential way to achieve herd immunity during the
ongoing COVID-19 pandemic, even for the convalescents.

\textbf{Conflict of interest}

The authors have no financial conflicts of interest.

\textbf{Author Contributions}

W.J.L. and G.F.G. designed and supervised the study. MS.L., L.L., J.T.,
J.Z., M.L., S.D., J.Z., K.C, Y.J., and S.X. collected the samples.
MS.L., L.L., M.Y., X.W., Y.L., and B.S. conducted the experiments. J.Z.,
M.L., H.L., C.Y., S.Y., Y.G., K.Z., D.Z., and Y.Z. provided technical
support and experimental assistance. MS.L. and W.J.L. analyzed and
interpreted data. MS.L., J.Z., and W.J.L. wrote the initial draft of the
manuscript. All authors contributed intellectually and approved the
manuscript.

\textbf{Acknowledgments}

The study was supported by the National Key Research and Development
Program of China (2022YFC2604105 and 2021YFC2301400), the National
Natural Science Foundation of China (82161148008 and 81971501), and CAMS
Research Units of Adaptive Evolution and Control of Emerging Viruses
(2018RU009).

\textbf{Ethics Statement}

The study was approved by the Ethics Committee of National Institution
for Viral Disease Control and Prevention, China CDC (Ethical approval
No. IVDC2020-021).

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\textbf{Table 1}

Characteristics of participants in this study.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\par\null\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Convalescents-6months\textsuperscript{a} (n=23)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Convalescents-1year\textsuperscript{a} (n=23)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Convalescents-2 year (Vaccination) (n=51)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Health control (Vaccination) (n=35)\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Health control (Vaccination) (n=26)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Sex\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Male\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9(39.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9(39.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
25(49.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
25(71.4\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
14(53.8\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Female\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
14(60.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
14(60.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26(51.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10(28.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
12(46.2\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Age(years)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Median (IQR)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
57(45,69.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
57(45,69.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
51(44,60)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
47(42.5,51.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
37.5(26.25,60)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Range\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
37-76\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
37-76\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
31-76\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
21-68\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
22-71\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Severity\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Asymptomatic\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1(4.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1(4.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2(3.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Mild\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9(39.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9(39.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
23(45.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Moderate\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10(43.5\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10(43.5\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
19(37.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Severe\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3(13.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3(13.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7(13.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Vaccination\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Unvaccination\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1 dose\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5(9.8\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2 doses\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
36(70.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3(8.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3 doses\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10(19.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
32(91.4\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Days since last dose (Median, IQR)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1dose\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
151(147,153)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2 doses\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
124(106,175.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
150(142.5,163)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3 doses\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.5(1.25,31.25)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
62.5(52.75,65.25)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Days post after symptoms onset\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Median (IQR)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
177(172.5,179.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
353(348.5,355.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
696(691,699)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
---\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a} 23 of 51 2-year convalescents were also enrolled at
the 6-month and 1-year follow-up visits.

IQR: interquartile range.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. IgG and IgA antibody responses to SARS-CoV-2
invaccinated 2-year convalescents.}

A, Schematic overview of workflow to detect the immune memory in
vaccinated 2-year convalescents and healthy controls with vaccination. A
total of 112 individuals were recruited for the analysis in Macheng and
Beijing, and they were classified by whether they had prior exposure to
SARS-CoV-2 and vaccination. 23 of 51 COVID-19 convalescents were
enrolled in the follow-up study at 6 months, 1 year, and 2 years after
symptoms onset.

B-I, IgG and IgA antibody levels were measured by MCLIA and ELISA in
healthy individuals after being vaccinated (HC-Vac, green, n=35) or
unvaccinated (HC-Unvac, blue, n=26), and in COVID-19 convalescents
following at 6 months (6m, orange, n=23), 1 year (1Y, green, n=23) and 2
years with vaccination (2Y-Vac, red, n=51). B-C, Anti-SARS-CoV-2 IgG
antibody titers determined by ELISA (B) or MCLIA (C). D-E, The
longitudinally dynamic changes of IgG levels in convalescents at 6
months, 1 year, and 2 years in the same individuals after symptoms
onset, using ELISA (D, n=23) or MCLIA (E, n=18). F-G, Correlation of IgG
titers and different disease severity in 51 vaccinated 2-year
convalescents (2Y-Vac), including Asym-Mild (Asymptomatic and Mild, red,
n=25) and Mod-Sev (Moderate and Severe, dark green, n=26). H-I, IgA
antibody levels measured by MCLIA were shown in HC-Vac and 2Y-Vac groups
(H, HC-Vac: n=35; 2Y-Vac: n=51). The dynamic changes of IgA antibody
responses during the 2-year follow-up (I, n=18). The dotted lines
reflect the cut-off values for positive antibody titers. Significant
differences in antibody titers were tested using a Mann-Whitney U-test
(B-C, F-H). A Wilcoxon matched-pairs signed rank test was used to
compare IgG antibody titers at three time points (D-E, I). * p
\textless{} 0.05, ** p \textless{} 0.01, *** p \textless{} 0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. IgM antibody responses to SARS-CoV-2 in vaccinated
2-year convalescents.}

IgM antibody levels were measured by MCLIA and ELISA in healthy
individuals after being vaccinated (HC-Vac, green, n=35) or unvaccinated
(HC-Unvac, blue, n=26), and in COVID-19 convalescents following at 6
months (6m, orange, n=23), 1 year (1Y, green, n=23) and 2 years with
vaccination (2Y-Vac, red, n=51). A-B, Anti-SARS-CoV-2 IgM antibody
titers determined by ELISA (A) or MCLIA (B). C-D, The longitudinally
dynamic changes of IgM levels in convalescents at 6 months, 1 year, and
2 years in the same individuals after symptoms onset, using ELISA (C,
n=23) or MCLIA (D, n=18). E-F, Correlation of IgM titers and different
disease severity in 51 vaccinated 2-year convalescents (2Y-Vac),
including Asym-Mild (Asymptomatic and Mild, red, n=25) and Mod-Sev
(Moderate and Severe, dark green, n=26). Black bars denote median
responses. Each multi-colored dot or connective line indicates one
individual. The dotted lines reflect the cut-off values for positive
antibody titers. Significant differences in antibody titers were tested
using a Mann-Whitney U-test (A-B, E-F). A Wilcoxon matched-pairs signed
rank test was used to compare IgM antibody titers at three time points
(C-D). * p \textless{} 0.05, ** p \textless{} 0.01, *** p \textless{}
0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Cellular immune responses to SARS-CoV-2 in vaccinated
2-year convalescents detected by ELISpot.}

A, Cross-sectional T cell responses to the Wild type (WT) overlapping
peptide pools encompassing the S1, S2, N, and M proteins, which were
detected using IFN-\selectlanguage{greek}γ \selectlanguage{english}ELISpot assay in vaccinated 2-year convalescents
(2Y-Vac, n=51) and vaccinated healthy controls without prior infection
(HC-Vac, n=35). B-C, The magnitude of T cell responses in HC-Vac (blue,
n=35) and 2Y-Vac (red, n=51) groups. Freshly isolated PBMCs (B) and
cultured PBMCs (C) after 9 days of\emph{in vitro} expansion were
stimulated with one of four peptide pools (S1, S2, M, and N). D-E,
Comparison of the magnitude of T cell responses in Asym-Mild
(Asymptomatic and Mild, pink, n=25) and Mod-Sev (Moderate and Severe,
dark green, n=26) convalescents for \emph{ex vivo} (D) and\emph{in
vitro} experiments (E). F, The proportion of positive T cell responses
in 2Y-Vac and HC-Vac individuals found in nil (0 response, white), one
(1 response, pink), two (2 response, red), three (3 response, orange),
or four (4 response, reseda) peptide pools. Each dot reflects an
individual donor. The solid lines represent the mean \selectlanguage{ngerman}± SEM. Statistical
significance was performed with a Mann-Whitney U-test (B-E). * p
\textless{} 0.05, ** p \textless{} 0.01, *** p \textless{} 0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Cellular immune responses to SARS-CoV-2 in vaccinated
2-year convalescents detected by ICS.}

A, Gating strategy for cytokines (IL-2, TNF-\selectlanguage{greek}α, \selectlanguage{english}and IFN-\selectlanguage{greek}γ\selectlanguage{english}) expressing in
SARS-CoV-2-specific CD4\textsuperscript{+} and CD8\textsuperscript{+} T
cells, enumerated by Intracellular cytokine staining (ICS). B,
Representative dot plots showing IFN-\selectlanguage{greek}γ \selectlanguage{english}production of
CD4\textsuperscript{+} T cell against S1, S2, M, and N peptide pools in
vaccinated 2-year convalescents (2Y-Vac) and vaccinated healthy controls
(HC-Vac). C, The proportion of mean protein-specific responses of
CD4\textsuperscript{+} and CD8\textsuperscript{+} T cells secreting
IL-2, TNF-\selectlanguage{greek}α, \selectlanguage{english}or IFN-\selectlanguage{greek}γ \selectlanguage{english}from individuals in groups HC-Unvac (blue, n=5),
HC-Vac (green, n=21), and 2Y-Vac (red, n=29). The solid lines represent
the mean \selectlanguage{ngerman}± SEM. Statistical significance was performed with a
Mann-Whitney U-test (C). * p \textless{} 0.05, ** p \textless{} 0.01,
*** p \textless{} 0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Cross-responses of neutralizing antibodies to Omicron
in vaccinated 2-year convalescents and healthy controls.}

A, Competitive inhibition of anti-RBD neutralizing antibodies against
Wild Type (WT) (red, n=23), Delta (blue, n=23), BA.1 (green, n=23), BA.2
(purple, n=23), and BA.4/5 (pink, n=23) was measured at 6-month, 1-year,
and 2-year convalescent individuals after symptoms onset. B, Comparison
of the inhibition levels of neutralizing antibodies against the above
five strains for HC-Unvac (yellow, n=26), HC-Vac (purple, n=35), and
2Y-Vac (red, n=51) groups. C-D, Changes of neutralization capacity
toward WT (red), Delta (blue), BA.1 (green), BA.2 (purple), and BA.4/5
(pink) were detected using sera from vaccinated 2-year convalescents
(2Y-Vac, n=51) (C) and healthy controls after vaccination (HC-Vac, n=35)
(D). Pie charts summarized the positive inhibition ratios of
neutralizing antibodies (NAb) against different strains. E, NAb
inhibition rates against four SARS-CoV-2 variants (Delta, BA.1, BA.2,
and BA.4/5) were normalized using the geometric mean ratio of NAb
inhibition rates versus WT (HC-Vac: orange; 2Y-Vac: blue). F, The
seropositivity of neutralizing antibodies, in 2Y-Vac and HC-Vac
individuals, identified in naught (no response, white), one (WT,
yellow), two (WT + Delta, purple), three (WT + Delta + BA.2, blue), four
(WT + Delta + BA.1/2, green) or five (WT + Delta + BA.1/2/4/5, red)
strains. Each dot represents a unique donor. Black dotted lines and bars
represent the threshold for positive and medians of responders.
Significance was assessed using the Wilcoxon matched-pairs signed rank
test (A, C, D) and Mann-Whitney U-test (B). * p \textless{} 0.05, ** p
\textless{} 0.01, *** p \textless{} 0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6. T cell cross-response to Omicron in vaccinated 2-year
convalescents and healthy controls.}

A, Representative ELISpot responses against RBD, S1, and S2 protein
pools of wild type (WT) and Omicron BA.1. B, Comparative T cell
responses in vaccinated 2-year convalescents (2Y-Vac, red, n=51) and
healthy controls without COVID-19 infection (HC-Vac, blue, n=35) to
peptide pools of WT and BA.1 RBD/S1/S2. C-D, Frequency of spike-reactive
CD4\textsuperscript{+} and CD8\textsuperscript{+} T cells producing
IL-2, TNF-\selectlanguage{greek}α, \selectlanguage{english}or IFN-\selectlanguage{greek}γ \selectlanguage{english}cytokines after PBMCs of vaccinated 2-year
convalescents (C) and healthy controls (D) stimulated with S1 (C, n=5;
D, n=7) and S2 (C, n=4; D, n=3) peptide pools from WT (red) and Omicron
BA.1 (blue). Each dot represents an individual. A Mann-Whitney U-test
was used to calculate statistical significance (B). * p \textless{}
0.05, ** p \textless{} 0.01, *** p \textless{} 0.001.\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-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`Table1.docx`  available at \url{https://authorea.com/users/489422/articles/629139-inactivated-vaccine-fueled-adaptive-immune-responses-to-omicron-in-2-year-covid-19-convalescents}

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

