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\begin{document}

\title{Durability of humoral and cellular immunity six months after the
BNT162b2 bivalent booster}



\author[1]{Julien Favresse}%
\author[1]{Constant Gillot}%
\author[2]{Mélanie Closset}%
\author[3]{Julien Cabo}%
\author[3]{Loris Wauthier}%
\author[4]{Clara David}%
\author[3]{Marc Elsen}%
\author[1]{Jean-Michel Dogné}%
\author[1]{Jonathan Douxfils}%
\affil[1]{Universite de Namur}%
\affil[2]{CHU UCL Namur Departement de Medecine}%
\affil[3]{Clinique Saint-Luc Bouge}%
\affil[4]{Qualiblood sa Research and Development Department}%


\vspace{-1em}



  
  \date{May 28, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Introduction} Studies about the duration of the humoral and
cellular response following the bivalent booster administration are
still scarce. We aimed at assessing the humoral and cellular response in
a cohort of healthcare workers that received this booster.
\textbf{Material and methods} Blood samples were collected before the
administration of the bivalent booster from Pfizer-BioNTech and after
14, 28, 90, and 180 days. Neutralizing antibodies against either the
D614G strain, the delta variant, the BA.5 variant, or the XBB.1.5
subvariant were measured. The cellular response was assessed by
measurement of the release of interferon gamma (IFN\selectlanguage{greek}γ\selectlanguage{english}) from T cells in
response to an \emph{in vitro} SARS-CoV-2 stimulation. \textbf{Results}
A substantial waning of neutralizing antibodies was observed after 6
months (23.1-fold decrease), especially considering the XBB.1.5
subvariant. The estimated T \textsubscript{1/2} of neutralizing
antibodies was 16.1 days (95\% CI=10.2--38.4 days). Although most
participants still present a robust cellular response after 6 months
(i.e., 95\%), a significant decrease was also observed compared to the
peak response (0.41 versus 0.95 UI/L, p=0.0083). \textbf{Conclusion} A
significant waning of the humoral and cellular response was observed
after 6 months. These data can also help national competent authorities
in their recommendation regarding the administration of an additional
booster.%
\end{abstract}%



\sloppy


\textbf{Durability of humoral and cellular immunity six months after the
BNT162b2 bivalent booster}

Julien Favresse\textsuperscript{1,2}, Constant
Gillot\textsuperscript{1}, M\selectlanguage{ngerman}élanie Closset\textsuperscript{3}, Julien
Cabo\textsuperscript{2}, Loris Wauthier\textsuperscript{2}, Clara
David\textsuperscript{4}, Marc Elsen\textsuperscript{2}, Jean-Michel
Dogné\textsuperscript{1}, Jonathan Douxfils\textsuperscript{1,4}.

\emph{\textsuperscript{1}Department of Pharmacy, Namur Research
Institute for Life Sciences, Namur Thrombosis and Hemostasis Center,
University of Namur, Namur, Belgium.}

\emph{\textsuperscript{2}Department of Laboratory Medicine, Clinique
St-Luc Bouge, Namur, Belgium.}

\emph{\textsuperscript{3}Department of Laboratory Medicine, Université
catholique de Louvain, CHU UCL Namur, Namur, Belgium.}

\emph{\textsuperscript{4}Qualiblood s.a., Research and Development
Department, Namur, Belgium.}

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

Department of Pharmacy, University of Namur B-5000 Namur, Belgium Phone
+32 81 72 43 91 Email: julien.favresse@slbo.be

\textbf{Running title:} Humoral and cellular response 6 months after the
bivalent booster.

\textbf{Keywords:} SARS-CoV-2, bivalent booster, humoral response,
cellular response, Omicron, BA.5, XBB.1.5.

\textbf{Words (manuscript):} 1,051.

\textbf{Number authors:} 9.

\textbf{Number of references} (main text): 10.

\textbf{Number of references} (appendix): 6.

\textbf{Number of tables:} 0.

\textbf{Number of figures:} 1.

\textbf{Supplemental figures:} 2.

\textbf{Competing Interests:} The authors declare that there are no
competing interests.

\textbf{Author contribution:} Conceptualization: JF and JD; methodology:
JF, CG, MC, JC, LW, CD and JD; software: JF, CG and JD; validation: JF
and JD; formal analysis: JF, CG and JD; investigation: JF, CG, MC, JC,
LW, and JD; resources: ME, JMD, and JD; data curation: JF, CG, MC, JC,
LW, and ME; writing--original draft preparation, JF; writing--review and
editing, JF, CG, MC, JC, LW, CD, ME, JMD, and JD; supervision: JD;
project administration: JF, MC, ME and JD; funding acquisition: JF, MC,
ME, JMD and JD. All authors have read and agreed to the published
version of the manuscript.

\emph{Abstract}

\emph{Introduction}

Studies about the duration of the humoral and cellular response
following the bivalent booster administration are still scarce. We aimed
at assessing the humoral and cellular response in a cohort of healthcare
workers that received this booster.

\emph{Material and methods}

Blood samples were collected before the administration of the bivalent
booster from Pfizer-BioNTech and after 14, 28, 90, and 180 days.
Neutralizing antibodies against either the D614G strain, the delta
variant, the BA.5 variant, or the XBB.1.5 subvariant were measured. The
cellular response was assessed by measurement of the release of
interferon gamma (IFN\selectlanguage{greek}γ\selectlanguage{english}) from T cells in response to an \emph{in vitro}
SARS-CoV-2 stimulation.

\emph{Results}

A substantial waning of neutralizing antibodies was observed after 6
months (23.1-fold decrease), especially considering the XBB.1.5
subvariant. The estimated T\textsubscript{1/2} of neutralizing
antibodies was 16.1 days (95\% CI=10.2--38.4 days). Although most
participants still present a robust cellular response after 6 months
(i.e., 95\%), a significant decrease was also observed compared to the
peak response (0.41 versus 0.95 UI/L, p=0.0083).

\emph{Conclusion}

A significant waning of the humoral and cellular response was observed
after 6 months. These data can also help national competent authorities
in their recommendation regarding the administration of an additional
booster.

\emph{}

\emph{Highlights}

\begin{itemize}
\tightlist
\item
  Substantial waning of the humoral response was observed 6 months after
  the bivalent booster administration, especially considering the
  XBB.1.5 subvariant.
\item
  A significant decay in the cellular response was also observed over
  time.
\item
  The proportion of samples still able to release IFN\selectlanguage{greek}γ \selectlanguage{english}in response to
  an\emph{in vitro} SARS-CoV-2 stimulation remained however high.\emph{}
\end{itemize}

\emph{Introduction}

Neutralizing antibodies against omicron variants and subvariants, which
represents a strong correlate of protection from SARS-CoV-2 infection,
have been showed to significantly increase after bivalent booster
administration \textsuperscript{1-4}. Accumulating evidence suggests
that T cell response, i.e. helper CD4+ and cytotoxic CD8+ T cells, plays
a key role in the protection against severe disease \textsuperscript{5}.
In contrast to neutralizing antibodies, T cells are more resilient
against highly mutated emerging variants, with \textgreater{}80\% of
epitopes conserved among T cells \textsuperscript{5,6}. Currently, the
long-term kinetics of the humoral and cellular immunity has been poorly
explored.

\emph{Material and methods}

In this study, we present the 6-month humoral and cellular results from
the participants of the multicenter and prospective CRO-VAX HCP study
who received the bivalent booster (ethical approval number:
2020-006149-21; \textbf{Appendix} ). Thirty-six were females (median age
= 51.0 years; IQR = 43.0--58.8) and 15 were males (median age = 51.0
years; IQR = 35.0--59.0). Ages were non-significantly different between
females and males (p = 0.88). The majority of the participants (45/51;
88.2\%) had a history of SARS-CoV-2 (\textbf{Appendix} ).

A pseudovirus-neutralization test was used to assess the neutralization
potency of vaccines-elicited antibodies against either the D614G strain,
the delta variant, the BA.5 omicron variant, or the XBB.1.5 omicron
subvariant. The antibody titer is determined as the dilution of serum at
which 50\% of the infectivity is inhibited (IC\textsubscript{50}) as
determined by a nonlinear sigmoid regression model. A sample with a
titer of less than 1:20 is considered negative (\textbf{Appendix} ).
Neutralizing antibodies against BA.5 were measured at each timepoint but
neutralizing antibodies against the D614G strain, the delta variant and
the XBB.1.5 omicron subvariant were only measured at 6 months in a
subset of 30 participants randomly selected.

Total antibodies against the NCP (Roche Diagnostics) were measured using
the Elecsys Anti-SARS-CoV-2 assay. Results above 0.165 cut-off index
were considered positive (\textbf{Appendix} ).

The T cell-mediated immune response was assessed using the cobas IGRA
SARS-COV-2 Tubes and the Elecsys IGRA SARS-CoV-2 assay (Roche
Diagnostics). The test measures the release of interferon gamma (IFN\selectlanguage{greek}γ\selectlanguage{english})
from T cells in response to an \emph{in vitro} SARS-CoV-2 stimulation in
whole blood samples which have been formerly in contact with the
SARS-CoV-2 coated antigens. More than 180 different SARS-CoV-2 antigens
(structural (spike, membrane and nucleocapsid) and non-structural) are
coated on the antigen tube, enabling a substantial coverage of commonly
occurring HLA subtypes for stimulation of both CD4+ and CD8+ T cells.
The assay is therefore robust to detect different variants. A result
above 0.013 IU/mL was considered positive (\textbf{Appendix} ).

The detailed statistical analysis process is presented in
the\textbf{Appendix} .

\emph{Results}

The highest measured neutralizing capacity against the BA.5 variant was
reached at day 28 with a GMT of 1,095 (95\% CI = 903.4--1,327),
representing a significant 7.0-fold increase from baseline (i.e., 157;
95\% CI = 112--219, p \textless{} 0.0001). A substantial decrease was
then observed up to 180 days with an observed GMT of 47.4 (95\% CI =
36.6--61.6, p \textless{} 0.0001), which represents a 23.1-fold
decrease. The neutralizing capacity at 180 days was significantly lower
compared to baseline (p = 0.0004). The proportion of detectable
neutralizing antibodies (i.e., \textless{}1:20) was 93.6\%, 100\%,
100\%, 98.0\%, and 85.4\% at baseline and 14, 28, 90, and 180 days after
the administration of the vaccine (\textbf{Figure 1A} ). The fold-change
in the neutralizing capacity against BA.5 was similar between
participants who received the BA.1 or the BA.4/5 booster (p
\textgreater{} 0.05) (\textbf{Appendix} ). The estimated
T\textsubscript{1/2} of neutralizing antibodies was 16.1 days (95\% CI =
10.2--38.4 days). According to the model, a mean time of 137 days (95\%
CI = 76--170) would be needed to cross the dilution titer threshold of
1:20 (\textbf{Figure 1B} ).

At 6 months, neutralizing antibodies against the Delta variant, the BA.5
omicron variant and the XBB.1.5 omicron subvariant were 1.97, 5.20, and
10.81 lower compared to the D614G strain. The proportion of detectable
neutralizing antibodies was 100\%, 100\%, 91.3\%, and 66.6\%,
respectively (\textbf{Figure 1C} ).

The highest T-cell response was observed after 14 days with a GMT of
0.95 UI/mL (95\% CI = 0.72--1.24), representing a significant 1.97-fold
increase from baseline (i.e., 0.48; 95\% CI = 0.30--0.77, p = 0.0306). A
significant decrease was then observed up to 180 days with an observed
GMT of 0.41 (95\% CI = 0.21--0.82, p = 0.0083), representing a 2.28-fold
reduction compared to day 14 and a 1.17-fold decrease from baseline. The
IFN\selectlanguage{greek}γ \selectlanguage{english}responses at 90 and 180 days were not different from baseline (p =
0.91 and 0.95). The proportion of detectable levels of IFN\selectlanguage{greek}γ \selectlanguage{english}was 98\%,
100\%, 100\%, 100\%, and 95\% at baseline, 14, 28, 90, and 180 days
(\textbf{Figure 1D} ). The fold-change in the IFN\selectlanguage{greek}γ \selectlanguage{english}response against BA.5
was similar between participants who received the BA.1 or the BA.4/5
booster (\textbf{Appendix} ).

Eleven participants (21.6\%) developed a breakthrough infection between
90 and 180 days; which is consistent with the drop of neutralizing
antibodies. The infection was associated with a significant raise in
BA.5 neutralizing antibodies (fold-increase of 2.55, p = 0.0039). The
impact on the IFN\selectlanguage{greek}γ \selectlanguage{english}release was not significant in these patients (p =
0.4961) (\textbf{Appendix} ).

\emph{Discussion and conclusion}

The increase of neutralizing antibodies following the administration of
the bivalent booster we documented in our study (i.e., 7.0--fold
increase) was consistent with the conclusions of other
studies\textsuperscript{4,7}. As for the humoral response following the
first two doses of BNT162b2 and the homologous booster
\textsuperscript{8,9}, a substantial waning of the humoral response was
observed 6 months after the administration of the BNT162b2 bivalent
booster. This decrease was especially important considering the XBB.1.5
subvariant. Moreover, the drop of neutralizing antibodies over time
coincides with the decrease of bivalent booster effectiveness against
infection in the recent report of Lin \emph{et al.}
\textsuperscript{10}.

A significant decay in the cellular response was also observed over
time. Nevertheless, the proportion of samples still able to generate
IFN\selectlanguage{greek}γ \selectlanguage{english}in response to an \emph{in vitro} SARS-CoV-2 stimulation remained
high. This observation is consistent with the maintained and superior
effectiveness against severe disease in the report of Lin \emph{et
al.}\textsuperscript{10}. The fact that the release of IFN\selectlanguage{greek}γ \selectlanguage{english}could
represent a good surrogate of the risk of severe infection still remains
to be evaluated. Importantly, these data can also help national
competent authorities in their recommendation regarding the
administration of an additional booster.

\textbf{References:}

1. Seekircher L, B\selectlanguage{ngerman}ánki Z, Kimpel J, et al. Immune response after two
doses of the BNT162b2 COVID-19 vaccine and risk of SARS-CoV-2
breakthrough infection in Tyrol, Austria: an open-label, observational
phase 4 trial. \emph{The Lancet Microbe} 2023.

2. Nilles EJ, Paulino CT, De St Aubin M, et al. Tracking immune
correlates of protection for emerging SARS-CoV-2 variants. \emph{The
Lancet Infectious Diseases} 2023; \textbf{23} (2): 153-4.

3. Khoury DS, Cromer D, Reynaldi A, et al. Neutralizing antibody levels
are highly predictive of immune protection from symptomatic SARS-CoV-2
infection. \emph{Nat Med} 2021; \textbf{27} (7): 1205-11.

4. Carr EJ, Wu MY, Gahir J, et al. Neutralising immunity to omicron
sublineages BQ.1.1, XBB, and XBB.1.5 in healthy adults is boosted by
bivalent BA.1-containing mRNA vaccination and previous Omicron
infection. \emph{Lancet Infect Dis} 2023.

5. Wherry EJ, Barouch DH. T cell immunity to COVID-19
vaccines.\emph{Science} 2022; \textbf{377} (6608): 821-2.

6. 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. \emph{Cell} 2022; \textbf{185} (5): 847-59 e11.

7. Tan NH, Geers D, Sablerolles RSG, et al. Immunogenicity of bivalent
omicron (BA.1) booster vaccination after different priming regimens in
health-care workers in the Netherlands (SWITCH ON): results from the
direct boost group of an open-label, multicentre, randomised controlled
trial. \emph{Lancet Infect Dis} 2023.

8. Bayart JL, Douxfils J, Gillot C, et al. Waning of IgG, Total and
Neutralizing Antibodies 6 Months Post-Vaccination with BNT162b2 in
Healthcare Workers. \emph{Vaccines (Basel)} 2021; \textbf{9} (10).

9. Favresse J, Gillot C, Bayart JL, et al. Vaccine-induced binding and
neutralizing antibodies against Omicron 6 months after a homologous
BNT162b2 booster. \emph{J Med Virol} 2023; \textbf{95} (1): e28164.

10. Lin DY, Xu Y, Gu Y, Zeng D, Sunny SK, Moore Z. Durability of
Bivalent Boosters against Omicron Subvariants. \emph{N Engl J Med}
2023;\textbf{388} (19): 1818-20.

\textbf{Figure 1: (A)} Evolution of neutralizing antibodies against the
BA.5 omicron variant before and after the bivalent booster with a
6\selectlanguage{english}-month follow\selectlanguage{english}-up in a population of 51 healthy-volunteers. GMT were 157
(95\% CI: 112--219), 598 (470--761), 1,095 (903--1,327), 106
(83.4--134), and 47.4 (36.6--61.6) at baseline and after 14, 28, 90, and
180 days. (\textbf{B} ) Kinetic models of the neutralizing capacity
against the BA.5 omicron variant. (\textbf{C} ) Comparison of the
neutralizing capacity against the D614G strain, the delta and BA.5
omicron variants, and the XBB.1.5 omicron subvariant in a population of
30 healthy-volunteers 6 months after having received the bivalent
booster. GMT were 319 (95\% CI: 241--423), 162 (119--220), 61.4
(42.7--88.2), and 29.5 (21.4--40.6) for the D614G strain, the delta
variant, the BA.5 omicron variant, and the XBB.1.5 omicron subvariant.
The dotted line represents the positivity cut\selectlanguage{english}-off for neutralizing
antibodies (dilution titer of 1:20). (\textbf{D} ) Evolution of the
cellular response by means of the measurement of IFN\selectlanguage{greek}γ. \selectlanguage{english}GMT were 0.53
UI/mL (95\% CI: 0.37--0.75), 0.95 (0.72--1.24), 0.87 (0.65--1.17), 0.65
(0.48--0.87), and 0.52 (0.34--0.79) at baseline and after 14, 28, 90,
and 180 days. The positivity cut-off for IFN\selectlanguage{greek}γ \selectlanguage{english}was 0.013 IU/mL. Geometric
means and 95\% CIs are represented. Only p values \textless{}0.05 were
graphically represented.\selectlanguage{english}
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