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\begin{document}

\title{Impaired systemic nucleocapsid antigen clearance in severe COVID-19}



\author[1]{Christian Bauer}%
\author[1]{Elisabeth Mack}%
\author[1]{Véronique Hefter}%
\author[1]{Alexandra Fischer}%
\author[1]{Kirsten Volland}%
\author[1]{Chrysanthi Skevaki}%
\author[1]{Andreas Neubauer}%
\author[1]{Thomas Gress}%
\author[1]{Stephan Becker}%
\author[1]{Christian Keller}%
\affil[1]{Philipps-Universitat Marburg}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Objectives:} Circulating nucleocapsid (NCP) antigen of
SARS-CoV-2 is increased in severely ill COVID-19 patients. However,
clinical deterioration of COVID-19 often happens about one week after
benign initial presentation. The role of NCP antigenemia as a biomarker
in those cases remains unclear. We investigated NCP clearance kinetics
in hospitalized patients as a risk assessment tool for predicting
necessity of intensive care treatment of COVID-19 patients.
\textbf{Methods:} Serum NCP was quantified using a commercial
NCP-specific ELISA in hospitalized COVID-19 patients (n=63) during their
hospital stay. Results were correlated to COVID-19 disease severity,
inflammation parameters, antibody response and results of SARS-CoV-2 PCR
from nasopharyngeal swabs. ~ \textbf{Results:} We demonstrate that NCP
antigen levels in serum remained elevated in 45.6\% of patients
requiring treatment on intensive care units (ICU) after \textgreater{}8
days post positive SARS-CoV-2 PCR, compared to complete clearance in all
non-ICU patients. This was in contrast to mucosal clearance of virus as
measured by PCR. Antigen clearance was associated with higher IgG
against S1 but not NCP. ~ \textbf{Conclusions:} Detection of NCP
antigenemia after 8 days post COVID-19 diagnosis identifies patients who
will require intensive care. Lack of NCP clearance after one week can
thus help to assess the risk to develop severe COVID-19.%
\end{abstract}%



\sloppy


\textbf{Impaired systemic nucleocapsid antigen clearance in severe
COVID-19}

Running head: NCP antigenemia in severe COVID-19

Christian Bauer\textsuperscript{a}, Elisabeth Mack\textsuperscript{b},
V\selectlanguage{ngerman}éronique Hefter\textsuperscript{c}, Alexandra
Fischer\textsuperscript{c}, Kirsten Volland\textsuperscript{c},
Chrysanthi Skevaki\textsuperscript{d}, Andreas
Neubauer\textsuperscript{b}, Thomas Gress\textsuperscript{a}, Stephan
Becker\textsuperscript{c}, Christian Keller\textsuperscript{c}

\textsuperscript{a} Department of Gastroenterology, Endocrinology,
Metabolism and Infectiology, University Hospital Marburg, Philipps
University Marburg, Germany, Baldingerstraße, D-35043 Marburg, Germany.

\textsuperscript{b} Department of Internal Medicine, Hematology and
Oncology, University Hospital Marburg, Philipps University Marburg,
Germany, Baldingerstraße, D-35043 Marburg, Germany.

\textsuperscript{c} Institute of Virology, University Hospital Marburg,
Philipps University Marburg, Germany, Hans-Meerwein-Str. 2, D-35043
Marburg.

\textsuperscript{d} Institute of Laboratory Medicine, Universities of
Giessen and Marburg Lung Center (UGMLC), Philipps University Marburg,
German Center for Lung Research (DZL) Marburg, Baldingerstraße, D-35043
Marburg, Germany.

Correspondence to: Dr. Christian Keller, Institute of Virology, Philipps
University Marburg, Hans-Meerwein-Str. 2, D-35043 Marburg, Germany.

Mail: christian.keller@staff.uni-marburg.de. Phone: +49 6421 28 21977.

\textbf{Abstract}

\textbf{Objectives}

Circulating nucleocapsid (NCP) antigen of SARS-CoV-2 is increased in
severely ill COVID-19 patients. However, clinical deterioration of
COVID-19 often happens about one week after benign initial presentation.
The role of NCP antigenemia as a biomarker in those cases remains
unclear. We investigated NCP clearance kinetics in hospitalized patients
as a risk assessment tool for predicting necessity of intensive care
treatment of COVID-19 patients.

\textbf{Methods}

Serum NCP was quantified using a commercial NCP-specific ELISA in
hospitalized COVID-19 patients (n=63) during their hospital stay.
Results were correlated to COVID-19 disease severity, inflammation
parameters, antibody response and results of SARS-CoV-2 PCR from
nasopharyngeal swabs.

\textbf{Results}

We demonstrate that NCP antigen levels in serum remained elevated in
45.6\% of patients requiring treatment on intensive care units (ICU)
after \textgreater{}8 days post positive SARS-CoV-2 PCR, compared to
complete clearance in all non-ICU patients. This was in contrast to
mucosal clearance of virus as measured by PCR. Antigen clearance was
associated with higher IgG against S1 but not NCP.

\textbf{Conclusions}

Detection of NCP antigenemia after 8 days post COVID-19 diagnosis
identifies patients who will require intensive care. Lack of NCP
clearance after one week can thus help to assess the risk to develop
severe COVID-19.

\textbf{Key words}

COVID-19, nucleocapsid antigen, ELISA, SARS-CoV-2, biomarker

\textbf{Word count abstract:} 200

\textbf{Word count text:} 1284

\textbf{Introduction}

Circulation of viral antigen in blood is a mainstay of diagnostic
testing in systemic viral infections, including HBV, HCV or HIV.
However, in respiratory infections such as SARS-CoV-2, antigenemia in
serum is harder to interpret {[}1, 2{]}. Systemic presence of
nucleocapsid (NCP) as well as spike (S) protein has been described
{[}3{]}. A high level of serum NCP antigen was consistently found to be
associated with serious illness {[}4-7{]}. It was suggested that the
serum NCP antigen level may serve as an early biomarker for predicting
the maximum disease grade of severe COVID-19, using NCP antigenemia as a
tool for allocation of ICU options on admission {[}8{]}. However, some
patients progress to more severe disease not before the second week of
Covid-19; for those patients, no biomarker has been established yet.
Here, we demonstrate a delay of NCP antigen clearance in patients who
required intensive care during hospitalization.

\textbf{Patients and Methods}

\textbf{Study Cohort}

Patients (n=63) diagnosed with SARS-CoV-2 infection by Real-Time PCR
from respiratory specimens (Altona Diagnostics, Hamburg, Germany) were
enrolled into the study at Marburg University Hospital (Germany) between
March and November 2020. The baseline characteristics of the study
population are shown in Table S1.

\textbf{Antibody and antigen testing}

Serum IgG antibodies against S1 and NCP antigens of SARS-CoV-2 were
measured by commercial ELISAs (Anti-SARS-CoV-2-ELISA {[}IgG{]} and
Anti-SARS-CoV-2-NCP-ELISA {[}IgG{]}; Euroimmun, Lübeck, Germany),
according to the manufacturer's instructions. Upon manual dilution, sera
were processed in a BEPIII analyzer (Siemens, Eschborn, Germany).
Results are indicated as Ratio (cutoff: \textless{}0.8; borderline:
\selectlanguage{english}[?]0.8, \textless{}1.1; positive: \textgreater{}1.1). Circulating NCP
antigen in serum was quantified using the ScheBo(r)
SARS-CoV-2\textsuperscript{TM} Antigen ELISA (ScheBo Biotech, Giessen,
Germany). Values \selectlanguage{english}[?]2.97 pg/mL were considered positive.

\textbf{Statistical analysis}

Continuous variables are presented as means +- SD or min/max as
indicated. Unpaired t tests and linear correlations were performed by
GraphPad PRISM 7.0e. A two-sided p value of \textless{}0.05 was
considered significant.

\textbf{Ethics statement}

The study protocol was reviewed and approved by the Ethics Commission of
the Medical Faculty of Philipps University Marburg (vote 57/20).
Informed consent was obtained from all study participants.



\textbf{Results}

\textbf{NCP antigenemia and SARS-CoV-2-specific antibody responses}

In sera of hospitalized COVID-19 patients without detectable antibodies,
NCP antigen was detected in high concentrations \textgreater{}100 pg/mL
(Fig. S1A,B). As expected, a subset of patients with detectable
antibodies against S1 or NCP had NCP antigen levels \textless{}2.97
pg/mL, consistent with successful clearance of the infection. We assumed
that the appearance of NCP-specific IgG would show a strong association
with the reduction of NCP antigenemia. Instead, we found that only
33.0\% of anti-NCP-positive, but 40.6\% of anti-S1-positive sera had an
NCP antigen concentration below the cutoff. Simultaneous detection of
antibodies and NCP antigen was also found (29.3\% of S1 IgG-positive
sera, 38.3\% of NCP IgG-positive sera, Fig. S1A,B). Accordingly, NCP
antigen was lower in anti-S1-positive compared to anti-NCP-positive sera
(Fig. S1C), suggesting that S1-specific IgG is more relevant than
NCP-specific IgG to reduce NCP antigenemia.

\textbf{Inflammatory response and NCP antigenemia in ICU and non-ICU
patients}

Patients were grouped into those requiring intensive care (ICU patients)
versus those without need for ventilatory support (non-ICU patients).
Higher levels of CRP and IL-6 were found in ICU compared to non-ICU
patients (Fig. S2A,B). Based on this differentiation, we found that NCP
antigenemia correlated positively with both CRP and IL-6 in ICU, but not
in non-ICU patients (Fig. S2C,D). NCP antigenemia was significantly
higher in patients \selectlanguage{english}[?]75 years of age (Table S1). Sex was not associated
with increased antigenemia.

\textbf{Time course of systemic NCP antigenemia in COVID-19 patients}

Next, we compared NCP serum concentrations between non-ICU and ICU
patients. During the first week, NCP antigen was detected in 36/43
samples from ICU patients (83.7\%) and 23/25 samples from non-ICU
patients (92.0\%, Fig. 1A) with similar peak values (Fig. 1B). After day
8 post COVID-19 PCR diagnosis, non-ICU patients had invariably cleared
NCP (14/14, 100\%). In contrast, NCP was still elevated in 26/57
(45.6\%) samples from ICU patients obtained during the late phase of
illness (\textgreater{}8 days, Fig. 1B). Mean NCP antigen concentrations
remained significantly higher in ICU compared to non-ICU patients after
the 8\textsuperscript{th} day of illness (Fig. 1B). Importantly, the Ct
values in swab PCRs from ICU patients above vs. below the NCP cutoff did
not differ significantly (Fig. S3A-C), suggesting that NCP antigenemia
is independent of mucosal clearance. Thus, while high NCP antigenemia
was a phenomenon in all hospitalized COVID-19 patients during the first
week of illness, prolonged systemic circulation of NCP beyond the first
week was detected only in ICU patients.

\textbf{Antibody responses in NCP-positive ICU patients}

To understand the possible defect in ICU patients that have prolonged
NCP antigenemia (after 8 days of COVID-19), ICU patients were grouped
into those with NCP levels above and those below the cutoff, and the S1-
and NCP-specific antibody responses were analyzed. ICU patients without
detectable antigenemia had antibodies against S1 (Fig. 2A) and NCP (Fig.
2B) with ratios similar to non-ICU patients, suggesting normal antibody
responses. In contrast, ICU patients with antigenemia after day 8 of
COVID-19 had significantly lower S1-specific antibodies compared to ICU
patients without antigenemia. This difference was restricted to
S1-specific antibodies, while no difference was found for IgG against
NCP (Fig. 2B). Thus, the prolonged NCP antigenemia in COVID-19 patients
on ICU was associated with decreased antibody responses against S1, but
not NCP.

\textbf{Discussion}

The present study investigated the role of circulating SARS-CoV-2 NCP in
COVID-19 with relation to antibody status, disease severity, time post
diagnosis and inflammation markers. We show that high systemic
concentrations of NCP antigen are a consistent feature of the early
phase of hospitalized COVID-19 patients, independent of disease
severity. Remarkably, NCP antigen clearance to levels below the
detection threshold was impaired after the 8\textsuperscript{th} day of
disease in COVID-19 patients needing intensive care compared to non-ICU
patients, suggesting a reduced systemic antigen clearance in severe
disease.

Circulating NCP antigen is detected at higher concentrations in severely
ill COVID19 patients {[}7, 9-11{]}. Our study identified a moderate but
significant correlation between NCP antigenemia and CRP / IL-6 in ICU
patients. Earlier investigations had observed an association of higher
NCP antigenemia with CRP {[}6{]}, IL-10, IP-10 and RAGE {[}10{]}. As a
prognostic marker, NCP concentrations of \textgreater{}1000 pg/mL upon
hospitalization were associated with later ICU admission and delayed
hospital discharge {[}10, 12{]}. Our study now adds that clearance of
NCP antigenemia is delayed in \textgreater{}40\% of severely ill COVID19
patients who required intensive care. Thus, if NCP is detectable after 1
week of illness, a potentially severe course of disease is likely,
emphasizing its role as a prognostic biomarker.

The biological basis of NCP antigenemia is still ill-defined. Plasma NCP
antigenemia was found not to correlate with mucosal Ct values {[}8{]}.
However, it is unresolved whether NCP antigen in plasma reflects ongoing
systemic viral replication {[}12{]}, or rather dissemination from the
respiratory tract {[}10{]}. Our study is the first to show that
prolonged antigenemia in ICU patients is associated with lower S1- but
not NCP-specific IgG after 8 days post diagnosis. Reduced S1-specific
antibodies could thus result in less efficient neutralization and permit
systemic dissemination of virions.

Of note, the present study found that PCR results from nasopharyngeal
swabs did not differ significantly between ICU and non-ICU patients, as
also described by Yilmaz et al. {[}13{]}. In contrast, systemic NCP
antigenemia could differentiate both groups, suggesting independent
regulation of viral clearance from the mucosa and systemic clearance of
viral antigen. Consequently, detection of systemic NCP antigen after 8
days post COVID-19 diagnosis identifies patients that progress to
clinical deterioration requiring intensive care, while those who clear
antigenemia after this time are likely to recover. Thus, prolonged
antigenemia can serve as a novel biomarker to recognize severe courses
of COVID-19 infection.

\textbf{Conflict of interest}

CB reports funding by the European Fund of Regional Development (EFRE).
CK reports funding by the European Fund of Regional Development (EFRE)
and the Pandemic Network of the State of Hessen as well as lecture
honorarium from Roche (unrelated to present study).

\textbf{Funding}

This work was supported by the European Fund of Regional Development
(EFRE) to CB and CK, and by the Pandemic Network of the State of Hessen
to CK.

\textbf{Acknowledgments}

We gratefully acknowledge the outstanding technical expertise of all
technical assistants at the Institute of Virology, Marburg, for
molecular SARS-CoV-2 testing.

\textbf{Contribution}

CB: investigation, formal analysis, writing -- original draft. EM:
conceptualization, patient recruitment. VH: investigation,
writing-review and editing. AF: investigation. KV: investigation. CS:
conceptualization, patient recruitment, writing-review and editing. AN:
patient recruitment, writing-review and editing. TG: writing-review and
editing, supervision. SB: writing-review and editing, supervision. CK:
conceptualization, investigation, formal analysis, writing -- original
draft. All authors read and approved the final manuscript.

\section*{References}

{\label{references}}

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{[}6{]} Perna F, Bruzzaniti S, Piemonte E, Maddaloni V, Atripaldi L,
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{[}7{]} Verkerke HP, Damhorst GL, Graciaa DS, McLendon K, O'Sick W,
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https://doi.org/10.1093/infdis/jiac225

{[}8{]} Yokoyama R, Kurano M, Nakano Y, Morita Y, Ohmiya H, Kishi Y, et
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Microbiol 2021; 12:791489. https://doi.org/10.3389/fmicb.2021.791489

{[}9{]} Favresse J, Bayart JL, David C, Gillot C, Wieers G, Roussel G,
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{[}10{]} Wick KD, Leligdowicz A, Willmore A, Carrillo SA, Ghale R,
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{[}11{]} Zhang Y, Ong CM, Yun C, Mo W, Whitman JD, Lynch KL, et al.
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{[}13{]} Yilmaz A, Marklund E, Andersson M, Nilsson S, Andersson LM,
Lindh M, et al. Upper Respiratory Tract Levels of Severe Acute
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https://doi.org/10.1093/infdis/jiaa632

\textbf{Figure legend}

\textbf{Fig. 1. The clearance of NCP antigen is delayed in ICU patients}

Antigen concentration of systemic NCP in patient sera was measured by
ELISA (see Supplementary Methods). Samples were grouped based on ICU
admission. (A) NCP concentration at indicated days after positive
SARS-CoV-2 PCR. (B) Samples were grouped into those collected before or
after 8 days since COVID-19 diagnosis. ICU and non-ICU patients
exhibited high NCP levels in the early phase of COVID-19 infection. NCP
antigenemia remained significantly higher in ICU compared to non-ICU
patients after 8 days since diagnosis. Means +- SD. ** p\textless{}0.01;
ns=not significant by t test.

\textbf{Fig. 2. ICU patients with impaired NCP antigen clearance show
lower antibody concentrations against S1 but not NCP}

(A) S1 IgG concentrations of individuals after day 8 post first positive
PCR, from ICU (red) and non-ICU patients (grey). The ICU patients were
split into the participants with NCP antigenemia above and below the
test threshold defined by the manufacturer (2.97 pg/ml). S1-specific
antibody concentrations (n=20, 24 and 10, respectively). (B)
NCP-specific antibody concentrations (n=19, 24 and 11, respectively).
Means +- SD. ** p\textless{}0.01; ns=not significant by t test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Bauer-NCP-JMV-Figure-1/Bauer-NCP-JMV-Figure-1}

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

\end{center}
\end{figure}

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