\documentclass[10pt]{article}

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





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


\usepackage{natbib}




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

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


\usepackage{authblk}


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

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

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



\usepackage{float}








\begin{document}

\title{Maintenance of neutralizing SARS-CoV-2 antibodies over five months in
convalescent SARS-CoV-2 afflicted patients.}



\author[1]{Sissy Sonnleitner}%
\author[2]{Martina Prelog}%
\author[1]{Bainca Jansen}%
\author[3]{Chantal Rodgarkia-Dara}%
\author[1]{Sarah Gietl}%
\author[1]{Carmen Schönegger}%
\author[4]{Stephan Koblmüller}%
\author[4]{Christian Sturmbauer}%
\author[5]{Wilfried Posch}%
\author[2]{Giovanni Almanzar}%
\author[2]{Hanna  Jury}%
\author[6]{Norbert Nowotny}%
\author[1]{Gernot Walder}%
\affil[1]{Dr. Gernot Walder GmbH}%
\affil[2]{University Hospital Wurzburg}%
\affil[3]{THP Medical products}%
\affil[4]{University of Graz}%
\affil[5]{Medical University of Innsbruck}%
\affil[6]{Mohammed Bin Rashid University of Medicine and Health Sciences}%


\vspace{-1em}



  
  \date{May 08, 2024}


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





\selectlanguage{english}
\begin{abstract}
Level and duration of protective immunity against SARS-CoV-2 after
primary infection is of crucial importance for preventive approaches. In
order to provide evidence for the longevity of specific antibodies, we
investigated the generation and maintenance of neutralizing antibodies
of convalescent SARS-CoV-2-afflicted patients over a five month period
post primary infection using an immunofluorescence assay, a commercial
chemiluminescent immunoassay and an in-house enzyme-linked
plaque-reduction neutralization assay. We present the successful
application of an improved version of the plaque-reduction
neutralization assay, which can be analyzed optometrically,
significantly simplifying the interpretation of the results. Based on
the results of the plaque-reduction neutralization assay, neutralizing
antibodies were maintained in 85.3\% of convalescent individuals without
significant decay over five months. Furthermore, a positive correlation
between severity of infection and neutralizing titer was shown. In
conclusion, SARS-CoV-2-afflicted individuals have been proven to be able
to establish and maintain neutralizing antibodies over a five months'
period after primary infection which allows to hope for long-lasting
presumably protective humoral immunity after wild-type infection or even
after vaccination.%
\end{abstract}%



\sloppy


\textbf{Maintenance of neutralizing SARS-CoV-2 antibodies over five
months in convalescent SARS-CoV-2 afflicted patients.}

Short title: Serologic study on the longevity of humoral immunity
against SARS-CoV-2.

Sissy Therese Sonnleitner\textsuperscript{1,3}, Martina
Prelog\textsuperscript{4}, Bianca Jansen\textsuperscript{1}, Chantal
Rodgarkia-Dara\textsuperscript{5,} Sarah Gietl\textsuperscript{1},
Carmen Maria Sch\selectlanguage{ngerman}önegger\textsuperscript{1}, Stephan
Koblmüller\textsuperscript{2}, Christian Sturmbauer\textsuperscript{2},
Wilfried Posch\textsuperscript{3,} Giovanni Almanzar\textsuperscript{4},
Hanna Jury\textsuperscript{4}, Norbert Nowotny\textsuperscript{6,7},
Gernot Walder\textsuperscript{1}.

\textsuperscript{1} Dr. Gernot Walder GmbH, Medical Laboratory,
Department of Virology, 9931 Ausservillgraten 30, Austria.
sissy.lamprecht@infektiologie.tirol

\textsuperscript{2} Institute of Biology, University of Graz,
Universitaetsplatz 2, 8010 Graz, Austria.

\textsuperscript{3} Division of Hygiene and Medical Microbiology,
Medical University of Innsbruck, 6020 Innsbruck, Austria.

\textsuperscript{4} University Hospital Wuerzburg, Department of
Pediatrics, University of Wuerzburg, Josef-Schneider-Str. 2, Wuerzburg,
Germany.

\textsuperscript{5} THP Medical products, Shuttleworthstrasse 19, 1210
Vienna, Austria.

\textsuperscript{6} Viral Zoonoses, Emerging and Vector-Borne Infections
Group, Institute of Virology, University of Veterinary Medicine Vienna,
Veterinaerplatz 1, 1210 Vienna, Austria.

\textsuperscript{7} Department of Basic Medical Sciences, College of
Medicine, Mohammed Bin Rashid University of Medicine and Health
Sciences, Building 14, Dubai Healthcare City, Dubai, United Arab
Emirates.

ABSTRACT

Level and duration of protective immunity against SARS-CoV-2 after
primary infection is of crucial importance for preventive approaches. In
order to provide evidence for the longevity of specific antibodies, we
investigated the generation and maintenance of neutralizing antibodies
of convalescent SARS-CoV-2-afflicted patients over a five month period
post primary infection using an immunofluorescence assay, a commercial
chemiluminescent immunoassay and an in-house enzyme-linked
plaque-reduction neutralization assay.

We present the successful application of an improved version of the
plaque-reduction neutralization assay, which can be analyzed
optometrically, significantly simplifying the interpretation of the
results. Based on the results of the plaque-reduction neutralization
assay, neutralizing antibodies were maintained in 85.3\% of convalescent
individuals without significant decay over five months. Furthermore, a
positive correlation between severity of infection and neutralizing
titer was shown.

In conclusion, SARS-CoV-2-afflicted individuals have been proven to be
able to establish and maintain neutralizing antibodies over a five
months' period after primary infection which allows to hope for
long-lasting presumably protective humoral immunity after wild-type
infection or even after vaccination.

Keywords: SARS-CoV-2, immunity, longevity, neutralization test, IgG
antibodies

INTRODUCTION

Little is known about persistence of the immunologic footprint left in a
convalescent SARS-CoV-2 afflicted patient and first publications showed
discrepancies concerning the longevity of specific IgG antibodies . The
development of specific antibodies in the acute phase of COVID-19 is
well documented and most patients display specific antibody responses
between day 10 and day 21 post infection (Kellam \& Barclay, 2020; Long
et al., 2020). Whereas milder courses of disease may result in a delayed
generation of antibodies, in a small number of cases, patients may even
stay antibody-negative after infection with SARS-CoV-2 . In comparable
studies, seropositivity rate reached up to 90\% and 100\% Studies
investigating the persistence of the antibody response are rare, but it
is known that antibodies to other coronaviruses wane over time, in the
range of 12 -- 52 weeks from the onset of symptoms onwards (. Short
follow-up studies showed preservation of SARS-CoV-2 IgG antibody levels
over the course of seven weeks (Xiao et al., 2020), at least in 80\% of
patients (Jianyun et al., 2020). In comparison, 90\% and 50\% of
SARS-CoV-1 infected patients have been shown to maintain IgG antibodies
for two and three years, respectively (.

Short-term immunity is defined as the wane of specific antibodies after
a period of about 40 weeks, which leads to annual or otherwise periodic
outbreaks as is well-known from nonSARS-like human coronaviruses
(nonSARS-like hCoVs) as well as influenza viruses . The knowledge of
duration of protective immunity against SARS-CoV-2 after primary
infection is of great importance for the understanding of possible
future scenarios of herd protection after wild-type infection or
vaccination.

In this study, we accompanied 34 volunteers representing the first
SARS-CoV-2 afflicted patients in East Tyrol and investigated the course
of specific antibody responses beginning in convalescence 21-43 days
after the onset of symptoms until 5 months (22 weeks) later.

In order to analyze potentially protective neutralizing IgG antibodies
against SARS-CoV-2, we developed and introduced a fast and reliable
novel enzyme-linked neutralization assay (ELNA) that allows the
determination of neutralization antibodies within 30 hours.

MATERIAL \& METHODS

\emph{Enrolment and sample collection}

Thirty-four volunteers in East Tyrol with PCR-confirmed SARS-CoV-2
infections and mostly clinically manifested COVID-19 symptoms (31/34)
were accompanied over 22 weeks post infection after informed consent was
taken from the volunteers. Ethical approval of the study was obtained by
the Ethics Committee of the University Hospital Wuerzburg no.
20201105\_01.

Following the time range of initial data collection, we conducted
serological follow-up examinations to evaluate possible recurrences and
seroconversion rates. First blood draw occurred 21 -- 43 days (median 35
days, standard deviation 15.6 days) after the onset of symptoms in the
first week of April 2020, followed by further assessments during the
investigative period, in the following called T1 -- T4, and a final
blood draw 5 and 6 months post infection in August and September 2020,
respectively (T5). Clinical data were obtained using a standardised data
collection form.

The presence of IgG-anti-spike1/spike2 and neutralizing antibodies was
analyzed in follow-up serum samples by four different serologic methods
to ensure validity of results: IgG in-house immunofluorescence assay
(IFA), IgM in-house IFA, CLIA IGG® SARS-CoV-2 S1/S2 IgG (DiaSorin
S.p.A., Saluggia, Italy) (CLIA IGG; clinical sensitivity 97.1\%; 95\%
CI: 85.5 -- 99.5\%, clinical specificity 100\%; 95\% CI: 96.5 -- 100\%
as reported by the manufacturer) and enzyme-linked neutralization assay
(ELNA).

\emph{RNA extraction and RT-qPCR:}

Sample drawing was performed by trained healthcare personnel via
nasopharyngeal swab. Immediately after collection, viral RNA was
extracted using the Indimag Pathogen kit (Indical Bioscience GmbH,
Germany)~and tested for SARS-CoV-2 by RT-qPCR using the Bio-Rad CFX96
system (Roche, Switzerland) with a LightMix Modular Assay kit in
accordance with the modified Charité guidelines . 10 \selectlanguage{greek}µ\selectlanguage{english}l of extracted RNA
were added into 15\selectlanguage{greek}µ\selectlanguage{english}l reaction mixture (mastermix). Each 15\selectlanguage{greek}µ\selectlanguage{english}l mastermix
contained 12.5 \selectlanguage{greek}µ\selectlanguage{english}l buffer solution, 0.25 \selectlanguage{greek}µ\selectlanguage{english}l enzyme mix, 1.75 \selectlanguage{greek}µ\selectlanguage{english}l of
nuclease-free water and 0.5 \selectlanguage{greek}µ\selectlanguage{english}l primer probe wHCoV (ORF1ab: E-Gene,
occasionally N-Gene/Rd-Gene). Reactions were incubated at 55\selectlanguage{ngerman}°C for 5
minutes and 95°C for 5 minutes in order to conduct reverse transcription
of viral RNA, sample denaturation and enzyme activation. These steps
were followed by PCR-amplification with 45 cycles at 95°C for 5s, 60°C
for 15s and 72°C for 15s. Cooling was implemented at 10°C for 30s.

Results were interpreted based on the Second derivative maximum (SDM)
method (Tichopad et al., 2003). Positive results were confirmed by RdRp-
and N-gene (Corman et al., 2020) RT-qPCR. Samples with an initial Cq
value lower than or equal to 37 were assigned to repeated testing
including extraction. A Cq value higher than 40 was defined negative.

\emph{Isolation of SARS-CoV-2:}

Isolation of SARS-CoV-2 was performed from nasopharyngeal swabs after
obtaining positive RT-qPCR results by inoculation on VeroB4 (no. ACC-33,
DSMZ) in T25 tissue culture flasks (Sarstedt, Germany) for 1 h at 35° C.
After incubation, the sample was removed and Medium199 (Gibco, USA) with
2.5\% fetal calf serum (FCS; Gibco, USA) and a mixture of antibiotics
(streptomycin, vancomycin, penicillin, each 1\selectlanguage{greek}µ\selectlanguage{english}g/ml) was added. We
monitored virus cultures daily for cytopathic effects and tested for
specific viral RNA every three days. Isolation was considered successful
when cytopathic effect was 80 to 100\% in passage 0 as well as passage 1
and/or Cq value in qPCR was lower than 20. Highly positive supernatants
were harvested, centrifuged at 3,400g for 5 minutes and stored at -80\selectlanguage{ngerman}° C
in 10\% FCS. A further passage of diverse isolates was performed to
obtain the highest possible concentration, which was Cq 14 on average.
All work involving infectious SARS-CoV-2 was carried out in a BSL3
facility, following the institutional guidelines and regulations. Whole
genome sequencing was carried out by Eurofins Genomics, Germany, and a
frequent local genotype (no. 6893) was used for neutralization assay.

\emph{In-house immunofluorescence assays (IFA)} were assembled as
described elsewhere . In brief, VeroB4 cells were infected with local
SARS-CoV-2 strain 6893 and fixed on IFA slides after three days using
ice-cold acetone-methanol (1:1).

\emph{The LIAISON® SARS-CoV-2 S1/S2 IgG} (DiaSorin S.p.A., Saluggia,
Italy) (LIAISON) is a CLIA (Chemiluminescent Immunoassay) which detects
IgG antibodies reactive with the spike protein (S1/S2 domain). The assay
was performed on the LIAISON® XL Analyzer according to the
manufacturer's instructions. The diagnostic sensitivity was 97.9\%
(89.1\% - 99.6\%; Wilson 95\% Cl) according to the manufacturer, the
specificity in laboratory routine was 99.0\% (96.4\% - 99.7\%; Wilson
95\% Cl).

\emph{Enzyme-Linked Neutralization Assay (ELNA):}

VeroB4 cells (ACC-33, DSMZ) were seeded in flat-bottom 96 well plates
(Sarstedt, Germany) with Medium199 (Thermo Scientific Gibco, USA) and
10\% fetal calf serum (Thermo Scientific Gibco, USA) at a density of
about 10\textsuperscript{6} cells/ml to receive a confluent monolayer.
Next day, an infectivity titration was carried out to determine 100
tissue culture infectious dose 50\% (100 TCID\textsubscript{50}).

Inactivated serum samples were titrated in duplicate in twofold dilution
steps, starting at a dilution of 1:4 in Medium199 containing 3\% fetal
calf serum. Equal volumes of virus (1 x
10\textsuperscript{5}TCID\textsubscript{50}) and serum dilutions in
Medium199 were mixed and subsequently incubated for 1 to 1.5 hours at
room temperature in U-bottom 96 well plates (Thermo Scientific Thermo
Scientific Nunc, USA). After incubation, a pre-seeded flat-bottom 96
well plate with confluent VeroB4 cells was used, medium was discarded,
the incubated mixture of patient´s serum and defined virus solution was
transferred to each corresponding well of the flat-bottom plate and the
plate was incubated for 24 hours at room temperature. Incubation was
stopped by discarding supernatant, cells were washed in PBS twice, fixed
with ice-cold acetone-methanol (1:1) and frozen for at least 15 minutes.
All steps were performed under strict observation and in compliance with
biosafety level 3. The analysis was carried out like an enzyme-linked
immunosorbent assay using a BEP III (Siemens, Germany) according to the
following steps: blocking (45 minutes, 37° C, Candor Biosciences,
Germany), washing 3 x (wash pod, Siemens, Germany), anti-SARS-CoV-2
nucleocapsid protein IgG (Bioss bsm-41413M, dilution 1:5000) for 30 min.
at 37° C), washing 3 x, adding of horseradish-peroxidase-conjugated goat
anti mouse IgG (ABIN376241, dilution 1:5000) for 30 min. at 37° C),
washing 3 x, adding substrate tetramethylbenzidine (TMB) and stop
solution (Siemens, Germany). 24 hours post infection the reading of the
results was done.

The cut-off titer was set by titrating defined negative human sera from
volunteers out of healthy Tyrolean blood donors from the year 2009 and
was set at 1:4 in combination with the viral dose of
1x10\textsuperscript{5} TCID\textsubscript{50} and calculated as median
optic density minus the standard deviation. A sample was considered
positive when the given optic density was higher than the cut-off titer.

Titers of single 1:4 in ELNA were valued as borderline, titers of 1:4 in
duplicate as weak neutralization ability, 1:8 to 1:16 as moderate/good
and \textgreater{} 1:16 as strong neutralizing ability.

\emph{Definition of antibody development:}

In ELNA and IFA, a change in antibody titer of more than one dilution
step over the investigation period from time point 1 (T1) to time point
5 (T5) was defined as an increase or decrease of the titer. In the IgG
CLIA assay, the development of antibodies was defined as a change in
chemoluminescence of more than 50\%.

\emph{Statistics:}

Dichotomous data were tested by a chi-squared test. A two-sided
significance level of \emph{p} = 0.05 was used for determining
statistical significance. The Pearson correlation coefficient was used
to analyze correlation of titers between CLIA and ELNA (Microsoft®
Excel®, Microsoft 395 MSO, Windows 2010). To determine the predominant
titers in a group of patients, the median was calculated (Microsoft®
Excel®, Microsoft 395 MSO, Windows 2010).

RESULTS

\emph{Characteristics of the study group:}

The individual severity of disease was assessed clinically using a
standardized questionnaire including age, gender, pre-existing as well
as acute physical condition and rated as asymptomatic, mild, moderate
and severe course of disease according to the definitions by Gietl et
al., under review).

Within our study group, three patients (8.8\%) were asymptomatically
infected with SARS-CoV-2, 20 patients (58.8\%) showed a mild, six
(18.8\%) a moderate and five (14.7\%) a severe course of the disease.

In total, the group consisted of 18 women and 16 men. Gender-related
differences in the course of disease was not significant (\emph{p} =
0.11), although only one (5.6\%) female had a severe course compared to
four male patients (25\%).

The median age was 49.0 years (range 11 to 77 years; \emph{sd} = 16.4).
Due to different expectations of disease course (Gietl et al., under
review), the age groups were divided into three groups of 11 -- 30, 31
-- 60 and 61 -- 77 years.

\emph{Seroconversion assessed by different methods:}

90.9\% (30 of 33) SARS-CoV-2-positive patients in the convalescent phase
(\textgreater{} 21 d after onset of the symptoms) tested had
seroconverted at T1 in April and in total, 85.3\% of the patients (29 of
34 samples) had seroconverted until September, as determined by
neutralization test (table 1).

Overall, 85.3\% of SARS-CoV-2-infected persons seroconverted and
maintained constant neutralizing antibodies over the study period of
five months.

The highest number of positives was found in the IgG IFA one month post
infection (32 of 34; 94.1\%), the lowest number of seroconversions was
found in CLIA IgG (28 of 34; 82.4\%). In 11 cases (32.4\%), IgM was
detectable by IFA 28 -- 41 days after onset of the symptoms.

Four patients had no specific antibody response at all and eleven
patients developed only weak antibody responses five months post
infection. All four negative patients had a mild course of disease;
their mean age was 51 (\emph{sd} = 12.4) years.

\emph{Comparison CLIA IgG -- ELNA}

Both methods showed that specific antibodies stayed constant over the
observation period and that a stronger disease severity led to a more
stable antibody response.

CLIA IgG recognized two sera from the first time point as positive
(5.9\%), which turned out as negative in ELNA. This result goes along
with the manufacturer´s information concerning 95\% specificity and 0\%
false-negative results. Repeated serologic investigations at T5
exhibited one patient (3.0\%) false-negative in CLIA IgG.

\emph{Comparison IFA -- ELNA}

IFA gave 94\% positive results, 6 more than ELNA, in the sera of T1 and
the highest decrease of antibodies until T5 (n = 7; 65.6\%; decrease of
28.4\%). Two of the seven sera, which proved negative in IFA, were also
negative in ELNA, the others were weak positive with titers around 1:4
in ELNA, as shown in table 2.

\emph{Longevity of IgG antibodies:}

In one 65 year old male patient with a mild course of disease the
neutralizing antibody titer dropped from 1:32 at T1 to \textless{}1:8 at
T5.

Two patients showed an increase in neutralizing antibodies over the
sample period. In both patients, the first blood sample showed weak or
moderate neutralizing ability with titers of 1:4 and 1:8, which
increased to titers of 1:32 and 1:64, respectively. The courses of
disease in these cases were mild and severe, respectively.

In 83.9\% of the patients, the neutralizing antibody titers against
SARS-CoV-2 stayed constant and did not change more than two dilution
steps or more than 50\% change of optic density in CLIA IgG.

Two patients were negative in the CLIA IgG assay but showed weak
neutralizing activities with antibody titers of 1:4.

\emph{Influence of the course of disease on neutralizing antibodies:}

The median neutralizing antibody titer of symptomatic patients was two
levels higher than in asymptomatic patients (n = 31 versus 3
patients;\textbf{Figure 1} ). However, there was no statistically
significant correlation between the individual titers and the severity
of infection (\emph{k} = -0.09).

Our data also reveal a relation between the severity of infection and
neutralizing activity by ELNA (3 and 31/34; \emph{p} = 0.07).

Moderate or severe SARS-CoV-2 infections led to the development of
neutralizing antibodies significantly more frequently than in
asymptomatic or mild infections (\emph{p} = 0.03). There was no
significant difference in neutralization activities between asymptomatic
and mild courses of disease (\emph{p} = 0.17). Interestingly, 60\% of
the patients who suffered severe infections (3/5), developed low
neutralizing antibodies (\textbf{Figure 2} ).

\emph{Age and disease severity:}

There was no correlation between age and disease severity (\emph{k} =
0.09) or neutralizing antibodies and disease severity (\emph{k} =
-0,0951), nor could we find significant predominance of severe cases in
older age groups or male patients (\emph{p} = 0.53).

The mean age of severe cases was 46 (\emph{sd} 15.1, n = 5) versus 49
years (sd 16.8; \emph{p} = 0.36, not significant) in cases of other
severity categories. All patients with severe infections had
neutralizing antibodies at T1. However, only 2 (40\%) developed
constantly neutralizing antibodies, and 60\% in this group showed low
titers (\textless{}1:8) 5 months post infection.

DISCUSSION

Specific neutralizing antibodies were detected in 90.0\% of 34
laboratory confirmed cases of SARS-CoV-2 infected patients in the early
convalescent phase \textasciitilde{}21 days post infection and stayed
constant in 85.3\% of the patients in the convalescent stage five months
later, confirming recently published data of high prevalence of
neutralizing antibodies in most SARS-CoV-2 afflicted individuals

In our study, neutralization activity was correlated with disease
severity. 86.7\% of the symptomatic patients (21 of 31) as well as
66.7\% of the asymptomatic patients (1 of 3) maintained neutralizing
antibodies over the observation period of 5 months. Investigations of
IgG levels and neutralizing antibodies in early phase of convalescence
gave a similar result (. 60\% of the study members who suffered severe
infections (3/5) developed low neutralizing antibody titers, which may
allow the speculation that impaired immune responses may contribute to
severe disease manifestation or an unevently observed B cell lymphopenia
in COVID-19 (Melenotte et al., 2020; Ni et al., 2020; Q. X. Long et al.,
2020; Wu et al., 2020; Qin et al., 2020). Although, concomitant
treatments e. g. with glucocorticoids or other immunosuppressants
recommended for COVID-19 may mitigate the generation of an efficient
humoral immune response (Bamoulid et al., 2015; Thaunat et al., 2016).

In accordance with another study , the IgG levels in the symptomatic
group (n=31) were significantly higher than those in the asymptomatic
group (n=3; \emph{p} = 0.008).

Two patients were negative in the early stage of the convalescent phase
four weeks post infection (T1), one - a patient with a mild course of
disease - stayed negative in all tests, whereas another one with an
asymptomatic infection developed a weak neutralization response in the
late convalescent phase (T5). This also fits well with published data,
where milder courses of disease may cause longer periods to generate
specific antibodies and in a low number of cases, patients did not
seroconvert at all after infection with SARS-CoV-2

A recently published study (Wajnberg et al., 2020) investigated the
persistence of neutralizing antibodies and is in accordance with our
results of relatively stable maintenance of neutralizing antibodies in
convalescent COVID-19 patients over a study period of 5 months.

The comparison of different serologic methods shows that the IgG CLIA
reflects the results of the neutralization assay well with concordances
of 95\%, giving overall 5.9\% false-positive and 3\% false-negative
results. The discrepancies between the two methods may be found in the
coating with Spike S1 and S2, but the Receptor Binding Domaine (RBD),
which is expressed in the NT results, is of great significance, too. It
is also discussed by others (Suthar et al., 2020).

Many commercially available SARS-CoV-2 ELISAs are coated with Spike 1, 2
or both, but neutralizing antibodies are directed against the RBD or
other targets and more unspecific antibodies tend to decline in the
convalescent phase of an infection (Suthar et al., 2020). A high ELNA
titer does not necessarily mean a high CLIA titer and vice versa.

The CLIA IgG is a fast and easy to handle diagnostic tool, which is
known to have limited specificity. The high number of positives at the
first time point might be due to the low specificity of the method and
suggests a high degree auf cross-reactivity, presumably with endemic
nonSARS-hCoVs like OC43.

Our results show that a high proportion of convalescent SARS-CoV-2
afflicted patients maintain constant neutralizing antibody titers at
least five months post-infection, high enough to give hope for a
protective and longer-lasting immunity after wild-type SARS-CoV-2
infection, which may also be relevant to estimate the long-term efficacy
of a vaccination. Furthermore, our data reveal that individuals who have
recovered from symptomatic COVID-19 generated more robust neutralizing
antibody responses than those with asymptomatic infections.

ACKNOWLEDGEMENTS

We gratefully acknowledge the financial support of the Österreichische
Forschungsförderungsgesellschaft (FFG), grant no. 35863961. The authors
report no potential conflict of interest. We also thank DDI Martin
Lamprecht, Stefanie Sonnleitner and Mag. Eva Hinterbichler for excellent
technical assistance and valuable suggestions.

ETHICAL STATEMENT

The authors confirm that the ethical policies of the journal, as noted
on the journal's author guidelines page, have been adhered to and the
appropriate ethical review committee approval has been received (Ethics
Committee of the University Hospital Wuerzburg no. 20201105\_01).

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

REFERENCES

\textbf{Table 1:} Comparison of different serologic methods for antibody
detection in 34 COVID-19 patients one respectively five months post
infection.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\textbf{T1} & \textbf{positive} & \textbf{{[}\%{]}} & \textbf{negative}
& \textbf{{[}\%{]}} & \textbf{borderline} & \textbf{{[}\%{]}} &
\textbf{n. a.} & \textbf{total}\tabularnewline
\midrule
\endhead
ELNA & 30 & 90.9 & 2 & 5.0 & 1 & 3.0 & 1 & 34\tabularnewline
IFA & 32 & 94.1 & 2 & 4.9 & 0 & 0.0 & 0 & 34\tabularnewline
CLIA IgG & 28 & 82.4 & 5 & 7.9 & 1 & 2.9 & 0 & 34\tabularnewline
IFA IgM & 11 & 32.4 & 23 & 67.6 & 0 & 0 & 0 & 34\tabularnewline
Total & 34 & 100.0 & & 2.9 & & 0.0 & &\tabularnewline
\textbf{T5} & \textbf{positive} & \textbf{{[}\%{]}} & \textbf{negative}
& \textbf{{[}\%{]}} & \textbf{borderline} & \textbf{{[}\%{]}} &
\textbf{n. a.} & \textbf{total}\tabularnewline
ELNA & 29 & 85.3 & 5 & 14.7 & 0 & 0.0 & 0 & 34\tabularnewline
IFA & 21 & 65.6 & 5 & 15.6 & 6 & 18.8 & 2 & 34\tabularnewline
CLIA IgG & 27 & 81.8 & 5 & 15.2 & 1 & 3.0 & 1 & 34\tabularnewline
IFA IgM & 0 & 0.0 & 34 & 100.0 & 0 & 0.0 & 0 & 34\tabularnewline
\bottomrule
\end{longtable}

n.a.: not assessed.

\textbf{Table 2:} Development of SARS-CoV-2 specific humoral immunity
within the first five months (22 weeks) post infection.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
& \textbf{loss} & \textbf{{[}\%{]}} & \textbf{increase} &
\textbf{{[}\%{]}} & \textbf{constant} & \textbf{{[}\%{]}} &
\textbf{total (without negatives)}\tabularnewline
\midrule
\endhead
NT & 1 & 3.2 & 2 & 6.5 & 28 & 90.3 & 31\tabularnewline
IFA IgG & 11 & 34.4 & 0 & 0.0 & 21 & 65.6 & 32\tabularnewline
CLIA IgG & 7 & 21.9 & 7 & 21.9 & 18 & 56.3 & 32\tabularnewline
IFA IgM & 11 & 100 & 0 & 0.0 & 0 & 0.0 & 11\tabularnewline
\bottomrule
\end{longtable}

Only patients with borderline or positive results in the corresponding
tests were used for calculation. In NT, one patient (3.2\%) lost
antibodies, two (6.5\%) developed higher, more stable IgG titers, the
antibody titers of 28 patients (90.3\%) stayed constant during the first
five months post infection.

\textbf{Figure 1:} The median titer of \textbf{s} ymptomatic patients
was two levels higher than those of asymptomatic patients.

\textbf{Figure 2:} Development of neutralizing antibodies goes along
with disease severity. Neutralization activity is more pronounced in
patients with more severe infections. Moderate and severe courses of
disease led to neutralizing antibodies in 100\% of the patients, whereas
33\% and 20\% of persons with an asymptomatic course of infection did
not constantly develop neutralizing antibodies.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}

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

