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\begin{document}

\title{Assessment of timing of transplacental antibody transfer after
SARS-CoV-2 vaccination against the Omicron variant: A prospective cohort
study}



\author[1]{Chia-Jung Chiang}%
\author[1]{Wei-Lun Hsu}%
\author[1]{Meitsz Su}%
\author[1]{Wen-Chien Ko}%
\author[1]{Keng-Fu  Hsu}%
\author[1]{Pei-Yin  Tsai}%
\affil[1]{National Cheng Kung University Hospital}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Objective} To determine the optimal timing of SARS-CoV-2
vaccination during pregnancy by investigating the transplacental
transfer of SARS-CoV-2 antibodies from maternal SARS-CoV-2 vaccination
or infection. \textbf{Design} Prospective cohort study \textbf{Setting}
Samples were collected between June 6, 2022 and September 20, 2022 in
Taiwan. \textbf{Sample} Seventy-five maternal-cord pairs, including 38
pairs with a primary maternal SARS-CoV-2 infection by the Omicron
variant (BA.2.3.7) and 37 pairs without maternal infection.
\textbf{Methods} SARS-CoV-2 antibodies against the nucleocapsid
(anti-N), receptor-binding domain of the spike protein (anti-S), and the
neutralizing antibody (nAb) titers against different SARS-CoV-2 variants
were measured for the maternal-cord pairs. The participants were
categorized based on the timing of their last vaccination, including
during the third (T3) and second trimesters (T2) and before/during the
first trimester (T1). Comparison of anti-spike protein antibody and
neutralizing antibody concentrations was analyzed using the
Kruskal-Wallis test followed by a post-hoc Mann-Whitney U test.
\textbf{Main Outcome Measures} Anti-S concentrations of maternal and
cord serum among T1, T2, and T3 groups. \textbf{Results} Among
participants without SARS-CoV-2 infection, the highest anti-S levels (
\emph{p} value for anti-S levels in maternal and cord plasma were
\textless{}0.001 and \textless{}0.001, respectively) and highest nAb
potency against both the Wuhan and Omicron strains ( \emph{p} value for
nAb against Omicron strain were both \textless{}0.001 for maternal and
cord plasma) were observed in maternal and cord plasma in the T3 group.
\textbf{Conclusions} A booster vaccine dose during the third trimester
can provide maximum transplacental protection against the Wuhan
wild-type strain and Omicron variant. Pregnant women are encouraged to
receive vaccinations during pregnancy, ideally in the third trimester,
for the highest level of neonatal protection.%
\end{abstract}%



\sloppy


\textbf{Article type:} Original Research

\textbf{Title:} \textbf{Assessment of timing of transplacental antibody
transfer after SARS-CoV-2 vaccination against the Omicron variant: A
prospective cohort study}

Chia-Jung Chiang, MD\textsuperscript{*1}; Wei-Lun Hsu,
MD\textsuperscript{*1}; Mei-Tsz Su, MD, PhD\textsuperscript{1} Wen-Chien
Ko, MD\textsuperscript{2,3}; Keng-Fu Hsu, MD, PhD\textsuperscript{1};
Pei-Yin Tsai, MD\textsuperscript{1}

\textsuperscript{*} Chia-Jung Chiang and Wei-Lun Hsu contributed equally
to this paper.

\textsuperscript{1} Department of Obstetrics and Gynecology, National
Cheng Kung University Hospital, College of Medicine, National Cheng Kung
University, Tainan, Taiwan

\textsuperscript{2} Department of Internal Medicine, National Cheng Kung
University Hospital, College of Medicine, National Cheng Kung
University, Tainan, Taiwan

\textsuperscript{3} Center for Infection Control, National Cheng Kung
University Hospital, College of Medicine, National Cheng Kung
University, Tainan, Taiwan

\textbf{* Correspondence to: Pei-Yin Tsai, MD}

Department of Obstetrics and Gynecology, National Cheng Kung University
Hospital, College of Medicine, National Cheng Kung University, Tainan,
Taiwan

Address: 138 Sheng-Li Road, Tainan, Taiwan 704, ROC

Phone numbers: +886-6-2353535 (5221)

E-mail address: tsaipy@ncku.edu.tw

\textbf{Running title:} Optimal Timing of SARS-CoV-2 Vaccination in
Pregnancy

\textbf{Disclosure of interest}

The authors have declared no conflicts of interest.

\textbf{Contribution to Authorship}

CJC involved in conceptualization, methodology, and writing the original
draft, which was reviewed and critically revised by all authors. WLH
involved in analysis and visualization. PYT involved in
conceptualization, validation, review of manuscript, and funding
acquisition. MTS, WCK, KFH involved in resources and review of
manuscript. All authors approved the final manuscript as submitted.

\textbf{Funding}

This work was supported by the NCKU Epidemic Prevention Scientific
Research Center, Ministry of Science and Technology of Taiwan (MOST
111-2321-B006-009) and funded by grant from the National Cheng Kung
University Hospital (NCKUH -11109022). The funders had no role in
designing and conducting the study, in collecting and interpreting the
data or in the decision to submit the work for publication.

\textbf{Details of Ethics Approval}

The study was approved by the Institutional Review Board of NCKUH (IRB:
B-ER-111-164), and all the participants provided informed consent.

\textbf{Abstract}

\textbf{Objective}

To determine the optimal timing of SARS-CoV-2 vaccination during
pregnancy by investigating the transplacental transfer of SARS-CoV-2
antibodies from maternal SARS-CoV-2 vaccination or infection.

\textbf{Design}

Prospective cohort study

\textbf{Setting}

Samples were collected between June 6, 2022 and September 20, 2022 in
Taiwan.

\textbf{Sample}

Seventy-five maternal-cord pairs, including 38 pairs with a primary
maternal SARS-CoV-2 infection by the Omicron variant (BA.2.3.7) and 37
pairs without maternal infection.

\textbf{Methods}

SARS-CoV-2 antibodies against the nucleocapsid (anti-N),
receptor-binding domain of the spike protein (anti-S), and the
neutralizing antibody (nAb) titers against different SARS-CoV-2 variants
were measured for the maternal-cord pairs. The participants were
categorized based on the timing of their last vaccination, including
during the third (T3) and second trimesters (T2) and before/during the
first trimester (T1). Comparison of anti-spike protein antibody and
neutralizing antibody concentrations was analyzed using the
Kruskal-Wallis test followed by a post-hoc Mann-Whitney U test.

\textbf{Main Outcome Measures}

Anti-S concentrations of maternal and cord serum among T1, T2, and T3
groups.

\textbf{Results}

Among participants without SARS-CoV-2 infection, the highest anti-S
levels (\emph{p} value for anti-S levels in maternal and cord plasma
were \textless{}0.001 and \textless{}0.001, respectively) and highest
nAb potency against both the Wuhan and Omicron strains (\emph{p} value
for nAb against Omicron strain were both \textless{}0.001 for maternal
and cord plasma) were observed in maternal and cord plasma in the T3
group.

\textbf{Conclusions}

A booster vaccine dose during the third trimester can provide maximum
transplacental protection against the Wuhan wild-type strain and Omicron
variant. Pregnant women are encouraged to receive vaccinations during
pregnancy, ideally in the third trimester, for the highest level of
neonatal protection.

\textbf{Keywords}

SARS-CoV-2 vaccine, COVID-19, Omicron, Pregnancy, Transplacental
antibody transfer

\textbf{Introduction}

The global outbreak of severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) in December 2019 has been a worldwide concern. Taiwan,
being an isolated island in East Asia, had a low incidence of SARS-CoV-2
infection until 2020.(1, 2) In Taiwan, the pandemic surged in April 2022
with the Omicron variant (BA.2.3.7) being identified as the major
lineage.(3)

Pregnant individuals are encouraged to receive the SARS-CoV-2 vaccine
because of the higher risk of adverse outcomes, such as death,
intubation, admission to an intensive care unit, preterm birth,
preeclampsia, venous thromboembolism, and other severe
comorbidities.(4-6) The safety of SARS-CoV-2 vaccination during
pregnancy has been supported by current evidence.(7, 8)

Newborns have immature immune systems and are vulnerable to infectious
diseases. Neonatal passive immunity mainly depends on the maternal
concentration of respective antibodies for most viral diseases.(9) Low
or early disappearance of maternal-derived antibody concentrations can
lead to insufficient protection for newborns. Maternal-derived,
vertically transferred immunity provides protection for newborns against
vaccine-preventable infectious diseases, such as measles, mumps, and
rubella, which cannot be administered to infants before 12 months.(10)
Similarly, no SARS-CoV-2 vaccine is currently available for newborns.
For the window period before routine vaccination of the newborn, passive
immunization for neonates from vertical transmission can be present for
up to six months.(11)

Transplacental transfer of SARS-CoV-2 antibodies, specifically against
the receptor binding domain (RBD) of the spike protein (anti-S) and
nucleocapsid antigen (anti-N), has been observed in previous studies of
both infected pregnant individuals and those vaccinated against
COVID-19.(12-17) However, the relationship between the timing of
vaccination or infection and transplacental antibody transfer is
unclear.

Given that newborn protection from infectious diseases depends on
antibodies acquired transplacentally,(9) we investigated the timing of
transplacental passage of SARS-CoV-2 antibodies through vaccines and
maternal infections. We focused on a specific, highly vaccinated
pregnant population with or without SARS-CoV-2 infection, solely by the
Omicron strain.

\textbf{Methods}

A prospective cohort study was conducted at the National Cheng Kung
University Hospital (NCKUH), a medical center in Southern Taiwan,
between June 6, 2022 and September 20, 2022. Eligible participants
included the following: pregnant women who delivered at the NCKUH; women
with or without a primary SARS-CoV-2 infection officially reported to
the Central Epidemic Command Center (CECC) of Taiwan; women with cord
blood sample available upon delivery and maternal blood sample available
within three days from delivery; and participants who provided informed
consent. Our study population did not have a second SARS-CoV-2
infection.

The participants were categorized based on the timing of their last
vaccination, including during the third trimester (T3), second trimester
(T2), and before and during the first trimester (T1). The diagnosis of
SARS-CoV-2 was confirmed by real-time polymerase chain reaction (RT-PCR)
or antigen testing of respiratory tract specimens, according to the
guidelines of the CECC of Taiwan. The study was approved by the
Institutional Review Board of NCKUH (IRB: B-ER-111-164), and all the
participants provided informed consent.

Clinical and laboratory data were collected using questionnaires and
electronic medical records. Cord blood was obtained from the umbilical
cord immediately after delivery. Blood samples were centrifuged at 1000
g for 10 min at room temperature, and plasma samples were aliquoted into
dedicated pre-coded tubes and stored at --80\selectlanguage{ngerman}°C until analysis.

\textbf{Serological Testing}

Severe acute respiratory syndrome coronavirus 2 antibody levels were
measured in the maternal and fetal cord plasma using the Elecsys
Anti-SARS-CoV-2 and Anti-SARS-CoV-2 S kits (Roche Diagnostics,
Switzerland). Immunoglobulin M and G antibodies against the nucleocapsid
(anti-N) and receptor-binding domain~of the spike (anti-S) of SARS-CoV-2
were detected.~The cut-off for positivity was \selectlanguage{english}[?]1.0 or the anti-N assay
and \selectlanguage{english}[?]0.8 U/mL for the anti-S\_RBD assay. The value of the anti-S\_RBD
assay between 0.4--250 U/mL represents the linear range, and samples
\textgreater{}250 U/mL were diluted with a Diluent~Universal provided by
the kit. The cPass\textsuperscript{TM}~surrogate virus neutralization
test (GenScript Biotech~Corp., USA) was conducted to measure
neutralizing antibody (nAb) titers against different~SARS-CoV-2
variants. Ninety-six-well plates coated with recombinant hACE2 and
recombinant S-RBD~from Wuhan and Omicron BA.2 variants were used in the
experiments, according to the manufacturer's instructions. An inhibition
percentage \selectlanguage{english}[?]30\% was considered seropositive for SARS-CoV-2 nAbs.
Additionally, the percent inhibition of the Wuhan strain was converted
to IU/mL using a previously proposed formula.(18)

\textbf{Statistics}

Differences between participants with and without SARS-CoV-2 infection
were analyzed using the Mann-Whitney U and chi-square tests with a
two-sided P value. Demographic variables and antibody responses were
also evaluated. Continuous outcome measures are summarized as median and
interquartile range (IQR), and antibody concentrations were
log10-transformed for the analysis. A P value of \textless{}0.05 was
considered significant.

The comparison of anti-S antibody and neutralizing antibody
concentrations was analyzed using the Kruskal-Wallis test followed by a
post-hoc Mann-Whitney U test. Statistical analysis was conducted using
SPSS version 28 and Prism version 7 (GraphPad). Temporal relationships
between antibody concentrations were plotted as quadratic curves using a
polynomial regression approach.

\textbf{Results}

Between June 6, 2022 and September 20, 2022, there were 89 women
admitted to the NCKUH Maternal Unit for delivery. Of these, 46 were
either previously diagnosed with SARS-CoV-2 infection during pregnancy
or had a positive nasopharyngeal RT-PCR test result upon admission. Of
the 46 women with SARS-CoV-2 infection, 42 had paired maternal and cord
blood samples available for the analysis. Four women without SARS-CoV-2
vaccination had negative maternal plasma anti-S antibody levels;
therefore, they were not eligible for further analysis of transplacental
antibody transfer and were excluded from the SARS-CoV-2 infection group.
Of the 89 women, 43 had no history of SARS-CoV-2 infection and had
negative nasopharyngeal RT-PCR test results upon admission with paired
maternal and cord blood samples. Six women were excluded from the
SARS-CoV-2 negative group due to reactive anti-N antibody levels, which
indicated a possible previous asymptomatic SARS-CoV-2 infection without
documentation. Ultimately, 75 maternal-cord pairs were enrolled for
analysis, including 38 pairs from the SARS-CoV-2 infection group and 37
pairs from the SARS-CoV-2 negative group (Fig. S1).

Demographic, obstetric, and clinical characteristics of the participants
are summarized in Table 1. The median ages of the participants in the
SARS-CoV-2 and SARS-CoV-2 negative group were 32.5 (IQR, 29.75--38) and
34 (IQR, 30--37), respectively. Both groups had similar profiles in
terms of body mass index (BMI), gravidity, and gestational age at birth.
Cesarean section rate was higher in the SARS-CoV-2 negative group (49\%
vs. 21\%, p=0.012).

Both groups had similar vaccination profiles, and all the participants
included in the analysis received a vaccine. Approximately 60\% of the
patients from both groups (58\% in the SARS-CoV-2 group and 60\% in the
non-SARS-CoV-2 group) were fully vaccinated with a primary series plus
one booster dose. Most of the patients (58\% in the SARS-CoV-2 group and
62\% in the non-SARS-CoV-2 group) received various types of vaccines,
including mRNA (NT162b2, Pfizer/BioNTech, or mRNA-1273, Moderna), viral
vector (ChAdOx1 nCoV-19, Oxford-AstraZeneca), and protein subunit
(MVC-COV1901, Medigen) vaccines. The difference in median intervals from
the last vaccination to delivery was not significant with median
intervals of 125 days in the SARS-CoV-2 group and 105 days in the
non-SARS-CoV-2 group (p=0.824). (Table 1)

The participants were categorized based on the timing of their last
vaccination: during the T3, T2, and before/during the T1 (Fig. 1). The
highest concentration of maternal and cord plasma anti-S antibodies was
observed in the T3 group among all the participants (median 10627.5
U/mL, IQR 6102.75--18021.5 U/mL in maternal plasma; median 16082.5, IQR
10805.5--19823.75 U/mL in cord plasma; p\textless{}0.001 and
\textless{}0.001, respectively) (Fig. 1A). For the SARS-CoV-2 negative
pregnant women, the anti-S titer was significantly higher in the T3
group (median 10442 U/mL, IQR 9535--13033 U/mL for maternal plasma;
median 14999 U/mL, IQR 12085--19042 U/mL for cord plasma;
p\textless{}0.001 and \textless{}0.001, respectively) than in the T1
(median 578.5 U/mL, IQR 160.25--6425 U/mL for maternal plasma; median
576.1 U/mL, IQR 184--3757 U/mL for cord plasma) and T2 (median 4565
U/mL, IQR 1749--10213.25 U/mL for maternal plasma; median 6017 U/mL, IQR
3838.75--11717.25 U/mL for cord plasma) groups (p\textless{}0.001 for
maternal plasma and \textless{}0.001 for cord plasma) (Fig. 1B).
However, the significance of vaccine timing on both maternal and cord
plasma anti-S titer was diminished when comparing the SARS-CoV-2
positive T1, T2, and T3 groups (Fig. 1C), which emphases the effect of
recent infection on the anti-S titer. (Table S1)

The nAbs response at different times after the last vaccination were
similar to those of anti-S antibodies. Maximum neutralizing responses
against both the Wuhan wild-type strain and Omicron variant were found
in the maternal and cord sera of the T3 group (Fig. 1D, 1G). The
neutralizing responses of the SARS-CoV-2 vaccine against the Omicron
variant were more pronounced in the SARS-CoV-2 negative participants,
and the neutralizing responses increased significantly across the T1,
T2, and T3 groups (T3 group of cord sera with median 98.15\%, IQR
98.04--98.22\% for the Wuhan strain; median 88.94, IQR 79.36--91.98\%
for the Omicron strain; p \textless{}0.001) (Fig. 1E, 1H). However, the
nAb response rates in both maternal and cord sera were not significant
for participants with SARS-CoV-2 infection across the T1, T2, and T3
groups (Fig. 1F, 1I). (Table S2)

The levels of maternal and cord serum anti-S antibody concentrations
increased significantly seven days after SARS-CoV-2 infection (Fig. S2)
(Table S3) and peaked around 50 days from the day of the last
vaccination (Fig 2A) or SARS-CoV-2 infection (Fig. 2B). Delayed peaks
and declines were observed in the cord sera of both groups. The anti-S
antibodies were measurable in maternal and cord sera up to one year
after the last vaccination (Fig. 2A). A delayed appearance of anti-N
antibody in cord sera was also found following SARS-CoV-2 diagnosis.
While the anti-N antibodies were detected in 5 out of the total 46
maternal serum samples within 7 days from diagnosis, a delayed response
was observed in cord serum, with a rise in anti-N antibodies not
observed until 18 days after SARS-CoV-2 diagnosis. (Figure S3)

The transplacental transfer ratio (TR), which was calculated as the cord
serum antibody concentration divided by the maternal serum antibody
concentration measured the transplacental transfer of antibodies. Time
variable was presented as the time interval from delivery to the last
antigen exposure, either the last vaccination or infection, which ever
occurred last. The TRs of anti-S from the post-infection and
non-infection groups and anti-N from the post-infection group showed a
similar linear correlation with the time variable before 100 days (Fig.
3). The TRs peaked at approximately 100 days, reaching as high as 3--4,
then plateaued at approximately 1--2 after 150 days.

\textbf{Discussion}

\textbf{Main Findings}

Our study has shown that a booster dose of the SARS-CoV-2 vaccine during
the third trimester can help achieve maximum transplacental protection
against SARS-CoV-2.

\textbf{Strengths and Limitations}

Our study presents some significant advantages. First, the study
population was unique in that it enabled us to analyze only patients
infected with the Omicron variant and without any previous SARS-CoV-2
exposure. Second, our study cohort of pregnant women differed from those
of previous studies with an extremely high rate (95.5\%) of vaccine
coverage. (7) All of the included 85 pregnant women had received one or
more vaccinations. Third, a clear temporal relationship was established
between maternal and cord serum antibodies. Both the timing of maternal
SARS-CoV-2 infection and maternal SARS-CoV-2 vaccination were well
documented in all our included cases. Fourth, a similar rising trend in
the TR was observed in both the anti-S and anti-N from vaccinated or
infected maternal-fetal pairs, suggesting that the previously proposed
lower TRs in the third trimester or in infected individuals may be
influenced by time. Lastly, our results showed that mothers and newborns
would receive the greatest protection at birth if the mother received
her last vaccination during the third trimester, as indicated by
maternal and cord serum anti-S antibody levels and neutralizing
antibodies.

However, our study has some limitations. The sample size was relatively
small, and data on serial follow-up of antibody titers were not
available; therefore, we could not address the dynamic changes in
antibody titers in individuals. Additionally, maternal-acquired passive
immunity includes not only immunoglobulin but also cellular responses,
such as the vertical transfer of pathogen-specific T cells.(9) Our focus
was solely on the measurable immunoglobulin titer from maternal and cord
serum. Further research that specifically examines cellular level and on
functional activity may provide additional insights into the neonatal
immunity.

\textbf{Interpretation}

The safety of SARS-CoV-2 vaccination during pregnancy has been
established in current short-term reports.(7, 8) Despite this, more than
50\% of pregnant women have reported hesitancy towards COVID-19
vaccination,(19, 20) and the coverage rate among this population is only
approximately 30--50\%.(7, 21)

Our study focused on a highly vaccinated pregnant population, where only
four out of 89 participants were unvaccinated and excluded from the
analysis. Seventy-two participants received one or more vaccinations
during pregnancy, and half of them completed the primary series with a
booster dose. All the participants received first-generation vaccines
designed for the Wuhan strain.

Participants with and without SARS-CoV-2 infection showed similar
characteristics in terms of vaccination history and type. Our high
vaccination coverage rate allowed for a focused analysis of the effects
of vaccination and infection in the pregnant population. Other
characteristics, including gestational age, maternal general condition,
comorbidities, patient age, and BMI, were similar between the two
groups. The cesarean rates differed because our hospital serves as a
tertiary center where patients without SARS-CoV-2 infection were mostly
admitted because of high-risk pregnancy.

The timing of vaccination is a critical factor that affects the level of
cord serum antibodies, particularly in pregnant individuals without
prior SARS-CoV-2 infection. Previous reports have demonstrated the
safety of SARS-CoV-2 vaccination during the third trimester and its
ability to reduce the risk of neonatal adverse outcomes.(7) However,
optimal trimester for maternal vaccination remains a topic of discussion
due to limited cord samples and follow-up intervals in previous
studies.(22) Our study, on the other hand, revealed that neonates born
to mothers who received a booster dose of SARS-CoV-2 vaccine during the
third trimester, especially those without prior SARS-CoV-2 infection,
had the highest level of cord serum anti-S concentration (Fig. 2) and
the most effective protection against both Wuhan and Omicron strains.
(Fig. 3)

In the immunocompetent general population, anti-S and anti-N antibodies
appear 8--14 days after the onset of symptoms.(23) In our study, delay
in the elevation of cord serum anti-S antibody levels was also observed
in comparison to maternal serum levels, both following vaccination and
infection. Our study found that the transmission ratios of both anti-S
and anti-N in maternal-fetal pairs had a comparable association with the
time variable, up to 150 days from the last vaccination or infection.
Previous observations of a negative correlation between transmission
ratios and viral load (12, 24) may be attributed to the decline in viral
load over time. Based on these findings, we can conclude that recent
SARS-CoV-2 infection may induce a booster vaccine-like effect on
antibody titers in pregnant women and their fetuses. Furthermore, our
study suggests that the interval between antigen exposure and blood
sampling may be the primary determinant of the transplacental antibody
transmission ratio.

Our results are consistent with previous experience with the combined
tetanus-diphtheria-pertussis vaccine. Tetanus-diphtheria-pertussis
immunization 8--12 weeks prior to delivery provides newborns with the
highest antibody titer,(25) similar to the suggestion for optimal timing
of SARS-CoV-2 vaccination in the third trimester. We believe that our
results can be replicated for future variants of the SARS-CoV-2.

\textbf{Conclusion}

Our findings showed that the level of antibody peaked at two months
after either vaccination or infection, and a booster-like effect was
observed following a recent SARS-CoV-2 infection. Our results suggest
that a booster dose of the vaccine during the third trimester can
provide maximum transplacental protection against both the Wuhan
wild-type strain and Omicron variant. Based on these results, we
encourage pregnant women to receive vaccination during pregnancy with a
focus on the third trimester for optimal protection.

\textbf{Acknowledgement}

The authors are grateful for the support from the NCKU Epidemic
Prevention Scientific Research Center and Core Research Laboratory,
College of Medicine, National Cheng Kung University. We also thank the
technical services provided by the ``Biosafety Level 3 Core Laboratory
(MOST 111-2740-B006-003) of the National Core Facility for
Biopharmaceuticals (MOST 111-2740-BA49-002), National Science and
Technology Council, Taiwan. We would also like to express our gratitude
to all the team members involved in the COVID-19 specialized maternity
unit, including obstetricians, obstetric nurses, infectious disease
physicians, and the Center for Infection Control of NCKUH.

\textbf{Conflicts of interest}

The authors have declared no conflicts of interest.



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