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\begin{document}

\title{Virology and immune dynamics reveal high household transmission of
ancestral SARS-CoV-2 strain}



\author[1]{Shidan Tosif}%
\author[2]{Ebene R. Haycroft}%
\author[1]{Sohinee Sarkar}%
\author[1]{Zheng Quan Toh}%
\author[1]{Do Lien Anh-ha}%
\author[1]{Celeste M. Donato}%
\author[2]{Kevin J. Selva}%
\author[3]{Monsurul Hoq}%
\author[3]{Isabella Overmars}%
\author[3]{Jill Nguyen}%
\author[4]{Lai-Yang Lee}%
\author[1]{Vanessa Clifford}%
\author[4]{Andrew Daley}%
\author[2]{Francesa L. Mordant}%
\author[2]{Jodie McVernon}%
\author[1]{Kim Mulholland}%
\author[2]{Adrian Marcato}%
\author[2]{Miranda Smith}%
\author[1]{Nigel Curtis}%
\author[1]{Sarah McNab}%
\author[1]{Richard Saffery}%
\author[2]{Katherine Kedzierska}%
\author[2]{Kanta Subarrao}%
\author[1]{David Burgner}%
\author[1]{Andrew Steer}%
\author[1]{Julie E. Bines}%
\author[1]{Philip Sutton}%
\author[1]{Paul Licciardi}%
\author[2]{Amy W. Chung}%
\author[1]{Melanie Neeland}%
\author[1]{Nigel W. Crawford}%
\affil[1]{Department of Paediatrics The University of Melbourne}%
\affil[2]{The Peter Doherty Institute for Infection and Immunity}%
\affil[3]{Murdoch Children's Research Institute}%
\affil[4]{The Royal Children's Hospital Melbourne}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
\textbf{Background} Household studies are crucial for understanding the
transmission of SARS-CoV-2 infection, which may be underestimated from
PCR testing of respiratory samples alone. We aim to combine assessment
of household mitigation measures; nasopharyngeal, saliva and stool PCR
testing; along with mucosal and systemic SARS-CoV-2 specific antibodies,
to comprehensively characterise SARS-CoV-2 infection and transmission in
households. \textbf{Methods} Between March and September 2020, we
obtained samples from 92 participants in 26 households in Melbourne,
Australia, in a 4-week period following onset of infection with
ancestral SARS-CoV-2 variants. \textbf{Results} The secondary attack
rate was 36\% (24/66) when using nasopharyngeal swab (NPS) PCR
positivity alone. However, when respiratory and non-respiratory samples
were combined with antibody responses in blood and saliva, the secondary
attack rate was 76\% (50/66). SARS-CoV-2 viral load of the index case
and household isolation measures were key factors that determine
secondary transmission. In 27\% (7/26) of households, all family members
tested positive by NPS for SARS-CoV-2 and were characterised by lower
respiratory Ct-values than low transmission families (Median 22.62 vs
32.91; IQR 17.06 to 28.67 vs 30.37 to 34.24). High transmission families
were associated with enhanced plasma antibody responses to multiple
SARS-CoV-2 antigens and the presence of neutralising antibodies. Three
distinguishing saliva SARS-CoV-2 antibody features were identified
according to age (IgA1 to Spike 1, IgA1 to nucleocapsid protein (NP),
suggesting that adults and children generate distinct mucosal antibody
responses during the acute phase of infection. \textbf{Conclusion}
Utilising respiratory and non-respiratory PCR testing, along with
measurement of SARS-CoV-2 specific local and systemic antibodies,
provides a more accurate assessment of infection within households and
highlights some of the immunological differences in response between
children and adults.%
\end{abstract}%



\sloppy


\textbf{Virology and immune dynamics reveal high household transmission
of ancestral SARS-CoV-2 strain}

Shidan Tosif\textsuperscript{1,2,3}, Ebene R.
Haycroft\textsuperscript{4}, Sohinee Sarkar\textsuperscript{1,2}, Zheng
Quan Toh\textsuperscript{1,2}, Lien Anh Ha Do\textsuperscript{1,2},
Celeste M. Donato\textsuperscript{1,2}, Kevin J.
Selva\textsuperscript{4}, Monsurul Hoq\textsuperscript{5}, Isabella
Overmars\textsuperscript{2}, Jill Nguyen\textsuperscript{2}, Lai-Yang
Lee\textsuperscript{6}, Vanessa Clifford\textsuperscript{1,2,6,8},
Andrew Daley\textsuperscript{6}, Francesa L. Mordant\textsuperscript{4},
Jodie McVernon\textsuperscript{7}, Kim Mulholland\textsuperscript{1,2},
Adrian Marcato \textsuperscript{7}, Miranda Smith\textsuperscript{7},
Nigel Curtis\textsuperscript{1,2,8}, Sarah McNab\textsuperscript{1,2,3},
Richard Saffery\textsuperscript{1,2}, Katherine
Kedzierska\textsuperscript{4}, Kanta Subarrao\textsuperscript{4,9},
David Burgner\textsuperscript{1,2,8}, Andrew
Steer\textsuperscript{1,2,8}, Julie E. Bines\textsuperscript{1,2, 10},
Philip Sutton\textsuperscript{1,2}, Paul V.
Licciardi\textsuperscript{1,2}, Amy W. Chung\textsuperscript{4}, Melanie
R. Neeland PhD\textsuperscript{1,2}* and Nigel W. Crawford
PhD\textsuperscript{1,2,3}*

*Contributed equally

\textbf{Affiliations}

\textsuperscript{1} Department of Paediatrics, The University of
Melbourne

\textsuperscript{2} Infection and Immunity, Murdoch Children's Research
Institute

\textsuperscript{3} Department of General Medicine, The Royal Children's
Hospital

\textsuperscript{4} Department of Microbiology and Immunology,
University of Melbourne, at the Peter Doherty Institute for Infection
and Immunity, Victoria, Australia

\textsuperscript{5} Clinical Epidemiology and Biostatistics Unit,
Murdoch Children's Research Institute

\textsuperscript{6} Department of Microbiology, The Royal Children's
Hospital

\textsuperscript{7} Department of Infectious Diseases, University of
Melbourne, at the Peter Doherty Institute for Infection and Immunity

\textsuperscript{8} Infectious Diseases Unit, Department of General
Medicine, The Royal Children's Hospital

\textsuperscript{9} WHO Collaborating Centre for Reference and Research
on Influenza

\textsuperscript{10} Department of Gastroenterology and Clinical
Nutrition, RCH

\textbf{Running Title}

SARS-CoV-2 household infection and immunity

\textbf{Corresponding Author}

Dr Shidan Tosif

Murdoch Children's Research Institute

The Royal Children's Hospital Melbourne

3 West Clinical Offices

50 Flemington Road Parkville VIC 3052

T +61 3 9345 5729

P +61 3 9345 5522 (5582)

F +61 9345 4751

E shidan.tosif@rch.org.au

\textbf{Word Count} 3694

\textbf{Number of tables} 0

\textbf{Number of figures} 2

\textbf{Conflict of interest}

All authors have indicated they have no conflicts to declare relevant to
this study.

\textbf{Financial support}

Funding for the laboratory analysis and recruitment of participants was
provided by the Royal Children's Hospital Foundation, DHB Foundation and
the Infection and Immunity Theme, MCRI. KK was supported by a NHMRC
Leadership Investigator Fellowship (1173871). ST was supported by a
Clinician Scientist Fellowship (MCRI).

\textbf{ABSTRACT}

\textbf{Background}

Household studies are crucial for understanding the transmission of
SARS-CoV-2 infection, which may be underestimated from PCR testing of
respiratory samples alone. We aim to combine assessment of household
mitigation measures; nasopharyngeal, saliva and stool PCR testing; along
with mucosal and systemic SARS-CoV-2 specific antibodies, to
comprehensively characterise SARS-CoV-2 infection and transmission in
households.

\textbf{Methods}

Between March and September 2020, we obtained samples from 92
participants in 26 households in Melbourne, Australia, in a 4-week
period following onset of infection with ancestral SARS-CoV-2 variants.

\textbf{Results}

The secondary attack rate was 36\% (24/66) when using nasopharyngeal
swab (NPS) PCR positivity alone. However, when respiratory and
non-respiratory samples were combined with antibody responses in blood
and saliva, the secondary attack rate was 76\% (50/66). SARS-CoV-2 viral
load of the index case and household isolation measures were key factors
that determine secondary transmission. In 27\% (7/26) of households, all
family members tested positive by NPS for SARS-CoV-2 and were
characterised by lower respiratory Ct-values than low transmission
families (Median 22.62 vs 32.91; IQR 17.06 to 28.67 vs 30.37 to 34.24).
High transmission families were associated with enhanced plasma antibody
responses to multiple SARS-CoV-2 antigens and the presence of
neutralising antibodies. Three distinguishing saliva SARS-CoV-2 antibody
features were identified according to age (IgA1 to Spike 1, IgA1 to
nucleocapsid protein (NP), suggesting that adults and children generate
distinct mucosal antibody responses during the acute phase of infection.

\textbf{Conclusion}

Utilising respiratory and non-respiratory PCR testing, along with
measurement of SARS-CoV-2 specific local and systemic antibodies,
provides a more accurate assessment of infection within households and
highlights some of the immunological differences in response between
children and adults.

\textbf{Key words}

Immunology; COVID-19; children; novel coronavirus; SARS-CoV-2, household
transmission

\textbf{INTRODUCTION}

The COVID-19 pandemic has uniformly identified households as the highest
risk setting for SARS-CoV-2 transmission \textsuperscript{1}, even when
community transmission is reduced \textsuperscript{2, 3, 4}. Occupants
of a household face higher risk through sharing a closed space, being in
close contact without personal protective equipment, and potential
crowding \textsuperscript{2, 5}. Numerous household transmission studies
have identified factors which contribute to higher secondary attack
rates, including a symptomatic index case, spouses compared with other
household members, and that adults are more likely to transmit than
children \textsuperscript{6} \textsuperscript{4}.

Transmission dynamics vary within households for reasons that are still
not well understood. Clustering of infection in the household can occur,
where transmission is characterised by higher secondary transmission
rates, whilst in other households there may be no
transmission\textsuperscript{4}. SARS-CoV-2 is transmitted primarily by
exposure to respiratory fluids when individuals cough or breathe,
through contact and droplet or airborne transmission \textsuperscript{7,
8}. Individuals who are symptomatic often have higher nasopharyngeal
viral RNA concentrations early in the course of symptomatic
infection\textsuperscript{9}. In addition to respiratory fluid,
SARS-CoV-2 has been detected in other biological samples, such as
saliva, stool and urine\textsuperscript{10, 11}. Prolonged excretion has
been shown to occur following negative respiratory viral testing
\textsuperscript{12}. These factors may account for higher transmission
in household settings and testing from multiple sample types may improve
sensitivity in detection of transmission routes.

Understanding the host immune responses to SARS-CoV-2 in controlling the
infection are important in determining susceptibility. The immune
responses to SARS-CoV-2 differ with age; children are less likely to
experience severe disease as compared to adults, and both children and
adults can mount an immune response to SARS-CoV-2 without virological
confirmation of infection \textsuperscript{13, 14}. Immune differences
and endothelial/clotting function are proposed hypotheses for the age
related severity of COVID-19 \textsuperscript{15}. Emerging variants of
concern (VOC) may induce different immune responses and cause varying
severity of disease.

Most transmission studies have relied on SARS-CoV-2 PCR testing of
nasopharyngeal swabs (NPS) and symptoms in contacts to describe
secondary infection and clinical attack rates \textsuperscript{4}.
However, timing of NPS, host viral load, and swab collection quality may
miss the pervasive nature of the infection and underestimate
transmission routes. Higher density analyses of multiple biological
specimens at numerous timepoints, together with the antibody-mediated
immune response following COVID-19, may provide a more comprehensive
profile of SARS-CoV-2 transmission. In this study, we describe the
extent of SARS-CoV-2 infection and host immune responses behind
transmission dynamics with ancestral SARS-CoV-2 in households.

\textbf{METHODS}

\emph{Study Design}

This study was aligned with the Australian FFX study which ran
concurrent to this project and is aligned to the WHO First Few X
Protocol.\textsuperscript{26} \textsuperscript{27}. The Australian FFX
study was led by the Doherty Institute, The University of
Melbourne\textsuperscript{28}. Families were invited to join either, or
both studies at time of first contact, for more intensive biosampling
and follow-up. This study was approved by the Royal Children's Hospital
Research and Ethics Committee (\#63666 and 63101).

Suspected SARS-CoV-2 cases and close contacts were tested by PCR of
nasopharyngeal swabs (NPS) at The RCH from March 2020 to September 2020.
These dates correspond to the first two epidemiological peaks of
SARS-CoV-2 in Melbourne, Australia. Confirmed cases and their household
members were recruited if all household members consented to
participate.

\emph{Clinical Data and Sample Collection}

Daily symptoms and household isolation measures (e.g. mask use,
household separation) were recorded in a standardized diary and disease
severity was classified according to WHO criteria \textsuperscript{29}.
Serial samples of saliva, NPS, and stool were self-collected by all
family members, every week for one month following the date of the first
positive swab of the index case. Blood samples were collected
approximately at baseline and 28 days after onset of infection. Data
were compiled for each participant, for SARS-CoV-2 (positive/negative),
case (index/secondary), symptoms (symptomatic/asymptomatic), stool
(positive/negative), saliva (positive/negative), salivary antibodies
(positive/negative) and serology (positive/negative) (Supplementary
Table 1). The household secondary attack rate was calculated as the
total number of secondary cases over the total number of household
contacts.

\emph{Viral Identification}

NPS were processed by the lab and their extraction was processed using
the automated MagNA Pure system (Roche, Basel, Switzerland). The
majority of samples were tested with the LightMix\selectlanguage{ngerman}® Modular SARS and
Wuhan CoV E-gene kit (targeting the E-gene; TIB Molbiol, Berlin,
Germany) for the SARS-CoV-2 PCR. Some were tested using the
AusDiagnostics Respiratory Pathogens 16-well assay (Mascot, Australia),
on the AusDiagnostics High-Plex 24 system (the SARS-CoV-2 target of this
assay is the ORF-1 gene). Respiratory panel testing was by Roche
LightCycler 480 Instrument II viral panel\textsuperscript{30}.

Viral RNA was manually extracted from 140\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}L of NPS, saliva and 140 \selectlanguage{greek}μ\selectlanguage{english}L
of 20\% (w/v) faecal suspension \textsuperscript{31}~and then eluted in
60 \selectlanguage{greek}μ\selectlanguage{english}L sterile, molecular grade water (Life Technologies, Australia),
using the QIAamp viral RNA kit (QIAGEN, Hilden, Germany) according to
the manufacturer's instructions. The Centers for Disease Control and
Prevention (CDC) developed a real-time reverse transcription PCR panel
targeting nucleocapsid protein genes, N1 and N2\textsuperscript{32}.
CDC's validated platform was selected for saliva and stool analyses.
SARS-CoV-2 standard (Exact Diagnostic, USA) was used as the standard
curve in each assay to determine viral load.

Whole genome sequencing was conducted on a subset of 12 participants
from 7 households. Briefly, viral RNA from saliva or stool (extracted as
described above) was amplified using the ARTIC version 3 primers and
published protocols and subjected to Illumina sequencing as previously
described\textsuperscript{33}. Following quality trimming, reads were
aligned to the reference genome (Wuhan Hu-1; GenBank MN908947.3) and
consensus sequences generated utilising Geneious Prime. Samples were
classified into the recognised SARS-CoV-2 lineages using
Pangolin\textsuperscript{34}.

\emph{Salivary antibodies}

Parents self-collected saliva in a 50 mL conical Falcon tube. Children
were provided a SalivaBio swab and Salimetrcs swab-storage tube.
Children produced between 0.1-1 mL of saliva from the swab, and parents
provided on average 2 mL. After centrifugation, saliva samples were
aliquoted and stored at -80 \selectlanguage{ngerman}°C until analysis. Immuno MaxiSorp 96-well
ELISA plates (Thermo Fisher Scientific, USA) were coated overnight at
4\selectlanguage{english} degC with 2\selectlanguage{english} \selectlanguage{greek}µ\selectlanguage{english}g/mL recombinant SARS-CoV-2/2019-nCoV S1 protein (Sino
Biologicals) diluted in PBS. Wells were blocked with 10\% skim milk in
PBST (PBS + 0.1\% Tween 20) at room temperature for 1 h. Two-fold serial
dilutions of saliva samples in PBST were transferred to the ELISA plates
(in duplicate) and incubated at room temperature for 1 h. Saliva from an
asymptomatic individual confirmed negative for SARS-CoV-2 by clinical
testing was used as a negative control. Saliva from a convalescent
individual recently infected with SARS-CoV-2 was used as a positive
control and pre-COVID saliva samples were used as negative controls.
Antibody binding was detected with anti-human secretory IgA (sIgA,
1:10,000; Merck; followed by 1h incubation with biotinylated anti-mouse
IgG detection antibody, 1:1,000; Southern Biotech) and biotinylated IgG
(1:10,000; Assay Matrix) for 1 h at room temperature, then
Streptavidin-HRP (1:5000; Life Technologies) in PBST for 45 min at room
temperature. Colour was developed with TMB solution (Sigma-Aldrich) and
H\textsubscript{2}O\textsubscript{2} with the reaction stopped using 2 M
H\textsubscript{2}SO\textsubscript{4}. Absorbance at 450 nm was read on
a microplate reader and used to calculate end point titres of samples.
Cut-off values for each antibody class was defined as two standard
deviations above the maximum titre from the corresponding negative
controls.

\emph{Serological Immunity}

\emph{Plasma S1 and RBD ELISA}

The ELISA method used to measure IgG, IgM, and IgA levels to SARS-COV-2
S1 and RBD protein was based on the Mount Sinai Laboratory method
previously described Briefly, 96-well high-binding plates were coated
with receptor binding domain (RBD) or S1 (Sino Biological, China)
antigen diluted in PBS at 2 \selectlanguage{greek}µ\selectlanguage{english}g/mL. Serum samples were first screened
with RBD antigen, and potential seropositive samples were then confirmed
with S1 antigen. Goat anti-human IgG- (1:10,000) horseradish peroxidase
(HRP) conjugated secondary antibody was used, and the plates were
developed using 3.3', 5.5'-tetramethylbenzidine substrate solution.
Seropositive samples were titrated and calculated based on a World
Health Organization (WHO) SARS-CoV-2 pooled serum standard (National
Institute of Biological Standards and Controls, United Kingdom). Results
were reported in International Units/mL. The cut-off for seropositivity
was 8.36 IU/mL based on pre-pandemic samples, while seronegative samples
were given half of the seropositive cut-off value.

\emph{Coronavirus antibody multiplex assay (blood and saliva)}

A novel coronavirus multiplex bead array was designed as previously
described\textsuperscript{19} consisting of SARS-CoV-2 spike 1 (Sino
Biological), spike 2 (ACRO Biosystems), spike trimer (kind gift from
Adam Wheatley), RBD (BEI Resources) and nucleoprotein (ACRO Biosystems).
Tetanus toxoid (Sigma-Aldrich), influenza hemagglutinin (H1Cal2009; Sino
Biological) and SIV gp120 (Sino Biological) were also included in the
assay as positive and negative controls respectively. Antigens were
covalently coupled to magnetic carboxylated beads (Bio Rad) using a
two-step carbodiimide reaction and blocked with 0.1\% BSA, before being
resuspended and stored in PBS 0.05\% sodium azide till use.

The antigen-coupled beads were combined to form a coronavirus multiplex
bead cocktail to investigate serological signatures from plasma and
saliva samples. Briefly, 20 \selectlanguage{greek}µ\selectlanguage{english}L of working bead mixture (1000 beads per
bead region) and 20 \selectlanguage{greek}µ\selectlanguage{english}L of diluted plasma (final dilution 1:200) or 20 \selectlanguage{greek}µ\selectlanguage{english}L
of diluted saliva (final dilution 1:50) were added per well in 384 well
plates and incubated overnight at 4\selectlanguage{ngerman}°C on a shaker. Fourteen different Fc
detectors were used to assess coronavirus-specific antibodies as
previously described\textsuperscript{35}, including phycoerythrin
(PE)-conjugated mouse anti-human pan-IgG, IgG1-4 and IgA1-2 (Southern
Biotech; 1.3 \selectlanguage{greek}µ\selectlanguage{english}g/mL, 25 \selectlanguage{greek}µ\selectlanguage{english}L/well). IgM (biotinylated mouse anti-human IgM
(mab MT22; Mabtech; 1.3 \selectlanguage{greek}µ\selectlanguage{english}g/mL, 25 \selectlanguage{greek}µ\selectlanguage{english}L/well), C1q protein (MP Biomedicals)
and Fc\selectlanguage{greek}γ\selectlanguage{english}R dimers (higher affinity polymorphisms Fc\selectlanguage{greek}γ\selectlanguage{english}RIIa-H131, lower
affinity polymorphisms Fc\selectlanguage{greek}γ\selectlanguage{english}RIIa-R131, Fc\selectlanguage{greek}γ\selectlanguage{english}RIIb, higher affinity
polymorphisms Fc\selectlanguage{greek}γ\selectlanguage{english}RIIIa-V158, lower affinity polymorphisms Fc\selectlanguage{greek}γ\selectlanguage{english}RIIIa-F158;
1.3 \selectlanguage{greek}µ\selectlanguage{english}g/mL, 25 \selectlanguage{greek}µ\selectlanguage{english}L/well; kind gifts from Bruce Wines and Mark
Hogarth\textsuperscript{36} were first added to the beads, washed, and
followed by the addition of PE-conjugated streptavidin (1.3 \selectlanguage{greek}µ\selectlanguage{english}g/mL, 25
\selectlanguage{greek}µ\selectlanguage{english}L\selectlanguage{greek}µ/\selectlanguage{english}well). Assays were read on a Flexmap 3D with x-PONENT 4.2 software
and performed in duplicate. Antibody levels are reported as median
fluorescent intensity (MFI) of the PE signal associated with each bead.
Pre-SARS-CoV-2 pandemic samples were used as controls in multiplex
assay. SARS-CoV-2 plasma antibodies (positive/negative) and salivary
antibodies (positive/negative) cut-off thresholds were determined by
calculating the average plus two-standard deviations of respective
pre-pandemic control data.

\emph{Systems Serology Analysis}

To holistically examine the spectrum of antibody signatures obtained via
the above mentioned SARS-CoV-2 multiplex array, multivariate analysis
techniques were utilised using MATLAB version 9.6 (including machine
learning and statistical toolbox) (The MathWorks, Inc., Natick, MA) and
Eigenvector PLS toolbox (Eigenvector, Manson, WA). Heatmaps were
generated using Morpheus (https://software.broadinstitute.org/morpheus).
Prism GraphPad version 9.0.2 (GraphPad Software, San Diego, CA) were
used to illustrate final figures and to conduct any univariate analysis.
For univariate analysis \emph{p} value of 0.05 was set as the level for
statistical significance, unless otherwise stated.

\emph{Data Normalization}

For all multivariate analysis, positive antigens were removed (Tetanus
and H1Cal2009). If any antibody feature included negative values,
right-shifting was performed (by adding the minimum value for each
respective feature back to all samples). Data was then log transformed
to ensure that the majority of features were normally distributed, by
using the following equation log\textsubscript{10} (x+1). Values were
subsequently normalized by mean centering and variance scaling by
calculating respective \emph{z} -scores.

\emph{LASSO and PCA}

To determine the minimal number of antibody features that distinguish
between different groups, a least absolute shrinkage and selection
operator (LASSO) feature reduction method was employed as previously
described\textsuperscript{37}. Cross validation was performed
iteratively (repeated 1,000 times; 10-fold cross validation) to identify
the optimal value of the regularized parameters. Unsupervised principal
component analysis (PCA) was then performed on LASSO-selected antibody
features (which resolves multiple variables into principal components
that describe the variance within the data set). The contribution of
each variable in describing the variance within each principal component
is represented on loading plots.

\emph{ElasticNet and PLS-R}

To identify the key contributing antibody signatures from the data set,
elasticNet regression was utilised as previously
described\textsuperscript{19, 38} The Elastic Net hyperparameter was set
to have equal weights between L1norm and L2norm i.e. \selectlanguage{greek}α\selectlanguage{english}=0.5. Model
performance was evaluated iteratively (1000 iterations, 4 fold cross
validation). Partial least squares regression (PLS-R) was performed on
ElasticNet-selected antibody features to visualize the relationship
between antibody signatures with continuous variables. i.e. Ct (cycle
threshold) PCR values (determined via Roche LightCycler 480 Instrument
II).

\emph{Neutralising Antibodies}

Microneutralization assay

SARS-CoV-2 isolate CoV/Australia/VIC01/2020\textsuperscript{39} passaged
in Vero cells was stored at -80 \selectlanguage{ngerman}°C. Serial two-fold dilutions of
heat-inactivated plasma were incubated with 100 TCID50 of SARS-CoV-2 for
1\selectlanguage{english} h and residual virus infectivity was assessed in quadruplicate wells
of Vero cells; viral cytopathic effect was read on day 5. The
neutralizing antibody titer was calculated using the Reed/Muench
method\textsuperscript{40}.

\emph{Statistical analysis for factors associated with transmission and
correlation between immune parameters}

Associations between household members being positive to SARS-CoV-2
detected by NPS with demographic characteristics, clinical parameters
and preventive measures were assessed using generalising estimating
equations (assuming an exchangeable correlation structure and
distribution of dependent variable as binomial) controlling for the
number of contacts within a household (Figure 2 A). The correlation
between immune parameters were assessed using tetrachoric correlation as
the immune parameters are dichotomous (Figure 2 B).



\textbf{RESULTS}

\emph{Demographics and Transmission Dynamics}

We included 92 participants from 26 households, recruited between
1\textsuperscript{st} March and 30\textsuperscript{th} September 2020.
The median family size was 3.5 (inter quartile range (IQR) 3.0 to 4.0).
Overall, 47\% (43/92) of participants were female, and 43\% (40/92)
children with a median age of 3.9 years (IQR 1.9 to 7.6). SARS-CoV-2 was
detected in 54\% (50/92) of participants (25 children, 25 adults) on NPS
(Supplementary Figure 1). There were 26 index cases and 24 secondary
cases, hence the secondary attack rate using NPS results alone was 36\%
(24/66). Genetic analysis was available from 15 samples which reflected
the circulating community original Wuhan strain or `Alpha' variants of
SARS-CoV-2 at the time of recruitment, all of lineage D.2 except one
which was B.1.338.

Twelve participants from 8 households also tested positive by PCR for
SARS-CoV-2 in stool, with fifteen out of 92 participants testing
positive in saliva (Supplementary Figure 1). Those who tested positive
in saliva or stool were also positive on NPS PCR for SARS-CoV-2. NPS
positivity rate decreased over time with 13\% (6/46) positive 28 days
following onset of infection. SARS-CoV-2 in stool was detected for the
longest period of all virological samples, with 42\% (5/12) positive at
day 28.

\emph{Higher transmission vs lower transmission families}

In 27\% (7/26) of households, all family members tested positive by NPS
for SARS-CoV-2, which we have termed high transmission families
(families 3, 4, 6, 11, 12, 13, 20). High transmission families were
positive for most respiratory and non-respiratory samples (Supplementary
Figure 1). High transmission families were also largely characterised by
lower respiratory Ct-values than low transmission families
(Supplementary Figure 2A; High vs low transmission families; Median
22.62 vs 32.91; IQR 17.06 to 28.67 vs 30.37 to 34.24; p = 0.007).
Feature selection analysis identified antibody signatures associated
with lower Ct-values (Figure 1A-B). A heatmap including only these
selected antibody features illustrates that individuals from high
transmission households (green) generally had higher plasma antibody
responses to SARS-CoV-2 antigens (indicated by red heat signatures) and
clustered separately from the low transmission households which had
largely lower SARS-CoV-2 plasma antibody levels (indicated by blue
signatures). As before, this pattern of clustering between high and low
transmission families largely coincided with their differences in
measured Ct-values (low to high; white to purple) (Figure 1C).
Interestingly, all family members from 6/7 high transmission families
demonstrated evidence of neutralising antibodies as determined by
microneutralization assay (denoted by the asterisk on Supplementary
Figure 1). Furthermore, neutralising antibodies were only detected in
participants from high transmission families.

\emph{Factors associated with transmission}

The probability of transmission to household members, detected by NPS,
increased if the index case was an adult compared to a child (62\% vs
12\%, odds ratio (OR) 12.4, 95\% confidence interval (95 Cl) 1.8 to
84.8, p = 0.10), or had a Ct-value below 32 compared to \selectlanguage{english}[?]32 (54\% vs
21\%, OR 4.4, 95 CI 1.1 to 17.1, p = 0.034) (Figure 2A). The probability
of transmission among household members increased by 10\% in cases who
were symptomatic, relative to asymptomatic cases (34\% vs 44\%, OR 1.5,
95 CI 0.7 to 3.4, p = 0.329). Households who employed any
non-pharmaceutical intervention measure were less likely to have
household transmission compared to those without (31\% vs 46\%, OR 0.5,
95 CI 0.1 to 2.0, p = 0.358), specifically, household separation (27\%
vs 45\%, OR 0.46, 95 CI 0.1 to 2.2, p = 0.330) and mask wearing (16\% vs
43\%, OR 0.2, 95 CI 0.0 to 1.6, p = 0.149) were associated with lower
secondary transmission (Figure 2A). \emph{Correlation between immune
parameters}

SARS-CoV-2 virus detection in saliva positively correlated with evidence
of symptoms (r = 1, p = 0.002) (Figure 2B). Detection of SARS-CoV-2 in
saliva also correlated with SARS-CoV-2 specific plasma IgG (r = 1.0, p
\textless{}0.001) and IgM/IgA (r = 1.0, p\textless{}0.001) (Figure 2B).
Similarly, detection of SARS-CoV-2 in stool positively correlated with
SARS-CoV-2-specific plasma IgG (r = 1.0, p \textless{}0.001) and IgM/IgA
(r = 1.0, p = 0.0015), and showed concordance with detection of
SARS-CoV-2 in saliva (r = 0.83, p\textless{}0.001) (Figure 2B). In
SARS-CoV-2 positive children, 72\% (13/18) produced Spike 1-specific
salivary antibodies but had no detected serum antibodies (Supplementary
Figure 1).

\emph{Differences in humoral responses between children and adults}

Multiplex systems analysis of antibody responses in saliva found
distinct SARS-CoV-2 specific antibody responses in infected children
compared to adults during the acute phase of infection (Figure 3A).
Three SARS-CoV-2 antibody features (IgA1 to Spike 1, IgA1 to
nucleocapsid protein (NP), IgA2 to NP, Figure 3B) were identified by
dimensionality reducing analysis (least absolute shrinkage and selection
operator (LASSO)) as significantly elevated responses in adult saliva
compared to children (Supplementary Figure 3, p =0.0023, p
\textless{}0.001, p \textless{}0.001 respectively), thus suggesting that
adults and children generate distinct mucosal antibody responses during
the acute phase of infection.

Given we observed different antibody profiles in the saliva between
adults and children, multiplex systems analysis was also conducted on
acute children and adult plasma samples. This analysis identified three
elevated antibody features that were unique to adult plasma (C1q Trimer
S C1q (Marker of antibody-mediated complement activation) to Trimer S,
IgG4 to RBD, IgA2 to NP, Supplementary Figure 4).

\emph{Secondary attack rate when including comprehensive virological and
antibody assessment} Evidence of SARS-CoV-2 exposure was observed in
saliva and plasma antibody responses in 62\% of household contacts who
tested negative by NPS (26/42: 7/42 serology, 24/42 saliva antibodies,
Supplementary Figure 1). Therefore, the secondary attack rate when
respiratory (NPS) and non-respiratory measures were included (saliva
PCR, stool PCR, plasma antibodies, saliva antibodies) was 76\% (50/66).
There was no onward transmission from participants who tested negative
by NPS, even if they were PCR positive in other biological samples.

\textbf{DISCUSSION}

This study provides a detailed virological and immunological profile of
families exposed to SARS-CoV-2 `Alpha' variants in 2020-2021 in a low
COVID-19 incidence country, Australia. A key finding was detection of
widespread infection with higher secondary attack rates when a
comprehensive set of biological specimens were analysed compared with
NPS alone. Enhanced plasma antibody levels were observed in individuals
from high transmission families compared to low transmission families.
We show that SARS-CoV-2 specific salivary antibodies were detected in a
high proportion of participants and that antibody features in blood and
saliva differ between children and adults.

Whilst new VOC, such as the Delta and Omicron variants, are associated
with higher reproductive numbers compared with the ancestral
strain\textsuperscript{16}, our data suggests that high levels of
household transmission of the ancestral virus are detected when
extensive virology and immune assessments are collected. This highlights
that dense sampling protocols are more likely to identify infected
household members and could be used as a more accurate assessment of
secondary attack rate\textsuperscript{17}. With emergence of new VOC,
determining the true extent of infection will be important in comparing
virulence and transmission dynamics associated with each variant.

In this study, IgA responses in saliva, especially to the SARS-CoV-2 NP
antigen, were identified during the acute phase of infection in adults
but not in children. Interestingly, elevated antibody responses to NP
were observed in both adult saliva and plasma. Previous studies have
demonstrated that SARS-CoV-2 NP is highly cross-reactive with NP from
other human coronaviruses, thus cross-reactive antibodies are more
rapidly induced upon SARS-CoV-2 exposure within the blood, especially
amongst adults and elderly in comparison to children due to pre-existing
memory\textsuperscript{18, 19, 20}. Our study suggests that this
cross-reactive antibody priming occurs for both mucosal and systemic
antibody responses.

Like other studies, we identified prolonged faecal shedding beyond
respiratory sample detection \textsuperscript{21, 22, 23}. Fifteen out
of 22 patients in an Italian paediatric cohort had RNA detected in stool
at diagnosis, independently from gastrointestinal symptoms. Similarly,
prolonged SARS-CoV-2 positivity was detected in a study by Xu \emph{et
al} ., 8 out of 10 children persistently tested positive on rectal swabs
even after nasopharyngeal testing was negative\textsuperscript{24}.
Stool specimens in this study remained positive when NPS were negative,
with a median duration of 14 days (range 10-15) from onset of symptoms
compared with 8 days (range 2-17) for NPS, providing an opportunity for
diagnosing SARS-CoV-2 beyond the period of acute
infection\textsuperscript{25}.

This study has some limitations. Transmission to household contacts was
assumed to have occurred within the household, and not due to infections
acquired outside the household. This assumption was made due to
quarantine rules restricting movement from identification of first
positive case, however, a family may have had a shared external
exposure. This study includes lineages D.2 and B.1.338, and the
applicability of our findings following the emergence of the Delta and
Omicron strains with higher transmission rates\textsuperscript{16} is
unclear. Comparative analyses between our data and VOC in the future
will be important.

\textbf{CONCLUSION}

Utilising multiple virological and immunological specimens, it is
possible to show evidence of infection much greater than those detected
from SARS-CoV-2 NPS alone. High transmission in families is associated
with detection of SARS-CoV-2 in saliva and stool, and an acute and
robust blood and saliva response which is only detected following
comprehensive assessment of biological samples. Denser sampling methods
provide a more comprehensive assessment of infection and highlights some
of the immunological differences in response between children and
adults. This profile of infection within households provides a basis for
comparison in future studies as VOC emerge.

\textbf{Acknowledgements}

We thank the study participants and families for their involvement in
this study. We also acknowledge the MCRI Biobanking service for their
help in processing the samples. Reagents were supplied by Adam Wheatley,
Bruce Wines and Mark Hogarth, Burnet Institute

The following reagent was produced under HHSN272201400008C and obtained
through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding
Domain (RBD) from SARS-Related Coronavirus 2, Wuhan-Hu-1 with C-Terminal
Histidine Tag, Recombinant from HEK293F Cells, NR-52366.''

Stephen J. Kent provided pre-pandemic healthy saliva samples which were
used to create a positive threshold for saliva studies.

\textbf{Impact Statement}

When respiratory and non-respiratory samples were combined with antibody
responses in blood and saliva, a much higher secondary attack rate of
SARS-CoV-2 in houoseholds was identified. Lower viral load and
mitigation measures reduced transmission. Saliva and serum antibody
analyses show differences in immune responses between adults and
children.

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