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\begin{document}

\title{LEVEL OF MATERNAL ANTIBODIES AGAINST RESPIRATORY SYNCYTIAL VIRUS (RSV)
NUCLEOPROTEIN AT BIRTH AND RISK OF RSV VERY-SEVERE LOWER RESPIRATORY
TRACT INFECTION}



\author[1]{Matthieu RECEVEUR}%
\author[2]{Michele Ottmann}%
\author[3]{Jean-Marc Reynes}%
\author[4]{Jean-François Eleouet}%
\author[4]{Marie Galloux}%
\author[5]{Aurore Receveur}%
\author[1]{Dominique Ploin}%
\author[3]{Sylvie Fiorini}%
\author[3]{Nathalie Rivat}%
\author[1]{Martine Valette}%
\author[2]{Bruno Lina}%
\author[6]{jean-sebastien casalegno}%
\affil[1]{Hospices Civils de Lyon}%
\affil[2]{Université de Lyon, Université Lyon 1}%
\affil[3]{Université de Lyon}%
\affil[4]{Université Paris-Saclay}%
\affil[5]{Pacific Community}%
\affil[6]{Hospices Civils de Lyon, National Influenza Centre (South of France)}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: The nucleoprotein (N protein) of respiratory syncytial virus
(RSV) is a candidate antigen for new RSV vaccine development. The aim of
the present study was to investigate the association between maternal
antibody titers against the RSV N protein at birth and the newborns'
risk of developing very-severe lower respiratory tract infection
(VS-LRTI). Methods: In this single-center prospective cohort study, 578
infants born during the RSV epidemic season in France were included.
Among these, 36 were hospitalized for RSV VS-LRTI. A generalized linear
model was used to test the occurrence of a VS-LRTI in function of sex,
mode of delivery, parity of the mother, type of pregnancy, date of birth
in relation to the peak of the epidemic, and antibody titer against N
protein. Results: All cord blood samples had detectable antibodies
against N protein. The mean titers were significantly lower in newborns
with risk factors for RSV severe LRTI (preterm infants, birth before the
peak epidemic, multiparous mother). There was no association between
antibody titer against the N protein and a protection against VS-LRTI.
Conclusions The present study found that transfer of maternal antibodies
against the RSV N protein may not provide a significant immune
protection early in infancy. Clinical Trials Registration. NCT04144816.%
\end{abstract}%



\sloppy


\textbf{Level of Maternal ANTIBODIES AGAINST Respiratory Syncytial Virus
(RSV) NUCLEOPROTEIN AT BIRTH AND RISK OF RSV VERY-SEVERE LOWER
RESPIRATORY TRACT INFECTION}

Authors: Matthieu Receveur\textsuperscript{1,2,3}, Mich\selectlanguage{ngerman}èle
Ottmann\textsuperscript{4}, Jean-Marc Reynes\textsuperscript{4,5},
Jean-François Eleouet\textsuperscript{6}, Marie
Galloux\textsuperscript{6}, Aurore Receveur\textsuperscript{7,8},
Dominique Ploin\textsuperscript{1,4}, Sylvie Fiorini\textsuperscript{4},
Nathalie Rivat\textsuperscript{4}, Martine Valette\textsuperscript{2,9},
VRS~study group in Lyon\textsuperscript{1}, Bruno
Lina\textsuperscript{2,3,9} , Jean-Sébastien
Casalegno\textsuperscript{2,3,9}

Affiliations:

1 Hospices Civils de Lyon, Hôpital Femme Mère Enfant, Service de
Réanimation Pédiatrique et d'Accueil des Urgences, 69500 Bron, France

2 Hospices Civils de Lyon, Hôpital de la Croix-Rousse, Institut des
Agents Infectieux, 69004 Lyon, France

3 Université de Lyon, Université Claude Bernard Lyon 1, Faculté de
médecine Lyon Est, 69008 Lyon, France

4 Université de Lyon, Université Claude Bernard Lyon 1, Laboratoire de
Virologie et Pathologies Humaines Virpath, École Normale Supérieure de
Lyon, Centre International de Recherche en Infectiologie (CIRI), Inserm
U1111, CNRS, UMR5308, 69007 Lyon, France

5 Unité Environnement et Risques Infectieux, Institut Pasteur, Paris,
France

6 Université Paris-Saclay, INRAE, Unité de Virologie et Immunologie
Moléculaires, 78350 Jouy-en-Josas, France

7 Pacific Community, SPC, OFP/FEMA, 98848 Noumea, New~Caledonia

8 Université de la Réunion, IRD, CNRS, UMR9220, ENTROPIE, Université de
La Nouvelle-Calédonie, Ifremer, 98848 Noumea, New~Caledonia

9 Hospices Civils de Lyon, Hôpital de la Croix-Rousse, Institut des
Agents Infectieux (IAI), Laboratoire de Virologie, Centre National de
Référence des virus des infections respiratoires, Lyon 69004, France

Corresponding author:

Matthieu Receveur

Hôpital Femme Mère Enfant, Service de Réanimation Pédiatrique et
d'Accueil des Urgences,

59 Boulevard Pinel

69500 Bron, France

Tel: +33 4 27 85 56 42

Fax: +33 4 27 86 92 63

Mail: matthieu.receveur@chu-lyon.fr

Short running title: RSV antibodies at birth and risk of RSV VS-LRTI

Key words: Respiratory Syncitial Virus; Nucleoprotein; Lower respiratory
tract infection~(LRTI); Maternal antibodies; Vaccine; Infants

Abstract: 204/250 words; Text: 3560/5000 words; Tables: 2; Figures: 2

Conflict of interest: none

Financial support: none

\emph{Abstract}

\textbf{Background} : The nucleoprotein (N protein) of respiratory
syncytial virus (RSV) is a candidate antigen for new RSV vaccine
development. The aim of the present study was to investigate the
association between maternal antibody titers against the RSV N protein
at birth and the newborns' risk of developing very-severe lower
respiratory tract infection (VS-LRTI).

\textbf{Methods:} In this single-center prospective cohort study, 578
infants born during the RSV epidemic season in France were included.
Among these, 36 were hospitalized for RSV VS-LRTI. A generalized linear
model was used to test the occurrence of a VS-LRTI in function of sex,
mode of delivery, parity of the mother, type of pregnancy, date of birth
in relation to the peak of the epidemic, and antibody titer against N
protein.

\textbf{Results:} All cord blood samples had detectable antibodies
against N protein. The mean titers were significantly lower in newborns
with risk factors for RSV severe LRTI (preterm infants, birth before the
peak epidemic, multiparous mother). There was no association between
antibody titer against the N protein and a protection against VS-LRTI.

\textbf{Conclusions} The present study found that transfer of maternal
antibodies against the RSV N protein may not provide a significant
immune protection early in infancy.

Clinical Trials Registration . NCT04144816.

\emph{Introduction}

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis
in infants and of pneumonia in children younger than 5 years of age in
infants {[}1,2{]}, and in this population it is responsible each year
for approximately 33 million cases of lower respiratory tract infections
(LRTI) worldwide {[}1{]}. It is recognized to cause substantial
mortality in low and middle-income countries {[}2{]}, and burden in
high-income countries {[}3,4{]}; the highest rate of hospitalization is
in infants aged less than 3 months {[}5{]}. Despite this, preventive
measures are to date limited to the monthly injection of RSV-specific
neutralizing antibodies for children at a high risk of severe
complications, the cost of which may be prohibitive {[}6{]}. There are,
however, vaccines currently in development that have the potential to be
a more affordable option to reduce the worldwide RSV burden~{[}6{]}.
Most candidate vaccines target the fusion protein, which is an envelope
protein highly conserved between different subtypes or RSV, stabilized
in its prefusion form {[}7,8{]}. However, RSV nucleoprotein (N protein),
which it is implicated in nucleocapsid-RNA complex formation that allows
RSV replication and transcription {[}9{]}, is one of the most conserved
viral protein among strains {[}10{]} and is also considered as a
potential target for candidate vaccines inducing T CD8+
response~{[}11,12{]}. These vaccines may be good candidates for maternal
immunization strategy; an approach that appears as safe because of the
particular vulnerability of infants and their inability to produce
effective antibodies {[}13{]}. Such a strategy has already demonstrated
its ability to provide a passive humoral protection for infants against
tetanus, influenza, and pertussis {[}14{]}. Humoral protection is
conferred at birth by the transfer of maternal IgG, the titer of which
depends on the maternal level of RSV antibodies and the effectiveness of
transplacental RSV antibody transfer from mother to infant {[}15{]}.
Although the protection conferred to infants by neutralizing antibodies
induced by natural exposure to RSV of mothers has been suggested,
published data is conflicting {[}16--20{]}; furthermore there is no
published data concerning the protection provided by maternal antibodies
against the N protein. The aim of the present study was therefore to
investigate the association between maternal antibody titers against the
RSV N protein at birth and the newborns' risk of developing RSV VS-LRTI
early in infancy.

\emph{Methods}

\emph{Study design}

All infants born at the Hôpital Femme Mère Enfant (HFME; part of the
university hospitals of Lyon, France) from August 26, 2019 to February
27, 2020 (during the RSV epidemic season in France {[}4{]}) with a cord
blood sample available were eligible for inclusion in this single-center
prospective observational cohort study. Cord blood samples were
collected for all included patients. Stillborn children, those living
outside the Auvergne-Rhône-Alpes region or those with insufficient
sample were excluded.

The administrative registry of all infants born in the university
hospitals of Lyon (Hospices Civils de Lyon, HCL) was used to recover the
following variables of interest: sex (male/female), month of birth,
gestational age (weeks of amenorrhea, WA), maternal parity
(primiparity/multiparity), type of pregnancy (simple/multiple
gestation), mode of delivery (vaginal birth/caesarean section), and
birth weight. Preterms were classified as either ``moderate-to-late
preterm'' {[}from 32 to 36(+6) WA{]}, ``very preterm'' {[}28 to 31(+6)
WA{]}, and ``extremely preterm'' {[}22 to 27(+6) WA{]}, as defined by
the World Health Organization (WHO) {[}21{]}. Low birth weight was
defined as less than 2500 g {[}22{]}.

Infants aged 3 months or younger who visited the emergency department of
the HFME hospital (either directly or transfer from another hospital)
between August 26, 2019 to May 27, 2020 with laboratory-confirmed RSV
bronchiolitis recognized by the International Statistical Classification
of Diseases and Related Health Problems (ICD-10) codes (J21.0, J12.1
and/or J20.5) were identified in the informatics' database of the
hospital. A 3-month follow-up seemed to be optimal to detect a
protection purposed by maternal antibodies as children under 3 months of
life experience the greatest risk of hospitalization and mortality
{[}5,23{]}, and as previous studies have shown that maternal antibody
concentrations against RSV decreased between 2 and 6 months {[}1,24{]},
with a half-life of approximately 27~days {[}25{]}. According to local
protocols, all infants hospitalized with an LRTI diagnosis were tested
for RSV on a nasopharyngeal sample. Laboratory-confirmed RSV infection
were those diagnosed by real-time reverse transcriptase (RT)-PCR, as
previously described {[}26{]}. Clinical records were reviewed to further
classy these as VS-LRTI, as defined by the WHO: cough or difficulty
breathing, associated with fast breathing or peripheral capillary oxygen
saturation (SpO2)~\textless{}~90\% or inability to feed or unconscious
{[}13{]}. The peak of the epidemic was defined as December 9, 2019 based
on both pediatric emergency admissions for bronchiolitis and RSV
detection data in Lyon, in accordance with data from previous years.

\emph{Laboratory procedures}

Cord bloods were collected at birth in heparinized tubes, centrifuged
and stored at -20°C until testing. IgG against RSV N protein were
measured in 1:100 diluted plasma using an in-house enzyme-linked
immunosorbent assay (ELISA) performed according to Roux \emph{et
al.}{[}27{]} and using recombinant N protein from RSV A long strain and
anti-human polyvalent Ig G/A/M peroxidase antibody produced in goat
(Sigma-Aldrich®, Saint-Louis, MA, US). In order to establish a standard
curve, 8 two-fold dilutions (starting at 1:100) of a positive control
(BEI Resources NR-4020, Manassas, VA, US) were used. Negative control
was obtained from the IgM/IgG RSV ELISA kit (VIROTECH Diagnostics,
Dietzenbach, HE, DE). Titers were calculated using a Michaelis-Menten
equation applied to the values of the standard curve and expressed as
relative unit (RU)/mL.

\emph{Statistical analysis}

Mean and standard deviation (SD) were calculated to describe clinical
continuous variables. Concerning antibody titers against RSV N, both
mean (SD) and median (interquartile range, IQR) were calculated.
Comparisons between mean antibody titers were tested using a 2-sided
parametric Student t-test or an ANOVA for more than 2 groups. The birth
weight and the gestational age were treated as categorical variables. A
generalized linear model (GLM) was used to test the occurrence of a
VS-LRTI in function of sex, mode of delivery, parity of the mother, type
of pregnancy, date of birth in relation to the peak of the epidemic, and
titer antibodies against RSV N (quantitative variable). Preterm infants
as well as patients who contracted a VS-LRTI but for whom the result for
RT-PCR RSV detection was negative were excluded from the GLM.
Statistical analyses were performed using R~software version~4.0.3.

\emph{Ethics}

This study was approved by the review board of university hospitals of
Lyon, is registered on ClinicalTrials.gov (NCT04144816), and was
declared to national data protection commission (19-198).\emph{}

\emph{Results}

\emph{Characteristics of the cohort}

During the study period 1719 infants were born at the HFME. Cord bloods
were available for 1574 of them. Because it was not financially feasible
to test all samples, two days, every two months, were allocated to
process study samples, and then 578 were randomly included for analysis.
Among those included, there were 71 infants aged 0-3 months who visited
the emergency department with a diagnosis of LRTI and 43 hospitalized
with a diagnosis of VS-LRTI; of the latter, 36 tested positive for RSV
(Fig. 1).

The total cohort comprised 578 infants; 53.8\% (n=311) were boys, 6.1\%
(n=35) from multiple births, 23.0\% (n=133) born by cesarean section,
from 60.7\% (n=351) multiparous mothers, 9.9\% (n=57) born preterm, and
the mean (SD) birth weight was 3179 (633) g. Among the 36 hospitalized
in their first 3 months of life with a laboratory-confirmed RSV VS-LRTI
infection, 50.0\% (n=18) were boys, none were premature, 88.9\% (n=32)
were born to multiparous mothers, and none were from multiple births
(Table 1).

\emph{Maternal antibody titers against the RSV N protein}

The mean~(SD) value of maternal antibody titers against the RSV N
protein among newborns was 502 (255) RU/mL. There was no significant
difference in mean values according to sex (Figure 2A), type of
pregnancy (Figure 2B), or mode of delivery (Figure 2C). The mean value
was significantly different according to the term of pregnancy
(p~\textless{}~0.001; Figure 2D); it was significantly lower in preterm
newborns (i.e. extremely, very, and moderate-to-late combined) than in
those born at term (377~RU/mL vs. 516 RU/mL, p~\textless{}~0.001), in
newborns with a low birth weight than in those with a birth weight \selectlanguage{english}[?]
2500g (401 RU/mL vs. 515~RU/mL, p~\textless{}~0.001; Figure 2E), in
newborns from primiparous mother than those from multiparous mothers
(470~RU/mL vs. 522~RU/mL, p~\textless{}~0.05; Figure 2F) and in newborns
whose birth occurred before the peak of the epidemic than in those born
after (458~RU/mL vs. 542 RU/mL, p~\textless{}~0.001; Figure 2G). There
was no significant difference in mean antibody titers between infants
who experienced a VS-LTRI (irrespective of RSV detection) and those who
did not (466~RU/mL vs. 505~RU/mL, p~=~0.37; Figure 2H).

\emph{Prediction of VS-LRTI using clinical variables and serological
status}

Preterm infants were excluded from the GLM given that a 3-month
follow-up was not sufficient to analyze their risk of being admitted to
hospital for VS-LRTI (they might have been hospitalized since birth at 3
months of age). In multivariate analysis, maternal multiparity (Relative
Risk, RR: 2.34, 95\%CI {[}1.58; 3.01{]}) and a date of birth before the
peak of the epidemic (RR: 2.84, 95\%CI {[}2.08; 3.59{]}) were
significantly associated with the occurrence of VS-LRTI; there was no
significant association between antibody titer and VS-LRTI (Table 2).

\emph{Discussion}

In the present study no association was found between antibody titer
against the RSV N protein measured in cord blood and a protection
against RSV VS-LRTI. This result differs from the largest cohort studies
that analyzed cord blood titers; these studies, conducted in Denmark,
Mali, and on American Indian infants found that a higher concentration
of neutralizing antibodies at birth was associated with a lower risk of
contracting an RSV LRTI and of being hospitalized for this
{[}14,15,23{]}. Considering the power of the present study; the sample
size is comparable with the study reported by Buchwald \emph{et al.}(587
infants) {[}18{]}, and greater than that reported by Eick \emph{et al.}
(372 infants) {[}16{]}, which both concluded to a protection conferred
by neutralizing antibodies (while the study conducted in Alaska {[}155
infants{]} and Kenya {[}90 infants{]} did not find any association
{[}17,18{]}). Therefore, only a weak protection might have been missed.
In addition, the statistical power was sufficient to detect a robust
association between known risk factors of RSV VS-LRTI (parity of the
mother and a date of birth in the first part of the epidemic season
{[}29{]}) and the occurrence of VS-LRTI. For the present study, owing to
the lack of data available regarding antibody titers against RSV N
protein, there was no determination of number of subjects required; the
data provided herein will therefore help for the power calculation in
future studies.

It is of note that the mean antibody titers against RSV N protein at
birth were lower in premature infants, those with a low birth weight,
born to a primiparous mother, and born before the peak of the epidemic.
This profile is not surprising; for instance, it is well known that
majority of transplacental transfer of IgG occurs during the third
trimester of pregnancy, and that preterm newborns have a compromised
passive immunity {[}28--30{]}. Furthermore, multiparous mothers are
likely to have been exposed to RSV, as well as those delivering at the
end of the epidemic season. These factors are also known to be risk
factors for severe RSV infection {[}31--33{]}. The multivariate analysis
indicates that this risk is mostly mediated through another pathway that
the lack of RSV N antibodies.

The study does have some limitations. The most important is that only
patients admitted to the hospital they were born in were considered for
the analysis of VS-LRTI, thus any cases visiting elsewhere may have been
missed. However, there are few other pediatric emergency departments in
the region, the HFME university hospital is the largest pediatric
hospital in the area and the only one with an intensive care unit; thus
it is likely that most cases of RSV VS-LRTI occurring before 3 months of
age were captured from either direct admission or from transfers from
other hospitals. Furthermore, detection used ELISA that allows
standardization of result but does not quantify neutralizing activity.
However, a neutralization assay would not have specifically detect
antibodies against N protein which were investigated herein. Finally,
the severity of LRTI episodes might have been overestimated; a third of
cases (n=12/36) were classified as VS-LRTI only on the ``inability to
feed'' criterion, and patients hospitalized for LRTI were very likely to
have enteral nutrition, sometimes for less than twenty-four hours
(\emph{data not shown} ). However it is unlikely to have led to a
differential selection according to the antibody titer level, and the
classification of all episodes of LRTI according to the WHO standardized
severity criteria allows comparison with future studies.

In conclusion, the findings presented herein suggest that transfer of
maternal antibodies against the RSV N protein does not provide a
significant immune protection early in infancy against RSV VS-LRTI.
Maternal vaccination may therefore not be a suitable strategy for an N
protein candidate vaccine.

Conflict of interest: none

Financial support: none

\emph{References:}

1. Shi T, McAllister DA, O'Brien KL, et al. Global, regional, and
national disease burden estimates of acute lower respiratory infections
due to respiratory syncytial virus in young children in 2015: a
systematic review and modelling study. Lancet. \textbf{2017} ;
390(10098):946--958.

2. Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower
respiratory infections due to respiratory syncytial virus in young
children: a systematic review and meta-analysis. Lancet. \textbf{2010} ;
375(9725):1545--1555.

3. Zhou H, Thompson WW, Viboud CG, et al. Hospitalizations associated
with influenza and respiratory syncytial virus in the United States,
1993-2008. Clin Infect Dis. \textbf{2012} ; 54(10):1427--1436.

4. Kramer R, Duclos A, VRS study group in Lyon, Lina B, Casalegno J-S.
Cost and burden of RSV related hospitalisation from 2012 to 2017 in the
first year of life in Lyon, France. Vaccine. \textbf{2018} ;
36(45):6591--6593.

5. Stockman LJ, Curns AT, Anderson LJ, Fischer-Langley G. Respiratory
syncytial virus-associated hospitalizations among infants and young
children in the United States, 1997-2006. Pediatr Infect Dis
J.\textbf{2012} ; 31(1):5--9.

6. Simoes EAF, Bont L, Manzoni P, et al. Past, Present and Future
Approaches to the Prevention and Treatment of Respiratory Syncytial
Virus Infection in Children. Infect Dis Ther. \textbf{2018} ;
7(1):87--120.

7. Karron RA. Preventing respiratory syncytial virus (RSV) disease in
children. Science. \textbf{2021} ; 372(6543):686--687.

8. Graham BS. Vaccine development for respiratory syncytial virus. Curr
Opin Virol. \textbf{2017} ; 23:107--112.

9. Bakker SE, Duquerroy S, Galloux M, et al. The respiratory syncytial
virus nucleoprotein--RNA complex forms a left-handed helical
nucleocapsid. J Gen Virol. \textbf{2013} ; 94(Pt 8):1734--1738.

10. Collins PL, Fearns R, Graham BS. Respiratory syncytial virus:
virology, reverse genetics, and pathogenesis of disease. Curr Top
Microbiol Immunol. \textbf{2013} ; 372:3--38.

11. Abarca K, Rey-Jurado E, Munoz-Durango N, et al. Safety and
immunogenicity evaluation of recombinant BCG vaccine against respiratory
syncytial virus in a randomized, double-blind, placebo-controlled phase
I clinical trial. EClinicalMedicine. \textbf{2020} ; 27:100517.

12. Green CA, Scarselli E, Voysey M, et al. Safety and immunogenicity of
novel respiratory syncytial virus (RSV) vaccines based on the RSV viral
proteins F, N and M2-1 encoded by simian adenovirus (PanAd3-RSV) and MVA
(MVA-RSV); protocol for an open-label, dose-escalation, single-centre,
phase 1 clinical trial in healthy adults. BMJ Open. \textbf{2015} ;
5(10):e008748.

13. Modjarrad K, Giersing B, Kaslow DC, Smith PG, Moorthy VS, WHO RSV
Vaccine Consultation Expert Group. WHO consultation on Respiratory
Syncytial Virus Vaccine Development Report from a World Health
Organization Meeting held on 23-24 March 2015. Vaccine. \textbf{2016} ;
34(2):190--197.

14. Jones CE, Calvert A, Le Doare K. Vaccination in Pregnancy-Recent
Developments. Pediatr Infect Dis J. \textbf{2018} ; 37(2):191--193.

15. Wilcox CR, Holder B, Jones CE. Factors Affecting the FcRn-Mediated
Transplacental Transfer of Antibodies and Implications for Vaccination
in Pregnancy. Frontiers in Immunology. \textbf{2017} ; 8:1294.

16. Eick A, Karron R, Shaw J, et al. The role of neutralizing antibodies
in protection of American Indian infants against respiratory syncytial
virus disease. Pediatr Infect Dis J. \textbf{2008} ; 27(3):207--212.

17. Stensballe LG, Ravn H, Kristensen K, et al. Respiratory syncytial
virus neutralizing antibodies in cord blood, respiratory syncytial virus
hospitalization, and recurrent wheeze. J Allergy Clin
Immunol.\textbf{2009} ; 123(2):398--403.

18. Buchwald AG, Graham BS, Traore A, et al. RSV neutralizing antibodies
at birth predict protection from RSV illness in infants in the first
three months of life. Clin Infect Dis. \textbf{2020} ; ciaa648.

19. Bulkow LR, Singleton RJ, Karron RA, Harrison LH, Alaska RSV Study
Group. Risk factors for severe respiratory syncytial virus infection
among Alaska native children. Pediatrics. \textbf{2002} ;
109(2):210--216.

20. Nyiro JU, Sande CJ, Mutunga M, et al. Absence of Association between
Cord Specific Antibody Levels and Severe Respiratory Syncytial Virus
(RSV) Disease in Early Infants: A Case Control Study from Coastal Kenya.
PLoS One. \textbf{2016} ; 11(11):e0166706.

21. Blencowe H, Cousens S, Oestergaard MZ, et al. National, regional,
and worldwide estimates of preterm birth rates in the year 2010 with
time trends since 1990 for selected countries: a systematic analysis and
implications. Lancet. \textbf{2012} ; 379(9832):2162--2172.

22. Kramer MS, Papageorghiou A, Culhane J, et al. Challenges in defining
and classifying the preterm birth syndrome. Am J Obstet
Gynecol.\textbf{2012} ; 206(2):108--112.

23. Giersing BK, Karron RA, Vekemans J, Kaslow DC, Moorthy VS. Meeting
report: WHO consultation on Respiratory Syncytial Virus (RSV) vaccine
development, Geneva, 25-26 April 2016. Vaccine. \textbf{2019} ;
37(50):7355--7362.

24. Capella C, Chaiwatpongsakorn S, Gorrell E, et al. Prefusion F,
Postfusion F, G Antibodies, and Disease Severity in Infants and Young
Children With Acute Respiratory Syncytial Virus Infection. J Infect
Dis.\textbf{2017} ; 216(11):1398--1406.

25. Buchwald AG, Graham BS, Traore A, et al. RSV neutralizing antibodies
at birth predict protection from RSV illness in infants in the first
three months of life. Clin Infect Dis. \textbf{2020} ; ciaa248.

26. Gaymard A, Bouscambert-Duchamp M, Pichon M, et al. Genetic
characterization of respiratory syncytial virus highlights a new BA
genotype and emergence of the ON1 genotype in Lyon, France, between 2010
and 2014. J Clin Virol. \textbf{2018} ; 102:12--18.

27. Roux X, Dubuquoy C, Durand G, et al. Sub-nucleocapsid nanoparticles:
a nasal vaccine against respiratory syncytial virus. PLoS
One.\textbf{2008} ; 3(3):e1766.

28. Linder N, Ohel G. In utero vaccination. Clin Perinatol.\textbf{1994}
; 21(3):663--674.

29. Mammas IN, Drysdale SB, Rath B, et al. Update on current views and
advances on RSV infection (Review). Int J Mol Med. \textbf{2020} ;
46(2):509--520.

30. Malek A, Sager R, Kuhn P, Nicolaides KH, Schneider H. Evolution of
maternofetal transport of immunoglobulins during human pregnancy. Am J
Reprod Immunol. \textbf{1996} ; 36(5):248--255.

31. Homaira N, Mallitt K-A, Oei J-L, et al. Risk factors associated with
RSV hospitalisation in the first 2\selectlanguage{english} years of life, among different
subgroups of children in NSW: a whole-of-population-based cohort study.
BMJ Open. \textbf{2016} ; 6(6):e011398.

32. Bont L, Checchia PA, Fauroux B, et al. Defining the Epidemiology and
Burden of Severe Respiratory Syncytial Virus Infection Among Infants and
Children in Western Countries. Infect Dis Ther. \textbf{2016} ;
5(3):271--298.

33. Aikphaibul P, Theerawit T, Sophonphan J, Wacharachaisurapol N,
Jitrungruengnij N, Puthanakit T. Risk factors of severe hospitalized
respiratory syncytial virus infection in tertiary care center in
Thailand. Influenza Other Respir Viruses. \textbf{2021} ; 15(1):64--71.

\textbf{Table 1: Clinical characteristics of the population.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Total cohort (N = 578)\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Cases of VS-LRTI (N = 36)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Sex\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Female\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
267 (46.2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
18 (50.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Mode of delivery\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Vaginal\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
445 (77.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
29 (80.6\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Caesarean\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
133 (23.0\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
7 (19.4\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Parity\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Monoparity\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
227 (39.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
4 (11.1\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Multiparity\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
351 (60.7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
32 (88.9\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Gestational age at delivery (weeks of amenorrhea)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Mean (SD+)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
38.7 (2.5)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
39.1 (1.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Term\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
521 (90.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
36 (100.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Moderate-to-late preterm\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
43 (7.4\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Very preterm\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
9 (1.6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Extremely preterm\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
5 (0.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Infant birth weight (g)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Mean (SD+)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
3179 (633)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
3301 (337)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Low birth weight (\textless{} 2500 g)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
65 (11.3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Type of pregnancy\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Singleton\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
543 (93.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
36 (100.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Multiple\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
35 (6.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
0 (0.0\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Date of birth in relation to the peak of the epidemic\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Birth before the peak\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
277 (47.9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
31 (86.1\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Birth after the peak\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
301 (52.1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
5 (13.9\%)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

+SD = Standard Deviation

Table 2. Multivariate analysis of the factors associated with VS-LRTI\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& Level & Relative risk (RR) of VS-LRTI+ & {[}95\% CI{]}++ & p-value &
Significance\tabularnewline
\midrule
\endhead
Sex & Female & Ref & & 0.75 & -\tabularnewline
& Male & 0.89 & {[}0.34; 1.45{]} & &\tabularnewline
Type of pregnancy & Singleton & Ref & & 0.99 & -\tabularnewline
& Multiple & 0.14 & {[}-1.42; 1.40{]} & &\tabularnewline
Mode of delivery & Vaginal & Ref & & 0.79 & -\tabularnewline
& Caesarean & 0.88 & {[}0.14; 1.62{]} & &\tabularnewline
Parity & Monoparity & Ref & & \textless{} 0.01 & **\tabularnewline
& Multiparity & 2.34 & {[}1.58; 3.01{]} & &\tabularnewline
Date of birth in relation to the peak of the epidemic & Birth after the
peak & Ref & & \textless{} 0.001 & ***\tabularnewline
& Birth before the peak & 2.84 & {[}2.08; 3.59{]} & &\tabularnewline
Titer of antibodies & Slope & -0.00049 & {[}-0.00167; 0.000692{]} & 0.50
& -\tabularnewline
\bottomrule
\end{longtable}

**p\textless{} 0.01, ***p\textless{} 0.001. Relative risk and p values
are calculated using a generalized linear model

+VS-LRTI = very severe low respiratory tract infection.

++95\% CI = 95\% confidence interval



\textbf{Figure 1: Flow chart. LRTI: Lower Respiratory Tract Infection.}

Figure 2: Titers of antibodies against N protein in different subgroups
A. Sex, B. type of pregnancy, C.Mode of delivery, D. Gestational age at
delivery, E. Infant birth weight, F.~Parity, G. Date of birth (epidemic
peak), H. VS-LTRI. Boxplots present medians and interquartile ranges
{[}IQR{]}, wiskers 1.5 times the IQR, and outliers are
\textgreater{}~1.5 times and \textless{}~3 times the interquartile range
beyond end of the box. *p~\textless{} 0.05, **p~\textless{}~0.01,
***p~\textless{}~0.001 in Student t-test or ANOVA.

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

