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Humancoronavirusreinfectiondynamics:lessonsforSARS‐CoV‐2
Authors
ArthurW. D.Edridge1,JoannaKaczorowska1, AlexisC.R.Hoste2,Margreet Bakker1,Michelle Klein1,
Maarten F. Jebbink1, Amy Matser3, Cormac M. Kinsella1, Paloma Rueda2, Maria Prins3,4, Patricia
Sastre2,MartinDeijs1,LiavanderHoek1,*
Affiliations
1. Laboratory of Experimental Virology, Department of Medical Microbiology and Infection
Prevention,AmsterdamInfection&ImmunityInstitute,AmsterdamUMC,UniversityofAmsterdam,
Meibergdreef15,1105AZAmsterdam,TheNetherlands.
2.INGENASA,InmunologíayGenéticaAplicadaS.A.,Av.delaInstitución Libre de Enseñanza,39,
28037Madrid,Spain
3.DepartmentofInfectiousDiseases,PublicHealthServiceofAmsterdam,NieuweAchtergracht100,
1018WTAmsterdamTheNetherlands.
4. Amsterdam UMC, University of Amsterdam, Department of Infectious Diseases, Amsterdam
Infection& ImmunityInstitute,AmsterdamUMC,University ofAmsterdam,Meibergdreef15,1105
AZAmsterdam,TheNetherlands.
*Correspondingauthor
ABSTRACT
InthecurrentSARS‐CoV‐2pandemicakeyunsolvedquestionisthequalityanddurationofacquired
immunity in recovered individuals. This is crucial to solve, however SARS‐CoV‐2 has circulated for
underfivemonths,precludingadirectstudy.Wethereforemonitored10subjectsoveratimespanof
35years(1985‐2020), providinga total of2473follow upperson‐months,and determineda)their
antibodylevelsfollowinginfectionbyanyofthefourseasonalhumancoronaviruses,andb)thetime
periodafterwhichreinfectionsbythesameviruscanoccur.Analarminglyshortdurationofprotective
immunitytocoronaviruseswasfoundbybothanalyses.Wesawfrequentreinfectionsat12months
post‐infectionandsubstantialreductioninantibodylevelsassoonas6monthspost‐infection.
ONESENTENCESUMMARY:Coronavirusprotectiveimmunityisshort‐lasting
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MAINTEXT
SARS‐CoV‐2isanovelcoronavirusresponsibleforanongoingpandemic.Itsrapidtransmissionismost
probably caused bythefactthatthe virus entered a grossly naive, thus highly susceptible, human
population, combinedwiththecapacity ofthevirusto transmit duringtheasymptomaticphaseof
infection.Sincenopharmaceuticalinterventionsareuniversallyavailableorapplied,currentpolicies
tolimitthespreadofSARS‐CoV‐2revolvearoundcontainment,socialdistancing,andtheassumption
thatrecovered patientsdevelopprotectiveimmunity.Itisstillunclearwhetherprotectiveimmunity
isindeed inducedafterinfection, andforhowlong.Thedurationofprotectionwillimpactnotonly
theoverallcourseofthecurrentpandemic,butalsothepost‐pandemicperiod.Todate,noconcrete
evidenceofreinfectionbySARS‐CoV‐2isavailable,norisanyexampleofreinfectionbySARS‐CoV‐1or
MERS‐CoV,yetthisislikelyinfluencedbytherecentemergenceofSARS‐CoV‐2andthelimitedscale
of the SARS‐CoV‐1 and MERS‐CoV epidemics. The limited available data on potential protective
immunityagainstcoronavirusreinfectionisderivedfromoneexperimentalinfectionstudyin
volunteers(1),fromwhichitisgenerallyassumedthatreinfectionbycoronavirusescanoccur.Natural
coronavirusinfectionsandsusceptibilitytoreinfectionhavenotbeen investigatedthusfar. If they
occur,reinfectionswillprobablybedictatedbytwovariables:exposuretothevirus,andthequality
ofsustainedimmunity(2).
Even though it is not possible to investigate SARS‐CoV‐2 reinfections yet, the seasonal
coronavirusesmayserveasareliablemodel.Therearefourspeciesofseasonalcoronaviruses,HCoV‐
NL63,HCoV‐229E,HCoV‐OC43,andHCoV‐HKU1.Allareassociatedwithmostlymildrespiratorytract
infections. However, aside from being etiological agents of common cold, the four viruses are
biologically dissimilar. Two belong to the genus Alphacoronavirus, and two to the genus
Betacoronavirus.The virusesuse characteristicreceptormoleculestoentera targetcell,andbased
onreceptordistributiontheydonotallenterthesameepithelialcelltypeinthelungs(3).Giventhis
variability,theseasonalcoronavirusesarethemostrepresentativeviru sgroupfr omwhichtoco nclude
generalcoronaviruscharacteristics,particularlycommondenominatorslikehostprotectiveimmunity
and susceptibility to reinfection. Since most people experience their first seasonal coronavirus
infectionduringearlychildhood(4–6),reinfectionslaterinlifecanbeinvestigated.
Theaimofthisstudywastoinvestigatethetimeperiodbetweencoronavirusreinfectionsand
thedynamicsofantibodydeclinefollowinginfection.Theseparameterswereassessedbymeasuring
theimmuneresponsetoeachindividualseasonalcoronavirus,overanextendedperiod.Wefollowed
healthysubjectsformultipledecadesatregularintervals.AsIgGlevelsonlyincreaseaftersuccessful
infection and—importantly—remain constant after unsuccessful viral challenge (1), increased
antibodylevelsidentifyaninfection.Weusedthecarboxylterminalofthenucleocapsidprotein(NCt)
as the target antigen in ELISA, as this protein is immunogenic, relatively low in interspecies
conservation,yethighinintraspeciesconservationwhencomparedtotheSpikeprotein(4,7–10).
Frequencyofinfectionsandreinfections.
Fromaprospectivecohortstudyfollowingadultmales(seeM&Mand (11)), ten subjects were
randoml yselected.Foll ow‐upof subject sstartedin1985 and,besidesagapinfollowupbetween1997
and2003,continueduntil2020atregularintervals(every3monthspriorto1989,andevery6months
afterwards,Table1).Thecumulativeperiodatwhichsubjectswerecontinuouslyfollowedtotaled
morethan200person‐years(2473months).Atstartofthestudy,subjectagerangedfrom27to40
years; by the end of follow‐up , subjects were 49 to 66 years old.Thestudywasapprovedbythe
MedicalEthicsCommitteeoftheAmsterdamUniversityMedicalCenter of the University of
Amsterdam,theNetherlands(MEC07/182).
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Infections by seasonal coronaviruses were defined as 1.4‐fold increase in antibody levels
occurringbetweentwovisits(1),andadditionallythestandard deviationsofthemeanatbothvisits
notoverlapping.Atotalof132events,rangingfrom3to22persubject,metthesecriteriaandwere
thusconsideredinfections(Table1).Theincidenceratesofseasonalcoronavirusreinfectionsper100
person‐yearsof follow‐upwas15.3 (95%CI10.0‐23.4);20.1(95% CI14.2‐28.7);16.4(95%CI 10.4‐
26.1);6.3(95%CI2.4‐16.8),forHCoV‐NL63,HCoV‐229E,HCoV‐OC43andHCoV‐HKU1respectively.To
test whether serological infection criteria represented symptomatic infections , we compared self‐
reported influenza like illnesses in the interval directly preceding the antibody peaks. Indeed,
reportingoffeverandcoughweresignificantlyassociated(Fisher’sexacttestp=0.031,supplementary
tableS1).
Table1.Studysubjectsandseasonalcoronavirusinfectionsduringfollowup.
Subject Year Age
Continuous
follow‐up
period
CoronavirusInfections
Start End Start End Months Years Total* NL63 229E HKU1 OC43
1 1985 2017 32 64 265 22.1 14 4 3 1 6
2 1985 2019 30 64 310 25.9 15 5 6 3 1
3 1985 2020 29 64 340 28.3 6 2 3 0 1
4 1985 2010 33 59 230 19.2 22 3 12 2 5
5 1985 2010 27 53 232 19.3 9 5 3 1 0
6 1985 1997 37 49 144 12.0 3 1 1 0 1
7 1985 2003 32 49 138 11.5 13 3 5 1 4
8 1986 2014 34 62 256 21.3 10 3 5 0 2
9 1985 2010 40 75 342 28.6 22 7 5 3 7
10 1985 2011 35 60 233 19.4 18 4 6 2 6
Total 2473 205.6 132 37 49 13 33
Medianreinfectiontimesof33(IQR18–60),31(IQR15–42),27(IQR21–49),and46(IQR
36–68)monthswerefoundforHCoV‐NL63,HCoV‐229E,HCoV‐OC43andHCoV‐HKU1,respectively,
and30(IQR18–54)monthsforallvirusescombined(Fig.1A).Therewasnostatisticallysignificant
differencebetweentheinfectionintervallengthsoftheindividualviruses(Kruskal‐Wallistest,P=0.74).
Inafewcases,re‐infectionsoccurredasearlyas6months(twotimesforHCoV‐229Eandonetimefor
HCoV‐OC43)and9months(twotimesforHCoV‐NL63).Themostfrequentobservedreinfectiontime
was 12 months. For reinfections occurring as early as 6 months, we observed no reduction in
antibodies between infections (Fig. 1A, white circles). At longer infection intervals, intermediate
reductionsinantibodylevelswereobserved.
Theability todetect short‐termreinfectionsislimitedbythesamplinginterval.Importantly
though,noreinfectionwasobservedatthefirstsubsequentfollow‐upvisitaftera 3monthinterval
(Fig.1A).Wedidobserveseveralreinfectionsatsubsequentvisitswitha6‐monthinterval,suggesting
that reinfections within 6 month s do not occur. To further support this point, weexamined ratios
betweenantibodylevelsbetweentwovisits,bothfor3and6monthfollow‐ups.AsshowninFig.1B,
only ratios below 1 were found in the observations with every 3month‐sampling,andwecan
thereforesafelyconcludethatreinfectionsoccurfrom6monthson,yetmostprobablynotearlier.
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Figure1Infectionandreinfectioncharacteristics,andwaningimmunity for seasonal coronaviruses.(A) The
intervaldurationsbetweenreinfections.Onlyreinfectionswhich were observed within a continuous follow‐u p
periodareshown.Whitedotsindicatereinfectionsforwhichnointermediatedecreaseinantibodylevelscouldbe
observed. Black vertical lines describe median reinfection times. (B) Changes in antib ody levels post‐infection
relativetothefollow‐upintervalduration.Eachcirclerepresentsaninfection. Thex‐axisdescribesthetimeuntil
thenext follow‐upvisit post‐infection.The y‐axisdescribesthechange inantibody levelatthesubsequentvisit.
Largercircles representsahigherratio‐riseinantibodylevelsattheinitialinfection.Thehorizontallineindicates
theborderbetweenincreases(>1.0)ordecreases(<1.0)inantibodylevelsatthenextstudyvisit(C)Kaplan‐Meier
curve showing decline of antibodies post infe ction (100%, 75% and 50%). The visit at which the infection wa s
establishedwas countedas timepoint0,caseswere subsequentlyfollowed. Anevent isdefinedwhen antibody
levels drop below the indicated level. When the antibody level did not decrease to the indicated levels, an
observationwascensoredatthelasttimepointofactivefollow‐up.
Antibodydynamicsafterinfection.
Onsomeoccasions,antibodyrisesreachedhighlevels(uptoratio11),representedinFig.1Baslarger
circles,yetthesehighvalueswereneversustainedatthenextstudyvisit(Fig.1B).Toinvestigatethese
dynamicsfurther,wecalculatedthetimeuntila50%,75%,orfullreturnofantibodylevelstobaseline
(pre‐infectionantibodylevels).AscanbeseeninFig.1C,within6monthspost‐infectionthemajority
ofpeoplelost50%oftheirantibodies,whereasafterayearthemajoritylost75%.Acompletereturn
tobaselinelevelsoccurredwithin4yearsforhalfofinfections(Fig.1C).
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Simultaneousinfectionsbyalpha‐andbetacoronaviruses.
AlthoughourELISAtestsusingtheC‐terminalpartoftheNproteinwerecautiouslydesignedtobe
specificforeachindividualvirus,wecannotruleoutthatacertaindegreeofantibodycross‐reactivity
occurred.Wethereforeinvestigatedhowofteninfectionscoincided,sincecross‐reactivitymayhave
ledtofalselabelingofinfections. We observed no significant simultaneous infections for
Alphacoronavirus(HCoV‐NL63orHCoV‐229E)alongsideBetacoronavirus(HCoV‐HKU1orHCoV‐OC43),
however, we did see that infections by the betacoronaviruses HCoV‐OC43 and HCoV‐HKU1 often
coincided (38.5%, Table 2).Likewise,foralphacoronaviruses,HCoV‐229Einfectionscoincided with
HCoV‐NL63infectionsin59.5%ofthecases,andviceversain44.9%ofthecases.Hence,thereisarisk
thatweoverestimated the numberinfectionsandthus reinfections. We thereforere‐analyzedthe
datawithamorestringentdefinitionofinfection,includingonlythestrongestantibodyriseinduced
bya BetacoronavirusorAlphacoronavirusatagiventimepoint.Underthisdefinitionwestillfound
infectionintervalscomparabletotheoriginaldata(supplementaryFig.S2),withminimuminfection
intervals as short as 6 months and frequent reinfections at 12 months, although the number of
reinfectionswasobviouslyreduced.
Table2.Coincidingcoronavirusinfections
Simultaneousinfections(%)
Infection HCoV‐NL63 HCoV‐229E HCoV‐OC43 HCoV‐HKU1
HCoV‐NL63 NA 44.9 3.0 7.7
HCoV‐229E 59.5 NA 18.2 23.1
HCoV‐OC43 2.7 12.2 NA 38.5
HCoV‐HKU1 2.7 6.1 15.2 NA
Broadly acting antibodies recognizing SARS‐CoV‐2. In theory, antibodies induced by (repeated)
coronavirus infections may have broad coronavirus‐recognizing characteristics. We therefore
performedanadditionalELISAonall10subjects,thistimeusingthecompleteNproteinofSARS‐CoV‐
2, to allow detection of broadly reacting antibodies. Visual inspection suggested that broadly
recognizing antibodies wer e produced, and were most likely induced by combined infections with
HCoV‐NL63andHCoV‐HKU1(subjects2,9and10,supplementaryFig.S1).
Coronavirusinfectionsinchangingseasons.
To date it is uncertain whether SARS‐CoV‐2 will share the same winter prevalence peak that is
observedforseasonalcoronavirusesinnon‐equatorialcountries.However,itisimportanttoconsider
thatwinterpreferenceof seasonal coronaviruses has only beendetermined by testingrespiratory
samplesofpeoplethatexperienceddisease(12).Samplingandstorageisthereforedictatedbyhaving
symptomsandnotbystudyprotocol.Ifco ronavirusspreadcontinuesunabatedinsummer,yetpeople
rarely display symptoms (e.g. because of higher vita min D levels) and are therefore not sampl ed,
infectionswillremainundetected.Ourserologicalstudyisuniquebecauseitavoidsthissamplingbias.
TheNetherlandshas a typicaltemperateclimate,andour study samples werecollectedatregular
intervals.Thesamplingofeachsubjectstartedatdifferenttimesoftheyear,and,becauseofthe3or
6monthsregimeofvisits,sampleswerecollectedthroughoutallseasons.Consequently,wecanfor
thefirsttimevisualizeinanunbiasedmannertheseasonalityofcoronavirusinfections.Asshownin
Fig.2,May,June,July,AugustandSeptemberindeedshowthelowestprobabilityofinfectionsforall
fourseasonalcoronaviruses(Wilcoxonsigned‐ranktest,p=0.005).
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Fig.2Seasonalityofinfections.Theprevalenceof thefour seasonal coronavirusesshown as theprobability of infection
onsetforaspecificmonth.Themonthsbetweenandincludingthemonthsofthefollow‐upvisitsatwhichtheinfectionwas
establishedandthepriorfollow‐upvisitwerecounted.Thevaluepermonthwasdividedbythenumberofmonthsbetween
follow‐upvisits.
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DISCUSSION
Weshow,forthefirsttime,thatreinfectionswithallseasonal coronaviruses occur in nature.The
majorityofreinfectionsoccurredwithin3years.IfSARS‐CoV‐2willbehavelikeaseasonalcoronavirus
inthefuture,asimilarpatternmaybeexpected.However,thistimespanbetweeninfectionsdoesno t
indicatethatan individual’sprotectiveimmunitylastsforthesameperiodoftime,asreinfectionis
alsodependentonre‐exposure.Infact,basedontheminimuminfectionintervalsandthedynamics
ofantibodywaningthatweobserved,theprotectiveimmunity maylast aslittle as6 to12 months.
Recently Kissler et al.modeledthe protective immunity andreinfectiondynamicsHCoV‐OC43 and
HCoV‐HKU1 and estimated a 45 week period of protective immunity(13).Ourserologicalstudy
confirmsthisprediction.
WhenweviewourfindingsinlightofthecurrentcontrolactionstakenforSARS‐CoV‐2,itis
clearthatcoronavirusreinfectionriskiskeytopublichealthpolicy.Herewerevealariskthatinthe
nearfuture,serologybasedteststhatmeasurepreviousinfectionsforSARS‐CoV‐2mayhavelimited
use if that infection has occurred >1 year prior to sampling. Additionally, vaccine studies should
anticipate that sustained protective immunity may be uncertain for coronaviruses, and repeated
yearlyorhalf‐yearlyvaccinationsmaybeneededtocircumventongoingtransmission.
ThereisongoingdiscussionregardingherdimmunityforSARS‐CoV‐2control.Herdimmunity
occurswhenathresholdproportion ofapopulation isimmunetoacertainpathogen,andprotects
evennon‐immune individuals against theinfectionbylimitingoverall spread. This effect has been
observedforavarietyofviruses,mainlythosethatarepartiallycontrolledviavaccinationprograms,
suchashepatitisAvirus(14),influenzaAvirus(15), andhuman papillomavirus(16).Inthecaseof
SARS‐CoV‐2,achievingherdimmunitymaybechallengingduetorapidlossofprotectiveimmunity.It
wasrecentlysuggestedthatrecoveredindividualsshouldreceiveaso‐called“immunitypassport”(17)
whichwouldallowthemtorelaxsocialdistancingmeasuresandprovide governmentswith dataon
herdimmunitylevels inthepopulation. However,asprotective immunitymaybelostby6 months
post infection, the prospe ct of reaching functional herd immunity by natural infection seems very
unlikely.
It is generally assumed that exposure to each seasonal coronavirus is not equal. The
BetacoronavirusHCoV‐HKU1is consideredtohave thelowestprevalence (4,13)andwefoundthe
same. We also observed that antibody levels to HCoV‐HKU1 were low over the whole range (all
samplestested).WhetherthisindicatesHCoV‐HKU1infectionelicitsapoorinductionofantibodiesis
intriguing,yetnot answerableinourstudy. Weuseda relativelysmallpartoftheNproteinasthe
antigeninourtests,tomostspecificallyidentifyinfectionsbyeachvirusindividually.Thissmallpart
oftheproteinmaynotcontainthemostimmunodominantepitopesofHCoV‐HKU1.Aneutralization
testwouldideallybedonetosolvetheissueofprotectiveimmunityraisedbyHCoV‐HKU1;however,
unfortunatelynoculturingsystemsuitableforneutralizationtestingisavailableforHCoV‐HKU1(18).
WenoticedthreesubjectstocarryantibodiesrecognizingSARS‐CoV‐2N protein atcertain
timepoints.ItisunlikelythattheyhadbeeninfectedwithaSARS‐CoV‐2‐likevirusin1985(subject10),
1992(subject2),or 2006(subject 9),and wethereforesuggestthatbroadly actingantibodiesmay
havebee ninduce dbycoin cidin ginfect ionsofanAlpha‐andaBetacoronavirus(inoursub ject(s) HCoV‐
HKU1andHCoV‐NL63).Toexplorethisfindingwelookedat thegenetic distanceandconsequently
aminoacid differencesinthestructuralproteinofthevariouscoronaviruses(supplementaryTable
S2).Notably,SARS‐CoV‐2Nproteinhasonly32%and34%identityontheaminoacidlevelwiththeN
proteinofHCoV‐OC43andHCoV‐HKU1respectively,andonly26%and24%identitywithHCoV‐NL63
andHCoV‐229Erespectively. Similarly,thedistancebetweenAlphacoronavirusandBetacoronavirus
Nproteinislarge (only 24% to26%aminoacid identity). Still, we cannot exclude the presenceof
conserved(conformational) epitopesin HCoV‐HKU1andHCoV‐NL63 Nproteinthat mayresultin a
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more broadly acting antibody response, due to simultaneous exposure in concurrent infections.
Additionalscreening,includingmoresubjects,isrequiredforconfirmation.
Ourstudywassubjecttolimitations.Oneistheinabilitytosequencethevirusgenomeduring
infection.Intheory,strainvariationcouldplayaroleinsusceptibilitytoreinfection.HCoV‐NL63,HCoV‐
OC43,andHCoV‐HKU1allshowdifferentco‐circulatinggeneticclusters(19–21).ForHCoV‐229E,there
arenomajorgeneticsubtypesknown,likethecurrentSARS‐CoV‐2situation(22,23).Itcouldbethat
ourobservationofshort‐termimmunityisinfluencedbygenotypevariation,andastudyonprotective
immunitywouldthereforeideallyallowsequencingofre‐infectingstrainsfromrespiratorymaterial;
however,thisisintractableinanaturalinfectionstudybecausevirussheddinginreinfectionscanbe
asshortasoneday(1).Anotherlimitationisthatthestudiessubjectswereallmales.ForCOVID‐19,
andalsoHCoV‐NL63,menhaveahigherincidenceofdisease(25,26),anditisthereforeofinterestto
determinethedynamicsofprotectiveimmunityalsoinacohortofhealthywomen.
In conclusion, seasonal human coronaviruses have little in common, apart from causing
commoncold.Still,theyallseemtoinduceashort‐lastingimmunitywithrapidlossofantibodies.This
maywellbeageneraldenominatorforhumancoronaviruses.
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Acknowledgments
TheauthorsgratefullyacknowledgetheAmsterdamCohortStudies(ACS)onHIVinfectionandAIDS,acollaborationbetween
the Public Health Service of Amsterdam, the Amsterdam UMC of the University of Amsterdam, Sanquin B lood Supply
Foundation,MedicalCenter JanvanGoyen, andthe HIVFocusCenteroftheDC‐Clinics.ItispartoftheNetherlandsHIV
MonitoringFoundation andfinanciallysupportedbytheCenterforInfectiousDiseaseControlof theNetherlandsNational
InstituteforPublicHealthandtheEnvironment.TheauthorsthankallACSparticipantsfortheircontribution,aswellasthe
ACS study nurses, data‐managers, and lab technicians. This work was supported by a grant from the European Union's
Horizon2020researchandinnovationprogramme,undertheMarieSkłodowska‐CurieActionsgrantagreementno.721367
(HONOURs),AmsterdamUMCfundingconnectedtoHONOURs,andtheAmsterdamUMCPhDscholarshipofA.W.D.Edridge.
Authorcontributions:A.W.D.E:Conceptualization,Writing– originaldraft,review andediting,Investigation,Visualization,
Formal Analysis, Validation; J.K.: Investigation, Writing – original draft, review and editing, A.C.R.H.: Resources; M.B.:
Investigation,Resources;M.K.: Investigation;M.F.J.: Investigation,Methodology;A.M.: FormalAnalysis;C.M.K.:Writing–
originaldraft,reviewandediting,FormalAnalysis; P.R.:Resources; M.P.:Resources;P.S.: Resources;M.D.:Investigation,
Methodology;L.v.d.H.:Conceptualization,Writing–originaldraft,Writing‐reviewandediting,Supervision.Competing
interests:Authorsdeclarenocompetinginterests.
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 18, 2020. .https://doi.org/10.1101/2020.05.11.20086439doi: medRxiv preprint