Dynamics of SARS-Cov-2 and the Adaptive Immune Response

Dynamics of Executive Summary • SARS-CoV-2, the virus that causes COVID-19, is a beta-coronavirus that is closely related SARS-CoV-2 and to SARS-CoV, the virus that caused Severe Acute Respiratory Syndrome (SARS). • Manifestations of COVID-19 include: asymptomatic infection; a self-limiting illness the Adaptive comprised of fever, cough, and various non-specific symptoms; pneumonia, typically with ground-glass opacities on CT images; a variety of thrombotic or vasculitic Immune Response syndromes; and life-threatening acute respiratory distress syndrome (ARDS), viral sepsis and cytokine storm. Oren Cohen, M.D., F.I.D.S.A. • Antibodies to SARS-CoV-2 begin to appear during the first week after onset of symptoms. For a variable period of time (generally weeks 2-3 after onset of symptoms), Marcia Eisenberg, Ph.D. rising antibody titers and declining levels of viral load in the respiratory tract are Brian Caveney, M.D., J.D., M.P.H. detectable. Paul Kirchgraber, M.D., M.B.A. • Virus-specific neutralizing antibodies and T cell responses emerge during Dorothy Adcock, M.D. seroconversion; however, the relationship between the presence of these immune responses, viral clearance, and the durability of this response are just beginning to be Steve Anderson, Ph.D. explored. • In the early phase of COVID-19, there is a strong correlation between the presence of viral RNA detected by polymerase chain reaction (PCR) and the ability to isolate replication-competent, presumably transmissible, virus. Emerging data from a very small sample of patients suggest that although viral RNA may be detectable in the respiratory tract for a prolonged period, the ability to isolate replication-competent virus becomes increasingly difficult as antiviral antibodies appear. More data are urgently needed to determine whether shedding of viral RNA during convalescence represents infectious virus or non-infectious but detectable SARS-CoV-2 nucleic acid. • The correlates of protective natural immunity to infection with SARS-CoV-2 are yet to be defined; however, a critical role for neutralizing antibodies is suggested by converging lines of evidence. • A broad variety of serological tests for detection of antibodies to SARS-CoV-2 is available, including mostly rapid lateral flow assays and more conventional enzyme linked immunosorbent assays (ELISA). Interpretation of results is dependent on the sensitivity and specificity of the test, as well as the prevalence of COVID-19 in the population. • Detection of antibodies to the S1 protein or the receptor binding domain (RBD) of the S1 protein of SARS-CoV-2 is the most specific indicator of past infection. Antibodies to other immunogenic viral components, such as the nucleocapsid protein, are also useful indicators of past infection. A confounding factor with regard to specificity of antibody tests is the degree to which they detect cross-reactivity with antibodies directed at the endemic human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63). • The nature of the interaction between the virus and the host immune response may result in a variety of outcomes. Whereas mild infections may not stimulate a robust and durable immune response, in severe cases an over-exuberant immune response may lead to a spectrum of immunopathologic phenomena. Understanding these dynamics has important implications for the diagnosis, treatment and prevention of COVID-19.

1 Dynamics of SARS-CoV-2 and the Adaptive Immune Response Oren Cohen, M.D., F.I.D.S.A.; Marcia Eisenberg, Ph.D.; Brian Caveney, M.D., J.D., M.P.H.; Paul Kirchgraber, M.D., M.B.A.; Dorothy Adcock, M.D.; Steve Anderson, Ph.D. 2011]. 2011]. patients 2007].Most becomeN EnglJMed seronegative for SARS between years infection[Tang, 3-5after JImmunol antibodies were in15%ofpatients nolongerdetectable andoverall meantiters geometric haddecreased by 97%[Cao, in25%ofpatientswas nolongerdetectable andoverall meantiters geometric haddecreased by 90%,andneutralizing over thisperiod, itremained stablefor neutralizing antibodies; however, by 3years onsetofillness, after virus-specificIgG and neutralizing antibodies over a16month period. meantiter Although thegeometric ofIgGdecreased significantly [Chan, 2005].Cao ClinDiagn etalshowed LabImmunol that 100%ofSARSpatients virus-specific IgG haddetectable by 93%by onsetofillness andIgAby 7months after 64%,althoughIgGlevels were sustainedover thistimeperiod decline significantly withtime. Chanetaldemonstrated thatmean titers geometric ofSARS-CoV-specific IgM declined ofantibodydurability responses in SARSismore robust compared withtheendemiccoronaviruses, however levels do and importantly, neutralizing antibodies appeared 2004]. inthis same timeframe Infect [Hsueh,Clin Microbiol The 2004]. Infect 2005;Hsueh,ClinMicrobiol Diagn LabImmunol The IgGresponse emerged 1-2days before IgAandIgM, Antibody responses reliably develop inSARSpatients, withameantimeto seroconversion of11-15days [Chan, Clin isolated from stool 6weeks after despite continued RNAsheddingfor more than14weeks (Liu, Dis2004). Emerg Infect viralinstool; RNAwas however, seeninsomepatients,PCR-detectable particularly noculturable live viruscould be ofviral nucleicacid in sputumwas 21days,time ofdetectability andinstool itwas 27days. Prolonged sheddingof respiratory illness, affecting 26 countries with over 800deaths. 8000casesandnearly Liuetalshowed that themedian istheetiologic agentSARS-CoV ofSevere Acute Syndrome Respiratory (SARS),whichemerged in2002asasevere explain therelativepart protection ofpeopleintheseagegroups from COVID-19. adults, itispossiblethat immuneresponses to protection thesevirusesafford againstSARS-CoV-2. partial This could in asendemiccoronavirus 2005].Insofar Clin Diagn LabImmunol infectionspredominantly affectchildren and young existing antibodies to HCoV-OC43 and-229E, andtheseantibody titers were boosted [Chan, by infectionwithSARS-CoV (Edridge, medRxiv2020;Ng, bioRxiv2020).Similarly, inastudyofpatients withSARS,itwas shown that allhadpre- endemic coronavirus infection demonstrate withthespikeandnucleocapsidproteins cross-reactivity from SARS-CoV-2 year, mosthave ofcases(8-30%),antibodies generated lost75%[Edridge, aminority inresponse medRxiv2020].In to an by infection,mostpeoplehave 6months after lost50% of theirendemiccoronavirus-specific antibody levels,1 and by coronaviruses over time[Callow, medRxiv2020;Edridge, thisregard, 1990;Galanti, medRxiv2020].In EpidemiolInfect is relatively short-lived, andindividualsexperience multiplerepeated infectionswiththesameordifferent endemic [Gao, 2015]. JInfect These infectionsgenerally resolve inassociation withemergence ofantibodies; however, immunity infections are common, especially amongchildren and young adults, declinein incidence theageof45 with asharp after (HCoV-229E and–NL63)causeseasonal, generally mildupperrespiratoryinfections. Endemichumancoronavirus Four endemichumancoronaviruses, including2alphacoronaviruses (HCoV-OC43, and-HKU1),2betacoronaviruses caused Severe Acute Syndrome istheseventh Respiratory coronavirus (SARS).It to infecthumans. known SARS-CoV-2, thevirusthat causesCOVID-19, isabetacoronavirus that iscloselyrelated to SARS-CoV, thevirusthat Lessons From Other Coronaviruses that takestimefarbeyond havedisease andtransmissibility theinitialscientific observations beenmade to date. gaining adeepunderstandingofthecomplex interplay between virusandhosthow thistranslates to expression of connect.biorxiv.org/relate/content/181, andhttps://www.ncbi.nlm.nih.gov/pubmed/ respectively. Ontheotherhand, endof2019,there arevery already many available andpublications at thousandsofmanuscripts onthesubject https:// thecaseofCOVID-19, informationIn isaccumulating at anunprecedented rate. Although theoutbreak beganonlyat the ofthediseaseisalmostuniformly fatal. history specific cellular immune responses; however, thevirusisnever cleared by theseimmune responses, andthenatural infection withHIVgives to rise awidearray ofimmuneresponses, includingneutralizing antibodies aswell asvirus- antibodies correlates well withviral clearance andclinicalrecovery. There are, however, notableexceptions. thisregard, In that develop inpatients whorecover ofviral from diseases, thedisease. themajority theappearance In ofneutralizing influenza, hepatitis A,hepatitis B, andothers),gooddata are available regarding the correlates ofprotective immunity for whichpreventative vaccines are available (e.g., measles, mumps, rubella,diphther 2 Dynamics of SARS-CoV-2 and the Adaptive Immune Response Oren Cohen, M.D., F.I.D.S.A.; Marcia Eisenberg, Ph.D.; Brian Caveney, M.D., J.D., M.P.H.; Paul Kirchgraber, M.D., M.B.A.; Dorothy Adcock, M.D.; Steve Anderson, Ph.D. vasculitis. disparate vascular complications may by beunderpinned anantiviral immune response that results inaninflammatory [Teuwen,on endothelial cells and/or pericytes 2020;He, thiscontext, Nat Immunol seemingly bioRxiv2020].In Rev disease manifestations may have acommon pathogenesis underlying related to receptors expression ofviral entry syndrome Lancet inchildren Lancet 2020].Anumberofthese 2020;Riphagen, [Verdoni, 2020];andaKawasaki-like Med and venous thrombotic events, endothelialitis andmicroangiopathy [Thomas, Thromb 2020;Ackermann, Res NEnglJ 2020];arterial Int Kidney hypogeusia [Hirsch, andhyposmia [Whittaker, injury Neurol Acta 2020];acute Scand kidney sequelae oftheantiviral immuneresponse. ofthesecomplications Some includemeningoencephalitis, stroke, effectsofviraldirect types that factors, infectioninthebroad of as expresscell well asinflammatory variety viral entry ofCOVID-19. ofcomplications aspart An expandingisbeingdescribed spectrum This islikelydueto acombination of 2020]. strongest factor, rates withcasefatality inexcess of15%for patients over theageof80years [Dowd, Proc Natl Acad Sci to widelyfrom country, country vary andare driven by alarge however,mortality numberoffactors; ageremains the phenomenon, rather thanaresult ofuncontrolled viral replication [Lescure, Dis2020]. Estimates Lancet Infect ofoverall this delayed onsetdecompensation isnotwell data suggestthat defined, itisanimmunologically-driven preliminary syndrome JAMA [Wang, 2020;Xiong, Lancet 2020;Mehta, Emerg 2020].Although thepathogenesis Infect of Microbes second 2020]. week NEnglJMed onsetofsymptoms after [Berlin, release This syndrome ofcytokine hascharacteristics of COVID-19, there canbesuddenonsetofARDSandrespiratorydecompensation that tends to occur the during medRxiv 2020; Wei, 2020;Foy, JInfect mild-to-moderate cases medRxiv2020;Cummings, otherwise medRxiv2020].In Petrilli, medRxiv2020;Zhang, Nature medRxiv2020; Chen,JClinInvest 2020;Kim, 2020;Liu Y, 2020;Ebinger, JInfect ratio,neutrophil-to-lymphocyte and elevated red bloodcell width[Zhou, distribution Lancet 2020; Wu, JAMA 2020; (i.e., C-reactive markers protein,levelsD-dimer, interleukin-6, ofinflammatory andprocalcitonin),ferritin elevated obesity, disease, chronic andimmunosuppression;parameters, kidney andlaboratory includinghypoxemia, elevated disease, chronic lungdisease,conditions, failure, diabetes, congestive suchashypertension, artery coronary heart for factors severeRisk diseaseandpooroutcomes includedemographic factors, includingageandmalesex; comorbid occurs inapproximately JAMA 5%ofcases[Wang, 2020;Huang, Lancet 2020;Chen,Lancet 2020]. develops inapproximately 14%ofcases. disease, Critical dysfunction, withrespiratoryfailure, ormulti-organ shock, weeks. More severe disease, withdyspnea, hypoxemia, involvement orextensive pulmonary demonstrated by imaging, infection withfever,respiratory tract cough andsore throat. dry Uncomplicated disease resolves withoutsequelaein1-2 2020; Lauer, mostsymptomatic 2020].In cases(>80%), thediseasepresents Med Ann Intern asanon-specificupper 4-5 days, and97.5%develop symptoms by 12days 2020;Chan,Lancet 2020;Guan,NEnglJMed [Li,NEnglJMed thosewhodevelopIn symptomatic diseasefollowing infection with SARS-CoV-2, themedianincubationis period presentations inCOVID-19. of SARS-CoV-2 isquite broad 2020],whichlikelyexplains Nat someoftheheterogeneous Med [Sungnak, clinical J Virol 2010;Hoffman, asyndrome,Cell 2020]. Although itcausesprimarily respiratory the cellular andtissuetropism of theviral andcell membranes facilitated by theS2component oftheviral spikeprotein [Li,Nature 2003;Matsuyama, (RBD) oftheS1region (ACE-2); oftheviral spikeprotein andfinallyfusion withhumanangiotensin converting enzyme-2 into humancells following event apriming by ahostprotease, TMPRSS2; interaction ofthereceptor bindingdomain cells [Menachery, 2015].SARS-CoV, Nat Med SARS-CoV-2, and(theoretically) virusessuchasBatCoV RaTG13, gainentry the potential oftheviral spikeprotein to oftheability infect humans, to by interact withareceptor virtue onhuman 2020].Bat coronavirusesEngl JMed circulate widely, etal. in2015ononesuchisolate reported that had andMenachery SARS-CoV-2 iscloselyrelated to SARS-CoV, andeven more closelyrelated to abat coronavirus, BatCoV RaTG13 [Zhu, N Natural History of COVID-19 andpossiblypangolins(SARS-CoV-2). camels(MERS-CoV), humans includecivets (SARS-CoV), speciesreservoir.with bats astheprimary speciesfrom whichinfections haveThe likelysecondary “spilled over” into All 3highlypathogenic humancoronaviruses, SARS-CoV, MERS-CoV, andSARS-CoV-2 emerged aszoonotic infections, antibody response isrobust [Payne, Dis2016]. Emerg Infect poorprognosticvery sign [Ko, Dis2017].For Diagn Infect Microbiol patients whodoseroconvert, ofthe thedura 3 Dynamics of SARS-CoV-2 and the Adaptive Immune Response Oren Cohen, M.D., F.I.D.S.A.; Marcia Eisenberg, Ph.D.; Brian Caveney, M.D., J.D., M.P.H.; Paul Kirchgraber, M.D., M.B.A.; Dorothy Adcock, M.D.; Steve Anderson, Ph.D. healthy blooddonors(N =45),patients withnon-coronavirus respiratory infections(N=76), andpatients withendemic authors showed that ELISAassays for antibodies to SARS-CoV-2 S1,RBDand Nproteins yielded negative results from Dis2020].Using Emerg anin-houseELISA,the [Okba, Infect andnot at allwithMERS-CoV minimally onlywithSARS-CoV andSARSCoV-2,of SARS-CoV andto alesser extent, MERS-CoV. With theS1protein asatarget, thesera cross-reacted etal. showedOkba that sera from asmallnumberofCOVID-19 patients tested positive inan ELISAusingtheSprotein OC43, HCoV-HKU1, HCoV-229E, andHCoV-NL63). SARS-CoV-2 andotherantigens, includingthosefrom other humancoronaviruses (i.e., SARS-CoV, MERS-CoV, HCoV- well underthetechnical conditions oftheassay. islargely dependent Specificity todiscriminate ontheability between immunogenicproteinsprevalence dependsonselecting orpeptidesthat perform of infectionisonly50%.Sensitivity positive predictive value ofapositive test from anassay inapopulation witha5%true that has95% specificity immunoassay [ELISA], lateral flow assay,enzyme-linked orother)isbothsensitive and specific.Inthis regard, the to antibodiesWhen ensure to detecting SARS-CoV-2, that itisimportant theassay (whetheritisimmunofluorescence, more generally, are quite immunogenic. protein into mediates humancells viral viabindingto entry ACE-2, that theRBD, itisnotsurprising andtheSprotein membrane (M)protein, andthenucleocapsid(N)protein. thereceptor Because bindingdomain(RBD) oftheviral spike Antibody responses to SARS-CoV-2 infectionare ofviral directed atproteins, avariety includingthespike(S)protein, the Humoral Response Immune an indicator ofsevere diseaseandpoorprognosis [Fang, 2020;Hogan,medRxiv2020]. JInfect 2020; Fang, 2020; JInfect Tan, addition,thepresence medRxiv2020;Lui, 2020].In ofviral JInfect nucleicacidinserumis disease [Lescure, Dis2020;Liu, Lancet Dis2020;Huang, Lancet Infect Infect Care Crit Am JRespir 2020;Zheng, Med BMJ levels and/ordelayedHigher ofSARS-CoV-2 RNA intherespiratory tract viral clearance are associated withmore severe who tested positive were asymptomatic [Sutton,2020]. NEnglJMed CoV-2. the 211 women withoutsymptoms, Of 29(13.7%)tested positive for SARS-CoV-2; thus, 29/33 (88%)ofwomen admitted for delivery. Four (1.9%)women hadsymptoms suggestive ofCOVID-19 and all 4tested positive for SARS- SARS-CoV-2 for allwomen admitted to 2hospitals’ wards. laboranddelivery a2-week Over period, 215women were peak oftheCOVID-19 outbreak inNew York inMarch 2020,Sutton -April City etal. adopted universal PCRtesting for The ratio ofsymptomatic to asymptomatic infectionswithSARS-CoV-2 remains at largely thistime. unknown Nearthe subsequently become symptomatic Dis2020;Xiao, [Zhou, JInfect Int medRxiv 2020]. individuals have lower durations levels ofviral ofviral sheddingcompared nucleicacidandshorter withpatients who to theonsetofsymptoms, andpeaked0.7days to prior theonsetofsymptoms [He, 2020].Asymptomatic Nat Med individuals.infection by pre-symptomatic The authorsinferred thatofinfectiousnessbegan2-3days theperiod prior 2020; He, 2020].Heetal. 2020;Li,Nat Nat casesofCOVID-19 Med Med estimated resulted that 44%ofsecondary from transmission from Rep suchindividualshasbeendocumented Mortal Wkly [Rothe, 2020;Kimball, Morb NEnglJMed individualsinfected withSARS-CoV-2pre-symptomatic have levels and detectable ofvirusintheirrespiratorytract, contrast2020]. In to thesituation inSARS, wherein theappearance ofsymptoms precedes thepeakofinfectiousness, [Tan,serum, andurine medRxiv2020;Hogan,Zheng, BMJ2020;Fang, 2020;HuangJ(1),medRxiv JInfect Rothe, 2020; NEnglJMed Williams, 2020].Less JClinMicrobiol frequently, viral instool, nucleicacidcanbedetected sputum from thelower [Zou, respiratory tract 2020;Pan, NEnglJMed Dis2020; Lancet Infect Yu, Dis2020; ClinInfect saliva, aswell stagesofdisease, theearly as viralIn andnasopharynx, nucleicacidisreadily intheoro- detectable a signal above background –thegreater theviral load, thefewer neededfor PCRcycles thesampleto become positive. employed methodfor involves semi-quantitation recording numberofthePCRat whichthesampleproduced thecycle isalsodifficult tonormalize thePCRsignal It totheamounttract). onthe ofthesampleswab. collected A commonly viral replication to compared support oftheepitheliuminupperairway withthelower ability respiratory spartan asadequate surrogatesserve for anumberofreasons (e.g., operator inswab technique variability andtherelatively difficultcritically to samplethe tissue(i.e., and affected lung),andsamplingthenot naresthroat, may nasopharynx who are infected withSARS-CoV-2. to Anumberofcaveats PCRtesting inpatients withCOVID-19. pertain Firstly, itis PCR testing for thepresence ofviral nucleic acidisthemostreliable methodfor andpractical identifying individuals Ac 4 Dynamics of SARS-CoV-2 and the Adaptive Immune Response Oren Cohen, M.D., F.I.D.S.A.; Marcia Eisenberg, Ph.D.; Brian Caveney, M.D., J.D., M.P.H.; Paul Kirchgraber, M.D., M.B.A.; Dorothy Adcock, M.D.; Steve Anderson, Ph.D. neutralizing antibody titer andage, ofthetiter was andstability demonstrated inasubsetof47patients whohadpaired that 165/175 (94%)haddeveloped SARS-CoV-2 neutralizing antibodies. There was apositive correlation between and second weeks onsetofillness. after from Samples all175patients at the timeofhospitaldischarge demonstrated and S2proteins aswell asthereceptor bindingdomain. These antibody responses mostlypeakedbetween thefirst contemporaneous appearance ofSARS-CoV-2 neutralizing antibodies withantibodies directed againsttheviral S1 F,course [Wu analysis serial 6ofthesesubjects, plasmasamples was possible, medRxiv2020].In demonstrating related to theemergence ofneutralizing antibodies in175COVID-19 patients whohadallrecovered from amild disease may beassociated withmore milddisease[Dahlke, medRxiv2020]. Wu etal. anumberofinteresting reported findings antibodies [Liu, 2020], andthere JClinMicrobiol isasuggestionfrom asmalldataset that anearly, robust IgA response onestudy,medRxiv 2020].In levels ofantiviral IgAcorrelated somewhat better thanIgGwithlevels ofneutralizing inantiviralthe rise antibody Dis2020; titers Emerg [Okba, Infect To, Dis2020; Lancet Infect Wolfel, Nature 2020;Dong, Neutralizing antibodies, whichare ableto prevent the virusfrom infecting susceptible cells, emerge concurrently with Nat2020]. Res bioRxiv2020;Nielsen, bioRxiv2020;Galson, individuals [Robbiani, responses were identical aminoacidsequences were seeninwhichantibodies withvirtually isolated from multiple were shown to bepredominantly IgG1andIgA, were mostlyspecific Interestingly,for RBDandS. convergent specific B cells emerge, withskewing toward utilization ofseveral chain heavy variable regions. Virus-specific antibodies Several investigators have probed theBcell receptor repertoire from patients withCOVID-19. Clonallyexpanded virus- infection withtheendemic coronaviruses. develop durable immunity. thissense, In suchcasesmay resemble thetransient immuneresponses associated with thatpeaklevelsraises early thepossibility protect these patients from developing severe disease, butthat theydonot [Li, medRxiv2020]. The steep declineinSARS-CoV-2-specific antibodies inpatients withmild-to-moderate disease a delayed intheseantibody rise levels, whichthenremain significantly higher compared withthelesssevere patients peakintheseantibodyearly levels followed by diseasehave asteep decline. contrast, In patients withsevere-to-critical cases. For IgG, patients withmild-to-moderate andRBD-specific disease severe-to-critical bothS-specific have ahigh ofthehumoral immuneresponseThe are kinetics quite different inmild-to-moderate casesof compared COVID-19 with Dis2020]. Infect ofantibodies todetection theviruscompared withthenucleocapsid(N)protein [Liu, 2020; JClinMicrobiol To, Lancet 2020;Zeng,J Infect 2020;Li,medRxiv2020].Using theviral JInfect Sprotein, oritscomponent RBDismore sensitive for IgG titers andplateau rise 3-8weeks, after andIgMtiters begin to week declineafter 3-5[Liu, 2020;Xiao, JClinMicrobiol symptoms, 100%seroconversion withnearly by week 3[seetablebelow]. of immunoassays, demonstrate that themediantimeto seroconversion isapproximately oneweek onsetof after is contemporaneous withIgGandIgAresponses rather thanpreceding them. The largest data sets, usingavariety ofthehumoral immuneresponseThe to kinetics SARS-CoV-2 are atypical, inthatpeaktheIgMresponse theearly among thesera from patients Dis2020]. withnon-coronavirus Emerg respiratoryinfections [Okba, Infect sera, whereas theIgAassay alsohad1(2.2%)falsepositive amongthehealthy blooddonorsand3(3.9%)falsepositives antibodies, and5/75(6.7%)for IgAantibodies. onlyshowedThe IgGassayfalsepositive otherwise results withSARS 4/75 (5.3%)ofthesera from theendemiccoronavirus panel(OC43, HKU1,229E, andNL63)hadapositive result for IgG inall3assays.cross reactivity Using acommercially available ELISAfor ofantibodies to detection SARS-CoV-2 S1protein, human coronavirus (OC43, HKU1,229E, andNL63)infections(N=75).Stored sera from 2patients withSARSdidshow symptom onset Time after 11-15 days 11-13 days 11-14 days 8-10 days 6-10 days ≥14 days 5-7 days 2-4 days Seroconversion 100% 89% 90% 50% 75% 50% 60% 30% rate To, Dis2020; Lancet Infect Zhao, Dis2020 ClinInfect Liu, 2020 JClinMicrobiol Liu, 2020 JClinMicrobiol Long, medRxiv2020 Long, medRxiv2020 Long, medRxiv2020 Long, medRxiv2020 Xiao, 2020 JInfect Reference 5 Dynamics of SARS-CoV-2 and the Adaptive Immune Response Oren Cohen, M.D., F.I.D.S.A.; Marcia Eisenberg, Ph.D.; Brian Caveney, M.D., J.D., M.P.H.; Paul Kirchgraber, M.D., M.B.A.; Dorothy Adcock, M.D.; Steve Anderson, Ph.D. cells [Wolfel, Nature inwhichSARS-CoV-2 2020].Asimilar singlecasewas could reported notbecultured beyond day levels subgenomicviral RNA,noreplication-competent ofdetectable viruscould inculture bedetected with E6 VERO day swabs andafter day 9from low after detected 5from sputum. Although 2stool nasopharyngeal sampleshadvery swab inthecourse samplesandsputumtakenearly ofdisease,RNA was found butcould notbe innasopharyngeal were found by analyzingsamplesfor subgenomicSARS-CoV-2 RNA,whichindicates viral active replication. Subgenomic could day after not be detected 8 even E6cellVERO PCRofsputumwas co-cultures) whendirect positive. Similarresults COVID-19 patients from Germany, Wolfel etal. found that infectious, replication-competent virus(by RT-PCR analysis of Care Crit Respir 2020;Zheng, Med BMJ2020;Fang, 2020; JInfect Tan, medRxiv2020].Importantly, inasmallstudyof9 shedding inpatients withsevere disease[Lescure, Dis2020; Liu, Lancet Dis2020;Huang, Lancet Infect Infect Am J with milddisease. additionto thestudyby In Xu etal. noted above, otherstudieshave alsofound prolonged viral occur [Li,JMed Virol 2020].Interestingly, prolonged sheddinginthislatter studyoccurred predominantly inpatients have found that inapproximately 10%ofpatients, prolonged sheddingofviral nucleic acid(median53.5days) may with confirmed becamePCR-negativeCOVID-19 by day [Xu, 35 fromInfect Dis 2020],others theonsetofillness Clin ventilation Dis2020;Xiao, [Xu, ClinInfect Dis2020).Although Xu ClinInfect etalfound that essentially all113patients shedding ofviral RNAincludeolderage, malesex, comorbidities, delayed hospitaladmission,andinvasive mechanical individual andanegative PCRtest convalescence during for was factors 26days prolonged 2020].Risk [Du, JInfect this range, etal. Du casesofCOVID-19, found withaninfected themediantimebetween contact that insecondary Xu, Dis2020;Chen X,medRxiv2020;HuangJ(1),Xiao, ClinInfect Dis2020].Consistent ClinInfect with orthroat swabs,(nasopharyngeal orsputum)range from 9.5-24days [Ling, J2020;Lo, ChinMed 2020; JBiolSci Int (seeFigureto detect 1).Estimates ofthemediantimefrom theonsetofillnessto having anegative viral PCRtest As antibody weeks titers during theonsetofsymptoms, rise 2-3after levels ofvirusdecrease andbecome more difficult Viral Clearance: Convalescence andBeyond (CD45RA- CD62L+) memory T cells medRxiv2020]. [Odak, correlation between convalescence andreappearance CD62L-) (CD45RA- ofeffector/effector memory and central ofthecellular immuneresponseevidence inCOVID-19 oftheimportance ofastrong comes from theobservation (TH17-like) ofS-specific frequency CCR6+CXCR3- T [Juno,cells andneutralizing antibody titers medRxiv2020]. Further IgGtitersT cells andtiters ofRBD-specific [Grifoni, Cell 2020].Junoetal. found anegative correlation between the responses have alsobeenmapped. Grifoni etal. found astrong correlation CD4+ ofS-specific between thefrequency components [Grifoni, Cell 2020].Relationshipsbetween cellular 2020;Juno, andhumoral Immunity medRxiv2020;Ni, andRBD-specificS- T cell responses predominate; however, responses are againstmultipleother viral alsodetectable All COVID-19 patients have virus-specificCD4+ detectable T cell responses, andmosthave CD8+ T cell responses as well. Cellular Response Immune ADE [Quinlan, bioRxiv2020]. immunization withtheSARS-CoV-2 RBDinananimalmodelleadsto apotent neutralizing antibody response without well J [Jaume, Virol 2011; Wang, data ACS come Dis2016].Reassuring from etalwhohave Infect Quinlan shown that 1977;Dejnirattisai, Science 2010]. J ExpMed This phenomenonwas at leasttheoretically as possiblewithSARS-CoV their Fc receptor. This “Trojan horse” phenomenonleadsto infectionofmacrophages, hence theterm ADE[Halstead, virus, thecaseofDengue ADE. somevirus-specificantibodies In deliver boundvirusinto of macrophages by virtue One oftheconcerns aboutvaccine candidates enhancement, isthetheoretical ofantibody-dependent or possibility inhibition ofviral entry, andalsosuggestmultiplecandidate vaccine targets. 2020; Brouwer, bioRxiv2020]. These data indicate ofcombining monoclonalantibodies thepossibility for synergistic compete withACE-2, microscopy electron [Wu shows that different epitopes antibodies bind to distinct Y, medRxiv bioRxiv 2020; Wu Y, medRxiv2020;Brouwer, bioRxiv2020].Even neutralizing antibodies that amongstRBD-specific ACE-2, andothersdonotcompete Cell withACE-2 2020; HostMicrobe [Wu, Wan, bioRxiv2020;Cao, Cell 2020;Seydoux, neutralizing antibodies,Among RBD-specific some compete with ACE-2 competefor binding, with somepartially neutralizing antibodiesMost isolated from COVID-19 patients bindto RBD, althoughsomebindto Sandnotto RBD. medRxiv 2020]. antibody 2020;Juno,neutralizing titers antibodyImmunity titers andRBD-specific andS- Ni, [Amana 6 Dynamics of SARS-CoV-2 and the Adaptive Immune Response Oren Cohen, M.D., F.I.D.S.A.; Marcia Eisenberg, Ph.D.; Brian Caveney, M.D., J.D., M.P.H.; Paul Kirchgraber, M.D., M.B.A.; Dorothy Adcock, M.D.; Steve Anderson, Ph.D. endemic coronaviruses. may have inadequate orinsufficiently inamanner that durable issomewhat analogous immunity toinfectionwiththe (as noted above) againraises the question ofwhetherasubsetpeoplewhohave beeninfected withSARS-CoV-2 potential infectiousnessofsuchpatients. Arecurrent positive test associated withlow levels ofvirus-specificantibodies intermittently at around thelimitof detectability. These different scenarios have different implications with regardto possible that there isnoongoing viral replication, andthat nucleicacidfrom neutralized virusisshed slowly or membranes, microangiopathy infiltrates andinflammatory consistent with COVID-19 [Yao, Itisalso Res2020]. Cell inbronchiolar andalveolar epithelialcells.particles Histopathological examination showed alveolar damage, hyaline PCR was positive for SARS-CoV-2 nucleicacidinlungtissue, microscopy andelectron demonstrated coronavirus tests for SARS-CoV-2. Ontheday shewas to bedischarged, sheexperienced sudden cardiac arrest anddied. At autopsy, from COVID-19 andwas awaiting discharge from 3consecutive thehospitalafter swab PCR negative nasopharyngeal thispossibility.results. from Acasereported China supports The caseinvolved a78-year-old woman whorecovered isaroundin theupperrespiratory tract thelimitofdetectability. This could give to rise intermittently positive PCRtest low levels (i.e., ofvirusreplication thelower compartments andthat are viral tract), respiratory load incertain occurring recurrent. thenegative If tests preceding the “recurrent” positive test are truenegatives, itisstillpossible that ongoing scenario, low levels ofviral replication may becontinuous for aprolonged oftime, period butmay falselyappearto be that thenegative inthis tests were falselynegative test sensitivity: actually dueto inadequate samplingorimperfect Recurrence ofpositive PCRtests following convalescence hasseveral possibleexplanations. somecases, In itispossible 2020]. recurrent positive PCRtests were associated withlow levels ofvirus-specificantibodies [Li,medRxiv2020;Hu, medRxiv orworseningbiomarkers ofchestimaging studies. authorshave Some asmallnumberofcasesinwhich reported are afew casesinwhichmore significant symptoms have occurred, orthat involve modestincreases ininflammatory patients appearwell orhave minornon-specificsymptoms parameters. alongwithbenign laboratory However, there variously beenreferred to asreinfection, recurrence, reactivation andrecrudescence. Among thecasedescriptions, most Dis2020;Cao, JInfect 2020; Chen,Int JMed Virol 2020;ChenJ, medRxiv2020]. This phenomenonisnotrare, andithas convalescence [Tang, Control Infect HospEpidemiol2020;Jiang, 2020;HuangJ(2),medRxiv JInfect Ye, JInfect Finally, there isaphenomenonofPCRtests becoming positive againfollowing at least2negative tests during necessarily indicative viral ofactive replication andinfectivity. be replicated onalarger scale, provide someevidence that prolonged for positivity viral by RNAdetected RT-PCR isnot theonsetofsymptoms18 after despite persistently positive PCR tests [Liu 2020]. WD JInfect These data, whichmust Exposure to virus Quantity SARS-CoV-2 RNA Symptom Onset Infectious Figure ofviral replication 1.Kinetics andthehumoral immuneresponse. 1 Infectious Probably

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