key: cord-0967851-izzwxcq2 authors: Chen, X.; Pan, Z.; Yue, S.; Yu, F.; Zhang, J.; Yang, Y.; Li, R.; Liu, B.; Yang, X.; Gao, L.; Li, Z.; Lin, Y.; Huang, Q.; Xu, L.; Tang, J.; Hu, L.; Zhao, J.; Liu, P.; Zhang, G.; Chen, Y.; Deng, K.; Ye, L. title: Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19 date: 2020-07-30 journal: nan DOI: 10.1101/2020.07.29.20164285 sha: b39ddac4c08f92e35e50dc56da438b3159c84458 doc_id: 967851 cord_uid: izzwxcq2 COVID-19 patients exhibit differential disease severity after SARS-CoV-2 infection. It is currently unknown as to the correlation between the magnitude of neutralizing antibody (NAb) responses and the disease severity in COVID-19 patients. In a cohort of 59 recovered patients with disease severity including severe, moderate, mild and asymptomatic, we observed the positive correlation between serum neutralizing capacity and disease severity, in particular, the highest NAb capacity in sera from the patients with severe disease, while a lack of ability of asymptomatic patients to mount competent NAbs. Furthermore, the compositions of NAb subtypes were also different between recovered patients with severe symptoms and with mild-to-moderate symptoms. These results reveal the tremendous heterogeneity of SARS-CoV-2-specific NAb responses and their correlations to disease severity, highlighting the needs of future vaccination in COVID-19 patients recovered from asymptomatic or mild illness. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 Introduction& As(of(July (28,(2020 ,(the(pandemic(of(coronavirus(disease(2019 ( infection, ( has( claimed( 16, 341, 920( clinically( confirmed( cases( and( 650, 805( deaths( worldwide 1 .( The( infected(patients(show(heterogeneous(clinical(manifestations, (which(can(be(generally( classified( into( four( groups, ( including( severe, ( moderate, ( mild( and( asymptomatic, ( according(to(the(severity(of(symptoms 2 .(Despite(daily(increasing(confirmed(cases(and( death, (currently(no(medical(agents(are(approved(to(prevent(SARS_CoV_2(infection(or( treat(COVID_19(patients.( ( A(growing(body(of(evidence(shows( that(recovered(COVID_19(patients(can(generate( IgG_type(antibodies(specifically(binding(to(various(structure(proteins(of(SARS_CoV_2( particles( shortly( after( the( onset( of( disease, ( albeit( at( variable( levels 3_6 .(Among( these( virus(specific(antibodies, (only(those(capable(of(blocking(SARS_CoV_2(spike((S) (protein( mediated( viral( attachment( and/or( entry( of( host( cells,( called( neutralizing( antibodies( (NAbs) , (can(effectively(curtail(infection 7 .(The(convalescent(plasma(or(sera(containing( NAbs( harvested( from( recovered( patients( have( shown( promising( results( in( treating( COVID_19(patients(of(critical(illness(in(several(small_scale(clinic(trials 8_11 .(In(addition, (a( variety( of( human( monoclonal( antibodies( (mAbs)( of( potent( SARS_CoV_2( neutralizing( activities(has(been(cloned(from(memory(B(cells(from(recovered(COVID_19(patients 12_ 21 , (holding(great(potentials(for(prophylactic(or(therapeutic(use.(However, (little(is(known( regarding(the(relationship(between(disease(severity(and(the(magnitude(of(SARS_CoV_ 2_specific( NAbs( responses( in( patients( recovered( from( COVID_19.( Defining( the( association(of(disease(severity(to(NAb(responses(will(facilitate(the(screening(of(COVID_ 19( recovered( patients( as( therapeutic( plasma( donors( as( well( as( memory( B( cell( providers(for(cloning(high_affinity(human(neutralizing(mAbs(to(prevent(or(treat(COVID_ 19.( ( ( The( circulation( of( high_titer( NAbs( provides( the( immediate( protection( against( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 To(explore(the(potential(association(between(SARS_CoV_2(S(protein_specific(antibody( responses(and(the(disease(severity(in(recovered(COVID_19(patients, (we(included(a( cohort( of( 59( adult( patients, ( 48( of( mild( (n=4), ( moderate( (n=34)( and( severe( (n=10)( symptoms( admitted( to( Guangzhou( Eighth( People's( Hospital( and( 11( asymptomatic( adult(patients(admitted(to(Chongqing(Public(Health(Center(Hospital.(The(median(age( of( these( patients( was( 47( (33_62, ( interquartile( range( (IQR) ) ( years( oldm( 50.8%( of( the( patients( were( femalem( Serum( samples( were( collected( at( the( day( of( discharge( after( symptom(resolution(and(SARS_CoV_2(nucleotide(testing(negative(twice(by(RT_PCR.( The(median(time(between(the(onset(of(symptom(to(sample(collection(was(20((12_30, ( IQR)(days.(The(asymptomatic(patients(were(identified(by(screening(those(with(close_ contact( history( to( COVID_19( patients( and( confirmed( by( SARS_CoV_2( RT_PCR.( The( disease( severity( was( stratified( into( asymptomatic, ( mild, ( moderate( and( severe, ( according(to(the(national(diagnosis(and(treatment(guideline(of(COVID_19((7 th ( (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 domain((RBD)(within(S1 (subunit(is(essential(for(virus(attachment(to(host(cell(receptor, ( human(angiotensin_converting(enzyme(2((ACE2), (while(S2(is(critical(for(virus(entry(by( mediating( viral( membrane( fusion( to( host( cell( membrane 26_28 .( Both( S1_RBD( and( S2( represent(important(potential(targets(of(NAbs 7 .(We(first(compared(the(antibodies(that( specifically(binding(to(S1, (RBD(and(S2(of(SARS_CoV_2(in(sera(of(COVID_19(recovered( patients( with( different( illness( severity( by( IgG( ELISA.( Notably, ( severe( and( moderate( symptomatic( patients( mounted( the( most( and( second( robust( S1, ( S1_RBD( and( S2( specific( antibodies, ( respectively, ( while( mild( and( asymptomatic( patients( exhibited( significantly(lower(abundances(of(S1_, (S1_RBD_(and(S2_specific(antibodies((Fig.1a_c), ( highlighting(the(disease(severity(as(a(key(determinant(factor(of(the(levels(of(antibodies( specific(to(SARS_CoV_2(S(proteins.( ( ( Next, (we(assessed(the(abilities(of(antibodies(in(these(COVID_19(recovered(patients(to( block(the(interaction(between(RBD(and(ACE2.(Similar(to(SARS_CoV_2(RBD(binding( antibodies, ( the( sera( from( recovered( patients( with( severe( symptoms( displayed( the( highest( scores( of( blocking( RBD_ACE2( engagement, ( followed( by( patients( with( moderate(symptoms, (while(antibodies(from(mild(and(asymptomatic(patients(showed( much(inferior(capacity(to(inhibit(RBD_ACE2(interaction((Fig.(2a, (b) .( ( ( Subsequently, ( we( conducted( SARS_CoV_2( S_protein( pseudotyped_lentiviral( based( neutralization( assay( to( examine( the( neutralization( capacity( of( sera( from( COVID_19( recovered(patients.(Such(assay(has(been(proven(to(be(free(of(biosafety(issue(but(as( reliable(as(the(canonical(plaque(assay(with(authentic(SARS_CoV_2 17, 19, 21 . (We(observed( that(the(sera(neutralization(capacity(was(positively(correlated(to(disease(severity((Fig.( 2c) . ( Specifically, ( 80%( of( patients( with( severe( symptoms( and( 47.1%( of( patients( with( moderate( symptoms( generated( antibodies( capable( of( completely( neutralizing( pseudovirus( infection, ( while( only( 25%( of( serum( samples( from( patients( with( mild( symptoms(were(able(to(block(pseudovirus(infectionm(strikingly, (sera(from(asymptomatic( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. . https://doi.org/10. 1101 /2020 .( Next, ( we( performed( neutralization( assay( with( the( authentic( SARS_CoV_2( to( explore( the(neutralization(capacity(of(sera(from(COVID_19(recovered(patients.(In(accordance( with( the( pseudotyped_lentiviral( based( neutralization( assay,( we( found( that( the( sera( neutralization(capacity(of(severe(patients(was(the(highest(and(was(61.1_fold,(1319 .1_ fold(and(2972 .( ( Furthermore,(the(binding(abilities(of(S1, (RBD(and(S2(were(positively(correlated(to(each( other( (Supplementary( Fig.( 1a_c)( and( also( positively( correlated( to( both( pseudovirus( neutralizing( capacities( (Supplementary( Fig.( 1d_f)( and( authentic( SARS_CoV_2( virus( neutralizing(capacities((Supplementary(Fig.(1g_i Given(that(NAbs(can(potentially(target(both(S1(and(S2(to(block(viral(infection 7 , (we(set( out(to(distinguish(S1(and(S2_specific(NAbs(in(COVID_19(patients(with(aforementioned( pseudovirus( neutralization( assay.( To( this( end, ( we( used( biotin_labeled( S1( or( S2( recombinant( protein( to( deplete( corresponding( antibodies( in( sera( from( 25( COVID_19( patients( that( were( confirmed( to( be( highly( neutralizing( in( Fig.( 2c( and( Fig.( 2e( (Supplementary(Fig.(2a, (b) .(After(depletion, (we(found(that(across(all(neutralizing(sera, ( 40%(of(patients(generated(both(competent(S1_(and(S2_NAbs((i.e., (post(either(S1_(or( S2_specific( antibody( depletion, ( sera( can( still( completely( neutralize( pseudovirusesm( labeled(as("S1/S2_NAbs")m(while(40%(of(patients(only(generated(S1_competent(NAbs( ("S1_NAbs( only"),( and( 4%( of( patients( only( generated( S2_specific( NAbs( ("S2_NAbs( only")m(interestingly,(16%(of(serum(samples(strictly(depended(on(the(collaboration(of( S1_ (and(S2_specific(NAbs(to(effectively(neutralize(pseudovirus(infection((i.e .,(either(S1_( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. . https://doi.org/10. 1101 /2020 or(S2_specific(antibody(depletion(in(the(serum(can(result(in(the(failure(of(neutralizationm( labeled(as("(S1+S2)_NAbs") ((Fig.(3a, (b) . (Among(NAbs(in(severe(symptomatic(patients,( the( majority( of( sera( (62.5%)( potently ( neutralized( both( S1_mediated( receptor( attachment( and( S2_mediated( membrane( fusion, ( while( 37.5%( only( blocked( S1_ mediated(receptor(engagement((Fig.(3c) . (For(mild(to(moderate(symptomatic(patients, ( NAb( features( were( more( diverse:( 41.2%( of( them( consisted( of( only( S1_neutralizing( NAbs, ( 29.4%( possessed( the( abilities( to( block( both( receptor( engagement( and( membrane(fusion.(Notably, (23.5%(of(these(sera(required(the(combination(of(S1_(and( S2_specific(NAbs(to(effectively(neutralize(pseudovirus(infection((Fig.(3c) . (Collectively, ( our(data(revealed(the(highly(heterogeneous(nature(of(NAb(responses(against(SARS_ CoV_2( S( protein( and( such( diversity( seemed( to( be( closely( associated( with( disease( severity.( The( immune( mechanisms( underlying( the( diversity( of( NAbs( responses( in( COVID_19(patients(with(different(degree(of(symptoms(warrant(further(investigations.( ( ( ( ( Finally, (we(investigated(whether(NAbs(depleted(by(S1_recombinant(protein(are(actually( targeting( RBD( for( their( neutralizing( capacity.( For( this( purpose, ( we( depleted( RBD_ specific(antibodies(in(10(serum(samples(showing(S1_specific(neutralization(by(biotin_ conjugated(RBD(protein(mediated(pull_down((Supplementary(Fig.(2c) . (Antibodies(post( RBD_depletion(were(shown(to(lose(RBD_binding(ability, (but(still(keep(their(binding(to( both(S1(and(S2(proteins, (suggesting(the(efficiency(and(specificity(of(RBD(Ab(depletion( (Supplementary( Fig.( 2c_e) . ( Notably, ( all( sera( with( S1_specific( neutralization( failed( to( neutralize( pseudovirus( infection( after( RBD_specific( NAb( depletion( (Fig.( 3d, ( e), ( demonstrating( the( strict( dependency( of( RBD_specific( NAbs( to( disengage( viral( attachment(to(the(host(receptor.(These(data(provided(the(rationale(for(exclusively(using( RBD(as(S1_immunogen(in(vaccine(design, (in(particular, (given(that(several(reports(have( shown(the(enhanced(disease(after(whole(S1(immunization 29, 30 .( ( ( The( COVID_19( patients( show( stratified( symptoms,( including( asymptomatic,( mild,( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. . https://doi.org/10.1101/2020.07.29.20164285 doi: medRxiv preprint moderate( and( severe 2 . ( Using( RBD_ACE2( blockade, ( pseudovirus( neutralization( and( authentic(virus(neutralization, (we(observed(that(disease(severity(positively(correlates( to( NAb( responses.( The( patients( recovered( from( severe( illness( mounted( the( most( robust( NAb( responses.( Strikingly, ( asymptomatic( patients( fail( to( generate( competent( NAbs.( The( mechanisms( underlying( disease( severity( associated( NAb( responses( are( elusive.(One(possible(explanation(is(that(the(induction(of(SARS_CoV_2_specific(NAb( responses(requires(the(strengthened(and(prolonged(B_cell(receptor((BCR)(stimulation.( Indeed, ( enhanced( BCR( rearrangement( was( observed( in( COVID_19( patients( with( severe( disease( symptom 31 .( This( may( provide( important( insights( into( the( COVID_19( vaccine(design, (in(which(the(vaccine(regimens(should(release(enough(SARS_CoV_2( immunogens(in(an(extended(period.( ( ( Given( the( critical( role( of( NAbs( in( protecting( viral( infection( in( airways, ( the( recovered( asymptomatic(patients(may(suffer(from(SARS_CoV_2(re_infection.(In(this(circumstance, ( these(patients(need(to(be(vaccinated(whenever(the(effective(vaccines(are(available.( Thus( far, ( it( is( unknown( as( to( the( protective( immunity( that( prevents( asymptomatic( patients(from(progressing(to(more(severe(disease.(Probably, (these(patients(can(mount( robust(SARS_CoV_2_specific(CD8 + (T(cell(responses, (which(may(confer(the(protection( by(directly(clearing(virus_infected(target(cells.(However, (this(hypothesis(needs(to(be( confirmed(in(future(investigations.( ( ( Our( results( also( demonstrated( the( tremendous( heterogeneous( NAb( responses( in( patients( capable( of( inducing( high_titer( NAbs. ( The( majority( (80.7%)( of( patients( can( produce( S1_specfic ( NAbs, ( and( half( patients( are( able( to( generate( S2_specific( NAbs.( However, (only(around(40%(of(patients(generated(both(S1_(and(S2_specific(competent( NAbs.( Particularly, ( approximate( 7%( patients( had( to( depend( on( the( collaboration( between( S1_( and( S2_specific( antibodies( for( efficient( viral( neutralization.( The( mechanisms( underlying( the( heterogeneous( NAb( responses( in( recovered( patients( remains( unknown( and( warrant( further( studies. ( Notably,( all( S1_specific( NAbs( were( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. . https://doi.org/10.1101/2020.07.29.20164285 doi: medRxiv preprint strictly ( RBD( dependent( and( deletion( of( RBD_specific( antibodies( led( to( the( failure( in( neutralization(in(S1_specific(sera.(These(results(highlighted(the(importance(of(S1_RBD( itself, (but(not(other(parts(of(S1(protein, (in(inducing(competent(NAbs.( ( ( In(conclusion, (we(have(demonstrated(the(positive(correlation(between(the(magnitude( of(NAb(responses(and(disease(severity(in(patients(recovered(from(COVID_19.(We(have( also( found( that( disease( severity( also( influences( the( neutralization( heterogeneity( of( SARS_CoV2_specific(antibodies.(Our(results(highlight(the(needs(to(include(mild_illness( and(asymptomatic(patients(for(future(vaccination, (and(also(suggest(the(collection(of( plasma(from(COVID_19(recovered(patients(should(be(restricted(to(those(with(moderate( to( severe( symptoms( for( passive( antibody( therapy.( Our( data( also( provide( important( rationale( for( exclusively( using( SARS_CoV_2( RBD( as( S1_immunogen( hour( with( 100( μl( blocking( buffer( (5%( FBS( and( 0.1%( Tween( 20( in( PBS) ( and( then( incubated(with(diluted(patient(or(healthy(control(sera(in(100(μl(blocking(buffer(for(1(hour.( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. ((Sino(Biological, (40591_V08H)(or(SARS_CoV_2(RBD(protein((Sino(Biological, ( 40592_V08B)(or(SARS_CoV_2(S2(protein(was(conjugated(with(biotin(by(following(the( manufacture's( protocol( (Thermo( Fisher( Scientific, ( A39257) . ( Then,( biotin_conjugated( proteins(were(incubated(with(BeaverBeads(Mag(Streptavidin(Matrix((Beaver,(22305)( at(4 o C(for(1.5(hours.(After(washing(with(PBS,(the(SARS_CoV_2(S(protein(coupled(beads( were( next( incubated( with( diluted( patient( sera( at( 4 o C( for( 1.5( hours.( Then,( the( supernatants(were(harvested(and(quality(controlled(by(ELISA(assays( belonging(to(patients(with(different(severity(were(compared(with(the(one_way(ANOVA( test( (Tukey's( multiple( comparisons( test) . ( The( cutoff( value( in( each( pseudovirus( neutralizing(function(assay(was(determined(by(the(ROC(curve(analysis(and(was( of(the( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 The( data ( sets( in( the( study( are( available( from( the( corresponding( authors( upon( reasonable(request.( & Acknowledgements& This(work(was(supported(by(grants(from(the(National(Science(and(Technology(Major( Project((No. 2017ZX10202102_006_002(to(L.Y.) , (the(National(Natural(Science(Fund(for( Distinguished( Young( Scholars( (No.( 31825011( to( L.Y.) ( and( the( Chongqing( Special( Research( Project( for( Novel( Coronavirus( Pneumonia( Prevention( and( Control( (No.( cstc2020jscx_2(to(L.Y.m(No.(cstc2020jscx_fyzx0074(to(Y.C.m(cstc2020jscx_fyzx0135( (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 30, 2020 . . https://doi.org/10.1101 /2020 patients'with'different'symptom'severity.! a5c,!ELISA!binding!assays!of!1004fold!diluted!COVID419!patient!sera!to!ELISA! plates!coating!of!SARS4CoV42!S1!(a),!RBD!(b)!and!S2!(c)!proteins.!The!dashed! lines!in!a4c!represent!the!average!values!of!healthy!control!groups.!*P! 50%) in 24-48 hours. The moderate patients were diagnosed with respiratory symptoms, fever and imageological evidence of pneumonia.The mild patients were diagnosed with mild clinical symptoms and no imageological evidence of pneumonia. The asymptomatic patients were those without clinical symptoms.All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 30, 2020. . https://doi.org/10. 1101 /2020