Site Of Vulnerability On SARS-CoV-2 Spike Induces Broadly Protective Antibody Against Antigenically Distinct Omicron Subvariants
Nov 30, 2023
Kev hloov pauv sai sai ntawm tus mob hnyav ua pa nyuaj ua rau tus mob coronavirus 2 (SARS-CoV-2) Omicron variants tau hais txog qhov yuav tsum tau txheeb xyuas cov tshuaj tiv thaiv uas muaj peev xwm ua kom tsis zoo los qhia rau yav tom ntej kev kho mob monoclonal thiab cov tswv yim txhaj tshuaj. Ntawm no, peb tau txheeb xyuas S728-1157, qhov dav dav neutralizing antibody (bnAb) lub hom phiaj ntawm qhov chaw receptor-binding site (RBS) uas tau muab los ntawm ib tus neeg yav dhau los kis WT SARS-CoV-2 ua ntej kev sib kis ntawm variants ntawm kev txhawj xeeb (VOCs). S728-1157 tau qhia dav dav hla-tsis nruab nrab ntawm txhua qhov sib txawv, suav nrog D614G, Beta, Delta, Kappa, Mu, thiab Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/ BL.1/XBB). Tsis tas li ntawd, S728-1157 tiv thaiv hamsters tiv thaiv hauv vivo cov kev sib tw nrog WT, Delta, thiab BA.1 cov kab mob. Cov qauv kev soj ntsuam pom tau tias cov tshuaj tiv thaiv kab mob no tsom mus rau chav kawm 1 / RBS-A epitope hauv receptor binding domain los ntawm ntau yam hydrophobic thiab polar kev sib cuam tshuam nrog nws cov saw hnyav ntxiv kev txiav txim siab hauv cheeb tsam 3 (CDR-H3), ntxiv rau cov motifs hauv CDR-H1 / CDR-H2 ntawm chav kawm 1 / RBS-A cov tshuaj tiv thaiv. Qhov tseem ceeb, qhov epitope no tau yooj yim nkag mus rau hauv lub xeev qhib thiab prefusion, lossis hauv hexaproline (6P)-stabilized spike constructs, piv nrog diproline (2P) constructs. Zuag qhia tag nrho, S728-1157 qhia txog kev kho mob dav dav thiab tuaj yeem qhia cov phiaj xwm tsim cov tshuaj tiv thaiv tiv thaiv yav tom ntej SARS-CoV-2 variants.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Taw qhia
Txij li thaum pib muaj kev sib kis thoob plaws lub Kaum Ob Hlis 2019, tus kab mob ua pa hnyav heev tus mob coronavirus 2 (SARS-CoV-2) tus kab mob tau ua rau ntau dua 660 lab tus neeg mob tus kabmob coronavirus 2019 (COVID-19) thiab tshaj 6.5. lab tuag thoob ntiaj teb. Txawm hais tias kev txhim kho sai thiab kev faib tawm ntawm cov tshuaj tiv thaiv thiab kev kho mob tau txwv cov txiaj ntsig ntawm COVID-19 mus rau qhov loj, qhov tshwm sim ntawm kev sib txawv ntawm kev txhawj xeeb (VOCs) txuas ntxiv sawv cev rau qhov kev hem thawj loj vim muaj peev xwm tiv thaiv kev tiv thaiv kab mob ntxiv. thiab txhim kho pathogenicity. D614G variant yog qhov hloov pauv ntxov tshaj plaws los tshwm sim thiab dhau los ua neeg thoob ntiaj teb tom qab ntawd. Piv nrog WT, qhov sib txawv ntawm D614G tau nthuav tawm ntau dua kev sib kis ntau dua li qhov ua rau muaj kab mob ntau ntxiv thiab yog li ntawd tsis zoo li yuav txo qis qhov ua tau zoo ntawm cov tshuaj tiv thaiv hauv kev sim tshuaj (1). Nruab nrab ntawm qhov tshwm sim ntawm D614G txog Lub Kaum Hli 2021, 4 qhov tseem ceeb VOCs tau hloov zuj zus mus thoob ntiaj teb, suav nrog Alpha, Beta, Gamma, thiab Delta. Ntawm cov kev hloov pauv no, Delta tau dhau los ua qhov kev hem thawj loj thoob ntiaj teb vim tias nws kis tau tus kab mob ntau ntxiv, thiab kev tiv thaiv kab mob ib nrab, raws li pom los ntawm kev txo qis ntawm polyclonal serum thiab mAbs los ua kom tsis txhob muaj kab mob no (2–6). Tsis ntev tom qab ntawd, thaum lub Kaum Ib Hlis 2021, Omicron variant tau txheeb xyuas thiab tshaj tawm tias yog VOC tshiab. Qhov kev hloov pauv no muaj qhov hloov pauv loj tshaj plaws rau hnub tim thiab tshwm sim kom kis tau sai dua li cov kab mob yav dhau los (7, 8). Tam sim no, muaj ntau yam ntawm Omicron sublineages ua rau COVID{21}} kis tshiab, nrog BQ.1, BQ.1.1, thiab XBB.1.5 dhau los ua tus tseem ceeb tshaj BA.5 thiab suav rau feem ntau cov mob tshiab thoob ntiaj teb thaum lub sijhawm ntawm kev sau ntawv. Cov tshuaj tiv thaiv Omicron tuaj yeem khiav dim kev lees paub los ntawm COVID-19 tshuaj tiv thaiv kab mob cuam tshuam rau ntau qhov sib txawv, yog li txo qis lub zog ntawm cov tshuaj tiv thaiv kab mob ntshav qab zib los ntawm convalescent, tag nrho cov tshuaj tiv thaiv mRNA thiab cov tib neeg txhawb nqa nrog WT / BA.5 bivalent tshiab. tshuaj tiv thaiv mRNA (9, 10). Ib yam li ntawd, Omicron variants muaj peev xwm dim ntawm kev khi ntawm ntau yam kev siv thaum muaj xwm txheej ceev (EUA) kho mAbs, txawm tias cov no tau pom yav dhau los ua tau zoo tiv thaiv VOCs ua ntej (10–12). Vim qhov txo qis nruab nrab ntawm Omicron thiab txuas ntxiv kev hem thawj ntawm VOCs yav tom ntej, muaj qhov xav tau ceev los txheeb xyuas qhov dav thiab muaj zog tiv thaiv kab mob uas tuaj yeem tiv thaiv ntau yam, hloov kho SARS-CoV{38}} kab mob. Hauv txoj kev tshawb no, peb tau txheeb xyuas qhov muaj zog receptor-binding domain-reactive (RBD-reactive) mAb los ntawm cov ntshav peripheral ntawm SARS-CoV-2-convalescent tus neeg uas ua tau zoo neutralized Alpha, Beta, Kappa, Delta, Mu, thiab Omicron variants (BA.1, BA.2, BA.2.75, BA.4, BA.5, BL.1 thiab XBB). Qhov no mAb, S728-1157, txo qis BA.1 Omicron, Delta, thiab WT cov kab mob hauv lub ntsws thiab qhov ntswg mucosa tom qab hauv vivo kev sib tw hauv hamsters. S728-1157 khi qhov chaw receptor binding site (RBS) uas tau nthuav tawm tag nrho thaum RBD ntawm qhov ntsia hlau loj yog nyob rau hauv kev sib haum xeeb. Lub mAb siv cov motifs pom nyob rau hauv CDR-H1 thiab CDR-H2 uas muaj ntau rau IGHV3-53/3-66 chav kawm 1/RBS-A cov tshuaj tiv thaiv (13, 14), tab sis kuj los ntawm kev sib cuag nrog CDR. -H3 kom hla kev hloov pauv hauv VOCs spikes. Qhov no qhia tias kev tsim qauv tsim nyog ntawm cov tshuaj tiv thaiv yav tom ntej txhawb nqa npog Omicron variants yuav tsum tau hloov kho kom nthuav tawm qhov kev ruaj khov nyob rau hauv feem ntau ntawm kev teeb tsa kom ua tau zoo txhawb cov chav kawm 1 / RBS-A mAbs uas muaj cov yam ntxwv zoo sib xws CDR-H3.
cistanche cov txiaj ntsig-ua kom muaj zog tiv thaiv kab mob
Cov txiaj ntsig
Kev rho tawm ntawm RBD-reactive mAbs uas nthuav tawm cov qauv sib txawv ntawm qhov nruab nrab thiab muaj zog. Ua ntej kev sib kis ntawm Omicron cov kab mob, peb yav dhau los ua cim 43 mAbs tsom cov epitopes txawv ntawm cov protein ntau, suav nrog N-terminal domain (NTD), RBD, thiab subunit 2 (S2). Tsis muaj ib qho ntawm cov tshuaj tiv thaiv no tuaj yeem ua rau tsis zoo rau SARS-CoV-2 variants circulating thaum lub sijhawm ntawd (15). Nyob rau hauv txoj kev tshawb no, ib tug ntxiv vaj huam sib luag ntawm RBD-reactive mAbs tau qhia los ntawm 3 high-responder cov neeg uas mounted robust anti-spike IgG cov lus teb, raws li tau hais yav dhau los (16) (Cov Lus Qhia Ntxiv 1 thiab 3; cov khoom siv ntxiv muaj nyob hauv online nrog rau tsab xov xwm no; https://doi.org/10.1172/JCI166844DS1). Txawm hais tias qhov feem pua ntawm cov kab mob RBD-binding B hlwb zoo sib xws hauv cov neeg teb siab piv nrog cov neeg teb nruab nrab thiab qis (Daim duab 1, A-C), cov saw hnyav ntawm somatic hypermutation tus nqi tau ntau dua nyob rau hauv pawg neeg teb siab (Daim duab 1. , D, thiab E), tawm tswv yim tias cov tib neeg no yuav muaj peev xwm ua tau zoo tshaj plaws los ua kom muaj zog hla-reactive mAbs (16). Cov tshuaj tiv thaiv no tau raug tshawb xyuas ntxiv tawm tsam RBD mutants txhawm rau txheeb xyuas lawv cov kev faib tawm epitope (17). Ntawm 14 RBD-reactive mAbs, peb tau txheeb xyuas 4 chav kawm 2 mAbs, 2 chav kawm 3 mAbs, thiab 8 tsis muaj npe mAbs uas pom me ntsis kom tsis muaj kev txo qis hauv kev khi tawm tsam ib qho tseem ceeb RBD mutants kuaj (Daim duab 1F). Yuav tsum tau sau tseg, chav kawm 2, chav kawm 3, thiab chav kawm 4 cov tshuaj tiv thaiv kwv yees sib xws rau RBS BD, S309, thiab CR3022 epitopes tau teev tseg hauv cov kev tshawb fawb yav dhau los (13, 18). Chav Kawm 2 thiab 3 RBD mAbs tsis paub txog ntau yam RBD mutant uas muaj K417N / E484K / L452R / N501Y hloov, ib qho RBD tsim tsim los suav nrog cov kev hloov pauv tseem ceeb rau kev khiav tawm ntawm tus kab mob (17, 18), thiab lawv tsis tau ua kom pom kev cuam tshuam rau qhov RBD of SARS-CoV-1 and Middle Eastern respiratory syndrome (MERS)-CoV (Figure 1F). Ua haujlwm tau zoo, chav kawm 2 thiab 3 RBD mAbs muaj peev xwm neutralized D614G thiab Delta variants, tab sis kev ua haujlwm nruab nrab tau txwv ntau dua rau Beta, Kappa, thiab Mu (Daim duab 1G). Tsis muaj ib qho ntawm cov chav kawm 2 lossis 3 cov tshuaj tiv thaiv kab mob uas tau txheeb xyuas tsis muaj kev sim Omicron variant.
Hauv qhov sib piv, feem ntau ntawm mAbs uas tsis tau muab cais ua ke rau RBD ntau yam sib txawv thiab hla-reacted rau SARS-CoV-1 RBD (Figure 1F). Ntawm cov no, peb tau txheeb xyuas 3 mAbs, S451-1140, S626-161, thiab S728-1157, uas tau pom tias muaj kev ua haujlwm siab nruab nrab tiv thaiv D614G thiab cross-neutralized Beta, Delta, Kappa, Mu, thiab Omicron BA.1 nrog 99% inhibitory concentration (IC99) nyob rau hauv thaj tsam ntawm 20–2,500 ng / mL (Daim duab 1G). Muab lub zog nruab nrab ntawm cov 3 mAbs no, ntxiv rau cov plaque assay platform, peb kuj tau ua qhov kev ua haujlwm tsis zoo tiv thaiv cov qhabnias BA.2.75, BL.1 (BA.2.75+R346T), BA.4, BA.5, thiab XBB cov kab mob siv tsom txo qis nruab nrab kev sim (FRNT) (Daim duab 1G). Ntawm cov no, S728-1157 tau nthuav tawm cov kev ua haujlwm siab nruab nrab tiv thaiv lub vaj huam sib luag ntawm Omicron sib txawv xws li BA.1, BA.2, BA.4, thiab BA.5, nrog rau IC99 txog 100 ng / mL raws li ntsuas los ntawm a plaque assay. Ib qho xwm txheej zoo sib xws tau pom siv FRNT, qhov twg S728-1157 khaws nws cov kev ua haujlwm siab nruab nrab tiv thaiv BA.2.75, BL.1, BA.4, BA.5, thiab XBB nrog IC50 hauv thaj tsam ntawm 8-300 ng /mL (Daim duab 1G). S451-1140 neutralized BA.1, BA.2, BA.2.75, thiab BL.1 muaj zog, tab sis tsis yog BA.4 thiab BA.5, raws li pom nyob rau hauv ob qho tib si neutralization assay platforms. Ntawm qhov tod tes, S626-161 tsis tau ua kom pom kev ua haujlwm tsis zoo tawm tsam Omicron variants dhau ntawm BA.1 variant (Daim duab 1G). Txawm hais tias S626-161 muaj qhov tsis txaus ntseeg qis dua tawm tsam VOCs uas tau sim dua li lwm 2 cov tshuaj tiv thaiv kab mob, nws tsuas yog mAb uas pom tias muaj kev cuam tshuam tsis zoo rau SARS CoV-1 RBD tab sis kuj tseem tuaj yeem ua rau cov bat coronaviruses WIV-1 thiab RsSHC014 (Daim duab 1, F thiab G). Cov ntaub ntawv no qhia tias S626-161 lees paub qhov kev txuag epitope uas tau sib koom ntawm cov kab mob arbovirus no tab sis tsis muaj nyob hauv BA.2 thiab tom qab hom kab mob. Tsis tas li ntawd, piv nrog S728-1157 thiab S451-1140, S626-161 muaj CDR-H3 ntev dua uas tuaj yeem muab qhov muaj peev xwm txhim kho kom paub txog qhov kev txuag tau zoo ntawm cov khoom seem sib koom thoob plaws arboviruses, raws li tau piav qhia hauv kev kawm yav dhau los (19) (Cov duab ntxiv 1). Thaum muab piv rau immunoglobulin hnyav (IGHV) thiab lub teeb sawb (IGLV lossis IGKV) cov noob sib txawv ntawm cov 3 mAbs nrog cov muaj SARS-CoV-2 neutralizing mAbs database (13, 15, 20–27), peb pom tias cov saw hnyav. Cov noob hloov pauv tau siv los ntawm S728-1157 (IGHV3-66), S451-1140 (IGHV3-23), thiab S626-161 (IGHV4-39) muaj yav dhau los tau tshaj tawm los encode ntau lub zog tsis zoo SARS-CoV-2 cov tshuaj tiv thaiv lub hom phiaj rau RBD (21, 22, 28, 29). Txawm li cas los xij, tsuas yog S728-1157 muaj qhov tshwj xeeb hnyav thiab lub teeb saw sib txawv ntawm cov noob sib txawv uas tsis tau tshaj tawm hauv cov ntaub ntawv (Cov Lus Qhia Ntxiv 3), qhia tias nws tsis yog pej xeem clonotype.
Daim duab 1. Tus cwj pwm ntawm RBD-reactive mAbs cais los ntawm COVID-19-cov neeg muaj hnub nyoog
Cov 3 mAbs no (S451-1140, S626-161, thiab S728-1157) tau tus cwj pwm ntxiv los txiav txim siab lawv txoj kab dav dav rau SARSCoV-2 VOCs (Daim duab 2, A thiab B) . Qhov prefusion-stabilized spike uas muaj 2-proline hloov pauv hauv S2 subunit (2P; diproline) tau pom tias yog ib qho tshuaj tiv thaiv zoo tshaj piv nrog WT spike thiab yog lub hauv paus ntawm ntau yam tam sim no SARS-CoV-2 cov tshuaj tiv thaiv, suav nrog cov tshuaj tiv thaiv mRNA (30, 31). Tsis ntev los no, spike protein stabilized nrog 6 prolines (6P; hexaproline) tau tshaj tawm los txhawb kev qhia thiab ua kom ruaj khov dua li qub diproline tsim; Raws li qhov tshwm sim, nws tau raug thov kom siv rau tiam tom ntej ntawm COVID{17}} tshuaj tiv thaiv (32, 33). Txhawm rau txiav txim siab seb puas muaj qhov sib txawv antigenicity ntawm diproline thiab hexaproline spike constructs, ob qho tib si immunogens tau suav nrog hauv peb pawg kuaj. Raws li kev ntsuas los ntawm ELISA, peb pom tias 3 mAbs khi 6P-WT spike antigen mus rau qhov ntau dua piv nrog WT-2P spike (Daim duab 2, A thiab B). Tag nrho 3 mAbs tau pom zoo sib piv rau cov kab mob ntawm Alpha, Beta, Gamma, thiab Delta, txheeb ze ntawm WT-2P (Daim duab 2, A thiab B). Txawm li cas los xij, qhov kev sib koom ua ke ntawm cov 3 mAbs no tau txo qis rau lub vaj huam sib luag ntawm Omicron-tsev neeg antigens (Daim duab 2, B, thiab C). S451-1140 khi tau nkag siab rau cov kev hloov pauv uas pom hauv BA.1 thiab BA.2, ua rau muaj qhov txo qis hauv kev khi thiab 31-fold txo qis hauv nruab nrab ntawm cov kev hloov pauv no piv nrog WT-2 P antigen thiab D614G tus kab mob, feem (Daim duab 2B). Tus kab mob sarbecovirus-cross neutralizing mAb, S626-161, kuj tau pom 1.2- rau 3.5-fold txo kev khi rau spike BA.1 antigen, uas yuav suav tau rau {{45} }fold txo qis hauv kev ua haujlwm nruab nrab tiv thaiv BA.1 (Daim duab 1G thiab daim duab 2, B thiab C). Rau qhov muaj zog tshaj plaws ntawm cov tshuaj tiv thaiv kab mob (bnAb), S728-1157, khi rau Omicron antigens raug txo kom tsawg dua (xws li 1.1- txog 4.4-fold) piv nrog WT-2P nce siab thiab tsis muaj kev cuam tshuam hauv kev ua haujlwm nruab nrab (Daim duab 1G thiab daim duab 2, B thiab C). Qhov txo qis hauv Omicron-neutralizing mAb khi rau BA.1 spike tej zaum yuav yog vim muaj kev hloov pauv hauv nws txoj kev txav mus los thiab cuam tshuam nrog kev ntim nruj ntawm Omicron 3-RBD-down qauv thiab nyiam rau 1- nce RBD uas pab tiv thaiv cov tshuaj tiv thaiv, raws li tau tshaj tawm los ntawm kev kawm dhau los (34). Lub 2P thiab 6P stabilizing kev hloov pauv kuj muaj qhov sib txawv ntawm Omicron variants qhov twg tag nrho 3 mAbs tau pom ntau dua 2.8-fold nce binding rau spike BA.1-6P piv nrog BA.1-2P version, tab sis tsuas yog marginally nce binding rau spike BA.2 thiab BA.4/5 6P versions piv nrog lawv 2P versions los ntawm 1.2 × rau 1.4 ×, tawm tswv yim me ntsis kev nkag tau zoo ntawm Omicron-neutralizing mAbs rau hexapole versions, tshwj xeeb tshaj yog rau cov spike BA.1 tsim. Ntxiv nrog rau ELISA, biolayer interferometry (BLI) tau siv los ntsuas qhov sib npaug thiab qhov sib npaug (kon, koff, thiab KD) ntawm 3 mAbs rau lub vaj huam sib luag ntawm cov kab mob sib kis (Cov duab ntxiv 2). Kev paub txog cov nqi ntawm cov fragment antigen binding (Fab) rau hexaproline spikes yog 1.5- rau 3.3-fold sai dua thaum piv nrog diproline spikes (Cov duab ntxiv 2, B thiab C), qhia tias cov tshuaj tiv thaiv khi rau 6P tsim sai dua li 2P tsim. Qhov no yuav xav tau yog tias cov epitopes tau nkag tau ntau dua ntawm RBD hauv lub xeev qhib ntawm hexaproline spike. Tshwj tsis yog rau S626- 161, Fabs dissociated los ntawm hexaproline spike qeeb qeeb (muaj qis dua koff) dua li diproline spike, xws li tag nrho KD pom tias S728-1157 thiab S451-1140 khi rau hexaproline spike nrog ntau dua affinity (Ntxiv daim duab 2, B thiab C). Qhov nce hauv kev khi rau hexaproline spike yog qhov tseem ceeb tshaj rau qhov tsis zoo IgG los ntawm 1: 2 kev sib cuam tshuam qauv raws li qhia los ntawm S728-1157 thiab S451- 1140 mAbs, zoo ib yam nrog rau ntau qhov epitopes nrog 6P stabilization tso cai. txhim kho avidity (Cov duab ntxiv 2, A thiab C). Muab ua ke, cov txiaj ntsig no qhia tias cov epitopes tsom mus rau qhov sib piv tau yooj yim dua ntawm 6P-stabilized spike thaum RBD nyob rau hauv lub xeev qhib. Cov qauv kev txheeb xyuas tau ua tom ntej los txheeb xyuas qhov kev xav no.
Daim duab 2. Kev khi dav ntawm Omicron-neutralizing mAbs
Kev txheeb xyuas cov qauv ntawm qhov dav neutralizing mAbs. Raws li thawj qhov kev kwv yees ntawm epitopes khi, ib qho kev sib tw ELISA tau siv los txiav txim siab seb 3 qhov kev cuam tshuam dav dav ntawm mAbs tau sib koom ua ke nrog peb lub vaj huam sib luag tam sim no ntawm mAbs, ib qho kev sau ntawm mAbs nrog cov paub epitope tshwj xeeb los ntawm cov kev tshawb fawb yav dhau los (15, 25, 35) , thiab 2 lwm mAbs tam sim no nyob rau hauv kev kho mob siv, LY-CoV555 (Eli Lilly) (36) and REGN10933 (Regeneron) (37). Cov chaw khi ntawm S451-1140 thiab S{11}} ib feem sib tshooj nrog CC12.3 (23, 25), chav kawm 1 tsis muaj tshuaj tiv thaiv kab mob, thiab cov tshuaj tiv thaiv kab mob hauv chav kawm 2 feem ntau, suav nrog LY-CoV555 thiab REGN10933, tab sis tsis nrog rau chav kawm 3 thiab chav kawm 4 cov tshuaj tiv thaiv (Daim duab 3A). S626-161 tau qhia txog qhov sib tshooj tseem ceeb hauv cheeb tsam kev khi nrog chav kawm 1 CC12.3, ntau chav kawm 4 cov tshuaj tiv thaiv, suav nrog CR3022, thiab lwm yam tshuaj tiv thaiv tsis tau cais tawm, thaum muaj qee qhov sib tshooj nrog ntau chav kawm 2 thiab ib chav kawm 3 cov tshuaj tiv thaiv (Daim duab 3 A). Analogously, kev sib tw BLI kev soj ntsuam tau qhia tias S451-1140 thiab S728-1157 sib tw nrog ib leeg rau kev khi rau qhov hnyav WT-6P, qhov S626-161 tsis tau (Daim duab ntxiv 3). Zuag qhia tag nrho, cov ntaub ntawv no qhia tias S451-1140 thiab S728-1157 paub cov epitopes zoo sib xws uas txawv ntawm S626-161.
Daim duab 3. Mechanism ntawm broad neutralization ntawm S728-1157
Antibody S728-1157 tau encoded los ntawm IGHV3-66 thiab muaj ib tug luv luv complementarity txiav txim cheeb tsam 3 (CDR-H3). Qhov tseem ceeb, mAbs uas khi RBS hauv kev khi hom 1 (xws li RBS-A lossis chav kawm 1 qhov chaw), typified los ntawm CC12.1, CC12.3, B38, thiab C105 (13, 18, 23, 29, 38, 39), nyiam siv IGHV3-53 lossis 3-66 thiab muaj kev cuam tshuam rau VOC kev hloov pauv (40). Txawm li cas los xij, thaj tsam CDR-H3 ntawm S728-1157 yog qhov txawv ntawm lwm cov tshuaj tiv thaiv kab mob hauv chav kawm no, uas muaj feem cuam tshuam rau nws txoj haujlwm dav dav. Txhawm rau kom nkag siab txog lub hauv paus ntawm kev tsis sib haum xeeb dav dav los ntawm S728-1157 ntawm qhov epitope no, peb tau daws cov qauv cryo-electron microscopy (cryo-EM) qauv (Daim duab 3B) ntawm IgG S728-1157 nyob rau hauv complex nrog spike WT{ {31}}P-Mut7, ib tug qauv ntawm spike WT-6P muaj ib tug interprotomer disulfide daim ntawv cog lus ntawm C705 thiab C883, ntawm kwv yees li 3.3 Å ntiaj teb no daws teeb meem (Ntxiv daim duab 4E). Siv symmetry expansion, tsom faib, thiab refinement txoj kev, peb ua tiav cov kev daws teeb meem hauv zos ntawm RBD-Fv interface rau kwv yees li 4 Å (Cov duab ntxiv 4E thiab Cov Lus Ntxiv 8). Cov qauv siv lead ua ntawm S728-1157 Fab tau txiav txim siab ntawm 3.1 Å daws teeb meem thiab siv los tsim cov qauv atomic ntawm RBD-Fv interface. Peb cov qauv lees paub tias S728-1157 khi RBS-A (los yog chav kawm 1) epitope hauv RBD-up conformation (Daim duab 3B thiab ntxiv daim duab 4E), zoo ib yam li lwm yam IGHV3-53/{{55} } cov tshuaj tiv thaiv (Daim duab 3C). Steric blockage ntawm angiotensin-converting enzyme 2 (ACE2) binding site los ntawm S728-1157 piav qhia nws lub zog nruab nrab siab tiv thaiv SARS-CoV-2. Lub 32NY33 motif thiab 53SGGS56 motif (23) hauv S728-1157 CDR-H1 thiab -H2 cuam tshuam nrog RBD yuav luag tib yam li CC12.3 (Cov duab ntxiv 4, B thiab C). Txawm li cas los xij, piv nrog VH 98DF99 hauv CC12.3, VH 98DY99 hauv S728-1157 CDR-H3 tsim kev sib cuam tshuam ntau dua, suav nrog ob qho tib si hydrophobic thiab polar kev sib cuam tshuam, nrog RBD, uas tuaj yeem suav rau qhov dav nruab nrab ntawm VOCs (Daim duab 3D thiab Cov Lus Qhia Ntxiv 6 thiab 7). Lub diglycine VH 100GG101 hauv S728- 1157 CDR-H3 kuj tseem tuaj yeem pab txhawb kev sib txuas ntau dua piv nrog VH Y100 hauv CC12.3, yuav yog vim qhov yooj yim ntawm cov glycine residues uas ua rau muaj qhov sib txawv ntawm qhov kawg ntawm CDR. -H3 lub voj voog thiab cov txheeb ze hloov ntawm cov seem ntawm 98DY99.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Nyem qhov no mus saib Cistanche Enhance Immunity khoom
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Txawm hais tias Omicron VOCs muaj ntau qhov kev hloov pauv hauv RBD, feem ntau ntawm cov seem no tsis cuam tshuam nrog S728-1157, raws li kev khi tseem raug pom (Cov duab ntxiv 4A). Los ntawm peb cov spike WT-6P-Mut7 + Fab S728-1157 qauv, Y505 rau VL Q31, thiab E484 rau VH Y99 raug kwv yees los ua cov ntawv cog lus hydrogen (Cov duab ntxiv 4D thiab Cov Lus Ntxiv 6 ), uas muaj peev xwm cuam tshuam los ntawm Omicron kev hloov pauv Y505H thiab E484A. Txawm li cas los xij, kev hloov pauv Y505H tseem yuav tso cai rau kev sib cog lus hydrogen nrog VL Q31, thiab kev hloov pauv E484A yuav ntxiv lwm qhov kev sib txuas ntawm hydrophobic nyob ze hydrophobic residues VL Y99, F456, thiab Y489. Cov kev sib cuag no tuaj yeem piav qhia, ib feem, cov txheej txheem uas ua rau S728-1157 khaws cov kev ua haujlwm nruab nrab, txawm hais tias txo qis, tawm tsam tus kab mob BA.1 antigen (Daim duab 1G thiab daim duab 2B). Lub BA.1 antigen, nyob rau hauv lem, tejzaum nws muaj feem xyuam rau lub Omicron mutations hloov lub conformational toj roob hauv pes ntawm spike protein (34). Txawm li cas los xij, ntau yam somatically mutated residues, piv txwv li, VH L27, L28, R31, F58, thiab VL V28 thiab Q31, hauv S728-1157 tau koom nrog hauv kev sib cuam tshuam nrog SARS-CoV-2 RBD (Supplemental Figure 1 thiab Supplemental Table 7), uas kuj tseem tuaj yeem ua rau nws qhov kev ua haujlwm dav dav piv nrog CC12.3. Zuag qhia tag nrho, peb cov kev tshawb fawb txog kev tsim qauv tau nthuav tawm lub hauv paus ntawm kev tsis sib haum xeeb dav dav ntawm S728-1157 uas tuaj yeem ua rau muaj kev hloov pauv feem ntau hauv SARS-CoV-2 VOCs.
Daim duab 4. Kev tiv thaiv zoo ntawm bnAbs tiv thaiv SARS-CoV-2 kab mob hauv hamsters.
S728-1157 txo replication of SARS-CoV-2 BA.1 Omicron, Delta, and WT SARS-CoV-2 in Syrian hamsters. Txhawm rau ntsuas qhov kev tiv thaiv kev ua tau zoo ntawm peb qhov nruab nrab mAbs dav dav, peb siv tus qauv kub Syrian hamster kab mob uas tau siv dav rau SARS-CoV-2. Hamsters tau txais 5 mg / kg ntawm peb qhov kev sim mAbs lossis kev tswj isotype tsom rau cov tshuaj tiv thaiv tsis sib xws (ebolavirus glycoprotein) los ntawm kev txhaj tshuaj intraperitoneal 1 hnub tom qab kis kab mob SARS CoV-2 viruses. Lub ntsws thiab qhov ntswg tau sau 4 hnub tom qab kis kab mob (Daim duab 4A). Kev tswj hwm kev kho mob ntawm S728-1157 ua rau txo qis titers ntawm WT, BA.1 Omicron, thiab Delta variants nyob rau hauv ob qho tib si lub qhov ntswg turbinates thiab ntsws ntawm tus kab mob hamsters (Daim duab 4, B-D). Interestingly, cov nyhuv ntawm S728-1157 nyob rau hauv lub ntsws yog heev, txo WT thiab BA.1 Omicron viral loads los ntawm kwv yees li 104PFU, nrog rau cov kab mob titers ntawm BA.1 Omicron variant raug tshem tawm tag nrho (Daim duab 4C). Nyob rau hauv sib piv rau hauv vitro neutralization (Daim duab 1G), S451-1140 tsis txo BA.1 Omicron viral replication nyob rau hauv lub ntsws thiab qhov ntswg turbinates, qhia ib tug disconnect ntawm in vitro neutralization thiab nyob rau hauv vivo tiv thaiv rau lub clone (Daim duab 4E) . Hauv kev sib piv, S626-161 kev tswj hwm tau ua rau muaj qhov txo qis hauv cov kab mob ntsws ntsws loj heev tom qab WT thiab BA.1 kev sib tw (Daim duab 4, F, thiab G). Cov ntaub ntawv no qhia meej tias, txhawm rau txheeb xyuas qhov dav dav tiv thaiv mAbs, ntsuas kev tiv thaiv kev ua tau zoo nyob rau tib lub sijhawm nrog kev ua haujlwm nruab nrab yog xav tau. Tsiv mus tom ntej, nws yuav txaus siab los tshuaj xyuas seb qhov muaj peev xwm tiv thaiv ntawm S728-1157 yog Fc-dependent. Zuag qhia tag nrho, S728-1157 sawv cev rau kev cog lus mAb nrog kev ua haujlwm nruab nrab dav dav tiv thaiv SARS CoV-2 variants uas muaj peev xwm txo qis WT, Delta, thiab BA.1 replication hauv vivo.
SARS-CoV-2 kab mob tsis tshua muaj zog S728-1157-zoo li cross-neutralizing mAbs. Muab qhov cross-neutralization thiab prophylactic muaj peev xwm ntawm S728-1157, peb tau nrhiav los tshuaj xyuas seb S728–1157-zoo li cov tshuaj tiv thaiv kab mob feem ntau tshwm sim ntawm cov lus teb polyclonal hauv SARS-CoV-2 cov neeg mob. Txhawm rau soj ntsuam qhov no, peb tau ua kev sib tw ELISAs siv cov tshuaj tiv thaiv kab mob txhawm rau txhawm rau txhawm rau tiv thaiv RBD cov tshuaj tiv thaiv kab mob uas tuaj yeem sib tw rau kev khi nrog S728-1157 (Daim duab 5A). Cov ntsiab lus tau muab faib ua 3 pawg raws li qhov loj ntawm cov tshuaj tiv thaiv kab mob, raws li tau hais tseg yav dhau los (15, 16). Txawm hais tias cov neeg teb siab thiab nruab nrab muaj cov titers siab dua ntawm S728-1157-cov tshuaj tiv thaiv kab mob sib tw piv nrog cov neeg teb qis (Daim duab 5B), cov titers tau qis heev hauv txhua pab pawg, qhia tias nws tsis yooj yim kom tau txais qib siab S{{ 18}}-zoo li cov tshuaj tiv thaiv kab mob hauv polyclonal serum tom qab WT SARS-CoV-2 kab mob. Ntxiv rau S728-1157, peb tau sim cov kev sib tw ntawm convalescent serum nrog lwm cov mAbs, suav nrog S451- 1140, S626-161, LY-CoV555, REGN10933, CR3022, thiab CC12.3. Zoo ib yam li S728-1157, peb tau pom cov titers qis ntawm cov tshuaj tiv thaiv sib tw nrog S451-1140, S626-161, LY-CoV555, REGN10933, thiab CC12.3 hauv polyclonal serum los ntawm feem ntau ntawm cov convalescent. cov tib neeg (Daim duab 5, C–F thiab H). Txawm li cas los xij, cov neeg teb siab tau zoo li muaj cov titers siab dua tiv thaiv cov neeg nruab nrab mAbs dua li cov neeg teb qis (Daim duab 5, B-F thiab H). Hauv qhov sib piv, cov tshuaj tiv thaiv lub hom phiaj ntawm CR3022 epitope qhov chaw tau tshaj tawm ntau dua hauv cov tib neeg convalescent, qhia txog kev ua kom muaj txiaj ntsig ntawm chav kawm 4 RBD cov tshuaj tiv thaiv hauv polyclonal serum (Daim duab 5G). Qhov tshwj xeeb, tsis muaj qhov sib txawv tseem ceeb hauv titers ntawm CR3022 thoob plaws 3 pawg neeg teb, tawm tswv yim tias CR3022-cov tshuaj tiv thaiv kab mob hauv chaw tau raug cuam tshuam tsis tu ncua thaum WT SARS-CoV-2 kab mob hauv cov neeg feem coob. Qhov zoo siab, piv nrog CC12.3, S728-1157 tau kuaj pom ntawm 4-fold qis qis hauv cov ntshav ntawm cov neeg teb siab. Yog li, txawm hais tias chav kawm 1 cov tshuaj tiv thaiv feem ntau raug cuam tshuam los ntawm kev kis tus kab mob thiab txhaj tshuaj tiv thaiv (14, 20, 28, 29, 41–43), peb cov ntaub ntawv qhia tias S728-1157-zoo li cov tshuaj tiv thaiv uas sawv cev rau ib pawg ntawm cov chav kawm no tsis tshua muaj sib piv.
Tsis tas li ntawd, peb tau tshuaj xyuas qhov sib txawv ntawm qhov tshwm sim rau 2P piv rau 6P-stabilized spike nyob rau hauv peb cov convalescent cohort sera (Daim duab 5, I–K). Peb pom tias tag nrho 3 pawg neeg teb tau teeb tsa cov tshuaj tiv thaiv kab mob tiv thaiv kab mob tiv thaiv 6P-stabilized spike WT kom ntau dua li 2P-stabilized spike WT, los ntawm qhov tseem ceeb ntawm 6-rau-11--fold (Daim duab 5J), qhia tias loj antigenic epitopes tau pom zoo los yog stabilized ntawm 6P-stabilized antigen. Siv tib cov qauv, cov lus teb siab thiab nruab nrab kuj tseem muaj qis dua ntawm cov tshuaj tiv thaiv kab mob sib kis tiv thaiv BA.1-2P dua BA.{18}}P, los ntawm 4–to–5–fold (Daim duab 5K). Ntawm qhov ceeb toom, cov neeg teb tsis tshua muaj qhov hloov me me hauv kev sib khi reactivity tiv thaiv spike BA.1 Omicron-2P thiab 6P (2-fold txo) piv nrog WT-2P thiab 6P spike ({ {28}}fold txo) (Daim duab 5, J thiab K), qhia tias cov tshuaj tiv thaiv kab mob ntshav qab zib tau tawm tsam BA.1 Omicron-reactive epitopes tej zaum yuav raug txwv ntau dua hauv cov lus teb tsawg. Zuag qhia tag nrho, cov ntaub ntawv no qhia tias muaj kev txhim kho polyclonal binding induced los ntawm tej yam ntuj tso kab mob rau 6P-stabilized spikes, ob qho tib si rau WT thiab Omicron kab mob.
S728-1157-zoo li cov tshuaj tiv thaiv yog qhov ua tau zoo hauv cov ntsiab lus ntawm kev tiv thaiv kab mob sib xyaw. Thawj tus kab mob SARS-CoV-2 yam tsis muaj tshuaj tiv thaiv tau dhau los ua qhov tsis tshua muaj nyob hauv lub ntiaj teb tam sim no, thiab ntau qhov kev tshawb fawb tau tshaj tawm tias SARS-CoV-2 kev tiv thaiv txawv ntawm cov tib neeg uas muaj cov tshuaj tiv thaiv tshwj xeeb / kab mob keeb kwm. Raws li qhov tshwm sim, peb txuas ntxiv mus tshawb xyuas qhov twg tshwm sim, ib sab ntawm WT kab mob nrog poj koob yawm txwv SARS-CoV-2 ib leeg, yuav ua rau muaj txiaj ntsig zoo S728-1157-zoo li cov tshuaj tiv thaiv kab mob hauv ntshav los ntawm cov tshuaj tiv thaiv kab mob monovalent mRNA nrog thiab tsis ua ntej. kab mob. Peb tau txais cov qauv biospecimen uas tsim nyog los ntawm Kev Tiv Thaiv Kev Tiv Thaiv Kev Tiv Thaiv Kev Tiv Thaiv Kev Ruaj Ntseg rau SARS-CoV-2 (PARIS) kev tshawb fawb pawg, uas tau ua raws li cov neeg ua haujlwm kho mob ntev txij li pib muaj kev sib kis (44). Peb tau xaiv cov qauv ntshav plasma los ntawm kev txhaj tshuaj tiv thaiv tag nrho (2 × txhaj tshuaj tiv thaiv) cov neeg koom nrog thiab tsis muaj kab mob nrog rau cov neeg koom nrog txhawb nqa (3 × txhaj tshuaj tiv thaiv) nrog thiab tsis muaj kab mob. Tsis tas li ntawd, peb kuj suav nrog cov qauv los ntawm cov neeg koom nrog kev kawm uas tau txais cov tshuaj tiv thaiv mRNA bivalent (cov poj koob yawm txwv WA1/2020 ntxiv rau Omicron BA.5) (Daim duab 6A thiab Cov Lus Ntxiv 2). Cov kab mob sib kis tau zoo hauv cov neeg koom nrog uas tau txais cov tshuaj tiv thaiv tshuaj tiv thaiv tau tshwm sim thaum lub sijhawm Omicron cov kab mob tau hloov pauv tag nrho lwm cov kab mob SARS-CoV-2 hauv New York cheeb tsam. Peb pom tias cov tib neeg uas tau txhaj tshuaj ob zaug muaj qhov qis tshaj plaws ntawm S728-1157 cov tshuaj tiv thaiv kab mob sib tw hauv cov 5 pawg ntawm cov qauv kuaj (Daim duab 6B). Qhov tseem ceeb, cov qib no tau zoo ib yam li qhov tau pom rau peb pawg neeg tsis muaj tshuaj tiv thaiv kab mob (txhua tus neeg teb; Daim duab 5B). Hauv kev sib piv, cov tib neeg uas muaj keeb kwm ntawm kev kis tus kab mob ntuj, suav nrog cov neeg uas muaj tus kab mob uas muaj 2 ntawm 3 koob tshuaj tiv thaiv, thiab cov tib neeg uas tau ntsib tus kab mob sib kis thiab tau txais ib qho tshuaj tiv thaiv bivalent, tau pom ntau dua ntawm S728-1157 elicitation piv nrog cov tsis muaj kab mob. tab sis cov neeg tau txhaj tshuaj tiv thaiv (Daim duab 6B). Txawm hais tias cov tshuaj tsis muaj kab mob 3- pawg tshuaj pom tsuas yog qhov nce tsis tseem ceeb piv nrog 2- pawg koob tshuaj, ua ke sib cais los ntawm hom tshuaj tiv thaiv tau qhia tias homologous thib peb koob ntawm BNT162b2 thiab mRNA-1273 nce S{{ 38}}zoo li neutralizing antibody titers los ntawm 2.72 × thiab 2.85 ×, ntsig txog (Daim duab 6, C thiab D). Yuav tsum nco ntsoov, ntawm cov neeg koom nrog 3 tag nrho cov kev sib cuag nrog spike los ntawm txhua txoj kev, S728-1157-zoo li cov tshuaj tiv thaiv kab mob yog 3 × siab dua hauv cov tshuaj tiv thaiv ob zaug piv nrog cov tshuaj tiv thaiv kab mob-naive triple-cov tshuaj tiv thaiv, qhia tias SARS-CoV{{ 51}} kev kis kab mob zoo tshaj plaws ua rau qhov clonotype no. Ntawm cov pab pawg tiv thaiv kab mob sib xyaw, peb tau sau tseg tias feem ntau ntawm cov neeg txhawb nqa uas tau txais cov tshuaj tiv thaiv kab mob bivalent tsuas muaj ntau dua S728-1157 cov tshuaj tiv thaiv kab mob sib piv nrog cov tshuaj tiv thaiv kab mob ua ntej omicron convalescent, qhia tias S{53 }} titer tau zoo li mus txog toj siab tom qab 3 raug. Peb kuj tau tshawb xyuas cov titers ntawm polyclonal antibodies uas sib tw nrog CC12.3 thiab CR3022 ntxiv rau S728-1157. Txhua tus neeg tau nthuav tawm cov titers siab heev ntawm CC12.3- thiab CR3022-zoo li cov tshuaj tiv thaiv kab mob, tsis muaj tus lej thiab hom kis tau tus mob (Cov duab ntxiv 5), tsis zoo li peb tau pom rau S728-1157- zoo li cov tshuaj tiv thaiv. Zuag qhia tag nrho, cov ntaub ntawv no qhia tau hais tias SARS-CoV-2 kev kis kab mob thiab kev txhaj tshuaj mRNA ob leeg ua rau S728-1157-zoo li cov tshuaj tiv thaiv kab mob, nrog rau kev kis kab mob ua lub luag haujlwm tseem ceeb hauv cov neeg tau txhaj tshuaj.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Thaum kawg, hauv kev sib piv cov lus teb tawm tsam 2P- piv rau 6P-stabilized spike hauv mRNA-cov tshuaj tiv thaiv kab mob, peb pom tias feem ntau pab pawg tau tshem tawm cov tshuaj tiv thaiv zoo sib xws rau ob qho tib si tsim. Qhov tshwj xeeb rau qhov no yog pab pawg uas tsis tau txhaj tshuaj tiv thaiv kab mob triple, uas tau ua pov thawj ntau dua, txawm hais tias tsuas yog nce me ntsis, qhov tshwm sim ntawm 2P piv nrog 6P-stabilized spike (Daim duab 6E). Cov ntaub ntawv no qhia tias qhov sib txawv ntawm cov kab mob ntuj tsim (Daim duab 5, J, thiab K), kev txhaj tshuaj ib leeg ua rau cov lus teb polyclonal uas txwv ntau dua rau cov epitopes hauv Spike-2P tsim, nyob rau hauv kab nrog Spike{12 }}P tsim cov tshuaj tiv thaiv tam sim no. Thaum kawg, cov kev tshawb pom no txhawb lub tswv yim tias 6P-stabilization ntawm yav tom ntej SARS-CoV{16}} cov tshuaj tiv thaiv tuaj yeem muaj txiaj ntsig zoo hauv kev tsim cov tshuaj tiv thaiv kab mob dav dav xws li S728-1157.
Daim duab 5. Convalescent serum antibody kev sib tw nrog broad neutralizing RBD-reactive mAbs thiab kev sib piv ntawm cov tshuaj tiv thaiv kab mob hauv cov ntshav tawm tsam 6P- tiv thaiv 2P-stabilized spikes
Daim duab 6. mRNA-tiv thaiv cov tshuaj tiv thaiv kab mob sib tw nrog S728-1157 neutralizing RBD-reactive mAbs thiab kev sib piv ntawm cov tshuaj tiv thaiv kab mob hauv cov ntshav tawm tsam 6P- tiv thaiv 2P-stabilized spikes.
Kev sib tham
Hauv txoj kev tshawb no, peb txheeb xyuas qhov muaj zog bnAb cais tawm ntawm lub cim xeeb B cell ntawm ib tus neeg uas tau zoo los ntawm SARS-CoV-2 tus kab mob thaum lub sij hawm pib nthwv dej ntawm COVID-19 muaj thoob qhov txhia chaw. Qhov no bnAb, S728- 1157, khaws cia muaj txiaj ntsig zoo sib xws thiab muaj kev ua haujlwm tsis tu ncua tawm tsam txhua qhov kev sim SARS-CoV-2 VOC, suav nrog Omicron BA.1, BA.2, BA.2.75, BL.1 ( BA.2.
Peb pom tias cov ntshav qab zib convalescent los ntawm peb pawg neeg muaj cov tshuaj tiv thaiv qis qis uas sib tw nrog S728-1157 (ib chav kawm 1 / RBS-A antibody) thiab chav kawm 2 epitope mAbs. Qhov no qhia tias S728-1157 yog qhov tshwj xeeb me ntsis los ntawm lwm cov tshuaj tiv thaiv kab mob uas tsom rau chav kawm 1 epitopes thiab tsis tshua muaj tshwm sim hauv RBD tshwj xeeb nco B cell pas dej. Hloov chaw, peb cov kab mob sib kis tau tshwm sim los ua rau cov tshuaj tiv thaiv kab mob tsom rau CR3022 (chav kawm 4) epitope; Cov tshuaj tiv thaiv kab mob ntawm qhov tshwj xeeb no feem ntau cuam tshuam tab sis tsis tshua muaj peev xwm nruab nrab dua li RBS-targeting antibodies (14, 17). Cov ntaub ntawv no yog ntxiv rau peb qhov kev tshawb pom yav dhau los uas qhia tau tias muaj ntau ntawm chav kawm 3 / S309 cov tshuaj tiv thaiv kab mob hauv convalescent sera tuaj yeem ua rau muaj kev cuam tshuam tsis zoo rau Alpha thiab Gamma variants, qhov tsis muaj chav kawm 2 cov tshuaj tiv thaiv yuav suav rau txo qis kev muaj peev xwm tiv thaiv Delta (15) . Txawm li cas los xij, qhov dav ntawm cov haujlwm ntawm feem ntau ntawm cov RBS-targeting antibodies (RBS-A / chav kawm 1, RBS-B, C / chav kawm 2, thiab RBS-D, S309 / chav kawm 3) tawm tsam Omicron variants tau tshaj tawm tias muaj kev txwv ntau. (11, 40, 45).
Qhov kev sib tw tseem ceeb mus rau pem hauv ntej yuav txiav txim siab yuav ua li cas txhawm rau txhim kho kev tshem tawm ntawm cov kab mob sib kis-reactive antibodies kom txuag tau RBS epitopes. Hauv qhov no, peb tau pom ntawm no tias cov tib neeg uas muaj kev tiv thaiv kab mob sib kis tau nce siab dua titers ntawm S728-1157-zoo li cov tshuaj tiv thaiv ntau dua li cov neeg tau txhaj tshuaj yam tsis muaj kab mob ua ntej. Qhov tseem ceeb, qhov tshwm sim no tau raug sau tseg txawm tias thaum muaj pes tsawg tus kis tau raug tswj hwm (xws li, hauv cov tshuaj tiv thaiv ob zaug piv rau cov tshuaj tiv thaiv peb zaug uas tsis muaj kab mob), qhia tias qee lub ntsiab lus ntawm kev tiv thaiv kab mob sib kis (los yog cov tshuaj tiv thaiv uas tuaj yeem ua raws li hom kev tiv thaiv no) Nws yog ib qho tseem ceeb rau kev tshem tawm cov clonotype no. Qhov no yog ua raws li cov ntaub ntawv pov thawj kev sim uas cov neeg uas muaj kev tiv thaiv kab mob sib kis tau dav dua cov tshuaj tiv thaiv kab mob-reactivity piv nrog cov uas tsuas yog txhaj tshuaj tiv thaiv kab mob lossis thawj kis kab mob-xws li lub cev tiv thaiv kab mob (9).
Cov qauv ntawm no qhia tau hais tias S728-1157 khi RBS-A/class 1 epitope nyob rau hauv up-conformation RBD. Cov epitope no zoo li nkag tau yooj yim dua ntawm 6P-stabilized spikes, uas tau tshaj tawm los nthuav tawm 2 RBDs nyob rau sab saud, piv nrog 2P spikes, uas tam sim no tsuas yog 1 (30, 33, 46, 47), thiab peb cov tshuaj tiv thaiv. tshwj xeeb rau up-conformation spike qhia txhim kho kev khi. S728-1157 raug cais tawm tom qab muaj kab mob; nyob rau hauv xws li cov ntsiab lus, qhov txawv ntawm inducing S728-1157-zoo li clones yuav muaj ntau dua vim hais tias RBD yuav tsum muaj peev xwm los txais yuav ib tug zoo conformation, txawm transiently, khi rau ACE2, yog li nthuav tawm no epitope. Tsis zoo li feem ntau ntawm IGHV3-53/3-66 RBS-A/class 1 antibodies, S728-1157 tuaj yeem ua raws li cov kev hloov pauv tseem ceeb hauv VOC spikes siv kev sib cuam tshuam ntawm CDR-H3 thiab RBD (29, 48–50). S728-1157 kuj tseem siv cov saw hlau sib txawv (IGLV3-9) piv nrog rau lwm cov tshuaj tiv thaiv qis dua xws li CC12.3 (IGKV3-20), uas yuav cuam tshuam rau tag nrho cov kev sib raug zoo; Txawm li cas los xij, peb qhov kev soj ntsuam qhia tias muaj kev sib txuas ntawm hydrogen tsawg dua ntawm S728-1157 lub teeb sawb thiab RBD piv nrog CC12.3 (Cov Lus Ntxiv 7). Txawm hais tias feem ntau ntawm CDR-H3 kev sib cuag residues tseem ceeb rau VOC cross-reactivity nyob rau hauv qhov kev sib tshuam no yog germline-encoded thiab tsis qhia los ntawm somatic mutations, ob peb somatically mutated residues nyob rau hauv lub moj khaum cheeb tsam los yog CDR-H1, CDR-H2, thiab CDR-L1 yog. koom nrog kev sib tham nrog SARS-CoV-2 RBD. Ntawm qhov tod tes, qhov no qhia tias nco B hlwb encoding IGHV3-53/66 chav tshuaj tiv thaiv kab mob tuaj yeem tau txais qib zoo sib xws ntawm kev sib cuam tshuam los ntawm kev sib txuas ntxiv. Ntawm qhov tod tes, qhov no kuj tseem qhia tau tias muaj peev xwm tsim cov kab mob tiv thaiv kab mob uas tiv thaiv kab mob S728-1157-zoo li naive B hlwb. Txawm hais tias nws yuav nyuaj rau tsim cov tshuaj tiv thaiv uas tuaj yeem cuam tshuam tshwj xeeb S728-1157-zoo li cov tshuaj tiv thaiv nrog xaiv CDR-H3s muaj peev xwm kov yeej VOC kev hloov pauv, nws txhawb kom IGHV-gene txwv tau pom nyob rau hauv lwm yam muaj zog SARS-CoV{ {58}} neutralizing mAbs kev tshawb fawb (13, 15, 20–27). Xwb, qhov no kuj tseem tuaj yeem ua tau los ntawm kev txhaj tshuaj tiv thaiv kab mob zoo dua nrog cov tshuaj tiv thaiv kab mob RBD zoo, raws li tau tshaj tawm yav dhau los rau lwm cov kab mob (51-55).
Txawm hais tias muaj ntau yam kev hloov pauv tau pom nyob rau hauv RBS-A / chav kawm 1 antigenic site (18), hais txog S728-1157 epitope, 13 ntawm 15 tag nrho RBD tiv tauj residues thiab 2 ntawm 3 CDR-H{{9} }bound RBD contact residues yog khaws cia hauv Omicron thiab tag nrho lwm yam VOCs. Qhov no qhia tias thaj tsam RBD qhov twg S728-1157 epitope pom tuaj yeem suav nrog cov khoom seem tseem ceeb rau nws txoj haujlwm tsis muaj zog thiab kis kab mob thiab yog li ntawd yuav tsis kam lees ntawm kev hloov pauv thiab cov tshuaj tiv thaiv kab mob ntau dua li nyob ib puag ncig RBS-A / class 1 site residues. Yog tias qhov no tshwm sim, qhov kev nyiam rau qhov tshwj xeeb epitope no yuav ploj mus raws li cov kab mob sib kis hloov zuj zus yuav tsum raug txo kom tsawg, ua tus cwj pwm ntawm S728-1157 thiab cov tshuaj tiv thaiv zoo sib xws thiab cov epitopes tseem ceeb rau cov tshuaj tiv thaiv kab mob sib txawv lossis kev kho mAb. Hauv cov ntsiab lus, peb txoj kev tshawb fawb txheeb xyuas bnAbs uas tuaj yeem qhia txog kev tsim cov tshuaj tiv thaiv kab mob rau cov tshuaj tiv thaiv kab mob coronavirus tom ntej los yog ua haujlwm li mAb kho mob uas tiv taus SARS CoV-2 evolution. Tshwj xeeb, nyob rau hauv cov nqe lus ntawm kev sib xyaw potency thiab dav, S728-1157 zoo nkaus li yog qhov zoo tshaj plaws-hauv-chav tshuaj tiv thaiv cais tawm rau hnub tim. Muab hais tias cov tshuaj tiv thaiv kab mob no khi tau yooj yim dua nrog 6P-stabilization, nws tau kwv yees tias yuav raug nyiam los ntawm 6P-stabilized recombinant spike proteins lossis tag nrho cov kab mob, uas qhia tias kev hloov kho hexaproline tuaj yeem muaj txiaj ntsig rau yav tom ntej cov tshuaj tiv thaiv txhawm rau txhawm rau tiv thaiv SARS-CoV yav tom ntej. -2 variants thiab lwm yam arboviruses.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Cov txheej txheem
Monoclonal antibody cais. PBMCs raug cais tawm ntawm cov lim dej leukoreduction thiab khov raws li tau piav qhia dhau los (24). B hlwb tau enriched los ntawm PBMCs ntawm FACS. Cells tau stained nrog CD19, CD3, thiab antigen probes conjugated rau oligo-fluorophore; cov hlwb ntawm kev txaus siab raug txheeb xyuas raws li CD3– CD19+ Antigen+. Txhua mAbs tau tsim los ntawm oligo-tagged, antigen bait-sorted cells txheeb xyuas los ntawm ib leeg-cell RNA-Seq, raws li tau piav qhia yav dhau los (15, 24). Cov ntaub ntawv B cell ib leeg tsim tawm hauv txoj kev tshawb no tau muab tso rau Gene Expression Omnibus: GSE171703 thiab GSM5231088–GSM5231123.
Antigen-specific B hlwb raug xaiv los tsim mAbs raws li antigen-kev sojntsuam kev siv tshuaj ntsuam xyuas los ntawm JMP Pro 15. Antibody hnyav thiab lub teeb saw noob tau tsim los ntawm Integrated DNA Technologies (IDT) thiab cloned rau hauv tib neeg IgG1 thiab tib neeg κ lossis λ lub teeb-chain qhia vectors los ntawm Gibson los ua ke, raws li tau piav qhia yav dhau los (56). Cov saw hnyav thiab lub teeb ntawm cov sib txuas mAb tau hloov pauv mus rau HEK293T hlwb (ATCC). Tom qab hloov mus rau 18 teev, cov hlwb hloov pauv tau ntxiv nrog cov protein-dawb hybridoma nruab nrab supernatant (PFHM-II, Gibco). Lub supernatant uas muaj cov zais cia mAb tau sau rau hnub 4 thiab ua kom huv si siv cov protein A-agarose hlaws (Thermo Fisher Scientific) raws li tau piav qhia yav dhau los (56). Sequences of heavy and light chains of the well-characterized antibodies were derived from Protein Data Bank (PDB), LY-CoV555 (PDB ID: 7KMG), CR3022 (PDB ID: 6W7Y), and REGN1{{ 125}}933 (PDB ID: 6XDG) thiab tau ua ke raws li tau piav qhia saum toj no. CC12.3 mAb (PDB ID: 6XC4) yog muab los ntawm Meng Yuan ntawm Scripps Research Institute (San Diego, California, USA). Recombinant spike protein qhia. The recombinant D614G SARS-CoV-2 full-length (FL) spike, BA.2-6P, BA.4/5-6P, BQ.1-6P, BQ. 1.1-6P, XBB-6P, WT RBD, ib leeg RBD mutants (R346S, K417N, K417T, G446V, L452R, S477N, F486A, F486Y, N487Q, Y489F, Q493N, Y505A, and Y505F), ua ke RBD mutant (K417N/E484K/L452R/NN501Y), SARS-CoV-1 RBD, and MERS-CoV RBD were generated in-house. Luv luv, cov recombinant antigens tau qhia siv Expi293F hlwb (Thermo Fisher Scientific). Cov noob ntawm kev txaus siab tau cloned rau hauv cov tsiaj txhu qhia vector (hauv tsev hloov kho AbVec) thiab hloov pauv siv cov khoom siv ExpiFectamine 293 (Thermo Fisher Scientific) raws li cov chaw tsim khoom raws tu qauv. Lub supernatant tau sau nyob rau hnub 4 tom qab transfection thiab incubated nrog Ni-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen). Kev lim dej tau ua tiav los ntawm kev siv lub ntiajteb txawj nqus ntws thiab eluted nrog imidazole-muaj tsis raws li tau piav qhia yav dhau los (57, 58). Lub eluate tau buffered thiab pauv nrog PBS siv lub Amicon centrifugal unit (Millipore). Cov recombinant FL spikes stabilized los ntawm 2P kev hloov pauv ntawm cov variants B.1.1.7 Alpha, B.1.351 Beta, P.1 Gamma, B.1.617.2 Delta, BA.1, BA.2, thiab BA.4 Omicron thiab yog tsim nyob rau hauv Sather Laboratory ntawm Seattle Children's Research Institute. K417V, N439K, thiab E484K RBDs thiab recombinant FL spike WT-2P thiab 6P tau tsim nyob rau hauv Krammer lub chaw kuaj mob ntawm Icahn Tsev Kawm Ntawv Tshuaj ntawm Mount Sinai. SARS-CoV-2-6P-Mut7 and spike BA.1-6P tau tsim thiab tsim tawm raws li tau piav qhia hauv kev tshawb fawb yav dhau los (59). Cov protein ntau thiab cov peev txheej rau txhua qhov antigen muaj nyob rau hauv Daim Ntawv Ntxiv 4. ELISA. Recombinant SARS-CoV-2 spike/RBD proteins were coated to high protein-binding microtiter plates (Costar) at 2 ug/mL in PBS at 50 μL/well and overnight at 4 degree . Cov phaj tau ntxuav nrog PBS uas muaj 0.05% Tween 20 (PBS-T) thiab thaiv nrog 150 μL ntawm PBS uas muaj 20% FBS rau 1 teev ntawm 37 degree. Monoclonal cov tshuaj tiv thaiv tau serially diluted 3-fold pib ntawm 10 ug/mL hauv PBS thiab incubated nyob rau hauv lub qhov dej rau 1 teev ntawm 37 degree . Tom qab ntawd cov phaj raug ntxuav thiab incubated nrog HRP-conjugated tshis los tiv thaiv tib neeg IgG antibody (Jackson ImmunoResearch; 109- 035-098), 1: 1, 000) rau 1 teev ntawm 37 degree . Tom qab ntxuav, 100 μL ntawm Super AquaBlue ELISA substrate (eBioscience) tau ntxiv rau ib qhov dej. Absorbance tau ntsuas ntawm 405nm ntawm microplate spectrophotometer (Bio-Rad). Cov kev ntsuam xyuas tau raug tsim los siv cov tshuaj tiv thaiv kab mob S144-509 (15), nrog rau kev paub txog cov yam ntxwv ntawm txhua lub phaj, thiab cov phiajcim tau tsim kom txog thaum lub absorbance ntawm kev tswj mus txog OD ntawm 3.0. Tag nrho mAbs tau sim ua dua, thiab txhua qhov kev sim tau ua ob zaug.
Serum ELISA. High protein-binding microtiter daim hlau tau coated nrog recombinant SARS-CoV-2 spike antigens ntawm 2 ug/mL hauv PBS thaum hmo ntuj ntawm 4 degree. Cov phaj tau ntxuav nrog PBS 0.05% Tween thiab thaiv nrog 200 μL PBS 0.1% Tween + 3% skim mis hmoov rau 1 teev ntawm chav tsev kub (RT). Cov plasma kuaj tau kub-ua haujlwm rau 1 teev ntawm 56 degree ua ntej ua qhov kev sim serology. Plasma yog serially diluted 2-fold hauv PBS 0.1% Tween + 1% skim mis hmoov. Cov phaj tau incubated nrog cov ntshav dilutions rau 2 teev ntawm RT. HRP-conjugated tshis los tiv thaiv tib neeg Ig cov tshuaj tiv thaiv kab mob thib ob diluted ntawm 1: 3, 000 nrog PBS 0.1% Tween + 1% skim mis hmoov tau siv los kuaj xyuas cov tshuaj tiv thaiv kab mob. Tom qab 1 teev ntawm incubation, daim hlau tau tsim nrog 100 μL SigmaFast OPD tov (Sigma-Aldrich) rau 10 feeb. Tom qab ntawd, 50 μL 3M HCl tau siv los nres cov tshuaj tiv thaiv kev loj hlob. Absorbance tau ntsuas ntawm 490 nm ntawm microplate spectrophotometer (Bio-Rad). Endpoint titers tau extrapolated los ntawm sigmoidal 4PL (qhov twg x yog log concentration) tus qauv nkhaus rau txhua tus qauv. Qhov txwv ntawm kev kuaj pom (LOD) yog txhais raws li qhov txhais tau tias + 3 SD ntawm OD teeb liab sau tseg siv ntshav los ntawm pre-SARS-CoV-2 cov tib neeg. Txhua qhov kev suav tau ua tiav hauv GraphPad Prism software (version 9.0).
Kev sib tw ELISA. Txhawm rau txiav txim siab lub hom phiaj epitope kev faib tawm ntawm RBD-reactive mAbs, kev sib tw ELISAs tau ua los ntawm lwm cov mAbs uas paub cov epitope khi cov yam ntxwv raws li cov neeg sib tw mAbs. Cov neeg sib tw mAbs tau biotinylated siv EZ-Link sulfo-NHS-biotin (Thermo Fisher Scientific) rau 2 teev ntawm RT. Cov biotin ntau dhau ntawm biotinylated mAbs raug tshem tawm nrog 7k molecular weight-cutoff (MWCO) Zeba spin desalting txhua (Thermo Fisher Scientific). Cov phaj tau coated nrog 2 ug / mL RBD antigen thaum hmo ntuj ntawm 4 degree. Cov phaj tau thaiv nrog PBS–20% FBS rau 2 teev ntawm RT, thiab 2-fold dilution ntawm mAbs ntawm cov chav kawm tsis tau txiav txim siab lossis cov ntshav qab zib tau ntxiv, pib ntawm 20 ug/mL ntawm mAbs thiab 1:10 dilution ntawm cov ntshav. Tom qab cov tshuaj tiv thaiv kab mob incubation rau 2 teev ntawm RT, biotinylated tus neeg sib tw mAb tau ntxiv ntawm qhov concentration ob zaug ntawm nws qhov kev sib cais tsis tu ncua (KD) thiab incubated rau lwm 2 teev ntawm RT ua ke nrog mAb los yog ntshav qab zib uas tau ntxiv yav dhau los. Cov phaj raug ntxuav thiab incubated nrog 100 μL HRP-conjugated streptavidin (Southern Biotech) ntawm ib tug dilution ntawm 1: 1, 000 rau 1 teev ntawm 37 degree . Cov phiajcim tau tsim nrog Super AquaBlue ELISA substrate (eBioscience). Txhawm rau normalize qhov kev ntsuam xyuas, tus neeg sib tw biotinylated mAb tau ntxiv rau hauv qhov dej tsis muaj kev sib tw mAbs lossis cov ntshav dej raws li kev tswj hwm. Cov ntaub ntawv raug kaw thaum lub absorbance ntawm kev tswj zoo mus txog OD ntawm 1.0-1.5. Qhov feem pua ntawm cov kev sib tw ntawm mAbs tau muab xam los ntawm kev faib ib qho piv txwv ntawm kev soj ntsuam OD los ntawm OD mus txog los ntawm kev tswj qhov zoo, rho tawm tus nqi no los ntawm 1, thiab muab faib los ntawm 100. Rau cov ntshav, ODs tau log10 hloov pauv thiab tshuaj xyuas los ntawm nonlinear regression los txiav txim siab 50. % inhibition concentration (IC50) qhov tseem ceeb siv GraphPad Prism software (version 9.0). Cov ntaub ntawv tau hloov mus rau Log1P thiab npaj rau hauv ib daim duab sawv cev ntawm kev sib txuas ntawm cov ntshav ntshav ntawm IC50 ntawm cov ntshav dilution uas tuaj yeem ua tiav 50% kev sib tw nrog cov neeg sib tw mAb ntawm kev txaus siab. Tag nrho mAbs tau sim ua dua, txhua qhov kev sim tau ua 2 zaug ntawm nws tus kheej, thiab qhov txiaj ntsig los ntawm 2 qhov kev sim ywj pheej tau nruab nrab.
Plaque kev soj ntsuam. Plaque assays tau ua nrog SARS-CoV-2 variant viruses ntawm Vero E6/TMPRSS2 cells (Japanese Collection of Research Bioresources (JCRB)) (Supplemental Table 5). Cells raug coj los ua kom tiav 90% confluency ua ntej yuav trypsinized thiab noob ntawm ib tug ceev ntawm 3 × 104 hlwb/zoo nyob rau hauv 96-zoo daim hlau. Hnub tom qab, 102 PFUs ntawm SARS-CoV{11}} variant tau incubated nrog 2-fold-diluted mAbs rau 1 teev. Cov tshuaj tiv thaiv kab mob sib xyaw ua ke tau tsim nrog Vero E6 / TMPRSS2 hlwb rau 3 hnub ntawm 37 degree. Cov phaj tau kho nrog 20% methanol thiab tom qab ntawd stained nrog crystal violet tov. Qhov ua tiav inhibitory concentrations (IC99) tau suav nrog siv lub cav (inhibitor) piv rau cov lus teb zoo li qub (qhov sib txawv ntawm txoj kab nqes), ua hauv GraphPad Prism (version 9.0). Tag nrho mAbs tau sim ua dua, thiab txhua qhov kev sim tau ua ob zaug. Ua kom pom tseeb txo qis neutralization xeem. Kev ntsuam xyuas kom txo qis nruab nrab (FRNTs) tau siv los txiav txim siab txog kev ua haujlwm nruab nrab raws li lub platform ntxiv ib sab ntawm cov tshuaj plaque. Serial dilutions ntawm cov ntshav pib ntawm qhov kawg siab ntawm 1:20 tau sib xyaw nrog 103 tsom-tsim cov kab mob ntawm ib qhov dej thiab incubated rau 1 teev ntawm 37 degree. Ib qho piv txwv ntawm prepandemic serum tau txais kev tswj hwm. Cov tshuaj tiv thaiv kab mob sib xyaw ua ke tau muab tso rau hauv Vero E6 / TMPRSS2 hlwb (JCRB) hauv 96- cov phiaj zoo thiab incubated rau 1 teev ntawm 37 degree . Ib qhov sib npaug ntawm cov tshuaj methylcellulose tau ntxiv rau txhua qhov dej. Cov hlwb raug incubated rau 16 teev ntawm 37 degree thiab tom qab ntawd kho nrog formalin. Tom qab cov formalin raug tshem tawm, cov hlwb raug txhaj tshuaj nrog nas mAb tiv thaiv SARS-CoV-1/2 nucleoprotein [clone 1C7C7 (Sigma-Aldrich)], tom qab ntawd los ntawm HRP-labeled tshis anti-nas immunoglobulin (Sigma- Aldrich; A8924). Cov kab mob kis tau raug stained nrog TrueBlue Substrate (SeraCare Life Sciences) thiab tom qab ntawd ntxuav nrog dej distilled. Tom qab ziab, cov lej tsom xam tau ntsuas los ntawm kev siv ImmunoSpot S6 Analyzer, ImmunoCapture software, thiab BioSpot software (Cellular Technology). Lub IC50 tau suav los ntawm tus nqi sib cuam tshuam los ntawm lub cav (inhibitor) piv rau cov lus teb zoo ib yam, siv qhov sib txawv ntawm txoj kab nqes (4 tsis) tsis muaj kab rov tav ua hauv GraphPad Prism (version 9.0).
Cov ntaub ntawv
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