Dissection Of Antibody Responses Of Gam-COVID-Vac Vaccinated Subjects suggests Involvement Of Epitopes Outside RBD in SARS-CoV-2 Neutralization
Oct 16, 2023
Abstract: Ntau lab tus tib neeg tau txhaj tshuaj tiv thaiv Gam-COVID-Vac tab sis qhov tshwj xeeb zoo ntawm cov tshuaj tiv thaiv kab mob tsis tau kawm tiav. Plasma los ntawm 12 naïve thiab 10 tus kab mob coronavirus 2019 (COVID-19) cov neeg mob tau txais kev kho mob ua ntej thiab tom qab ob qhov kev txhaj tshuaj nrog Gam-COVID-Vac. Antibody reactivity nyob rau hauv cov plasma cov qauv (n=44) tau kawm nyob rau hauv ib lub vaj huam sib luag ntawm micro-arrayed recombinant folded thiab unfolded mob hnyav ua pa mob tus mob coronavirus 2 (SARS-CoV-2) proteins thiab 46 peptides spanning spikes. protein (S) thiab immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). Lub peev xwm ntawm Gam-COVID-Vac-induced antibodies los inhibit binding ntawm receptor-binding domain (RBD) rau nws cov receptor angiotensin-hloov enzyme 2 (ACE2) tau tshawb xyuas hauv kev tshuaj ntsuam xyuas molecular (MIA). Tus kab mob-tsis muaj peev xwm ntawm cov tshuaj tiv thaiv tau kawm los ntawm pseudo-typed virus neutralization test (pVNT) rau Wuhan-Hu-1 thiab Omicron. Peb pom tias kev txhaj tshuaj Gam-COVID-Vac ua rau muaj kev nce ntxiv ntawm IgG1 tab sis tsis yog ntawm lwm cov IgG subclasses tiv thaiv folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), thiab RBD nyob rau hauv ib qho kev sib piv hauv naïve thiab convalescent. yam. Tus kab mob neutralization muaj feem cuam tshuam nrog kev txhaj tshuaj tiv thaiv kab mob tshwj xeeb rau folded RBD thiab cov peptide tshiab (piv txwv li, peptide 12). Peptide 12 tau nyob ze rau RBD nyob rau hauv N-terminal ib feem ntawm S1 thiab tej zaum yuav muaj feem cuam tshuam rau kev hloov pauv ntawm kev hloov ua ntej mus rau tom qab fusion conformation ntawm spike protein. Hauv cov ntsiab lus, kev txhaj tshuaj Gam-COVID-Vac induced S-specific IgG1 antibodies nyob rau hauv naive thiab convalescent kawm nyob rau hauv ib tug piv txwv. Dhau li ntawm cov tshuaj tiv thaiv tshwj xeeb rau RBD, cov tshuaj tiv thaiv kab mob cuam tshuam rau peptide ze rau N-terminus ntawm RBD kuj tseem cuam tshuam nrog tus kab mob-tsis zoo.
Keywords: SARS-CoV-2; COVID-19; Gam-COVID-Vac; epitope; tshuaj tiv thaiv; tus kab mob-neutralization; Kev soj ntsuam kev sib cuam tshuam ntawm molecular; Omicron
cistanche ntxiv cov txiaj ntsig-nce kev tiv thaiv
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1. Taw qhia
Tom qab thawj kis ntawm tus kab mob Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) thaum kawg ntawm xyoo 2019 hauv Wuhan, Tuam Tshoj, tus kab mob tshiab coronavirus (COVID-19) sai sai kis thoob plaws. lub ntiaj teb thiab loj hlob mus rau thoob ntiaj teb kev sib kis [1–4]. Kev loj hlob sai ntawm COVID-19 cov tshuaj tiv thaiv thiab kev qhia txog kev txhaj tshuaj tiv thaiv thoob ntiaj teb ua ke nrog cov tsos mob ntawm cov kab mob tsawg dua SARS-CoV-2 variants yog suav tias yog qhov tseem ceeb rau kev txo qis ntawm COVID-19- cuam tshuam kev tuag thiab qhov hnyav ntawm kev sib kis [1,5]. Thawj cov tshuaj tiv thaiv uas tau dhau los yog cov tshuaj tiv thaiv kab mob raws li kev hloov pauv ntawm adenovirus-mediated hloov ntawm cov gene coding rau SARS-CoV-2 surface protein S [6–8]. SARS-CoV-2 S protein muaj S1 subunit uas muaj cov receptor binding domain (RBD) txuas rau nws cov receptor ACE2 ntawm tib neeg lub hlwb thiab S2 subunit uas ua kom cov kab mob txuas mus rau lub cell membrane los ntawm proteolytic cleavage ntawm lub fusion peptide (FP) thaum kis tus kab mob [9]. S gene siv los hloov cov noob hauv qee yam tshuaj tiv thaiv kab mob adenovirus (piv txwv li, COVID-19 Vaccine Janssen) tuaj yeem hloov kho kom ruaj khov cov protein thiab cuam tshuam rau nws qhov kev sib cais tab sis qhov no tsis yog rau lwm tus [10]. Tib neeg cov hlwb uas kis tau los ntawm adenoviruses uas muaj S-encoding gene tsim ntau qhov sib txawv ntawm S antigen uas tau tso tawm thiab nthuav tawm los ntawm qhov loj histocompatibility complex (MHC) chav kawm II rau T hlwb ua rau CD4+ T cell pab. rau zus tau tej cov S-specific antibodies. S protein qhia los ntawm cov kab mob sib kis kuj tau nthuav tawm los ntawm MHC chav kawm I nyob rau saum npoo ntawm cov kab mob ntawm tes thiab ua rau muaj kev ua haujlwm ntawm S-specific cytotoxic CD8+ T hlwb, uas tuaj yeem paub S antigen-tsim hlwb thiab ua kom puas. Lub hauv paus ntsiab lus ntawm kev txhaj tshuaj tiv thaiv, piv txwv li, kev qhia txog S-encoding genetic cov ntaub ntawv rau hauv cov tswv tsev thiab tom qab tsim S antigen los ntawm cov kab mob uas muaj kab mob, zoo ib yam li cov tshuaj tiv thaiv kab mob messenger ribonucleic acid (mRNA), txawm hais tias cov thev naus laus zis ntawm cov noob hloov pauv txawv ntawm adenovirus. - cov tshuaj tiv thaiv. Yog li, "cov tshuaj tiv thaiv caj ces" ua rau qee qhov kev kis tus kabmob SARS-CoV-2. Kev txheeb xyuas cov epitopes lees paub los ntawm cov tshuaj tiv thaiv los ntawm cov tshuaj tiv thaiv kev koom tes hauv kev tiv thaiv tus kab mob yog qhov tseem ceeb tsis yog rau kev nkag siab txog cov txheej txheem ntawm kev ua ntawm cov tshuaj tiv thaiv uas twb muaj lawm tab sis kuj rau kev txhim kho cov tshuaj tiv thaiv zoo thiab cov tswv yim tiv thaiv kab mob tiv thaiv COVID-19 [11 , 12] ib. Feem ntau ntawm cov tshuaj tiv thaiv tam sim no tau tsom mus rau qhov tau txais S-cov tshuaj tiv thaiv kab mob tshwj xeeb nrog kev saib xyuas tshwj xeeb rau RBD-cov tshuaj tiv thaiv kab mob tshwj xeeb vim nws tau pom tias RBD-cov tshuaj tiv thaiv tshwj xeeb muaj feem cuam tshuam nrog kev ua haujlwm siab nruab nrab tiv thaiv SARS-CoV-2 [ 13–15] ib. Txawm li cas los xij, tus naj npawb ntawm kev hloov pauv tau los ntawm txhua qhov kev txhawj xeeb tshiab (VOC), qhia qhov tseem ceeb ntawm kev txheeb xyuas cov epitopes khaws cia ntawm VOCs tsom los ntawm cov tshuaj tiv thaiv kab mob uas tsis yog nyob hauv RBD nkaus xwb tab sis kuj sab nraum RBD [16]. Qhov tseeb, muaj cov kev tshawb fawb qhia tias muaj qhov nruab nrab ntawm cov epitopes sab nraum RBD, uas tau lees paub los ntawm cov tshuaj tiv thaiv tom qab txhaj tshuaj [17-20]. Xyoo 2020 cov tshuaj tiv thaiv vector-raws li Gam-COVID-Vac, tseem hu ua Sputnik-V, tau dhau los siv rau hauv kev kho mob thiab tam sim no tau pom zoo siv ntau dua 70 lub teb chaws (https://sputnikvaccine.com/about-vaccine / ( nkag mus rau 17 Lub Ib Hlis 2023)). Raws li Peb Lub Ntiaj Teb Hauv Cov Ntaub Ntawv (OWID) cov peev txheej, 79 lab tus tib neeg hauv tebchaws Russia tau txais tsawg kawg ib qho tshuaj tiv thaiv, thiab 72 lab tau txhaj tshuaj tiv thaiv tag nrho (https://ourworldindata.org/ covid-vaccinations?country= RUS (mus txog 22 Lub Peb Hlis 2022)). Gam-COVID-Vac muaj ob hom tshuaj tiv thaiv, uas yog ua raws li ob hom tib neeg adenovirus vectors (hAd26 thiab hAd5) nqa cov ntaub ntawv haiv neeg ntawm S-protein ntawm Wuhan-Hu-1 strain SARS-CoV{{ 73}} ib. Sputnik-V suav nrog ob txoj kev txhaj tshuaj, ib qho nrog hAd26 thiab ib qho thib ob nrog hAd5 thiab Sputnik lub teeb, tsuas yog hAd26. Ob qhov sib txawv adenovirus vectors yog siv rau kev txhaj tshuaj thawj zaug thiab thib ob txhawm rau txo qhov muaj peev xwm uas cov neeg txhaj tshuaj tiv thaiv kev tiv thaiv kab mob S protein tiv thaiv kab mob adenovirus. Qhov tseeb, adenovirus-cov tshuaj tiv thaiv tshwj xeeb yuav txwv qhov ua tau zoo ntawm kev txhaj tshuaj tiv thaiv kab mob los ntawm kev ua kom tsis zoo ntawm adenovirus vector tsheb. Yog li ntawd, nyob rau hnub 21 tom qab thawj koob tshuaj txhaj tshuaj tiv thaiv nrog rAd26, rov xa cov tshuaj tiv thaiv antigen yog nqa tawm nrog rAd5 vector [8,21]. Ob peb txoj kev tshawb fawb tau tshawb xyuas txog qhov muaj txiaj ntsig zoo li S- thiab RBD-cov tshuaj tiv thaiv tshwj xeeb cov lus teb thiab cov kab mob cuam tshuam rau kev ua haujlwm hauv sera los ntawm Gam-COVID-Vac cov tshuaj tiv thaiv [10,22,23]. Kev nce ntawm RBD-specific IgG cov tshuaj tiv thaiv peb lub hlis tom qab Gam-COVID-Vac cov thawj coj tau tshaj tawm thiab nws tau pom tias cov tshuaj tiv thaiv kab mob sib kis nrog Alpha, Beta, Delta, thiab Omicron variants ntawm kev txhawj xeeb [24–26]. Gam-COVID-Vac kuj tau muab piv nrog ntau lwm cov tshuaj tiv thaiv muaj ntawv tso cai tiv thaiv SARS-CoV-2, uas tau pom tias muaj tus kab mob tsis zoo thiab muaj txiaj ntsig zoo tiv thaiv COVID-19- cuam tshuam txog kev tuag rau Sputnik-V, txawm tias me ntsis qis dua rau qee cov tshuaj tiv thaiv mRNA hais txog tus kab mob-tsis pom zoo tau pom [24]. Thaum kawg, nws tau pom tias kev txhaj tshuaj tiv thaiv kab mob human immunodeficiency virus-positive (HIV+) antiretroviral therapy-kho (ART-kho) cov neeg mob Gam-COVID-Vac qhia tau hais tias muaj kev sib kis tau zoo tiv thaiv cov tsiaj qus qub thiab Delta variant ntawm SARS-CoV{{ 113}} [27] ib. Txawm li cas los xij, txog tam sim no tsis muaj cov ncauj lus kom ntxaws txog kev paub txog epitope los ntawm cov tshuaj tiv thaiv hauv Gam-COVID-Vac cov tshuaj txhaj tshuaj tau ua thiab cov ntaub ntawv xav tau kom nkag siab txog immunoglobulin isotype thiab IgG subclass cov lus teb hauv Gam-COVID-Vacvaccinated subjects. Ntawm no peb tau tshawb xyuas ob pawg ntawm cov neeg uas tau txhaj tshuaj tiv thaiv Gam-COVID-Vac, ib tus uas tau muaj tus kab mob SARS-CoV-2 dhau los, thiab lwm pab pawg ntawm SARS-CoV-2 tsis zoo. Peb tau ua qhov kev soj ntsuam nruj me ntsis ntawm cov epitope tshwj xeeb ntawm induced antibodies siv micro-arrayed SARS-CoV-2- muab folded thiab unfolded proteins thiab peptides spans tag nrho S protein, suav nrog RBD. Tsis tas li ntawd, IgG subclass teb tau raug tshuaj xyuas. Peb txoj kev tshawb fawb nthuav tawm tias Gam-COVID-Vac-cov tshuaj txhaj tshuaj tiv thaiv kab mob nce RBD-cov tshuaj tiv thaiv tshwj xeeb muaj peev xwm thaiv kev cuam tshuam RBD-ACE2. Interestingly, peb pom tias tus kab mob neutralization kuj tseem cuam tshuam nrog cov tshuaj tiv thaiv kab mob tiv thaiv peptide sab nraum RBD, uas tej zaum yuav cuam tshuam rau kev hloov pauv ntawm preto-post-fusion conformation ntawm spike protein.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
2. Cov txiaj ntsig
2.1. Gam-COVID-Vac-Vaccinated Subjects suav nrog COVID-19 Cov Ntsiab Lus Tsis Txaus Siab thiab Tsis Txaus Siab
Convalescent cov neeg mob tau kis los ntawm Wuhan-Hu-1 strain raws li nws qhov kev nthuav dav thiab cov ntaub ntawv sib txuas tau txais thaum lub sijhawm kis kab mob hauv cheeb tsam Moscow. Cov neeg tsis paub qab hau tsis muaj tsos mob ntawm COVID-19 thiab tsis muaj SARS-CoV-2- cov tshuaj tiv thaiv tshwj xeeb thaum lub sijhawm nrhiav neeg ua haujlwm. Cov kev kawm suav nrog 15 tus poj niam thiab 7 tus txiv neej uas muaj hnub nyoog nruab nrab ntawm 60 (xws li, 25–70 xyoo) (Table 1).
Table 1. Cov neeg pej xeem, chaw kho mob, thiab serological yam ntxwv ntawm cov kev kawm.
Cov qauv ntshav tau txais los ntawm txhua qhov kev kawm ntawm lub sijhawm taw tes 0 (piv txwv li, kab hauv paus) (Daim duab 1A), tom qab ntawd txhua qhov kev kawm tau txhaj tshuaj tiv thaiv Sputnik-V nrog lub sijhawm kwv yees li 21 hnub ntawm thawj thiab thib ob. txhaj tshuaj (lub sij hawm cov ntsiab lus 1 thiab 2). Thaum lub sij hawm taw tes 3 tau sau cov plasma thib ob los txiav txim cov tshuaj tiv thaiv kab mob tiv thaiv kab mob (Daim duab 1A). Cov ntsiab lus raug soj ntsuam ntxiv rau 120 hnub ntxiv. Hnub 110 tom qab txhaj tshuaj tiv thaiv zaum kawg ib qho kev kawm (piv txwv li, cov ntsiab lus 22) muaj COVID-19 thaum lwm tus tsis tau tsim cov tsos mob ntawm COVID-19 (Table 1).
Daim duab 1. Kawm tsim thiab SARS-CoV-2-cov tshuaj tiv thaiv tshwj xeeb ua ntej thiab tom qab txhaj tshuaj tiv thaiv Sputnik-V. (A) Kawm tsim qauv. Cov qauv ntshav ntshav tau txais los ntawm COVID-19-naïve (n=12) thiab cov ntsiab lus convalescent (n=10) ntawm lub sij hawm taw tes 0 (baseline), ces cov neeg kawm tau txais ob Sputnik-V Kev txhaj tshuaj tiv thaiv ntawm lub sijhawm cov ntsiab lus 1 thiab 2, raws li thiab tau txais cov qauv ntshav plasma ntawm lub sijhawm 3 (piv txwv li, hnub 80-85 tom qab txhaj tshuaj tiv thaiv thawj zaug). (B) Nucleocapsid-specific IgG antibody qib (y-axis: ISU) hauv cov ntsiab lus tsis zoo thiab convalescent (x-axis) ntawm lub hauv paus. Kab rov tav qhia qhov kev txiav tawm rau qhov zoo. (C) IgG antibody qib (y-axes: ISU) tshwj xeeb rau RBD, S, S1, thiab S2 hauv cov ntsiab lus ntawm lub sijhawm taw tes 0 thiab 3 (x-axes). (D) Los ntawm sab laug mus rau sab xis IgG1 , IgG2 , IgG3 , thiab IgG4 antibody qib (y-axes: optical density OD values) rau S (sab sauv) thiab RBD (qis dua) ntawm lub sij hawm cov ntsiab lus 0 thiab 3 ( x-axes). Qhov sib txawv tseem ceeb ntawm cov pab pawg raws li tau txiav txim los ntawm Wilcoxon xeem yog qhia: ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns — tsis tseem ceeb.
2.2. Gam-COVID-Vac Vaccination Induces Broad IgG Responses to S suav nrog S1, RBD, thiab S2
cistanche ntxiv cov txiaj ntsig-nce kev tiv thaiv
Peb tau ua qhov kev soj ntsuam ntxaws ntxaws ntawm cov tshuaj tiv thaiv cov lus teb rau lub vaj huam sib luag loj ntawm micro-arrayed folded thiab nthuav tawm SARS-CoV-2 proteins thiab peptides hla S protein nyob rau hauv cov plasma cov qauv los ntawm SARS-CoV-2-naïve thiab convalescent kawm Tom qab kev txhaj tshuaj tag nrho nrog Gam-COVID-Vac (Daim duab 1A,B; Table 1). Thaum muab piv cov ntshav plasma los ntawm lub sij hawm taw tes 0 thiab lub sij hawm taw tes 3 tom qab txhaj tshuaj, ib qho tseem ceeb induction ntawm IgG cov lus teb tawm tsam folded tab sis tsis nthuav tawm RBD, S, S1, thiab S2 tau pom (Daim duab 1C). Peb lub hlis tom qab txhaj tshuaj tiv thaiv cov neeg mob tau pom tias muaj 21-Pib nce ntawm RBD-specific IgG cov tshuaj tiv thaiv (p < 0.{{20}}001) thiab ib qho {{ 18}}fold nce ntawm S-specific IgG cov tshuaj tiv thaiv (piv txwv li, lub sij hawm taw tes 0: nruab nrab 0.291, lub sij hawm taw tes 3: nruab nrab 5.778) (p < 0.0001), feem. IgG cov tshuaj tiv thaiv rau S1 nce 3.7- zaug (p=0.0023) thiab IgG cov tshuaj tiv thaiv rau S2 nce 2.8-x (p=0.0094) (Daim duab 1C ). Kev txhaj tshuaj tiv thaiv nrog Gam-COVID-Vac induced feem ntau IgG cov lus teb rau folded S, S1, thiab RBD piv rau hauv cov ntsiab lus tsis zoo thiab convalescent (Figures 1C, S2 thiab S3), qhia tias nws tuaj yeem siv los txhawb SARS-CoV{-2- cov tshuaj tiv thaiv kab mob tshwj xeeb kuj tseem nyob hauv cov neeg mob convalescent. Qhov tseem ceeb boosts ntawm IgG cov tshuaj tiv thaiv rau RBD, S, thiab S1 tau pom nyob rau hauv cov ntsiab lus tsis muaj zog thiab tsis muaj zog (Daim duab S2) thiab tsis muaj qhov sib txawv tseem ceeb ntawm IgG qib rau RBD, S, S1, thiab S2 hauv cov ntsiab lus tsis zoo thiab convalescent thaum lub sij hawm taw tes 3 (Daim duab S3).
2.3. Gam-COVID-Vac Vaccination Induces Mainly IgG1 Subclass Response to S thiab RBD
Nws tau raug tshaj tawm tias cov tshuaj tiv thaiv kab mob tshwj xeeb (AIT) ua rau muaj qhov ua xua tshwj xeeb IgG1 thiab IgG4 cov lus teb uas txawv tsis yog hais txog lawv txoj haujlwm ua haujlwm (IgG1 tuaj yeem kho cov tshuaj tiv thaiv kab mob-nyob ntawm tes cellular cytotoxicity (ADCC) thiab ua kom muaj zog ntxiv thaum IgG4 tsis muaj. effector functions) tab sis kuj hais txog lawv cov kinetics (IgG1: pib sai thiab ntxov poob: IgG4: qeeb pib tab sis txhawb nqa qib) [28–30]. Yog li peb tau tshawb xyuas IgG subclass (piv txwv li, IgG1–IgG4) cov lus teb rau S thiab RBD hauv Sputnik-V-cov tshuaj txhaj tshuaj. Daim duab 1D qhia tau hais tias kev txhaj tshuaj tiv thaiv yuav luag tsuas yog IgG1 subclass teb rau S thiab RBD. Qhov tseem ceeb nce qib IgG1 tshwj xeeb rau S thiab RBD (p=0.0002 thiab p=0.0071, raws li)) tau pom tom qab txhaj tshuaj tiv thaiv. Txawm li cas los xij, tsis muaj qhov cuam tshuam rau IgG2, IgG3, lossis IgG4 subclass cov lus teb tshwj xeeb rau S thiab RBD tuaj yeem kuaj pom (Daim duab 1D).
2.4. Induction of Virus-Neutralizing Antibodies and Antibodies Inhibiting the RBD-ACE2 Interaction by Gam-COVID-Vac Vaccination
Rau kev kuaj pom tus kab mob neutralizing cov tshuaj tiv thaiv, peb siv pVNT kuaj, qhov twg tus kab mob zoo li cov kab mob tau pseudo-typed nrog S-protein los ntawm tus thawj Wuhan-Hu-Hu hom kab mob thiab tsis ntev los no tau piav VOC Omicron. Cov tshuaj tiv thaiv nrog kev ua haujlwm tsis zoo rau Wuhan Hu-1 strain tau pom nyob rau hauv tag nrho tab sis ib qho (piv txwv li, cov ntsiab lus 22) ntawm 22 yam (Daim duab 2A, Table 2) whereas neutralizing antibody titers rau Omicron tau qis dua (Daim duab 2B) thiab pom tsuas yog hauv 17 ntawm 20 yam kev kawm (Daim duab 2A, Table 2). Tsis muaj qhov cuam tshuam rau qhov nruab nrab ntawm cov ntsiab lus 6, 7, 10, 11, 15 (Daim duab 2A, Table 2). Tus kab mob neutralization titers rau Omicron yog 25 npaug qis dua rau WT (qhov nruab nrab 141.9 thiab 5.7 rau Wuhan-Hu-1 thiab Omicron, feem) (Daim duab 2B).
Daim duab 2. Virus-neutralizing kev ua haujlwm hauv plasma 3 lub hlis tom qab txhaj tshuaj Sputnik-V thiab koom nrog cov tshuaj tiv thaiv IgG rau RBD thiab S-derived peptides. (A) Tus kab mob nruab nrab (ID50) (y-axis) tawm tsam Wuhan-hu-1 strain (ntsuab) thiab Omicron (txiv kab ntxwv) tau txais cov ntshav los ntawm naïve (xiav) thiab convalescent (liab) cov ntsiab lus (x-axes). (B) Kev sib piv ntawm tus kab mob nruab nrab (y-axis: ID50 tau txais cov ntshav ntshav los ntawm cov kev sim, cov kab nruab nrab nruab nrab nrog cov kab sib txuas (IQR) Wuhan Hu-1 (ntsuab) thiab Omicron (txiv kab ntxwv) . Cov kab kab rov tav sawv cev rau cov txiaj ntsig tau los ntawm cov ntshav ntshav los ntawm kev noj qab haus huv, tsis muaj kab mob (kev tswj hwm kev noj qab haus huv, HC). {{30}}001. (C) Localization ntawm RBD thiab peptides correlated nrog tus kab mob neutralization nyob rau hauv ib tug tswvyim ntawm S (S1, S2). (D) Kev sib raug ntawm tus kab mob neutralization titers rau Wuhan-Hu{{19 }} (WT) lossis Omicron (y-axes) thiab IgG qib tshwj xeeb rau RBD thiab spike-derived peptides 12, 32, thiab 46A (x-axes: ISU). Cov cim xiav thiab xim liab qhia txog cov ntsiab lus tsis zoo thiab convalescent, ntsig txog. qhia txog kev sib raug zoo raws li qhov kev ntsuam xyuas nonparametric Spearman, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001, ns=tsis tseem ceeb.
Table 2. Kev sib piv ntawm IgG qib tshwj xeeb rau S, S1, S2, thiab RBD ntsuas los ntawm micro-array nrog inhibitions ntawm RBD khi rau ACE2 thiab tus kab mob neutralization titers (Wuhan-Hu-1, Omicron) txiav txim siab rau cov ntshav los ntawm cov tshawb xyuas cov ntsiab lus. Kev ntsuas kub tau ua rau txhua qhov ntsuas cais siv Excel. Qhov siab tshaj plaws tau suav tias yog xim liab tsaus thiab qhov qis tshaj yog dawb.
Qhov tseeb, nyob rau hauv tag nrho tab sis ib qho kev kawm (piv txwv li, cov ntsiab lus 22) tus kab mob neutralizing cov tshuaj tiv thaiv kab mob tau nce ntxiv tom qab txhaj tshuaj tiv thaiv qhia tau tias lawv raug txhaj tshuaj tiv thaiv (Table 2). Cov ntsiab lus 21 tau pom tias twb muaj kev thaiv siab ntawm RBD-ACE2 kev cuam tshuam hauv MIA thiab kev tiv thaiv kab mob siab ntawm lub hauv paus (Table 2). Qhov tseem ceeb, cov qauv plasma los ntawm Gam-COVID-Vac txhaj tshuaj tiv thaiv kab mob convalescent cov neeg mob uas muaj tus kab mob nruab nrab (#1, 2, 17, 18, 21) muaj cov kab mob tsis zoo siab dua li cov neeg mob uas muaj kab mob me me (#22) lossis tsis muaj tsos mob. (#4, 5, 7, 9) (Table 1 thiab 2). Txawm li cas los xij, qhov kev tshawb pom tseem ceeb yog qhov inhibition ntawm RBD khi rau ACE2 thiab tus kab mob nruab nrab tsis yog ib txwm cuam tshuam. Ntau tshaj 25% inhibition ntawm kev khi ntawm RBD Wuhan Hu-1 rau ACE2 tsuas yog tshwm sim rau 6 tus neeg mob uas tau pom tias muaj kev cuam tshuam hauv kev kuaj kab mob tsis zoo vim yog txhaj tshuaj tiv thaiv (piv txwv li, hom 1, 2, 3, 17, 18 , thiab 19) (Table 2). Interestingly, virus neutralizing kev ua si (50% kis kab mob (ID50) > 100) tau soj ntsuam rau 6 yam tab sis lawv tsis pom los yog tsis zoo inhibition ntawm RBD binding rau ACE2 (Table 2: Kawm 6, 10, 12, 13, 16, 20) .
2.5. Kab mob Neutralization hauv Gam-COVID-Vac-Vaccinated Subjects Muaj Kev Sib Raug Zoo nrog IgG Cov Lus Teb rau RBD thiab rau S-Derived Peptide 12
Raws li tau hais ua ntej, plasma los ntawm ntau yam Gam-COVID-Vac-cov tshuaj tiv thaiv (piv txwv li, hauv cov ncauj lus 6, 10, 12, 13, 16, 20; Table 2) tau pom tus kab mob tsis ua haujlwm tab sis tsis cuam tshuam rau RBD-ACE2 kev cuam tshuam. Yog li ntawd peb tau txheeb xyuas IgG cov tshuaj tiv thaiv kab mob rau cov peptides hla S protein los ntawm micro-array-raws li nti tsom xam [13] (Daim duab S4 thiab daim duab 2C) thiab sib cuam tshuam peptide-specific IgG cov lus teb nrog tus kab mob neutralization ntawm Wuhan-Hu{19} } strain thiab Omicron (Daim duab 2D). IgG teb rau peb peptides (piv txwv li, peptides 12, 32, 46A) xws li IgG cov lus teb rau folded RBD correlated nrog neutralization ntawm Wuhan-Hu -1 strain (Daim duab 2C, D). Peptide 12 sawv tawm vim hais tias cov tshuaj tiv thaiv cov lus teb rau unfolded peptide 12 (Daim duab S1), tab sis tsis yog rau unfolded peptides 32 thiab 46A tau txhawb nqa los ntawm Gam-COVID Vac hauv cov ntsiab lus tsis paub (Daim duab 3A) thiab tseem muaj feem cuam tshuam nrog neutralization ntawm Omicron2D (Daim duab. ). Tsis muaj kev txhawb nqa tseem ceeb rau cov neeg mob convalescent rau peptides 12, 32, lossis 46A (Daim duab 3A). Qhov sib lawv liag ntawm peptide 12 tau txais txiaj ntsig zoo ntawm SARS-CoV-2 strains (Daim duab 4) thiab nyob hauv S1 ze rau N-terminus ntawm RBD thiab yog li qhia txog thaj tsam uas yuav cuam tshuam rau kev hloov pauv ntawm lub sijhawm ua ntej. mus rau tom qab fusion conformation ntawm S protein (Daim duab 3B).
Daim duab 3. Cov qib nruab nrab IgG cov tshuaj tiv thaiv kab mob (y-axes: ISU) rau S-derived peptides (P12, P32, P46A) nyob rau hauv cov tshuaj tiv thaiv tsis zoo thiab convalescent ntawm lub sijhawm cov ntsiab lus 0 thiab 3 (x-axes) (A) . Qhov sib txawv tseem ceeb ntawm cov pab pawg raws li tau txiav txim los ntawm Wilcoxon xeem yog qhia: * p < 0.05, ns=tsis tseem ceeb. (F) (B) Daim duab 3. Qhov nruab nrab IgG antibody theem (y-axes: ISU) mus rau S-derived peptides (P12, P32, P46A) hauv cov tshuaj tiv thaiv tsis zoo thiab convalescent ntawm lub sijhawm cov ntsiab lus 0 thiab 3 (x -axes) (A). Qhov sib txawv tseem ceeb ntawm cov pab pawg raws li tau txiav txim los ntawm Wilcoxon xeem yog qhia: * p < 0.05, ns=tsis tseem ceeb. (B) Visualization ntawm peptides 12, 32, thiab 46A hauv qhov chaw sawv cev ntawm SARS-CoV-2 spike protein trimer (sab saib, pre-fusion conformation) generated nrog PyMOL. Peptide 12 yog qhov tseem ceeb hauv S-trimer qauv hauv xim liab, peptide 32 hauv xiav, thiab 46A hauv txiv kab ntxwv. Carbohydrates pom nyob rau hauv lub teeb grey.
Daim duab 4. Protein sequence alignment of Wuhan-Hu-1 (top) and Omicron (below) of SARS-CoV-2 S-protein. RBD yog xim daj thiab peptides 12, 22, 25, 32, 33, 46A thiab 47 yog underlined. Wuhan S-protein sequence sib raug rau WA1 cais (GeneBank accession Nr.: BCN86353.1), Omicron S-protein sequence Omicron yog clade GRA, kab B.1.1.529 BA.1 raws li GISAID database ( https://www.gisaid.org/ ( nkag mus rau 15 Lub Peb Hlis 2022)). Amino acid hloov pauv yog xim-coded raws li lawv cov hydropathy thiab cov nqi zog (liab=hydrophobic, ntsuab=nruab nrab, lub teeb xiav=yooj yim hydrophilic, thiab lub teeb liab=acidic hydrophilic).
3. Kev sib tham
Vector-based vaccine Gam-COVID-Vac kuj hu ua Sputnik-V, yog ib qho ntawm thawj SARS-CoV-2 cov tshuaj tiv thaiv uas tau los siv rau hauv kev kho mob. Txawm li cas los xij, muaj cov ntaub ntawv me me hais txog qhov tshwj xeeb ntawm cov tshuaj tiv thaiv kab mob hauv cov neeg uas tau txhaj tshuaj tiv thaiv COVID-19. Hauv txoj kev tshawb no, peb tau ua qhov kev soj ntsuam ntxaws ntxaws ntawm cov tshuaj tiv thaiv kab mob rau cov micro-arrayed folded thiab nthuav tawm SARS-CoV-2 proteins thiab peptides spanning S protein nyob rau hauv cov ntshav plasma cov qauv los ntawm SARS CoV{11}}naïve thiab convalescent kawm tom qab kev txhaj tshuaj tag nrho nrog Gam COVID-Vac. Gam-COVID-Vac induced mas IgG1 tab sis tsis muaj lwm yam IgG subclass teb rau folded S, S1, S2, thiab RBD nyob rau hauv ib tug zoo sib piv nyob rau hauv naïve thiab convalescent kawm, qhia tias nws yuav siv tau los txhawb SARS-CoV-2- cov tshuaj tiv thaiv kab mob tshwj xeeb kuj tseem nyob hauv cov neeg mob convalescent. Ib yam li lwm cov tshuaj tiv thaiv kab mob, Gam-COVID-Vac-induced antibody teb nyob hauv IgG1 subclass uas muaj ib nrab-lub neej luv dua piv rau cov lus teb ntawm IgG4 subclass uas muaj kev ruaj ntseg dua. Piv txwv li, cov lus teb tshwj xeeb IgG4 subclass yog txuam nrog kev tiv thaiv mus sij hawm ntev thaum lub sij hawm ua xua rau cov tshuaj tiv thaiv kab mob tshwj xeeb thaum cov lus teb tshwj xeeb IgG1 nce sai tab sis ploj tom qab ob peb lub hlis [28-30]. Yog li, nquag txhaj tshuaj Gam-COVID-Vac thiab lwm yam tshuaj tiv thaiv caj ces yuav tsum tau ua kom tsis txhob muaj cov tshuaj tiv thaiv kab mob nyob rau theem siab. Kev txhaj tshuaj tiv thaiv nrog Gam-COVID-Vac ua rau muaj cov tshuaj tiv thaiv kab mob uas ua rau muaj ntau yam uas pom tom qab muaj kab mob SARS-CoV-2 [13]. Tsis tas li ntawd, cov lus teb rau cov tshuaj tiv thaiv kab mob tom qab kev kis tus kab mob yog feem ntau yog IgG1 subclass thiab raug qhia tawm tsam folded S, S1, S2, thiab RBD [13]. Qhov kev tshawb nrhiav tshiab thiab nthuav dav ntawm peb txoj kev tshawb fawb yog tias cov ntshav ntshav los ntawm ntau yam Gam-COVID-Vac - txhaj tshuaj tiv thaiv kab mob (piv txwv li, hauv cov ncauj lus 6, 10, 12, 13, 16, 20; Table 2) tau pom cov kab mob tsis ua haujlwm tab sis tsis txwv cov Kev sib cuam tshuam ntawm RBD-ACE2. Yog li ntawd, peb tau txheeb xyuas IgG cov tshuaj tiv thaiv kab mob rau cov peptides nthuav dav S protein ua tiav los ntawm micro-array-raws li nti tsom xam [13] (Figures 2D thiab S4) thiab sib txheeb peptide-specific IgG cov lus teb nrog tus kab mob neutralization ntawm Wuhan-Hu{{62} } strain thiab Omicron BA.1 (Daim duab 2D). IgG cov lus teb rau peb peptides (piv txwv li, peptides 12, 32, 46A) xws li IgG cov lus teb rau RBD cuam tshuam nrog kev tsis sib haum xeeb ntawm Wuhan-Hu-1 strain (Daim duab 2C, D). Txawm li cas los xij, peptide 12 yog qhov tshwj xeeb vim tias cov tshuaj tiv thaiv cov lus teb rau peptide 12 nkaus xwb, tab sis tsis yog rau peptides 32 thiab 46A tau txhawb nqa los ntawm kev txhaj tshuaj tiv thaiv (Daim duab 3A) thiab tseem muaj feem cuam tshuam nrog neutralization ntawm Omicron (Daim duab 2D). Qhov loj ntawm kev txhaj tshuaj tiv thaiv kev txhawb nqa ntawm IgG cov lus teb rau peptide 12 yog qhov me me tab sis tseem ceeb hauv cov ntsiab lus tsis zoo thiab zoo sib xws rau kev txhawb nqa ntawm IgG cov tshuaj tiv thaiv rau S thiab S1. Txawm li cas los xij, IgG cov tshuaj tiv thaiv kab mob tiv thaiv cov peptide cais tawm (Daim duab S1) tuaj yeem sawv cev tsuas yog ib feem ntawm cov tshuaj tiv thaiv kab mob tiv thaiv peptide 12 hauv cov ntaub qhwv protein. Tsis tas li ntawd, tsis yog tsuas yog qib tab sis kuj tseem muaj cov tshuaj tiv thaiv kab mob tseem ceeb. Qhov sib lawv liag ntawm peptides 12 tau txais txiaj ntsig zoo ntawm Wuhan-Hu-1 strain thiab BA.1 Omicron VOC (Daim duab 4) thiab yog muab los ntawm thaj chaw uas tej zaum yuav cuam tshuam rau kev hloov pauv ntawm kev ua ntej mus rau tom qab fusion. conformation ntawm S protein (Daim duab 3B). Ntxiv mus, kev sib piv ntawm cov hloov pauv tshiab ntawm Omicron (BA.1, BA.2, BA.4/5, BA.2.12.1, BA.2.75, BQ.1), qhia tias peptide 12 (284–313 aa. ) tseem tsis hloov pauv ntawm cov amino acid ib ntus, sib piv rau, piv txwv li, peptide 32 (https://covariants.org/shared-mutations, ( nkag mus rau 28 Kaum Ib Hlis 2022)). Qhov tseem ceeb, cov tshuaj tiv thaiv kab mob monoclonal nrog tus kab mob-tsis muaj peev xwm tiv thaiv thaj tsam N-terminal domain (NTD) uas peptide 12 yog ib feem tau piav qhia [31], thiab nws tau tshaj tawm tias lwm cov tshuaj tiv thaiv tuaj yeem ua rau muaj cov tshuaj tiv thaiv kab mob tiv thaiv thaj tsam no. [18–20,32], qhia tias thaj av tau teev tseg los ntawm peptide 12 yuav yog ib qho tseem ceeb rau inducing neutralizing antibodies. Yog li ntawd, ib tus yuav xav tias cov tshuaj tiv thaiv kab mob tshwm sim los ntawm Gam-COVID-Vac txhaj tshuaj tiv thaiv tuaj yeem ua rau SARS-CoV-2 los ntawm ob lub tswv yim, ib qho yooj yim los ntawm inhibiting RBD-ACE2 kev cuam tshuam thiab lwm yam los ntawm kev tsom mus rau thaj av uas muaj peptide 12 ntawm S- cov protein uas tuaj yeem koom nrog hauv cov qauv kev hloov pauv (piv txwv li, kev hloov pauv ntawm pre-fusion rau hauv fusion conformation ntawm S). Peb txoj kev kawm muaj kev txwv. Piv txwv li, peb tau siv qhov kev ntsuam xyuas tsis zoo raws li cov kab mob pseudo-typed lentiviruses tab sis cov kev ntsuam xyuas no tau siv dav thiab txhim khu kev qha [33]. Nws kuj yog ib qho kev txwv ntawm peb txoj kev tshawb fawb tias peptides ntawm peb cov micro-array tau nthuav tawm thiab yog li yuav tau txheeb xyuas tsuas yog ib feem ntawm cov tshuaj tiv thaiv kab mob uas tau lees paub cov peptides unfolded whereas ib feem loj ntawm cov tshuaj tiv thaiv kab mob tuaj yeem raug khi rau conformational epitopes hauv kev txheeb xyuas. cheeb tsam. Tsis tas li ntawd, nws yog ib qho kev txwv ntawm peb txoj kev tshawb fawb uas peb tau txheeb xyuas tsuas yog qee qhov me me ntawm cov kev kawm. Txawm li cas los xij, peb txoj kev tshawb fawb qhia meej tias cov tshuaj tiv thaiv kab mob sib txawv nrog cov epitope tshwj xeeb tuaj yeem ua rau muaj kev tiv thaiv ntawm Gam COVID-Vac txhaj tshuaj tiv thaiv. Tsis tas li ntawd, txoj kev tshawb no qhia tau hais tias cov lus teb rau qhov muaj peev xwm khaws cia peptide uas tuaj yeem siv los tsim cov tshuaj tiv thaiv kab mob dav dav.
cistanche ntxiv cov txiaj ntsig-yuav ua li cas ntxiv dag zog rau lub cev
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Kawm Cov Lus Qhia Txog Pej Xeem thiab Ethics
Cov kev noj qab haus huv nrog (n {{0}}) lossis tsis muaj (n=12) ua ntej SARS-CoV-2 kab mob tau tso npe rau hauv txoj kev tshawb fawb thaum Lub Kaum Ob Hlis 2020 ntawm National Research Center Institute of Immunology ntawm Tsoom Fwv Teb Chaws Kho Mob Biological Agency ntawm Russia (tus naj npawb Ethics: #12-1, 29 Kaum Ob Hlis 2020). Txhua tus neeg tuaj koom tau sau ntawv tso cai. Yav dhau los SARS-CoV-2 tus kab mob no tau lees paub los ntawm kev kuaj PCR yav dhau los thiab cov tsos mob ntawm COVID-19 lossis los ntawm muaj SARS-CoV-2- cov tshuaj tiv thaiv tshwj xeeb qhia rau nucleocapsid antigen thiab/los yog RBD thiab/los yog S antigen raws li tau piav qhia [13]. Txhua tus neeg mob tau txhaj tshuaj intramuscularly nrog ob koob tshuaj ntawm Sputnik-V (Gam-COVID-Vac, Biocard, Moscow, Russia) ntawm lub sijhawm peb lub lis piam. Cov ntshav kuaj tau ib hnub ua ntej txhaj tshuaj tiv thaiv thawj zaug (T0, Daim duab 1A) thiab 80 mus rau 85 hnub tom qab txhaj tshuaj thawj zaug (T3, daim duab 1A).
4.2. Detection of SARS-CoV-2-Specific Antibody Responses
Immunoglobulin cov lus teb ntawm kev tsis zoo thiab COVID{{0}} cov neeg mob convalescent raug soj ntsuam hauv cov qauv ntshav plasma ua ntej thiab tom qab txhaj tshuaj nrog Sputnik-V (Daim duab 1A). Tshwj xeeb IgG rau lub vaj huam sib luag ntawm SARS-CoV-2 proteins thiab 46 peptides (25–30 mers) spanning S protein tau ntsuas hauv 1: 50 diluted plasma kuaj los ntawm microarray-chip technology raws li yav dhau los piav qhia [13]. Cov amino acid sequences, tus naj npawb ntawm cov amino acids, thiab qhov hnyav molecular rau tag nrho cov hluavtaws peptides ntawm micro-arrayed nti tau nthuav tawm hauv Table 3. Rau peptides 12 thiab 32 thiab RBD qhia hauv Escherichia coli (E. coli) (unfolded) thiab hauv Human Embryonic raum 293 (HEK293) hlwb (folded) ncig dichroism (CD) spectra tau muab (Daim duab S1). Peptides 12 thiab 32 qhia tsis muaj cov qauv tshwj xeeb raws li kev txiav txim siab los ntawm cov voj voog dichroism (CD) raws li qhia hauv daim duab S1. Hauv kev nthuav dav, iav slides uas muaj microarrays nyob ib puag ncig los ntawm Epoxy thav duab (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Lub teb chaws Yelemees) tau qhib nrog amine reactive complex organic polymer, MCP-2 (Lucidant Polymers, Sunnyvale, CA , Tebchaws USA) txhawm rau txhawm rau txhawm rau txhawm rau khi cov protein thiab peptides. SARS-CoV-2 antigens/peptides were spotted using a concentration of 0.5–1 mg/mL in (75 mM Na2HPO4, pH=8.4) in triplicates using a SciFlexArrayer S12 (Scienion AG, Berlin, Lub teb chaws Yelemees) [13]. Kev qhia thiab kev ua kom huv ntawm cov protein thiab peptide synthesis tau piav qhia hauv [13]. IgG antibody reactivity rau micro-arrayed antigens tau ntsuas los ntawm kev ntxuav thawj cov microarrays rau 5 min nrog phosphate-buffered saline nrog 0.5% Tween 20 (PBST) thiab ziab lawv los ntawm centrifugation siv Sigma 2-7 centrifuge thiab MTP-11113 rotor (ob leeg Sigma Laborzentrifugen GmbH, Osterode am Harz, Lub teb chaws Yelemees). Tom qab ntawd 35 µL aliquots ntawm 1:50 diluted plasma kuaj (cov qauv diluent, Thermofisher, Waltham, MA, USA) tau ntxiv thiab incubated rau 2 h ntawm 22 ◦C. Tom qab lwm cov kauj ruam ntxuav, 30 µL ntawm cov tshuaj tiv thaiv kab mob thib ob (DyLight 550 (Pierce, Rockford, IL, USA)) sau npe los tiv thaiv tib neeg IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA)) tau thov thiab incubated rau 30 min ntawm 22 ◦ C RT. Cov chips raug ntxuav, qhuav, thiab tom qab ntawd luam tawm siv lub tshuab laser confocal (Tecan, Männedorf, Switzerland). Cov duab tsom xam tau ua los ntawm MAPIX microarray duab nrhiav thiab tshuaj xyuas software (Innopsys, Carbonne, Fabkis) thiab hloov pauv cov ntsuas fluorescence units rau ISAC standardized units (ISU) tau ua raws li tau piav qhia [13]. Cov tshuaj tiv thaiv kab mob tshwj xeeb tau qhia hauv ISAC standardized units (ISU). S thiab RBD-specific IgG1-4 antibody cov lus teb tau ntsuas hauv cov qauv ntshav (1:50 diluted) hauv qhov sib npaug los ntawm ELISA raws li tau piav qhia yav dhau los [13] nrog kev hloov pauv ntawm<5% for their average values.
Table 3. Synthetic SARS-CoV-2 spike protein-derived peptides §.
4.3. Far UV Circular Dichroism (CD) Spectra
Rau peptides, P12 thiab P32 nrog rau RBD qhia hauv E. coli thiab HEK293 hlwb CD tsom xam tau ua los ntawm Jasco J-180 spectropolarimeter (Nyiv Spectroscopic Co., Tokyo, Nyiv) yav dhau los tau piav qhia [34]. CD spectra ntawm peptides thiab proteins (Daim duab S1) tau ntsuas ntawm qhov concentration ntawm 0.1 mg/mL hauv 10 mM NaH2PO4 (pH 8.0), uas yog siv rau pom cov antigens.
4.4. Pseudo-Virus-Based Virus Neutralization Assay (PVT)
Tus kab mob-neutralization kev ua haujlwm hauv cov ntshav plasma tau txiav txim siab los ntawm pseudo-virus-raws li tus kab mob nruab nrab ntawm kev soj ntsuam raws li tau piav qhia yav dhau los [35]. Los tsim SARS-CoV-2 spike virus-like particles (VLPs), HEK293T hlwb tau sib txuas nrog 3 plasmids: lentiviral ntim plasmid pCMV∆8.2R (Addgene, Teddington, UK), pUCHR-GFP, thiab a pCAGGS-S∆19 plasmid encoding the wild-type SARS-CoV-2 spike protein (zoo tib yam rau qhov siv Wuhan-Hu-1 thiab WA1 cais) lossis pCAGGS-S∆19-Om plasmid encoding the SARS-CoV-2 Omicron spike protein (zoo khoom plig los ntawm Andrey Gorchakov, Laboratory of Immunogenetics, Institute of Molecular thiab Cellular Biology, Siberian ceg ntawm Lavxias teb sab Academy ntawm Sciences, Novosibirsk, Russia). Ua ntej siv hauv pVNT, VLPs tau titrated los ntawm kev txwv cov dilution thiab incubated rau 4 hnub nrog hACE2-hloov HEK293T hlwb. Ib koob tshuaj tiv thaiv kab mob uas muab 50% ntsuab fluorescent protein (GFP)-zoo hlwb raug xaiv los siv rau hauv kev sim. Rau pVNT, tag nrho cov plasma kuaj tau kub-ua haujlwm rau 30 min ntawm 56 ◦C ua ntej siv. Hnub 4, cov kab mob tom qab tau raug ncua, thiab feem pua ntawm GFP-zoo hlwb raug ntsuas los ntawm kev ntws cytometry. ID50 qhov tseem ceeb tau suav nrog siv qhov nkhaus sigmoidal (GraphPad Prism 9.2.0 software, Sigmoidal, 4PL), rov tsim dua los ntawm feem pua ntawm qhov nruab nrab ntawm qhov sib txawv plasma concentrations.
4.5. Molecular Interaction Assay (MIA)
Txhawm rau txheeb xyuas qhov muaj peev xwm ntawm cov plasma cov qauv ntawm COVID{{0}}} cov kab mob convalescent lossis tsis zoo (Table 1), ua ntej thiab tom qab txhaj tshuaj tiv thaiv Sputnik-V, txhawm rau tiv thaiv kev khi ntawm 50 ng/mL ntawm RBD-hu-1 rau ACE2 receptor kev soj ntsuam kev sib cuam tshuam ntawm molecular (MIA) tau ua raws li tau piav qhia [13,36–39]. Qhov kev ntsuam xyuas no tuaj yeem ntsuas qhov inhibition ntawm RBD rau ELISA phaj-kawg ACE2 los ntawm cov tshuaj tiv thaiv lossis lwm yam kev cuam tshuam ntawm RBD-ACE2 kev cuam tshuam [38]. Hauv luv luv, recombinant ACE-2 (GenScript, Piscataway, NJ, USA) tau coated (2 µg/mL) hmo ntuj rau NUNC Maxisorb 96 lub qhov dej zoo (Thermofisher). Tom qab ntawd, peb lub voj voog ntxuav tau ua nrog kev ntxhua khaub ncaws thiab tom qab ntawd cov phaj raug thaiv rau 3 teev ntawm RT nrog kev thaiv tsis pub. Cov qauv ntshav tau diluted 1: 2 hauv PBS, 0.05% Tween 20, 1% BSA, thiab pre-incubated rau 2 h nrog 50 ng Nws-tagged recombinant RBD (GenScript). Tom qab ntawd cov qauv kuaj ua ntej incubated tau muab tso rau hauv daim hlau uas muaj ACE-2 rau 3 h thiab cov phaj tau ntxuav thiab incubated ib hmos nrog ib tug 1:1000 diluted nas monoclonal anti-Nws tag antibody (Dianova, Hamburg, lub teb chaws Yelemees) . Tom qab 3 kauj ruam ntxuav, 1: 1000 diluted HRP-txuas los tiv thaiv nas IgG1 antibody (GE Healthcare, Chicago, IL, USA) tau ntxiv rau 2 h thiab kuaj pom tau ua nrog ABTS. Lub ntsiab lus ntawm qhov muag pom qhov muag (OD) qhov tseem ceeb sib xws rau khi RBD tau ntsuas ntawm 405 nm thiab 492 nm (siv) hauv TECAN Infinite F5 ELISA nyeem ntawv nrog cov software sib xyaw i-control 2.0 (Tecan Group Ltd., Männedorf, Switzerland). Kev tswj tsis tau (overlay tsis RBD) raug rho tawm los ntawm txhua qhov tshwm sim. Kev txiav txim siab tau ua nyob rau hauv duplicates thiab cov txiaj ntsig tau pom raws li qhov tseem ceeb nrog kev hloov pauv ntawm<5%.
cistanche ntxiv cov txiaj ntsig-yuav ua li cas ntxiv dag zog rau lub cev
4.6. Kev pom ntawm Peptides hauv Spike Protein Structure
Surface sawv cev ntawm SARS-CoV-2 spike protein was generated in PyMOL (PyMOL Molecular Graphics System, Version 2.5.0a0, Schrödinger, LLC, New York, NY, USA) ntawm PDB nkag 6XR8.
4.7. Kev txheeb cais
Kev txheeb cais tau ua tiav siv GraphPad Prism (version 9.2.0 GraphPad Software, La Jolla California). Kev xeem Wilcoxon (Figures 1C, D, thiab 3A), Mann–Whitney test (Figures 2B thiab S3), thiab Friedman test (Figure S2) tau siv los sib piv ntawm ob lossis ntau pawg. p < 0.05 tau suav tias yog qhov tseem ceeb. Kev sib raug zoo ntawm ob pawg tau txiav txim siab los ntawm Spearman qeb xeem (Daim duab 2D). Ib qho normalized nonlinear regression tau ua los ntawm GraphPad Prism software (Sigmoidal 4PL). Cov ntaub ntawv tau nthuav tawm raws li qhov nruab nrab ± IQR. asterisks qhia qhov sib txawv ntawm pawg, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns — tsis tseem ceeb.
5. Cov lus xaus
Hauv cov ntsiab lus, peb txoj kev tshawb fawb yog thawj zaug los kawm hauv qhov tob ntawm epitope thiab IgG subclass tshwj xeeb ntawm cov tshuaj tiv thaiv kab mob tshwm sim los ntawm Gam-COVID-Vac thiab nthuav tawm cov txheej txheem sib txawv ntawm Gam-COVID-Vac-induced virus neutralization los ntawm cov tshuaj tiv thaiv. Tshwj xeeb, peb tau txais cov pov thawj tias cov tshuaj tiv thaiv kab mob coj mus rau peptide (piv txwv li, peptide 12) nyob ze rau tab sis sab nraum RBD hauv N-terminal ib feem ntawm S1 yog txuam nrog tus kab mob neutralization. Qhov muaj peev xwm ua kom tus kab mob tsis zoo ntawm peptide 12- cov tshuaj tiv thaiv tshwj xeeb yuav yog vim lawv lub peev xwm los cuam tshuam nrog kev hloov pauv ntawm qhov ua ntej mus rau tom qab fusion conformation ntawm spike protein. Peb hais kom xav txog kev suav nrog hauv cheeb tsam uas tau teev tseg los ntawm peptide 12 hauv daim ntawv tiv thaiv kab mob hauv cov tshuaj tiv thaiv kom ua rau cov tshuaj tiv thaiv kab mob tiv thaiv thaj tsam epitope no thiab sim tshuaj tiv thaiv kab mob los ntawm cov tshuaj tiv thaiv kab mob no. Nyob rau hauv rooj plaub uas tus kab mob nruab nrab ntawm cov tshuaj tiv thaiv tuaj yeem txhim kho, suav nrog cov tshuaj tiv thaiv kab mob peptide 12 epitopes tuaj yeem txhim kho kev ua tau zoo ntawm cov tshuaj tiv thaiv COVID-19. Tsis tas li ntawd, ib tus tuaj yeem txiav txim siab tsim cov tshuaj tiv thaiv kab mob tiv thaiv peptide 12-txhais epitope rau kev txhaj tshuaj tiv thaiv kab mob. Vim tias peptide 12-txhais epitope tau khaws cia hauv tam sim no paub SARS-CoV-2 variants, tshuaj tiv thaiv thiab tshuaj tiv thaiv kab mob qhia rau peptide 12 Tej zaum yuav tiv thaiv SARS-CoV-2 variants. Yog li peb muab cov kev paub tshiab uas yuav pab tau rau kev txhim kho cov tswv yim txhaj tshuaj tiv thaiv kab mob tshiab thiab tsis zoo rau COVID-19.
Cov ntaub ntawv
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