Mechanisms Thiab Lub Hom Phiaj Ntawm Fc -receptor Mediated Immunity rau Malaria Sporozoites

Ib qho tshuaj tiv thaiv zoo heev yuav pab txhawb kom ua tiav thiab txhawb nqa kev tshem tawm malaria. Kev nkag siab txog cov txheej txheem ntawm kev tiv thaiv kab mob sib kis yog qhov tseem ceeb heev rau kev tsim cov tshuaj tiv thaiv uas muaj txiaj ntsig zoo. Ntawm no, peb txheeb xyuas lub luag haujlwm tseem ceeb hauv kev tiv thaiv kev lom zem rau Fc -receptor (Fc R) kev sib cuam tshuam thiab opsonic phagocytosis ntawm sporozoites.

Peb txheeb xyuas lub luag haujlwm tseem ceeb rau neutrophils hauv kev sib kho phagocytic tshem tawm ntawm sporozoites hauv cov ntshav peripheral, thaum monocytes pab txhawb lub luag haujlwm me. Cov tshuaj tiv thaiv kuj txhawb kev ua haujlwm ntawm tes tua neeg. Mechanistically, antibody kev sib cuam tshuam nrog Fc RIII tshwm sim tseem ceeb, nrog Fc RIIa kuj xav tau rau kev ua haujlwm siab tshaj plaws. Tag nrho cov cheeb tsam ntawm circumsporozoite protein yog lub hom phiaj ntawm cov tshuaj tiv thaiv kev ua haujlwm tiv thaiv sporozoites, thiab N-terminal antibodies muaj kev ua haujlwm ntau dua hauv qee qhov kev ntsuam xyuas. Cov tshuaj tiv thaiv kab mob ua haujlwm tau maj mam tau tom qab raug tus kab mob malaria, muaj nyob hauv qee cov neeg laus, tab sis tsis tshua muaj tshwm sim ntawm cov menyuam yaus. Peb qhov kev tshawb pom tau nthuav tawm lub hom phiaj thiab cov txheej txheem ntawm kev tiv thaiv kab mob uas tuaj yeem siv rau hauv kev tsim tshuaj tiv thaiv kom ua tau zoo tshaj plaws.

Malaria tseem muaj kev txhawj xeeb txog kev noj qab haus huv thoob ntiaj teb, nrog P. falciparum malaria ua rau ~ 219 lab tus neeg mob thiab 435,000 neeg tuag txhua xyoo. Txawm hais tias muaj kev tswj hwm thoob ntiaj teb, lub nra malaria tseem nyob tas li nyob rau xyoo tas los no1. Muaj qhov xav tau muaj zog rau cov tshuaj tiv thaiv kab mob malaria uas muaj txiaj ntsig zoo rau kev tswj hwm thiab tshem tawm cov kab mob malaria, uas tau hais ntxiv los ntawm kev nce cov ntawv ceeb toom ntawm cov tshuaj tiv thaiv kab mob malaria thiab tshuaj tua kab 1. Lub Koom Haum Saib Xyuas Kev Noj Qab Haus Huv Ntiaj Teb thiab cov koom tes pab nyiaj tau teeb tsa lub hom phiaj los tsim cov tshuaj tiv thaiv nrog Ntau dua lossis sib npaug li 75 feem pua ​​​​ntawm kev ua tau zoo los ntawm 20302, uas tau ua pov thawj nyuaj kom ua tiav 3,4. Kev nkag siab ntau dua ntawm cov txheej txheem uas kho kom muaj kev tiv thaiv kab mob yog xav tau los tsim cov tswv yim uas tsim kom muaj zog tiv thaiv kab mob teb ntau dua.

Kev txhim kho kev tiv thaiv kab mob hauv lub neej niaj hnub xav kom peb tswj hwm tus cwj pwm zoo. Tsim thiab tswj lub sijhawm rau kev mus pw thiab sawv tuaj yeem txhawb nqa kev ua haujlwm ib txwm muaj ntawm circadian atherosclerosis. Tsis txhob noj cov zaub mov uas muaj calorie ntau ntau, thiab tsis txhob noj cov zaub mov uas muaj kua qaub los yog ntsim uas tuaj yeem ua rau zom tau yooj yim. Xaiv rau noj hmo nrog cov khoom tshiab. Txwv tsis pub siv cov khoom siv hluav taws xob. Lub teeb pom kev tuaj yeem ua rau peb sawv ntau zaus hauv ib hmo los ntawm kev hloov pauv kev pw tsaug zog thiab pw tsaug zog zoo. Tsis txhob tawm dag zog thaum yav tsaus ntuj. Yam tsawg kawg yog ob teev yog pom zoo. Kev tawm dag zog txhawb kev tso tawm adrenaline thiab nce lub plawv dhia, uas ua rau peb tsaug zog. Tsis tas li ntawd, peb tuaj yeem noj Cistanche deserticola. Cov polysaccharides thiab verbascoside hauv Cistanche deserticola tuaj yeem txhim kho kev tiv thaiv. Cov polysaccharides thiab verbascoside ntawm Cistanche deserticola tuaj yeem ua kom cov enzyme kev ua haujlwm ntawm lub plawv thiab lub hlwb, thiab txhim kho phagocytic kev ua haujlwm ntawm peritoneal hlwb thiab cov lus teb ntawm lymphocytes proliferation.

Nyem thaum coj cistanche

Tus kab mob malaria pib los ntawm tus kab mob Anopheles yoov tshaj cum tom, thaum lub sij hawm uas sporozoites raug inoculated rau hauv dermis5. Sporozoites tom qab ntawd tsiv mus rau cov hlab ntsha, ncig hauv cov hlab ntsha mus rau lub siab thiab tsim cov kab mob hauv hepatocytes. Sporozoites sawv cev rau lub hom phiaj tseem ceeb ntawm cov tshuaj tiv thaiv kab mob malaria vim tias kev tshem tawm sporozoites yuav txwv tsis pub kis tus kab mob ua ntej pib kho mob malaria3. Kev tshawb fawb hauv cov qauv tsiaj thiab kev sim tshuaj tiv thaiv tau tsim tias cov tshuaj tiv thaiv kab mob yog tus neeg nruab nrab tseem ceeb ntawm kev tiv thaiv kab mob rau sporozoites (saib hauv ref. 3). Txawm li cas los xij, yuav ua li cas cov tshuaj tiv thaiv ua haujlwm ua haujlwm kom tshem tawm sporozoites thiab tiv thaiv kev kis kab mob, thiab cov hom phiaj tshwj xeeb ntawm cov tshuaj tiv thaiv kab mob ua haujlwm, tsis to taub zoo. Kev nkag siab tshiab rau cov lus nug no yog xav tau los pab txhawb kev tsim cov tshuaj tiv thaiv zoo dua.

Lub circumsporozoite protein (CSP) yog cov protein ntau tshaj plaws nyob rau saum npoo ntawm sporozoites6 thiab lub hom phiaj tseem ceeb ntawm tib neeg cov tshuaj tiv thaiv 6. CSP-raws li cov tshuaj tiv thaiv tau ua tau zoo dua lwm cov tshuaj tiv thaiv antigens rau hnub tim, txawm hais tias kev ua tau zoo tseem yog qhov zoo tshaj 3. Cov tshuaj tiv thaiv kab mob malaria zoo tshaj plaws, RTS, S, yog ua raws li daim ntawv txiav tawm ntawm CSP (tsuas yog thaj chaw rov qab hauv nruab nrab thiab C-terminal cheeb tsam) thiab tau txais txiaj ntsig me me tiv thaiv kab mob malaria (29–36 feem pua) hauv theem III kev sim tshuaj 7. Lub hauv paus rov qab cheeb tsam ntawm CSP yog suav hais tias yog lub hom phiaj tseem ceeb ntawm cov tshuaj tiv thaiv kab mob, thiab yog li ntawd, qhov tseem ceeb ntawm cov tshuaj tiv thaiv kab mob rau cov cheeb tsam uas tsis yog rov ua dua tau raug understudied8 tab sis kuj tseem ceeb heev. Cov tshuaj tiv thaiv rau thaj tsam C-terminal tau cuam tshuam nrog kev tiv thaiv hauv RTS thiab S tshuaj tiv thaiv kev sim hauv menyuam yaus 9.

Hais txog cov tshuaj tiv thaiv kab mob ua haujlwm, feem ntau ntawm cov kev tshawb fawb luam tawm tau tsom mus rau qhov ncaj qha inhibitory kev ua haujlwm ntawm cov tshuaj tiv thaiv rau hauv nruab nrab ntawm thaj tsam ntawm CSP, uas tuaj yeem cuam tshuam sporozoite motility thiab hepatocyte ntxeem tau hauv vitro10-17. Tsis tshua paub txog lwm yam tseem ceeb uas muaj peev xwm thiab cov hom phiaj ntawm kev tiv thaiv kab mob.

Ib qho tseem ceeb ntawm kev tiv thaiv kab mob uas tau txais kev pom zoo hauv kev tiv thaiv kab mob thiab kab mob kab mob18-21 yog lub peev xwm ntawm cov tshuaj tiv thaiv kom cuam tshuam nrog Fc receptors (Fc R) ntawm ntau hom cell ua rau opsonic phagocytosis lossis ncaj qha cytotoxicity. Cov tshuaj tiv thaiv rau sporozoite antigens feem ntau yog tswj hwm los ntawm IgG1 thiab IgG3 subclasses13, uas muaj peev xwm cuam tshuam nrog Fc Rs22. Txawm li cas los xij, tam sim no tsis tshua paub txog cov txheej txheem muaj peev xwm tiv thaiv kab mob sporozoites. Cov phagocytic cell hom muaj xws li monocytes thiab neutrophils (2-10 feem pua ​​​​thiab 45-70 feem pua ​​​​ntawm cov qe ntshav dawb peripheral, feem).

Ntxiv rau phagocytosis, neutrophils, thiab natural killer (NK) hlwb kuj tuaj yeem tua cov hlwb los ntawm cov tshuaj tiv thaiv kab mob hauv cellular cytotoxicity (ADCC) 23,24, thiab ob qho tib si monocytes thiab neutrophils kuj muaj kev tiv thaiv kab mob ntau dua los ntawm kev qhia cov cim ua kom pom, cytokines, thiab tshuaj chemokines (saib xyuas hauv refs. 25–27).

Fc R cov qauv qhia txawv ntawm monocytes thiab neutrophils, uas cuam tshuam rau lawv txoj haujlwm hauv kev tiv thaiv. Txij li thaum sporozoites sawv cev rau thawj kauj ruam ntawm kev pib kis tus kab mob malaria, sporozoites feem ntau yuav ntsib cov phagocytic hlwb nyob rau hauv so, los yog homoeostatic, lub xeev es tsis yog lub xeev activated.

Resting-stage monocytes feem ntau nthuav qhia Fc RI thiab Fc RIIa, nrog ib qho me me qhia Fc RIIIa28, thaum so-theem neutrophils nthuav qhia Fc RIIa thiab Fc RIIIb29, thiab qis qis ntawm Fc RIIIa30 (uas yog qhov tseem ceeb ua haujlwm). Kev ntsuam xyuas kev sib cuam tshuam ntawm cov tshuaj tiv thaiv thiab sib txawv Fc Rs yog li ntawd tseem ceeb rau kev nkag siab txog lub luag haujlwm ntawm cov phagocytes sib txawv hauv kev tiv thaiv kab mob. IgG subclass, glycosylation, thiab epitope tshwj xeeb tuaj yeem cuam tshuam kev cuam tshuam nrog Fc sib txawv Rs22.

Kev txhais cov Fc R-mediated mechanisms tsom rau sporozoites yuav qhia tau qhov kev nkag siab uas tuaj yeem siv los txhim kho kev tiv thaiv kev tiv thaiv ntawm cov tshuaj tiv thaiv sporozoite. Hauv txoj kev tshawb no, peb nthuav tawm cov txheej txheem tseem ceeb thiab lub hom phiaj ntawm kev tiv thaiv kab mob sporozoites sib kho los ntawm Fc Rs tshwj xeeb ntawm lub cev tiv thaiv kab mob. Peb tsim cov tswv yim los ntsuas kev sib cuam tshuam ntawm cov tshuaj tiv thaiv kab mob thiab cov Fc Rs tshwj xeeb thiab txheeb xyuas cov tshuaj tiv thaiv kab mob tshwj xeeb, cov hom phiaj antigenic, thiab Fc Rs uas yog qhov tseem ceeb rau kev ua haujlwm zoo. Tsis tas li ntawd, peb txheeb xyuas qhov tau txais kev ua haujlwm ntawm Fc R-dependent antibody cov lus teb rau cov menyuam yaus thiab cov neeg laus uas raug mob malaria. Peb qhov kev tshawb pom nthuav tawm cov txheej txheem tiv thaiv kab mob tseem ceeb tiv thaiv sporozoites thiab qhia cov tswv yim thiab cov hom phiaj antigenic rau kev tsim cov tshuaj tiv thaiv kab mob malaria uas muaj txiaj ntsig zoo.

Cov txiaj ntsig

Opsonic phagocytosis ntawm sporozoites feem ntau yog kho los ntawm neutrophils hauv cov ntshav peripheral.

Txhawm rau tshawb xyuas qhov cuam tshuam ntawm Fc R-mediated mechanisms tiv thaiv sporozoites, peb thawj zaug tsim opsonic phagocytosis kev soj ntsuam siv cov THP undifferentiated -1 cell kab (pro-monocytic hlwb), nyob rau hauv tus txheej txheem tej yam kev mob31,32 (Supplementary Fig. 1A). Txhawm rau opsonize sporozoites, peb siv cov tshuaj tiv thaiv kab mob los ntawm Kenyan cov neeg laus nyob hauv thaj av uas muaj kev sib kis mob malaria siab thiab muaj kev tiv thaiv tseem ceeb rau malaria thiab cov tshuaj tiv thaiv rau CSP33. Opsonized P. falciparum sporozoites tau zoo phagocytosed los ntawm THP-1 hlwb nyob rau hauv ib tug concentration-dependent yam nrog me ntsis phagocytosis siv cov tshuaj tiv thaiv los ntawm malaria-naive pub (cov neeg nyob hauv Melbourne) (Supplementary Fig. 1B). Opsonic phagocytosis mediated los ntawm ib tug neeg sera yog heev reproducible (r=0.925, P < 0.001; Supplementary Fig. 1C).

Txhawm rau kov yeej cov kev cov nyom hauv kev tsim cov sporozoites loj loj rau opsonic phagocytosis assays, peb tsim kev siv cov hlaws fluorescent coated nrog tag nrho-ntev CSP, cov antigen loj qhia rau sporozoites, raws li kev ntsuas surrogate ntawm opsonic phagocytosis ntawm sporozoites. CSP-coated hlaws opsonized nrog xaiv cov qauv tshuaj tiv thaiv los ntawm cov neeg laus Kenyan sib txawv tau zoo phagocytosed los ntawm THP-1 hlwb, thiab opsonic phagocytosis ntawm CSP-coated hlaws muaj kev cuam tshuam nrog opsonic phagocytosis ntawm P. falciparum sporozoites (r {{4}). }}.831, P=0.0015, Ntxiv Fig. 1D). Yog li ntawd, CSP hlaws dai tau muab lub platform zoo rau kev kawm cov txheej txheem thiab cov hom phiaj ntawm cov tshuaj tiv thaiv kab mob opsonic phagocytosis.

Tom qab ntawd, peb tau tshawb xyuas cov xovtooj ntawm tes uas kho opsonic phagocytosis hauv kev soj ntsuam tag nrho-leukocyte nrog cov ntshav tshiab, pib siv CSP hlaws opsonized nrog polyclonal luav antibody rau CSP. Interestingly, tus nqi ntawm phagocytosis (tus naj npawb ntawm cov hlaws phagocytosed ib lub xov tooj ntawm tes) yog ntau dua los ntawm neutrophils dua li ntawm monocytes (Fig. 1A, B). Vim tias peb ntsuas tus nqi phagocytosis, qhov kev ua haujlwm siab dua ntawm neutrophils tsis yog piav qhia los ntawm lawv cov kev nplua nuj ntau dua piv rau monocytes. Opsonic phagocytosis nce nrog nce cov tshuaj tiv thaiv kab mob, thiab ntau dua cov txheeb ze ntawm neutrophils piv rau monocytes tau pom thoob plaws cov tshuaj tiv thaiv kab mob thiab ntau cov hlaws: cell ratios. Qhov kev ua haujlwm ntawm phagocytosis ntau dua ntawm neutrophils tau lees paub tias siv cov sporozoites tshiab uas tau cais tawm (peb siv cov transgenic P. berghei sporozoites uas qhia P. falciparum CSP, uas hloov cov endogenous P. berghei CSP gene12, hu ua PfCSP-P. berghei). Opsonic phagocytosis ntawm sporozoites opsonized nrog cov tshuaj tiv thaiv los ntawm Kenyan malaria-exposed pub yog feem ntau kho los ntawm neutrophils nrog tsuas yog theem qis ntawm phagocytosis los ntawm monocytes, thiab neutrophils uas qhia phagocytosis ntau dua (Fig. 1C). Peb kuj tau ua qhov kev soj ntsuam kom tso cai rau ncua sij hawm rau phagocytosis (txog 60 min), tab sis peb tseem pom ntau dua phagocytosis los ntawm neutrophils piv rau monocytes (Cov Lus Ntxiv 2A).

Kev soj ntsuam ntxiv ntawm cov pej xeem monocyte tau qhia tias feem ntau ntawm phagocytosis yog los ntawm cov txheej txheem classical (CD14high CD16−; Fig. 1D thiab Supplementary Fig. 2B-D). Txhawm rau txhawb peb qhov kev tshawb pom, peb tau qhia ntxiv tias cov neutrophils purified tuaj yeem ua tau zoo phagocytose antibody-opsonized P. falciparum sporozoites (Fig. 1E). Depletion ntawm CSP-specific antibodies, los ntawm pre-incubation nrog recombinant CSP, txo phagocytosis ntawm sporozoites los ntawm 70 feem pua ​​​​piv rau cov tsis-depleted qauv (Fig. 1F thiab Supplementary Fig. 3). Peb tau siv ib txoj hauv kev ntxiv kom tau txais cov pov thawj ntawm qhov tseem ceeb ntawm CSP raws li lub hom phiaj ntawm kev ua haujlwm ntawm cov tshuaj tiv thaiv kab mob hauv tib neeg kev tiv thaiv los ntawm kev sim mAb (MGG4) cais tawm ntawm ib tus neeg raug tus kab mob sporozoite rov qab 34. Lub mAb induced phagocytosis ntau heev ntawm sporozoites, whereas muaj me ntsis phagocytosis nrog tsis-immune human antibodies, los yog siv anti-CSP MAb (2A10) uas tau hloov nyob rau hauv cheeb tsam Fc los inhibit Fc R kev sib cuam tshuam (Fig. 1G).

Txhais Fc receptors koom nrog hauv opsonic phagocytosis ntawm sporozoites.

Peb opsonized tag nrho P. falciparum sporozoites thiab CSP hlaws dai nrog cov tshuaj tiv thaiv los ntawm Kenyan cov neeg laus thiab ntsuas opsonic phagocytosis los ntawm neutrophils nyob rau hauv lub xub ntiag ntawm tshwj xeeb Fc R-blocking antibodies. Neutrophils tau txo qis opsonic phagocytosis ntawm ob qho tib si CSP hlaws thiab P. falciparum sporozoites nyob rau hauv lub xub ntiag ntawm Fc RIIa lossis Fc RIII blockers (Fig. 2A, B thiab Supplementary Fig. 4A), qhia tias ob qho tib si receptors tseem ceeb.

Qhov tseem ceeb, Fc RIII blockade yog qhov ua tau zoo tshaj plaws ntawm inhibiting phagocytosis ntawm P. falciparum sporozoites lossis CSP hlaws, nrog rau qhov tsis muaj zog ntawm Fc RIIa blockade. Opsonic phagocytosis los ntawm neutrophils tsis cuam tshuam los ntawm kev thaiv Fc RI. Titrating qhov concentration ntawm thaiv cov tshuaj tiv thaiv tau pom tias peb tau ua tiav cov txiaj ntsig siab tshaj plaws rau kev thaiv ntawm txhua Fc R (Cov Lus Qhia Ntxiv 4B).

Yog li ntawd, peb ua ke Fc RIIa thiab Fc RIII thaiv cov tshuaj tiv thaiv kab mob thiab pom qhov no tau muab ntau dua inhibition ntawm phagocytosis dua li siv los thaiv cov tshuaj tiv thaiv, ntxiv qhia qhov tseem ceeb ntawm ob hom Fc R rau kev pom phagocytosis (Fig. 2B). Cov txiaj ntsig no zoo ib yam nrog kev qhia ntawm Fc Rs ntawm so neutrophils, uas feem ntau nthuav qhia Fc RIIa thiab Fc RIIIa / b, tab sis tsis muaj Fc RI35 (Cov Lus Qhia Ntxiv 2C). Qhov tseem ceeb ntawm Fc RIII hauv kev kho kom haum xeeb opsonic phagocytosis ntawm CSP hlaws thiab sporozoites tuaj yeem piav qhia qee qhov kev ua haujlwm qis ntawm monocytes txij li qhov loj monocyte subsets qhia me ntsis Fc RIIIa rau ntawm qhov chaw hauv lub xeev tsis ua haujlwm thiab tsis qhia Fc RIIIb35. THP-1 hlwb tau dav siv los ua tus qauv ntawm monocyte phagocytosis thiab nthuav qhia Fc RI36, uas yog khi los ntawm monomeric IgG (thaum Fc RIIa thiab Fc RIII qhia los ntawm neutrophils tsuas yog khi rau lub cev tsis muaj zog).

Raws li, peb pom tias opsonic phagocytosis los ntawm THP-1 hlwb nyob rau hauv cov txheej txheem (muaj 10 feem pua ​​​​FCS nkaus xwb thiab tsis muaj tib neeg cov ntshav) tau muaj zog inhibited los ntawm Fc RI blocker nrog txwv inhibition los ntawm Fc RIIa blocker, thiab tsis muaj Fc. RIII thaiv (Fig. 2C). Qhov no tau qhia tias kev sib cuam tshuam ntawm IgG thiab Fc RI yog qhov tseem ceeb tshwj xeeb rau THP-1 hlwb, sib piv cov txiaj ntsig pom nrog neutrophils. Yog li ntawd, peb tau hloov kho tus qauv THP-1 kev soj ntsuam kom ua rau nws muaj kev cuam tshuam ntau dua los ntawm kev suav nrog tib neeg cov ntshav hauv cov tshuaj ntsuam xyuas.

Nyob rau hauv lub xub ntiag ntawm ntxiv 2.5 feem pua ​​​​ntawm tib neeg cov ntshav (raws li siv nyob rau hauv tag nrho cov leukocyte thiab neutrophil kev soj ntsuam), muaj ntau tsawg opsonic phagocytosis (Fig. 2D), xav tias yog vim lub binding ntawm non-immune monomeric tib neeg IgG tam sim no nyob rau hauv tib neeg cov ntshav. rau Fc RI. Cov kev tshawb pom no tuaj yeem piav qhia ntxiv txog qhov qis phagocytic kev ua haujlwm ntawm thawj monocytes pom nyob rau hauv tag nrho cov leukocyte kev soj ntsuam nyob rau hauv lub xub ntiag ntawm tib neeg cov ntshav; bovine IgG (tam sim no hauv FCS) tsis khi tib neeg Fc RI37.

Muab qhov pom tseeb qhov tseem ceeb ntawm Fc RIII, peb ntxiv ntsuas qhov ua haujlwm ntawm cov tshuaj tiv thaiv kab mob rau CSP ntawm Kenyan cov neeg laus hauv ADCC kev soj ntsuam, txij li Fc RIII kev sib cuam tshuam ua lub luag haujlwm tseem ceeb hauv kev kho ADCC ntawm NK hlwb. Peb tau sim xaiv cov qauv los ntawm Kenyan cov neeg laus pawg (Kanyawegi cohort) los sawv cev siab, nruab nrab, thiab cov neeg teb qis (n=31) hauv kev ntsuam xyuas siv ADCC tus xov tooj ntawm tes uas tsuas yog qhia Fc RIIIa. Cov tshuaj tiv thaiv ib txwm tau txais ntawm Kenyan cov neeg laus muaj cov haujlwm tseem ceeb, thaum cov tshuaj tiv thaiv los ntawm Melbourne malaria-naive pub tsis tau (Daim duab 2E, F). Cov kev tshawb pom no qhia tias cov tshuaj tiv thaiv rau CSP kuj tseem tuaj yeem txhawb nqa ADCC cov haujlwm los ntawm kev koom nrog Fc RIIIa. Tom qab ntawd, peb tau sim cov haujlwm ntawm thawj NK hlwb hauv cov ntshav tshiab uas siv qhov kev ntsuam xyuas degranulation 38. Incubation ntawm NK hlwb nrog CSP hlaws opsonized los ntawm Kenyan cov neeg laus cov tshuaj tiv thaiv ua rau degranulation ntawm thawj NK hlwb (Fig. 2G, H). Kev ua tau txawv ntawm cov qauv kuaj, thiab muaj feem cuam tshuam nrog Fc RIII khi (r=0.538, P=0.002, Fig. 2H), tab sis tsis muaj zog cuam tshuam nrog IgG rau CSP (r {{10}) }.127, P=0.489, n=31).

Kev khi ncaj qha ntawm Fc RIII thiab Fc RIIa los ntawm cov tshuaj tiv thaiv-antigen complexes.

Peb tsim Fc R-binding kev soj ntsuam siv Fc RIIa thiab Fc RIII uas tau qhia thiab purified li dimers yog li lawv tuaj yeem cuam tshuam nrog antigen-antibody complexes39. Cov neeg laus hauv Kenyan muaj qhov ntau thiab qhov loj ntawm cov tshuaj tiv thaiv rau CSP uas tuaj yeem cuam tshuam nrog Fc RIIa thiab Fc RIII dimers (Daim duab 3A). Zuag qhia tag nrho, 67.3 thiab 61.5 feem pua ​​​​ntawm cov qauv (n=104) tau pom zoo rau Fc RIIa thiab Fc RIII khi. Thaum txoj kev khi ntawm ob qho tib si Fc Rs muaj kev sib raug zoo, kev sib raug zoo ntawm cov qauv (r=0.71, P< 0.0001, n = 104) (Fig. 3B), some individual samples demonstrated high binding to one FcγR and low binding to the other. Furthermore, the ability of antibodies to CSP to promote direct binding of FcγRIII was significantly correlated with ADCC tested by the ADCC reporter cell line (r = 0.405, P = 0.022, Fig. 2F) and primary NK cells (r = 0.538, P = 0.002, Fig. 2H). FcγRs occur in populations as different alleles40. 

Yog li ntawd, peb tau sim cov alleles sib txawv ntawm Fc RIIa (R131, H131) thiab Fc RIII (F158, V158) rau IgG-mediated binding39. Cov ntaub ntawv tshaj tawm qhia tias cov alleles no muaj nyob hauv peb cov neeg kawm 41. Cov tshuaj tiv thaiv rau CSP tau txhawb nqa kev khi ntawm ob qho tib si ntawm Fc RIIa thiab Fc RIII. Muaj kev sib raug zoo ntawm Fc R khi ntawm cov alleles sib txawv; rho=0.8371 rau Fc RIIa, thiab rho=0.78 rau Fc RIII (Fig. 3C, D).

Cov cheeb tsam ntawm CSP tsom los ntawm cov tshuaj tiv thaiv ua haujlwm.

Peb cov ntaub ntawv dhau los tau qhia tias CSP yog lub hom phiaj tseem ceeb ntawm tib neeg cov tshuaj tiv thaiv kab mob uas txhawb nqa phagocytosis (Fig. 1F, G). Yog li ntawd, peb txuas ntxiv mus soj ntsuam cov cheeb tsam sib txawv ntawm CSP raws li lub hom phiaj ntawm cov tshuaj tiv thaiv kab mob hauv cov neeg mob malaria uas siv peb qhov tsim ntawm CSP: (i) hluavtaws peptide sawv cev rau thaj tsam NT (uas tsis suav nrog RTS, S tshuaj tiv thaiv), (ii. ) ib qho hluavtaws peptide sawv cev rau thaj tsam rov ua dua (NANP rov ua dua), (iii) cov protein sib txuas uas sawv cev rau thaj tsam CT. Txhua lub tsev tsim tau nyias coated rau cov hlaws fluorescent (ntawm saturating concentrations) thiab opsonized nrog lub pas dej ntawm Kenyan sera. Cov tshuaj tiv thaiv zoo txhawb nqa neutrophil phagocytosis ntawm cov hlaws coated nrog txhua tus tsim (Fig. 4A).

Tom qab ntawd peb tau soj ntsuam ib txwm tau txais cov tshuaj tiv thaiv rau thaj tsam ntawm CSP rau lawv lub peev xwm los txhawb kev koom tes ntawm Fc RIIa thiab Fc RIII. Tib neeg cov tshuaj tiv thaiv khi rau txhua cheeb tsam CSP nrog qhov ntom txaus los txhawb kev khi ntawm Fc RIIa thiab Fc RIII (Fig. 4B). Raws li tag nrho cov theem ntawm IgG binding varied ntawm txawv CSP constructs (Ntxiv daim duab 5), peb standardized Fc RIIa thiab Fc RIII binding txheeb ze rau theem ntawm IgG reactivity (lub sij hawm Fc R-binding efficiency). Peb cov ntaub ntawv qhia tias cov tshuaj tiv thaiv rau thaj tsam NT muaj peev xwm ntau dua los koom nrog Fc Rs dua li hauv nruab nrab rov ua dua thiab CT cheeb tsam (kev ua tau zoo dua rau NT piv rau CT lossis NANP cheeb tsam, P < 0.001 rau txhua qhov sib piv) .

Peb tau soj ntsuam ntxiv qhov tseem ceeb ntawm cov cheeb tsam sib txawv raws li lub hom phiaj ntawm cov tshuaj tiv thaiv kev ua haujlwm uas siv cov tshuaj tiv thaiv kab mob tsim tawm rau cov cheeb tsam tshwj xeeb thiab tag nrho cov sporozoites hauv phagocytosis kev soj ntsuam. Peb thawj zaug tau lees paub tias luav anti-CSP cov tshuaj tiv thaiv tuaj yeem txhawb phagocytosis ntawm sporozoites. P. falciparum sporozoites opsonized nrog cov tshuaj tiv thaiv no tau muab qhov ntsuas phagocytosis siab (40.5 feem pua) piv rau kev tswj hwm (Daim duab 4C). Tom qab ntawd, peb tau tsim kom muaj ib tug paub mAb tiv thaiv lub hauv paus NANPrepeat cheeb tsam ntawm P. falciparum CSP (mAb 2H8, nas IgG2a subclass) 33 txhawb nqa phagocytosis tseem ceeb (17.4 feem pua, Fig. 4D) uas ntau dua li pom nrog kev tswj tsis zoo mAb 3D11 uas. Lub hom phiaj CSP ntawm P. berghei uas tsis muaj NANPrepeat epitope (Fig. 4D). Lub cheeb tsam NANP-repeat encodes ib qho immunodominant epitope, thaum tsawg dua paub txog qhov tseem ceeb ntawm NT thiab CT cheeb tsam raws li lub hom phiaj ntawm cov tshuaj tiv thaiv.

Txhawm rau txheeb xyuas lub luag haujlwm ntawm cov tshuaj tiv thaiv kab mob hauv thaj tsam CT thiab NT, thiab ntxiv cov ntaub ntawv siv tib neeg cov tshuaj tiv thaiv kab mob, peb tsim cov kab mob luav polyclonal antibodies rau txhua cheeb tsam (Ntxiv daim duab 6) thiab siv cov tshuaj tiv thaiv no los opsonize transgenic PfCSPP. berghei sporozoites rau phagocytosis los ntawm neutrophils. Luav polyclonal antibodies rau ob thaj tsam CT thiab NT tau txhawb phagocytosis ntawm PfCSP-P. berghei sporozoites (Fig. 4E). Txij li thaum cov concentration ntawm IgG mus rau CT thiab NT los ntawm cov tshuaj tiv thaiv kab mob luav sib txawv (Ntxiv daim duab 6), peb tau soj ntsuam cov phagocytosis efficiency thiab cov tshuaj tiv thaiv kab mob, piv rau qib IgG (xws li phagocytosis kev ua ub no faib los ntawm IgG reactivity txiav txim los ntawm ELISA). Qhov no tau qhia tias cov tshuaj tiv thaiv kab mob rau thaj tsam N-terminal tau muaj txiaj ntsig zoo dua phagocytosis piv rau CT cov tshuaj tiv thaiv.

Txij li cov tshuaj tiv thaiv kab mob rau thaj tsam NT tau ua haujlwm tau zoo hauv kev ua kom Fc Rs thiab txhawb nqa opsonic phagocytosis, peb tau ua daim ntawv qhia epitope ntawm luav polyclonal antibodies tiv thaiv thaj tsam NT siv qhov sib tshooj peptide array (Fig. 4F thiab Supplementary Fig. 6D). Ib qho peptide array yog tsim nyog rau epitope daim ntawv qhia vim hais tias thaj tsam NT tau kwv yees tias yuav tsis muaj teeb meem thiab tsis muaj cysteine ​​(PlasmoDB, https://plasmodb.org/plasmo/app/ record/gene/PF3D7_0304600). Cov tshuaj tiv thaiv kab mob tau rov ua haujlwm nrog peptides npog thaj tsam 21-aa (DDGNNEDNEKLRKPKHKKLKQ) uas nyob ze rau qhov sib txuas ntawm thaj av N-terminal thiab thaj tsam NANP/NVPD rov ua dua.

Tau txais cov tshuaj tiv thaiv kab mob uas txhawb Fc R kev sib cuam tshuam thiab opsonic phagocytosis los ntawm kev raug ntuj tsim.

Peb tau txheeb xyuas cov qauv kuaj ntshav uas tau sau los ntawm ib pawg ntawm cov menyuam yaus uas raug mob malaria thiab cov neeg laus nyob rau sab hnub poob Kenya hauv ib cheeb tsam uas muaj kab mob malaria siab heev (los ntawm cheeb tsam Chulaimbo, Daim Ntawv Ntxiv 1). Hauv qhov chaw no, feem ntau ntawm malaria morbidity thiab tuag tshwm sim hauv cov menyuam yaus hnub nyoog<5 years. 

Hauv qhov sib piv, cov neeg laus muaj kev tiv thaiv ntau heev, pom los ntawm qhov tsis muaj kev txhim kho ntawm cov tsos mob malaria, txawm hais tias asymptomatic parasitemia yuav tshwm sim42. Peb ntsuas lub peev xwm ntawm CSP-cov tshuaj tiv thaiv tshwj xeeb los cuam tshuam nrog Fc RIIa thiab Fc RIII nrog rau txhawb nqa opsonic phagocytosis ntawm CSP hlaws los ntawm neutrophils (Fig. 5A–D). Tag nrho cov tshuaj tiv thaiv kab mob nrog rau txhua qhov kev ua haujlwm yog neutrophil phagocytosis ntawm 16 feem pua ​​​​, (95 feem pua ​​​​CI, 9.19, 26.38 feem pua ​​), Fc RIIa khi ntawm 20 feem pua ​​(95 feem pua ​​​​CI, 12.28, 30.84 feem pua), thiab Fc1 RIII feem pua. (95% CI, 8.19, 24.85%). Qib ntawm IgG reactivity rau CSP, Fc R binding, thiab opsonic phagocytosis los ntawm neutrophils yog tsawg ntawm cov menyuam yaus thiab muaj hnub nyoog nce ntxiv.

Los ntawm qhov sib txawv, cov neeg laus uas raug mob mus ib txhis tau txais cov tshuaj tiv thaiv zoo dua qub piv rau cov menyuam yaus. Peb kuj tau soj ntsuam cov tshuaj tiv thaiv kab mob uas txhawb nqa phagocytosis ntawm cov ntshav-theem merozoites, uas muaj qhov muaj ntau dua thiab qib ntawm cov menyuam yaus piv rau cov tshuaj tiv thaiv kab mob rau CSP (Fig. 5B, E, F). Qhov no txhawb nqa lub tswv yim uas ib txwm tau txais kev tiv thaiv kab mob rau sporozoites tau txais qeeb dua thiab yuav tsum muaj kev nthuav dav ntxiv, piv rau kev tiv thaiv cov ntshav. Peb tau tshawb xyuas seb qhov kev ua haujlwm siab dua ntawm cov neeg laus tsuas yog los ntawm qhov siab dua ntawm CSP-specific IgG lossis tseem qhia txog kev ua haujlwm siab dua ntawm cov tshuaj tiv thaiv ntawm cov neeg laus. Peb tau txheeb xyuas cov tshuaj tiv thaiv kab mob ua haujlwm ntawm tus txheeb ze rau IgG reactivity tiv thaiv CSP; Txhawm rau ua qhov no, peb tsis suav cov tib neeg uas muaj IgG qis heev rau CSP (hauv qab qhov nruab nrab OD ntawm 0.140), thiab peb suav cov kev ua ub no txheeb ze rau qib IgG rau txhua tus neeg.

Interestingly, qhov no qhia tau hais tias tus txheeb ze phagocytosis efficiency tau ntau dua nyob rau hauv cov neeg laus piv rau cov me nyuam (Daim duab. 5G) (Mann–Whitney test, P=0.022). Tsis tas li ntawd, muaj kev sib raug zoo ntawm lub hnub nyoog thiab Fc R binding efficiency (r=0.354, P=0.031 rau Fc RIIa binding thiab r=0}.572 rau Fc RIII khi, P <0.001).

Opsonic phagocytosis los ntawm neutrophils correlated nrog Fc R khi thiab cytophilic IgG subclasses

Peb tau txuas ntxiv peb qhov kev tshuaj xyuas mus rau pawg neeg loj dua ntawm cov neeg laus tiv thaiv kab mob nyob rau sab hnub poob Kenya (Kanyawegi cohort, n=104) uas tau kis tus kab mob malaria mus ntev hauv ib cheeb tsam uas muaj kab mob malaria siab. Peb tau xav tias feem ntau ntawm cov neeg laus no yuav muaj cov tshuaj tiv thaiv kab mob ua haujlwm rau CSP, pab txhawb kev tsom xam ntawm cov tshuaj tiv thaiv kab mob thiab cov yam ntxwv tshwj xeeb uas txiav txim siab ua haujlwm opsonic phagocytosis. Tseeb, ntau cov qauv qhia tau hais tias muaj ntau yam phagocytosis thiab Fc R binding kev ua. Feem ntau ntawm cov tshuaj tiv thaiv nrog txhua qhov kev ua haujlwm yog neutrophil phagocytosis 47.1 feem pua ​​(95 feem pua ​​CI, 37.6, 56.9 feem pua), Fc RIIa khi 67.3 feem pua, (95 feem pua ​​​​CI, 57.6, 75.8 feem pua) thiab Fc 65.95 feem pua ​​(Fc RIII). , 51.7, 70.5 feem pua).

Opsonic phagocytosis los ntawm neutrophils tau sib raug zoo nrog Fc RIIa khi (r {{0}}.50, P < 0.001) thiab Fc RIII khi (r=0.43, P < 0.001) (Fig. 6A). Multivariate regression tsom xam pom tias Fc RIII yog thawj yam cuam tshuam nrog neutrophil opsonic phagocytosis (regression coefficient=10.15, P < 0.001, Table 1), whereas Fc RIIa tsis tseem ceeb. Yav dhau los peb tau tshaj tawm tias cov tshuaj tiv thaiv rau CSP hauv pawg neeg laus no feem ntau yog IgG1 thiab IgG3, nrog rau qib qis ntawm IgG2, thiab IgG433 tsawg heev. IgG1 thiab IgG3 yog cov subclasses tseem ceeb uas cuam tshuam nrog Fc RIIa thiab Fc RIII los txhawb phagocytosis, thiab IgG2 muaj kev ua haujlwm tsis muaj zog. Ntawm cov neeg laus hauv Kenyan, neutrophil phagocytosis tau sib raug zoo nrog IgG1 (r=0.32, P < 0.001), IgG2 (r=0.26, P=0.007), thiab ntau dua. muaj zog nrog IgG3 (r=0.50, P < 0.001) (Fig. 6B, C, Ntxiv Fig. 7 thiab Supplementary Table 2).

Tsis muaj kev sib raug zoo nrog IgG4 (r=0.09, P = 0.365), tab sis qib ntawm IgG4 tsawg. Hauv kev hloov pauv ntau yam, IgG3 tseem muaj feem cuam tshuam nrog neutrophil phagocytosis, qhia tias nws yog tus neeg nruab nrab ntawm qhov kev ua no (Cov Lus Qhia Ntxiv 3). Tsis tas li ntawd, ob qho tib si Fc RIIa thiab Fc RIII khi tau sib raug zoo nrog tag nrho cov theem ntawm IgG thiab IgG subclasses, feem ntau IgG1 thiab IgG3 (Cov Lus Ntxiv 2 thiab 3). Zuag qhia tag nrho, cov qauv cais raws li muaj kev ua haujlwm siab phagocytosis (txhais tau tias yog siab tshaj qhov nruab nrab) muaj ntau dua ntawm Fc RIIa thiab Fc RIII khi thiab IgG3, thiab muaj ntau dua IgG rau txhua cheeb tsam ntawm CSP, piv rau cov qauv uas tsis tshua muaj phagocytosis. kev ua si (Fig. 6D, E).

Cov kev soj ntsuam zoo sib xws tau pom ntawm Chulaimbo pawg ntawm cov menyuam yaus thiab cov neeg laus. Opsonic phagocytosis cuam tshuam nrog Fc R khi, thiab IgG1 thiab IgG3, nrog kev sib raug zoo dua rau Fc RIII thiab IgG3 (Cov Lus Ntxiv 4 thiab 5). Peb tau siv cov kev ntsuam xyuas no hauv theem 1/2a kev sim tshuaj tiv thaiv RTS, S. Qhov no tau qhia tias cov tshuaj tiv thaiv uas txhawb nqa opsonic phagocytosis los ntawm neutrophils thiab khi ntawm Fc RIII thiab Fc RIIa tau siab dua ntawm cov kev tiv thaiv ntau dua li cov uas tsis muaj kev tiv thaiv43.

Kev sib tham

Peb muab cov txiaj ntsig txhais cov txheej txheem thiab lub hom phiaj ntawm kev tiv thaiv kab mob tiv thaiv kab mob rau sporozoites los ntawm kev sib cuam tshuam nrog cov Fc Rs tshwj xeeb qhia ntawm cov kab mob tiv thaiv kab mob. Peb qhov kev tshawb pom txhawb nqa lub luag haujlwm tseem ceeb rau cov neutrophils hauv kev tshem tawm cov sporozoites hauv cov ntshav, kho los ntawm Fc RIIa thiab Fc RIII kev cuam tshuam nrog IgG1 thiab IgG3 tsom rau cov sporozoite antigen loj, CSP, nrog kev koom tes ntxiv los ntawm monocytes (classical subset) thiab NK hlwb. Siv cov sporozoites tsis tu ncua thiab antigen-coated hlaws, peb tau tsim tias phagocytosis, hauv cov ntshav tag nrho, feem ntau yog cov neutrophils, sib piv cov kev ua haujlwm qis uas pom nrog monocytes. Peb tau txhais cov txheej txheem tseem ceeb cuam tshuam los ntawm kev thaiv cov Fc Rs tshwj xeeb ntawm cov xov tooj ntawm tes thiab los ntawm kev siv Fc R dimers ua kev sojntsuam los khi cov tshuaj tiv thaiv kab mob antigen-antibody complexes.

Qhov no tau txheeb xyuas qhov tseem ceeb ntawm Fc RIIa thiab Fc RIII, thiab cov ntaub ntawv tshwj xeeb taw tes rau qhov tseem ceeb ntawm Fc RIII. Peb tau txhais cov hom phiaj tiv thaiv kab mob ntawm qhov kev ua haujlwm no siv cov tshuaj tiv thaiv rau thaj tsam sib txawv ntawm CSP, thaj tsam CSP sib txawv coated rau cov hlaws, thiab chimeric P. berghei sporozoites qhia P. falciparum CSP. Peb tau tsim tias CSP yog lub hom phiaj tseem ceeb ntawm cov tshuaj tiv thaiv kab mob no thiab tag nrho peb thaj tsam ntawm CSP tuaj yeem raug tsom los ntawm cov tshuaj tiv thaiv kom koom nrog Fc R thiab txhawb nqa phagocytosis.

Tshwj xeeb, cov tshuaj tiv thaiv rau thaj tsam NT muaj cov haujlwm tseem ceeb thoob plaws ntau qhov kev ntsuam xyuas sib txawv, muab qhov muaj peev xwm ua rau kev tsim tshuaj tiv thaiv txij li thaj tsam NT tsis suav nrog CSP-raws li cov tshuaj tiv thaiv RTS, S (tam sim no hauv kev tshawb fawb txog kev siv), lossis R21. (hauv theem 2 kev sim). Tsis tas li ntawd, peb tau tsim kom muaj cov tshuaj tiv thaiv kab mob uas muaj peev xwm los txhawb opsonic phagocytosis ntawm sporozoites. Kev nrhiav tau ntawm cov tshuaj tiv thaiv kev ua haujlwm yog nyob ntawm hnub nyoog thiab qib siab ntawm opsonic phagocytosis kev ua haujlwm thiab kev khi ntawm Fc RIIa thiab Fc RIII tsuas yog pom nrog cov qauv ntawm cov neeg laus tab sis tsis yog cov menyuam yaus. Kev ua haujlwm muaj feem cuam tshuam nrog IgG1 thiab IgG3 subclasses.

Kev ua haujlwm ntawm phagocytosis ntau dua ntawm neutrophils, tab sis qhov kev ua haujlwm qis heev ntawm monocytes, tuaj yeem cuam tshuam nrog kev muaj thiab siv cov Fc Rs sib txawv; neutrophils muaj tus nqi ntau dua ntawm phagocytosis, uas tsis yog tsuas yog piav qhia los ntawm ntau dua ntawm neutrophils piv rau monocytes. Interestingly, blocking Fc RIIa los yog Fc RIIIa / b inhibited phagocytosis kev ua los ntawm neutrophils, qhia tias ob qho tib si receptors yuav tsum tau rau phagocytosis, uas tau txais kev txhawb nqa los ntawm kev ua tiav inhibition ntau dua nrog thaiv cov tshuaj tiv thaiv rau Fc RIIa thiab Fc RIII siv ua ke. Thaiv Fc RI tsis cuam tshuam rau phagocytosis los ntawm neutrophils. Ntau qhov kev soj ntsuam pom tias qhov tseem ceeb dua ntawm Fc RIII; feem ntau muaj ntau dua inhibition ntawm sporozoite phagocytosis los ntawm Fc RIII blockade, thiab Fc RIII khi los ntawm cov tshuaj tiv thaiv tau muaj kev cuam tshuam nrog phagocytosis ntau dua li Fc RIIa hauv kev tshuaj xyuas ntau yam.

Fc RIIIb, uas yog GPI-anchored, yog ntau tshaj Fc RIII hom qhia ntawm neutrophils, tab sis tsis ntev los no cov kev tshawb fawb tau qhia tias neutrophils kuj qhia Fc RIIIa30, uas muaj ib tug transmembrane thiab cytoplasmic domain rau intracellular signaling. Rau phagocytosis, intracellular signaling tuaj yeem tshwm sim ntawm Fc RIIIa lossis Fc RIIa, nrog rau kev khi rau Fc RIIIb kuj tseem ceeb, raws li qhia los ntawm peb qhov kev tshawb pom. Yog tias Fc RIII thiab Fc RIIa ua kev koom tes hauv phagocytosis, qhov no yuav piav qhia vim li cas thiaj li muaj kev txwv ntawm Fc RIIa hauv phagocytosis los ntawm monocytes. NK hlwb nthuav qhia Fc RIIIa, thiab peb tau tsim cov tshuaj tiv thaiv rau CSP tuaj yeem txhawb nqa NK ua haujlwm los ntawm kev cuam tshuam nrog Fc RIIIa. Qhov no tuaj yeem ua rau kev tua ntawm sporozoites nrog rau kev tso tawm ntawm pro-inflammatory cytokines44.

Txawm li cas los xij, NK hlwb tsuas yog tsuas yog 1-5 feem pua ​​​​ntawm cov leukocytes hauv cov ntshav qhia tias lawv yuav ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv sporozoites dua li neutrophils, uas suav nrog 50-70 feem pua ​​​​ntawm cov leukocytes. Sporozoites kuj tseem raug rau cov kab mob dermal macrophages thiab daim siab Kupffer hlwb, uas tuaj yeem koom nrog cov txheej txheem ntxiv ntawm Fc R-mediated clearance thiab lav kev tshawb nrhiav hauv kev tshawb fawb yav tom ntej, tab sis cov txheeb ze zaus ntawm cov hlwb thiab lawv cov kev qhia ntawm Fc Rs txawv rau phagocytes hauv cov ntshav. .

Cov kev tshawb fawb uas twb muaj lawm ntawm cov tshuaj tiv thaiv kab mob opsonic phagocytosis hauv malaria feem ntau siv THP-1 kab xov tooj ntawm tes ua qauv, siv cov qauv txheej txheem nrog FCS (tab sis tsis suav nrog tib neeg cov ntshav). Nyob rau hauv cov xwm txheej no, peb pom tias phagocytosis tau zoo inhibited los ntawm kev thaiv ntawm Fc RI, tab sis qhov tsawg kawg nkaus inhibition tau pom nrog thaiv Fc RIIa lossis Fc RIII. Ib txoj kev tshawb fawb tsis ntev los no tau tshaj tawm tias opsonic phagocytosis los ntawm THP-1 hlwb tsis cuam tshuam nrog kev tiv thaiv hauv RTS, S theem I / IIa sim hauv malaria-naive cov neeg laus45. Peb tau pom tias tsis muaj IgG tshwj xeeb hauv tib neeg cov ntshav tuaj yeem txo THP-1 cell phagocytosis kev ua haujlwm vim Fc RI kev cuam tshuam cuam tshuam los ntawm monomeric IgG nthuav tawm hauv tib neeg cov ntshav, thaum monomeric bovine IgG tsis khi tib neeg Fc RI. Ua ke, cov kev tshawb pom no qhia tias THP-1 hlwb tsis yog ib qho qauv zoo rau kev kawm opsonic phagocytosis tiv thaiv sporozoites.

Peb tau tsim tias tag nrho peb thaj tsam ntawm CSP tuaj yeem raug tsom los ntawm cov tshuaj tiv thaiv uas koom nrog Fc RIIa thiab Fc RIII los txhawb phagocytosis ntawm sporozoites los ntawm neutrophils, yog li sau cov haujlwm rau cov tshuaj tiv thaiv rau cov cheeb tsam no uas tsis tau tshaj tawm yav dhau los. Nco ntsoov, muaj cov haujlwm tseem ceeb ntawm cov tshuaj tiv thaiv kab mob rau thaj tsam NT raws li pom hauv phagocytosis kev soj ntsuam siv tib neeg cov tshuaj tiv thaiv nrog antigens-coated hlaws, phagocytosis ntawm sporozoites los ntawm luav cov tshuaj tiv thaiv kab mob tsa rau thaj tsam sib txawv, thiab Fc R khi los ntawm tib neeg cov tshuaj tiv thaiv. Cov kev tshawb pom no qhia tias kev tsom mus rau thaj tsam NT ntau dua hauv kev tsim tshuaj tiv thaiv tej zaum yuav muaj txiaj ntsig, ntxiv rau kev suav nrog hauv nruab nrab rov ua dua thiab CT cheeb tsam. Cov tshuaj tiv thaiv tam sim no tsis suav nrog thaj tsam NT3,46. Peb mapped antibody epitopes mus rau ib cheeb tsam 21-aa uas nyob ze rau ntawm qhov sib txuas ntawm NT thiab hauv nruab nrab cov cheeb tsam rov ua dua, tab sis tsis suav nrog qhov tsis ntev los no tau piav qhia txog junctional epitope34 thiab txawv ntawm Thaj Chaw I epitope, uas yog qhov chaw rau N-terminal cleavage thaum lub sij hawm ntawm hepatocyte invasion47,48.

Peb cov kev soj ntsuam uas cov tshuaj tiv thaiv tau txais los ntawm kev kis tus kab mob malaria tuaj yeem txhawb nqa Fc R-mediated phagocytosis los ntawm neutrophils ntxiv txhawb lub tswv yim tias qhov no yog ib qho txheej txheem uas tshwm sim hauv vivo ntawm cov tib neeg uas txhim kho kev tiv thaiv qib siab. Hauv kev teeb tsa ntawm kev kis tus kabmob malaria siab hauv Kenya, cov tshuaj tiv thaiv kev ua haujlwm no tsawg heev ntawm cov menyuam yaus uas feem ntau muaj kev pheej hmoo kis tus kabmob thiab mob npaws hnyav tab sis tshwm sim rau cov neeg laus. Cov kev tshawb fawb ua ntej hauv cov pej xeem no pom tias muaj cov tshuaj tiv thaiv kab mob siab rau CSP ntawm cov neeg laus tau cuam tshuam nrog kev txo qis ntawm cov neeg laus49. Hauv qhov sib piv, ib pawg ntawm cov menyuam yaus feem ntau pom tsis muaj kev koom tes hauv Mali, qhov chaw kis tus kabmob malaria qis dua peb cov neeg kawm 50. Interestingly, cov txheeb ze ntawm kev sib khi ua haujlwm nrog txhua Fc R sib txawv ntawm cov tib neeg, uas yuav cuam tshuam rau kev ua phagocytosis. Cov kev hloov pauv hauv qhov tshwj xeeb Fc R khi yuav cuam tshuam los ntawm IgG subclass profiles, epitopes tsom los ntawm cov tshuaj tiv thaiv, thiab IgG glycosylation22. IgG1 thiab IgG3 predominated ntawm tau txais IgG subclasses thiab muaj feem cuam tshuam nrog Fc R-binding kev ua.

Hauv txoj kev tshawb no, peb tau hloov kho kev siv cov antigen-coated microspheres los kawm txog opsonic phagocytosis kev ua haujlwm ntawm cov tshuaj tiv thaiv rau CSP, lub hom phiaj tseem ceeb tiv thaiv kab mob P. falciparum sporozoites. Qhov kom zoo dua ntawm txoj hauv kev no yog tias nws kov yeej cov teeb meem ntawm kev tsim cov sporozoites loj rau kev soj ntsuam immunologic, uas yog qhov nyuaj rau kev ua tiav rau cov pej xeem loj thiab kev sim tshuaj. Qib ntawm opsonic phagocytosis ntsuas siv CSP-coated hlaws tau zoo sib xws nrog qhov ntsuas uas siv cov sporozoites tsis zoo. Lub platform no kuj tau ua rau peb nthuav tawm cov tshuaj tiv thaiv kab mob ua haujlwm rau txhua cheeb tsam ntawm CSP. Peb kuj tau lees paub tias phagocytosis siv cryopreserved sporozoites (kev vam meej> 85 feem pua ​​51), thiab tshiab sporozoites dissected.

Cov kev tshawb fawb ua ntej tau pom tias cov tshuaj tiv thaiv rau sporozoites tuaj yeem cuam tshuam sporozoite motility, cell traversal, thiab hepatocyte ntxeem tau raws li lub peev xwm tiv thaiv kab mob. Txawm li cas los xij, tam sim no tsis muaj pov thawj pom zoo uas qhia txog cov txheej txheem no cuam tshuam nrog kev tiv thaiv kab mob Plasmodium hauv kev tshawb fawb soj ntsuam 3. Hauv cov qauv txhaj tshuaj tiv thaiv kab mob hauv cov tsiaj, qib siab ntawm IgG rau CSP tuaj yeem tiv thaiv kev sim kab mob 3,4. Txawm li cas los xij, kev tiv thaiv qib siab zoo sib xws, txhawb nqa lub sijhawm, tseem tsis tau ua tiav los ntawm kev txhaj tshuaj tiv thaiv kab mob hauv kev sim tshuaj 52-56. RTS, S induces siab heev CSP IgG qib, tab sis tsuas yog muab kev tiv thaiv me ntsis 53,56, tawm tswv yim tias yuav tsum muaj lwm cov tswv yim los tsim cov tshuaj tiv thaiv uas muaj kev tiv thaiv kev ua haujlwm siab dua 3,4. Peb cov ntaub ntawv muab kev nkag siab rau hauv cov kev tiv thaiv kab mob loj heev ntawm cov tshuaj tiv thaiv kab mob hauv phagocytic tshem tawm ntawm sporozoites thiab qhia tias kev siv Fc Rmediated mechanisms tuaj yeem yog lub tswv yim los tsim kom muaj zog tiv thaiv ntau dua rau kev tiv thaiv qib siab. Qhov no tuaj yeem ua tiav los ntawm cov tswv yim tshuaj tiv thaiv uas ua kom zoo dua IgG subclass cov lus teb, txo IgM uas tuaj yeem cuam tshuam Fc R kev sib cuam tshuam, thiab txhim kho lub hom phiaj ntawm cov haujlwm tshwj xeeb epitopes. Cov kev nce qib no tuaj yeem ua tiav los ntawm kev siv cov tshuaj tiv thaiv tshwj xeeb adjuvants thiab immunogen tsim. FC r kev cuam tshuam nrog iGG khi rau antigens kuj tseem tuaj yeem cuam tshuam los ntawm kev ua kom zoo nkauj thiab kev qhia ntawm cov epitopes57,58. Cov txheej txheem biology yuav muaj txiaj ntsig kom nkag siab qhov no thiab qhia txog kev tsim tshuaj tiv thaiv. Nws kuj tseem muaj peev xwm hais tias cov cheeb tsam sib txawv ntawm CSP kho qhov kev tiv thaiv kab mob los ntawm kev tiv thaiv kab mob sib txawv.

Qhov no tau tshwm sim rau tus mob khaub thuas hemagglutinin, nrog Fc Rmediated mechanisms yog ib qho tseem ceeb rau cov tshuaj tiv thaiv kab mob rau thaj tsam stalk khaws cia, tab sis tsis tseem ceeb rau cov kab mob tshwj xeeb rau lub taub hau cheeb tsam18,21.

Cov kev tshawb fawb yav tom ntej uas tshawb xyuas qhov cuam tshuam ntawm cov tshuaj tiv thaiv kev ua haujlwm no thiab lawv cov kev sib raug zoo nrog kev tiv thaiv tau lav nyob rau theem II thiab theem III kev sim ntawm RTS, S, thiab lwm yam tshuaj tiv thaiv CSP. Peb tau ua qhov kev tshawb fawb thawj zaug hauv theem I / IIa sim ntawm RTS, S56. Qhov no tau tsim tias RTS, S txhaj tshuaj tiv thaiv tiag tiag ua rau cov tshuaj tiv thaiv rau CSP uas txhawb kev khi ntawm Fc RIIa thiab Fc RIII, nrog rau phagocytosis los ntawm neutrophils. Qhov tseem ceeb, cov haujlwm ua haujlwm no tau nce siab dua ntawm cov neeg kawm uas tau txais kev tiv thaiv tiv thaiv kab mob malaria piv rau cov uas tau kis kab mob43. Thaum CSP yog lub hom phiaj ntawm kev tiv thaiv tib neeg, suav nrog cov tshuaj tiv thaiv kev ua haujlwm, tam sim no muaj kev nkag siab tsis txaus ntawm qhov sib txawv ntawm qhov tau txais thiab txhaj tshuaj tiv thaiv kab mob sporozoites. Kev tshawb fawb ntxiv ntawm qhov sib txawv no tuaj yeem nthuav qhia cov kev pom tseem ceeb los qhia txog kev tsim tshuaj tiv thaiv.

Hauv cov ntsiab lus, peb cov kev tshawb fawb tau txhais cov txheej txheem tseem ceeb thiab lub hom phiaj ntawm Fc R-mediated tiv thaiv tiv thaiv sporozoites hauv cov ntshav. Peb qhov kev tshawb pom taw qhia rau lub luag haujlwm tseem ceeb rau neutrophils hauv phagocytic tshem tawm ntawm sporozoites, txhais lub luag haujlwm tseem ceeb rau Fc RIIa thiab Fc RIII, thiab tsim CSP ua lub hom phiaj tseem ceeb rau kev ua haujlwm no. Monocytes kuj ua rau phagocytosis tab sis muaj kev ua haujlwm qis dua, thiab cov tshuaj tiv thaiv kuj tuaj yeem txhawb nqa NK cell ADCC kev ua haujlwm los ntawm kev koom tes ntawm Fc RIIIa. Kev nkag siab thiab txhais cov txheej txheem uas kho kev tiv thaiv kab mob yog qhov tseem ceeb los pab txhim kho cov tshuaj tiv thaiv zoo dua. Cov kev tshawb fawb yav tom ntej yuav tsum tshawb xyuas seb cov tshuaj tiv thaiv tam sim no zoo li cas hauv kev sim tshuaj ntsuam xyuas tsim cov tshuaj tiv thaiv nrog cov haujlwm no. Kev siv cov kev paub no hauv kev tsim tshuaj tiv thaiv tej zaum yuav yog ib lub tswv yim zoo rau kev tsim cov tshuaj tiv thaiv kab mob uas muaj zog tiv thaiv kab mob malaria.

Cov txheej txheem

Kawm cov pej xeem thiab cov lus hais txog kev coj ncaj ncees.

Plasma los ntawm tus kab mob malaria-pom zoo, asymptomatic cov neeg laus nyob hauv Kanyawegi sub-koog tsev kawm ntawv ntawm Kisumu County, Kenya (n=104, hnub nyoog 18–79; 26.9 feem pua ​​​​txiv neej) tau sau thaum Lub Yim Hli 2007 (Kanyawegi pawg) 59; tsis muaj cov neeg koom nrog cov tsos mob ntawm tus kab mob malaria thiab 46.15 feem pua ​​​​ntawm cov neeg muaj asymptomatic P. falciparum parasitemia (kuaj los ntawm microscopy). Plasma los ntawm cov menyuam yaus noj qab nyob zoo, asymptomatic cov menyuam yaus thiab cov neeg laus nyob hauv Chulaimbo sub koog tsev kawm ntawv ntawm Kisumu County, Kenya (n=75, hnub nyoog thaj tsam 0.3–5.9 xyoo rau cov menyuam yaus, hnub nyoog 19.6–69.2 xyoos rau cov laus; 55 feem pua ​​​​poj niam) yog sau nyob rau lub Ob Hlis-Lub Peb Hlis 2007 (Chulaimbo cohort)42; Cov ntsiab lus ntawm cohort tau sau tseg nyob rau hauv Cov Lus Qhia Ntxiv 1. Kev sib kis mob hnyav heev thaum lub sijhawm sau cov ntshav (qhov feem ntau ntawm asymptomatic parasitemia hauv cov menyuam yaus tsawg dua lossis sib npaug li 10 xyoo yog 70–80 feem pua) 60. Cov ntshav tau sau los ntawm cov txiv neej thiab poj niam cov neeg laus noj qab haus huv rau kev siv tag nrho cov leukocyte, neutrophil, monocyte, thiab NK kev soj ntsuam.

Kev pom zoo rau kev coj ncaj ncees tau txais los ntawm Alfred Tsev Kho Mob Human Research thiab Ethics Committee (txoj cai 385-18), Lub Tsev Haujlwm Saib Xyuas Kev Tshawb Fawb Txog Tib Neeg ntawm University Hospitals of Cleveland rau Case Western Reserve University, USA (txoj cai 02-04-04) thiab Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Kenya Medical Research Institute, Kenya ( raws tu qauv SSC867). Sau ntawv tso cai tau txais los ntawm txhua tus neeg koom nrog kev kawm lossis lawv niam lawv txiv lossis cov neeg saib xyuas raug cai.

Antigens.

Cov antigens siv nyob rau hauv txoj kev tshawb no yog tag nrho raws li 3D7 siv ib ntus ntawm P. falciparum CSP (Gene ID PF3D7_0304600; www.plasmodb.org). Lub recombinant puv-ntev CSP tau yuav los ntawm Sanaria (Sanaria, Rockville, USA).

Cov protein tau qhia hauv Pichia pastoris pib ntawm amino acid residue 50 thiab muaj (NANP) 22 rov ua dua thiab (NVPD) 4 rov ua dua. C-terminal cheeb tsam ntawm CSP (CT) thiab fusion ntawm N-terminal cheeb tsam thiab C-terminal cheeb tsam ntawm CSP (N ntxiv rau C) tau tsim los ua cov protein sib txuas siv HEK293F hlwb (yuav los ntawm Invitrogen, Australia). N-terminal thaj tsam ntawm CSP (amino acid 19-104, NT) tau tsim los ua hluavtaws peptides, thiab lub hauv paus rov qab cheeb tsam ntawm CSP (NANP) kuj tau tsim los ua hluavtaws peptides (NANP × 15) los ntawm LifeTein (LifeTein, NJ, USA).

Kev tsim cov tshuaj tiv thaiv rau cov protein CSP sib txawv.

ua los ntawm Walter Eliza Hall Institute antibody chaw; Kev pom zoo tau muab los ntawm Pawg Saib Xyuas Kev Ncaj Ncees Tsiaj ntawm Walter thiab Eliza Hall Institute (ref: 2020.019, 2014.009). Luv luv, luav (n=2) tau txhaj tshuaj tiv thaiv nrog ib qho ntawm cov antigens hauv qab no: tag nrho-ntev CSP, N ntxiv rau C recombinant protein, thiab NT peptide. Txhua tus luav tau txais peb koob tshuaj antigen (200 µg / koob) koom nrog Freud's adjuvant hnub 0, hnub 28, thiab hnub 56. Luav sera tau sau tom qab txhaj tshuaj thib peb rau hnub 68, thiab luav IgG tau huv los ntawm protein A. - xaiv qhov zoo. Cov luav anti-N ntxiv rau C IgG tau pib sim, thiab pom tias feem ntau lees paub thaj tsam C-terminal ntawm CSP, thiab tsis yog thaj tsam N-terminal (Ntxiv daim duab 6A). Tus luav tiv thaiv NT IgG tau lees paub kom paub txog N-terminal cheeb tsam ntawm CSP (Sab Ntxiv Fig. 6B). Ob leeg NT thiab CT cov tshuaj tiv thaiv tau hnov ​​​​mob nrog CSP tag nrho (Ntxiv Fig. 6C). Monoclonal antibody (mAb) MGG434, uas tau raug cais tawm ntawm ib tus neeg tom qab rov kis tus kab mob nrog cov sporozoites attenuated, tau qhia thiab ua kom huv si raws li cov tshuaj tiv thaiv IgG1 recombinant hauv HEK293 hlwb siv cov txheej txheem tsim. Lub mAb tau sim tawm tsam CSP recombinant kom paub meej meej. Anti-CSP mAb 2A1061 tau nthuav tawm nrog LALA kev hloov pauv hauv cheeb tsam Fc62 los tiv thaiv Fc R kev sib cuam tshuam raws li kev tswj hwm cov tshuaj tiv thaiv.

Kev rho tawm ntawm PBMC, neutrophils, thiab tag nrho cov leukocytes los ntawm cov ntshav peripheral.

Peripheral blood mononuclear cells (PBMC) thiab neutrophils raug cais tawm ntawm cov ntshav peripheral siv txoj kev tsim yav dhau los63,64. Luv luv, cov ntshav peripheral los ntawm cov neeg noj qab haus huv tau sib cais siv Ficoll gradient centrifugation. Cov khaubncaws sab nraud povtseg, uas muaj PBMCs, thiab RBC pellet uas muaj cov neutrophils, tau cais cais. Rau kev cais PBMC, cov hlwb uas tau sau los ntawm Buffy txheej tau ntxuav plaub zaug nrog PBS-1 feem pua ​​NCS ntawm 4 degree . PBMCs tau rov qab ua dua hauv RPMI1640-10 feem pua ​​​​fetal bovine serum (FCS) thiab khaws cia rau hauv dej khov. Neutrophils tau enriched los ntawm dextran segmentation los ntawm RBC pellet post Ficoll gradient centrifugation, ua raws li hypotonic lysis63.

Tag nrho cov leukocytes raug cais los ntawm tag nrho cov ntshav los ntawm kev pib ua kom muaj txiaj ntsig nrog dextran segmentation ua raws li hypotonic lysis. Cov neutrophils cais thiab tag nrho cov leukocytes tau hloov kho rau 5 × 105 neutrophils / ml lossis 5 × 106 phagocytes (monocytes thiab neutrophils) / ml thiab tom qab ntawd rov ua dua hauv RPMI1640 ntxiv nrog 10 feem pua ​​​​FCS thiab 2.5 feem pua ​​​​ntawm tib neeg cov cua sov-inactivated. Cells tau phenotyped rau Fc R qhia siv cov tshuaj tiv thaiv rau Fc RI (Clone 10.1 BD Bioscience, 1:50), Fc RIIa (Clone 8.26, BD Bioscience, 1:25), thiab Fc RIII (Clone 3G8 BD Bioscience, 1:50) . Cov tshuaj tiv thaiv rau CD14 (Clone M5E2, BD Bioscience, 1:20) kuj tau siv los txiav txim cov kab mob monocyte.

Kev tu ntawm THP-1 cell.

Cells ntawm THP{{0}}} promonocytic cell kab (los ntawm ATCC) tau khaws cia hauv RPMI-1640 nrog 0.002 mol/L L-glutamine, 0.01 mol/L HEPES , thiab 10 feem pua ​​​​fetal calf serum (FCS). Cell ceev tau saib xyuas zoo thiab khaws cia ntawm 1 × 105 thiab 1 × 106 cell / ml. Cells tau kis txhua txhua 6 hnub los yog thaum cell ceev mus txog 1 × 106 hlwb / ml.

Kab lis kev cai thiab kev sib cais ntawm P. berghei sporozoites.

Anopheles stephensi yoov tshaj cum tau noj ntawm Swiss Webster nas kis tus kab mob chimeric CSP qhia P. berghei ANKA kab mob kab 12. Cov qog ua kua qaub tau raug txiav tawm 18-22 hnub tom qab noj zaub mov kis kab mob thiab homogenized hauv PBS. Cov sporozoites tau dhau los ntawm 70-μm cov ntaub so ntswg lim (Falcon) thiab suav nrog hemocytometer.

Kab lis kev cai thiab kev cais ntawm P. falciparum merozoites.

D10 kab ntawm P. falciparum tau khaws cia hauv RPMI-HEPES ntxiv nrog 0.5 feem pua ​​​​ Albumax (Life Technologies) thiab 0.18 feem pua ​​NaHCO3. Kab lis kev cai tau khaws cia qis dua 10 feem pua ​​​​ntawm cov kab mob parasitemia thiab synchronized los ntawm kev kho sorbitol. Cov merozoites raug cais los ntawm cov txheej txheem uas tau tshaj tawm yav dhau los 32. luv luv, theem schizont ntawm cov kab mob erythrocytes tau ntxiv los ntawm kev sib nqus purification thiab txuas ntxiv mus rau hauv ib qho nruab nrab ntxiv nrog transepoxysuccinyl-L-leucylamido(4-guanidino) butane (E64, Sigma). -Aldrich) rau 8 teev ua ntej dhau cov schizonts paub tab los ntawm 1.2-μm lim kom tso cov merozoites. Cov merozoites tau hloov kho rau 5 × 107 / ml rau opsonic phagocytosis assay lossis 1 × 107 / ml rau txheej rau ELISA phaj rau Fc R-binding assay.

Covalent coupling ntawm antigens rau fluorescent latex hlaws.

Amine-modified fluorescent latex hlaws ntawm 2.0-µm loj (Sigma-Aldrich) tau ntxuav ob zaug nrog 400 µl ntawm PBS thiab centrifuged ntawm 2000 × g rau 3 min. Glutaraldehyde ntawm 8 feem pua ​​​​hauv PBS tau ntxiv rau cov hlaws thiab incubated rau ib tug menyuam hmo ntuj ntawm 4 degree. Tom qab ntxuav nrog PBS, 1 mg / ml ntawm recombinant CSP tau ntxiv rau cov hlaws dai thiab incubated ntawm lub vortex rau 6 h. Tom qab ntawd, cov hlaws dai tau centrifuged thiab resuspended hauv 200 µl ntawm ethanolamine thiab incubated rau 30 feeb ntawm ib tug vortex kom tua tag nrho cov seem amine pawg. Cov hlaws dai tom qab tau ntxuav hauv PBS thiab thaiv nrog 1 feem pua ​​BSA thaum hmo ntuj ntawm 4 degree. Cov hlaws dai antigen-coated tau khaws cia rau hauv ib da dej sonicating rau 20 min ntawm 4 degree kom txo tau cov aggregation thiab tom qab ntawd hloov mus rau 5 × 107 hlaws dai / ml. Cov hlaws tau khaws cia ntawm 4 degree nyob rau hauv muaj 0.1 feem pua ​​SDS thiab 0.02 feem pua ​​NaN3.

Opsonic phagocytosis kev soj ntsuam.

Opsonic phagocytosis assay tau ua nyob rau hauv qhov chaw sib txawv rau ntau lub hom phiaj raws li tau piav qhia hauv qab no thiab hauv Cov Lus Ntxiv 6.

Rau opsonic phagocytosis ntawm P. falciparum thiab P. berghei sporozoites siv cov hlwb cais, cryopreserved P. falciparum sporozoites tau muab los ntawm PATHMVI (Samaria, Rockville, USA) thiab khaws cia hauv cov kua nitrogen, thiab tau thawed ntawm 37 degree rau 40 in s raws li piav qhia. cov neeg tsim khoom siv phau ntawv. Freshly dissected P. berghei sporozoites uas tau kis tau los qhia txog PfCSP12 kuj tau siv hauv txoj kev tshawb no. Tib txoj hauv kev tau siv rau P. falciparum thiab P. berghei sporozoites hauv kev sim. Cov sporozoites tau stained nrog 10 µg / ml ethidium bromide rau 1 h ntawm cov dej khov tom qab peb ntxuav nrog RPMI-HEPES. Rau opsonization, 50, 000 sporozoites tau incubated nrog 1 ul ntawm cov ntshav kuaj rau 1 teev ntawm dej khov, ua raws li kev sib koom ua ke nrog 5000 neutrophils ntawm qhov concentration ntawm 5 × 105 hlwb / ml hauv RPMI-1640 ntxiv nrog 10 feem pua ​​​​FCS thiab 2.5 feem pua ​​​​ntawm cov neeg tsis muaj zog thaum kub hnyiab los ntawm cov neeg mob malaria-naive Melbourne pub. Ib qho ntxiv 1ul ntawm cov tshuaj ntsuam xyuas ntshav kuj tau ntxiv rau tus account rau qhov ntim hloov pauv los ntawm kev ntxiv neutrophils thiab tswj tib lub sijhawm kuaj tshuaj tiv thaiv kab mob thaum lub sijhawm phagocytosis.

Lub coincubation tau tso cai rau 15 min ntawm 37 degree hauv 5 feem pua ​​CO2 incubator rau phagocytosis tshwm sim. Cells raug centrifuged ntawm 4 degree thiab ntxuav nrog txias PBS-1 feem pua ​​NCS–0.04 feem pua ​​NaN3 (FACS tsis) kom tsis txhob muaj phagocytosis. Cov theem ntawm phagocytosis tau ntsuas los ntawm kev ntws cytometry (FACS CantoII, BD Biosciences) thiab tshuaj xyuas siv FlowJo software (version 10). Cov ntaub ntawv tau nthuav tawm raws li phagocytosis index (PI), uas txhais tau tias yog qhov feem pua ​​​​ntawm neutrophils (Supplementary Fig. 4A) los yog THP-1 hlwb (Supplementary Fig. 1A) uas muaj phagocytosed sporozoites. Rau kev sim ntawm Kanyawegi thiab Chulaimbo cohort, PI tau ntxiv tus qauv raws li tus txheeb ze phagocytosis index (RPI), uas tau txhais tias yog feem pua ​​​​ntawm PI rau txhua tus qauv txheeb ze rau kev tswj zoo. Unopsonized sporozoites tau suav nrog kev tswj tsis zoo hauv txhua qhov kev ntsuam xyuas. Standard neutrophil phagocytosis assays suav nrog 2.5 feem pua ​​​​ntawm tib neeg cov ntshav los ntawm cov neeg mob malaria uas tsis pub pub dawb. Txheem THP-1 phagocytosis kev soj ntsuam tau ua los ntawm cov txheej txheem tsim uas suav nrog 10 feem pua ​​FCS.

Rau phagocytosis ntawm antigen-coated hlaws dai los yog merozoites siv cov hlwb cais, phagocytosis assay tau yoog los ntawm cov txheej txheem luam tawm yav dhau los nrog kev hloov kho 32. Luv luv, 1 × 106 antigen-coated fluorescent latex hlaws lossis merozoites tau opsonized nrog cov qauv ntshav rau 1 h. Cov hlaws los yog merozoites tau ntxuav peb zaug nrog RPMI-1640 ua ntej co-incubation nrog 1 × 105 neutrophils rau phagocytosis. Phagocytosis raug tso cai tshwm sim rau 20 min rau cov hlaws dai lossis 10 min rau merozoites ntawm 37 degree thiab cov hlwb tau tom qab ntxuav nrog FACS tsis nyob ntawm 300 × g thiab 4 degree rau 4 min. Qhov kev faib ua feem ntawm cov neutrophils uas muaj cov hlaws fluorescent lossis merozoites tau soj ntsuam los ntawm cov cytometry ntws (FACS CantoII, BD Biosciences) thiab tshuaj xyuas siv FlowJo software (Cov duab ntxiv 4A). Hauv qee qhov kev ntsuam xyuas, THP-1 kab xov tooj tau siv nrog cov txheej txheem uas suav nrog 10 feem pua ​​FCS; Hauv lwm qhov kev ntsuam xyuas, ib qho ntxiv 2.5 feem pua ​​​​ntawm tib neeg cov ntshav los ntawm malaria naive Melbourne cov neeg pub dawb tau siv los sib piv.

Rau phagocytosis ntawm antigen-coated hlaws dai los yog P. berghei sporozoites siv tag nrho cov ntshav leukocyte npaj, CSP-coated hlaws tau opsonized nrog ib tug luav polyclonal antibody tsa tawm tsam CSP. Phagocytosis tau ua los ntawm kev sib koom ua ke cov hlaws opsonized nrog tag nrho cov leukocyte feem ntawm tag nrho cov ntshav uas muaj 1 × 106 phagocytes (monocytes thiab neutrophils) rau 20 min ntawm 37 degree hauv 5 feem pua ​​CO2 incubator. Cov kev sim no tau ua los ntawm kev siv cov tshuaj tiv thaiv kab mob sib txawv rau opsonization lossis sib txawv ntawm cov hlaws-rau-phagocyte piv. Rau kev sim opsonic phagocytosis ntawm P. berghei sporozoites los ntawm tag nrho-leukocyte npaj, 5 × 105 sporozoites tau opsonized nrog lub pas dej ua ke ntawm malaria-exposed Kenyan neeg laus sera ntawm ib tug dilution ntawm 1:100, ua raws li co-incubation nrog tag nrho cov leukocytes. 104 phagocytes. Cov hlwb tau centrifuged ntawm 4 degree rau 4 min kom tsis txhob muaj phagocytic kev ua si thiab cov phagocytes sib txawv (monocytes thiab neutrophils) tau sau nrog fluoresce-conjugated antibodies rau kev tshuaj xyuas ntxiv. Monocytes tau sau npe nrog anti-CD14-Alexa647 (MφP9 BD Bioscience, 1:33) thiab anti-CD16- BV421 (3G8 BD Bioscience, 1:20), thiab neutrophils tau sau nrog anti-CD66bAPC -H7 (G10F5 BD Bioscience, 1:33).

Rau FACS tsom xam, phagocytosed hlaws tau raug xaiv raws li PEhigh thiab FSChigh pejxeem. Cov hlaws uas tau phagocytosed los ntawm monocytes thiab neutrophils tau ntxiv qhov txawv siv CD14 thiab CD66b staining. Tus naj npawb ntawm cov hlaws phagocytosed los ntawm monocytes thiab neutrophils raug xam raws li kev siv fluorescence (Ntxiv daim duab 8). Tus lej no tau tsim kho ntxiv siv tag nrho cov naj npawb ntawm phagocytes (monocytes thiab neutrophils) thiab qhia tias yog hlaws rau 100 phagocytes, sawv cev rau phagocytosis tus nqi ntawm ntau hom cell.

Rau inhibiting opsonic phagocytosis los ntawm thaiv Fc receptors lossis depleting CSP cov tshuaj tiv thaiv, sib txawv Fc Rs raug thaiv siv cov tshuaj tiv thaiv tshwj xeeb. Fc RI raug thaiv nrog 50 µg / ml thaiv cov tshuaj tiv thaiv (mAb 10.1 Merck), Fc RIIa raug thaiv nrog 50 µg / ml thaiv cov tshuaj tiv thaiv (mAb IV.3), Fc RIII raug thaiv nrog 50 µg / ml thaiv cov tshuaj tiv thaiv (mAb 3G8) . THP-1 hlwb thiab neutrophils raug kho nrog txhua Fc R blocker, raws li, ntawm 4 degree rau 30 min ua ntej co-incubation nrog CSP-coated latex hlaws, uas tau opsonized nrog cov ntshav los ntawm cov neeg laus tiv thaiv kab mob. Kev ntsuam xyuas phagocytosis tau ua raws li tau hais los saum no. Txhawm rau ntsuas lub luag haujlwm ntawm cov tshuaj tiv thaiv rau CSP hauv kev txhawb nqa phagocytosis kho los ntawm tib neeg cov tshuaj tiv thaiv kab mob, lub pas dej ntawm Kenyan cov neeg laus cov ntshav tau ua ntej incubated rau 30 min nrog recombinant CSP thiab NT cheeb tsam peptide ntawm ib tug concentration ntawm 20 ug / ml los khi CSP tshwj xeeb. Cov tshuaj tiv thaiv kab mob, ua ntej siv hauv kev tshuaj ntsuam phagocytosis.

Ntsuas tag nrho IgG thiab IgG subclasses los ntawm ELISA.

Tag nrho IgG thiab IgG subclasses rau recombinant proteins tau ntsuas los ntawm ELISA raws li tau piav qhia dhau los33. Luv luv, CSP tau coated ntawm 1 µg/ml ntawm 96-zoo Maxisorp microtiter daim hlau (NUNC, Roskilde, Denmark) thiab incubated hmo ntuj ntawm 4 degree . Cov phaj tau ntxuav nrog PBS uas muaj 0.05 feem pua ​​Tween20 (PBS-Tween) thiab thaiv nrog PBS -1 feem pua ​​BSA ntawm 37 degree rau 2 teev. Tib neeg cov ntshav los yog luav IgG tau diluted mus rau qhov xav tau concentration thiab ntxiv rau daim hlau hauv duplicates. Rau tag nrho IgG nrhiav pom, HRP-conjugated tshis tiv thaiv tib neeg IgG tau siv ntawm 1: 2500 dilution tom qab ABTS substrate rau kev tsim xim. Cov tshuaj tiv thaiv tau nres nrog 1 feem pua ​​SDS, thiab absorbance yog ntsuas ntawm 405 nm. Rau IgG subclass nrhiav pom, mAbs rau tib neeg IgG1, IgG2, IgG3, thiab IgG4 tau siv ntawm 1: 1000 dilution, ua raws li HRP conjugated tshis tiv thaiv nas IgG siv ntawm 1: 2500 dilution. Thaum kawg, 3,3′,5,5′-Tetramethylbenzidine (TMB, Life Technologies) substrate tau siv rau kev tsim xim, thiab cov tshuaj tiv thaiv raug tso tseg nrog 1 M H2SO4. Lub absorbance yog ntsuas ntawm 450 nm. Lub pas dej ntawm Kenyan cov neeg laus cov ntshav nrog siab IgG reactivity rau CSP tau siv los ua kev tswj xyuas zoo.

Mapping antibody epitopes ntawm thaj av NT.

Ib qho array uas muaj 70 biotinylated 15-mer peptides sawv cev rau cov kab sib lawv liag ntawm CSP (3D7/NF54 strain) tau ua ke (Mimotopes, Australia). Txhua peptide muaj luv SGSG spacer nruab nrab ntawm CSP peptide ib ntus thiab biotin tag (uas tau ntxiv rau kev pab cuam immobilization), thiab 12 aa sib tshooj ntawm peptides. Cov tshuaj tiv thaiv kab mob tau ua los ntawm cov txheej txheem uas tau tsim yav dhau los 65,66; 50 ul ib qhov dej ntawm peptides tau ntxiv rau ELISA daim hlau uas tau ua ntej coated nrog 1 ug / ml streptavidin (Sigma-Aldrich). Saturation ntawm peptide khi tau ua tiav thiab paub tseeb los ntawm kev txheeb xyuas qhov seem ntawm biotin-binding qhov chaw ntawm streptavidin siv biotin-HRP. ua raws li thaiv nrog 10 feem pua ​​​​cov mis nyuj, thiab ntxuav nrog PBS nrog Tween 0.05 feem pua. Luav polyclonal IgG tau tsa tawm tsam thaj tsam NT ntawm CSP tau ntxiv rau txhua qhov dej ntawm qhov concentration ntawm 1: 500. Qib ntawm luav IgG khi tau kuaj pom los ntawm HRPconjugated tshis los tiv thaiv luav IgG (Millipore, 1: 2500) thiab suav nrog siv TMB substrate.

Fc receptor-binding assay.

Fc receptor-binding assay tau yoog los ntawm cov kev tshaj tawm yav dhau los nrog kev hloov kho me me39. Luv luv, 5{{20}} ul ntawm recombinant CSP ntawm ib tug concentration ntawm 1 ug/ml yog coated rau Maxisorp™ daim hlau thiab incubated ib hmos ntawm 4 degree, ua raws li peb ntxuav nrog PBS-Tween. Cov phiajcim tau raug thaiv nrog 200 ul ntawm 1 feem pua ​​​​BSA-PBS ntawm 37 degree rau 2 h tom qab peb ntxuav nrog PBS-Tween. Cov qauv ntshav tau diluted ntawm 1:50 hauv PBS-BSA thiab 50 ul ntawm txhua tus qauv tau ntxiv rau hauv daim hlau sib npaug thiab incubated ntawm chav tsev kub rau 2 h, ua raws li peb ntxuav hauv PBS-Tween. Tsib caug microliters ntawm biotin-conjugated recombinant soluble Fc RIIa H131 ectodomain dimers (0.2 ug / ml) los yog Fc RIII V158 ectodomain dimers (0.1 ug / ml) tau ntxiv rau txhua qhov dej thiab incubated ntawm 37 degree rau 1 h tom qab ntawd peb ntxuav nrog PBS. -Tween. Rau qee qhov kev ntsuam xyuas, R131 allele ntawm Fc RIIa thiab F158 allele ntawm Fc RIII tau siv tib qhov concentration. Qhov no tau ua raws li theem nrab HRP-conjugated streptavidin (Thermo Fisher Scientific, 1:10,000) hauv PBS-BSA ntawm 37 degree rau 1 h tom qab peb ntxuav nrog PBS-Tween.

Thaum kawg, 50 ul ntawm TMB kua substrate tau ntxiv rau 20 feeb los ntsuas cov tshuaj tiv thaiv enzymatic. Cov tshuaj tiv thaiv tau nres nrog 50 ul ntawm 1 M H2SO4 tov. Qib ntawm kev khi tau ntsuas raws li qhov muag pom ntawm 450 nm. Cov tib neeg IgG sib xyaw los ntawm cov neeg mob malaria-exposed Kenyan cov neeg laus (1: 100) thiab luav polyclonal IgG rau CSP (1: 500) tau siv los ua kev tswj xyuas zoo. Tus kheej sera los ntawm cov neeg laus uas tsis paub lus Melbourne (1:50) tau siv los ua kev tswj tsis zoo.

Fc receptor-binding assay tau yoog los ntawm cov kev tshaj tawm yav dhau los nrog kev hloov kho me me39. Luv luv, 5{{20}} ul ntawm recombinant CSP ntawm ib tug concentration ntawm 1 ug/ml yog coated rau Maxisorp™ daim hlau thiab incubated ib hmos ntawm 4 degree, ua raws li peb ntxuav nrog PBS-Tween. Cov phiajcim tau raug thaiv nrog 200 ul ntawm 1 feem pua ​​​​BSA-PBS ntawm 37 degree rau 2 h tom qab peb ntxuav nrog PBS-Tween. Cov qauv ntshav tau diluted ntawm 1:50 hauv PBS-BSA thiab 50 ul ntawm txhua tus qauv tau ntxiv rau hauv daim hlau sib npaug thiab incubated ntawm chav tsev kub rau 2 h, ua raws li peb ntxuav hauv PBS-Tween. Tsib caug microliters ntawm biotin-conjugated recombinant soluble Fc RIIa H131 ectodomain dimers (0.2 ug / ml) los yog Fc RIII V158 ectodomain dimers (0.1 ug / ml) tau ntxiv rau txhua qhov dej thiab incubated ntawm 37 degree rau 1 h tom qab ntawd peb ntxuav nrog PBS. -Tween. Rau qee qhov kev ntsuam xyuas, R131 allele ntawm Fc RIIa thiab F158 allele ntawm Fc RIII tau siv tib qhov concentration. Qhov no tau ua raws li theem nrab HRP-conjugated streptavidin (Thermo Fisher Scientific, 1:10,000) hauv PBS-BSA ntawm 37 degree rau 1 h tom qab peb ntxuav nrog PBS-Tween. Thaum kawg, 50 ul ntawm TMB kua substrate tau ntxiv rau 20 feeb los ntsuas cov tshuaj tiv thaiv enzymatic. Cov tshuaj tiv thaiv tau nres nrog 50 ul ntawm 1 M H2SO4 tov. Qib ntawm kev khi tau ntsuas raws li qhov muag pom ntawm 450 nm. Cov tib neeg IgG sib xyaw los ntawm cov neeg mob malaria-exposed Kenyan cov neeg laus (1: 100) thiab luav polyclonal IgG rau CSP (1: 500) tau siv los ua kev tswj xyuas zoo. Tus kheej sera los ntawm cov neeg laus uas tsis paub lus Melbourne (1:50) tau siv los ua kev tswj tsis zoo.

Antibody-dependent cell-mediated cytotoxicity assay.

ADCC-inducing kev ua ntawm ib qho kev xaiv ntawm cov qauv ntshav tau soj ntsuam siv cov khoom siv ADCC Reporter Bioassay (Promega, Cat. No. G7010) raws li cov chaw tsim khoom cov lus qhia, nrog rau cov kev hloov kho hauv qab no. CSP-coated hlaws tau opsonized nrog tib neeg cov ntshav ntawm 1:50 dilution. Cov xov tooj xov tooj tau muab plated ntawm 72,000 cov cell ntawm ib qhov zoo ua ke nrog CSP-coated fluorescent hlaws ntawm 10: 1 piv. Tom qab 6 teev ntawm incubation, chemiluminescence substrate tau ntxiv thiab luminescence tau kuaj pom thiab ntsuas kwv yees li ntawm 5 min rau 40 min (CLARIOstar, BMG LabTech). Cov ntaub ntawv nthuav tawm tau sau tseg txog 30 min tom qab ntxiv cov chemiluminescence substrate.

Qib ntawm ADCC kuj tau lees paub hauv thawj NK hlwb siv txoj kev tshaj tawm yav dhau los 38. Luv luv, PBMCs uas muaj NK hlwb raug coj los ua hauv RPMI1640 ntxiv nrog 10 feem pua ​​FCS thiab 100 IU / ml ntawm interleukin-2 (IL{{5 }}) hmo ntuj. CSP-coated hlaws tau opsonized nrog tib neeg cov ntshav ntawm 1:50 dilution, ces co-cultured nrog primed PBMCs nyob rau hauv lub xub ntiag ntawm anti-CD107a-AF647 (H4A3, BD Bioscience) rau 1 h. Tom qab ntawd, brefeldin A (Sigma-Aldrich) thiab protein thauj inhibitor (BD Bioscience) tau ntxiv rau kev coj noj coj ua ntawm ib qho concentration ntawm 5 ug / ml thiab 1/1500, raws li, thiab txuas ntxiv mus rau 3 teev. Tom qab incubation, cov hlwb raug centrifuged ntawm 300 × g rau 4 min ntawm 4 degree thiab stained nrog anti-CD3-APC-H7 (SK7, BD Bioscience, 1:33), anti-CD56- PE-Cy7 (B159, BD Bioscience, 1:33) and anti-CD16-BV421 (3G8, BD Bioscience, 1:20). NK hlwb raug txhais raws li CD3−, CD56 ntxiv rau cov lymphocytes thiab qib ntawm ADCC tau suav tias yog feem pua ​​​​ntawm NK hlwb nrog CD107a staining los ntawm cov cytometry ntws (LSR Fortessa X-20, BD Bioscience) (Sab Ntxiv Fig. 9).

Cov ntaub ntawv tsom xam.

Kev txheeb xyuas txheeb cais tau ua tiav siv Prism version 7 (GraphPad Software Inc) thiab STATA version 13.1 (StataCorp). Kev xeem Statistics thiab P qhov tseem ceeb yog qhia hauv daim duab dab neeg. Kev sib raug zoo ntawm phagocytosis, Fc R binding, thiab IgG qib tau raug soj ntsuam siv Spearman's correlation coefficients thiab ntau hom kab rov tav. Qhov sib txawv ntawm theem ntawm phagocytosis, Fc R khi thiab IgG qib thoob plaws cov hnub nyoog thiab cov xwm txheej sib txawv tau raug soj ntsuam siv ANOVA ib-txoj kev nrog ntau qhov sib piv.

Txhawm rau ntsuas cov tshuaj tiv thaiv kab mob ua haujlwm tsis hais txog tag nrho IgG titer rau CSP, Fc R khi thiab phagocytosis cov txiaj ntsig tau raug xam raws li qib ntawm Fc R khi lossis qib ntawm opsonic phagocytosis txheeb ze rau IgG reactivity rau CSP. Spearman qhov kev sib raug zoo thiab kev xeem Mann-Whitney tau siv los tshuaj xyuas cov tshuaj tiv thaiv kab mob ua haujlwm zoo ntawm cov menyuam yaus thiab cov neeg laus. Ob txoj kev ANOVA tau siv los sib piv cov opsonic phagocytosis los ntawm neutrophils thiab monocytes hauv tag nrho cov leukocyte kev soj ntsuam. Kruskal-Wallis test thiab Mann-Whitney test tau ua rau cov ntaub ntawv uas tsis yog ib txwm faib. Tag nrho cov kev txheeb xyuas txheeb cais tau ua raws li kev ntsuam xyuas ob sab. Cov qauv tau muab cais ua qhov zoo rau cov tshuaj tiv thaiv kab mob yog tias cov tshuaj tiv thaiv ntau dua li qhov nruab nrab ntxiv rau 3 tus qauv sib txawv ntawm qhov tseem ceeb ntawm cov tshuaj tiv thaiv kab mob malaria uas tsis muaj kev cuam tshuam.

Tshaj tawm cov ntsiab lus.

Cov ntaub ntawv ntxiv ntawm kev tshawb fawb tsim muaj nyob rau hauv Kev Tshawb Fawb Txog Kev Tshawb Fawb Txog Kev Tshawb Fawb Txog Kev Tshawb Fawb txuas rau kab lus no.

Cov ntaub ntawv muaj.

Cov ntaub ntawv siv los tsim cov duab yog muaj los ntawm cov neeg sau ntawv thov. Cov ntaub ntawv pov thawj uas tau txheeb xyuas hauv kev npaj ntawm daim ntawv no thiab nthuav tawm hauv cov duab thiab cov ntxhuav yog muaj los ntawm tus neeg sau ntawv raws li qhov kev thov tsim nyog thiab tseem tos kev pom zoo los ntawm pawg neeg saib xyuas kev ncaj ncees (rau cov ntaub ntawv kho mob). P. falciparum sequences tau txais los ntawm pej xeem cov ntaub ntawv ntawm PlasmoDB.org.

Tau txais: 2 Cuaj hlis 2019; Tau Txais: 24 Lub Ob Hlis 2021.

Cov ntaub ntawv

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Kev lees paub

Peb ua tsaug rau cov neeg koom nrog txoj kev tshawb fawb thiab cov neeg ua haujlwm ntawm Kenyan Medical Research Institute rau cov qauv ntawm Chulaimbo cohort thiab Kanyawegi cohorts. P. falciparum sporozoites thiab mAb 2H8 tau ua siab zoo los ntawm PATH Malaria Vaccine Initiative (MVI). Lub mAb 3D11 thiab hybridoma tau ua siab zoo los ntawm Miguel Prudêncio ntawm Instituto de Medicina Molecular. P. berghei parasites qhia P. falciparum CSP tau muab los ntawm Chris Janse, Shahid Khan, thiab Hans Kroeze ntawm Leiden University Medical Center. Covalent coupling ntawm antigen rau fluorescent hlaws tau txais kev txhawb nqa zoo heev los ntawm Paul Ramsland ntawm RMIT, Paul Sanders ntawm Burnet Institute, thiab Geoffrey Pietersz ntawm Baker Heart thiab Diabetes Institute.

Peb kuj ua tsaug Michelle Wong ntawm Burnet lub koom haum ua peb tus kws kho mob phlebotomist thiab Emily Locke rau cov lus pom zoo thiab cov lus qhia. Txoj kev tshawb no kuj tau txais kev txhawb nqa los ntawm AMREP Flow cytometry core chaw. Txoj haujlwm no tau txais kev txhawb nqa los ntawm National Health and Medical Research Council (NHMRC) ntawm Australia (Program Grant and Senior Research Fellowship to JG Beeson; Project Grants to JGB, GI McFadden, and PM Hogarth; Career Development Fellowship to MJB), PATH-MVI (Grant to JGB), CASS Foundation (grant to GF), Australian Research Council Laureate Fellowship to GI McFadden, thiab NIH International Centers for Excellence in Malaria Research (U19AI129326). Lub koom haum Burnet tau txais kev txhawb nqa los ntawm NHMRC rau Independent Research Institutes Infrastructure Support Scheme thiab Victorian State Government Operational Infrastructure Support. JGB, GF, MJB, JAC, SC, thiab LK yog cov tswv cuab ntawm NHMRC Australian Center for Research Excellence in Malaria Elimination.

Sau cov nyiaj pab

JGB, GF, thiab BDW coj txoj kev kawm tsim nrog cov tswv yim los ntawm PMH, GIM, LK, JAC, thiab lwm tus neeg sau ntawv; GF, BDW, LK, PB, DML, SC, VM, thiab AC ua kev sim thiab txhais cov txiaj ntsig; GF, PB, thiab JAC tau soj ntsuam cov ntaub ntawv nrog cov tswv yim los ntawm JGB, LK, thiab MJB; JGB, BDW, PMH, JWK, AED, KC, GIM, AC, VM, DRD, thiab RJC muab los yog tsim cov reagents tseem ceeb, cov ntaub ntawv thiab / lossis cov peev txheej; GF, JGB, LK, JAC, thiab DRD tau pab txhawb rau kev npaj ntawv nrog cov tswv yim los ntawm tag nrho lwm tus kws sau ntawv.

Kev nyiam sib tw

Cov kws sau ntawv tshaj tawm tsis muaj kev sib tw nyiam.

Cov ntaub ntawv txheeb xyuas

Nature Communications ua tsaug rau Katie Ewer, Photini Sinnis, thiab lwm tus, tsis qhia npe, tus neeg saib xyuas rau lawv txoj kev koom tes rau cov phooj ywg txheeb xyuas cov haujlwm no. Cov ntawv ceeb toom ntawm cov neeg txheeb xyuas muaj nyob.

Publisher daim ntawv

Springer Nature tseem nyob nruab nrab ntawm kev txiav txim plaub ntug hauv daim duab qhia kev tshaj tawm thiab cov koom haum koom nrog.

Qhib Access

Kab lus no tau tso cai raws li Creative Commons Attribution 4.0 Daim ntawv tso cai thoob ntiaj teb, uas tso cai siv, sib koom, hloov kho, faib, thiab luam tawm hauv ib qho nruab nrab lossis hom ntawv, tsuav yog koj muab credit rau tus thawj sau ) thiab qhov chaw, muab qhov txuas mus rau Creative Commons daim ntawv tso cai, thiab qhia seb puas tau hloov pauv. Cov duab lossis lwm yam khoom siv thib peb hauv tsab xov xwm no suav nrog hauv tsab xov xwm Creative Commons daim ntawv tso cai tshwj tsis yog tau qhia lwm yam hauv kab qiv nyiaj rau cov khoom siv. Yog tias cov ntaub ntawv tsis suav nrog hauv tsab xov xwm Creative Commons daim ntawv tso cai thiab koj qhov kev npaj siv tsis raug tso cai los ntawm txoj cai lij choj lossis tshaj qhov kev tso cai siv, koj yuav tsum tau txais kev tso cai ncaj qha los ntawm tus tuav ntaub ntawv. Txhawm rau saib daim ntawv tso cai no, mus saib http://creativecommons.org/ licences/by/4.0/.

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