Ukuhlaziywa kwedatha yomphakathi
Imininingwane emigqeni ye-SARS-CoV-2 nosuku lapho isampula lathathwa khona kulandelana olutholakala eChile lwatholakala kusiza seConsorcio Genomas CoV2 esitholakala https://auspice.cov2.cl/ncov/chile-global. Imininingwane yokugoma itholakele kwimininingwane yomphakathi evela eMnyangweni Wezesayensi, Ubuchwepheshe, Ulwazi kanye Nokusungula etholakala ku- https://github.com/MinCiencia/Datos-COVID19 (Umkhiqizo 83).
Ukuhlolwa kokungenwa yisifo
Amagciwane abizwa ngokuthi yi-pseudotyped aphethe amaprotheni we-spike ahlukile we-SARS-CoV-2 alungiswa njengoba sichaze ngaphambilini12. Kafushane, ama-pseudotypes asuselwa ku-HIV-1 akhiqizwa kumaseli we-HEK2T ngokudlulisa i-pNL293-ΔEnv-Luc kanye ne-pCDNA-SARS-CoV-4.3 Spike coding vector e-2: 1 molar ratio. Ama-Plasmids abhala i-spike elungiselelwe i-codon entula ama-amino acid wokugcina we-C-terminal end (SΔ1) aziwa ngokugwema ukugcinwa ku-endoplasmic reticulum12 zitholwe ngokuhlanganiswa kwezakhi zofuzo noma i-mutagenesis eqondiswe kusayithi eyenziwe ngokwezifiso (i-GeneScript) futhi iqukethe ukuguqulwa okulandelayo: uhlu lozalo A (ukulandelana kwezinkomba), uhlu lozalo B (D614G), uhlu lozalo B.1.1.7 (-69-70, -144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), uhlu lozalo P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I) nohlu lozalo C.37 (G75V, T76, T246, 252, L452Q, F490S, D614G, T859N). Ukulungiselelwa ngakunye kwe-pseudotype kususwe yi-centrifugation ngo-3,000 rpm ekamelweni lokushisa, kwalinganiswa kusetshenziswa i-HIV-1 Gag p24 Quantikine ELISA Kit (R&D Systems), ebhalwe ku-50% fetal bovine serum (Sigma-Aldrich) yagcinwa ku -80 ° C kuze kube sebenzisa. Inani elihlukile lamagciwane asetshenzisiwe (njengoba kunqunywa amazinga we-HIV-1 p24 protein) asetshenziselwe ukuthelela amaseli we-HEK-ACE2 namahora angama-48 kamuva, umsebenzi we-firefly luciferase walinganiswa kusetshenziswa i-Luciferase Assay Reagent (Promega) kwi-Glomax 96 Microplate i-luminometer (Promega).
Ukuhlolwa kwe-neutralization
Izilingo ze-Pseudotyped virus neutralization zenziwa ngendlela ebesichaze ngayo phambilini12. Ngamafuphi, ukuxutshwa okulandelanayo kwamasampuli e-plasma (1: 4 kuya ku-1: 8748) kwalungiswa ku-DMEM nge-10% serum bovine serum futhi kwafakwa ne-5 ng ye-p24 yegciwane ngalinye elibizwa nge-pseudotyped ngesikhathi se-1h ku-37 ° C bese, i-1 × 104 kwamaseli we-HEK-ACE2 engezwe emthonjeni ngamunye. Amaseli we-HEK293T (angakhombisi i-ACE2) afakwe ngegciwane le-pseudotyped (uhlu lozalo A) asetshenziswa njengokulawulwa okungekuhle. Amaseli afakwa ama-lys amahora angama-48 kamuva, kwathi umsebenzi we-firefly luciferase walinganiswa kusetshenziswa i-Luciferase Assay Reagent (Promega) ku-Glomax 96 Microplate luminometer (Promega). Amaphesenti we-neutralization wokuhlanjululwa ngakunye abaliwe futhi i-ID50 yesampula ngayinye ibalwe kusetshenziswa inguqulo ye-GraphPad Prism 9.0.1.
Ukuhlaziya kwesitatimende
Ukuhlaziywa kwezibalo kwenziwa kusetshenziswa inguqulo yesoftware ye-GraphPad Prism 9.1.2. Ukuqhathaniswa kwamaqembu amaningi okwehlisa amandla ama-antibody titers (NAbTs) ngokumelene nephaneli lama-virus we-SARS-CoV-2 aqanjiwe kanye nokuqhathanisa izimpendulo ze-NAbs ngokobulili nangesimo sentuthu kwenziwa kusetshenziswa uhlolo olulinganisiwe olusayinwe nguWilcoxon. Ushintsho lwe-factor lubalwe njengomehluko we-titometry mean titer ku-ID50 uma kuqhathaniswa nalelo le-Wild type pseudotyped virus. Ukuhlaziywa kokuhlobana phakathi kwe-NAbTs nobudala noma i-BMI kwenziwa kusetshenziswa ukuhlolwa kukaSpearman. Ukuhlolwa kokuqhathanisa okuningi kwe-ANOVA noTukey kwenziwa ekuhlaziyweni kwezibalo zokungasebenzi. Inani le-p ≤0.05 lithathwa njengelibalulekile ngokwezibalo.
Ukugunyazwa kokuziphatha
Umthetho olandelwayo wamukelwa yiKomidi Lezimilo le-Faculty of Medicine e-Universidad de Chile (Amaphrojekthi uN ° 0361-2021 noN ° 096-2020) noClínica Santa Maria (Iphrojekthi N ° 132604-21). Bonke abaxhasi basayine imvume enolwazi, futhi amasampula abo awaziwa.
Umthelela wokushintshwa kwesipikha okwehlukanisweni kweLambda ekuthelelekeni nasekunciphiseni izimpendulo zamagciwane
Ukuhlaziywa kokulandelana okungama-3695 okuvela eChile kufakwe ku-GISAID kusukela ngoJuni 24th I-2021 ikhombisa ukubusa okucacile kokuhlukahluka kwe-SARS-CoV-2 iGamma neLambda ngesikhathi sokubalwa kwe-trimester yokugcina, ngokuhlangene, ngama-79% wakho konke ukulandelana.
Kuyathakazelisa ukuthi lesi sikhathi sibhekwe umkhankaso omkhulu wokugoma lapho i-65.6% yabantu ababhekisiwe (abaneminyaka eyi-18 nangaphezulu) bathole uhlelo oluphelele lokugoma kusukela ngoJuni 27th 2021
Njengoba kunikezwe ukuthi abantu abangama-78.2% abagonywe ngohlelo oluphelele bathole umuthi wokugomela igciwane ongasebenzi i-CoronaVac ovela eSinovac Biotech, siye safuna ukuphenya ngomthelela wokushintshwa kwezipikha okukhona okwahlukahlukaneni kweLambda kumandla okuqeda amandla ama-antibody atholwe yilo mgomo.
Ngalokhu, sikhiqize amagciwane e-pseudotyped asuselwa ku-HIV-1-based-SARS-CoV-2 aphethe amaprotheni we-spike avela ku-Wuhan-1 reference lineage (uhlobo lwasendle; u-A), ukuguqulwa kwe-D614G (uzalo B), kanye ne-Alpha (uhlu lozalo B .1.1.7), iGamma (uhlu lozalo P.1) kanye nokuhlukahluka kweLambda (lineage C.37).
Ngesikhathi sokulungiswa kwamagciwane, sihlale siqaphela ukuthi amaseli atheleleke ngegciwane elibizwa ngokuthi yi-Lambda spike akhiqize amanani aphakeme kakhulu we-bioluminescence uma kuqhathaniswa ne-D614G mutant noma i-Alpha ne-Gamma ehlukile ekhombisa ukwanda kokungenwa izifo okuqhutshwa yi-Lambda spike protein
Umfanekiso we-1.Ukungatheleleki okulamulelwe ngamaprotheni ahlukahlukene we-spike.
(A) Ukumelwa kwesikimu kwamaprotheni we-spike we-SARS-CoV-2 kanye nokuhlukahluka okusetshenziswe kulolu cwaningo. Imigqa ikhonjiswa kubakaki. I-RBD, isizinda esibopha i-receptor, i-CM; umsila we-cytoplasmic.
(B) Ukuhlukaniswa kwamapseudotypes ohlanga ngalunye kusetshenziswa inani elilinganayo le-HIV-1 p24. Umsebenzi we-firefly luciferase walinganiswa njengezinyunithi ezihlobene ne-luminescence (RLU) emahoreni angama-48 ngemuva kokutheleleka. Isilinganiso ne-SD kubalwe kusuka ekuhlolweni okumelwe okuphindwe kathathu.
Okulandelayo, sisebenzise amagciwane asetshenzisiwe ashiwo ngenhla ukwenza izivivinyo ze-neutralization sisebenzisa amasampula e-plasma angama-79 avela kubasebenzi bezempilo abaphuma eUniversidad de Chile naseClínica Santa María eSantiago, eChile.
Sikhiphe amasampula ama-4 njengoba singakwazanga ukubala i-ID50 titer. Kusukela kumasampula ahlaziyiweyo, ama-73% ahambelana nabesifazane, iminyaka engama-median engama-34 (IQR 29 - 43) kanye ne-body max index (BMI) yama-25 (IQR 22.7 - 27). Bangu-20.5% ababambe iqhaza ababike ukuthi bangabantu ababhemayo asebenzayo ngenkathi isikhathi sokugoma sisasebenza. Amasampula atholakele esikhathini esiphakathi sezinsuku ezingama-95 (IQR 76 - 96) ngemuva komthamo wesibili womuthi wokugoma weCoronaVac
Siqaphele ukuthi ukungathathi hlangothi kwegciwane elibizwa ngokuthi yi-pseudotyped virus eliphethe amaprotheni ohlobo lwe-Wild spike kuholele ekunciphiseni okungu-50% okuvimbelayo (ID50kusho titer ka-191.46 (154.9 - 227.95, 95% CI,), ngenkathi bekuyi-153.92 (115.68 - 192.16, 95% CI), 124.73 (86.2 - 163.2, 95% CI), 104.57 (75.02 - 134.11, 95% CI ) kanye no-78.75 (49.8 - 107.6, 95% CI) wamagciwane asetshenzisiwe athwala amaprotheni we-spike avela ku-D614G mutant noma okuhlukile kwe-Alpha, Gamma neLambda, ngokulandelana.
Siphinde sabona ukuthi i-geometric isho isihloko se-ID50 iziqu zehle ngo-3.05 (2.57 - 3.61, 95% CI) wegciwane elibizwa ngokuthi yi-Lambda spike, 2.33 (1.95 - 2.80, 95% CI) le-Gamma spike, 2.03 (1.71 - 2.41, 95% CI) kwe-Alpha spike ne-1.37 (1.20 - 1.55, 95% CI) ye-D614G spike uma kuqhathaniswa ne-Wild type spike.
Akukho kuhlangana phakathi kocansi, ubudala, inkomba yomzimba (BMI) noma isimo sentuthu nokunciphisa iziqu zama-antibody eqashelwe eqenjini lethu locwaningo.
Umfanekiso we-2.Ukuhlolwa kwe-Neutralization kusetshenziswa amasampula e-plasma avela emithini yokugoma yeCoronaVac
(A) Izinguquko kutayitela yokubuyisela emuva engu-50% (ID50) kumasampula e-plasma avela kubamukeli abangama-75 bomuthi wokugoma weCoronaVac olwa ne-D614G (uzalo B), i-Alpha (uhlu lozalo B.1.1.7),
Ukwahluka kweGamma (uzalo P.1) kanye neLambda (uhlu lozalo C.37) ngokuqhathaniswa negciwane lohlobo lwasendle. Imiphumela ikhonjiswa njengomehluko kuzitifiketi zokungathathi hlangothi zamasampuli afanayo. P amanani wokuqhathanisa i-ID50 zibalwa ngokuhlolwa kwesilinganiso esisayiniwe sikaWilcoxon.
(B) Iziza zamabhokisi zikhombise ububanzi obuphakathi nobubanzi be-ID (IQR) be-ID50 ngegciwane ngalinye elibizwa nge-pseudotyped. Izinguquko ze-factor zikhonjiswa njengomehluko we-titometry mean titer ku-ID50 uma kuqhathaniswa nalezo zohlobo lwe-Wild type pseudotyped virus. Ukuhlaziywa kwezibalo kwenziwa nge-Wilcoxon test-rank test-rank.
Ngokubambisana, imininingwane yethu iveze ukuthi iprotheni ye-spike yohlobo olusha olusanda kutholakala lwenzalo iLambda, olusakazeka kakhulu emazweni aseChile naseNingizimu Melika, ithwala izinguquko ezinikeza ukwanda kokutheleleka kanye namandla okubalekela ekunciphiseni amasosha omzimba afunwa yiCoronaVac.
