Public data analysis
Dhata pamusoro peSARS-CoV-2 mutsara uye zuva rakatorwa sample kubva kune akateedzana anowanikwa kubva kuChile akawanikwa kubva kuConsorcio Genomas CoV2 saiti iripo https://auspice.cov2.cl/ncov/chile-global. Data yekudzivirira yakatorwa kubva kuruzhinji data kubva kuBazi reSainzi, Tekinoroji, Ruzivo neKuvandudza zviripo https://github.com/MinCiencia/Datos-COVID19 (Chigadzirwa 83).
Infectivity assay
Pseudotyped mavhairasi akatakura akasiyana SARS-CoV-2 spike mapuroteni akagadzirwa sezvatakatsanangura kare.12. Muchidimbu, HIV-1-based SARS-CoV-2 pseudotypes yakagadzirwa mumaseru eHEK293T nekuchinjisa pNL4.3-ΔEnv-Luc pamwe chete neinowirirana pCDNA-SARS-CoV-2 Spike coding vector mune 1: 1 molar ratio. Plasmids inonyora codon-optimized spike inoshaya yekupedzisira 19 amino acids yeC-terminal end (SΔ19) inozivikanwa kudzivirira kuchengetwa pa endoplasmic reticulum.12 zvakawanikwa negene synthesis kana customized site-directed mutagenesis (GeneScript) uye ine zvinotevera kuchinja: mutsara A (reference sequence), mutsara B (D614G), mutsara B.1.1.7 (Δ69-70, Δ144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), mutsara P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027N, P37S, D75Y, R76S, K246T, E252K, N452Y, D490G, H614Y, T859N, P3,000S, D1Y, R24S, K50T, E80K, N1Y, D24G, H2Y, T48N, P96S mutsara, TXNUMXGXNUMX, TXNUMXGXNUMX) - XNUMX, LXNUMXQ, FXNUMXS, DXNUMXG, TXNUMXN). Imwe neimwe pseudotype gadziriro yakacheneswa ne centrifugation pa XNUMX rpm patembiricha yekamuri, yakaverengerwa uchishandisa HIV-XNUMX Gag pXNUMX Quantikine ELISA Kit (R&D Systems), yakatorwa muXNUMX% fetal bovine serum (Sigma-Aldrich) uye yakachengetwa pa -XNUMX ° C kusvika -XNUMX ° C kusvika. use. Huwandu hwakasiyana hwemavhairasi epseudotyped (seakatemwa nemazinga eHIV-XNUMX pXNUMX protein) akashandiswa kutapurira HEK-ACEXNUMX masero uye maawa makumi mana nemasere gare gare, firefly luciferase chiitiko chakayerwa uchishandisa Luciferase Assay Reagent (Promega) muGlomax XNUMX Microplate. luminometer (Promega).
Neutralization assay
Pseudotyped virus neutralization assays akaitwa chaizvo sezvatakatsanangura kare12. Muchidimbu, serial dilutions yemasampuli eplasma (1: 4 kusvika 1:8748) yakagadzirwa muDMEM ine 10% fetal bovine serum uye yakanyudzwa ne5 ng ye p24 yega yega pseudotyped virus panguva 1h pa37 ° C uyezve, 1 × 10.4 yeHEK-ACE2 maseru akawedzerwa kune rimwe nerimwe tsime. HEK293T maseru (asiri kuratidza ACE2) akaiswa neiyo pseudotyped hutachiona (mutsara A) akashandiswa senzira isina kunaka. Masero akaiswa lysed maawa 48 gare gare, uye firefly luciferase basa rakayerwa uchishandisa Luciferase Assay Reagent (Promega) muGlomax 96 Microplate luminometer (Promega). Iperesenti yekusagadzikana kweimwe neimwe dilution yakaverengerwa uye iyo ID50 yega yega sampu yakaverengerwa uchishandisa GraphPad Prism vhezheni 9.0.1.
Tsananguro inoongorora
Ongororo yenhamba yakaitwa pachishandiswa GraphPad Prism software shanduro 9.1.2. Mazhinji emapoka ekufananidza ekuita neutralizing antibody titers (NAbTs) kupesana nepaneru yeSARS-CoV-2 pseudotyped mavhairasi pamwe nekuenzaniswa kweNAbs mhinduro nebonde uye chimiro cheutsi chakaitwa pachishandiswa paired Wilcoxon yakasainwa-yakasainwa bvunzo. Factor shanduko yakaverengerwa semusiyano we geometric mean titer muID50 zvichienzaniswa neiyo yeWild type pseudotyped virus. Kuongororwa kwehukama pakati peNAbTs uye zera kana BMI yakaitwa uchishandisa bvunzo yaSpearman. Imwe-nzira ANOVA uye Tukey's yakawanda yekuenzanisa bvunzo yakaitwa yehuwandu hwekuongororwa kwekutapukira. A p kukosha ≤0.05 yaionekwa seyakakosha.
Kubvumidzwa kwetsika
Chirongwa chekudzidza chakabvumidzwa neEthics Committee yeFaculty of Medicine paUniversidad de Chile (Projects N ° 0361-2021 uye N ° 096-2020) uye Clínica Santa Maria (Project N ° 132604-21). Vese vanopa vakasaina mvumo yakaziviswa, uye masampuli avo haana kuzivikanwa.
Mhedzisiro yekuchinja kwe spike mumutsauko weLambda pane kusatapukira uye kusarerekera mhinduro dzemasoja ekudzivirira chirwere.
Ongororo ye3695 sequences kubva kuChile yakaiswa kuGISAID semuna Chikumi 24th 2021 inoratidza kutonga kwakajeka kweSARS-CoV-2 akasiyana Gamma neLambda panguva yekupedzisira trimester accounting, pamwe chete, ye79% yeese kutevedzana.
Sezvineiwo, nguva iyi yakaratidzwa nemushandirapamwe mukuru wejekiseni umo 65.6% yevanotarisirwa (vane makore gumi nemasere nevakuru) vakawana chirongwa chakazara chejekiseni semusi wa18 Chikumi.th 2021
Tichifunga kuti 78.2% yevanhu vakabayiwa nechirongwa chakazara vakagamuchira mushonga wekudzivirira hutachiona CoronaVac kubva kuSinovac Biotech, takatsvaga kuferefeta mabatiro ekuchinja kwespike kuripo mumutsauko weLambda pakusagadzikana kwemasoja ekudzivirira chirwere anowanikwa nejekiseni iri.
Kune izvi, takagadzira hutachiona hweHIV-1-based SARS-CoV-2 pseudotyped hutachiona hwakatakura spike protein kubva kuWuhan-1 referensi mutsara (Wild type; mutsara A), iyo D614G mutation (mutsara B), uye iyo Alpha (dzinza B. .1.1.7), Gamma (mutsara P.1) uye Lambda (mutsara C.37) akasiyana.
Munguva yekugadzirira hutachiona, takagara tichiona kuti masero akabatwa nehutachiona hwepseudotype akatakura Lambda spike akagadzira akakosha ebioluminescence hunhu kana achienzaniswa neiyo D614G mutant kana Alpha neGamma akasiyana anoratidza kuwedzera kutapukira kunofambiswa neLambda spike protein.
Mufananidzo 1.Infectivity inopindirana nemapuroteni akasiyana spike.
(A) Schematic inomiririra yeSARS-CoV-2 spike protein uye akasiyana anoshandiswa muchidzidzo ichi. Mitsara inotaridzwa mumaparenthesis. RBD, receptor-binding domain, CM; cytoplasmic muswe.
(B) Titration yepseudotypes yemutsara wega wega uchishandisa huwandu hwakaenzana hweHIV-1 p24. Iyo firefly luciferase chiitiko yakayerwa sehukama luminescence units (RLU) pamaawa makumi mana nemasere mushure mekutapukirwa. Avhareji uye SD zvakaverengerwa kubva kune inomiririra triplicate kuyedza.
Tevere, takashandisa mavhairasi emanyepo ataurwa pamusoro apa kuita bvunzo dzekusagadzikana tichishandisa makumi manomwe nepfumbamwe eplasma samples kubva kuvashandi vehutano vane hutano kubva kuUniversidad de Chile neClínica Santa María kuSantiago, Chile.
Takabvisa 4 samples sezvo tisina kukwanisa kuverenga ID50 titer. Kubva pamasampuli akaongororwa, 73% yaienderana nevakadzi, makore epakati 34 makore (IQR 29 - 43) uye body max index (BMI) ye25 (IQR 22.7 - 27). A 20.5% yevatori vechikamu vakazivisa kuva vanoputa fodya nguva yekubaiwa ichiripo. Masamples akawanikwa mukati memazuva makumi mapfumbamwe nemashanu (IQR 95 - 76) mushure memushonga wechipiri weCoronaVac vaccine.
Takaona kuti kusarerekera kwehutachiona hwepseudotyped hwakatakura Wild type spike protein yakakonzera 50% inhibitory dilution (ID.50) zvinoreva titer ye 191.46 (154.9 - 227.95, 95% CI,), apo yaiva 153.92 (115.68 - 192.16, 95% CI), 124.73 (86.2 - 163.2, 95% CI.104.57, 75.02% 134.11, 95, 78.75% 49.8, 107.6, 95, 614%. ) uye XNUMX (XNUMX - XNUMX, XNUMX% CI) yemavhairasi emanyepo anotakura spike protein kubva kuDXNUMXG mutant kana Alpha, Gamma neLambda akasiyana, zvichiteerana.
Isu takaona zvakare kuti geometric zvinoreva titer yeID50 titers dzakadzikira nechikamu che3.05 (2.57 - 3.61, 95% CI) yehutachiona hwepseudotyped inotakura Lambda spike, 2.33 (1.95 - 2.80, 95% CI) yeGamma spike, 2.03 (1.71 - 2.41% CI) yeAlpha spike uye 95 (1.37 - 1.20, 1.55% CI) yeD95G spike kana ichienzaniswa neWild type spike.
Hapana kuwirirana pakati pebonde, zera, body-mass index (BMI) kana chimiro cheutsi uye kusarerekera antibody titers zvakaonekwa muboka redu rekudzidza.
Mufananidzo 2.Neutralization assay uchishandisa plasma samples kubva kuCoronaVac vaccinees
(A) Shanduko mune inodzokororwa 50% neutralization titer (ID50) mumasampuli eplasma kubva kuvanhu makumi manomwe nevashanu vanowana mushonga weCoronaVac vachipikisa D75G (mutsara B), Alpha (mutsara B.614),
Gamma (mutsara P.1) uye Lambda (mutsara C.37) zvakasiyana kana zvichienzaniswa nehutachiwana hwemhuka. Mhedzisiro inoratidzwa semusiyano mune neutralization titers yeakafananidzwa samples. P kukosha kwekuenzanisa kweID50 anoverengerwa neWilcoxon akasaina-renji bvunzo.
(B) Zvirongwa zvebhokisi zvakaratidza iyo yepakati uye interquartile renji (IQR) yeID50 kune yega yega pseudotyped virus. Factor shanduko inoratidzwa semutsauko we geometric mean titer muID50 zvichienzaniswa neaya eWild type pseudotyped virus. Ongororo yezviverengero yakaitwa pachishandiswa Wilcoxon inofananidzwa-vaviri vakasaina-chinzvimbo bvunzo.
Pamwe chete, data redu rakaratidza kuti puroteni yepike yemhando ichangobva kuzivikanwa yekufarira Lambda, inotenderera zvakanyanya munyika dzeChile neSouth America, inotakura shanduko inopa kuwedzera kutapukira uye kugona kupukunyuka kubva mukusagadzikana masoja ekudzivirira chirwere anokonzerwa neCoronaVac.
