Synergistic Immunity And Protection in Mice Los ntawm Kev txhaj tshuaj tiv thaiv kab mob DNA nrog cov tshuaj tiv thaiv DNA Encoding Spike Protein Thiab Lwm Cov Proteins Cov Qauv Ntawm SARS-CoV-2
Dec 18, 2023
Abstract:Qhov tshwm sim ntawm qhov hloov pauv tshiab ntawm tus mob hnyav ua pa nyuaj ua pa mob coronavirus 2 (SARS CoV-2) tau ua rau muaj kev kis tus kabmob kis thoob ntiaj teb. Cov kev hloov pauv hloov pauv no txo qis kev ua tau zoo ntawm cov kab mob coronavirus tam sim no 2019 (COVID-19) cov tshuaj tiv thaiv, uas yog tsim los tsom tsuas yog cov kab mob loj (S) protein ntawm tus kab mob qub. Tsuas yog rau S ntawm SARS-CoV-2, lub peev xwm tiv thaiv kab mob ntawm lwm cov proteins (nucleocapsid, N; lub hnab ntawv, E; daim nyias nyias, M) raws li cov tshuaj tiv thaiv antigens tseem tsis meej thiab tsim nyog tshawb xyuas. Hauv txoj kev tshawb no, cov tshuaj tiv thaiv DNA synthetic encoding plaub SARS-CoV-2 structural proteins (pS, pN, pE, thiab pM) tau tsim, thiab nas tau txhaj tshuaj tiv thaiv peb zaug ntawm kev txhaj tshuaj intramuscular thiab electroporation. Qhov tshwj xeeb, kev txhaj tshuaj tiv thaiv nrog ob hom tshuaj tiv thaiv DNA uas qhia txog S thiab N cov proteins ua rau muaj cov tshuaj tiv thaiv kab mob siab dua thiab ua tau zoo dua los txo tus kab mob SARS-CoV-2 kab mob ntau dua li S protein ib leeg hauv cov nas. Tsis tas li ntawd, kev txhaj tshuaj pS nrog pN lossis pE + pM ua rau muaj kev tiv thaiv kab mob S protein ntau dua tom qab peb txhaj tshuaj tiv thaiv kab mob thiab ua rau muaj kev hloov pauv me me ntawm pS ib leeg tom qab kev sib tw. Lub luag haujlwm ntawm kev txuag cov proteins ntawm SARS-CoV{16}}, suav nrog N/E/M proteins, yuav tsum tau tshawb xyuas ntxiv rau lawv cov kev siv tshuaj tiv thaiv, xws li cov tshuaj tiv thaiv mRNA.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Keywords: COVID-19; SARS-CoV-2}}; kev txhaj tshuaj; tshuaj tiv thaiv DNA; protein ntau; structural protein
1. Taw qhia
Tus mob hnyav ua pa nyuaj ua rau tus mob coronavirus 2 (SARS-CoV-2) yog qhov ua rau tus mob coronavirus 2019 (COVID-19), uas tau ua rau ntau lab tus kab mob thiab tuag thoob ntiaj teb thiab ua rau muaj kev phom sij rau tib neeg kev noj qab haus huv thiab kev lag luam thoob ntiaj teb. . Txawm hais tias cov txheej txheem kho tau zoo tseem tsis tau muaj, lawv tau nce sai, suav nrog kev siv CAR-T cell therapy thiab nanotechnology [1,2]. Kev txhaj tshuaj tiv thaiv yog ib txoj hauv kev zoo los tswj kev sib kis, thiab ntau cov tshuaj tiv thaiv tau pom zoo siv los ntawm ntau lub koom haum tswj hwm kev noj qab haus huv [3,4]. Tus kab mob coronavirus genome encodes plaub lub ntsiab ntawm cov proteins, uas yog, cov kab mob (S), nucleocapsid (N), daim nyias nyias (M), thiab lub hnab ntawv (E) cov proteins, uas yog lub luag haujlwm rau kev sib sau virion thiab kev tawm tsam ntawm lub cev tiv thaiv kab mob [5 ]. S protein yog tsim los ntawm 1273 amino acid residues uas muaj ob subunits, namely, S1 thiab S2. Nws kho cov kab mob nkag mus thiab yog lub hom phiaj tseem ceeb rau kev tsim cov tshuaj tiv thaiv kab mob coronavirus [6–11]. Txawm li cas los xij, SARS-CoV-2 S protein muaj qhov hloov pauv ntau zaus. Tsis yog qhov xav tsis thoob, hauv SARS-CoV-2, tus kab mob RNA, kev hloov pauv tsis tu ncua thiab tsis muaj kev cuam tshuam. Muaj tsib SARS-CoV-2 variants of concern (VOC) uas tau tshwm sim txij lub Cuaj Hlis 2020, suav nrog B.1.1.7 (UK, Alpha), B.1.351 (South Africa, Beta), P.1 (Brazil, Gamma), B.1.617.2 (India, Delta), thiab B.1.1.529 (South Africa, Omicron) (Andreano thiab Rappuoli, 2021; Gupta, 2021). Lawv txhua tus muaj ntau yam kev hloov pauv hauv cov protein ntau [12]. Cov kev hloov pauv no cuam tshuam rau kev ua tau zoo ntawm cov tshuaj tiv thaiv COVID-19 tam sim no, uas yog tsim los tsom tsuas yog cov protein ntau.
N protein ntawm SARS-CoV-2 khi rau tus kab mob RNA los ntawm 140-amino-acid-long RNA-binding domain nyob rau hauv lawv cov tub ntxhais nyob rau hauv ib tug "pob hlaws ntawm ib txoj hlua". Nws tau txais txiaj ntsig zoo ntawm cov kab mob coronaviruses, sib koom ua ke ~ 90% cov cim qhia txog tus kheej nrog SARS-CoV, thiab nws tseem yog tib lub cev muaj protein nyob hauv virion [13]. Tsis tas li ntawd, nws ua lub luag haujlwm tseem ceeb hauv kev ntim cov kab mob RNA rau hauv ribonucleo capsid complex thiab tsim nyog rau cov kab mob RNA replication, virion los ua ke, thiab tso tawm los ntawm cov tswv tsev cell [14]. Raws li qhov sib npaug sib luag ntawm N protein nyob rau hauv tus mob coronaviruses, nws yuav raug pom zoo tias yog lub hom phiaj tiv thaiv kab mob sib kis. Peb yav dhau los pom tias kev txhaj tshuaj tiv thaiv nrog ob hom tshuaj tiv thaiv DNA uas qhia txog E thiab M cov proteins muab kev tiv thaiv ib nrab tiv thaiv SARS-CoV-2, thiab cov qauv no yuav tsum tau txiav txim siab thaum tsim cov tshuaj tiv thaiv [15]. Nyob ntawm cov ntaub ntawv toj roob hauv pes los ntawm WHO, feem ntau muaj xya lub tswv yim rau SARS-CoV-2 cov neeg siv tshuaj tiv thaiv, uas tuaj yeem muab faib ua peb pawg: thawj zaug, tshuaj tiv thaiv cov protein, suav nrog cov tshuaj tiv thaiv kab mob tsis ua haujlwm, zoo li tus kab mob. cov kab mob, thiab cov tshuaj tiv thaiv subunit protein; thib ob, cov tshuaj tiv thaiv kab mob, suav nrog cov tshuaj tiv thaiv kab mob, tshuaj tiv thaiv DNA, thiab tshuaj tiv thaiv mRNA; thib peb, kev sib xyaw ua ke ntawm ob qho tib si protein-based thiab gene-raws li txoj hauv kev, xws li cov tshuaj tiv thaiv kab mob muaj sia nyob. DNA thev naus laus zis, raws li cov tswv yim tshiab tshuaj tiv thaiv kab mob, tuaj yeem sib piv ntau yam tshuaj tiv thaiv thiab cov tswv yim sai sai thaum kuaj ua ntej [16,17]. Txoj kev xav, yuav luag tag nrho cov kab mob kis kab mob yog cov tshuaj tiv thaiv kab mob thiab cov hom phiaj tshuaj tiv thaiv. Txawm li cas los xij, rau qhov zoo tshaj plaws ntawm peb txoj kev paub, cov tshuaj tiv thaiv DNA hluavtaws 'immunogenicity thiab kev tiv thaiv muaj peev xwm nyob rau hauv encoding SARS-CoV-2 S proteins thiab lwm yam qauv proteins tseem tsis tau muaj kev tshaj tawm. Plaub hom tshuaj tiv thaiv DNA uas qhia txog SARS-CoV-2 S, N, E, thiab M l cov proteins tau raug soj ntsuam rau lawv cov kev tiv thaiv kab mob thiab kev tiv thaiv kev ua tau zoo hauv cov nas txhawm rau tshawb nrhiav cov tshuaj tiv thaiv kab mob S hauv kev sib xyaw nrog lwm cov proteins.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Cells
Huh7.5 hlwb thiab tib neeg lub raum embryonic raum 293T hlwb raug coj los ntawm 37 ◦C hauv qhov chaw humidified ntawm 5% CO2 thoob plaws hauv txoj kev tshawb no. Cov hlwb raug coj los ntawm DMEM nruab nrab (HyClone, Logan, UT, USA), ntxiv nrog 10% FBS (GEMINI Co., Shanghai, Tuam Tshoj) thiab 1% penicillin-streptomycin (Gibco, New York, NY, USA). Tag nrho cov kab ntawm tes tau lees paub qhov tsis zoo rau cov kab mob mycoplasma.
2.2. Construction of DNA Vaccines Coding SARS-CoV-2 S/N/E/M
SARS-CoV-2 S/N protein-encoding gene, uas muaj N-terminal Kozak sequence (GCCACC) ua raws li kev pib codon (ATG), tau tsim los siv cov tsiaj txhu zoo tshaj plaws codon (GenScript Co., Nanjing , Suav teb). Tom qab ntawd nws tau cloned rau hauv qhov kev qhia vector pcDNA3.1 (+) ntawm EcoRI thiab XbaI zom thiab hu ua pS/pN (DNA tshuaj tiv thaiv) (Daim duab 1A). Cov protein pE/PM tau tsim thiab txheeb xyuas raws li tau piav qhia yav dhau los [15]. Cov tshuaj tiv thaiv tau npaj siv cov khoom siv endotoxin-dawb Maxiprep (Qiagen, Beijing, Tuam Tshoj), thiab cov kab ke tau lees paub siv Sanger DNA sequencing. Qhov kev qhia ntawm S / N protein tau lees paub tias siv western blotting thiab anti-S (Sino Biological, Beijing, Tuam Tshoj) / anti-N antibodies diluted ntawm 1: 1000. Cov kev sim no tau ua raws li tau piav qhia yav dhau los [15,18].
Daim duab 1. Tsim thiab kev nthuav qhia ntawm recombinant DNA-based SARS-CoV-2 S/N protein vaccine constructs. (A) Schematic diagram of the recombinant DNA-based vaccines encoding SARS-CoV-2 spike (PS), nucleocapsid (pN), envelope (pE), and/or membrane (PM) proteins. (B) Lub hom phiaj qhia protein nyob rau hauv cov tshuaj tiv thaiv DNA tau raug lees paub los ntawm kev txheeb xyuas sab hnub poob ntawm 293T hlwb hloov nrog pS / pN / pE / pM plasmids.
2.3. Kev txhaj tshuaj tiv thaiv thiab kev sib tw
Poj niam BALB/c nas (Charles River Laboratories, Fabkis) thaum muaj hnub nyoog 6 lub lis piam tau nyob hauv National Institute of Occupational Health thiab Poison Control hauv 21 ◦C thiab cov av noo-tswj ib puag ncig nrog 12 teev lub teeb / tsaus nti. Lub caij no, zaub mov thiab dej tau muab ad libitum, thiab tag nrho cov kev sim tsiaj tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Txhaj Tshuaj ntawm Tuam Tsev Tiv Thaiv thiab Tiv Thaiv Kab Mob (China CDC). Kev tshawb fawb tau ua raws li cov cai tswj kev ncaj ncees.
Cov nas tau muab faib ua tsib pawg thiab txhaj tshuaj nrog pS / pN ib leeg lossis koom nrog kev txhaj tshuaj nrog pS + pN lossis pS + pE + PM rau hnub 0, 21, thiab 42 ntawm kev txhaj tshuaj intramuscular ntxiv rau electroporation (35 mg / 50). mL) (Daim duab 2) [19,20]. Luv luv, cov tshuaj tiv thaiv DNA tau txhaj rau hauv tibialis anterior (TA) cov leeg thiab tam sim ntawd rub nrog hluav taws xob siv 5 hli-sib nrug ob-kab koob electrode (ECM830; BTX) nrog koob. Sera los ntawm cov nas tau sau rau kev tshuaj xyuas kev tiv thaiv kab mob humoral, thiab nas spleens tau ua tiav los ntsuas qhov kev tiv thaiv kab mob ntawm tes (Daim duab 2).
Daim duab 2. Schematic of the immunization and SARS-CoV-2 challenge. Lub sijhawm txhaj tshuaj tiv thaiv, nyuaj, thiab kuaj ntshav / cov ntaub so ntswg. BALB/C nas tau muab faib ua pawg.
SARS-CoV-2 kev sim kev sim tau ua raws li tau piav qhia yav dhau los [15,21]. Luv luv, cov nas tsuag tau tshuaj loog thiab tom qab ntawd hloov mus rau intranasally nrog 2.5 × 108 PFU ntawm Ad5-hACE2 hauv tag nrho ntim ntawm 45 µL. Tsib hnub tom qab kev hloov pauv, cov nas tau txhaj tshuaj loog thiab tom qab ntawd intranasally challenged nrog 1 × 105 TCID50 of SARS-CoV-2 (Wuhan/IVDC HB-02/2019) in a total volume of 50 µL of saline tsis pub. Txhua qhov kev ua haujlwm nrog SARS-CoV-2 nyob hauv cov qauv nas tau ua nyob rau hauv Animal Biosafety Level 3 (ABSL{22}}) lub chaw soj nstuam.
2.4. Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assays (ELISA) tau ua raws li tau piav qhia yav dhau los [15]. Luv luv, S (muas los ntawm Sino Biological) / N proteins (ib qho khoom plig los ntawm Nkauj) diluted nyob rau hauv carbonate buffer (0.1 M, pH 9.6) tau coated rau 96-zoo EIA / RIA daim hlau (Thermo Fisher Scientific, Waltham, MA, USA) hmo ntuj ntawm 4 ◦C. Cov phaj raug thaiv nrog 200 µL ntawm 10% tshis ntshav hauv PBS ntawm 37 ◦C rau 2 h, tom qab ntawd ntxuav tsib zaug nrog PBST. Tom qab ntawd, cov qauv kuaj ntshav serially diluted hauv 2% tshis ntshav hauv PBS tau ntxiv thiab incubated rau 2 h ntawm 37 ◦C, ua raws li tsib zaug nrog PBST. HRP-conjugated tshis tiv thaiv nas IgG Ab (1:5000) tau ntxiv ntawm 37 ◦C rau 1 teev. Tag nrho ntawm 100 µL ntawm TMB substrate tau ntxiv rau txhua qhov dej thiab quenched nrog 50 µL ntawm 2M H2SO4. Lub absorbance tau nyeem ntawm lub wavelength ntawm 450 nm siv SPECTR Ostar Nano (BIO-GENE, Hong Kong, Suav).
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2.5. Pseudovirus Infection thiab Neutralization Experiments
Qhov kev ntsuam xyuas pseudovirus neutralization tau ua raws li tau piav qhia yav dhau los [21,22]. Lub plasmid qhia txog tus kab mob poj koob yawm txwv S protein tau tsim ua ntej [22]. The Omicron variant SARS-CoV-2 spike protein gene (GISAID: EPI_ISL_6590782.2) yog synthesized (a gift from Vazyme Biotech Co., Ltd., Nanjing, China) siv cov tsiaj txhu-optimized codon thiab cloned rau hauv pcDNA3.1 vector, raws li tau piav ua ntej [22]. Luv luv, plasmids qhia tus neeg sau xov xwm luciferase thiab plasmids qhia txog S protein tau sib koom ua ke hauv HEK 293T hlwb siv X-treme GENE HP DNA Transfection Reagent. Cov kab lis kev cai ntawm tes tau rov ua dua 6 teev tom qab hloov pauv, thiab cov kab mob pseudovirus-muaj supernatant tau sau tom qab 48 teev thiab khaws cia ntawm −70 ◦C. Nyob rau hauv qhov kev ntsuam xyuas pseudovirus neutralization, qhov sib npaug ntawm cov ntshav-cov kab mob sib xyaw yog tom qab ntawd incubated ntawm 37 ntim ntawm cov kab mob pseudovirus-muaj supernatant thiab tom qab ntawd ntxiv rau hauv cov ntshav diluted. ◦C rau 1 h. Tom qab ntawd Huh7.5 hlwb kab lis kev cai xov xwm tau hloov nrog 100 µL ntawm cov tshuaj tiv thaiv kab mob sib xyaw thiab tau muab tso rau ntawm 37 ◦C rau 12 teev. Cells cultured tsuas yog SARS-CoV-2 pseudoviruses tau khiav ua ke. Cov xov xwm tom qab ntawd tau hloov nrog DMEM (2% FBS), thiab qhov incubation yog incubated ntawm 37 ◦C rau 48 teev. Tom qab ntawd, lub teeb liab luciferase tau ntsuas siv cov khoom siv Bright-Glo firefly luciferase (Promega).
2.6. SARS-CoV-2 Neutralization Assay
SARS-CoV-2 (Wuhan/IVDC-HB-02/2019) tau siv hauv qhov kev sim no. Luv luv, cov sera tau diluted ob npaug los ntawm qhov pib dilution ntawm 1:10, tov nrog ib qho sib npaug ntim (10–15 pfu/well) ntawm SARS-CoV nyob -2, thiab incubated rau 1 h ntawm 37 ◦C, Tom qab ntawd lawv tau ntxiv rau cov noob Vero hlwb. Tom qab incubation ntawm 37 ◦C rau 48 h, cytopathic nyhuv (CPE) tau pom, thiab 100 µL ntawm kab lis kev cai supernatant tau sau rau nucleic acid extraction thiab real-time fluorescence reverse transcription PCR (RT-PCR). Qhov nruab nrab neutralization koob tshuaj (ND50) tau suav nrog txoj kev Reed-Munch [15].
2.7. IFN- ELISpot Assay
Cov pas dej peptide hla tag nrho S / N / E / M protein raws li sib law liag 15-mers dhau lapping los ntawm 10 amino acids tau tsim los ntawm Scilight Biotechnology, LLC. Kwv yees li ntawm 2.5 mg ntawm txhua qhov purified peptide nyob rau hauv lub pas dej peptide muaj nyob rau hauv ib lub vial. Qhov kev sim tau ua raws li tau piav ua ntej [18]. Luv luv, 96-cov phiaj zoo (BD ELISPOT Set, USA) tau coated nrog anti-IFN- ntes Ab thiab incubated hmo ntuj ntawm 4 ◦C. Cov phiajcim raug thaiv nrog cov kab lis kev cai ua tiav tom qab ntxuav peb zaug. Splenocytes tau sau tseg tom qab cov nas tau euthanized nyob rau hnub 35 thiab 120 Fresh ib leeg-cell suspensions los ntawm txhua pab pawg tau plated ntawm 5 × 106 ib qhov dej, thiab peptides tau ntxiv. Cov phiajcim tau muab tso rau ntawm 37 ◦C hauv 5% CO2 rau 22 h thiab kuaj pom siv ELISpot phaj nyeem ntawv (Biosys, So. Pasadena, CA, USA). Ib qho chaw tsim qauv (SFU) sawv cev rau T cell-secreting IFN- .
2.8. Kev ntsuas kev tiv thaiv hauv Mice Post-SARS-CoV-2 Challenge
Cov kev sim tau ua raws li tau piav ua ntej [15,21]. Luv luv, lub ntsws tau sau tom qab cov nas tau euthanized. Ib nrab ntawm cov ntaub so ntswg tau siv rau nucleic acid extraction, real-time fluorescence RT-PCR, thiab TCID50. Lwm ib nrab raug xa mus rau College of Veterinary Medicine, Tuam Tshoj Agricultural University rau kev ntsuam xyuas kab mob.
2.9. Kev txheeb cais
Unpaired t-tests, ob-txoj kev ANOVA xeem, thiab Dunnett ntau qhov kev sib piv xeem tau ua los ntawm GraphPad Prism 7.0 (GraphPad Software LLC). p-tus nqi < 0. ** p < 0.001; **** p < 0.0001).
3. Cov txiaj ntsig
3.1. Yam ntxwv ntawm DNA Vaccines
E thiab M cov protein ntau tau kuaj pom siv western blotting. Peb ntsuas qhov kev qhia ntawm encoded S/N/E/M proteins of SARS-CoV-2 in HEK-293T cells transfected with pS/pN/pE/pM plasmids via western blot analysis, using anti -S/anti-N cov tshuaj tiv thaiv thiab ib qho tshuaj tiv thaiv -6 x Nws cov tshuaj tiv thaiv, hauv cov cell lysates. Cov pab pawg tau kwv yees qhov kwv yees qhov hnyav molecular ntawm S (140–142 kDa), N (45 kDa), E (10 kDa), thiab M (22–25 kDa) cov proteins (Daim duab 1B).
3.2. Muaj zog thiab ruaj khov los tiv thaiv-S thiab / lossis tiv thaiv N IgG Ntau Lawm Los ntawm pS thiab / lossis pN DNA
Vaccines Serum tau sau los ntawm cov nas BALB/c ntawm 35, 56, 96, thiab 120 hnub (Daim duab 2). Anti S / anti-N IgG qib tau kuaj pom siv ELISA. Qhov loj ntawm S- lossis N-tshwj xeeb IgG cov lus teb raug ntxias los ntawm pS lossis pN tau nce hauv cov ntshav tom qab thawj zaug thiab thib ob boosts. Cov anti-S thiab anti-N IgG titers tau siab dua hauv pawg pS + pN dua li lawv nyob hauv lwm pab pawg; Txawm li cas los xij, qhov sib txawv tsis yog qhov tseem ceeb (Daim duab 3A, B). Tsis muaj zog E/M cov tshuaj tiv thaiv kab mob tshwj xeeb cov lus teb tau raug kuaj pom, uas zoo ib yam nrog cov txiaj ntsig ntawm kev tshawb fawb yav dhau los (cov ntaub ntawv tsis qhia) [15].
Figure 3. B-cell responses to SARS-CoV-2 in BALB/c mice. (A) Serum IgG binding endpoint titers for the SARS-CoV-2 S (A) and N proteins (B). (C) Neutralization titers tau txiav txim siab raws li SARS-CoV-2 pseudotyped-virus system. (D) Anti-SARS-CoV-2 neutralization titers tau txiav txim siab siv tus kab mob SARS-CoV-2. (E) Neutralization assay based on SARS-CoV-2 Omicron pseudotyped virus system. Qhov sib piv inhibition rau sera los ntawm kev thuam (xiav), pS (liab), pS + pN (ntsuab), pS + pE + pM (liab), thiab pN (txiv kab ntxwv) pawg tau qhia. Cov kab yuam kev sawv cev rau SEM, thiab p-tus nqi tau suav nrog ob txoj kev ANOVA thiab Sidak's post hoc tsom xam, qhov twg * p < 0.05
3.3. Qib siab ntawm Neutralizing Antibody Induced los ntawm Co-Immunization nrog pS thiab pN Vaccines
Cov titers neutralizing ntawm serially diluted serum kuaj tau txiav txim siv tus kab mob pseudotyped SARS-CoV-2. Cov qib siab tshaj plaws ntawm cov tshuaj tiv thaiv tsis zoo (nAbs) tau pom nyob rau hauv pawg pS + pN, nrog rau qhov sib npaug EC50 geometric txhais tau tias titers ncav cuag 2988 (hnub 35) thiab 3578 (hnub 56) (Daim duab 3C). Cov txiaj ntsig zoo sib xws tau raug soj ntsuam los ntawm kev soj ntsuam kab mob nyob hauv microneutralization (MN), qhov twg cov qib nAbs hauv pawg pS + pN tau siab dua cov hauv pawg S hnub 56 thiab 96 (p < 0.05; Daim duab 3D). Ntxiv mus, cov theem ntawm nAbs nyob rau hauv pawg pS + pN hnub 56 (thib ob boost) tau loj dua cov hnub 35 (p <0.05; Daim duab 3D).
Kev ua haujlwm nruab nrab ntawm txhua qhov tshuaj tiv thaiv tiv thaiv kab mob SARS-CoV-2 Omicron variant tau txiav txim siab ntxiv los ntawm kev siv pseudotyped platform thiab cov qauv ntshav. Qhov kev tsis sib haum xeeb tawm tsam tus kab mob Omicron rau hnub 35 thiab 56 tau zoo ib yam li qhov tiv thaiv kab mob poj koob yawm txwv (Daim duab 3E), qhia tias kev kho PS + pN tau hla qhov tsis muaj zog.
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3.4. T-Cell Responses Induced by DNA Vaccination
Raws li tau piav qhia dhau los, T-cell cov lus teb tawm tsam SARS-CoV-2 S/N/E/M antigens tau kwv yees siv IFN- ELISpot, raws li tau piav qhia yav dhau los [15]. Raws li qhov xav tau, ob qho tib si PS + pN thiab pS + pE + pM kev tswj hwm tau ua rau muaj ntau dua ntawm IFN + T hlwb tshwj xeeb rau S protein nyob rau hnub 120 dua li hnub 35 (p < 0. 05; Daim duab 4A). Ntxiv mus, tus naj npawb ntawm IFN + T hlwb tshwj xeeb rau N protein nyob rau hnub 120 (thib ob boost) tau ntau dua li hnub 35 hauv pawg pS + pN (p <0.05; Daim duab 4B). Thaum kawg, tus naj npawb ntawm IFN + T hlwb tshwj xeeb rau M protein nyob rau hnub 120 (thib ob boost) tau ntau dua li ntawm hnub 35 hauv ob pawg (p <0.05; Daim duab 4D).
Figure 4. T cell responses to SARS-CoV-2 individual structural proteins in BALB/c nas. (A) T-cell teb tau ntsuas siv IFN- ELISpot hauv splenocytes stimulated rau 20 h nrog sib tshooj peptide pools hla SARS-CoV-2 S, (B) N, (C) E, thiab (D) M proteins. Bars sawv cev qhov nruab nrab ± SD. Kev txheeb xyuas txheeb cais tau ua tiav siv ob txoj kev ANOVA thiab Sidak's post hoc test, qhov twg * p < 0.05, ** p < 0.01, *** p < 0.01, thiab **** p < 0.0001.
3.5. Synergistic Protection Induced los ntawm Co-Immunization nrog pS/pN los yog pS/pE/pM
Tom qab ntawd peb tau soj ntsuam qhov kev tiv thaiv zoo ntawm cov tshuaj tiv thaiv DNA siv hACE2 nas txhaj tshuaj tiv thaiv tom qab kev sib tw nrog cov poj koob yawm txwv SARS-CoV-2 tus kab mob. Tom qab qhov kev sib tw, cov nas nyob hauv pab pawg neeg thuam tau nthuav tawm qhov hnyav zuj zus. Hauv qhov sib piv, cov nas txhaj tshuaj nrog pS lossis pS + pom qhov hnyav me me tam sim tom qab kis kab mob, ua raws li kev rov zoo (Daim duab 5A). Tsis muaj tus kab mob nyob hauv cov nas uas tau txhaj tshuaj pS, pS + pN, lossis pS + pE + pM. Tsis tas li ntawd, qhov tshuaj tiv thaiv pS + pN txo qis tus kab mob RNA cov lej piv rau cov uas tau txais kev txhaj tshuaj pS ib leeg (p=0.0228; Daim duab 5B). Tsis tas li ntawd, lub ntsws histopathology tau pom tias cov nas nyob rau hauv ob qho tib si ntawm cov mock thiab pN pawg tau pom cov thaj tsam ntawm qhov mob, pleural invagination, alveolar vau, qib siab ntawm inflammatory cell infiltration, thiab hemorrhagic cheeb tsam. Hauv kev sib piv, cov nas tau kho nrog pS + pN lossis pS + pE + pM nthuav tawm cov kev hloov pauv ntawm histopathological thiab qis dua INHAND cov qhab nia tom qab sib tw dua li lwm pab pawg (Daim duab 5C).
Daim duab 5. Kev tiv thaiv kev tiv thaiv zoo tom qab kev sib tw nrog tus kab mob SARS-CoV-2 nyob. (A) Cov nas tau hnyav txhua hnub (txhais tau tias ± tus qauv yuam kev ntawm qhov nruab nrab (SEM), n=4) rau peb hnub tom qab kev sib tw. (B) Infectious SARS-CoV-2 titer nyob rau hauv lub ntsws homogenates nyob rau hnub peb tom qab kev sib tw, raws li tau txiav txim los ntawm TCID5{{10}} assay thiab RNA tus lej luam. Kev txheeb xyuas qhov sib txawv ntawm cov pab pawg tau txiav txim siab siv ib txoj kev ANOVA ua raws li Dunnett ntau qhov kev sib piv kho (* p < 0.05, ** p < 0.01, *** p < 0.001, thiab **** p <0.0001). (C) Lub ntsws histopathological tsom xam siv H&E staining.
4. Kev sib tham
Hauv txoj kev tshawb no, kev txhaj tshuaj tiv thaiv nrog ob hom tshuaj tiv thaiv DNA uas qhia txog S thiab N cov proteins ua rau muaj nAbs siab thiab ua tau zoo heev los txo tus kab mob SARS-CoV-2 kab mob hauv cov nas. Cov tshuaj tiv thaiv DNA uas qhia txog S protein induced nce S protein-specific cellular tiv thaiv theem tom qab peb txhaj tshuaj thaum nas tau txhaj tshuaj nrog N / E thiab M proteins thiab alleviated histopathological kev hloov tom qab sib tw. Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, qhov no yog thawj daim ntawv tshaj tawm qhia txog kev sib koom ua ke ntawm kev tiv thaiv kab mob thiab kev tiv thaiv hauv nas uas siv cov tshuaj tiv thaiv DNA encoding S protein thaum lawv koom nrog cov tshuaj tiv thaiv DNA encoding lwm cov proteins ntawm SARS-CoV{{8 }}.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Immunodominant B-cell epitopes hauv cheeb tsam N antigen tau pom nyob rau hauv ntau qhov kev tshawb fawb. Cov tshuaj tiv thaiv N-raws li feem ntau tsis tuaj yeem ua rau nAbs, zoo li vim tias N protein tsis tshwm sim rau ntawm tus kab mob. Qhov tseem ceeb, kev txhaj tshuaj tiv thaiv nrog S thiab N cov proteins ua rau muaj ntau dua nAbs tawm tsam cov poj koob yawm txwv thiab Omicron SARS-CoV-2 virus dua li lwm pab pawg. Kev nce nAb cov lus teb tau cuam tshuam nrog kev tshem tawm cov kab mob zoo dua thiab kev tiv thaiv zoo. Peb cov txiaj ntsig tau pom tias kev kho pS + pN tau zoo dua li kev kho pS ib leeg hauv kev txo tus kab mob SARS-CoV-2 kab mob tom qab kev sib tw. Ib txoj kev tshawb fawb yav dhau los tau tshaj tawm tias cov hamsters txhaj tshuaj tiv thaiv nrog cov tshuaj tiv thaiv uas qhia txog M thiab N cov proteins tau tiv thaiv qhov hnyav poob thiab lub ntsws pathology thiab tau txo qis tus kab mob titers hauv oropharynx thiab lub ntsws tom qab SARS-CoV-2 kev sib tw, uas yog raws li peb cov txiaj ntsig [23]. Hmoov tsis zoo, qhov txo qis ntawm tus kab mob titers tsis tuaj yeem raug ntaus nqi rau M lossis N protein, thiab qib nAb tsis raug ntsuas hauv txoj kev tshawb no. Ib txoj kev tshawb fawb SARS-CoV-2 mRNA txhaj tshuaj tiv thaiv tau tshaj tawm tias S + N kev txhaj tshuaj tiv thaiv kab mob ua rau muaj qhov ntxiv S-specific CD8+ T-cell teb thiab neutralizing cov tshuaj tiv thaiv kab mob, muab kev tiv thaiv zoo dua hauv lub ntsws tiv thaiv Delta. variant piv nrog S ib leeg, uas zoo ib yam nrog cov txiaj ntsig ntawm txoj kev tshawb no [24]. Lwm txoj kev tshawb fawb tau tshaj tawm tias N protein ntawm kis kab mob gastroenteritis tus mob coronavirus tau txhawb nqa cov synthesis ntawm neutralizing antibodies thaum porcine TGEV-IMMUNE hlwb raug txhawb nrog kev sib xyaw ntawm S thiab N proteins hauv vitro, thiab cov txiaj ntsig no yuav piav qhia los ntawm tus pab T-lymphocyte teb rau. N protein [25].
Immunodominant CD4+/CD8+ T-cell epitopes hauv cheeb tsam N-antigen tau raug txheeb xyuas yav dhau los. Ntau qhov kev tshawb fawb tau tshaj tawm tias cov tshuaj tiv thaiv raws li SARS-CoV-2 N protein ua rau muaj txiaj ntsig zoo rau lub cev tiv thaiv kab mob. Cov pab pawg S + N pom tau tias muaj S protein ntau ntawm cov kab mob tiv thaiv kab mob tom qab peb txhaj tshuaj. Ib txoj kev tshawb nrhiav SARS-CoV-2 mRNA txhaj tshuaj tiv thaiv tau tshaj tawm tias kev sib xyaw ua ke S + N ua rau muaj qhov ua kom muaj S-specific CD8+ T-cell teb piv nrog S ib leeg, uas zoo ib yam nrog peb cov txiaj ntsig [24]. Lwm txoj kev tshawb fawb tau tshaj tawm tias T-cell teb rau S thiab N antigens tom qab dual-antigen hAd5 S + N thawj koob tshuaj tiv thaiv ib leeg yog sib npaug rau cov neeg mob yav dhau los SARS-CoV-2-, thiab hauv silico kwv yees qauv ntawm T-cell. epitope HLA binding qhia tias T-cell cov lus teb rau hAd5 S + N tshuaj tiv thaiv yuav khaws lawv cov txiaj ntsig tawm tsam B.1.351 variant. Ntxiv mus, plasma los ntawm yav dhau los SARS-CoV-2- cov neeg mob kis tau pom tias muaj kev sib koom ua ke ntau dua rau cov hlwb uas qhia txog cov antigen dual S-Fusion + N-ETSD tsim dua li hAd5 S-Fusion ib leeg, ntxiv qhia tias kev tiv thaiv kab mob ntawm lub cev. S + N ob-antigen tshuaj tiv thaiv zoo dua li ntawm cov tshuaj tiv thaiv S ib leeg-antigen [26].
Tus kab mob nyob tsis tau kuaj pom hauv lub ntsws, thiab kev poob phaus tom qab kev sib tw tau txo qis hauv pawg pS, pS + pN, thiab pS + pE + pM, thaum kev kho pN tsis zoo txo tus kab mob titer. Cov kev tshawb pom no hais txog qhov tsis tseem ceeb thiab kev ua tau zoo ntawm S protein raws li lub hom phiaj tshuaj tiv thaiv. Qhov tseem ceeb, kev txhaj tshuaj nrog pS thiab pN muaj txiaj ntsig zoo dua li pS lossis pN ntawm kev tshem tawm cov kab mob. Cov pab pawg pS + pE + pM qhia tsawg dua cov kev hloov pauv hauv lub ntsws, uas zoo ib yam nrog cov txiaj ntsig ntawm peb txoj kev tshawb fawb yav dhau los [15]. Pab pawg S + N muaj cov ntawv theej RNA tsawg tsawg hauv lub ntsws, txo qhov hnyav, thiab lub sijhawm rov qab sai tom qab SARS-CoV-2 kev sib tw piv rau pab pawg tau txhaj tshuaj tiv thaiv S/N ib leeg, uas tau ua raws li cov cov txiaj ntsig ntawm qhov kev tshawb fawb no. Txawm li cas los xij, tsis muaj ib pawg twg tau kuaj pom cov tshuaj tiv thaiv kab mob tsis zoo, uas yuav piav qhia los ntawm qhov sib txawv ntawm cov tshuaj tiv thaiv ntau yam thiab cov tsiaj sim [26]. Ib qho SARS-CoV-2 adenovirus vector vaccine txoj kev tshawb fawb tau tshaj tawm tias cov tshuaj tiv thaiv S tsuas yog muab kev tiv thaiv lub hlwb thaum sib koom ua ke nrog tshuaj tiv thaiv N [27]. Lwm txoj kev tshawb fawb tau tsim Tri: ChAd, Bi: ChAd, thiab Mono: ChAd cov tshuaj tiv thaiv uas qhia txog S1 / N / RdRp, N / RdRp, thiab S1 proteins, raws li, thiab sim lawv hauv B.1.351 tsiaj qauv. Kev nthuav dav tag nrho cov kab mob tau pom nyob rau hauv Mono: ChAdlungs, whereas Bi: ChAd thiab Tri: ChAd ntsws tshwm sim ze li ntawm cov kab mob no [28].
Tsis tas li ntawd, cov tsiaj tsis tau txhaj tshuaj tiv thaiv kab mob ntsws muaj kab mob siab, qhov kev kho Tri: ChAd txo cov kab mob kis los ntawm 3.5 teev. Hauv kev sib piv, ob qho tib si Bi: ChAd thiab Mono: ChAd cov tshuaj tiv thaiv tsuas yog txo qis tus kab mob. Cov txiaj ntsig no qhia tias qhov kev tiv thaiv ntawm S / N ob npaug-antigen tshuaj tiv thaiv kab mob sib txawv yuav zoo dua li cov tshuaj tiv thaiv S ib leeg-antigen, uas zoo ib yam nrog peb cov txiaj ntsig [28]. Ob peb txoj kev tshawb fawb tau tshaj tawm tias N cov nas uas tau txhaj tshuaj tiv thaiv kab mob ua rau mob ntsws mob hnyav tom qab tus kab mob SARS-CoV [29–31]. Cov kev tshawb fawb yav dhau los kuj tau tshaj tawm tias kev txhaj tshuaj tiv thaiv kab mob adenovirus vector uas qhia txog Mouse Hepatitis Virus N protein tiv thaiv cov nas tiv thaiv kab mob tuag taus, uas qhia tau tias N protein tuaj yeem tsim kev tiv thaiv [32]. Tsis tas li ntawd, pab pawg tau txhaj tshuaj tiv thaiv kab mob CRT/N DNA tau muaj qhov txo qis ntawm tus kab mob titer tom qab kev sib tw, nrog rau cov tshuaj tiv thaiv kab mob uas qhia txog SARS-CoV N protein [33].
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Ib txoj kev tshawb fawb ntawm S protein tau pom tias cov tshuaj tiv thaiv DNA / protein sib xyaw ua rau ob qho tib si humoral thiab cellular tiv thaiv zoo dua li cov tshuaj tiv thaiv DNA / protein ib leeg [8]. Cov tshuaj tiv thaiv tsom rau tsuas yog S protein tau pom tias txo qis kev ua tau zoo hauv kev tiv thaiv tus kab mob me me mus rau nruab nrab-19 tshwm sim los ntawm kev hloov pauv tshiab. Lub luag haujlwm ntawm kev khaws cia SARS-CoV-2 cov qauv proteins, suav nrog N / E / M cov proteins, yog qhov tsim nyog rau kev saib xyuas hauv kev tsim tshuaj tiv thaiv thiab kev siv, vim tias cov tshuaj tiv thaiv T-cell cov lus teb tawm tsam cov epitopes khaws cia feem ntau tsis cuam tshuam los ntawm kev hloov pauv. . Ib txoj kev tshawb fawb tau tshaj tawm tias cov neeg mob SARS-rov qab los (n=23) tseem muaj lub cim xeeb ntev ntev T cells reactive rau SARS-CoV N protein 17 xyoo tom qab kev sib kis xyoo 2003, uas tau pom muaj kev cuam tshuam zoo rau SARS CoV{ {15}} N protein, ntxiv kev siv cov N protein ua cov tshuaj tiv thaiv kab mob sib kis [34]. Txoj kev tshawb no pom tau tias pS/pN kev txhaj tshuaj tiv thaiv kab mob cuam tshuam nrog nAb cov lus teb ntau dua, kev tshem tawm cov kab mob zoo dua, thiab txhim kho kev tiv thaiv kab mob ntawm tes thiab tuaj yeem muab kev tiv thaiv zoo dua tom qab SARS-CoV{20}} kev sib tw piv nrog pS ib leeg. Tsis tas li ntawd, SARS-CoV-2 variants tau pom tias kis tau rau ntau hom tsiaj, thiab kev sib kis ntawm tib neeg mus rau tsiaj tau pom hauv qee cov tsiaj qus thiab tsiaj txhu [7]. Yog li, cov tshuaj tiv thaiv kws kho tsiaj SARS-CoV-2 xav tau kev saib xyuas ntxiv. Tsis tas li ntawd, nanotechnology tej zaum yuav yog ib qho cuab yeej muaj zog hauv kev ua kom cov tshuaj tiv thaiv zoo thiab tsim nyog rau kev saib xyuas ntxiv [2].
Txoj kev tshawb no muaj ntau yam kev txwv. Ua ntej, peb tsuas yog soj ntsuam cov tshuaj tiv thaiv DNA hauv cov nas BALB / c, thiab cov kev tshawb fawb yav tom ntej yuav tsum ntsuas cov tshuaj tiv thaiv kab mob ntawm cov tshuaj tiv thaiv kab mob hauv lwm yam tsiaj. Qhov thib ob, kev tshawb fawb ntxiv yog xav tau kom nkag siab tag nrho cov txheej txheem molecular hauv qab ntawm augmented nAb- thiab S-specific CD8 T-cell cov lus teb tshwm sim los ntawm kev txhaj tshuaj tiv thaiv kab mob siv S thiab N cov proteins thiab siv cov kev paub no los txhim kho COVID-19 txhaj tshuaj tiv thaiv. Thaum kawg, kev ua haujlwm ntawm N cov tshuaj tiv thaiv tshwj xeeb tsim nyog tau txais kev kawm ntxiv.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Hauv kev xaus, qhov kev tshawb fawb no tau soj ntsuam qhov kev tiv thaiv kab mob tiv thaiv kab mob sib kis nrog SARS-CoV-2 S, N, E, thiab M proteins. Ntau cov tshuaj tiv thaiv uas tsom rau tsuas yog S protein muaj qhov txo qis kev tiv thaiv ntawm cov kab mob sib txawv. Peb cov txiaj ntsig yuav tsim lub hauv paus rau kev tsim cov tshuaj tiv thaiv kab mob khaub thuas tiv thaiv COVID-19 los tswj tam sim no thiab tshwm sim SARS-CoV-2 qhov sib txawv thiab tiv thaiv kev muaj peev xwm-coronavirus pandemics.
Cov ntaub ntawv
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