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Chimeric Human Papillomavirus-16 Cov kab mob zoo li tus kab mob nthuav tawm P18I10 Thiab T20 Peptides Los ntawm HIV-1 Lub hnab ntawv Induce HPV16 Thiab HIV-1-Tshwj xeeb Humoral Thiab T Cell-Mediated Immunity hauv BALB/c nas

Dec 07, 2023

Abstract:

Hauv txoj kev tshawb no, HIV-1 P18I10 CTL peptide tau muab los ntawm V3 voj ntawm HIV-1 gp120 thiab T20 anti-fusion peptide ntawm HIV-1 gp41 tau muab tso rau hauv HPV16 L1 capsid protein. los tsim cov tshuaj tiv thaiv kab mob HPV: HIV (L1: P18I10 thiab L1: T20) VLPs los ntawm kev siv cov kab mob mammalian cell nthuav qhia. HPV: HIV VLPs raug ntxuav los ntawm chromatography. Peb pom tau hais tias qhov ntxig ntawm P18I10 lossis T20 peptides rau hauv DE voj ntawm HPV16 L1 capsid proteins tsis cuam tshuam rau hauv vitro stability, nws tus kheej sib dhos, thiab morphology ntawm chimeric HPV: HIV VLPs. Qhov tseem ceeb, nws tsis cuam tshuam nrog HIV-1 cov tshuaj tiv thaiv kab mob tiv thaiv lub hom phiaj ua raws li P18I10 thiab T20 peptides nthuav tawm ntawm chimeric HPV: HIV VLPs lossis nrog induction ntawm HPV16 L1- cov tshuaj tiv thaiv tshwj xeeb hauv vivo. Peb tau pom tias cov tshuaj tiv thaiv chimeric L1:P18I10/L1:T20 VLPs tuaj yeem ua rau HPV16- tab sis tsis muaj zog HIV-1-cov tshuaj tiv thaiv kab mob tshwj xeeb thiab elicited HPV16- thiab HIV-1- tshwj xeeb T- cov lus teb ntawm tes hauv BALB/c nas. Tsis tas li ntawd, tuaj yeem yog qhov muaj peev xwm txhawb nqa kom nce cov kab mob HIV tshwj xeeb ntawm tes hauv kev txhaj tshuaj tiv thaiv heterologous tom qab priming nrog BCG.HIVA tshuaj tiv thaiv. Qhov kev tshawb fawb no yuav pab txhawb ib kauj ruam mus rau kev txhim kho tus tshiab chimeric HPV: HIV VLP-based tshuaj tiv thaiv platform rau kev tswj hwm tus kab mob HPV16 thiab HIV-1, uas yog qhov yuav tsum tau ua sai sai hauv cov teb chaws tsim thiab kev lag luam.

Desert ginseng-Improve immunity (12)

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Nyem qhov no mus saib Cistanche Enhance Immunity khoom

【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692

Ntsiab lus:

HIV-1; HPV 16; tshuaj tiv thaiv; cov kab mob zoo li cov kab mob; P18I10; T20 enfuvirtide; BCG.HIVA; kev tiv thaiv kev lom zem; T cell-mediated tiv thaiv kab mob

1. Taw qhia

Tib neeg kev tiv thaiv kab mob tiv thaiv kab mob -1 (HIV-1), uas ua rau kis tau tus kab mob tiv thaiv kab mob tiv thaiv kab mob (AIDS), tau tshawb pom thaum xyoo 1980s, thiab txij li ntawd los nws tau dhau los ua kev sib kis thoob ntiaj teb [1]. Txawm hais tias muaj kev siv tshuaj tiv thaiv kab mob tiv thaiv kab mob (HAART), nrog rau kev tiv thaiv kab mob ua ntej (PrEP) tuaj yeem muaj kev cuam tshuam tiag tiag rau kev tswj hwm tus kab mob HIV-1, kev txhaj tshuaj tseem yog ib qho tseem ceeb rau cov txiaj ntsig rau pej xeem thiab muab tso rau. xaus rau lub ntiaj teb HIV-1 kev sib kis [2]. Txawm hais tias muaj ntau tshaj li peb lub xyoos dhau los ntawm kev tshawb fawb txog HIV-1 thiab kev sim tshuaj tiv thaiv ntau heev, cov tshuaj tiv thaiv kab mob HIV -1 muaj ntawv tso cai los txog rau tam sim no tseem ua tsis tau. Qhov kev sim RV144 tau ua nyob rau Thaib teb yog thawj rooj plaub uas qhia txog qhov ua tau zoo ntawm 31.2% tiv thaiv kev kis tus kabmob HIV-1 [3]. Feem ntau ntawm lwm cov kab mob HIV-1 cov neeg sib tw tshuaj tiv thaiv uas tau mus kuaj mob feem ntau yog raws li DNA, cov kab mob sib kis, lossis cov qauv subunit protein [4,5]. Qhov zoo tshaj plaws, cov tshuaj tiv thaiv kab mob HIV ua tau zoo -1 muaj peev xwm ua rau muaj kev tiv thaiv kab mob hauv lub cev, ua rau cov tshuaj tiv thaiv kab mob tiv thaiv kab mob [6] nrog rau cytotoxic T lymphocyte (CTL) cov lus teb los tshem tawm cov kab mob [7]. Txawm li cas los xij, kev tshem tawm txhua qhov lus teb yuav xav tau cov tswv yim tshuaj tiv thaiv sib txawv, lav kev sib cais tab sis kev siv zog sib npaug. Kev xaiv cov tshuaj tiv thaiv kab mob thiab cov kab mob xa mus yuav muaj kev cuam tshuam loj rau kev ua haujlwm thiab qhov tshwj xeeb ntawm HIV-1 tshuaj tiv thaiv [8,9]. Kev rov ua tsis tiav uas siv cov qauv txheej txheem rau kev txhaj tshuaj tiv thaiv kab mob HIV-1 tau ua rau pom qhov tseem ceeb ntawm kev xa cov vector xaiv, kev tswj hwm kev txhawb nqa, thiab kev tiv thaiv kab mob tshwj xeeb hauv ob qho tib si humoral thiab cellular teb. Ntau tshaj 100 hom tib neeg papillomavirus (HPV) twb paub lawm thiab HPV genotypes 16 thiab 18 raug suav hais tias yog lub luag haujlwm rau kwv yees li 70% ntawm cov qog noj ntshav thoob ntiaj teb [10]. HPV L1 virus-like particles (VLPs), muab faib ua ib hom tshuaj tiv thaiv subunit, feem ntau tuaj yeem ua rau muaj qhov sib piv L1- cov lus teb tshwj xeeb rau cov tsiaj qus virion thiab T cell-mediated teb [11–13]. Tam sim no, peb cov tshuaj tiv thaiv HPV tau tso cai nyob rau hauv khw thiab tag nrho lawv yog raws li VLPs ntawm HPV L1 capsid protein. Ob tug ntawm lawv, Gardasil (Merck, Rahway, NJ, USA) thiab Gardasil-9 (Merck) yog tsim los ntawm cov poov xab (Saccharomyces cerevisiae) qhia system thaum lwm yam, Cervarix (GSK, Brentford, UK), yog tsim. los ntawm baculovirus qhia vector/insect cell (BEVS/IC) system [14]. Txog tam sim no, qhov zoo tshaj plaws ntawm kev tsim cov kab mob HPV16 L1 cov protein nyob rau hauv cov tsiaj txhu qhia tsis tau zoo. Yog li, kev tsim cov tshuaj tiv thaiv ua ke uas yuav tiv thaiv kab mob HPV thiab kab mob HIV yog kev siv dag zog hauv kev tawm tsam ob tus kab mob loj hauv ntiaj teb no.

Hauv peb cov ntawv tshaj tawm kev tshuaj xyuas yav dhau los hais txog kev tsim cov ntsiab lus ntawm tus kab mob zoo li tus kab mob (VLP)-raws li HIV-1 cov tshuaj tiv thaiv, peb tau hais tias VLPs uas tsis yog lub hnab ntawv, xws li papillomavirus VLPs, tuaj yeem ua lub luag haujlwm ua haujlwm raws li kev xa cov vectors los nthuav tawm. HIV-1 CTL lossis neutralizing antibody epitopes [15,16]. Qhov kev xav no tau lees paub nyob rau hauv ntau yam chimeric bovine papillomaviruses (BPV) L1 VLP nthuav qhia P18I10 CTL epitope los ntawm V3 voj ntawm gp120 HIV-1 lub hnab ntawv protein (Env) thiab 2F5 epitope lossis MPER cheeb tsam ntawm gp41 HIV-1 Env [17–22]. Cov yam ntxwv ntawm tib neeg papillomavirus hom-16 (HPV16) L1 capsid proteins zoo ib yam li BPV thiab tuaj yeem sib sau ua ke rau hauv ib txheej L1 VLPs [23]. Tsib ntawm HPV16 L1 cov proteins tsim ib qho pentamer thiab 72 ntawm cov pentamers nws tus kheej sib sau ua ke rau hauv HPV16 VLP [24]. Txawm li cas los xij, tseem tsis tau muaj pov thawj tseeb tias chimeric HPV16: HIV capsid proteins tuaj yeem ruaj khov hauv vitro thiab nws tus kheej sib sau ua ke rau hauv morphologically integral VLPs. Ntawm qhov tod tes, HPV16 L1 VLPs tau pom tias muaj kev tiv thaiv kab mob siab heev thiab muaj peev xwm ua rau cov tshuaj tiv thaiv kab mob tshwj xeeb T thiab B-cell tiv thaiv kab mob [11–13]. Nws tseem tseem yuav pom tias qhov kev nthuav qhia ntawm HIV-1 epitopes los ntawm HPV: HIV VLPs tuaj yeem yog immunogenic. Nyob rau hauv txoj kev tshawb no, peb tsom mus tsim ib tug chimeric VLP-raws li HPV: tshuaj tiv thaiv kab mob HIV los ntawm kev siv tib neeg 293F hlwb, ib tug zoo-tsim mammalian cell qhia system. HPV 16 L1 protein ua raws li cov qauv tshuaj tiv thaiv scaffold, thiab P18I10 thiab T20 peptides raug xaiv ua HIV-1 immunogens thiab muab tso rau hauv DE voj ntawm HPV 16 L1 protein. Lub immunodominant P18I10 CTL epitope suav nrog 10 amino acids (residues 311–320: RGP GRAFVTI) yog muab los ntawm qhov sib txawv thib peb (V3) ntawm HIV-1 hnab ntawv glycoprotein gp120. P18I10 peptide tau raug txheeb xyuas tias yog H-2Dd-txheej txheem MHC chav kawm-I molecule los ua rau cytotoxic T lymphocyte (CTL) cov lus teb [25,26]. Lub T20 peptide, hu ua Enfuvirtide thiab tsim los ua ib qho tshuaj tua kab mob multimeric fusion peptide, muaj 36 amino acid ib ntus (YTSLIHSLIEESQNQQEKNEQ ELLELDKWASLWNWF) ua raws li C-terminal heptad helix sib lawv liag ze rau ntawm daim nyias nyias.0 s proximal external region (MPER) ntawm HIV-1 hnab ntawv glycoprotein 41 (gp41) [27]. Ob tus kab mob HIV no -1 T (P18I10) thiab B (T20) cell-based epitopes tau raug xaiv los ua qhov pib thiab pov thawj ntawm kev sim tswv yim rau chimeric VLP-based HPV: HIV tshuaj tiv thaiv kab mob.

Ntau xyoo dhau los, ntau hom kev txhawb nqa tseem ceeb ntawm VLP-based HIV-1 tshuaj tiv thaiv tau raug sim [15]. Txawm hais tias feem ntau ntawm cov kab mob HIV yav dhau los -1 VLP [28] lossis chimeric BPV: HIV VLP [17–22] cov tswv yim tshuaj tiv thaiv tau tsom mus rau kev txhawb lub cev tiv thaiv kab mob los ntawm kev siv homologous prime-boost regimen, ob qho kev tshawb fawb yav dhau los qhia tias kev txhaj tshuaj heterologous. muaj cov recombinantMycobacterium bovisBacillus Calmette-Guérin (rBCG) qhia txog HIV-1 Gag prime thiab HIV-1 Gag VLP boost tuaj yeem pab txhawb T-cell tiv thaiv kab mob [29,30]. Hauv peb pab pawg tshawb fawb, peb tau ua pov thawj priming nrog rBCG qhia HIVA immunogen thiab boosting nrog recombinant viral vector MVA.HIVA muaj kev nyab xeeb thiab elicited HIV-1- tshwj xeeb T-cell tiv thaiv kab mob hauv BALB/c nas [31–33]. Cov tshuaj tiv thaiv kab mob HIVA, tsim los ntawm Dr. Tomas Hanke, yog tsim los ntawm tag nrho-ntev HIV-1 Gag protein ua ke nrog ntau CTL epitopes suav nrog P18I10 epitopes ntawm C-terminus [34]. Yog li ntawd, peb tsom mus soj ntsuam seb BCG.HIVA tuaj yeem txhawb T-cell tiv thaiv kab mob los ntawm HIV: HPV (L1:P18I10) VLPs hauv BALB/c nas.

Nyob rau hauv txoj kev tshawb no, chimeric HPV: HIV (L1: P18I10 thiab L1: T20) immunogens tau tsim thiab tsim los ntawm kev siv 293F qhia qhov system. Lub chimeric L1: P18I10 thiab L1: T20 protein qhia tau lees paub los ntawm kev tiv thaiv kab mob. HPV: HIV VLPs tom qab ntawd tau ua kom huv los ntawm 3-txoj kev chromatographic kauj ruam, suav nrog cation (CEC), qhov loj me (SEC), thiab heparin affinity (H-AC) chromatography. Tom qab ntawd, qhov kev ruaj ntseg hauv vitro, hauv vitro nws tus kheej sib sau ua ke, thiab morphology ntawm purified HPV: HIV VLPs tau lees paub los ntawm tsis txo qis SDS-PAGE, molecular mass assay, thiab kis electron microscopy (TEM), feem. Cov kab mob sib kis thiab ua raws li P18I10 thiab T20 peptides nthuav tawm ntawm chimeric HPV: HIV VLPs tau ntxiv tus cwj pwm los ntawm kev tiv thaiv HIV-1 gp120 V3 thiab 2F5 monoclonal antibodies hauv vitro los ntawm kev siv Western blot thiab indirect ELISA assay. Thaum kawg, qhov immunogenicity ntawm HPV: HIV VLPs raug soj ntsuam hauv BALB/c nas qauv. Peb tau pom tias cov tshuaj tiv thaiv kab mob L1: P18I10 thiab L1: T20 VLP tuaj yeem ua rau HPV16- thiab HIV-1- cov tshuaj tiv thaiv tshwj xeeb thiab chimeric L1:P18I10 VLPs tuaj yeem ua rau HPV16- thiab HIV{ {37}}cov lus teb tshwj xeeb T-cell hauv BALB/c nas. Vim tias kev txhim kho thiab tsim cov tshuaj tiv thaiv kab mob HPV: tshuaj tiv thaiv kab mob HIV tseem tsis tuaj yeem ua tiav, txoj kev tshawb no tau muab cov tswv yim hauv paus uas yuav tsim nyog los txhawb lub ntiaj teb kev siv zog los tsim cov tshuaj tiv thaiv kab mob VLP tshiab rau kev tswj hwm tus kab mob HPV thiab HIV-1.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Kev tsim kho ntawm BCG.HIVA2auxo.int Vaccine Strain

Recombinant BCG nthuav qhia HIVA immunogen yog yav tas los tsim siv E.coli-mycobacterial integrative shuttle vector p2auxo.int. Kev tsim kho ntawm E. coli/mycobacterial vector qhia txog HIVA antigen yav dhau los tau piav qhia [31–33]. BCG.HIVA2auxo.int tau diluted nyob rau hauv PBS-Tween20 rau 2 × 107 cfu/mL, sonicated los cuam tshuam cov kab mob clumps, thiab inoculated rau hauv lub rear zaub mov ncoo los yog BALB/c nas (50 µL, 106 cfu / nas).

Desert ginseng-Improve immunity (23)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

2.2. Kab lis kev cai thiab kev hloov pauv

Cells ntawm glycine auxotrophic strain ntawm E. coli, M151GlyA (Invitrogen, Waltham, MA, USA), muab los ntawm Dr. Pau Ferrer, tau coj mus rau hauv me me M9-derivative nruab nrab (M9-D: Na2HPO4, 6.78 g/L; KH2PO4, 3 g/L; NaCl, {{10}}.5 g/L; NH4Cl, 1 g/L, qabzib, 1{{2{23}} }} g/L; MgSO4, 2 mmol/L; CaCl2, 0.1 mmol/L; thiamine, 0.1 g/L; FeCl3, 0 0. }}25 g/L; AlCl3·6H2O, 0.13 mg/L; ZnSO4·7H2O, 2.6 mg/L; CoCl2·6H2O, 0.47 mg/L; CuSO4·H2O, 4.6 mg/L; H3BO3, 0. , ntxiv nrog glycine (70 μg / mL). E. coli M151Gly hlwb tau hloov nrog p2auxo.HIVA plasmids los ntawm electroporation. Rau qhov no, E. coli kab lis kev cai tau loj hlob mus rau qhov muag pom ntawm 0.9 ntawm 600 nm thiab hloov pauv siv Bio-Rad gene pulser electroporator ntawm 2.5 kV, 25 µF, thiab 200 Ω. Cov hlwb hloov pauv tau tom qab kab lis kev cai ntawm M9-D agar daim hlau (cov khoom raws li tau piav qhia yav dhau los, nrog 1.5% bactoagar ntxiv) yam tsis muaj glycine ntxiv rau kev xaiv lossis nrog glycine supplementation los tswj. Lub lysine auxotrophic BCG strain, BCG∆lys, ua siab zoo muab los ntawm WR Jacobs Jr., BR Bloom, thiab T. Hsu tau hloov pauv nrog p2auxo.HIVAint plasmid DNA los ntawm electroporation. Cov kab mob mycobacteria tau coj mus rau hauv Middlebrook 7H9 broth nruab nrab lossis ntawm Middlebrook agar 7H10 nruab nrab ntxiv nrog albumin-dextrose-catalase (ADC; Difco) uas muaj 0.05% Tween 80. L-lysine monohydrochloride (Sigma, Kawasaki, Nyiv) tau yaj hauv dej distilled. siv los ua ib qho ntxiv ntawm qhov kawg ntawm 40 µg / mL. Rau kev hloov pauv, BCG tau coj mus rau qhov pom qhov pom ntawm 1.5 ntawm 600 nm thiab hloov pauv siv Bio-Rad gene pulser electroporator ntawm 2.5 kV, 25 µF, thiab 1000 Ω. Cov kev hloov pauv tau raug coj mus rau hauv ADC-ntxiv Middlebrook agar 7H10 nruab nrab uas muaj 0.05% Tween 80 yam tsis muaj lysine supplementation.

2.3. Cell Kab thiab Cell Culture

Lub 293F hlwb (Tibco), muab tau los ntawm tib neeg lub raum embryonic (HEK) 293 hlwb, tau coj mus rau hauv FreeStyle 293 qhia nruab nrab (Tibco) ntxiv nrog 5 mL / L ntawm penicillin-streptomycin (Tibco) thiab incubated hauv 37 ◦C incubator. ib qho av noo ntawm 5% CO2 ntawm lub orbital shaker platform rotating ntawm 125 rpm.

2.4. Kev tsim tawm ntawm L1: P18I10 thiab L1: T20 Proteins Siv 293F Kev Tshaj Tawm System

Lub pCDNA3.1 tsim muaj L1:P18I10 los yog L1:T20 DNA coding sequences coj mus rau chimeric L1:P18I10 thiab L1:T20 proteins, feem. Tus kab mob HIV-1 P18I10 CTL peptide (RGPGRAFVTI) lossis T20 peptide (YTSLIHSLIEESQNQQEKNEQE LLELD KWASLWNWF) tau muab tso rau hauv DE voj ntawm HPV16 L1 capsid protein. HPV16 L1 DE lub voj kab sib txuas encoding 130-136 amino acids tau hloov nrog P18I10I10 lossis T20 peptide. Lub L1: P18I10 lossis L1: T20 DNA coding sequences tau hloov kho nrog Kozak ib ntus, ua kom zoo nrog tib neeg codon, flanked los ntawm kev txwv enzyme chaw ntawm HindIII thiab XbaI, thiab cloned rau hauv pcDNA3.1 (+) vector los ntawm kev siv GeneArt gene synthesis cov kev pabcuam ( Thermo Fisher, Waltham, MA, USA). Lub recombinant plasmid DNA (pDNA) tau hloov mus rau hauv E. coli DH5 cov hlwb muaj peev xwm (Invitrogen) rau kev nthuav dav thiab muab rho tawm los ntawm kev siv cov khoom siv plasmid Maxi (QIAGEN, Hilden, Lub teb chaws Yelemees). Lub 293F hlwb tau coj los ua ke nrog 30mL FreeStyle 293 qhia nruab nrab hauv 125mL Erlenmeyer flask (Corning, New York, NY, USA) mus rau qhov ntom ntawm 1.0 × 106 / mL thiab transiently kis nrog L1: P18I10 lossis LD1: T20 ceg. polyethyleneimine nrog ib MW ntawm 25 kDa (PEI-25K) (Polysciences) ntawm qhov ua tau zoo ntawm DNA rau PEI 1: 3 (w/w) thiab DNA rau kab lis kev cai nruab nrab 1: 1 (w/v), raws li rau cov chaw tsim khoom cov lus qhia [35]. 293F hlwb tau sau thaum 96 teev tom qab hloov pauv. 293F hlwb tuaj yeem ncav cuag qhov sib npaug ntawm 3.6 × 106 hlwb / mL nrog kwv yees li 50% kev muaj peev xwm.

2.5. Immunofluorescence Staining

Cov hlwb tau permeabilized ntawm iav swb nrog 100% txias acetone. Tom qab ntawd, cov hlwb ruaj khov tau raug soj ntsuam nrog cov tshuaj tiv thaiv HPV16 L1 antibody CAMVIR-1 (Abcam, Cambridge, UK) thiab ntes nrog cov tshuaj tiv thaiv nas IgG-FITC (Sigma). Immune-stained cell monolayers raug ntxuav kom huv si nrog PBS thiab npog nrog lub nruab nrab nruab nrab nrog DAPI (Abcam). Cov duab immunofluorescence raug tshuaj xyuas nyob rau hauv ib qho inverted microscope ntawm 40 × magnification. Transfection efficiency yog txiav txim los ntawm qhov piv ntawm FITC (ntsuab) - zoo hlwb rau DAPI (xiav)-stained hlwb.

2.6. Purification ntawm HPV: HIV (L1:P18I10 thiab L1:T20) VLPs

Tag nrho ntawm 108 kis tau 293F hlwb hauv 125 mL Erlenmeyer flask (30 mL kab lis kev cai nruab nrab / lub raj mis) tau sau los ntawm centrifugation ntawm 1500 rpm rau 5 min thiab ntxuav ob zaug nrog PBS. Cov pellets ntawm tes tau raug tshem tawm hauv lysis buffer formulated nrog 1% Triton X-100, protease inhibitor (1:100) (Millipore), thiab Benzonase (25 U / mL) (Millipore). Cell lysates tau qhia meej nrog 0.45 µm PVDF syringe lim (Millipore). Cov HPV: HIV (L1:P18I10 thiab L1:T20) VLP cov qauv tau raug purified siv cation txauv (Capto SP ImpRes, GE, Boston, MA, USA), qhov loj me (Capto Core 700, GE) thiab affinity (HiTrap Heparin HP , GE) chromatography. Cov txheej txheem chromatographic tau piav qhia hauv peb cov kev tshawb fawb yav dhau los [36,37] thiab ua raws li cov neeg tsim khoom raws tu qauv [38]. Lub teeb liab L1 protein nyob rau hauv txhua kauj ruam purification yog tus yam ntxwv los ntawm Western blot tsom xam thiab soj ntsuam nrog anti-HPV16 L1 antibody CAMVIR-1 [39].

2.7. Tsis-Txo SDS-PAGE

HPV16 L1, L1:P18I10, thiab L1:T20 VLPs tau sib xyaw nrog 2 × Laemmli cov qauv tsis zoo (BIO-RAD) thaum tsis muaj lossis muaj 5% (v/v) 2-mercaptoethanol (2- }ME) thiab hnov ​​​​mob ntawm chav tsev kub (RT) rau 24 teev. Cov qauv raug cais los ntawm 8-16% TGX stain-free protein gels (BIO-RAD). Tom qab ntawd, cov gels raug xa mus rau PVDF daim nyias nyias. Cov daim nyias nyias tau raug soj ntsuam nrog cov tshuaj tiv thaiv HPV16 L1 CAMVIR-1 mAb ntawm ib qho dilution ntawm 1:4000. Tom qab ntawd, cov membranes tau incubated nrog anti-nas IgG Peroxidase Conjugate (Sigma-Aldrich, St. Louis, MO, USA) ntawm ib tug dilution ntawm 1:4000. Lub teeb liab tau tsim thiab pom los ntawm chemiluminescence siv Western Blot ECL substrate kit (Bio-Rad, Hercules, CA, USA). Cov duab blot tau txais los ntawm kev siv Odyssey Fc imaging system.

2.8. Molecular Mass Analysis

HPV16 L1, L1:P18I10, thiab L1:T20 VLPs yam tsis muaj 2-ME kev kho mob tau lim tawm los ntawm 1000 kDa molecular weight cutoff (MWCO) ultrafiltration devices (SARTORIUS). HPV16 L1, L1:P18I10, thiab L1:T20 VLPs nrog 2-ME kev kho mob tau dhau los ntawm 100 kDa MWCO ultrafiltration devices (Amicon). Cov retentates tau rov ua dua tshiab rau qhov qub ntim thiab sau los ntawm cov khoom siv lim lim dej, thaum cov filtrates tau sau rau hauv qab ntawm lub raj centrifuge. Lub teeb liab L1 tau ntsuas los ntawm kev siv cov dot blot probed nrog anti-HPV16 L1 mAb thiab kuaj los ntawm anti-nas IgG-peroxidase conjugate (Sigma-Aldrich). Cov duab tau txais los ntawm Odyssey Fc Imaging System ntawm chemiluminescence channel.

Desert ginseng-Improve immunity

Cov txiaj ntsig ntawm cistanche tubulosa- ua kom muaj zog tiv thaiv kab mob

2.9. Tsis zoo Staining thiab Transmission Electron Microscopy

Tom qab them cov carbon-coated tooj liab daim phiaj (Sigma-Aldrich) nyob rau hauv ultraviolet lub teeb rau 5 min, coj mus muag HPV16 L1 (Abcam), purified L1: P18I10 thiab L1: T20 VLPs equilibrated nrog 20 mM Tris-HCl (pH 7.4, 137 mM. ) tau nqus ntawm daim phiaj rau 1 min thiab yaug peb zaug los ntawm dej miliQ. HPV: HIV VLPs tsis zoo-stained nrog 2% uranyl acetate ntawm pH 4.5 (Sigma-Aldrich) rau 1 min. Ntau cov staining agents raug tshem tawm los ntawm Whatman qualitative filter paper (Sigma-Aldrich). Cov kab sib txuas tau muab tso rau hauv chav dehumidifier tsawg kawg 2 teev ua ntej kev soj ntsuam. Cov duab tau txais los ntawm kev siv lub tshuab hluav taws xob xa hluav taws xob (Tecnai Spirit 120 kV) ntawm magnification SA135K (100 nm) thiab SA59000 (200 nm), feem.

2.10. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis thiab Western Blotting Analysis

Qhov sib npaug (500 ng) ntawm HPV16 L1 protein (Abcam), purified L1: P18I10, thiab L1: T20 VLPs tau tov nrog 2 × Laemmli cov qauv tsis muaj 5% 2-ME thiab boiled ntawm 95 ◦C rau 5 min . Cov qauv raug cais los ntawm 8–16% TGX Stain-free protein gels thiab tom qab ntawd xa mus rau PVDF daim nyias nyias (Millipore, Burlington, MA, USA) siv cov khoom siv Semi-Dry (Bio-Rad). Lub membrane tau thaiv nrog 5% skim mis nyuj hauv TBST. Tom qab ntawd, cov ntaub so ntswg tau raug soj ntsuam nrog cov tshuaj tiv thaiv HPV16 L1 CAMVIR-1 mAb ntawm dilution ntawm 1: 4000, anti-HIV-1 gp120 V3 voj mAb (NIBSC, EVA3012) ntawm dilution ntawm 1: 40 thiab HIV1 gp41 (2F5) mAb (NIBSC, ARP3063) ntawm ib tug dilution ntawm 1:4000, feem. Tom qab ntawd, cov membranes tau incubated nrog anti-nas IgG Peroxidase Conjugate (Sigma-Aldrich) ntawm ib tug dilution ntawm 1:4000. Cov khoom siv Western ECL substrate (BIO-RAD) tau siv rau kev txhim kho teeb liab. Cov duab blot tau txais los ntawm kev siv Odyssey Fc imaging system ntawm chemiluminescence channel.

2.11. Kev txhaj tshuaj ntawm cov nas, sau ntawm Sera, thiab cais tawm ntawm Splenocytes

Qhov no yog ib qho kev tshawb fawb ua pov thawj-ntawm-lub tswv yim los qhia txog kev tiv thaiv kab mob HPV: HIV VLPs (L1:P18I10 thiab L1:T20 VLPs). Cov koob tshuaj, txoj kev tswj hwm, thiab lub sijhawm tseem ceeb ntawm peb cov HPV: HIV VLPs raug xa mus rau cov kev tshawb fawb yav dhau los uas txhaj tshuaj tiv thaiv nas nrog bovine papillomavirus (BPV): HIV VLPs rau inducing antibody teb [20,21]. Purified HPV: HIV VLPs tau emulsified nrog qhov sib npaug ntawm qhov ntim ntawm (225 µg ib koob tshuaj 0.5 mL) txhuas hydroxyphosphate sulfate (Thermo Fisher), kom paub meej cov qauv zoo sib xws rau cov ntawv tso cai Gardasil-9 tshuaj tiv thaiv HPV [40]. Txhua pawg nas tau sib npaug ntawm poj niam txiv neej (txiv neej n=4 thiab poj niam n=4 ib pab). Hauv pawg A thiab B, BALB / c nas tau txhaj tshuaj intramuscularly (im) nrog 10 µg ntawm L1: P18I10 lossis L1: T20 VLPs, feem, los ntawm kev ua raws li homologous prime-boost regime. Hauv pab pawg C, cov nas tau inoculated nrog 106 cfu ntawm BCG.HIVA2auxo.int intradermally (id, ntawm cov khoom noj khoom haus) thiab txhawb nqa nrog 10 µg ntawm L1: P18I10 VLPs intramuscularly. Hauv pab pawg D, cov nas tswj tau zoo tau inoculated nrog Gardasil - 9 prime tom qab Gardasil -9 boost intramuscularly nrog 10 µg ntawm HPV16 L1 VLPs. Hauv pab pawg E, cov nas tswj tsis zoo tau txhaj tshuaj tiv thaiv ob zaug nrog PBS tsis. Lub sijhawm tseem ceeb ntawm kev txhawb nqa yog 2 lub lis piam. Cov nas tau muab txi rau hnub 28. Cov ntshav tau sau los ntawm nas lub siab. Sera tau rov qab los ntawm centrifugation thiab khaws cia ntawm −20 ◦C rau ELISA kev soj ntsuam. Murine spleens raug tshem tawm thiab nias ib tus zuj zus los ntawm lub xov tooj ntawm tes (Falcon) nrog 5 mL syringe roj hmab plunger. Tom qab tshem tawm cov qe ntshav liab nrog ACK lysing buffer (Lonza), splenocytes raug ntxuav thiab rov ua dua hauv lymphocyte nruab nrab R10 (RPMI 1640 ntxiv nrog 10% fetal calf serum (FCS), penicillin-streptomycin, 20 mM HEPMES thiab 15. 46}}ME) ntawm qhov concentration ntawm 2 × 107 cell / mL.

2.12. Enzyme-Linked Immunosorbent Assay

Txhawm rau kuaj HPV16 L1- thiab HIV-1- cov tshuaj tiv thaiv tshwj xeeb khi rau chimeric HPV: HIV VLP tsim hauv vitro, 50 µL ntawm qhov sib npaug (200 ng / mL) ntawm recombinant HPV16 L1 protein (Abcam, ab119880 ), purified L1:P18I10 thiab L1:T20 VLPs hauv 50mM carbonate bicarbonate tsis (pH=9.6) (Sigma) tau 2-fold serially diluted thiab coated rau Maxisorb daim hlau (Nunc). Cov phiajcim tau muab tso rau ntawm 4 ◦C thaum hmo ntuj. Cov phaj raug thaiv nrog qhov thaiv thaiv (5% skim mis nyuj hauv TBST) ntawm 37 ◦C rau tsawg kawg 2 teev. Tom qab ntxuav ob zaug nrog TBST, VLP-coated daim hlau tau incubated nrog anti-HPV16 L1 CAMVIR-1 mAb ntawm ib tug dilution ntawm 1:8000, 2F5 mAb (NIBSC, ARP3063) ntawm ib tug dilution ntawm 1:8000 thiab anti- HIV-1 gp120 V3 loop mAb (NIBSC, EVA3012) ntawm dilution ntawm 1:40 hauv kev thaiv qhov tsis sib haum, feem, ntawm 37 ◦C rau 2 h. Tom qab ntxuav peb zaug nrog TBST, cov phaj tau incubated nrog recombinant protein G peroxidase conjugate (Thermo Scientific, Waltham, MA, USA) ntawm ib tug dilution ntawm 1:4000 nyob rau hauv thaiv tsis pub ntawm 37 ◦C rau 1 h. TMB tau siv los tsim cov enzyme-linked immunosorbent assay (ELISA) teeb liab thiab nres nrog 50 µL ntawm 2M H2SO4. Optical density (OD) ntawm txhua qhov dej tau ntsuas thiab sau tseg ntawm lub wavelength ntawm 450 nm los ntawm kev siv EL × 800 absorbance microplate nyeem ntawv.

Txhawm rau ntsuas VLP-induced antibodies nyob rau hauv BALB/c nas, cov microtiter daim hlau tau coated nrog 50 µL ntawm 2 µg/mL recombinant HPV16 L1 protein (Abcam, ab119880), HIV-1 P18I10 peptide (NIBSC, ARP734), T20 peptide (NIBSC, ARP984), feem, nrog 50 mM carbonate-bicarbonate buffer (pH=9.6). Cov phiajcim tau muab tso rau ntawm 4 ◦C thaum hmo ntuj. Cov phaj raug thaiv nrog qhov thaiv thaiv (5% skim mis nyuj hauv TBST) ntawm 37 ◦C rau tsawg kawg 2 teev. Nyob rau tib lub sijhawm, sera sau los ntawm pab pawg AE txhaj tshuaj tiv thaiv nas tau diluted nrog 5% skim mis nyuj hauv TBST ntawm qhov sib piv ntawm 1: 50. Tom qab ntxuav ob zaug nrog TBST, cov phiajcim tau muab tso rau hauv qhov dej ntawm 37 ◦C rau 2 teev. Tom qab ntxuav peb zaug nrog TBST, cov phaj tau ntxiv nrog recombinant protein G HRP conjugate ntawm ib tug dilution ntawm 1:4000 nyob rau hauv thaiv tsis pub thiab incubated ntawm 37 ◦C rau 1 h. TMB tau siv los tsim ELISA teeb liab thiab nres nrog 50 µL ntawm 2M H2SO4. Lub OD ntawm txhua qhov dej tau ntsuas ntawm lub wavelength ntawm 450 nm los ntawm kev siv EL × 800 absorbance microplate nyeem ntawv (Biotek).

2.13. IFN- ELISpot Assay

Kev soj ntsuam enzyme-linked immune absorbent spot (ELISpot) tau ua los ntawm cov khoom siv murine IFN- ELISpot (Mabtech, Nacka Strand, Sweden), raws li cov chaw tsim khoom cov lus qhia. ELISpot daim hlau (MSISP4510, 96-cov phiaj zoo nrog polyvinylidene difluoride membranes, Millipore, Middlesex County, MA, USA) tau 70% EtOH kho thiab coated nrog purified anti-nas interferon- (IFN- ) ntes monoclonal antibody diluted hauv phosphate-buffered saline (PBS) mus rau qhov kawg siab ntawm 5 µg / mL ntawm 4 ◦C thaum hmo ntuj. Tom qab ntawd, 2.5 × 105 tshiab splenocytes tau ntxiv rau txhua qhov dej. Tom qab ntawd, cov hlwb los ntawm pawg A thiab B tau txhawb nqa nrog 2 µg/mL ntawm HPV16 L1 VLPs thiab HIV-1 P18I10 peptides, raws li. Tag nrho cov qauv thiab kev tswj hwm tau plated hauv cov qhov dej sib npaug. ELISpot kev soj ntsuam tau tsim rau 16 teev ntawm 37 ◦C, 5% CO2. Cov phaj tom qab tau ntxuav 5 × nrog PBS, incubated rau 2 h nrog biotinylated anti-IFN- monoclonal antibody (mAb) diluted hauv PBS 2% Fetal Calf Serum (FCS) mus rau qhov kawg concentration ntawm 2 µg / mL, ntxuav 5 zaug. hauv PBS, thiab incubated nrog streptavidin-alkaline phosphatase conjugate hauv PBS 2% FCS. Tom qab ntawd, daim hlau raug ntxuav 5 zaug nrog PBS ua ntej incubating nrog 100 µL ntawm 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate tov (Sigma-Aldrich , St. Louis, MO, USA). Tom qab 5-10 feeb, cov phiaj tau ntxuav nrog dej kais, qhuav, thiab cov txiaj ntsig tau suav nrog siv ELISPOT nyeem ntawv (AID, Autoimmun Diagnostika GmbH, Strasberg, Lub teb chaws Yelemees). Rau txhua tus tsiaj, lub ntsiab lus ntawm cov lus teb tom qab raug rho tawm ib tus zuj zus los ntawm tag nrho cov qhov dej kom muaj kev sib piv ntawm IFN-spot forming cells (SFC)/106 ntawm pawg. Txhawm rau txheeb xyuas cov lus teb zoo, qhov pib tau txhais tias tsawg kawg tsib qhov chaw hauv ib qhov dej, thiab cov lus teb ntau tshaj qhov nruab nrab ntawm cov chaw hauv qhov dej tswj tsis zoo ntxiv rau peb qhov kev sib txawv ntawm qhov dej tswj tsis zoo.

2.14. Kev txheeb cais

Txhua qhov kev txheeb cais tau ua tiav siv Prism 6 GraphPad software (CA, USA). Peb tau siv cov ntaub ntawv los ntawm kev sim ua ntau dua 5 qhov sib txawv ELISA phaj txheej txheej ntau (0, 50, 100, 150, 200 ng/mL) ntawm recombinant HPV16 L1 protein (Abcam, ab119880) thiab HPV: HIV VLPs. Peb tau sau cov ntaub ntawv los ntawm 2 pawg (HPV16 L1 thiab HPV: HIV VLPs) thiab sau peb qhov rov ua dua rau txhua txheej txheej. Daim duab hauv Prism pom cov ntaub ntawv raws li ib qho scatterplot qhia txheej txheej concentration (ng / mL) ntawm X-axis thiab OD450 ntawm Y-axis. Txij li thaum peb xav tias peb cov ntaub ntawv hauv vitro ELISA muaj feem cuam tshuam rau hauv cov qauv kab, peb tau ua qhov kev txheeb xyuas kab rov tav thiab muab piv rau cov kab nqes ntawm ob kab kom paub meej tias cov ntaub ntawv teeb tsa -1 thiab cov ntaub ntawv teeb -2 (antibody reactivity ntawm HPV16 L1 thiab HPV: HIV VLPs) txawv ntawm lawv cov kev ua. Peb xaiv 95% kev ntseeg siab ib ntus nrog rau txoj kab zoo tshaj plaws. Prism cia li overlaid linear regression kab ntawm ob peb cov ntaub ntawv teev thiab nws tau npaj ib tug dotted kab sawv cev rau 95% kev ntseeg siab intervals. Thaum lub sij hawm linear regression tsom xam, lub software xam tsuas yog qhov nruab nrab Y tus nqi ntawm peb cov ntaub ntawv teev uas qhia qhia qhov zoo ntawm haum. Tsis tas li ntawd, Prism tau muab cov lus qhia rau peb, yog li peb tuaj yeem xaus tias qhov sib txawv ntawm ob txoj kab nqes yog qhov tseem ceeb heev.

Peb muaj 5 pawg (ELISA) thiab 4 pawg (ELISPOT) ntawm cov ntaub ntawv sau los ntawm kev sim tshuaj tiv thaiv nas. Cov ntaub ntawv no muaj tus lej sib npaug (txiv neej n=4 thiab poj niam n=4) hauv txhua pab pawg. Peb tau siv Gardasil-9- txhaj tshuaj tiv thaiv nas los ua pab pawg tswj hwm zoo thiab PBS-cov nas tiv thaiv kab mob ua pab pawg tswj tsis zoo. Cov ntaub ntawv ntawm peb cov kev sim tsim tsis sib xws los yog ua ke. Peb tau ua ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA) kom pom seb cov txhais tau li cas txawv. Peb thawj zaug kuaj peb cov ntaub ntawv haum rau Gaussian ib txwm faib. Peb pom tias qee pawg ntawm cov ntaub ntawv hauv ELISA qhov kev xeem tsis dhau Gaussian kev faib khoom ib txwm muaj. Yog li ntawd, peb tau ua ib qho kev txheeb xyuas tsis yog parametric ntawm cov ntaub ntawv no. Los ntawm qhov sib txawv, txhua pawg ntawm cov ntaub ntawv hauv ELISPOT qhov kev xeem dhau los ntawm Gaussian ib txwm faib kev xeem. Yog li, peb tau ua qhov parametric txheeb cais ntawm cov ntaub ntawv no. Cov theem tseem ceeb thiab kev ntseeg siab tau teem rau 0.05 (sib npaug rau 95% kev ntseeg siab ib ntus). p tus nqi qhia feem ntau qhov tshwm sim uas muaj qhov sib txawv ntawm pawg. Txij li thaum peb qhov kev txhawj xeeb tseem ceeb rau qhov kev sim no yog qhov txawv ntawm peb pawg, ntau qhov kev sib piv hauv Prism tau muab peb cov txiaj ntsig ntawm kev sib piv hauv txhua pab pawg nrog txhua pab pawg.

2.15. Cov lus hais txog Ethics

Rau rau yim lub lis piam BALB/c nas tau yuav los ntawm Envigo (ib lub tuam txhab Inotiv, Chicago, IL, USA) thiab pom zoo los ntawm cov tub ceev xwm hauv zos (Generalitat de Catalunya, qhov project naj npawb 11157) thiab Universitat Autònoma de Barcelona Ethics Committee. Cov tsiaj thwmsim tau nruj me ntsis raws li txoj cai tsiaj noj qab haus huv ntawm Generalitat de Catalunya. Tag nrho cov kev sim no tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees hauv cheeb tsam (Cov Txheej Txheem 43.19, Tsev Kho Mob de la Vall d'Hebron, Universitat Autònoma de Barcelona).

3. Cov txiaj ntsig

3.1. Tsim L1:P18I10 thiab L1:T20 Immunogens thiab Kev Ntsuam Xyuas ntawm HPV: HIV Protein Expression los ntawm Kev Siv 293F Kev Tshaj Tawm

Lub P18I10 peptide los ntawm HIV-1 Env thib peb qhov sib txawv domain (V3) lub voj thiab T20 peptide los ntawm HIV-1 Env membrane's proximal external region (MPER) tau muab tso rau hauv HPV16 L1 DE voj protein los tsim chimeric L1 P18I10 thiab L1:T20 immunogens. Lub chimeric L1:P18I10 thiab L1:T20 DNA coding sequences tau cloned rau hauv pcDNA3.1 (+) plasmid DNA qhia vector rau transient transfection hauv 293F hlwb (Daim duab 1A). Cov qauv monomer ntawm HPV16 L1, chimeric L1:P18I10, thiab L1:T20 capsid proteins tau kwv yees ua ntej siv SWISS-model server (Daim duab 1B). HPV16 loj capsid protein L1 (7cn2.1.R) raug xaiv los ua tus qauv tsim los tsim HPV16 L1, L1:P18I10, thiab L1:T20 capsid protein homology qauv. Piv nrog rau kev ua raws li kev khi P18I10 peptide nyob rau hauv txoj kev tshawb no dhau los [41], N rau C terminus lub ntsiab saw hlau kev taw qhia ntawm P18I10 peptide ntawm peb chimeric L1: P18I10 protein khiav sab xis mus rau sab laug (Daim duab 1B, nruab nrab thiab sab saum toj vaj huam sib luag), thiab nthuav tawm P18I10 sab chains tuaj yeem cuam tshuam nrog T cell receptors (Daim duab 1B, nruab nrab thiab hauv qab vaj huam sib luag). Piv nrog rau cov qauv ntawm HIV-1 fusion inhibitor peptide hauv daim ntawv tshuaj xyuas yav dhau los [42], qhov ntxig T20 peptide ntawm HPV16 L1 capsid protein yog nthuav tawm raws li kev tsim -helix-zoo li (Daim duab 1B, sab xis thiab sab saum toj vaj huam sib luag ), thiab nthuav tawm T20 sab chains tuaj yeem lees paub los ntawm B cell receptors (Daim duab 1B, sab xis thiab hauv qab vaj huam sib luag). Txij li thaum HPV16 L1 capsid proteins tuaj yeem sib sau ua ke rau hauv T=7 icosahedral particle nrog 72 pentameric capsomeres [43], cov zaub siab ceev ntawm P18I10 lossis T20 peptides rau sab nrauv ntawm chimeric HPV: HIV VLPs muaj peev xwm ua rau muaj peev xwm ua tau zoo. txhawm rau txhawb cov kab mob epitope tshwj xeeb hauv lub cev.

Figure 1. L1:P18I10 and L1:T20 immunogen design and construction of chimeric HPV: HIV VLPs by using 293F expression system. (A) The chimeric L1:P18I10 and L1:T20 DNA coding sequences were cloned into pcDNA3.1+ expression vectors, respectively, for transient transfection in 293F cells. (B) Prediction of monomer structures of HPV16 L1 and chimeric HPV: HIV capsid proteins by using the SWISS-model server. HPV16 major capsid protein L1 (7cn2.1.R) was selected as the structural template to build the HPV16 L1 (left panel), L1:P18I10 (middle panel), and L1:T20 (right panel) capsid protein homology modeling. Secondary structural elements of HPV16 L1 capsid protein are labeled, with letters h1–h5 for the 5 α-helices. Loops of HPV16 L1 capsid protein between strands are labeled BC, CD, DE, EF, FG, and HI. Secondary structural elements of HIV-1 P18I10 and T20 peptides L1 are labeled by the arrows (top panel). The part of the P18I10 and T20 peptide that protrudes above the surface of the HPV16 L1 capsid protein is indicated by the arrows (bottom panel). (C) Immunofluorescence staining of L1 protein in 293F cells. The following pDNA.L1:P18I10 (left), pDNA.L1:T20 (middle), and pDNA.without insertion (right) were transfected in 293F cells. The transfected cells were probed with anti-HPV16 L1 mAb and detected with anti-mouse IgG-FITC (green channel). Cell nuclei were stained with DAPI (blue channel). Immunofluorescence images were merged by using Adobe Photoshop


Daim duab 1. L1:P18I10 thiab L1:T20 immunogen tsim thiab kev tsim kho ntawm chimeric HPV: HIV VLPs los ntawm kev siv 293F qhia qhov system. (A) Lub chimeric L1:P18I10 thiab L1:T20 DNA coding sequences tau cloned rau hauv pcDNA3.1+ qhia vectors, ntsig txog, rau transient transfection hauv 293F hlwb (B) Kev kwv yees ntawm cov qauv monomer ntawm HPV16 L1 thiab chimeric HPV: HIV capsid proteins los ntawm kev siv SWISS-model server. HPV16 loj capsid protein L1 (7cn2.1.R) raug xaiv los ua tus qauv qauv los tsim HPV16 L1 (sab laug vaj huam sib luag), L1: P18I10 (middle panel), thiab L1: T20 (txoj cai vaj huam sib luag) capsid protein homology qauv. Cov txheej txheem thib ob ntawm HPV16 L1 capsid protein tau sau npe, nrog cov ntawv h1–h5 rau 5 -helices. Loops ntawm HPV16 L1 capsid protein ntawm cov strands yog sau npe BC, CD, DE, EF, FG, thiab HI. Cov txheej txheem thib ob ntawm HIV-1 P18I10 thiab T20 peptides L1 tau sau los ntawm cov xub (saum toj kawg nkaus). Ib feem ntawm P18I10 thiab T20 peptide uas protrudes saum npoo ntawm HPV16 L1 capsid protein yog qhia los ntawm cov xub (hauv qab vaj huam sib luag). (C) Immunofluorescence staining ntawm L1 protein nyob rau hauv 293F hlwb. Cov nram qab no pDNA.L1:P18I10 (sab laug), pDNA.L1:T20 (nruab nrab), thiab pDNA.without insertion (txoj cai) tau kis nyob rau hauv 293F hlwb. Cov kab mob kis tau raug soj ntsuam nrog cov tshuaj tiv thaiv HPV16 L1 mAb thiab kuaj pom nrog cov tshuaj tiv thaiv nas IgG-FITC (ntsuab channel). Cell nuclei raug stained nrog DAPI (xiav channel). Cov duab Immunofluorescence tau sib koom ua ke los ntawm kev siv Adobe Photoshop

Txij li thaum HPV16 L1 protein C davhlau ya nyob twg ib ntus kho cov cellular nuclear ntshuam tshuab thaum kis kab mob [44], nuclear localization signals (NLS) ntawm HPV16 L1 protein tau raug txheeb xyuas hauv cov kev tshawb fawb ua ntej [45]. CAMVIR-1 monoclonal antibody raug xaiv los lees paub HPV16 L1 epitope (GFGAMDF, 230–236 aa) [39], thiab fluorescein-based dye FITC tau siv los ua tus tshaj tawm los saib xyuas qhov kev qhia ntawm L1:P18I10 thiab L1: T20 proteins. Immunofluo rpresenceimages qhia meej tias HPV16 L1 (hauv ntsuab) feem ntau nyob hauv lub nuclei (hauv xiav) ntawm 293F hlwb. Tsis muaj L1 teeb liab tau pom nyob rau hauv kev tswj plasmid-transected 293F hlwb (pcDNA3.1 plasmid tsis muaj ntxig) (Daim duab 1C). Cov txiaj ntsig tau qhia tias ob qho tib si chimeric L1: P18I10 thiab L1: T20 capsid proteins tuaj yeem qhia tau los ntawm kev siv polyethyleneimine (PEI)- mediated transfection thiab lees paub los ntawm HPV16 L1 CAMVIR-1 monoclonal antibody.

3.2. Purification ntawm L1: P18I10 thiab L1: T20 VLPs los ntawm Kev Siv Chromatographic Methods

The chimeric L1:P18I10 and L1:T20 proteins were produced by using the 293F expression system. The capture, intermediate purification, and polishing (CiPP) strategy to develop our chromatographic purification protocol is shown in Figure 2A. Flowthrough (FT) in each purification step was collected and the level of L1 protein expression was detected by Western blot analysis using anti-L1 mAb to trace intermediate HPV: HIV VLPs (Figure 2B, C). A cation exchange (CEC) column was selected as a capturing step to isolate HPV: HIV VLPs from host cell proteins (HCPs). The result of CEC FT revealed that most of the L1:P18I10 and L1T20 VLPs were lost in the FT over the CEC column (Figure 2B, C, lane 2). Traditional CEC matrices we used heavily rely on diffusion-limited mass transfer [46]. Large macromolecular complexes, such as our HPV: HIV VLP samples, might be inefficient for the VLP binding to CEC matrices. In order to reach the maximum binding capacities of the CEC column (~50 mg protein/mL resin), we loaded a double amount of soluble cell lysate containing approximately 2% of HPV: HIV VLPs into the CEC column. In the intermediate purification step, HPV: HIV VLPs were purified using a layered-bead size exclusion chromatography (SEC) resin [38]. Large HPV: HIV particles (>700 kDa) were eluted while most of the small impurities were trapped in the beads (Figure 2B, C, lane 5). Due to heparin having a similar structure as DNA and possibly binding to positively charged peptides of conformational HPV16 L1 VLPs, we selected a heparin affinity chromatography (H-AC) as a polishing step to remove heterogeneous or closely related particles [47]. Analysis of densitometry from Western blot analysis and bovine serum albumin (BCA) assay confirmed that purity of L1:P18I10 and L1:T20 VLPs after diafiltration step was high, over 76% (Figure 2B, C, lane 10). The purified L1:P18I10 and L1:T20 VLPs were detected as a band in size of approximately 56 kDa and 58 kDa, respectively. We found that the commercial HPV16 L1 protein and our purified chimeric HPV: HIV VLPs (L1:P18I10 and L1:T20) share a similar protein pattern (a target band >50 kDa thiab heterologous qis band<50 kDa). These heterologous lower bands could be detected, especially, when we loaded SDS-PAGE gel with a high amount of chimeric HPV: HIV VLP samples (Figure 2B, C, lane 1 to 10). It is probably caused by proteolytic degradation or heterogeneous formation of L1 proteins. A similar pattern (2 bands) was also found in many previous HPV16 L1 purification studies [23,48–51]. These data demonstrated that the 293F expression system and chromatographic purification methods are feasible approaches to engineer chimeric HPV: HIV VLPs.

Figure 2. Purification and characterization of L1:P18I10 and L1:T20 VLPs. (A) Schematic process flowchart of L1:P18I10 and L1:T20 VLP purification by chromatography. (B, C) Western blot analysis of L1:P18I10 and L1:T20 VLP samples from each purification step. The signal of L1 in each purification step was characterized by Western blot analysis probed with anti-HPV16 L1 mAb. The arrow indicates the molecular weight of ~56 kDa of L1:P18I10 and ~58 kDa of L1:T20 proteins. Lane 1: clarified cell lysate (CCL); Lane 2: flow-through (FT) from cation exchange chromatography (CEC) sample loading; Lane 3: CEC eluate; Lane 4: FT from CEC 2M NaCl regeneration step; Lane 5: size exclusion chromatography (SEC) FT-1; Lane 6: SEC FT-2; Lane 7: FT from heparin affinity chromatography (H-AC) sample loading; Lane 8: H-AC eluate; Lane 9: FT from H-AC 2M NaCl regeneration step; Lane 10: 10-fold diafiltration.


Daim duab 2. Purification thiab characterization ntawm L1:P18I10 thiab L1:T20 VLPs. (A) Schematic txheej txheem flowchart ntawm L1: P18I10 thiab L1: T20 VLP purification los ntawm chromatography. (B, C) Western blot tsom xam ntawm L1: P18I10 thiab L1: T20 VLP cov qauv ntawm txhua kauj ruam purification. Lub teeb liab ntawm L1 nyob rau hauv txhua kauj ruam purification yog tus cwj pwm los ntawm Western blot tsom xam nrog anti-HPV16 L1 mAb. Cov xub qhia qhov hnyav molecular ntawm ~ 56 kDa ntawm L1: P18I10 thiab ~ 58 kDa ntawm L1: T20 proteins. Txoj kab 1: clarified cell lysate (CCL); Txoj kab 2: ntws-dhau (FT) los ntawm cation exchange chromatography (CEC) qauv loading; Txoj kab 3: CEC eluate; Txoj kab 4: FT los ntawm CEC 2M NaCl regeneration kauj ruam; Txoj kab 5: qhov loj me tsis suav nrog chromatography (SEC) FT-1; Txoj kab 6: SEC FT-2; Txoj kab 7: FT los ntawm heparin affinity chromatography (H-AC) qauv thauj khoom; Txoj kab 8: H-AC eluate; Txoj kab 9: FT los ntawm H-AC 2M NaCl regeneration kauj ruam; Txoj kab 10: 10-fold diafiltration.

3.3. In Vitro Stability thiab Self-Assembly ntawm L1:P18I10 thiab L1:T20 VLPs

Txhawm rau kom paub meej tias HPV purified: HIV VLPs pom zoo ib yam hauv vitro ruaj khov rau HPV16 L1 VLPs, peb tau ua qhov tsis txo qis SDS-PAGE los ntsuas cov disulfide cross-linking ntawm HPV: HIV capsid proteins (Daim duab 3A). Nws paub tias pH, ionic zog, kub [52], thiab redox ib puag ncig tag nrho cuam tshuam nrog disulfide bonds ntawm HPV16 L1 capsid proteins [53]. HPV L1 VLPs zoo li nws tus kheej sib dhos ntawm pH qis thiab siab ionic zog. Qhov siab tshaj plaws disassembly ntawm VLPs feem ntau yuav tsum tau raug rau ib tug siab concentration ntawm txo tus neeg sawv cev, xws li 5% 2-mercaptoethanol (2-ME) rau lub sij hawm ntev [54]. Thaum tsis muaj cov tshuaj txo qis 2-ME, tsuas yog ib feem me me ntawm HPV-16 L1, L1:P18I10, thiab L1:T20 protein hloov mus rau monomers nrog qhov pom tseeb molecular hnyav (MW) ntawm 55 kDa . Kwv yees li ntawm 70% ntawm L1 proteins yog disulfide bonded rau hauv loj dua dimers los yog pentamers, nrog kwv yees MW ntawm 110 kDa thiab 280 kDa (Daim duab 3A, kab 2, 4, thiab 6). Los ntawm qhov sib piv, yuav luag tag nrho ntawm HPV-16 L1, L1:P18I10 thiab L1:T20 cov proteins hauv cov khoom siv tsis sib xws tau tshwm sim cov qauv monomeric hauv cov tsis txo SDS-PAGE (Daim duab 3A, txoj kab 3, 5, thiab 7). Cov txiaj ntsig no tau qhia tias nyob rau hauv vitro ruaj khov ntawm purified L1: P18I10 thiab L1: T20 VLPs nthuav tawm cov qauv sib txuas sib txuas zoo sib xws li HPV16 L1 VLPs nyob rau hauv tib lub pH, ionic zog, thiab thermal tej yam kev mob. Cov purified VLPs zoo li tau tawg mus rau qib ntawm pentamers, trimers, thiab dimers tom qab raug rau lub sij hawm ntev mus rau siab concentrations ntawm txo cov neeg ua hauj. Cov ntaub ntawv no tau sib haum nrog cov kev tshawb fawb yav dhau los [53,54] Txawm li cas los xij, kev tshem tawm ntawm HPV: HIV VLPs tseem nyob deb ntawm kev ua tiav (monomers).

Figure 3. In vitro stability of L1:P18I10 and L1:T20 VLPs. (A) Disulfide cross-linking of L1:P18I10 and L1:T20 VLPs in non-reducing SDS-PAGE. The HPV16 L1 VLPs, purified L1:P18I10, and L1:T20 VLPs were mixed with Laemmli sample buffer in the absence or presence of 2-mercaptoethanol (2-ME), respectively, and analyzed by non-reducing SDS-PAGE. The position of the L1 monomer (55 kDa), L1 dimer (110 kDa), and L1 pentamer (280 kDa) are indicated by the arrow. Lane 1: protein molecular weight marker; Lane 2: HPV16 L1 VLP; Lane 3: HPV16 L1 VLP treated with 2-ME; Lane 4: L1:P18I10 VLP; Lane 5: L1:P18I10 VLP treated with 2-ME; Lane 6: L1:T20 VLP; Lane 7: L1:T20 VLP treated with 2-ME. (B) Molecular mass analysis of L1:P18I10 and L1:T20 VLPs. Assembled VLPs untreated with 2-ME (lanes 2, 4, and 6) were filtered out through 1000kDa molecular weight cutoff (MWCO) diafiltration devices (upper panel). Disassembled VLPs treated with 2-ME (lanes 3, 5, and 7) were filtered out through 100kDa MWCO centrifugal filter devices (lower panel). Retentates (R) were collected from filter device sample reservoirs, while the filtrates (F) were collected at the bottom of centrifuge tubes. The L1 protein signal was detected by using a dot blot probed with anti-HPV16 L1 mAb.


Daim duab 3. Nyob rau hauv vitro stability ntawm L1:P18I10 thiab L1:T20 VLPs. (A) Disulfide cross-linking ntawm L1:P18I10 thiab L1:T20 VLPs hauv kev tsis txo SDS-PAGE. Cov HPV16 L1 VLPs, purified L1:P18I10, thiab L1:T20 VLPs tau tov nrog Laemmli cov qauv tsis nyob hauv qhov tsis muaj lossis muaj cov 2-mercaptoethanol (2-ME), feem, thiab tshuaj xyuas los ntawm kev tsis txo qis. SDS-PAGE. Txoj hauj lwm ntawm L1 monomer (55 kDa), L1 dimer (110 kDa), thiab L1 pentamer (280 kDa) yog qhia los ntawm xub. Txoj kab 1: protein molecular hnyav marker; Txoj kab 2: HPV16 L1 VLP; Txoj kab 3: HPV16 L1 VLP kho nrog 2-ME; Txoj Kev 4: L1:P18I10 VLP; Txoj Kev 5: L1:P18I10 VLP kho nrog 2-ME; Txoj Kev 6: L1:T20 VLP; Txoj Kev 7: L1:T20 VLP kho nrog 2-ME. (B) Molecular loj tsom xam ntawm L1:P18I10 thiab L1:T20 VLPs. Sib dhos VLPs tsis kho nrog 2-ME (kab 2, 4, thiab 6) tau lim tawm los ntawm 1000kDa molecular hnyav txiav tawm (MWCO) cov khoom siv diafiltration (saum vaj huam sib luag). Disassembled VLPs kho nrog 2-ME (txoj kab 3, 5, thiab 7) tau lim tawm los ntawm 100kDa MWCO centrifugal lim cov cuab yeej (hauv qab vaj huam sib luag). Retentates (R) tau sau los ntawm cov khoom siv lim dej, thaum cov filtrates (F) tau sau rau hauv qab ntawm cov raj centrifuge. Lub teeb liab L1 protein tau kuaj pom los ntawm kev siv cov dot blot probed nrog anti-HPV16 L1 mAb.

To demonstrate that purified HPV: HIV proteins by chromatography are able to self-assemble to icosahedral particles, we further performed molecular mass analysis under the same reducing condition (5% 2-ME). The commercial HPV16 L1, purified L1:P18I10, and L1T20 proteins without reducing agent treatment were filtered out through 1000 kDa molecular weight cut-off (MWCO) diafiltration devices individually (Figure 3B, top panel). The L1 monomers (55 kDa), oligomers (110~200 kDa), or pentameric capsomers (280 kDa) were expected to pass through an ultrafiltration membrane retaining the integral VLPs (MW~20,000 kDa). The L1 signal of commercial HPV16 L1 proteins was detected in both retentates and filtrates. Most of the purified L1:P18I10 and L1T20 proteins formed large particles (>1000 kDa) thiab tau khaws cia hauv retentates. Tus qauv yog coincidence nrog cov ntaub ntawv uas tau pom nyob rau hauv tsis-txo SDS-PAGE. Txawm hais tias tag nrho cov pab pawg VLP tau kho nrog 2-ME tau pom nyob rau hauv ib pawg monomeric (~55 kDa) hauv qhov tsis txo qis SDS-PAGE (Daim duab 3A, txoj kab 3, 5 thiab 7). Txawm li cas los xij, cov kev txo qis VLPs tsis raug lim tawm los ntawm 100kDa ultrafiltration membranes (Daim duab 3B, hauv qab vaj huam sib luag). Cov txiaj ntsig no tau qhia tias chimeric HPV: HIV cov proteins muaj peev xwm sib sau ua ke rau cov khoom loj dua, tab sis qhov siab tshaj plaws ntawm VLPs disassembly rau hauv monomers yuav tsum tsis yog tsuas yog txo cov disulfide bonds tab sis kuj muaj lwm yam denaturing, xws li pH lossis ionic zog.

3.4. Morphological Characterization ntawm L1: P18I10 thiab L1: T20 VLPs

Transmission electron microscopy (TEM) tau siv los tshuaj xyuas qhov kev sib haum xeeb ntawm HPV16 L1 thiab HPV: HIV VLPs. Cov kab mob HIV-1 P18I10 thiab T20 peptides tau muab tso rau hauv DE loops ntawm HPV16 L1 protein, feem. Cov kev lag luam HPV16 L1 thiab chimeric HPV: HIV capsid proteins tuaj yeem ua rau nws tus kheej sib sau ua ke hauv vitro rau hauv ib qho VLPs hauv txoj kab uas hla ntawm kwv yees li 50-60 nm (Daim duab 4A-C, txoj cai vaj huam sib luag). Piv rau electron micrographs ntawm HPV16 L1 VLPs luam tawm nyob rau hauv cov kev tshawb fawb yav dhau los [55,56], cov qauv icosahedral ntawm HPV16 L1, L1: P18I10, thiab L1: T20 VLPs ntawm no yog qhov sib piv thiab tsis meej. Nws tuaj yeem raug ntaus nqi rau qhov tsis zoo stain reagent uas peb siv hauv txoj kev tshawb no. Hauv peb txoj kev tshawb fawb tsis ntev los no tau luam tawm hauv kev tshawb fawb yav dhau los [37] thiab lwm daim ntawv txuas ntxiv nyob rau hauv kev tshuaj xyuas cov phooj ywg (cov ntaub ntawv tsis pom), peb tau txais qhov zoo thiab kev daws teeb meem ntawm electron micrographs thaum poov xab- thiab baculovirus-derived L1: P18I10 VLPs (tib yam chimeric tsim) tau sib npaug hauv PBS thiab tsis zoo-stained nrog phosphotungstic acid (PTA). Vim peb siv VLP ntau lawm thiab purification systems nyob rau hauv txoj kev tshawb no, mammalian cell-derived L1:P18I10 thiab L1:T20 VLPs tau equilibrated nrog Tris-HCl thiab tsis zoo-stained nrog uranyl acetate. Txawm hais tias cov electron micrographs tuaj yeem txhim kho, peb tseem tuaj yeem soj ntsuam tus qauv pom tseeb tias feem ntau ntawm HPV16 L1, L1: P18I10, thiab L1: T20 capsid protein tuaj yeem sib sau ua ke rau hauv morphological VLPs hauv qab TEM npo (Daim duab 4A-C, sab laug vaj huam sib luag. ). Yeej, HPV VLPs raug tiv thaiv kev sib sau ua ke hauv cov ntsev siab [57]. Qee qhov kev kuaj pom tau ntawm HPV16 L1 thiab HPV: HIV VLPs hauv cov ntsev tsawg Tris-HCl tsis tuaj yeem pom hauv qab qhov loj me (Daim duab 4A–C, sab laug vaj huam sib luag). Los ntawm cov txiaj ntsig no, peb tau txiav txim siab tias kev hloov pauv ntawm ib nrab L1 DE lub voj kab sib txuas los ntawm kev tso cov kab mob HIV-1 P18I10 lossis T20 peptides tsis cuam tshuam rau lub cev ntawm HPV: HIV VLPs.

Figure 4. Electron micrographs of L1:P18I10 and L1:T20 VLPs. (A) Morphology of HPV16 VLPs. (B) Morphology of L1:P18I10 VLPs. (C) Morphology of L1:T20 VLPs. Purified VLPs were absorbed on UV-charged carbon-coated copper grids, and negatively stained with 2% uranyl acetate. Images were acquired under transmission electron microscopy. The bar represents 200 nm at magnification 59,000 (left panel) and 100 nm at magnification 135K (right panels), respectively


Daim duab 4. Electron micrographs ntawm L1:P18I10 thiab L1:T20 VLPs. (A) Morphology ntawm HPV16 VLPs. (B) Morphology ntawm L1:P18I10 VLPs. (C) Morphology ntawm L1:T20 VLPs. Purified VLPs tau nqus los ntawm UV-them carbon-coated tooj liab daim phiaj, thiab tsis zoo stained nrog 2% uranyl acetate. Cov duab tau txais nyob rau hauv kis tau tus mob electron microscopy. Lub bar sawv cev rau 200 nm ntawm magnification 59, 000 (sab laug vaj huam sib luag) thiab 100 nm ntawm magnification 135K (txoj cai panels), feem.

3.5. Kev nthuav qhia thiab Kev tshwm sim ntawm HPV-16 thiab HIV-1 Epitopes 

Txhawm rau kom paub meej tias kab mob HIV ua ntu zus-1 P18I10 lossis 2F5 epitopes tau nthuav tawm hauv chromatography-purified L1:P18I10 lossis L1:T20 VLPs, Western blot tsom xam thiab tsis ncaj ELISA tau ua ntej siv epitope-specific mAbs. Peb tau xaiv cov tshuaj tiv thaiv kab mob monoclonal uas paub zoo (mAb), raug xaiv CAMVIR-1, kom paub txog qhov kev txuag epitope (GFGAMDF, aa 230–236) ntawm HPV16 L1 protein [39,58]. Ib tsab ntawv tshaj tawm yav dhau los mAb tsom rau HIV-1 gp120 V3 voj epitope (RIQRGPGRAFVTIGK, aa308–322) tau raug xaiv los txheeb xyuas cov kab mob P18I10 epitopes (RGPGRAFVTI, aa311–320) [59]. Ntawm qhov tod tes, qhov dav neutralizing antibody (bnAb) lees paub HIV-1 gp41 2F5 epitope (ELDKWA) tawm tsam ntau yam kab mob HIV-1 hom raug xaiv rau T20 peptide characterization [ 60, 61] ib. Western blot assay probed nrog HPV16 L1 mAb qhia bands 55, 56, thiab 58 kDa sib raug rau HPV16 L1, L1: P18I10, thiab L1: T20 protein, ntsig txog (Daim duab 5A, B, sab laug). Qhov hnyav molecular (MW) ntawm L1: P18I10 protein zoo ib yam li HPV16 L1 protein (Daim duab 5A, sab laug). Cov qhab sib raug rau L1:T20 protein tau pom me ntsis siab dua HPV16 L1 protein, raws li tau kwv yees los ntawm cov amino acid ntxiv (Daim duab 5B, sab laug). Cov bands ntawm kwv yees li 56 thiab 58 kDa, sib xws rau L1: P18I10 thiab L1: T20 protein, tau kuaj pom nyob rau hauv Western blot assay probed nrog anti-HIV-1 gp120 V3 thiab 2F5 mAb, ntsig txog (Daim duab 5A, B, txoj cai ). Peb xav tias qhov hnyav molecular (MW) ntawm anti-2F5-stained L1:T20 protein yog raug thiab haum rau qhov xav tau 58 kDa (Daim duab 5B, txoj cai). Txawm li cas los xij, MW ntawm kev tiv thaiv HIV-1 gp120 V3-stained L1:P18I10 protein yog qis me ntsis (Daim duab 5A, sab xis). Qhov no tuaj yeem raug ntaus nqi rau cov qauv heterogeneous ntawm chimeric L1: P18I10 proteins. Txij li thaum lub epitope conformation yuav ploj mus nyob rau hauv lub denaturing mob nyob rau hauv SDS-PAGE. Yog li ntawd, peb tau ua ntxiv tsis ncaj ELISA los ua kom pom qhov conformational P18I10 peptide kom raug nthuav tawm ntawm peb cov chimeric L1:P18I10 VLPs (Daim duab 5E). Ntawm qhov tod tes, cov tshuaj tiv thaiv HIV-1 gp120 V3 voj antibody uas peb siv tau lees paub tag nrho HIV-1 V3 voj es tsis yog P18I10 epitope. Yog li ntawd, tag nrho cov anti-V3 teeb liab tau qis dua (Daim duab 5A, B, txoj cai panels). Txawm li cas los xij, cov txiaj ntsig no tau qhia tias cov kab mob HIV txuas ntxiv -1 P18I10 thiab T20 peptides tau nthuav tawm hauv HPV: HIV VLPs.

Figure 5. Presentation of HPV-16 and HIV-1 epitopes. (A, B) Sequential epitope detection of chimeric L1:P18I10 and L1:T20 VLPs. The purified L1:P18I10 and L1:T20 VLPs were analyzed by Western blot, using anti-HPV16 L1, anti-HIV-1 gp120 V3 and 2F5 mAb. The HPV16 L1 VLPs were used as a control. The molecular weight of the L1 (55 kDa), L1:P18I10 (56 kDa), and L1:T20 (58 kDa) proteins are indicated by the arrow. Lane 1: protein molecular weight marker; Lane 2: HPV16 L1 protein; Lane 3: L1:P18I10 protein; Lane 4: L1:T20 protein. (C, D) Binding of HPV16 L1 mAb to chimeric L1:P18I10 and L1:T20 VLPs. (E) Binding of anti-HIV-1 gp120 V3 mAb to chimeric L1:P18I10 VLPs. (F) Binding of HIV-1 2F5 mAb to chimeric L1:T20 VLPs. The line graph of indirect ELISAs was performed to detect the conformational epitopes of recombinant HPV16 L1, L1:P18I10, and L1:T20 VLPs bound to anti-L1, anti-HIV-1 gp120 V3 or 2F5 mAbs, respectively. Data are representative of three independent experiments. A simple linear regression test was done to compare the line difference of purified L1:P18I10 and L1:T20 VLPs with the standard curve of commercial L1 VLPs; ns: not significant; ** p < 0.01.


Daim duab 5. Kev nthuav qhia ntawm HPV-16 thiab HIV-1 epitopes. (A, B) Sequential epitope nrhiav pom ntawm chimeric L1:P18I10 thiab L1:T20 VLPs. Cov purified L1:P18I10 thiab L1:T20 VLPs tau txheeb xyuas los ntawm Western blot, siv tshuaj tiv thaiv HPV16 L1, tiv thaiv HIV-1 gp120 V3 thiab 2F5 mAb. HPV16 L1 VLPs tau siv los ua kev tswj hwm. Qhov hnyav molecular ntawm L1 (55 kDa), L1: P18I10 (56 kDa), thiab L1: T20 (58 kDa) cov protein yog qhia los ntawm xub. Txoj kab 1: protein molecular hnyav marker; Txoj kab 2: HPV16 L1 protein; Txoj kab 3: L1: P18I10 protein; Txoj kab 4: L1: T20 protein. (C, D) Kev khi ntawm HPV16 L1 mAb rau chimeric L1: P18I10 thiab L1: T20 VLPs. (E) Kev khi ntawm kev tiv thaiv kab mob HIV-1 gp120 V3 mAb rau chimeric L1:P18I10 VLPs. (F) Kev khi ntawm HIV-1 2F5 mAb rau chimeric L1:T20 VLPs. Cov kab graph ntawm indirect ELISAs tau ua los xyuas kom pom cov epitopes conformational ntawm recombinant HPV16 L1, L1:P18I10, thiab L1:T20 VLPs khi rau anti-L1, anti-HIV-1 gp120 V3 los yog 2F5 mAbs, feem. Cov ntaub ntawv yog tus sawv cev ntawm peb qhov kev sim ywj pheej. Ib qho yooj yim linear regression xeem tau ua los sib piv cov kab sib txawv ntawm purified L1: P18I10 thiab L1: T20 VLPs nrog tus qauv nkhaus ntawm kev lag luam L1 VLPs; ns: tsis tseem ceeb; ** p <0.01.

Tsis tas li ntawd, txhawm rau txiav txim siab seb puas muaj kab mob HIV {{{{50}}}}}} cov kab mob sib kis tau nthuav tawm ntawm HPV: HIV VLPs tuaj yeem txheeb xyuas los ntawm gp120 V3 thiab 2F5 neutralizing antibodies hauv vitro, peb tau ua qhov kev ntsuam xyuas tsis ncaj ELISA los kuaj. lub epitope-binding specification thiab reactivity. Raws li pom hauv daim duab 5C, D, HPV16 L1, L1:P18I10, thiab L1:T20 VLPs tau lees paub los ntawm kev tiv thaiv L1 mAb. Peb tau ua qhov kev ntsuam xyuas linear regression los sib piv txoj kab nqes ntawm txhua kab dilution. Cov txiaj ntsig tau qhia tias L1 epitope-binding tshwj xeeb ntawm L1:P18I10 lossis L1:T20 VLPs tsis txawv ntawm HPV16 L1 VLPs. Ntxiv mus, tiv thaiv kab mob HIV-1 gp120 V3 mAb tau khi L1:P18I10 VLPs, tab sis tsis yog HPV16 L1 VLPs (Daim duab 5E). Ntxiv rau, 2F5 mAb tuaj yeem lees paub L1:T20 VLPs, tab sis tsis yog HPV16 L1 VLPs (Daim duab 5F). Tom qab kev txheeb xyuas kab rov tav, qhov sib txawv ntawm gp120 V3 epitope-binding tshwj xeeb ntawm L1:P18I10 thiab HPV16 L1 yog qhov tseem ceeb (p <0.01%). 2F5 epitope-binding tshwj xeeb ntawm L1:T20 VLPs tau txawv txav ntawm HPV16 L1 VLPs (p <0.01%). Cov qauv no tau qhia tias hydrophobic cellular lipids tsis tsim nyog rau kev khi ntawm 2F5-neutralizing antibodies rau HPV: HIV VLPs hauv vitro. Txawm hais tias qhov reactivity ntawm anti-HIV-1 gp120 V3 thiab 2F5 neutralizing antibodies rau HPV: HIV VLPs kuj yog me me, kev khi ntawm anti-HIV-1 gp120 V3 thiab 2F5 mAb rau HPV: HIV VLPs tau zoo heev. epitope tshwj xeeb.

3.6. Immunogenicity ntawm L1:P18I10 thiab L1:T20 VLPs tom qab BALB/c Mice txhaj

Peb tau soj ntsuam HPV16- thiab HIV-1- cov tshuaj tiv thaiv kab mob tshwj xeeb tom qab txhaj tshuaj nas BALB/c nrog L1:P18I10 thiab L1:T20 VLPs. Lub sijhawm txhaj tshuaj tiv thaiv muaj nyob hauv daim duab 6A. Vim tias VLP-induced immunogenicity tom qab kev tswj hwm mucosal feem ntau tsis muaj zog dua li kev tswj hwm lub cev, nas tau txhaj tshuaj intramuscularly nrog ib-rau ntawm Gardasil -9 HPV16 L1 koob [22,62]. Aluminium hydroxy phosphate sulfate adjuvant rau ib koob tshuaj (10 µg / 100 µL) ntawm chimeric HPV: HIV VLPs raug kho rau tib qhov concentration (1 mg / 1 mL) raws li Gardasil-9. Txhawm rau ntsuas qhov sib txawv ntawm kev sib deev hauv qhov tshwm sim ntawm kev txhaj tshuaj, kev sib piv ntawm cov tshuaj tiv thaiv kab mob ntawm cov txiv neej (n=4) thiab poj niam (n=4) nas raug soj ntsuam. Hauv pab pawg L1:P18I10 VLP, L1:T20 VLP thiab Gardasil-9, cov tshuaj tiv thaiv HPV16 L1 cov tshuaj tiv thaiv kab mob ntawm cov nas poj niam nyob nruab nrab siab dua cov txiv neej nas (Daim duab 6B). 2 tawm ntawm 4 (50%) L1: P18I10 VLP-cov poj niam txhaj tshuaj tiv thaiv kab mob, 3 tawm ntawm 4 (75%) L1: T20 VLP-cov poj niam txhaj tshuaj thiab tag nrho (100%) Gardasil-cov poj niam txhaj tshuaj tiv thaiv kab mob elicited siab dua titer ntawm cov tshuaj tiv thaiv-L1. tshaj txiv neej nas. Anti-L1 cov lus teb raug ntxias los ntawm poj niam nas hauv Gardasil-9 pab pawg tau siab dua li txiv neej nas (p=0.0041) (Daim duab 6B). Ib qib qis heev ntawm cov tshuaj tiv thaiv kab mob L1 tau pom nyob rau hauv pawg BCG.HIVA priming thiab L1:P18I10 VLP boosting. Cov qauv no sib haum nrog cov kev tshawb pom yav dhau los piav qhia tias BCG feem ntau ua rau T-cell cov lus teb ntau dua li IgG ntau lawm [63].

Figure 6. Induction of humoral immune responses by L1:P18I10 and L1:T20 VLPs in BALB/c mice. The immunization schedule is depicted in (A) All mouse groups had equal gender distribution (male n = 4 and female n = 4). Groups A and B: homologous prime-boost immunization with 10 µg L1:P18I10 or L1:T20 VLPs intramuscularly (i.m.); Group C: priming with 106 cfu rBCG.HIVA intradermally (i.d.) and boosting with 10 µg L1:P18I10 VLPs i.m.; Group D: homologous prime-boost vaccination with Gardasil-9 containing 10 µg of HPV16 L1 VLPs i.m.; Group E: immunization twice with PBS buffer. The prime-boost interval was 2 weeks. The endpoint of this trial was on day 28. Sera were collected and diluted at a titer of 1:50 for ELISA assay. (B) HPV L1-specific IgG in male and female mice. (C–E) Epitope-specific IgG induced by L1:P18I10 and L1:T20 VLPs. ELISA was performed to analyze anti-L1, anti-P18I10, and anti-T20 IgG induced by BALB/c mice following different prime-boost combinations as described above. Data are shown as mean ± S.D. One-way ANOVA (nonparametric) test was done to compare differences between groups. OD: opticaldensity. Ns not significant; ** p < 0.01


Daim duab 6. Induction ntawm humoral immune teb los ntawm L1:P18I10 thiab L1:T20 VLPs hauv BALB/c nas. Lub sijhawm txhaj tshuaj tiv thaiv kab mob tau piav qhia hauv (A) Txhua pab pawg nas muaj kev sib npaug ntawm poj niam txiv neej (txiv neej n=4 thiab poj niam n=4). Pawg A thiab B: homologous prime-boost txhaj tshuaj nrog 10 µg L1: P18I10 lossis L1: T20 VLPs intramuscularly (im); Pawg C: priming nrog 106 cfu rBCG.HIVA intradermally (id) thiab boosting nrog 10 µg L1: P18I10 VLPs im; Pawg D: homologous prime-boost txhaj tshuaj tiv thaiv nrog Gardasil -9 muaj 10 µg ntawm HPV16 L1 VLPs im; Pawg E: txhaj tshuaj tiv thaiv ob zaug nrog PBS tsis. Lub sijhawm tseem ceeb ntawm kev txhawb nqa yog 2 lub lis piam. Qhov kawg ntawm qhov kev sim no yog hnub 28. Sera tau sau thiab diluted ntawm titer ntawm 1:50 rau ELISA kev soj ntsuam. (B) HPV L1-IgG tshwj xeeb hauv cov nas txiv neej thiab poj niam. (C–E) Epitope-specific IgG induced los ntawm L1:P18I10 thiab L1:T20 VLPs. ELISA tau ua los tshuaj xyuas cov tshuaj tiv thaiv-L1, tiv thaiv-P18I10, thiab tiv thaiv T20 IgG vim los ntawm BALB / c nas tom qab sib txawv prime-boost ua ke raws li tau piav qhia saum toj no. Cov ntaub ntawv qhia tau hais tias txhais tau tias ± SD Ib-txoj kev ANOVA (nonparametric) kuaj tau ua los sib piv qhov sib txawv ntawm pawg. OD: opticaldensity. Ns tsis tseem ceeb; ** p <0.01

Peb tau soj ntsuam seb cov nas txhaj tshuaj L1: P18110 thiab L1: T20 VLPs tuaj yeem ua rau HPV-16 L1- tshwj xeeb thiab HIV-1 cov tshuaj tiv thaiv kab mob tshwj xeeb hauv cov nas BALB/c. VLP-induced IgG antibodies nyob rau hauv murine sera tau ntsuas los ntawm ELISA coated nrog recombinant HPV16 L1 protein, P18I10, los yog T20 peptides, feem. Qhov sib txawv ntawm qhov sib txawv hauv L1-IgG tshwj xeeb ntawm cov ntshav titer ntawm 1:50 tau kuaj pom hauv Gardasil-9 pab pawg hauv kev sib piv nrog pawg tswj hwm PBS (p=0.0039). Cov lus teb los tiv thaiv L1 ntawm Gardasil-9, L1:P18I10, thiab L1:T20 VLP-cov tshuaj tiv thaiv nas tau zoo sib xws thiab tsis txawv txav (Daim duab 6C). BCG.HIVA2auxo.int prime thiab L1:P18I10 VLP boost nas elicited qib qis heev ntawm anti-L1 IgG. Cov txiaj ntsig no tau qhia tias HPV: HIV VLP-cov nas txhaj tshuaj tiv thaiv kab mob ua rau tib theem ntawm cov tshuaj tiv thaiv L1 IgG li Gardasil-9-cov nas txhaj tshuaj (Daim duab 6C). Txawm hais tias pawg L1: P18I10 VLP cov naj npawb zoo li muaj qhov sib txawv mus rau qib siab dua ntawm P18I10 epitope-specific IgG dua li lwm pab pawg txhaj tshuaj, cov kev sib txawv no tsis tseem ceeb (Daim duab 6D). Hauv qee qhov L1:P18I10 VLP-cov tshuaj tiv thaiv nas (4 tawm ntawm 8, 50%), ntau dua los tiv thaiv P18I10 cov tshuaj tiv thaiv kab mob tau pom zoo piv rau Gardasil-9-cov nas txhaj tshuaj. Xwb, kev tshuaj ntsuam T-test tau qhia tias qhov sib txawv ntawm pawg L1:P18I10 VLP thiab Gardasil-9 yog qhov tseem ceeb (p=0.005) (cov ntaub ntawv tsis qhia). Hauv pawg L1:T20 VLP, cov tshuaj tiv thaiv kab mob siab dua tiv thaiv T20 peptide tau kuaj pom piv rau Gardasil-9 pawg (p=0.0083). Raws li kev cia siab, anti-T20 titers tsis tuaj yeem kuaj pom hauv Gardasil-9, PBS, L1:P18I10 VLP, thiab BCG.HIVA prime ua ke nrog L1:P18I10 VLP boost pawg (Daim duab 6E). Lub titer ntawm T20 peptide-specific antibody yog qhov tsawg. Qhov no yuav yog vim: (1) T20 yog ib qho subdominant peptide; (2) elicitation ntawm MPER los yog 2F5 neutralizing antibodies yuav tsum peptide-lipid conjugates (Daim duab 6E). Zuag qhia tag nrho, peb cov txiaj ntsig tau pom tias L1:P18I10 thiab L1:T20 VLPs tuaj yeem ua rau HPV16- tab sis tsis muaj zog HIV-1- cov tshuaj tiv thaiv tshwj xeeb hauv cov nas.

Desert ginseng-Improve immunity (2)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Txhawm rau soj ntsuam HPV thiab HIV tshwj xeeb T-cell tiv thaiv kab mob hauv cov nas, peb tau ua raws li lub sijhawm txhaj tshuaj tiv thaiv hauv daim duab 7A. Tsis tas li ntawd, heterologous BCG.HIVA2auxo.int priming thiab L1:P18I10 VLPs boosting tau muab piv nrog homologous L1:P18I10 VLP prime thiab txhawb kev txhaj tshuaj los ntsuas qhov zaus ntawm qhov tshwj xeeb-HPV16 thiab HIV-1 T-cell immune teb. Tsis muaj qhov sib txawv ntawm Gardasil-9 thiab PBS pawg hais txog IFN- zais cia tom qab splenocyte stimulation nrog HPV16 L1 VLPs. Qhov sib txawv hauv L1- tshwj xeeb IFN- zais cia kuj tsis tseem ceeb ntawm L1:P18I10 VLP thiab Gardasil-9 pawg. Peb tau txiav txim siab tias qhov tsis muaj zog L1- tshwj xeeb IFN- zais cia yuav raug ntaus nqi rau qhov tsis tshua muaj siab (2 µg/mL) ntawm HPV16 L1 VLPs siv los ua stimuli. Cov pab pawg ntawm BCG.HIVA2auxo.int prime thiab L1:P18I10 VLP txhawb nqa ntau zaus ntawm IFN- secreting splenocytes thiab qhov sib txawv tseem ceeb hauv IFN- secretion tau pom thaum piv nrog nas txhaj tshuaj tiv thaiv Gardasil -9 pab pawg (p {{36). }}.0103). 3 tawm ntawm 8 nas (~38%) elicited qhov siab tshaj L1- IFN- teb (Daim duab 7B). Qhov no tej zaum yuav raug ntaus nqi rau qhov tsis paub meej adjuvanticity ntawm BCG raws li peb cov kev tshawb fawb yav dhau los [64–66]. Cov txiaj ntsig tshwm sim tshwm sim, txawm tias los ntawm cov tsiaj qus BCG, yog nyob rau hauv txoj kab nrog lub peev xwm ntawm rBCG derivatives los ua qhov muaj zog adjuvants rau cov tshuaj tiv thaiv tom ntej [64,65]. Hais txog HIV-1-cov lus teb tshwj xeeb T-cell, qhov siab tshaj plaws tag nrho ntawm IFN- qhov chaw tsim cov hlwb (SFC)/106 splenocytes tau pom nyob rau hauv BCG.HIVA2auxo.int primed nas piv rau cov nas tau txais Gardasil{{54} } cov tshuaj tiv thaiv lossis L1:P18I10 VLPs. Cov nas tau pib nrog BCG.HIVA thiab txhawb nqa nrog L1:P18I10 VLPs elicited ntau dua IFN- zais cia piv nrog cov nas uas tau txhaj tshuaj L1:P18I10 VLP homologous prime-boost (p=0.0268). Tsis tas li ntawd, pom tias muaj ntau dua IFN- zais cia hauv cov nas uas tau txhaj tshuaj L1:P18I10 VLPs piv nrog cov nas tau txais Gardasil-9 cov tshuaj tiv thaiv (p=0.0157) (Daim duab 7C). Raws li qhov xav tau, IFN- zais zais tsis pom hauv Gardasil-9 thiab PBS pawg. Cov txiaj ntsig no tau pom tias (1) L1:P18I10 VLPs tau tshem tawm HIV-1-T-cell tiv thaiv kab mob tshwj xeeb; (2) L1:P18I10 VLPs elicited HPV tshwj xeeb T-cell tiv thaiv kab mob cov lus teb thiab (3) BCG.HIVA2auxo.int tuaj yeem txhawb cov kab mob HIV-1-cov tshuaj tiv thaiv T-cell tshwj xeeb uas tau txais los ntawm L1:P18I10 VLP.

Figure 7. Induction of HPV16 and HIV-1 specific T cell responses by chimeric L1:P8I10 VLPs and BCG.HIVA in BALB/c mice. The immunization schedule is depicted in (A). All mouse groups had equal gender distribution (male n = 4 and female n = 4). Group A: homologous prime-boost immunization with 10 µg L1:P18I10 VLPs intramuscularly (i.m.); Group B: priming with 106 cfu rBCG.HIVA intradermally (i.d.) and boosting with 10 µg L1:P18I10 VLPs i.m.; Group C: homologous prime-boost vaccination with Gardasil-9 containing 10 µg of HPV16 L1 VLPs i.m.; Group D: immunization twice with PBS buffer. The prime-boost interval was 2 weeks. The endpoint of this trial was on day 28. Splenocytes were isolated for IFN-γ ELISpot assay. T-cell immune responses to HPV16 and HIV-1 were assessed ex vivo by IFN-γ ELISpot after splenocyte stimulation with HPV16 L1 VLP and P18I10 peptide. (B, C) HPV16 L1- and HIV-1 P8I10-specific T-cell responses elicited by L1:P8I10 VLPs and BCG.HIVA prime combined with L1:P18I10 VLP boost. Data are shown as median ± S.D. A one-way ANOVA test was done to compare differences between groups. Ns not significant; * p < 0.05.


Daim duab 7. Induction ntawm HPV16 thiab HIV-1 tshwj xeeb T cell teb los ntawm chimeric L1:P8I10 VLPs thiab BCG.HIVA hauv cov nas BALB/c. Lub sijhawm txhaj tshuaj tiv thaiv kab mob tau piav qhia hauv (A). Txhua pawg nas tau sib npaug ntawm poj niam txiv neej (txiv neej n=4 thiab poj niam n=4). Pawg A: homologous prime-boost txhaj tshuaj nrog 10 µg L1: P18I10 VLPs intramuscularly (im); Pawg B: priming nrog 106 cfu rBCG.HIVA intradermally (id) thiab boosting nrog 10 µg L1: P18I10 VLPs im; Pawg C: homologous prime-boost txhaj tshuaj tiv thaiv nrog Gardasil -9 muaj 10 µg ntawm HPV16 L1 VLPs im; Pawg D: txhaj tshuaj tiv thaiv ob zaug nrog PBS tsis. Lub sijhawm tseem ceeb ntawm kev txhawb nqa yog 2 lub lis piam. Qhov kawg ntawm qhov kev sim no yog hnub 28. Splenocytes raug cais tawm rau IFN- ELISpot assay. T-cell immune teb rau HPV16 thiab HIV-1 raug soj ntsuam ex vivo los ntawm IFN- ELISpot tom qab splenocyte stimulation nrog HPV16 L1 VLP thiab P18I10 peptide. (B, C) HPV16 L1- thiab HIV-1 P8I10-cov lus teb tshwj xeeb T-cell tau txais los ntawm L1:P8I10 VLPs thiab BCG.HIVA prime ua ke nrog L1:P18I10 VLP txhawb nqa. Cov ntaub ntawv tau qhia raws li qhov nruab nrab ± SD Ib qho kev xeem ANOVA tau ua tiav los sib piv qhov sib txawv ntawm pawg. Ns tsis tseem ceeb; p <0.05.

4. Discushion

Ob leeg HPV16 thiab HIV-1 yog kab mob sib deev thiab tam sim no yog qhov tseem ceeb ntawm ntau qhov kev tshawb fawb tshuaj tiv thaiv. Txawm hais tias cov tshuaj tiv thaiv kab mob HPV tau ua lag luam thiab HIV-1 kis tau zoo heev los ntawm kev kho mob tiv thaiv kab mob (ART) thiab PrEP, muaj txiaj ntsig zoo, nyab xeeb, thiab pheej yig chimeric HPV: tshuaj tiv thaiv kab mob HIV tiv thaiv ob tus kab mob yog qhov xav tau ceev. Hauv txoj kev tshawb no, (1) peb tau pom tias 293F qhia qhov system thiab cov txheej txheem chromatographic purification tuaj yeem yog txoj hauv kev ua tau los tsim thiab lim dej chimeric L1: P18I10 thiab L1: T20 VLPs; (2) peb tau lees paub tias qhov kev tso ntawm P18I10 lossis T20 peptides rau hauv DE voj ntawm HPV16 L1 capsid proteins tsis cuam tshuam rau hauv vitro stability, nws tus kheej sib dhos thiab morphology ntawm chimeric HPV: HIV VLPs; (3) P18I10 lossis T20 peptides sib txuas ua ke uas raug rau DE loops ntawm chimeric HPV: HIV VLPs tuaj yeem kuaj pom los ntawm kev tiv thaiv HIV-1 gp120 V3 thiab 2F5 neutralizing antibodies hauv vitro; (4) Lub chimeric L1:P18I10 thiab L1:T20 VLPs tuaj yeem ua rau HPV tab sis tsis muaj zog HIV-1-cov tshuaj tiv thaiv tshwj xeeb hauv BALB/c nas. Tsis tas li ntawd, kev tso cov kab mob HIV-1 P18I10 lossis T20 peptides rau hauv HPV16 L1 protein tsis cuam tshuam rau HPV16 L1-cov tshuaj tiv thaiv tshwj xeeb hauv vivo; (5) L1:P18I10 VLPs tuaj yeem ua rau HPV16 thiab HIV-1-cov lus teb tshwj xeeb T-cell; (6) BCG.HIVA prime thiab L1:P18I10 VLP txhawb nqa qhov siab tshaj plaws ntawm IFN- tsim splenocytes piv nrog L1:P18I10 VLPs homologous prime-boost hauv BALB/c nas. Cov kev tshawb pom no txhawb kev txhim kho ntxiv ntawm HIV-1 cov tshuaj tiv thaiv raws li rBCG thiab chimeric HPV: HIV VLPs. Tag nrho txhua yam, txoj kev tshawb no muab cov tswv yim hauv paus uas yuav tsim nyog los txhawb lub ntiaj teb kev siv zog los tsim cov tshuaj tiv thaiv kab mob VLP tshiab rau kev tswj hwm tus kab mob HPV thiab HIV.

Hauv txoj kev tshawb no, L1:P18I10 thiab L1:T20 VLPs tuaj yeem ua rau tib theem ntawm kev tiv thaiv HPV16 L1 khi cov tshuaj tiv thaiv raws li cov tshuaj tiv thaiv HPV Gardasil-9. Txawm li cas los xij, L1: P18I10 thiab L1: T20 VLPs tsuas yog txo qis ntawm P18I10 thiab T20 epitope tshwj xeeb HIV-binding antibody teb. Peb xav tias tej zaum yuav yog vim ELISA daim phiaj ntsuam xyuas tau coated nrog cov protein ntau HPV16 L1, HIV-1 P18I10 peptide, thiab HIV-1 T20 peptide, raws li. Cov murine anti-L1 cov tshuaj tiv thaiv raug ntxias los ntawm peb cov L1: P18I10 thiab L1: T20 VLPs tuaj yeem tsom ntau qhov sib txuas ntawm cov protein L1. Los ntawm qhov sib txawv, murine anti-HIV-1 cov tshuaj tiv thaiv uas tau tawm los ntawm peb cov L1:P18I10 thiab L1:T20 VLPs tsuas yog tsom rau ib qho HIV peptide (P18I10 lossis T20) ntawm HPV: HIV VLPs. Yog li ntawd, tag nrho qhov siab tshaj OD450 qhov tseem ceeb ntawm kev tiv thaiv kab mob HIV-1 khi cov tshuaj tiv thaiv tau qis dua li cov tshuaj tiv thaiv HPV16 L1.

Yog vim li cas peb xaiv HPV16 L1 capsid protein DE voj raws li qhov chaw nkag ua ntej ntawm HIV-1 immunogen xa mus rau kev tshawb fawb yav dhau los uas txhaj tshuaj tiv thaiv nas nrog bovine papillomavirus (BPV): HIV VLPs rau inducing antibody teb [20]. Nws paub tias cov epitopes nyob rau hauv qhov chaw-exposed DE thiab FG loops ntawm HPV L1 loj capsid proteins dominantly pab rau cov tshuaj tiv thaiv-induced cross-neutralizing antibodies [67]. Tsis tas li ntawd, nws tau pom los ntawm cov kev tshawb fawb yav dhau los hais tias kev tso cov kab mob HIV-1 MPER rau hauv BPV L1 DE voj tuaj yeem ua rau cov tshuaj tiv thaiv kab mob tsis zoo uas tshwj xeeb lees paub cov haiv neeg ntawm MPER hauv HIV-1 Env [20]. Txawm li cas los xij, tsis muaj cov ntaub ntawv tau pom tias qhov recombinant BPV L1:MPER VLP cuam tshuam rau kev tiv thaiv kab mob thiab kev tsis sib haum xeeb tiv thaiv BPV tom qab tso cov kab mob HIV-1 MPER rau hauv BPV L1 protein. Hauv peb txoj kev tshawb fawb siv HPV16 L1 VLPs raws li HIV antigen tus me nyuam vector, peb tau pom tias HIV -1 peptide insertion tsis tau hloov HPV L1 qauv, thiab hais tias chimeric HPV: HIV proteins tseem tuav lub peev xwm los tsim VLPs. Tsis tas li ntawd, peb tau ua lub tswv yim ntawm kev tiv thaiv kab mob los ua kom pom cov kev tiv thaiv kab mob sib piv ntawm tus kab mob HPV: HIV VLP tus neeg sib tw (peb) thiab cov tshuaj tiv thaiv VLP-raws li HPV pom zoo ( ntawv tso cai Gardasil -9). Peb cov ntaub ntawv tau tshaj tawm tias kev tso cov kab mob HIV -1 P18I10 los yog T20 peptide rau hauv chimeric HPV: HIV VLPs tuaj yeem cuam tshuam ib qho zoo sib xws ntawm HPV16 L1-cov tshuaj tiv thaiv tshwj xeeb, piv rau HPV Gardasil-9 tshuaj tiv thaiv. Hom kab mob tshwj xeeb los tiv thaiv HPV16 L1 khi cov tshuaj tiv thaiv tau pom nrog cov ntawv tso cai HPV Gardasil-9 VLP tshuaj tiv thaiv piv nrog cov tshuaj tiv thaiv kab mob HPV Gardasil-9 VLP cov tshuaj tiv thaiv tau ua raws li peb cov ntaub ntawv.

Qhov ntev thiab qhov chaw ntawm qhov pom HIV zoo tshaj -1 cov tshuaj tiv thaiv txawv teb chaws tau muab tso rau hauv HPV16 L1 VLPs thiab kev ruaj ntseg hauv vitro ntawm qhov tshwm sim chimeric HPV: HIV VLPs yuav tsum tau txheeb xyuas ua ntej txhaj tshuaj nas. Peb txoj kev tshawb fawb tam sim no tau pom tias qhov tso ntawm P18I10 lossis T20 peptides rau hauv HPV16 L1 protein tsis cuam tshuam rau hauv vitro stability, nws tus kheej sib dhos, thiab morphology ntawm chimeric HPV: HIV VLPs. Cov txiaj ntsig no tau pom zoo nrog cov kev tshawb fawb yav dhau los qhia tias kev tso cov kab mob HIV -1 MPER domain rau hauv BPV L1 DE voj kab sib lawv liag tsis cuam tshuam lub peev xwm ntawm BPV L1 capsid protein nws tus kheej sib sau ua ke rau VLPs [20]. Yeej, cov epitopes nyob rau hauv qhov chaw-exposed DE thiab FG loops ntawm HPV L1 capsid proteins dominantly pab mus inducing L1- tshwj xeeb cross-neutralizing antibodies [67]. Ntawm no, peb tau ua pov thawj tias kev tso cov kab mob HIV-1 P18I10 lossis T20 peptides rau hauv HPV16 L1 DE voj tsis cuam tshuam rau L1-cov tshuaj tiv thaiv tshwj xeeb los ntawm chimeric HPV: HIV VLPs tom qab txhaj tshuaj nas. Tsis tas li ntawd, tus kab mob HIV-1 P18I10 lossis T20 epitopes mus rau HPV16 DE loops ntawm chimeric HPV: HIV VLPs tau kuaj pom hauv vitro thiab yog cov tshuaj tiv thaiv kab mob hauv vivo. HPV16 L1 VLPs ua ib qho chaw muaj peev xwm rau kev nthuav tawm ntawm HIV -1 epitope ntawm kev txaus siab. Nyob rau hauv txoj kev tshawb no, peb tau ua tsis ncaj ELISA los ua kom pom qhov conformational P18I10 thiab T20 peptide uas nthuav tawm ntawm peb cov chimeric HPV: HIV VLPs. Nyob rau hauv lub neej yav tom ntej, lub cev tiv thaiv kab mob-electron microscopy kuj tseem yog ib txoj hauv kev ntxiv los ua kom pom P18I10 lossis T20 antigen cov qauv hauv zos thiab koom haum hauv HPV: HIV VLPs.

Self-assembly yog ib tug sawv cev Performance index ntawm in vitro stability ntawm HPV16 L1 VLPs. Nws paub tias pH, ionic zog, kub [52], thiab redox ib puag ncig tag nrho cuam tshuam nrog disulfide bonds ntawm HPV16 L1 capsid proteins [53]. Cov ua haujlwm dhau los ntawm papillomavirus VLPs qhia qhov tseem ceeb ntawm cov nyiaj disulfide rau L1 loj capsid nws tus kheej sib dhos [68,69]. Kev tsim tawm ntawm Disulfide qhia tau hais tias ntau dua cysteine ​​​​ntawm L1 capsid protein nyob rau hauv qhov tsim nyog geometry [53]. Cov disulfide cross-links txuas cov residues 175 thiab 428 (rau HPV16) uas ruaj khov HPV virions thiab VLPs [70]. Hauv txoj kev tshawb no, peb tau txheeb xyuas cov qauv sib txuas ntawm cov tshuaj tiv thaiv kab mob sib txuas raws li cov pov thawj tsis ncaj ncees los ua pov thawj peb cov L1: P18I10 thiab L1: T20 capsid proteins zoo li nws tus kheej sib sau ua ke hauv vitro. Nyob rau hauv daim duab 3A, peb pom tau hais tias purified L1: P18I10 thiab L1: T20 VLPs tau nthuav qhia zoo sib xws intermolecular disulfide cross-linking qauv li HPV16 L1 VLPs nyob rau hauv tib pH, ionic zog, thiab thermal tej yam kev mob. Nyob rau hauv daim duab 3B, peb tau qhia ntxiv tias purified L1:P18I10 thiab L1:T20 proteins muaj peev xwm ntawm nws tus kheej-assembling rau cov khoom loj (loj dua L1 pentamer MW 280 kDa) hauv vitro. Yog li ntawd, ua ke nrog cov morphological self-assembly VLP qauv (txawm hais tias tus qauv icosahedral tsis zoo heev) pom nyob rau hauv daim duab 4, peb xaus peb cov purified chimeric HPV: HIV VLPs yog ruaj khov nyob rau hauv vitro. Dhau li ntawm VLP stability, ntau lwm yam tseem ceeb uas yuav cuam tshuam rau immunogenicity ntawm chimeric HPV: HIV VLPs yuav tsum tau txiav txim siab, xws li immunogen insertion site ntawm txawv loops ntawm VLPs [71], koob tshuaj, prime-boost intervals, thiab kev tswj txoj kev [22] , lwm.

P18I10 peptides muab tau los ntawm HIV-1 gp120 V3 lub voj yog nthuav tawm hauv cov kab mob HIV los ntawm qhov loj histocompatibility complex (MHC-I) chav kawm I molecules [26]. CD8+, cytotoxic T lymphocytes (CTL), tuaj yeem paub txog MHC-I-restricted P18I10 antigens thiab tso tawm ntau yam cytokines, xws li IFN- txhawm rau tshem tawm cov kab mob HIV [72–75]. Recombinant viral lossis plasmid DNA yog cov tshuaj tiv thaiv zoo tsheb los nthuav qhia P18I10 peptides nyob rau hauv cov cell thiab induce P18I10-cov lus teb tshwj xeeb ntawm tes los ntawm txoj kev MHC-I [76–79]. Piv txwv li, kev sib koom ua ke ntawm DNA prime thiab hloov kho cov tshuaj tiv thaiv kab mob Ankara (MVA) boost yog txaus rau induction ntawm IFN- thiab CTL cov lus teb tawm tsam P18I10 epitope [79]. Ntawm qhov tsis sib xws, exogenous P18I10 peptides tsis tau nthuav tawm zoo rau CD8+ T-cells los ntawm MHC-I txoj hauv kev [80,81] thiab yuav tsum muaj kev koom tes ntawm cov neeg tsim nyog adjuvants [82–85] lossis antigen cov cab, xws li VLPs. Piv txwv li, lub immunogenicity ntawm hluavtaws P18I10 peptides ntxiv nrog adjuvants yog marginal vim tsis muaj T-helper determinants [82-85]. Nws tau tshaj tawm yav dhau los tias HIV-1 Gag VLPs [86], kab mob siab B nto antigen (HBsAg) VLPs [87], parvovirus VP2 VLPs [88], thiab papillomavirus L1 VLPs [17–19] tuaj yeem ua tus xa khoom vectors rau MHC-I-txheej txheem CTL epitope kev nthuav qhia hauv vivo. Txawm hais tias cov txheej txheem ntawm VLP-induced MHC-I-txheej txheem T-cell cov lus teb tseem tsis tau meej, cov qauv ntawm VLPs tuaj yeem tau txais txiaj ntsig endocytic uptake ntawm macrophages lossis dendritic hlwb, yog li nkag mus rau cytosol thiab tom qab ntawd nkag mus rau MHC-I txoj hauv kev [89, 90] ib. Tsis tas li ntawd, MHC-I-txheej txheem P18I10 kev txiav txim siab tau raug soj ntsuam los txhawb CD4+ tus pab T-cell teb los ntawm txoj kev MHC-II [91,92]. Hybrid BPV1 L1 VLPs tuaj yeem siv los ua cov neeg nqa khoom antigenic epitope txhawm rau txhawm rau kho tus kab mob tshwj xeeb CTL cov lus teb los ntawm MHC-I thiab MHC-II txoj hauv kev [17,19], muab lub tswv yim zoo rau kev tsim tshuaj tiv thaiv los tswj kev kis kab mob. Qee qhov kev tshawb fawb thaum ntxov kuj tau qhia txog BPV L1 VLPs qhia HPV16 E7 epitope lossis P18I10 epitope ntawm HIV-1 gp120 V3 voj-induced mucosal nto thiab tseem muaj VLP epitope-specific humoral thiab T cell tiv thaiv [18]. Raws li cov kev tshawb fawb yav dhau los, peb tau pom ua ntej tias peb cov kab mob HPV: HIV (L1: P18I10) VLPs tuaj yeem ua rau HIV tshwj xeeb T-cell tiv thaiv kab mob hauv BALB / c nas tom qab splenocyte stimulation nrog P18I10 peptide. Tsis tas li ntawd, BCG.HIVA priming tau txhim kho cov kab mob HIV-1- tshwj xeeb T-cell tiv thaiv kab mob hauv cov nas. Txawm li cas los xij, ntau txoj haujlwm T-cell tiv thaiv kab mob tshwm sim los ntawm L1: P18I10 VLPs yuav xav tau kev tshawb fawb txog kev tiv thaiv kab mob ntxiv.

Dav dav CD8+ T-cell cov lus teb tawm tsam ntau CTL epitopes tau txais txiaj ntsig zoo los kov yeej HIV-1 kev sib txawv ntawm caj ces thiab khiav [7,93–100]. Kev tsim qauv tsim nyog ntawm HIV-1 T-cell immunogens, xws li HIVA, yuav tsum muaj peev xwm los teb rau ntau CTL epitopes [34]. Tus kab mob HIV-1 HIVA immunogen, tsim los ntawm Dr. Tomas Hanke, yog tsim los ntawm tag nrho-ntev HIV-1 Gag protein ua ke nrog ntau CTL epitopes suav nrog P18I10 epitopes ntawm C-terminus [34]. Cov DNA, MVA, thiab rBCG tau raug xaiv los ua lub tsheb thauj neeg mob HIVA immunogen thiab ua rau muaj qhov loj thiab dav ntawm CTL epitope cov lus teb tshwj xeeb ntawm tes los ntawm kev siv heterologous prime-boost regimes hauv nas thiab tsis yog tib neeg primate (NHP) qauv [34,101]. Peb cov kev tshawb fawb ua ntej tau pom tias BCG.HIVA thawj zaug ua ke nrog MVA.HIVA txhawb nqa HIV-1- tshwj xeeb IFN- tsim CD8+ T-cells hauv BALB/c nas [31–33,102]. Interestingly, VLPs tuaj yeem yog qhov muaj peev xwm txhawb nqa kom muaj cov kab mob HIV tshwj xeeb hauv cov tshuaj tiv thaiv kab mob heterologous nrog rBCG [29,30] lossis tshuaj tiv thaiv DNA [103,104]. Piv txwv li, rBCG qhia txog HIV-1 Gag protein tuaj yeem ua haujlwm zoo rau T-cell tiv thaiv kab mob rau kev txhawb nqa nrog Gag VLPs hauv NHP qauv [29,30]. Hauv kev tshawb fawb tam sim no, peb tau pom tias BCG.HIVA priming tuaj yeem txhawb T-cell tiv thaiv kab mob los ntawm HPV: HIV (L1:P18I10) VLPs. Peb yuav tshawb xyuas ntxiv txog qhov loj ntawm polyfunctional CD4+, CD8+, thiab nco T-cell cov lus teb tsim los ntawm rBCG prime thiab VLP boost regime.

Cistanche deserticola-improve immunity -

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Tam sim no, peb pab pawg tshawb fawb tau tsom mus rau kev txhim kho kev cog lus rBCG: tshuaj tiv thaiv kab mob HIV qhia txog kab mob HIV tshiab-1 T-cell immunogens, xws li tHIVconsvX thiab HIVACAT (HTI) T-cell immunogen, txhawm rau txhim kho HIV-1 variant match thiab T-cell teb dav. Lub 2nd-tiam HIVconsvX immunogens tau tsim los ntawm redefining pab pawg M khaws cia cheeb tsam thiab siv ib tug bivalent mosaic tsim los ua kom qhov sib tw ntawm tej zaum 9-mer T-cell epitopes nyob rau hauv cov tshuaj tiv thaiv mus rau lub ntiaj teb no variants [64]. HTI immunogen tau tsim los npog T-cell lub hom phiaj tiv thaiv T-cell cov lus teb feem ntau pom hauv HIV-1-cov neeg kis tus kab mob HIV tsawg-1 cov kab mob kis [65,66]. Vim tias papilloma VLPs tau raug pov thawj tias yog ntau tus neeg nqa khoom CTL epitope [17], peb tsom mus tsim chimeric HPV16 L1 VLPs nqa ntau yam kev tiv thaiv kab mob HIV-1 CTL epitopes ua ke nrog rBCG nthuav qhia ThivconsvX lossis HTI T-cell immunogens los ua kom dav dua. CTL tshuaj tiv thaiv kab mob HIV-1. Lub siab ntom ntom thiab ntau daim ntawv ntawm tus kabmob hb -1 CTL EMV: HIV VLP: HIV VLP: HIV VLP: HIV VLP: HIV Los ntawm qhov sib piv, recombinant BCG muaj feem ntau yuav tsim kom muaj tsawg zaus ntawm CTL cov lus teb vim qeeb replication hauv vivo. Vim hais tias BCG muaj qhov txawv txav ntawm kev sib txawv ntawm T hlwb, uas txhais tau hais tias BCG tuaj yeem ua rau lub cim xeeb ntawm CD8+ T cells los ntawm kev koom tes ntawm CD4+ T-helper cells [105], cov cuab yeej immunogenic. Tej zaum yuav ua rau BCG haum raws li tus neeg sawv cev priming hauv heterologous prime-boost regimens. Yog li, peb xav tias peb cov HPV: HIV VLPs yuav tshwm sim los ua ib qho kev cog lus txhawb kom nce qhov dav thiab dav ntawm HIV-1 CTL cov lus teb thaum priming nrog ib tug recombinant BCG qhia tshiab HIV-1 T-cell immunogen .

Lub T20 peptide muaj ib qho kev tiv thaiv zoo heev linear epitope 2F5 (ELDKWA). 2F5 cov tshuaj tiv thaiv uas tau sau los ntawm cov neeg mob HIV mus ntev tau tshaj tawm tias muaj kev cuam tshuam dav dav [106,107]. MPER ntawm gp41 yog suav tias yog cov tshuaj tiv thaiv tsis zoo, tej zaum muaj feem xyuam rau nws qhov chaw nyob ze ntawm lub cellular thiab viral phospholipid bilayer [108]. Kev siv DNA vectors nthuav qhia MPER hauv ib puag ncig lipid yog qhov muaj txiaj ntsig zoo rau kev txhawb nqa gp41- tshwj xeeb nAbs [109–111]. Piv txwv li, HIV-1 T20-encoding DNA tshuaj tiv thaiv, tsim los ntawm Dr. Britta Wahren, tau raug pom los ua kom muaj cov tshuaj tiv thaiv kab mob sib kis (nAb) cov lus teb [109]. Los ntawm qhov sib txawv, ntau qhov kev sim ua ntej ua rau nAbs lub hom phiaj gp41 los ntawm kev siv peptide lossis subunit cov tswv yim tshuaj tiv thaiv tau ua tsis tiav [112–116]. Tsis ntev los no, qee qhov kev tshawb fawb tau qhia BPV L1 VLPs qhia 2F5 epitope lossis MPER ntawm HIV-1 gp41 induced 2F5-cov tshuaj tiv thaiv tshwj xeeb hauv cov nas ua rau muaj kev cuam tshuam ntawm cross-clade neutralization [20,21]. Ib qho kev tiv thaiv kab mob zoo sib xws tau pom thaum tus kab mob siab B nto antigen (HBsAg) tau fused nrog HIV-1 2F5 epitope lossis MPER [59,117–119]. Ntawm no, peb tau ua pov thawj tias qhov kev nthuav qhia ntawm HIV-1 T20 thiab P18I10 peptide hauv peb cov kab mob HPV16 L1 VLP tuaj yeem ua rau cov tshuaj tiv thaiv kab mob tiv thaiv kab mob HIV-1 thiab HPV16. Neutralizing epitopes stabilized nyob rau hauv ib tug conformational scaffold, xws li HIV -1 functional spikes los yog VLPs, yuav yog lub ntsiab ntawm B-cell immunogen tsim rau kev ua tau zoo broad neutralizing antibodies (bnAbs) [93]. Txawm li cas los xij, feem ntau cov tshiab bnAb epitopes (kwv yees li 90%) yog cov cheeb tsam tsis txuas ntxiv thiab tsim los ua ke nyob rau hauv 3-seem configurations [120]. Txawm hais tias qhov kev nthuav qhia ntawm cov epitopes tsis tu ncua mus rau ib qho protein scaffold tuaj yeem kwv yees los ntawm kev ua qauv piv txwv [121], cov bnAb epitopes tej zaum yuav raug sib tw kom muab tso rau hauv ib lub hnab tsis muaj HPV16 L1 protein scaffold. Los ntawm qhov sib txawv, ib haiv neeg tsawg ntawm HIV-1 B-cell immunogens, xws li MPER (2F5) ntawm HIV-1 gp41 lossis V3 voj (P18IIB) ntawm gp120, muaj cov kab nruab nrab nruab nrab ntawm cov epitopes thiab tej zaum yuav haum rau cov HPV: HIV protein qaum. Yog li ntawd, peb xaiv cov linear 2F5 neutralizing epitope uas muaj nyob rau hauv ib tug txuas ntxiv T20 peptide ntawm HIV-1 MPER nyob rau hauv cov nqe lus ntawm cov qauv zoo rau -helix tsim [122]. T20 peptides tuaj yeem fused thiab ruaj khov ntawm L1 capsid scaffolds kom tshem tawm cov lus teb rau cov tshuaj tiv thaiv kab mob yog tias qhov kev teeb tsa ib txwm muaj ntawm 2F5 epitope tuaj yeem nthuav tawm. Hauv txoj kev tshawb no, peb pom tias 2F5 nAbs raug khi rau chimeric HPV: HIV (L1:T20) VLPs hauv vitro. Ntxiv mus, L1:T20 VLPs tuaj yeem ua rau T20-cov tshuaj tiv thaiv tshwj xeeb khi rau hauv cov nas BALB/c. Muaj cov pov thawj loj hlob tuaj tias HIV-1 fusion inhibitory (T20) peptide-induced antibodies muaj cov khoom zoo xws li tiv thaiv HIV-1 fusion peptide Enfuvirtide los khi hydrophobic trans-membrane T20 residue nyob hauv MPER ntawm gp41 thaum lub sijhawm HIV-1 fusion thiab pab txhawb kev tiv thaiv kab mob [109,123,124]. Raws li peb cov kev txheeb xyuas yav dhau los, kev tsim qauv tsim nyog ntawm cov tshuaj tiv thaiv kab mob VLP los txhawb HPV thiab HIV cov tshuaj tiv thaiv kab mob tshwj xeeb thiab CTL cov lus teb yuav tsum tau txiav txim siab txog kev xaiv cov tshuaj tiv thaiv kab mob, tshuaj tiv thaiv kab mob, thiab cov txheej txheem tseem ceeb. Hauv kev xaus, txoj kev tshawb fawb no tau qhia txog lwm txoj hauv kev ntawm cov tsiaj txhu hauv cell-based qhia platform thiab ib txoj hauv kev ua kom muaj chromatographic purification txoj kev rau engineer chimeric HPV: HIV VLPs. Peb txiav txim siab tias peb txoj kev ua kom huv tshiab yuav pab kom rov qab tau cov tshuaj tiv thaiv kab mob HPV: HIV VLPs los ntawm cov kab mob mammalian nrog lub hom phiaj ntawm kev txo lub sij hawm, nqi, thiab kev ua haujlwm thaum ua kom muaj peev xwm rau kev tsim khoom lag luam. kev xa khoom platform los nqa HIV-1 peptide antigen vim tias qhov ntxig ntawm P18I10 lossis T20 peptides rau hauv DE voj ntawm HPV16 L1 capsid proteins tsis cuam tshuam rau hauv vitro stability, nws tus kheej sib dhos, thiab morphology ntawm chimeric HPV: HIV VLPs thiab tau ua. tsis cuam tshuam HPV16 L1-cov tshuaj tiv thaiv tshwj xeeb induction hauv vivo. Ntawm qhov tod tes, chimeric HPV: HIV VLPs tuaj yeem cuam tshuam HPV16 thiab HIV tshwj xeeb B thiab T-cell tiv thaiv kab mob tawm tsam ob tus kab mob. Daim ntawv tshaj tawm no tau tshawb nrhiav qhov muaj peev xwm tsim cov tshuaj tiv thaiv kab mob HIV{111}} raws li cov tshuaj tiv thaiv kab mob BCG uas qhia txog HIV-1 immunogens thiab HPV: HIV VLPs, uas tuaj yeem siv rau kev txhaj tshuaj tiv thaiv menyuam yaus HIV{113}}. Txij li thaum kev txhim kho cov tshuaj tiv thaiv kab mob zoo tiv thaiv HPV16 thiab HIV-1 tseem yog ib qho kev sib tw, txoj haujlwm no txhawb nqa ib kauj ruam mus rau kev txhim kho cov tshuaj tiv thaiv kab mob HPV tshiab: HIV VLP-based tshuaj tiv thaiv platform rau tswj HPV16 thiab HIV{118 }} kab mob, uas yuav tsum tau ceev nrooj nyob rau hauv cov teb chaws tsim thiab industrialized.

Cov ntaub ntawv

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