Multi-component Prime-boost Chlamydia Trachomatis Vaccination Regimes Induce Antibody Thiab T Cell Responses Thiab Accelerate Clearance Of Infection in A Non-Human Primate Model Ⅱ

Cov ntaub ntawv thiab cov txheej txheem

Tshuaj tiv thaiv antigens thiab adjuvants

Cov tshuaj tiv thaiv sib txawv antigens thiab adjuvants tau siv rau hauv txoj kev tshawb no: CTH522 tshuaj tiv thaiv antigen, raws li MOMP (11) tau tswj hwm los ntawm txoj kev intramuscular (IM) nrog CAF01 adjuvant (42) lossis Aluminium hydroxide (AlOH) adjuvant, lossis los ntawm txoj kev intranasal ( IN) tsis muaj kev sib tw. Plasmid DNA tshuaj tiv thaiv, pcDNA3.1-MOMP, tau tsim los ntawm cloning kev pom zoo MOMP rau hauv plasmid vector pcDNA3.1 (14) thiab tswj los ntawm intra-dermal (ID) txoj hauv kev nrog electroporation (EP) hauv qab. Recombinant Tib Neeg Adenovirus serotype 5 (rHuAd5) tshuaj tiv thaiv qhia MOMP (HuAd5-MOMP) tau tsim los ntawm homologously recombining pom zoo MOMP (AAC45154.1) rau hauv E1 thiab E2 tshem tawm HuAd5 plasmid pAL11112 hauv vector thiab kev tswj hwm . Hloov kho cov tshuaj tiv thaiv Ankara-MOMP tshuaj tiv thaiv los ntawmrecombining kev pom zoo MOMP(AAC45154.1) rau hauv p3186 plasmid thiab attenuated MVA vector (14), thiab tswj los ntawmtxoj kev intramuscular.

Nyem qhov no Kom Tau Cistanche Rau Anti-inflammatory

C. trachomatis serovar D stock

Chlamydia trachomatis serovar D (Ct SvD; UW{{{0}}}/Cx, ATCC® VR-885™) tau txais los ntawm American Type Culture Collection (ATCC, Manassas, VA) thiab nthuav tawm hauv HeLa{ {2}} hlwb, sau, thiab purified raws li piav los ntawm Caldwell li al. (43). C. trachomatis SvD Tshuag tau khaws cia ntawm -80 degree hauv 0.2 M sucrose, 20 mM sodium phosphate (pH 7.4), thiab 5 mM glutamic acid (SPG) thiab cov concentration ntawm inclusions forming units (IFUs) tau txiav txim siab hauv HeLa{11}} hlwb thiab McCoy hlwb.

Tsis yog tib neeg primates

Peb caug cynomolgus macaques (Macaca fascicularis) hnyav 3 txog 5 kg tau suav nrog hauv txoj kev tshawb fawb txog biosafety qib 3 (ABSL3) NHP cov chaw ntawm IDMIT ("Cov Qauv Kab Mob thiab Kev Kho Tshiab" ntawm CEA "Commissariat à l'Energie Atomique," Fontenay-aux-Roses, Fabkis; daim ntawv pov thawj no. #D92- 032-02). Cov kev cai raug pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees "Comité d'Ethique en Expé rimentation Animale du Commissariat à l'Energie Atomique et aux Energies Alternatives" (CEtEA #44) raws li nqe lus A14-026. Txoj kev tshawb no tau tso cai los ntawm "Kev Tshawb Fawb, Kev Tsim Kho Tshiab, thiab Kev Kawm Txuj Ci" raws li tus lej APAFIS#720-201505281237660. Txhua tus tsiaj tau kuaj pom tsis zoo rau SIV, SHIV, STLV (simian Tlymphotropic virus), herpes B virus, filovirus, SRV-1, SRV-2 ua ntej txoj kev tshawb fawb thiab tau lees paub tias yog tus kab mob chlamydia. Tag nrho cov txheej txheem sim tau ua raws li European cov lus qhia rau kev saib xyuas tsiaj ("Journal Officiel de l'Union Europé enne", directive 2010/63/ UE, September 22, 2010).

Kev txhaj tshuaj tiv thaiv kab mob

30 macaques tau muab faib ua 6 pawg nrog 5 tus tsiaj hauv txhua tus. Qhov sib txawv tshuaj tiv thaiv prime-boost regimes rau 6 pawg yog piav qhia nyob rau hauv daim duab 1. Thaum lub sij hawm tag nrho cov tshuaj tiv thaiv, cov macaques tau sedated nrog ketamine chlorhydrate (10 mg / kg lub cev hnyav intramuscularly). CTH522 (85 µg ib tus tsiaj) tau tswj hwm los ntawm txoj kev intramuscular (IM) nrog CAF {{20}}1 adjuvant (625 µg / 125 µg) lossis AlOH adjuvant (0.425 mg. ), raws li pom hauv daim duab 1. Kev txhaj tshuaj IM tau ua nyob rau hauv 0.6 ml ntim ntawm tus ncej sab xis (M. quadriceps) ntawm tus tsiaj. CTH522 Intranasal (IN) txhaj tshuaj (30 µg ib tus tsiaj) tau ua nrog Vaxinator ntaus ntawv txuas nrog 1 ml syringe nrog ob txoj kev tswj hwm ntawm 0.25 ml, ib qho hauv txhua lub qhov ntswg. Cov tshuaj tiv thaiv DNA-MOMP tau muab tshuaj los ntawm txoj kev intradermal (ID) nrog electroporation (EP) hauv daim tawv nqaij ntawm sab nraub qaum. 6 rov qab ntim ntawm 0.1 ml tau txhaj ID (hauv tag nrho 1 mg) ua raws li EP siv lub Nepagen™ system (chaw: 6 square-ware pulses ntawm 10 ms nrog 90 ms intervals ntawm 110V, (300- 700mA)) . Cov tshuaj tiv thaiv hAd5-MOMP tau txhaj tshuaj IM nrog koob tshuaj 1011 kab mob hauv 1 ml rau ib tus tsiaj. Cov tshuaj tiv thaiv MVA-MOMP tau muab IM nrog ib koob ntawm 4 * 108 PFU rau ib tus tsiaj hauv 0.5 ml ntim **. Pab pawg neeg tsis ncaj ncees tsis tau txhaj tshuaj tiv thaiv thiab raug suav tias yog pawg tswj hwm.

Qauv sau

Thaum tag nrho cov txheej txheem kuaj, cov macaques tau sedated nrog ketamine chlorhydrate (10 mg / kg lub cev hnyav IM). Cov ntshav tau sau los ntawm femoral venipuncture ntawm lub lim tiam 0, 2, 4, 6, 8, 10, 16, 18, 20, 22, 24. Cervicovaginal fluid rau kev kuaj xyuas cov tshuaj tiv thaiv tau sau nrog Weck-Cel®spears (Medtronic Ophthalmics, Jacksonville, FL , Teb Chaws Asmeskas) ntawm lub lim tiam 4, 8, 16, 18, 20, 22 thiab 24. Cervicovaginal kuaj rau chlamydial load detection tau sau nrog tsis tu ncua flatlock swabs (Mast Diagnostic - ref.: 552C/10). Tom qab kev sib cuag nrog qhov chaw mos-cervical mucosa, lub swab tau muab tso rau hauv lub vial uas muaj UTM-RT nruab nrab (Mast Diagnostic - ref.: 330C/6) thiab khaws cia rau hauv dej khov.

Vaginal challenge infection with 5*107 IFUs of C. trachomatis SvD

Cov tsiaj tau sedated nrog ketamine chlorhydrate (10 mg / kg IM), muab tso rau hauv ventral recumbency nrog lawv lub duav elevated thiab 1 ml ntawm cov inoculum yog traumatically thov ncaj qha mus rau lub paum mucosa ntawm qhov chaw mos-cervical kev hloov pauv siv 1 ml syringe. Ib koob tshuaj 5 * 107 IFUs tau muab rau txhua tus tsiaj. Cov tsiaj raug tso cai nyob hauv ventral recumbency nrog lawv lub duav siab rau ib lub sij hawm luv luv tom qab inoculation.

Enzyme-linked immunosorbent assay

CTH522cov tshuaj tiv thaiv tshwj xeebNyob rau hauv cov ntshav thiab swab kuaj tau kuaj pom nrog qhov tsis ncaj qha ELISA. Maxisorp® daim hlau (NUNC A/S, Roskilde, Denmark) tau coated nrog CTH522 (0.5 µg/ml) hmo ntuj ntawm 4 degree . Cov isotypes IgG thiab IgA tau kuaj pom nrog HRP conjugated antibodies tshwj xeeb tiv thaiv tsis yog tib neeg primate IgG (43R-IG020HRP, Fitzgerald, Acton, MA, USA, 1:75.{10 }}) thiab IgA (43C-CB1631, Fitzgerald, USA, 1:50.{16}}). Cov tshuaj tiv thaiv tau pom nrog TMB PLUS substrate (KemEnTec, Taastrup, Denmark) thiab nres nrog 0.5 M sulfuric acid. Cov phiaj tau nyeem ntawm ELISA nyeem ntawv ntawm 450 nm nrog kev kho ntawm 650 nm. Kev tswj kom zoo (ntxhiab los ntawm kev kawm yav dhau los) tau siv los ua tus qauv sab hauv los kho rau cov phaj-rau-phaj variation. Ob lub qhov dej tau khiav tsis muaj substrate raws li kev tswj tsis zoo ntawm txhua lub phaj. Cov tshuaj tiv thaiv kab mob titers tau suav tias yog qhov sib npaug ntawm qhov siab tshaj plaws dilution nrog tus nqi kho qhov muag (OD) siab dua qhov kev txiav tawm. Qhov kev txiav tawm tau txiav txim siab txij hnub 0 tus qauv txhais tau tias ntxiv rau 2 * SD.

Peptide array thiab tsom xam

Lub CTH522 peptide array tau tsim nrog triplicates ntawm immobilized 15mer peptides overlapping los ntawm 14 amino acids uas tau luam tawm ntawm functionalized iav slides thiab incubated nrog serum kuaj los ntawm JPT Peptide Technologies, Berlin. Luv luv, cov array tau incubated nrog cov qauv kuaj hauv lub lis piam 24 thiab IgG peptide complexes tau pom pom siv biotinylated tshis anti-monkey IgG thiab Alexa Fluor® 647 Streptavidin. Slides tau luam theej duab nrog lub tshuab luam ntawv siab ntawm 635 nm thiab cov ntaub ntawv tau tshaj tawm tias yog fluorescence units. Qhov nruab nrab ntawm cov txiaj ntsig triplicate tau txiav txim siab, yog tias qhov sib txawv ntawm qhov sib txawv loj dua 0.5, tsuas yog siv 2 qhov ze tshaj plaws. 10 feem pua ​​​​ntawm cov teeb liab siab tshaj plaws (65,535) tau siv los ua kev txiav tawm rau ib qholus teb zoo, thiab txhais tau tias cov cim qhia hauv qab no tau muab tus nqi sib xws rau ib nrab ntawm qhov pib tus nqi (3277).

Kev ntsuam xyuas hauv vitro neutralization

Qhov kev ntsuam xyuas hauv vitro neutralization tau ua los ntsuas cov tshuaj tiv thaiv kab mob hauv cov tshuaj tiv thaiv kab mob uas muaj peev xwm los tiv thaiv kab mob Chlamydia ntawm HaK hlwb hauv vitro. Qhov kev ntsuam xyuas tau ua raws li cov txheej txheem tau piav qhia yav dhau los (44). Ua ntej, cov qauv kuaj ntshav los ntawm ib tus tsiaj tau muab tso rau ntawm 56 degree rau 30 feeb kom tsis txhob muaj qhov ntxiv. Chlamydia Tshuag (SvD, SvE, thiab SvF) tau muab sib xyaw thiab tsim nrog cov tshuaj tua kab mob ntawm cov tshuaj tiv thaiv kab mob los ntawm txhua tus tsiaj uas tau txhaj tshuaj rau 30 feeb thiab tom qab ntawd inoculated mus rau ib lub monolayer ntawm HaK hlwb hauv 96-zoo tiaj-hauv qab daim hlau hauv duplicates. Tom qab 30 teev ntawm incubation, hlwb, thiab inclusions tau fixated thiab pom nrog polyclonal luav anti-Ct043 cov ntshav thiab fluorescence-labeled thib ob antibody (Alexa Flour 488, tshis-tiv thaiv luav IgG, A11008, Life Technologies) nyob rau hauv lub dilution 5001: . Kev suav cov suav nrog tau ua manually hauv 20 thaj chaw saib ntawm 40x magnification (Olympus IX71 inverted fluorescence microscope). Neutralization raug xam raws li qhov feem pua ​​​​txo qhov nruab nrab ntawm IFUs piv rau lub pas dej ntawm sera los ntawm pawg tswj hwm tsiaj (naïve).

Intracellular PBMC cytokine staining thiab khub cytometry

PBMCs raug cais tawm thiab ({{0}}} x 106) tau rov ua dua hauv 150 µl ntawm qhov nruab nrab uas muaj 0.2 µg ntawm txhua costimulatory antibody CD28 thiab CD49b. Kev txhawb nqa tau ua nyob rau hauv 96 qhov dej / daim hlau siv 2 µM ntawm txhua lub pas dej peptide lossis SEB (raws li kev tswj zoo) lossis nruab nrab ib leeg (raws li kev tswj tsis zoo). Brefeldin A tau ntxiv rau txhua lub qhov dej ntawm qhov kawg ntawm 10 µg / ml thiab lub phaj yog incubated ntawm 37 degree C, 5 feem pua ​​CO2 thaum hmo ntuj. Tom qab ntawd cov hlwb raug ntxuav, stained nrog cov xim muaj zog (violet, fluorescent reactive dye, Invitrogen), tsau, thiab permeabilized nrog BD Cytofifix / Cytoperm reagent. Permeabilized cell kuaj tau muab khaws cia ntawm -80 degree ua ntej txheej txheem staining. Antibody staining tau ua nyob rau hauv ib kauj ruam tom qab permeabilization. Txhua yam sivtshuaj tiv thaivmuaj npe nyob rau hauv Table 1. Tom qab 30 feeb ntawm incubation ntawm cov dej khov nyob rau hauv qhov tsaus ntuj nti, hlwb raug ntxuav nyob rau hauv BD Perm/Wash tsis. Cells raug suav nrog LSR II (BD) tam sim ntawd tom qab cov txheej txheem staining thiab FlowJo software tau siv rau kev txheeb xyuas cov ntaub ntawv.

TAB 1 Antibodies siv rau staining ntawm lub cev tiv thaiv kab mob thiab intracellular cytokines

Kev kuaj pom ntawm qhov chaw mos chlamydial DNA los ntawm PCR zoo

Lub Abbott RealTime CT / NG assay (ABBOTT, Rungis, Fabkis) yog ib qho kev soj ntsuam hauv vitro polymerase saw cov tshuaj tiv thaiv (PCR) rau kev soj ntsuam ncaj qha, zoo ntawm plasmid DNA rau CT (C.trachomatis) thiab NG (N. gonorrhoeae) genomes . Nucleic acid tau muab rho tawm los ntawm UTM (universal transport media) cov qauv ntawm M2000SP thiab amplification tau ua nyob rau hauv M2000SP, raws li cov chaw tsim tshuaj paus cov lus qhia. Cov txiaj ntsig tau qhia raws li lub voj voog pib (Ct).

Kev txheeb cais

Tag nrho cov txheeb cais tau ua nrog GraphPad Prism software (version 8.1.2). Cov ntaub ntawv tsis yog ib txwm faib tau raug tshuaj xyuas nrog cov kev ntsuas uas tsis yog parametric; Kruskal–Wallis test thiab Dunn ntau qhov kev sib piv los txiav txim qhov tseem ceeb ntawm qhov sib txawv ntawm pawg. Curves qhia feem pua ​​​​ntawm PCR cov tsiaj tsis zoo tau muab piv nrog rau Log-rank (Mantel-Cox) statistical test. Cov kev sib piv tau suav tias yog qhov tseem ceeb yog tias tus nqi P yog < 0.05 (P < 0 05). Cov theem ntxiv ntawm qhov tseem ceeb yog qhia nrog lub hnub qub *P < 0.05, **P < 0.01, ***P < 0.001.

Cov ntaub ntawv muaj nyob

Thawj qhov kev koom tes uas tau nthuav tawm hauv txoj kev tshawb fawb no suav nrog hauv kab lus / Cov Khoom Siv Ntxiv. Cov lus nug ntxiv tuaj yeem xa mus rau tus kws sau ntawv.

Ethics nqe lus

Txoj kev tshawb nrhiav tsiaj raug tshuaj xyuas thiab pom zoo los ntawm "Comité d'Ethique en Expérimentation Animale du Commissariat à l'Energie Atomique et aux Energies Alternatives" (CEtEA #44) raws li nqe lus A14-026. Txoj kev tshawb no tau tso cai los ntawm "Kev Tshawb Fawb, Kev Tsim Kho Tshiab thiab Kev Kawm Txuj Ci" raws li tus lej APAFIS#720-201505281237660.

Sau cov nyiaj pab

Tsim thiab tsim kev sim: JD, FF, EL, PA, RG, RS. Kev sim ua: VC, DD, BD, SL, CD, CJ, ND-B, CB, BB, AT. Tsim cov tshuaj tiv thaiv thiab antigen tsim: IR, PM, AO. Kev txheeb xyuas cov ntaub ntawv: JD, EL, FF, IR, HJ. Drafted thiab kho cov ntawv: JD, EL, FF. Txhua tus kws sau ntawv sau ua ke tau txais txiaj ntsig zoo rau kev ua haujlwm los ntawm kev tsim thiab kev xav, thiab / lossis kev nrhiav tau, kev tshuaj xyuas, thiab kev txhais cov ntaub ntawv. Txhua tus kws sau ntawv tau pab txhawb rau tsab xov xwm thiab pom zoo rau cov ntawv xa tawm.

Kev lees paub

Peb ua tsaug rau Fondation Dormeur, Vaduz rau qhov pub dawb rau Viral Evolution thiab Transmission Unit rau cov cuab yeej kuaj ntsuas cuam tshuam rau txoj haujlwm no.

Kev tsis sib haum xeeb ntawm kev txaus siab FF, IR, PA thiab AWO yog cov neeg tsim khoom sib koom ua ke ntawm daim ntawv thov patent ntawm cov tshuaj tiv thaiv kab mob chlamydia [WO2014146663A1]. Txhua txoj cai tau raug xa mus rau Statens Serum Institut, lub koom haum Danish tsis tau txais txiaj ntsig los ntawm tsoomfwv. Cov kws sau ntawv ntxiv tshaj tawm tias qhov kev tshawb fawb tau ua thaum tsis muaj kev lag luam lossis kev sib raug zoo ntawm nyiaj txiag uas tuaj yeem txhais tau tias muaj kev tsis sib haum xeeb ntawm kev txaus siab.

Publisher's note Tag nrho cov lus thov hais hauv tsab xov xwm no tsuas yog cov neeg sau ntawv nkaus xwb thiab tsis tas yuav sawv cev ntawm lawv cov koom haum koom nrog, lossis cov tshaj tawm, cov neeg kho, thiab cov neeg tshuaj xyuas. Ib yam khoom uas yuav raug soj ntsuam nyob rau hauv tsab xov xwm no, los yog thov hais tias tej zaum yuav ua los ntawm nws cov chaw tsim tshuaj paus, yog tsis guaranteed los yog pom zoo los ntawm lub publisher.

Cov ntaub ntawv

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9. Armitage CW, O'Meara CP, Harvie MC, Timms P, Wijburg OL, Beagley KW. Kev ntsuam xyuas ntawm intra- thiab extra-epithelial secretory IgA hauv cov kab mob chlamydial. Immunology (2014) 143(4): 520–30. doi: 10.1111/ib.12317

10. Armitage CW, O'Meara CP, Harvie MC, Timms P, Blumberg RS, Beagley KW. Cov txiaj ntsig sib txawv tom qab transcytosis ntawm IgG tsom rau intracellular thiab extracellular chlamydial antigens. Immunol Cell Biol (2014) 92(5): 417–26. doi: 10.1038/icb.2013.110

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