Chlamydial Type III Secretion System Needle Protein ...downloads.hindawi.com/journals/bmri/2017/3865802.pdf · ResearchArticle Chlamydial Type III Secretion System Needle Protein

Research ArticleChlamydial Type III Secretion System Needle ProteinInduces Protective Immunity against Chlamydia muridarumIntravaginal Infection

Ekaterina A Koroleva Natalie V Kobets Dmitrii N ShcherbininNaylia A Zigangirova MaximM Shmarov Amir I TukhvatulinDenis Y Logunov Boris S Naroditsky and Alexander L Gintsburg

Gamaleya Institute of Epidemiology and Microbiology Ministry of Health of Russian Federation Gamaleya Street 18Moscow 123098 Russia

Correspondence should be addressed to Natalie V Kobets nkobets2000yahoocom

Received 16 December 2016 Accepted 19 February 2017 Published 26 March 2017

Academic Editor Lucia Lopalco

Copyright copy 2017 Ekaterina A Koroleva et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chlamydia trachomatis imposes serious health problems and causes infertility Because of asymptomatic onset it often escapesantibiotic treatmentTherefore vaccines offer a better option for the prevention of unwanted inflammatory sequelaeThe existenceof serologically distinct serovars of C trachomatis suggests that a vaccine will need to provide protection against multiple serovarsChlamydia spp use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is anessential virulence factor In this study we expressed the T3SS needle protein ofChlamydia muridarum TC_0037 an ortholog of Ctrachomatis CdsF in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginalChlamydia muridarum model For better immune responses we employed a heterologous prime-boost immunization protocolin which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-likereceptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion We found that immunization with TC_0037antigen induced specific humoral and T cell responses decreased Chlamydia loads in the genital tract and abrogated pathology ofupper genital organs Together our results suggest that TC_0037 a highly conserved chlamydial T3SS protein is a good candidatefor inclusion in a Chlamydia vaccine

1 Introduction

Chlamydia trachomatis is the most common sexually trans-mitted bacterial pathogen It imposes serious health problemsin humans and can cause severe complications such as pelvicinflammatory disease and ectopic pregnancy and infertility inwomen [1] Although antibiotic therapy effectively eliminatesacute infection it does not always moderate an establishedpathology and frequent asymptomatic courses of infectionpreclude early diagnosis and treatment To overcome theseproblems the development of a vaccine is highly desirableChlamydia muridarum has been extensively used to study themechanisms of C trachomatis pathogenesis and immunityin a mouse model [2] Intravaginal inoculation of mice with

C muridarum can lead to infection in the lower and uppergenital tract which closely mimics the pathology inducedby C trachomatis in humans [3] Both animal and humanstudies have established a vital role for T cell-mediatedimmunity predominantly that of IFN-120574-producing CD4+ Tcells and the complementary role of humoral immunity inhost resistance to chlamydial infection [4 5] CD8+ T cellshave been shown to protect against infection when culturedex vivo and transferred to naive animals and immunizationwith recombinant vaccinia viruses expressing CD8+ T cellantigens from C trachomatis conferred protection in mice[6] Chlamydia vaccine research has led to the discovery of alarge number of protective antigens [7ndash9] Major outer mem-brane protein (MOMP) and more recently polymorphic

HindawiBioMed Research InternationalVolume 2017 Article ID 3865802 14 pageshttpsdoiorg10115520173865802

2 BioMed Research International

membrane proteins (Pmps) both of which elicit antibodyand cell-mediated protective immune responses are widelystudied as potent vaccine candidates However the antigenicvariation in MOMP and Pmps suggests that evaluation ofconserved proteins as vaccine candidates could be valuableas this strategy has been successful for developing vaccinesagainst other pathogens [10]

The type three secretion system (T3SS) is the predom-inant virulence factor in Chlamydia It is required for cellinvasion and is active at all life stages [11 12] Some T3SSproteins are surface-exposed and can be targeted by neu-tralizing antibodies The T cell response to T3SS antigenswas recently shown to be associated with protection againstC trachomatis infection in highly exposed women [13] andT3SS components have recently attracted attention as vaccinecandidates against other pathogenic bacteria [14ndash17] The Ctrachomatis T3SS filament protein CdsF and its orthologsin other bacteria form the needles of injectisomes and arebelieved to facilitate the insertion of translocators into thehost cell membrane [11 12 18] CdsF is highly conservedshowing 95 sequence identity in the genus Chlamydia It isabundant on bacterial surfaces raising the possibility that aCdsF-based vaccine may induce a wide range of protectionagainst all medically significant strains

The ability of human adenoviruses to induce stronginnate and adaptive immune responses makes them a pow-erful delivery system to induce an immune response againstan encoded antigen Adenovirus has a natural tropism forthe mucosal epithelium which makes it an ideal vector forvaccination against infections acquired via mucosal surfaces[19] Intranasal immunization in different experimental set-tings has been reported to provide potent protection againstintravaginal challenge [18 20ndash22] Replication-defective ade-novirus vectors (rAds) are widely used for transgene expres-sion in different tissues and gene-based immunizationsEffective vaccines against tuberculosis malaria influenzaand other important infectious diseases have been designedfrom rAds The majority of rAd-based medical preparationsare currently in clinical trials [23ndash27] There is a large bodyof evidence indicating that rAd-derived vaccines are safe andefficacious [28ndash31] Combination of adenoviral-vectored andprotein-in-adjuvant vaccines in ldquoprime-boostrdquo regimenswiththe aim of enhancing both cellular and humoral immuneresponses has shown promising results in HIV and liver-stage malaria vaccine preclinical research [32ndash38] Toll-likereceptor (TLR) agonists are usually codelivered with antigensas adjuvants to enhance the immunogenicity of vaccinecomponents [39]

In this study we expressed the T3SS needle protein ofC muridarum TC_0037 a CdsF ortholog in a replication-defective adenoviral vector (AdTC_0037) and evaluated itsprotective efficacy in an intravaginal Chlamydia infectionmouse model To study the protective immunity of ourvaccine candidate we utilized a prime-boost immunizationprotocol with AdTC_0037 intranasal priming and subcu-taneous boosting with recombinant TC_0037 and TLR4agonist monophosphoryl lipid A (MPLA) mixed in asqualene nanoscale emulsion We found that immunizationwith TC_0037 antigen induced specific humoral and T cell

responses decreased Chlamydia loads in both the lower andupper genital tract and reduced the pathology of uppergenital organs

2 Materials and Methods

21 Bioinformatic Antigen Design The nucleotide sequenceof the C muridarum TC_0037 gene was obtained fromUniProtKB (Q9PLQ8) We performed an in silico analysisof the TC_0037 gene on the presence of a bacterial signalsequence (httpwwwcbsdtudkservicesSignalP) and nobacterial signal sequence was found We further optimizedTC_0037 gene sequences for expression in mouse cells bymodifying its codons with the two most frequent amino acidtriplets Frequent Mus musculus codons were defined usingthe httpwwwkazusaorjpcodon database As TC_0037 isabundant in bacterial cells and small in size (82 amino acids9 kDa) the N-terminal portion of this protein was boundto a 128-amino acid-sized N-terminal portion of mousemannose-binding lectin (MBL) to obtain TC_0037 hexamerscrosslinked with a collagen-like domain The nucleotidesequence of mouse MBL was obtained from UniProtKB(P41317) The MBL-TC_0037 gene was synthesized by Evro-gen (Moscow Russia) in plasmid pAL-TA-MBL-TC_0037

22 Generation of Recombinant Adenoviruses The NotI andHindIII sites of theMBL-TC_0037 fragment were cloned intoshuttle vector pShuttle-CMV (cytomegalovirus) to obtainthe shuttle plasmid pShuttle-CMV-MBL-TC_0037 Homol-ogous recombination was used to generate the replication-defective adenovirus Ad-MBL-TC_0037 (Figure 1) For thispurpose the plasmid pShuttle-CMV-MBL-TC_0037 was lin-earized by PmeI mixed with pAd-Easy (Adenoviral Vec-tor System StratoGen New York NY) and then cotrans-formed into Escherichia coli (BJ5183 strain) The obtainedrecombinant clones were used to extract plasmid DNAwhose molecular weight was later assessed E coli strainDH5alpha was transformed with plasmids larger than 20 kbpbecause this strain unlike BJ5183 allows one to producea sufficient amount of recombinant plasmids The purifiedplasmid clones were analyzed by both cleavage with theHindIII restriction endonuclease and polymerase chain reac-tion (PCR) Next we studied the infectivity of the describedplasmids in permissive cells HEK-293 cells were transfectedwith plasmid pAd-MBL-TC_0037 and linearized by PacITransfection was performed in a 24-well plate using Lipo-fectamine 2000 (Invitrogen Burlington Canada) Ten daysafter the transfection the cells were collected and subjectedto a freeze-thaw cycle the obtained lysate containing recom-binant adenoviruses was used to infect HEK-293 cells in a35mm dish After 5 days specific lysis caused by cytopathiceffect of the recombinant viruses was detectedThe lysate wasused to extract DNA and perform a PCR analysis The celllysate was shown to contain DNA of recombinant humanadenovirus serotype 5 (rAd5) carrying insertions that encodethe protective antigen

23 Virus Accumulation Recombinant adenovirus serotype5 (rAd5) was grown in HEK-293 cells as described elsewhere

BioMed Research International 3

ΔE1 ΔE3

100

pACMV

(a)

(b)

0

1 10 20 30 40 50 60

70 80 90 100 110 120

130 140 150 160 170

190 200 210 220 227

180

MBL

MBL

MBL GS-Spacer TC_0037

TC_0037QGTVDLGTMFNLQYRTQILCQYMEASSNILTAVHTEMITMARSAKGS

MSIFTSFLLLCVVTVVYAETLTEGVQNSCPVVTCSSPGLNGFPGKDGRDGAKGEKGEPGQ

GLRGLQGPPGKVGPTGPPGNPGLKGAVGPKGDRGDRAEFDTSEIDSEIAALRSELRALRN

WVLFSLSEGSPGSGSGSGSGSRSLEMAKSCSALNFNEMSEGVCKYVLGVQQYLTELETST

Figure 1 Design of Chlamydia muridarum TC_0037 expressed in an adenovirus construct (a) Human recombinant adenovirus serotype 5genome CMV cytomegalovirus promoter pA polyadenylation signal (b) The amino acid sequence of chimeric antigen MBL-TC_0037

[39] Cell monolayers at 50ndash70 confluence were infectedwith 107 PFU (plaque-forming units) of rAd5 per 15 cm plateAfter 48 h infected cells were collected concentrated by low-speed centrifugation resuspended in Tris HCl buffer (001MTrisHCl pH80 001MNaCl 5mMEDTA) anddisrupted bya triple freeze-thawThe obtained suspensionwas centrifugedand purified by cesium chloride equilibrium density gradientcentrifugation The concentration of adenovirus was deter-mined by a plaque-forming assay on HEK-293 cells

24 Construction of Recombinant TC_0037 The gene encod-ing the full-length TC_0037 protein along with six histi-dine tags (6x His) was cloned into the pET29b (NovagenMadison WI) expression vector at NdeIXhoI restrictionsites E coli BL21 (DE3) transformed with the designedplasmid was grown in Luria-Bertani broth supplementedwith ampicillin or kanamycin on a shaker at 37∘C to anOD

600

of 05 Protein expression from the pET29b-based plasmidwas induced by 1M isopropyl-120573-D-thiogalactopyranoside(Roth Karlsruhe Germany) for 3 h at 25∘CThe cleared lysatewas subjected to chromatography on a nickel-equilibratedchelating Sepharose Fast Flow column according to the man-ufacturerrsquos instructions (GEHealthcare Rahway NJ) ProteinTC_0037 production was analyzed by western blotting with amonoclonal antibody targeting the histidine tag

25 Vaccine Boost Preparation Theboosting squalene oil-in-water emulsion was prepared as described previously [40]Briefly a boosting vaccine formulation (200 120583L per dose)consisting of rTC_0037 protein (10120583gdose) immunostimu-latory molecule MPLA (1 120583gdose) (Sigma-Aldrich St LouisMO) 50 squalene (Sigma-Aldrich St Louis MO) 05Tween 80 and 05 Span 85 in an isotonic phosphate buffer(Sigma-Aldrich St Louis MO) was prepared by homoge-nization at 12000 psi with a microfluidizer (MicrofluidicsNewton MA) and passed through a polysulfone filter

(220 nm pore size GE Healthcare USA) for sterilizationTheaverage diameter (1083 +minus 87 nm) of the emulsion dropletswas determined by Nanoparticle Tracking Analysis (NTA)using a NanoSight NS300 (Malvern Ltd UK)

26 Chlamydia C muridarum (strain Nigg) ATCC VR-123was grown in cycloheximide-treated McCoy cells (a hybridcell line consisting of human synovial and mouse fibroblasts)as described previously [41 42] Chlamydial elementarybodies (EBs) were harvested purified quantified and storedat minus70∘C in SPG buffer (sucrosephosphateglutamic acid02M sucrose 20mMsodiumphosphate and 5mMglutamicacid)

27 Ethics Statement All animal work was undertaken instrict accordance with the recommendations of the NationalStandard of the Russian Federation (GOST R 53434-2009)The procedures used were approved by the GamaleyaResearch Center of Epidemiology and Microbiology Insti-tutional Animal Care Female 6ndash8-week-old BALBc miceobtained from the Animal Resource Center (Puschino breed-ing facility Puschino Moscow Russia) accredited by theAssociation for Assessment and Accreditation of LaboratoryAnimal Care (AAALAC International) were maintained atthe central animal facility of the Gamaleya Research Centerof Epidemiology and Microbiology

28 Immunization and Infection Mice (119899 = 20 per group)were intranasally (in) primed with 108 PFU of adenoviralvector-expressed TC_0037 (AdTC_0037) in 100120583L of sterilephosphate-buffered saline (PBS) or empty vector (Ad-nullcontrol) Prime-boost experimental groups of mice were fur-ther subcutaneously (sc) boosted with 200120583L of rTC_0037-MPLA two weeks after priming (Figure 2) To evaluatevaccine protection progesterone-treated mice (119899 = 10per group) were intravaginally (ivag) infected with 106 IFU


Priming inAdTC_0037

Boosting scrTC_0037-MPLA

Day 0 Day 14 Day 28

C muridarum ivag

Serum and spleen samples

Progesterone treatment sc

Day 21

Chlamydia DNA andpathology UGT

Day 58Day 31 Day 48

Vaginal swabs

Uterine samples

ELISA ELISPOT andneutralization

assay Detection IFU

Figure 2 Study design for animal experiments

(inclusion-forming units) of C muridarum in 40 120583L of PBSas described previously [42]

29 Mouse Sampling Blood (119899 = 5 per group) was collectedfrom tails and centrifuged 2 weeks after boost immunizationand serum was harvested and stored atndash20∘C To assess IFN-120574 T cell responses lymphocyte cultures from spleens (119899 =5 per group) were prepared as described previously [40]Briefly spleens were removed aseptically from mice at day 14after immunization Spleen cells from mice infected with Cmuridarum obtained at 14 days after infection were used as apositive control Spleen cells (2 times 106 per mL) were incubatedwith UV-killed C muridarum or rTC_0037 for 24 h incomplete RPMI-1640 (Gibco Carlsbad CA) containing 5fetal bovine serum 2mM l-glutamine and 1 penicillin-streptomycin (Gibco Carlsbad CA) Vaginal swabs wereobtained at 3 6 9 15 and 20 days after infection Uterinesamples for chlamydial DNA detection were collected in PBSand frozen at minus70∘C Gross uterine pathology presenting asa hydrosalpinx was examined at day 30 after intravaginal Cmuridarum challenge of immunized and naive mice

210 Detection of TC_0037-Specific Antibodies in Serum Theantibody (IgG1 and IgG2a subclasses) response in serumwas analyzed using an enzyme-linked immunosorbent assay(ELISA) with plates coated with rTC_0037 protein BrieflyrTC_0037 protein was diluted to 10 120583gmL inNa

2CO3(01M

pH 95) and coated ontomicrotiter plates (NUNC RochesterNY) Nonspecific binding was blocked by using 01 bovineserum albumin (BSA) in PBS for 30min at 37∘C Serum wasdiluted and titrated in 01 BSA in PBS and then incubatedovernight at 4∘C Biotinylated anti-mouse IgG1 and IgG2amonoclonal antibodies (BioLegend San Diego CA) used ata dilution of 1 1000 were added to plates and incubated for1 h at room temperature (20∘C) followed by the addition ofstreptavidin-HPR and TMB substrate (all from BioLegendSan Diego CA) The reaction was stopped 20min later byadding 50 120583Lwell of 1MH

2SO4 Absorbances at 450 nmwere

determined using aMultiskan EXmicroplate reader (ThermoFisher Scientific Oy Vantaa Finland)

211 In Vitro Serum Neutralization Assay A C muridarumneutralization assay was performed in McCoy cell cultures

[42] Briefly 100 120583L of C muridarum EB suspension (14times 105 IFUmL) in Dulbeccorsquos modified Eaglersquos medium(DMEM Paneco Moscow Russian) was added to 100120583Lof serial dilutions (from 1 16 to 1 256) of immune serafrom vaccinated mice and incubated for 30min at 37∘Con a slowly rocking platform Samples incubated with EBbut without serum were used as an infection control Onehundred microliters of each dilution were then inoculatedin duplicate into McCoy cells DMEM containing 10 fetalbovine serum and 1mgmL of cycloheximide were addedup to 1mL to infected cells in 24-well plates with coverslips(12mm in diameter) After centrifugation at 800timesg for 1 hinfected cultures were incubated at 37∘C for 48 h Afterthis ethanol-fixed monolayers were stained with fluoresceinisothiocyanate- (FITC-) conjugated monoclonal antibod-ies against chlamydial lipopolysaccharide (Nearmedic PlusMoscow Russia) Inclusion-containing cells were examinedusing a Nikon Eclipse 50i fluorescent microscope (NikonAmsterdamNetherland) at 1350xmagnification and countedto determine the percent infected cells in the monolayer

212 Detection of Chlamydia and TC_0037-Specific IFN-120574-Producing T Cells Chlamydia and TC_0037-specific IFN-120574-producing T cell enzyme-linked immunospot (ELISPOT)assays were performed on mouse splenocytes using a mouseIFN-gammaELISPOTReady-SET-Go Kit (eBioscience IncSan Diego CA) according to manufacturerrsquos instructionsBriefly IFN-120574 capture antibody was coated onto ethanol-activated MultiScreen-IP Filter Plates (Millipore BillericaMA) incubated overnight at 4∘C and then washed withsterile PBS CD4+ and CD8+ T cells were isolated from thespleens of vaccinated mice using CD4+ and CD8+ T cellseparation columns (Cederlane Burlington Canada) accord-ing to manufacturerrsquos protocols The final concentration ofenriched T cells was approximately 85 as determined byflow cytometry therefore we hypothesized that there wereenough antigen-presenting cells left to present antigens inthe study This assumption was confirmed in a separate setof experiments Enriched cell suspensions were added to thecoated plates at 2 times 105 cells per well (in 100 120583L of culturemedium) in the presence of UV-treated C muridarum EB(positive control) at a final concentration of 104 IFUmL andrTC_0037 at a final concentration of 10 120583gmL The plates


Table 1 Primers for the detection of Chlamydia muridarum

Name Gene 51015840-31015840 sequenceCmur-F

P MoPnForward 51015840-TGCGATAGAAACAATTCCCTGAGT -31015840

Cmur-R Reverse 51015840-TGCTTTAGAAAAGATTGGGCTATTG-31015840

Cmur_up_FAM Probe FAM-AGCTGCACGAACTTGTTTGGTGCCTTCT-RTQ1

were incubated for 24 h before further washing After thisplates were incubated with biotinylated anti-IFN-120574 mono-clonal antibodies The spots were visualized with Avidin-HRP reagent and freshly prepared 3-amino-9-ethyl carbazole(AEC) substrate solution Spots were calculated using an AIDELISpot Reader (Autoimmun Diagnostika GmbH Strass-berg Germany)

213 Quantification of the Bacterial Loads in Chlamydia-Challenged Mice Vaginal swabs were obtained at 3 6 915 and 20 days after infection The canal and exocervixwere vigorously scraped and swabbed material was trans-ferred to DMEM and frozen immediately at minus70∘C Bacterialloads were determined as previously described [41] Thawedundiluted samples (05mL) were plated onto McCoy cellson coverslips (12mm in diameter) in 24-well plates andthe infection was enhanced by centrifugation at 800timesg for1 h After centrifugation the inocula were removed 1mLof complete DMEM (DMEM with 10 fetal calf serum1 nonessential amino acids 2mM L-glutamine 1 vita-mins and 05 glucose) was added and the plates wereincubated in a humidified incubator with 5 CO

2at 37∘C

for 48 h Coverslips were incubated with FITC-conjugatedmonoclonal antibodies against chlamydial lipopolysaccha-ride (Nearmedic PlusMoscow Russia) Inclusion-containingcells were determined using a Nikon Eclipse 50i fluorescentmicroscope (Nikon Amsterdam Netherland) at 1350x mag-nificationThe results were expressed as log

10IFU per vaginal

swab

214 Detection of Chlamydial DNA in Uteruses ChlamydialDNA in uteruses was evaluated using a quantitative real-time PCR that allowed enumeration of the parasitersquos genomeequivalents in infected tissue Uterine samples were collectedat day 30 after infection homogenized in 1mL of physiolog-ical solution and frozen at minus70∘C The prepared suspensionswere lysed in 1mL of lysis buffer (bioMerieux Netherlands)and incubated with 40 120583L of proteinase K (Syntol MoscowRussia) at 60∘C for 1 h DNA was extracted with automatednucleic acid extractor NucliSENS easyMAG (bioMerieuxZaltbommel Netherlands) DNA contamination controlswere included The primers and TaqMan probe targetingthe cryptic plasmid region were selected using Primer 3(httpsimgenecomPrimer3) and Oligo7 programs and arepresented in Table 1C muridarumDNAwas amplified usinga Real-Time PCR Cycler CFX96 (Bio-Rad Irvine CA) Theresults are presented as percent positive mice in each group

215 Pathology The percent of mice with hydrosalpingesin each group was assessed at day 30 after C muridarumchallenge as described previously [43]

216 Statistical Analysis The results were analyzed with theaid of GraphPad Prism 70 software (GraphPad softwareSan Diego CA) ShapirondashWilk test was used to determinenormality of combined datasets from several similar exper-iments The MannndashWhitney 119880 test was used to evaluatedifferences in antibody titers in ELISA specific IFN-120574-producing CD4+ and CD8+ T cells and Chlamydia loadsAn analysis of variance (ANOVA) test was used to compareneutralizing antibodies in experimental groups

3 Results

31 Immunization with AdTC_0037 Results in the Productionof TC_0037-Specific Antibodies of IgG2a and IgG1 IsotypesThe immunogenicity of the TC_0037 antigen was assessedin BALBc mice immunized with TC_0037 expressed in anadenoviral vector (AdTC_0037) followed by subcutaneousimmunization with recombinant TC_0037 and MPLA in asqualene nanoscale emulsion (rTC_0037-MPLA) Sera frommice immunized with an empty vector with or withoutMPLA and sera from intact mice were used as controlsAdTC_0037 immunization induced TC_0037-specific IgG2aand IgG1 antibodieswith titers of 1 800 for both isotypes (119875 lt005) (Figure 3) Prime-boost immunization also resulted inan increase in specific IgG2a and IgG1 antibodies but thetiters were lower than those in the AdTC_0037-alone group(1 400 and 1 800 resp) Thus TC_0037 expressed in theadenovirus vector induced specific antibody responses inmice

32 Prime-Boost Immunization with AdTC_0037r TC_0037-MPLA Induces Antibodies That Neutralize C muridarumInfectivity In Vitro In the experiments described abovewe demonstrated that immunization with AdTC_0037rTC_0037-MPLA or AdTC_0037 alone induced TC_0037-specific antibodies of IgG2a and IgG1 isotypes Next weassessed the ability of serum antibodies to neutralizepathogen infectivity in vitro C muridarum EBs were prein-cubated with serum from immunized mice and then usedto infect McCoy cell monolayers Chlamydial inclusionswere counted under the microscope 48 h after incubationThe results are presented in Figure 4 We found that serafrom prime-boost immunized mice effectively neutralizedEB infectivity in vitro up to 90 at a dilution of 1 512In the group immunized with AdTC_0037 the reduc-tion in infectivity reached 65 (dilution 1 512) Overallour results suggest that immunization with AdTC_0037induces antibodies with high potential to neutralize Cmuridarum infection in vitro Interestingly the neutralizingpotential of specific antibodies from mice immunized with


lowastlowast

AdTC_0037Ad-nullIntact serum

00

02

04

06

08

10

12

14

16

18Se

rum

TC_

0037

spec

ific I

gG2a

200 400 800100Dilution

AdTC_0037 + rTC_0037-MPLA

Ad-null + MPLA(a)

lowast lowast

00

02

04

06

08

10

12

14

16

18

Seru

m T

C_00

37 sp

ecifi

c IgG

1

200 400 800100Dilution



Ad-null + MPLA(b)

Figure 3 IgG2a and IgG1 antibody responses to TC_0037 following prime-boost immunization with AdTC_0037rTC_0037-MPLA BALBcmice (119899 = 5 per group) were intranasally primed with Ad-TC_0037 followed by subcutaneous boosting with rTC_0037-MPLA Sera werecollected 2 weeks after the boost TC_0037-specific IgG2a (a) and IgG1 (b) titers were determined by enzyme-linked immunosorbent assayand expressed as the mean plusmn SEM for groups of 5 mice from two experiments (lowast119875 lt 005)

lowast

lowast

0

20

40

60

80

100

Neu

tral

izat

ion

()

64 128 256 51232Dilution

AdTC_0037 Ad-nullAdTC_0037 + rTC_0037-MPLA Ad-null + MPLA

Figure 4 Antibodies against Chlamydia muridarum TC_0037neutralize chlamydia infectivity in vitro C muridarum elementarybodies were opsonized with sera from immunized or control mice(119899 = 5) at different dilutions and used to infect McCoy cell mono-layers Inclusions were counted 48 h after infection Neutralizationactivity was determined by measuring the reduction in the numberof inclusions generated by antibody-opsonized elementary bodiesThe graphs present the percent neutralization as an average ofexperiments performed in triplicate with standard deviations (lowast119875 lt001)

AdTC_0037rTC_0037-MPLA was higher than that of anti-bodies in mice immunized with AdTC_0037 alone (119875 le001) This might contribute to higher protection with theprime-boost approach

33 Induction of Specific IFN-120574-Producing CD4+ and CD8+T Cells in Mice Immunized with AdTC_0037rTC_0037-MPLA and Infected with C muridarum Since IFN-120574 hasbeen found to play a major role in mediating controlof Chlamydia infection we measured IFN-120574 responses torTC_0037 and UV-killed C muridarum in immunized andinfected mice to evaluate the potential of TC_0037 to primeT cell responses to Chlamydia TC_0037 and C muridarum-specific IFN-120574+ CD4+ and CD8+ T cell responses wereassessed by ELISPOT 2 weeks after final immunization orinfection As shown in Figure 5 rTC_0037 and UV-killed Cmuridarum stimulation triggered robust IFN-120574 production inCD4+ and CD8+ T cells derived from mice immunized withAdTC_0037rTC_0037-MPLA or AdTC_0037 alone but notwith the control adenovirus (Ad-null+MPLA Ad-null) UV-killed C muridarum stimulation induced IFN-120574 CD4+ andCD8+ production in immunized mice at the same level asthat in C muridarum-infected mice (Figures 5(b) and 5(d))However CD4+ and CD8+ T cell responses to TC_0037 werehigher in the immunized groups (Figures 5(a) and 5(c)) Ourresults indicate that immunizationwith rTC_0037 resulted inthe induction of robust TC_0037-specific IFN-120574+CD4+ T cellresponses that were comparable to those induced in naturallyinfected mice (Figure 5(a)) Additionally it potentiates theinduction of higher levels of IFN-120574+ TC_0037-specific CD8+T cells compared to those in C muridarum-infected mice(Figure 5(c))

34 Immunization with AdTC_0037 and AdTC_0037rTC_0037-MPLA Enhances the Clearance of C muridarum in theVagina BALBcmicewere ivag infectedwithCmuridarumat day 14 after final immunization and cervicovaginal swabswere obtained from mice (Figure 2) on days 3 7 9 15


No stimulation rTC_00370

200

400

600

800IF

N-훾

SFU

2times10

5T

cells

lowastlowastlowastlowastlowastlowast

C muridarumAdTC_0037Ad-nullAdTC_003

PBS7 + rTC_0037-MPLA

Ad-null + MPLA

CD4+

(a)

No stimulation C muridarum0

200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast

AdTC_0037Ad-nullAdTC_003


Ad-null + MPLA

CD8+

(c)


200

400

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1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)

Figure 5 Prime-boost immunization with AdTC_0037rTC0037-MPLA induces specific IFN-120574-producing CD4+ and CD8+ responsesBALBc mice (119899 = 5 per group) were immunized intranasally with Ad-TC_0037 and boosted subcutaneously with rTC_0037-MPLA Miceinfected intravaginally with Chlamydia muridarum were used as controls Two weeks after the boost CD4+ and CD8+ T cells were isolatedand stimulated in vitrowith TC_0037 or UV-killed C muridarum ELISPOT counts (spot-forming units [SFUs]) in response to TC_0037 andC muridarum were analyzed Results are presented as the mean SFU per 2 times 105 splenocytes plusmn SEM (lowast119875 le 0005 lowastlowast119875 le 001 lowastlowastlowast119875 le 005relative to Ad-null-MPLA immunized mice)

and 20 following challenge To quantify resistance to infec-tion recoverable IFU were compared between the groups(Figure 6) There were no statistically significant differencesbetween the groups at days 3 and 7 (data not shown) andat day 9 (Figure 6(a)) after challenge However prime-boostimmunization with AdTC_0037rTC_0037-MPLA signifi-cantly reduced chlamydial IFUs at day 15 (Figure 6(b) lowast119875 lt0005) Almost complete eradication of infection (9 of 10micecleared infection in AdTC_0037rTC_0037-MPLA group)was observed at day 20 after infection (Figure 6(c) lowastlowast119875 lt001) whereas eradication following immunization withAdTC_0037 and in the infection control was not observedThese results show that prime-boost immunization with

AdTC_0037rTC_0037-MPLA reduced the magnitude andduration of C muridarum vaginal shedding after infection

35 Immunization with AdTC_0037rTC_0037-MPLA Redu-ces Chlamydia Loads in the Upper Genital Tract To assessthe effects of vaccination on control of ascending infectionwe analyzed the levels of chlamydial DNA in uterine sam-ples at day 30 after infection Immunization of mice withAdTC_0037 in both groups resulted in a decrease in Cmuridarum DNA in the upper genital tract of mice andthis effect was more pronounced in the mice immunizedwith AdTC_0037rTC_0037-MPLA (Figure 7)Therefore weconfirmed that prime-boost immunization with TC_0037


Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)

C muridarumAdTC_0037Ad-nullAdTC_0037 + rTC_0037-MPLA

Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowastlowastlowastlowast

(c)

Figure 6 Prime-boost immunization with Ad-TC_0037rTC0037-MPLA enhances clearance of intravaginal Chlamydia muridarum inBALBc mice Depo-Provera-treated BALBc mice (119899 = 10 per group) were infected intravaginally with 106 IFU of C muridarum strainNigg 2 weeks after the boost The vaginal vaults of mice were sampled using individual swabs at days 3 to 20 after challenge and vaginalchlamydial loads were quantified using an in vitro infection assay and immunofluorescent microscopy Individual and median values of Cmuridarum loads measured at days 9 (a) 15 (b) and 20 (c) after infection are presented (lowast119875 le 0005 lowastlowast119875 le 001)

antigen successfully inhibited upper genital C muridaruminfection in vivo

36 Immunization with AdTC_0037 Results in DecreasedPathology in the Upper Genital Tract One of the primarygoals for an effective chlamydial vaccine is to reduce orprevent upper genital tract pathology following lower tractinfection To assess the ability of TC_0037 immunization toreduce Chlamydia-induced immunopathology of the ovarianducts BALBc mice were immunized as shown in Figure 2and challenged ivag with 106 IFU of C muridarum Animalswere sacrificed at day 30 after infection to assess the develop-ment of hydrosalpinx As shown in Figure 8 mice vaccinatedwith AdTC_0037 or AdTC_0037rTC_0037-MPLA did not

develop upper genital tract pathology whereas mice in thecontrol groups did (Figure 8(c)) Thus immunization withAdTC_0037 resulted in complete abrogation of pathology inthe upper genital organs of BALBc mice infected ivag withC muridarum

4 Discussion

Despite decades of research and considerable progress madein recent years an efficacious C trachomatis vaccine remainselusive [44] As previous successful uses of an adenovirus-vectored vaccine against C muridarum (expressing CPAFand MOMP) have been reported [45ndash48] in this study wedeveloped a novel adenovirus-based vaccine expressing T3SS


Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA

Figure 7 Prime-boost immunization with AdTC_0037rTC_0037-MPLA reduces chlamydia loads in the upper genital tracts ofBALBc mice Uteruses were collected at day 30 after infection andChlamydia muridarum loads were determined by polymerase chainreaction A 100 reduction in bacterial infection was observed inthe prime-boost vaccinated group compared to the control groups(lowast119875 lt 001 lowastlowast119875 lt 0001)

Chlamydia antigen TC_0037 and evaluated its protectivepotential in a C muridarum mouse model To enhance pro-tection by our vaccine candidate we utilized a heterologousprime-boost immunization regimen using a combination ofintranasal priming with adenovirus-expressing TC_0037 andsubcutaneous boost with recombinant TC_0037 and MPLAmixed in squalene Prime-boost immunization of micewith adenovirus-expressed TC_0037 and rTC_0037MPLAelicited serum antibodies against TC_0037 of isotypes IgG2aand IgG1 (Figure 3) In addition serum from immunizedmice neutralized chlamydial infection in vitro (Figure 4)Mice vaccinated with TC_0037 and challenged with Cmuridarum showed a reduction in both bacterial sheddingand Chlamydia-induced fallopian tube pathology

TC_0037 protein forms a conserved and abundant TTSSneedle structure and may be a strong candidate protein forinclusion in a pan-serovar Chlamydia vaccine [12] We showhere for the first time that TC_0037 a T3SS needle proteinfrom C muridarum represents a good vaccine candidate asit is able to induce specific antibody and T cell responses anddecrease bacterial loads and pathology of the upper genitaltract

To date few studies have examined the use of T3SSproteins as antigens to vaccinate againstChlamydia Recentlyintranasal immunization with a mixture of three T3SS com-ponents CopB CopD and CT584 with CpG resulted in theproduction of specific neutralizing antibodies and decreasedchlamydial infection and Chlamydia-induced pathology [18]In contrast T3SS components have been used intensivelyas vaccine candidates against other Gram-negative bacteriawith considerable success Significant protection against invivo challenge with Shigella has been demonstrated after

vaccination with a Shigella CopB ortholog in combinationwith other T3SS antigens [49] Antibodies to the T3SS tipproteins LcrV in Yersinia spp and PcrV in Pseudomonasaeruginosa have been shown to block these infections [4748] In our study we provided evidence that immunizationwith another component of the chlamydial T3SS stimulatedstrong humoral and cellular immune responses and affordedprotection against intravaginal Chlamydia challenge

Cell-mediated immune responses have been documentedas critical for protection and clearance of Chlamydia infec-tion A large amount of data has been accumulated on therole of CD4+ T cells in protection against genital infec-tion in previous Chlamydia vaccine studies [50 51] Theimportance of IFN-120574 to Chlamydia control in vivo has alsobeen demonstrated [52 53] In our study immunization withAdTC_0037 and rTC_0037-MPLA induced robust specificIFN-120574 CD4+ and CD8+ T cell responses (Figure 5) In thesestudies we used CD4+ and CD8+ enriched T cell populationswith purity of 85 It should be noted that the remaining15 of non-T cells could also contain cells that produce IFN-gamma Besides differences in the remaining subpopulationsof antigen-presenting cells in different groups of mice couldalso affect the resulting IFN-gamma production Futurestudies employing more rigorous protocols of CD4+ andCD8+ T cells isolation will help to elucidate the role ofCD4+ and CD8+ T cells in the response to TC_0037 Prime-boost immunization also coinduced significant levels ofTC_0037-specific IgG2a and IgG1 antibodies that had higherneutralizing activity than those in response to AdTC_0037immunization and they afforded better protection

As reported in other studies [54 55] enhanced clearanceof Chlamydia is dependent upon CD4+ T cell responsesHowever antibodies play a contributing role in the resolutionof primary infections and protect from reinfection [56ndash58] CD4+ and CD8+ T cells have been reported to actcooperatively with antibodies [59ndash61] For example passivetransfer of immune serum protects mice against Chlamydiagenital infection only in the presence of CD4+ T cells [55]Several studies have linked antibodies to protection againstupper genital pathology [60ndash63] Antibodies of IgG2a isotypemediate effector functions including antibody-dependentcell-mediated cytotoxicity (ADCC) with evidence suggest-ing that this effector function facilitates early clearance ofchlamydial infections Furthermore ADCC is associatedwith enhanced antigen presentation with the potential toamplify CD4+ T cell responses [64] In our study wedid not elucidate the direct contribution of specific IgG2aantibodies to better protection from Chlamydia challengebut we observed that a higher level of neutralizing antibodyactivity in AdTC_0037rTC_0037-MPLA-immunized micewas associated with better protection This is in agreementwith previous findings in mice regarding the role of neutral-izing antibodies in protection against Chlamydia [63] Bet-ter neutralizing activity in AdTC_0037rTC_0037-MPLA-immunized mice is possibly connected with the boostingcomponent of our vaccine however further studies on therole of rTC_0037-MPLA in neutralization of chlamydialinfectivity and protection will be helpful Previously anti-bodies raised against T3SS translocators CopB and CopD


(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null

Ad-TC_0037Ad-null + MPLA

Ad-TC_0037 + MPLA + TC_0037

(c)

Figure 8 Prime-boost immunization with AdTC_0037rTC_0037-MPLA affects oviduct pathology in Chlamydia muridarum BALBcmice Mice were immunized with AdTC_0037rTC_0037-MPLA challenged intravaginally with C muridarum and sacrificed 30 days afterinfection Oviducts were analyzed for the presence of a hydrosalpinx (a) Hydrosalpinges in ovaries collected from unvaccinated micecompared to the complete absence of oviduct pathology in vaccinated mice (b) (c) Percent of mice with reduced pathology in AdTC_0037-immunized groups

were reported to inhibit chlamydial infection in vitro sug-gesting that antibodies directed at these proteins block anessential component of T3SS virulence [18] Serum collectedfrom mice immunized with TC_0037 reduced pathogeninfectivity suggesting that these antibodies were directedagainst surface-exposed epitopes on TC_0037 Although themechanism of neutralization remains to be elucidated it waspresumably because antibodies rendered the T3SS inactivepreventing host cell infection

An important characteristic of a Chlamydia vaccine isits ability to decrease bacterial shedding for reduced trans-mission and to produce an immune response that preventsChlamydia-induced immunopathology During the course ofa chlamydial infection in mice bacterial shedding occursfor approximately 14ndash35 days before being cleared from thelower genital tract [18] Based on mouse models of infectionpathology in the upper genital tract is believed to occur asa result of an ascending infection from the lower genitaltract To assess the ability of TC_0037 immunization toreduce bacterial shedding vaginal swabs were collected andbacterial loads were quantified Prime-boost immunizationwith AdTC_0037r TC_0037-MPLA significantly reducedchlamydial IFUs per swab at day 15 and caused almostcomplete eradication (9 of 10 mice) of infection at day 20

after infection (Figure 6) It also decreased bacterial loads inthe upper genital tract (Figure 7) Both results indicate thehigh potential of our vaccine candidate to provide protectionagainst intravaginal chlamydial infection

Previously our group has successfully used Ad5 fordesign of vaccines against influenza [10] Bacillus anthracis[65] Ebola virus [30 31] and some other pathogens Ad5vectors are commonly used among the adenoviruses due toa wide range of their well-described attractive features Theyare replication-defective and can be easily transfected andaccumulated in cell culture [66] They allow incorporationof large antigens that can be secreted by host cell They canbe additionallymodified by the components boosting naturalimmunity It was also shown that they are effective even aftersingle immunization [65] Overall Ad5 is a very promisingdelivery platform actively exploited worldwide In this studywe show that Ad5 can provide an effective delivery system forChlamydia vaccine candidate TC_0037 However we did notcompare the efficacy of our delivery system to other possiblevariants such as virus-like particles This question requiresadditional studies in future

It should be noted that we used adenovirus as a deliverysystem for our antigen only once in our protocol avoid-ing possible complications due to preexisting adenoviral


immunity However taking into account the fact that ouradenoviral delivery system is intended as a new platform for arange of novel vaccines to different pathogens the possibilityof preexisting immunity should be taken into considerationIn our study we employed Ad5 genome modification withmannose-binding lectin to improve the efficacy of our vac-cine and to escape limitations connected with preexistingadenoviral immunity However the practical efficacy of thisapproach expects confirmation in preclinical and clinicaltrials The safety of adenoviral vaccines is another questionthat should be addressed especially for widespread diseaseslike Chlamydia infection Recently our group published theresults of successful clinical trial of Ad5-based Ebola vaccinecandidate that confirmed safety of this vaccine [30 31]Adenovirus-based influenza vaccine has successfully passedphase I clinical trials in the USA and proved to be safe forhumans and highly immunogenic for the influenza A H5N2virus [67] Besides there are a great number of successfulsafety reports published recently by different groups Overallsafety of Ad-based vaccines can be accepted as reasonablyestablished at present

Because the development of oviduct pathology and com-plications are major concerns in individuals infected with Ctrachomatis we examined upper urogenital tracts followingpathogen challenge to determine whether vaccination couldprevent Chlamydia-induced pathology Indeed it completelyeradicated pathology in the upper genital tract in mice vac-cinated with AdTC_0037rTC_0037-MPLA or AdTC_0037alone (Figure 8)

Overall evaluation of AdTC_0037rTC_0037-MPLAvaccination in C muridarum infection model in micedemonstrates a potentially effective vaccine candidatewhich provides B and T cell immune responses and protectsfrom genital tract infection and pathology Howeverto proceed closer to a clinical trial there is a need fortesting of our vaccine candidate in a model employingC trachomatis Besides nonrodent animal models withbetter resemblance to Chlamydia infection in humans canafford higher level of immunological and physiologicalrelevance Finally long-term protection studies are alsohighly urgent to complete the profile of protection for ourvaccine candidate

5 Conclusion

The resolution of genital C muridarum infection coupledwith protection against Chlamydia-induced pathology sug-gests that prime-boost immunization with AdTC_0037rTC_0037-MPLA affords a significant degree of protectionand TC_0037 can be considered for further evaluation as avaccine candidate We are currently investigating the poten-tial of our vaccine candidate to protect against intravaginalC trachomatis infection in mice in our recently developedmurine model [42]

Conflicts of Interest

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Government Program ofthe Russian Federation for the development of a polyvalentadenoviral vaccine expressing C trachomatis target proteinsfor the prophylaxis of urogenital chlamydiosis

References

[1] World Health Organization Global Incidence and Prevalenceof Selected Curable Sexually Transmitted Infections-2008 WorldHealth Organization Geneva Switzerland 2012

[2] K Schautteet E De Clercq and D Vanrompay ldquoChlamydiatrachomatis vaccine research through the yearsrdquo InfectiousDiseases in Obstetrics and Gynecology vol 2011 Article ID963513 9 pages 2011

[3] A J Carey W M Huston K A Cunningham L M HafnerP Timms and K W Beagley ldquoCharacterization of in vitrochlamydia muridarum persistence and utilization in an invivo mouse model of chlamydia vaccinerdquo American Journal ofReproductive Immunology vol 69 no 5 pp 475ndash485 2013

[4] A K Murthy J P Chambers P A Meier G Zhong andB P Arulanandam ldquoIntranasal vaccination with a secretedchlamydial protein enhances resolution of genital Chlamydiamuridarum infection protects against oviduct pathology andis highly dependent upon endogenous gamma interferon pro-ductionrdquo Infection and Immunity vol 75 no 2 pp 666ndash6762007

[5] E K Naglak S G Morrison and R P Morrison ldquoIFN120574 isrequired for optimal antibody-mediated immunity against gen-ital chlamydia infectionrdquo Infection and Immunity vol 84 no 11pp 3232ndash3242 2016

[6] M D Picard J-L Bodmer T M Gierahn et al ldquoResolution ofChlamydia trachomatis infection is associated with a distinct Tcell response profilerdquo Clinical and Vaccine Immunology vol 22no 11 pp 1206ndash1218 2015

[7] S E M Howie P J Horner A W Horne and G EntricanldquoImmunity and vaccines against sexually transmitted Chlamy-dia trachomatis infectionrdquo Current Opinion in Infectious Dis-eases vol 24 no 1 pp 56ndash61 2011

[8] W Paes N Brown A M Brzozowski et al ldquoRecombinantpolymorphicmembrane proteinD in combinationwith a novelsecond-generation lipid adjuvant protects against intra-vaginalChlamydia trachomatis infection in micerdquo Vaccine vol 34 no35 pp 4123ndash4131 2016

[9] H Yu K P Karunakaran X Jiang and R C BrunhamldquoSubunit vaccines for the prevention of mucosal infection withChlamydia trachomatisrdquo Expert Review of Vaccines vol 15 no8 pp 977ndash988 2016

[10] I L Tutykhina D Y Logunov D N Shcherbinin et al ldquoDevel-opment of adenoviral vector-based mucosal vaccine againstinfluenzardquo Journal of Molecular Medicine vol 89 no 4 pp 331ndash341 2011

[11] H J Betts L E Twiggs M S Sal P BWyrick and K A FieldsldquoBioinformatic and biochemical evidence for the identificationof the type III secretion system needle protein of Chlamydiatrachomatisrdquo Journal of Bacteriology vol 190 no 5 pp 1680ndash1690 2008

[12] H J Betts-Hampikian andK A Fields ldquoThe chlamydial type IIIsecretionmechanism revealing cracks in a tough nutrdquo Frontiersin Microbiology vol 19 article 114 2010


[13] A N Russell X Zheng C M OrsquoConnell et al ldquoIdentificationof Chlamydia trachomatis antigens recognized by T cells fromhighly exposed women who limit or resist genital tract infec-tionrdquo Journal of Infectious Diseases vol 214 no 12 pp 1884ndash1892 2016

[14] J Sha M L Kirtley C Klages et al ldquoA replication-defectivehuman type 5 adenovirus-based trivalent vaccine conferscomplete protection against plague in mice and nonhumanprimatesrdquo Clinical and Vaccine Immunology vol 23 no 7 pp586ndash600 2016

[15] N Charro and L J Mota ldquoApproaches targeting the type IIIsecretion system to treat or prevent bacterial infectionsrdquo ExpertOpinion on Drug Discovery vol 10 no 4 pp 373ndash387 2015

[16] W Yang L Wang L Zhang J Qu Q Wang and Y ZhangldquoAn invasive and low virulent Edwardsiella tarda esrB mutantpromising as live attenuated vaccine in aquaculturerdquo AppliedMicrobiology and Biotechnology vol 99 no 4 pp 1765ndash17772015

[17] X Chen S P Choudhari F J Martinez-Becerra et al ldquoImpactof detergent on biophysical properties and immune response ofthe IpaDB fusion protein a candidate subunit vaccine againstShigella speciesrdquo Infection and Immunity vol 83 no 1 pp 292ndash299 2015

[18] D C Bulir S Liang A Lee et al ldquoImmunization with chlamy-dial type III secretion antigens reduces vaginal shedding andprevents fallopian tube pathology following live C muridarumchallengerdquo Vaccine vol 34 no 34 pp 3979ndash3985 2016

[19] S Afkhami Y Yao and Z Xing ldquoMethods and clinicaldevelopment of adenovirus-vectored vaccines against mucosalpathogensrdquo Molecular TherapymdashMethods amp Clinical Develop-ment vol 3 Article ID 16030 2016

[20] R Hadad E Marks I Kalbina et al ldquoProtection against genitaltract Chlamydia trachomatis infection following intranasalimmunization with a novel recombinant MOMP VS24 anti-genrdquo APMIS vol 124 no 12 pp 1078ndash1086 2016

[21] E Lorenzen F Follmann S Boslashje et al ldquoIntramuscular prim-ing and intranasal boosting induce strong genital immunitythrough secretory IgA in minipigs infected with Chlamydiatrachomatisrdquo Frontiers in Immunology vol 6 article 628 2015

[22] C V Nogueira X Zhang N Giovannone E L Sennott andM N Starnbach ldquoProtective immunity against Chlamydia tra-chomatis can engage both CD4+ and CD8+ T cells and bridgethe respiratory and genital mucosaerdquo Journal of Immunologyvol 194 no 5 pp 2319ndash2329 2015

[23] C Arama Y Assefaw-Redda A Rodriguez et al ldquoHeterologousprime-boost regimen adenovector 35-circumsporozoite proteinvaccinerecombinant Bacillus Calmette-Guerin expressing thePlasmodium falciparum circumsporozoite induces enhancedlong-termmemory immunity in BALBcmicerdquoVaccine vol 30no 27 pp 4040ndash4045 2012

[24] D F Hoft A Blazevic J Stanley et al ldquoA recombinant aden-ovirus expressing immunodominant TB antigens can signifi-cantly enhance BCG-induced human immunityrdquo Vaccine vol30 no 12 pp 2098ndash2108 2012

[25] C D Scallan D W Tingley J D Lindbloom J S Toomeyand S N Tucker ldquoAn adenovirus-based vaccine with a double-stranded RNA adjuvant protects mice and ferrets against H5N1avian influenza in oral delivery modelsrdquo Clinical and VaccineImmunology vol 20 no 1 pp 85ndash94 2013

[26] A Sharma M Tandon D S Bangari and S K MittalldquoAdenoviral vector-based strategies for cancer therapyrdquo CurrentDrug Therapy vol 4 no 2 pp 117ndash138 2009

[27] INGN 201 ldquoAd-p53 Ad5CMV-p53 adenoviral p53 p53 genetherapymdashintrogen RPRINGN 201rdquo Drugs in RampD vol 8 no3 pp 176ndash187 2007

[28] E Barnes A Folgori S Capone et al ldquoNovel adenovirus-basedvaccines induce broad and sustained T cell responses to HCVin manrdquo Science Translational Medicine vol 4 no 115 p 115ra12012

[29] O De Santis R Audran E Pothin et al ldquoSafety and immuno-genicity of a chimpanzee adenovirus-vectored Ebola vaccine inhealthy adults a randomised double-blind placebo-controlleddose-finding phase 12a studyrdquo The Lancet Infectious Diseasesvol 16 no 3 pp 311ndash320 2016

[30] I V Dolzhikova O V Zubkova A I Tukhvatulin et al ldquoSafetyand immunogenicity of GamEvac-Combi a heterologous VSV-and Ad5-vectored Ebola vaccine an open phase III trial inhealthy adults in Russiardquo Human Vaccines amp Immunotherapeu-tics pp 1ndash8 2017

[31] Y Shu S Winfrey Z-Y Yang et al ldquoEfficient protein boostingafter plasmid DNA or recombinant adenovirus immunizationwith HIV-1 vaccine constructsrdquoVaccine vol 25 no 8 pp 1398ndash1408 2007

[32] V R Gomez-Roman R H Florese B Peng et al ldquoAnadenovirus-based HIV subtype B primeboost vaccine regimenelicits antibodies mediating broad antibody-dependent cellularcytotoxicity against non-subtype B HIV strainsrdquo Journal ofAcquired Immune Deficiency Syndromes vol 43 no 3 pp 270ndash277 2006

[33] L Vinner DTherrien E Wee et al ldquoImmune response in rhe-sus macaques after mixed modality immunisations with DNArecombinant adenovirus and recombinant gp120 from humanimmunodeficiency virus type 1rdquo APMIS vol 114 no 10 pp690ndash699 2006

[34] S Zolla-Pazner M Lubeck S Xu et al ldquoInduction of neu-tralizing antibodies to T-cell line-adapted and primary humanimmunodeficiency virus type 1 isolates with a prime-boostvaccine regimen in chimpanzeesrdquo Journal of Virology vol 72no 2 pp 1052ndash1059 1998

[35] L J Patterson N Malkevitch J Pinczewski et al ldquoPotent per-sistent induction andmodulation of cellular immune responsesin rhesus macaques primed with Ad5hr-simian immunodefi-ciency virus (SIV) envrev gag andor nef vaccines and boostedwith SIV gp120rdquo Journal of Virology vol 77 no 16 pp 8607ndash8620 2003

[36] V A Stewart S M McGrath P M Dubois et al ldquoPrimingwith an adenovirus 35-circumsporozoite protein (CS) vac-cine followed by RTSSAS01B boosting significantly improvesimmunogenicity to Plasmodium falciparum CS compared tothat with either malaria vaccine alonerdquo Infection and Immunityvol 75 no 5 pp 2283ndash2290 2007

[37] C L Hutchings A J Birkett A C Moore and A V S HillldquoCombination of protein and viral vaccines induces potent cel-lular and humoral immune responses and enhanced protectionfrom murine malaria challengerdquo Infection and Immunity vol75 no 12 pp 5819ndash5826 2007

[38] N Gupta S Vedi D Y Kunimoto B Agrawal and R KumarldquoNovel lipopeptides of ESAT-6 induce strong protective immu-nity against Mycobacterium tuberculosis routes of immuniza-tion and TLR agonists critically impact vaccinersquos efficacyrdquoVaccine vol 34 no 46 pp 5677ndash5688 2016

[39] A V Bagaev A V Pichugin E S Lebedeva et al ldquoRegulationof the target protein (transgene) expression in the adenovirus


vector using agonists of toll-like receptorsrdquo Acta Naturae vol6 no 23 pp 27ndash39 2014

[40] A Tukhvatulin A S Dzharullaeva N M Tukhvatulina etal ldquoPowerful complex immunoadjuvant based on synergisticeffect of combined TLR4 and NOD2 activation significantlyenhances magnitude of humoral and cellular adaptive immuneresponsesrdquo PLoS ONE vol 11 no 5 Article ID e0155650 2016

[41] N A Zigangirova E A Kost L V Didenko et al ldquoA small-molecule compound belonging to a class of 24-disubstituted134-thiadiazine-5-ones inhibits intracellular growth and per-sistence of Chlamydia trachomatisrdquo Journal of Medical Microbi-ology vol 65 no 1 pp 91ndash98 2016

[42] E A Koroleva N V Kobets E S Zayakin S I Luyksaar LA Shabalina and N A Zigangirova ldquoSmall molecule inhibitorof type three secretion suppresses acute and chronic chlamydiatrachomatis infection in a novel urogenital Chlamydia modelrdquoBioMed Research International vol 2015 Article ID 4848532015

[43] H Yu X Jiang C Shen K P Karunakaran and R C BrunhamldquoNovel Chlamydia muridarum T cell antigens induce protectiveimmunity against lung and genital tract infection in murinemodelsrdquo The Journal of Immunology vol 182 no 3 pp 1602ndash1608 2009

[44] S L Gottlieb C D Deal B Giersing et al ldquoThe global roadmapfor advancing development of vaccines against sexually trans-mitted infections update and next stepsrdquo Vaccine vol 34 no26 pp 2939ndash2947 2016

[45] T H T Brown J David E Acosta-Ramirez et al ldquoComparisonof immune responses and protective efficacy of intranasalprime-boost immunization regimens using adenovirus-basedand CpGHH2 adjuvanted-subunit vaccines against genitalChlamydia muridarum infectionrdquo Vaccine vol 30 no 2 pp350ndash360 2012

[46] C Qiu J Zhou X-A Cao G Lin F Zheng and X GongldquoImmunization trials with an avian chlamydial MOMP generecombinant adenovirusrdquo Bioengineered Bugs vol 1 no 4 pp267ndash273 2010

[47] H Lu H Wang H-M Zhao et al ldquoDendritic cells (DCs)transfected with a recombinant adenovirus carrying chlamy-dial major outer membrane protein antigen elicit protectiveimmune responses against genital tract challenge infectionrdquoBiochemistry and Cell Biology vol 88 no 4 pp 757ndash765 2010

[48] A Badamchi-Zadeh P F McKay B T Korber et al ldquoA multi-component prime-boost vaccination regimen with a consensusMOMP antigen enhances Chlamydia trachomatis clearancerdquoFrontiers in Immunology vol 7 article 162 2016

[49] F J Martinez-Becerra J M Kissmann J Diaz-Mcnair et alldquoBroadly protective Shigella vaccine based on type III secretionapparatus proteinsrdquo Infection and Immunity vol 80 no 3 pp1222ndash1231 2012

[50] J Hansen K T Jensen F Follmann E M Agger M Theisenand P Andersen ldquoLiposome delivery of Chlamydia muridarummajor outer membrane protein primes a Th1 response thatprotects against genital chlamydial infection in amousemodelrdquoJournal of Infectious Diseases vol 198 no 5 pp 758ndash767 2008

[51] A W Olsen M Theisen D Christensen F Follmann and PAndersen ldquoProtection against Chlamydia promoted by a sub-unit vaccine (CTH1) compared with a primary intranasalinfection in a mouse genital challenge modelrdquo PLoS ONE vol5 no 5 Article ID e10768 2010

[52] T W Cotter K H Ramsey G S Miranpuri C E Poulsen andG I Byrne ldquoDissemination of Chlamydia trachomatis chronic

genital tract infection in gamma interferon gene knockoutmicerdquo Infection and Immunity vol 65 no 6 pp 2145ndash2152 1997

[53] J I Ito and J M Lyons ldquoRole of gamma interferon incontrolling murine chlamydial genital tract infectionrdquo Infectionand Immunity vol 67 no 10 pp 5518ndash5521 1999

[54] R M Johnson M S Kerr and J E Slaven ldquoPlac8-dependentand inducible NO synthase-dependent mechanisms clearchlamydiamuridarum infections from the genital tractrdquo Journalof Immunology vol 188 no 4 pp 1896ndash1904 2012

[55] L L Perry K Feilzer and H D Caldwell ldquoImmunity toChlamydia trachomatis is mediated by T helper 1 cells throughIFN-gamma-dependent and -independent pathwaysrdquoThe Jour-nal of Immunology vol 158 pp 3344ndash3352 1997

[56] S G Morrison H Su H D Caldwell and R P MorrisonldquoImmunity to murine Chlamydia trachomatis genital tractreinfection involves B cells and CD4+ T cells but not CD8+T cellsrdquo Infection and Immunity vol 68 no 12 pp 6979ndash69872000

[57] S G Morrison and R P Morrison ldquoA predominant role forantibody in acquired immunity to chlamydial genital tractreinfectionrdquo Journal of Immunology vol 175 no 11 pp 7536ndash7542 2005

[58] R P Morrison and H D Caldwell ldquoImmunity to murinechlamydial genital infectionrdquo Infection and Immunity vol 70no 6 pp 2741ndash2751 2002

[59] S Pal E M Peterson and L M De LaMaza ldquoVaccination withthe chlamydia trachomatis major outer membrane protein canelicit an immune response as protective as that resulting frominoculation with live bacteriardquo Infection and Immunity vol 73no 12 pp 8153ndash8160 2005

[60] S Pal O V Tatarenkova and L M de la Maza ldquoA vaccineformulated with the major outer membrane protein can protectC3HHeN a highly susceptible strain of mice from a Chlamy-dia muridarum genital challengerdquo Immunology vol 146 no 3pp 432ndash443 2015

[61] S Pal I Theodor E M Peterson and L M de la MazaldquoMonoclonal immunoglobulin A antibody to the major outermembrane protein of the Chlamydia trachomatis mouse pneu-monitis biovar protects mice against a chlamydial genitalchallengerdquo Vaccine vol 15 no 5 pp 575ndash582 1997

[62] T W Cotter Q Meng Z-L Shen Y-X Zhang H Su andH D Caldwell ldquoProtective efficacy of major outer membraneprotein-specific immunoglobulin A (IgA) and IgG monoclonalantibodies in amurinemodel of Chlamydia trachomatis genitaltract infectionrdquo Infection and Immunity vol 63 no 12 pp4704ndash4714 1995

[63] A W Olsen F Follmann K Erneholm I Rosenkrands andP Andersen ldquoProtection against Chlamydia trachomatis infec-tion and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of themajor outer membrane proteinrdquo The Journal of InfectiousDiseases vol 212 no 6 pp 978ndash989 2015

[64] K Rafiq A Bergtold and R Clynes ldquoImmune complex-mediated antigen presentation induces tumor immunityrdquo TheJournal of Clinical Investigation vol 110 no 1 pp 71ndash79 2002

[65] D N Shcherbinin I B Esmagambetov A N Noskov et alldquoProtective immune response against Bacillus anthracisinduced by intranasal introduction of a recombinantadenovirus expressing the protective antigen fused to theFc-fragment of IgG2ardquo Acta Naturae vol 6 no 20 pp 76ndash842014


[66] E S Sedova D N Shcherbinin A I Migunov et al ldquoRecom-binant influenza vaccinesrdquo Acta Naturae vol 4 pp 17ndash27 2012

[67] K R Van Kampen Z Shi P Gao et al ldquoSafety and immu-nogenicity of adenovirus-vectored nasal and epicutaneousinfluenza vaccines in humansrdquo Vaccine vol 23 no 8 pp 1029ndash1036 2005

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


membrane proteins (Pmps) both of which elicit antibodyand cell-mediated protective immune responses are widelystudied as potent vaccine candidates However the antigenicvariation in MOMP and Pmps suggests that evaluation ofconserved proteins as vaccine candidates could be valuableas this strategy has been successful for developing vaccinesagainst other pathogens [10]

The type three secretion system (T3SS) is the predom-inant virulence factor in Chlamydia It is required for cellinvasion and is active at all life stages [11 12] Some T3SSproteins are surface-exposed and can be targeted by neu-tralizing antibodies The T cell response to T3SS antigenswas recently shown to be associated with protection againstC trachomatis infection in highly exposed women [13] andT3SS components have recently attracted attention as vaccinecandidates against other pathogenic bacteria [14ndash17] The Ctrachomatis T3SS filament protein CdsF and its orthologsin other bacteria form the needles of injectisomes and arebelieved to facilitate the insertion of translocators into thehost cell membrane [11 12 18] CdsF is highly conservedshowing 95 sequence identity in the genus Chlamydia It isabundant on bacterial surfaces raising the possibility that aCdsF-based vaccine may induce a wide range of protectionagainst all medically significant strains

The ability of human adenoviruses to induce stronginnate and adaptive immune responses makes them a pow-erful delivery system to induce an immune response againstan encoded antigen Adenovirus has a natural tropism forthe mucosal epithelium which makes it an ideal vector forvaccination against infections acquired via mucosal surfaces[19] Intranasal immunization in different experimental set-tings has been reported to provide potent protection againstintravaginal challenge [18 20ndash22] Replication-defective ade-novirus vectors (rAds) are widely used for transgene expres-sion in different tissues and gene-based immunizationsEffective vaccines against tuberculosis malaria influenzaand other important infectious diseases have been designedfrom rAds The majority of rAd-based medical preparationsare currently in clinical trials [23ndash27] There is a large bodyof evidence indicating that rAd-derived vaccines are safe andefficacious [28ndash31] Combination of adenoviral-vectored andprotein-in-adjuvant vaccines in ldquoprime-boostrdquo regimenswiththe aim of enhancing both cellular and humoral immuneresponses has shown promising results in HIV and liver-stage malaria vaccine preclinical research [32ndash38] Toll-likereceptor (TLR) agonists are usually codelivered with antigensas adjuvants to enhance the immunogenicity of vaccinecomponents [39]

In this study we expressed the T3SS needle protein ofC muridarum TC_0037 a CdsF ortholog in a replication-defective adenoviral vector (AdTC_0037) and evaluated itsprotective efficacy in an intravaginal Chlamydia infectionmouse model To study the protective immunity of ourvaccine candidate we utilized a prime-boost immunizationprotocol with AdTC_0037 intranasal priming and subcu-taneous boosting with recombinant TC_0037 and TLR4agonist monophosphoryl lipid A (MPLA) mixed in asqualene nanoscale emulsion We found that immunizationwith TC_0037 antigen induced specific humoral and T cell

responses decreased Chlamydia loads in both the lower andupper genital tract and reduced the pathology of uppergenital organs

2 Materials and Methods

21 Bioinformatic Antigen Design The nucleotide sequenceof the C muridarum TC_0037 gene was obtained fromUniProtKB (Q9PLQ8) We performed an in silico analysisof the TC_0037 gene on the presence of a bacterial signalsequence (httpwwwcbsdtudkservicesSignalP) and nobacterial signal sequence was found We further optimizedTC_0037 gene sequences for expression in mouse cells bymodifying its codons with the two most frequent amino acidtriplets Frequent Mus musculus codons were defined usingthe httpwwwkazusaorjpcodon database As TC_0037 isabundant in bacterial cells and small in size (82 amino acids9 kDa) the N-terminal portion of this protein was boundto a 128-amino acid-sized N-terminal portion of mousemannose-binding lectin (MBL) to obtain TC_0037 hexamerscrosslinked with a collagen-like domain The nucleotidesequence of mouse MBL was obtained from UniProtKB(P41317) The MBL-TC_0037 gene was synthesized by Evro-gen (Moscow Russia) in plasmid pAL-TA-MBL-TC_0037

22 Generation of Recombinant Adenoviruses The NotI andHindIII sites of theMBL-TC_0037 fragment were cloned intoshuttle vector pShuttle-CMV (cytomegalovirus) to obtainthe shuttle plasmid pShuttle-CMV-MBL-TC_0037 Homol-ogous recombination was used to generate the replication-defective adenovirus Ad-MBL-TC_0037 (Figure 1) For thispurpose the plasmid pShuttle-CMV-MBL-TC_0037 was lin-earized by PmeI mixed with pAd-Easy (Adenoviral Vec-tor System StratoGen New York NY) and then cotrans-formed into Escherichia coli (BJ5183 strain) The obtainedrecombinant clones were used to extract plasmid DNAwhose molecular weight was later assessed E coli strainDH5alpha was transformed with plasmids larger than 20 kbpbecause this strain unlike BJ5183 allows one to producea sufficient amount of recombinant plasmids The purifiedplasmid clones were analyzed by both cleavage with theHindIII restriction endonuclease and polymerase chain reac-tion (PCR) Next we studied the infectivity of the describedplasmids in permissive cells HEK-293 cells were transfectedwith plasmid pAd-MBL-TC_0037 and linearized by PacITransfection was performed in a 24-well plate using Lipo-fectamine 2000 (Invitrogen Burlington Canada) Ten daysafter the transfection the cells were collected and subjectedto a freeze-thaw cycle the obtained lysate containing recom-binant adenoviruses was used to infect HEK-293 cells in a35mm dish After 5 days specific lysis caused by cytopathiceffect of the recombinant viruses was detectedThe lysate wasused to extract DNA and perform a PCR analysis The celllysate was shown to contain DNA of recombinant humanadenovirus serotype 5 (rAd5) carrying insertions that encodethe protective antigen

23 Virus Accumulation Recombinant adenovirus serotype5 (rAd5) was grown in HEK-293 cells as described elsewhere


ΔE1 ΔE3

100

pACMV

(a)

(b)

0

1 10 20 30 40 50 60

70 80 90 100 110 120

130 140 150 160 170

190 200 210 220 227

180

MBL

MBL









600








Priming inAdTC_0037


Day 0 Day 14 Day 28

C muridarum ivag



Day 21


Day 58Day 31 Day 48

Vaginal swabs

Uterine samples


assay Detection IFU





2CO3(01M















2at 37∘C


10IFU per vaginal

swab




3 Results




lowastlowast


00

02

04

06

08

10

12

14

16

18Se

rum

TC_

0037

spec

ific I

gG2a

200 400 800100Dilution


Ad-null + MPLA(a)

lowast lowast

00

02

04

06

08

10

12

14

16

18

Seru

m T

C_00

37 sp

ecifi

c IgG

1

200 400 800100Dilution



Ad-null + MPLA(b)


lowast

lowast

0

20

40

60

80

100

Neu

tral

izat

ion

()

64 128 256 51232Dilution








200

400

600

800IF

N-훾

SFU

2times10

5T

cells




Ad-null + MPLA

CD4+

(a)


200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast



Ad-null + MPLA

CD8+

(c)


200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)






Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ΔE1 ΔE3

100

pACMV

(a)

(b)

0

1 10 20 30 40 50 60

70 80 90 100 110 120

130 140 150 160 170

190 200 210 220 227

180

MBL

MBL









600








Priming inAdTC_0037


Day 0 Day 14 Day 28

C muridarum ivag



Day 21


Day 58Day 31 Day 48

Vaginal swabs

Uterine samples


assay Detection IFU





2CO3(01M















2at 37∘C


10IFU per vaginal

swab




3 Results




lowastlowast


00

02

04

06

08

10

12

14

16

18Se

rum

TC_

0037

spec

ific I

gG2a

200 400 800100Dilution


Ad-null + MPLA(a)

lowast lowast

00

02

04

06

08

10

12

14

16

18

Seru

m T

C_00

37 sp

ecifi

c IgG

1

200 400 800100Dilution



Ad-null + MPLA(b)


lowast

lowast

0

20

40

60

80

100

Neu

tral

izat

ion

()

64 128 256 51232Dilution








200

400

600

800IF

N-훾

SFU

2times10

5T

cells




Ad-null + MPLA

CD4+

(a)


200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast



Ad-null + MPLA

CD8+

(c)


200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)






Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Priming inAdTC_0037


Day 0 Day 14 Day 28

C muridarum ivag



Day 21


Day 58Day 31 Day 48

Vaginal swabs

Uterine samples


assay Detection IFU





2CO3(01M















2at 37∘C


10IFU per vaginal

swab




3 Results




lowastlowast


00

02

04

06

08

10

12

14

16

18Se

rum

TC_

0037

spec

ific I

gG2a

200 400 800100Dilution


Ad-null + MPLA(a)

lowast lowast

00

02

04

06

08

10

12

14

16

18

Seru

m T

C_00

37 sp

ecifi

c IgG

1

200 400 800100Dilution



Ad-null + MPLA(b)


lowast

lowast

0

20

40

60

80

100

Neu

tral

izat

ion

()

64 128 256 51232Dilution








200

400

600

800IF

N-훾

SFU

2times10

5T

cells




Ad-null + MPLA

CD4+

(a)


200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast



Ad-null + MPLA

CD8+

(c)


200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)






Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2at 37∘C


10IFU per vaginal

swab




3 Results




lowastlowast


00

02

04

06

08

10

12

14

16

18Se

rum

TC_

0037

spec

ific I

gG2a

200 400 800100Dilution


Ad-null + MPLA(a)

lowast lowast

00

02

04

06

08

10

12

14

16

18

Seru

m T

C_00

37 sp

ecifi

c IgG

1

200 400 800100Dilution



Ad-null + MPLA(b)


lowast

lowast

0

20

40

60

80

100

Neu

tral

izat

ion

()

64 128 256 51232Dilution








200

400

600

800IF

N-훾

SFU

2times10

5T

cells




Ad-null + MPLA

CD4+

(a)


200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast



Ad-null + MPLA

CD8+

(c)


200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)






Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast


00

02

04

06

08

10

12

14

16

18Se

rum

TC_

0037

spec

ific I

gG2a

200 400 800100Dilution


Ad-null + MPLA(a)

lowast lowast

00

02

04

06

08

10

12

14

16

18

Seru

m T

C_00

37 sp

ecifi

c IgG

1

200 400 800100Dilution



Ad-null + MPLA(b)


lowast

lowast

0

20

40

60

80

100

Neu

tral

izat

ion

()

64 128 256 51232Dilution








200

400

600

800IF

N-훾

SFU

2times10

5T

cells




Ad-null + MPLA

CD4+

(a)


200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast



Ad-null + MPLA

CD8+

(c)


200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)






Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















200

400

600

800IF

N-훾

SFU

2times10

5T

cells




Ad-null + MPLA

CD4+

(a)


200

400

600

800

IFN

-훾SF

U2

times10

5T

cells

lowast lowast

lowastlowast



Ad-null + MPLA

CD4+

(b)


C muridarum

0

200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowastlowastlowast

lowastlowastlowast



Ad-null + MPLA

CD8+

(c)


200

400

600

800

1000

IFN

-훾SF

U2

times10

5T

cells

lowast

lowast

lowastlowast



Ad-null + MPLA

CD8+

(d)






Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Day 90

1

2

3

4

5

6IF

Us p

er v

agin

al sw

ab(log10

)


Ad-null + MPLA(a)

Day 15

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA

lowast lowast

(b)

Day 20

IFU

s per

vag

inal

swab

0

1

2

3

4

5

6

(log10

)


Ad-null + MPLA


(c)





4 Discussion



Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Groups0

25

50

75

100

P

CR-p

ositi

ve o

f mic

egr

oup

lowastlowast

lowast


Ad-null + MPLA









(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

010 110

1010

0

20

40

60

80

100

of a

ffect

ed o

vidu

cts

C muridarumAd-null


Ad-TC_0037 + MPLA + TC_0037

(c)











5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion




Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Chlamydial Type III Secretion System Needle Protein ...downloads.hindawi.com/journals/bmri/2017/3865802.pdf · ResearchArticle Chlamydial Type III Secretion System Needle Protein