Vaccine 21 (2003) 1694–1703

RecombinantVibrio choleraeghosts as a delivery vehicle forvaccinating againstChlamydia trachomatis

Francis O. Ekoa,∗, Werner Lubitzb, Lucinda McMillana, Kiantra Rameya, Terri T. Moorea,Godwin A. Ananabac, Deborah Lyna, Carolyn M. Blackd, Joseph U. Igietsemea,d

a Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Dr. SW Atlanta, GA 30310, USAb Institute of Microbiology and Genetics, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria

c Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA 30314, USAd National Center for Infectious Disease, CDC, Atlanta, GA 30333, USA

Received 2 May 2002; received in revised form 12 September 2002; accepted 1 October 2002

Abstract

An efficacious vaccine is needed to control the morbidity and burden of rising healthcare costs associated with genitalChlamydiatrachomatisinfection. Despite considerable efforts, the development of reliable chlamydial vaccines using conventional strategies hasproven to be elusive. The 40 kDa major outer membrane protein (MOMP) ofC. trachomatisis so far the most promising candidate fora subunit vaccine. The lack of satisfactory protective immunity with MOMP-based vaccine regimens to date would suggest that eitherMOMP alone is inadequate as a vaccine candidate or better delivery systems are needed to optimize the effect of MOMP. RecombinantVibrio choleraeghosts (rVCG) are attractive for use as non-living vaccines because they possess strong adjuvant properties and areexcellent vehicles for delivery of antigens of vaccine relevance to mucosal sites. The suitability of the ghost technology for designing ananti-chlamydial vaccine was evaluated by constructing a rVCG vector-based candidate vaccine expressing MOMP (rVCG-MOMP) andassessing vaccine efficacy in a murine model ofC. trachomatisgenital infection. Intramuscular delivery of the rVCG-MOMP vaccineinduced elevated local genital mucosal as well as systemic Th1 responses. In addition, immune T cells from immunized mice couldtransfer partial protection against aC. trachomatisgenital challenge to naıve mice. These results suggest that rVCG expressing chlamydialproteins may constitute a suitable subunit vaccine for inducing an efficient mucosal T cell response that protects againstC. trachomatisinfection. Altogether, the potency and relatively low production cost of rVCG offer a significant technical advantage as a chlamydialvaccine.© 2002 Elsevier Science Ltd. All rights reserved.

Keywords:rVCG-MOMP; Vaccine; Protection

1. Introduction

Infection of the genitourinary tract of humans with theobligate intracellular bacterium,Chlamydia trachomatis, isa major cause of sexually transmitted diseases[1]. Geni-tal infection of women poses a significant risk often lead-ing to pelvic inflammatory disease, ectopic pregnancy andinfertility [2,3]. Most genital tract infections in women areasymptomatic with severe complications often being the firstsymptoms of an infection. Although antibiotic therapy ef-fectively eliminates chlamydial infection, it does not alwaysaffect established pathology[4], and the rampant asymp-tomatic infections make treatment of symptomatic individ-uals alone unlikely to be a successful control strategy. A

∗ Corresponding author. Tel.:+1-404-752-1584; fax:+1-404-752-1179.E-mail address:feko@msm.edu (F.O. Eko).

vaccine againstC. trachomatisis an attractive approach andthe most promising and effective strategy for controllingthese diseases.

Early human trials of candidate vaccines composed ofwhole chlamydial cells revealed that vaccinated individualssuffered exacerbated disease during subsequent infectionepisodes[5–8]. Thus, the use of whole chlamydial agentsas vaccine candidates appears to be unattractive due tothe potential existence of immunopathogenic components[9]. Besides, progress made in molecular immunology andbiotechnology in the last two decades has led to a gradualshift from the classical whole vaccines, consisting of inac-tivated and live-attenuated intact pathogens or their inacti-vated toxins, to peptide or subunit vaccines. Thus, currentresearch has focused on the development of vaccines basedon chlamydial subunit components. Although additional im-munogenic proteins have recently been predicted[10–13],

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.PII: S0264-410X(02)00677-1

F.O. Eko et al. / Vaccine 21 (2003) 1694–1703 1695

there are eight major serologically defined chlamydial anti-gens recognized during human infection by immunoblottinganalysis of sera from women withC. trachomatiscervical in-fections[9,14–16]. Among potential protective antigens, theimmunodominant 40 kDa MOMP protein is the best char-acterized, and the antigen that has shown most promise as acandidate vaccine. Purified MOMP and a number of otherimmunogenic subunits have been evaluated for their pro-phylactic and therapeutic potential in several animal models[17–20]. To date these efforts have only been partially ef-fective. This may suggest that MOMP alone is inadequate,calling for a multi-subunit approach, or that more effectivedelivery systems and adjuvants are needed to boost immuneresponses. As an alternative to purified antigens, clonedrecombinant fragments of chlamydial proteins[18,21–23]as non-replicating antigens, naked DNA or live recom-binant vectors[24,25] are other promising vaccine can-didates.

Genetic inactivation of pathogenic bacteria by the con-trolled expression of cloned bacteriophage PhiX174 lysisgeneE offers a promising new approach in non-living vac-cine technology[26,27]. Expression of plasmid-encodedgeneE leads to the formation of a transmembrane tunnelstructure through the cell envelope of Gram-negative bac-teria and the loss of cytoplasmic contents. The resultingbacterial ghosts share the functional and antigenic determi-nants of the envelope with their living counterparts[28].RecombinantVibrio cholerae ghosts (rVCG) are attrac-tive for use as non-living vaccines because they possessintrinsic adjuvant properties, maintain the structural andfunctional integrity of expressed antigens, and are excellentvehicles for delivery of foreign or heterologous proteinsand other antigens of vaccine relevance to the primaryantigen-presenting cells (APCs)[27]. It has been demon-strated that the cell targeting and adjuvant properties ofghosts are superior to Alum and complete Freund’s adjuvant[27]. Furthermore, the ghost technology can accommodatemultiple proteins that can lead to a multi-subunit vac-cine with greater potential for immunogenicity and protec-tion.

To test the adjuvant properties of rVCG in enhanc-ing the efficacy of an anti-chlamydial subunit vaccine,we have constructed a rVCG vector-based candidate vac-cine expressing MOMP (rVCG-MOMP) and evaluated itsability to induce a local genital Thl response and con-fer protection against aC. trachomatisgenital infectionin mice. The results showed that rVCG-MOMP admin-istered intramuscularly (IM) induced a genital mucosalchlamydial-specific Thl response. Also, the adoptive trans-fer of T cells from immunized mice into naıve recipientsconferred protection from significant genital chiamydialchallenge. These results suggest that appropriately atten-uated or inactivated bacterial vectors may be effectivealternative methods of delivering subunit chlamydial anti-gens that will elicit protective cellular and humoral immuneresponses.

2. Materials and method

2.1. DNA preparation and PCR amplification

Stock preparations ofC. trachomatisserovar D strainwere generated by propagating elementary bodies (EBs)in HeLa cells as previously described[29]. All stockswere titrated on HeLa cell monolayers followed by purifi-cation of EBs over renografin gradients[29] and storedat −70◦C. Genomic DNA was purified from 1× 108

chlamydial EBs using the QIAGEN DNeasy Tissue Kit(Qiagen, Valencia, CA) according to the manufacturer’sinstructions. The MOMP gene,omp1, was obtained bypolyrmerase chain reaction (PCR) amplification from puri-fied genomic DNA. The complete omp1 coding sequencewas amplified with the Expand High Fidelity PCR System(a unique mix of Taq and Pwo DNA polymerases) (Roche,Mannheim, Germany) using the oligonucleotide primersEF8F (5′-GGCCGGGCCCAGATG-AAAAAACTCT-3 ′)and EF6R (5′-ACAATCTGCAGTATTAGAAGCGGAA-3′),which containApa1 and Pst1 restriction sites (bold), re-spectively. The amplification reaction was carried out in aGeneAmp PCR System (Perkin-Elmer) and an amplifiedPCR product of the correct size (∼1200 bp) was isolatedfrom a 1% agarose gel and purified with the QlAquick PCRpurification kit (Qiagen, Valencia, CA).

2.2. Construction of the expression vector, pCOM12

Theomp1PCR product was digested withApa1 andPst1enzymes, purified and subcloned into theApa1-Pst1-cleavedmembrane-targeting vector, pKSEL5-2[30], such that thedirection of omp1 transcription was the same as that ofthe upstreamlacZ gene (Fig. 1). The resulting recom-binant expression plasmid, (designated pCOM12) con-tained theC. trachomatisomp1 gene inserted between theLacZ′ and L′ genes (i.e. L′ targeting). The plasmid wasrestriction-digested and sequenced to certify the size andorientation of the cloned insert.

2.3. Expression of recombinant MOMP (rMOMP) inV. cholerae 01

The pCOM12 was introduced intoV. cholerae01 strainH1 by electroporation and clones containing the plasmid,were isolated and designated HM12. The expression ofMOMP by HM12 cells was evaluated by immunoblottinganalysis. Cultures of HM12 and control HI (pKSEL5-2)were grown to mid-log phase under appropriate conditionsand rMOMP expression was induced by the addition of2 mM IPTG (isopropyl-�-d-thiogalactopyranoside) (RocheDiagnostics, IN, USA). Samples were removed at differenttime intervals, solubilized in sample buffer and separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) as previously described[31]. Purified MOMPsubjected to the same conditions was included as a positive

1696 F.O. Eko et al. / Vaccine 21 (2003) 1694–1703

Fig. 1. Construction of pCOM12. The serovar D MOMP gene,omp1, was amplified from genomic DNA with primers, which flanked either end of theomp1 open reading frame. The primers included the restriction sitesApa1 and Pst1 (underlined) used for cloning into the corresponding sites of themembrane targeting vector, pKSEL5-2, resulting in pCOM12. E′ and L′ are target sequences for membrane anchorage. Expression of rMOMP frompCOM12 is under the transcriptional regulation of thelac promoter (lacpo). Abbreviations: bla,�-lactamase gene;ColEl, replication origin;LacZ′,5′-sequences of�-galactosidase gene (lacz); E′, 5′-sequences of geneE from bacteriophage (PhiX174; L′, 3′-sequences of gene L from phage MS2;mcsl, mcs2, mcs3, different multiple cloning sites;omp1, gene coding for chlamydial major outer membrane protein.

control. Following protein transfer, rMOMP was detectedusing the mouse anti-MOMP antibody L2 1–10 kindly pro-vided by Dr. Harlan D. Caldwell, NIAID/NIH, Hamilton,MT.

2.4. Immunofluorescence of rMOMP-expressing cells

The expression of rMOMP by rVCG-MOMP was ana-lyzed by immunofluorescence staining. Purified EBs andVCG alone were included in the analysis as positive andnegative controls, respectively. EBs and lyophilized ghostpreparations were fixed with methanol on a microscope slideand washed with PBS. Fixed, air-dried smears were stainedwith the FITC conjugated monoclonal anti-MOMP antibodyYVS1653 (H1119) (Accurate Chemical & Scientific Corpo-ration, Westbury, NY, USA) for 35 min followed by Evansblue dye (0.5% in PBS) for 10 min. After washing withPBS, a drop of 90% glycerol was placed on each slide, cov-ered with a cover slip and MOMP protein expression wasdetected by fluorescence microscopy. Microscopy was car-ried out on an Olympus fluorescence microscope equippedwith an MTI DC-330 3CCD color camera (Dage-MTI Inc.,Michigan City, IN, USA) for image capture.

2.5. Production of recombinant V. cholerae ghostsexpressing MOMP (rVCG-MOMP)

V. cholerae 01 strain HM12 (containing the clonedomp1gene) was cotransfonned with the lysis plasmid pD-KLOI [32] to yield clone HM12E. Transcription of geneE from pDKLOI is directed by the activity of thePm/xylSpromoter–repressor system of thePseudomonas TOLplas-mid [32]. Bacteria were grown in brain heart infusion (BHI)broth containing ampicillin (100�g/ml) and kanamycin(25�g/ml) at 37◦C to mid-log phase and IPTG was addedto the growing culture to induce foromp1gene Expression.After 45 min of MOMP induction, cell lysis was achievedby adding 3-methyl benzoate (to 5 mM) to induce geneEexpression. At the end of lysis, cultures were harvested bycentrifugation, resuspended in a low ionic buffer or dis-tilled water and washed with PBS. Harvested ghosts wereresuspended in PBS and then lyophilized. The efficiency ofE-mediated killing of vibrios was estimated by plating sam-ples on BHI agar as previously described[33]. Results indi-cated a 99.99% killing efficiency; no colony-forming unitswere found on plates at any dilution. Lyophilized VCG wereweighed and the number of cfu/mg of VCG was calculated.

F.O. Eko et al. / Vaccine 21 (2003) 1694–1703 1697

2.6. Immunization protocol

Five- to eight-week-old female BALB/c mice were pur-chased from the Jackson Laboratory, Bar Harbor, Maine.The animals were housed in laminar flow racks underpathogen-free conditions at a constant temperature of 24◦Cwith a cycle of 12 h of light and 12 h of darkness andwere fed mouse chowder and water ad libitum. Immuniza-tions were carried out using a 1-ml syringe fitted with a27-guage needle. In this study, the intramuscular route ofimmunization was adopted since this route was previouslyshown to be most effective at inducing Thl response againstchlamydial genital infection[34]. Groups of mice (10 pergroup) were vaccinated intramuscularly with rVCG-MOMPor VCG alone as follows: groups 1 and 2 received 1 and0.01 mg, respectively, of lyophilized rVCG-MOMP whilegroups 3 and 4 received equivalent doses of VCG alone.Group 5 served as the negative control and received 50�lof PBS. The 1 mg of lyophilized rVCG-MOMP or VCGcorresponded to about 2× 109 cfu. All immunizations weregiven in 50�l of PBS while under phenobarbitol anes-thesia [29] and animals were boosted twice at 2 weeklyintervals.

2.7. Assessment of genital mucosal and systemicanti-chlamydia Thl response

Enzyme-linked immunosorbent assay (ELISA) kits forquantitating the amounts of immune cytokines in biolog-ical and culture fluids were purchased from BioSourceInternational, Camarillo, CA. The levels of genital mu-cosal and systemic Thl responses were determined bymeasuring the response of chlamydial-specific, gammainterferon (IFN-�)-secreting T cells from genital tissuesand spleens of immunized mice, as previously described[35].

2.7.1. Preparation of T cells from the genital tracttissue and spleens of immunized mice

Genital tracts (or the draining iliac lymph nodes) andspleens were harvested from immunized mice 2 weeks afterthe last immunization. Immune T cell-enriched cells wereprepared from the genital tract tissues by the collagenasedigestion and nylon wool enrichment methods as previouslydescribed[35,36]. Briefly, at the indicated time after im-munization, animals in each group were sacrificed, and thegenital tract between the vagina and ovaries (i.e. the cervix,uterus, and Fallopian tubes) was excised and placed in sterileHEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid)-buffered RPMI 1640 culture medium (Atlanta Bi-ologicals, Norcross, Ga.). Explants were transferred to7 ml of 0.6 mg/ml of filter-sterilized type I collagenase(Atlanta Biologicals, Norcross, GA). Minced tissues wereincubated at 37◦C for 45–60 min, then teased with for-ceps and passed through a cell strainer. After washing,enrichment for T cells was achieved by the nylon wool

adherence method[36,37]. Purified genital, lymph nodeor splenic cells contained at least 90% CD3+ cells, asdetermined by fluorescence-activated cell sorting analysis(FACS).

2.7.2. Assessment of Thl responseThe levels of mucosal and systemic Thl responses in-

duced by the immunizations were assessed by measuring theamounts of chlamydial-specific IFN-� produced by stimu-lated T cells[35]. Briefly, purified T cells and APCs wereseeded into 96-well tissue culture plates (Costar, Cambridge,MA) at 2× 105 cells per well, in the presence or absence ofUV-inactivated serovar D EBs as antigen. APCs were pre-pared by�-irradiation (∼2000 rad; Gammacell 1000 Elite,Nordian International, Kanata, Canada) of spleen cells fromnaıve mice. Following 5 days of incubation at 37◦C and 5%CO2, the supernatants were harvested and stored at−70◦Cuntil assayed for cytokine content by a quantitative ELISAprocedure. The amounts of IFN-� contained in supernatantsderived from culture-stimulated cells and controls were mea-sured by using a commercial ELISA kit (Cytoscreen Im-munoassay Kit; BioSource) according to the manufacturer’sinstructions. The concentration of each cytokine was ob-tained by extrapolation from a standard calibration curvegenerated simultaneously. Data were calculated as the meanvalues (±S.D.) of triplicate cultures for each experiment.Results were derived from at least three independent exper-iments.

Fig. 2. Western immunoblot of the 40-kDa MOMP detected with themouse anti-MOMP antibody, L2 1–10. Mid-log phase cultures were in-duced with IPTG for 45 min and cell lysates assayed for rMOMP expres-sion by Western blot. Lane 1:V. choleraeHI cells containing the plasmidvector, pKSEL5-2; lane 2: purified MOMP fromC. trachomatisEBs; lane3: V. choleraeH1 cells containing the recombinant plasmid, pCOM12, ex-pressing rMOMP. Numbers to the left are approximate molecular masses,in kilodaltons (kDa).

1698 F.O. Eko et al. / Vaccine 21 (2003) 1694–1703

2.8. Protection studies

Three groups of mice (six mice per group) were im-munized IM three times 2 weeks apart as follows: groups1 and 2 received, respectively, 1 mg of rVCG-MOMPor VCG alone while group 3 received 50�l of PBS.Three weeks after the last immunization, T cells wereisolated from the spleens by the nylon wool adherencemethod and adaptively transferred into natıve mice at2.5 × 107 cells per mouse (2.5 spleen equivalents). Themice were then challenged intravaginally with 104 ifuof live C. trachomatisserovar D in 30�l of PBS, 24 hafter the adoptive transfer while under phenobarbitolanesthesia. Infections were monitored by cervicovagi-nal swabbing of individual animals every 3 days andChlamydia was isolated from swabs in tissue culture asreported previously[29]. The experiment was repeatedtwice.

Fig. 3. Characterization ofV. choleraeand V. choleraeghosts (VCG): (A) field emission scanning electron micrograph (FESEM) ofV. cholerae01 HI;(B) transmission electron micrograph (TEM) of ultrathin sections ofV. cholerae01 HI (pDKLOl) prior to geneE induction; (C) protein E-lysedV.cholerae01 cells (VCG) with intact cellular morphology but devoid of cytoplasmic contents; (D) FESEM of a VCG showing the transmembrane lysistunnel (arrow) and surface structures.

2.9. Statistical analysis

The levels of IFN-� in samples from different experimentswere analyzed and compared by performing a Student’st-test, and the relationship between different experimentalgroups was assessed by analysis of variance. The level ofsignificance was judged atP < 0.05.

3. Results

3.1. Cloning and expression of chlamydial MOMP in V.cholerae 01

V. cholerae01 clone HM12 harbors plasmid pCOM12,which was obtained by cloning the MOMP gene,omp1intothe membrane-targeting vector pKSEL5-2 (Fig. 1). Restric-tion enzyme and sequencing analyses confirmed the plasmid

F.O. Eko et al. / Vaccine 21 (2003) 1694–1703 1699

to containomp1sequences downstream from thelac pro-moter. In pCOM12, rMOMP protein is expressed under thetranscriptional control of thelacpo promoter. The expressionand identity of rMOMP protein was confirmed by West-ern blotting using a mouse monoclonal antibody to MOMP(Fig. 2). Chlamydial MOMP was not detectable in the teststrain harboring the plasmid vector alone. Three forms ofrMOMP were detected. The major band comigrated with the40 kDa authentic mature MOMP purified from EBs. Smallerforms, approximately 36 and 23 kDa, possibly representingdegradation products were detected at lower concentrations.

3.2. Production and characterization of recombinant V.cholerae ghosts expressing MOMP (VCG-MOMP)

V. choleraeghosts were produced by protein E-mediatedlysis of bacterial cells[31]. Induction of thePmpromoter bythe addition of methyl benzoate to the growing bacterial cul-ture resulted in rapid de-repression and protein E-mediatedlysis. The extent and rate of lysis was quantified from thedecrease in turbidity per unit of time, measured by theabsorbance of the lysing culture at 600 nm. Electron mi-croscopic analysis of protein E-lysedV. cholerae01 cellsrevealed no gross alterations in cellular morphology in com-parison with unlysed cells (Fig. 3A–C). Fig. 3D shows ahigh-resolution field emission scanning electron micrographof a protein E-lysedV. cholerae01 cell; except for the lysispore (arrow), the morphology of the cell, including all cellsurface structures and appendages, is unaffected by the lysisevent.

The stability of rMOMP anchored on the membrane ofrVCG was investigated by staining ghost preparations todetect MOMP by immunofluorescence using anti-MOMPantibody (Fig. 4B). No fluorescence was found in samples ofVCG alone (Fig. 4C). MOMP-specific immunofluorescencestaining of EBs was also shown (Fig. 4A).

3.3. Induction of local genital mucosal and systemicchlamydial-specific Thl response by rVCG-MOMP

The current immunologic paradigm for designing andevaluating a potentially efficacious chlamydial vaccine is

Fig. 4. Expression of MOMP in VCG andChlamydiaEBs. The expression of MOMP was detected by indirect immunofluorescence staining using theFITC conjugated monoclonal anti-MOMP antibody YVS1653 (HI119) from (A)C. trachomatisserovar D EBs and (B) recombinantV. choleraeghostsexpressing MOMP (rVCG-MOMP). No fluorescence was observed in VCG alone (C).

Fig. 5. Induction of local genital mucosal chlamydial-specific Thl responseby rVCG-MOMP. Groups of mice were vaccinated intramuscularly, threetimes, 2 weeks apart with either rVCG-MOMP or VCG alone. Local Thlresponse was assessed, 2 weeks after immunization, by measuring theresponse of chlamydial-specific, IFN-�-secreting T cells from the genitaltract tissue of immunized mice. The amounts of IFN-� contained in cul-ture supernatants derived from culture-stimulated cells and controls weremeasured by ELISA. The concentration of the cytokine in each samplewas obtained by extrapolation from a standard calibration curve generatedsimultaneously. Data were calculated as the mean values± S.D. of trip-licate cultures for each experiment. The control cultures without antigendid not contain detectable levels of IFN-� and so the data were excludedfrom the results. The results are from three independent experiments.

based on the ability to induce a Thl response[38]. Lev-els of mucosal and systemic Thl responses induced weretherefore, evaluated in mice immunized with rVCG-MOMPand control groups (mice immunized with VCG alone).Within 2 weeks post-immunization, IM immunization withrVCG-MOMP induced a positive local genital mucosalThl response that measured 270.80± 5.49 pg/ml of IFN-�(Fig. 5).

The ability of rVCG-MOMP to induce a systemicchlamydial-specific Th1 response was also determined.Immunization with VCG-MOMP induced a specific Thl

1700 F.O. Eko et al. / Vaccine 21 (2003) 1694–1703

Fig. 6. Induction of chlamydial-specific systemic Thl response byrVCG-MOMP. Groups of mice were vaccinated intramuscularly, threetimes, 2 weeks apart with either rVCG-MOMP or VCG alone. The levelof Thl response induced was determined by measuring the response ofchlamydial-specific, IFN-�-secreting T cells from spleen cells of immu-nized mice. The amounts of IFN-� contained in culture supernatants de-rived from culture-stimulated cells and controls were measured by ELISA.The concentration of the cytokine in each sample was obtained by ex-trapolation from a standard calibration curve generated simultaneously.Data were calculated as the mean values (±S.D.) of triplicate culturesfor each experiment. The control cultures without antigen did not con-tain detectable levels of IFN-� and so the data were excluded from theresults. The results are from three independent experiments.

response measured by the level of IFN-� produced by Tcells from spleens of immunized mice (22.73± 0.60 pg/ml)at 2 weeks post-immunization (Fig. 6). To assess whetherTh2 responses were induced following immunization,antigen-specific IL-4 levels were evaluated in stimulated Tcells. IL-4 production was detected at very low levels and weobserved no significant difference in IL-4 levels producedin genital tract tissue (8.98 ± 0.41 pg/ml) and spleen cells(9.31± 2.62 pg/ml). Taken together, these data show that aVCG-MOMP vaccine formulation is capable of inducing achlamydial-specific genital mucosal as well as systemic Thlresponse indicating that VCG can function as an efficientdelivery vehicle for a chlamydial MOMP-based vaccine.

3.4. Induction of protective immunity by rVCG-MOMP

We hypothesized that chlamydial-reactive T cells fromimmunized animals showing vigorous mucosal Thl responsewould confer protection against aC. trachomatischallengeinfection. Therefore, the ability of rVCG-MOMP to induceprotective immunity that could be transferred by immune Tcells was determined. Purified splenic T cells from mice im-munized IM with rVCG-MOMP or VCG alone were adop-tively transferred into naıve animals and recipients werechallenged intravaginally, 24 h after transfer, with homolo-gous liveC. trachomatisserovar D. The level of protective

Fig. 7. Efficacy of protective immunity conferred by immune T cells frommice immunized with rVCG-MOMP or VCG. T cells from mice immu-nized with rVCG-MOMP, VCG alone or naıve mice were adoptively trans-ferred into naıve recipients at 2.5 spleen equivalents per mouse. The micewere challenged intravaginally with 104 ifu of live C. trachomatisserovarD after 24 h of cell transfer. The number of ifus recovered from cervicov-aginal swabs was determined at the indicated time points post-challenge.The data show the mean recoverable ifus, expressed as log10 ifu/ml, foreach experimental group± S.D. Student’s t-test was used to comparedifferences between control and experimental groups atP < 0.05.

immunity was assessed by cervicovaginal swabbing of theanimals and isolation of chlamydiae in tissue culture. Re-cipients of T cells from IM rVCG-MOMP-immunized micewere highly resistant to infection, shedding 182.8, 87.0, 12.7,0 and 0 ifu/ml on days 3, 6, 9, 12 and 15 after challenge,compared to mice immunized with VCG alone which shed2050.0, 3640.0, 3100.0, 2050.0 and 638.0 ifu/ml at thesetime points (Fig. 7). In addition, recipients of T cells fromnaıve control mice were not protected. These results suggestthat IM immunization of mice with rVCG-MOMP elicit Tcells capable of conferring protection against aC. trachoma-tis genital infection.

4. Discussion

A vaccine represents the best approach to protect thegreatest number of people from the widespread and oftendevastating consequences of genital chlamydial infections.In the continued search for an efficacious vaccine, it appearsthat the use of whole chlamydial agents is unattractive dueto the potential existence of pathologic reactions to certainchlamydial antigens[6,9]. With attention focused on design-ing vaccines based on subunit components, the challenge toovercome poor immunogenicity by designing effective ad-juvants and delivery vehicles to boost immunity has becomea priority [39]. The recombinantV. choleraeghost (rVCG)system is an effective carrier/delivery cum adjuvant systemthat targets specific immunogenic antigens to primary APCS,inducing both antigen-specific humoral and cellular immuneresponses[26,27], and therefore potentially capable of de-livering chlamydial proteins to induce a strong protective

F.O. Eko et al. / Vaccine 21 (2003) 1694–1703 1701

immunity. VCG are non-toxigenic, non-living bacterial cellsdevoid of cholera toxin and other cytoplasmic contents thatmaintain their natıve surface antigenic structures and cellu-lar morphology. They possess intrinsic adjuvant propertiesthat enhance immune responses against targeted antigens, in-cluding both mucosal humoral and T cell immune responses[27], making them ideal for delivering anti-chlamydial vac-cines.

To design a recombinant VCG-basedChlamydia vac-cine, C. trachomatis MOMP was expressed in theV.cholerae01 strain HI to yield clone HM12 that upon induc-tion expresses chlamydial MOMP, which was detectableby a MOMP-specific monoclonal antibody (Fig. 2). AVCG-MOMP vaccine candidate was then produced by pro-tein E-mediated lysis of strain HM12E. Transmission elec-tron microscopic evaluation of rVCG-MOMP preparationsrevealed that except for the lysis pore, the morphology ofthe cell, including all cell surface structures and appendages,is unaffected by the lysis event. Besides, immunofluores-cent staining of rVCG-MOMP demonstrated the stabilityof MOMP on the membrane of rVCG preparations (Fig. 4).Furthermore, immunologic and vaccine evaluation in vivosuggested that the rVCG system is likely to furnish aneffective carrier and delivery system for clonedC. tra-chomatis proteins, eliciting chlamydial-specific immuneresponses following immunization. Thus, following intra-muscular delivery, rVCG-MOMP elicited a strong mucosaland systemic Thl response, which is required for chlamydialimmunity [39]. Moreover, immunization also produced sig-nificant protective immunity against intravaginal challengeinfection with C. trachomatisserovar D in the absence ofadded adjuvants.

While other delivery systems such as viral[25,40] andnaked DNA[41,42] have previously been used to deliverchlamydial proteins in experimental vaccines, this studyrepresents the first report of protective immunity againstchlamydial genital infection induced by a genetically inacti-vated bacterial vector expressing a chlamydial protein. Thus,the results suggest that the use of rVCG may be an alter-native method of delivering chlamydial subunit antigens toelicit protective immune responses. Several degrees of im-munogenicity and levels of protective immunity have beenobserved from a number of MOMP-based subunit candi-date vaccines delivered with or without adjuvants or vec-tors [9,24,41–43]. Various strategies have also been usedto deliver chlamydial antigens to enhance immunogenic-ity and protective immunity. Vector-mediated immunizationwith naked DNA using genes encoding the MOMP protein[4,41,44]showed promise in models of respiratory chlamy-dial infection. It is interesting that the immunogenicity andprotective immunity induced by MOMP DNA vaccinationwere enhanced by use of an adjuvant[42]. This would sug-gest that proper selection of adjuvants and a delivery sys-tem to target immunity in the genital tract could enhancethe immunogenicity and protective immunity induced byMOMP-based formulations[39]. Data obtained in the cur-

rent study using rVCG-MOMP are encouraging and parallelthose obtained using purified MOMP protein in combinationwith lipophilic immune stimulating-complexes (ISCOMS)[34] suggesting that appropriately attenuated or inactivatedbacterial vectors may be effective alternative methods of de-livering subunit chlamydial antigens to elicit protective cel-lular and humoral immune responses. The previous report[34] highlighted the significance of adjuvants and an effec-tive delivery vehicle in the induction of protective immuneresponses to even seemingly immunogenic antigens. Thus,while the efficacy of a vaccine is influenced by the immuno-genicity of the antigen and the immune status of the host,the mucosal immune response to vaccines directed at mu-cosal pathogens is also affected by such factors as adjuvantsand delivery vehicles[39].

It is clear from these results that a sterilizing protectiveimmunity was not achieved with the rVCG-MOMP regi-men, and this has been observed with several MOMP-basedexperimental vaccines[39]. The role of MOMP conforma-tion in these preparations in relation to the partial immunityobtained is currently unclear but such factors have beenpredicted to influence immunogenicity[45]. Furthermore,the results may also suggest that MOMP alone may beinadequate as an anti-chlamydial vaccine, leading to theadvocation of a multi-component approach[39] in whichMOMP epitopes will be combined with other immunogenicprotein antigens to induce a higher frequency of immuneeffectors than MOMP alone. Recent advances in chlamy-dial genomics and proteomics have predicted a numberof potential antigens that can be incorporated into can-didate multi-subunit vaccines[46–48]. The recombinantghost platform technology offers an exceptional opportu-nity to develop a multi-subunit vaccine againstChlamydia.For this purpose, multiple chlamydial proteins can be ex-pressed on the membrane of VCG, which possess intrinsicadjuvant properties and mucosal tropism. Results fromstudies in progress in our laboratory relating to designing amulti-subunit experimental ghost vaccine expressing multi-ple proteins ofC. trachomatisin V. cholerae01 appear tobe promising[49].

Acknowledgements

This work was supported by PHS grants (AI4123 1, GM08248 and RRO3034) from the National Institutes of Health,and the Centers for Disease Control and Prevention (CDC).We thank Dr. Harlan D. Caldwell for kindly providing themonoclonal antibody L2 1–1 0.

References

[1] CDC & PHS. Chlamydia trachomatisgenital infections—UnitedStates, 1995. Morbid Mortal Wkly Rep 1997;46:193–8.

[2] Paavonen J, Wolner-Hanssen P.Chlamydia trachomatis: a majorthreat to reproduction. J Hum Reprod 1989;4:111–24.

1702 F.O. Eko et al. / Vaccine 21 (2003) 1694–1703

[3] Schachter J, Grayston JT. Epidemiology of human chlamydialinfections. In: Stamm WE, editor. Chlamydial infections. SanFrancisco/Berkeley, CA; 1998. p. 3–10.

[4] Stagg AJ. Vaccines againstChlamydia: approaches and progress.Mol Med Today 1998;4(4):166–73.

[5] Grayston JT, Wang S-P, Yeh LJ, Kuo CC. Importance of reinfectionin the pathogenesis of trachoma. Rev Infect Dis 1985;7:717–25.

[6] Grayston JT, Wang SP. The potential for vaccine against infectionof the genital tract withChlamydia trachomatis. Sex Trans Dis1978;5:73–7.

[7] Schachter J, Dawson, CR. In: Human chlamydial infections. Littleton,MA: PSG Publishing Company; 1978.

[8] Woolridge RL, Grayston JT, Chang IH, Yang CY, Cheng KH.Long-term follow-up of the initial (1959–1960) trachoma vaccinefield trial on Taiwan. Am J Ophthalmol 1967;63:1650–5.

[9] Brunham RC, Peeling RW.Chlamydia trachomatisantigens: role inimmunity and pathogenesis. Infect Agents Dis 1994;3:218–33.

[10] Kalman S, Mitchell W, Marathe R, et al. Comparative genomesof Chlamydia pneumoniaeand C. trachomatis. Nat Genet1999;21(4):385–9.

[11] Read TD, Brunham RC, Shen C, et al. Genome sequence ofChlamydia trachomatisMoPn andChlamydia pneumoniaeAR39.Nucl Acids Res 2000;28(6):1397–406.

[12] Stephens RS. Chlamydial genomics and vaccine antigen discovery.J Infect Dis 2000;181(Suppl 3):S521–3.

[13] Stephens RS, Kalman S, Lammel C, et al. Genome sequence of anobligate intracellular pathogen of humans:Chlamydia trachomatis.Science 1998;282(5389):754–9.

[14] Brunham RC, Peeling R, Maclean I, McDowell J, Persson K, OsserS. Post-abortalChlamydia trachomatis salpingitis: correlating riskwith antigen-specific serological responses and with neutralization.J Infect Dis 1987;155:749–55.

[15] Jones RB, Batteiger BE. Human immune response toChlamydiatrachomatis infections. In: Ward M, editor. Chlamydial infections.London: Cambridge University Press; 1986. p. 423–32.

[16] Ward ME. Chlamydial vaccine—future trends. J Infect 1992(Suppl1):11–26.

[17] Batteiger BE, Rank RG, Bavoil PM, Soderberg LSF. Partialprotection against genital reinfection by immunization of guinea-pigswith isolated outer-membrane proteins of the chlamydial agent ofguinea-pig inclusion conjunctivitis. J Gen Microbiol 1993;139:2965–72.

[18] Tuffrey M, Alexander I, Conlan W, Woods C, Ward M. Hetrotypicprotection of mice against chlamydial salpingitis and colonizationof the lower genital tract with a human serovar F isolate ofC.trachomatisby prior immunization with recombinant serovar Ll majorouter membrane protein. Eur J Immunol 1992;23:1169–72.

[19] Su H, Parnell M, Caldwell HD. Protective efficacy of a parenterallyadministered MOMP-derived synthetic oligopeptide vaccine in amurine model of Chlamydia trachomatisgenital tract infection:serum neutralizing IgG antibodies do not protect against genital tractinfection. Vaccine 1995;3(11):1023–32.

[20] Knight SC, Iqball S, Woods C, Stagg A, Ward ME, Tuffrey M.A peptide ofChlamydia trachomatisshown to be a primary T-cellepitope in vitro induces cell mediated immunity in vivo. Immunology1995;85:8–15.

[21] Brunharn RC, Zhang DJ. Transgene as vaccine forChlamydia. AmHeart J 1999;138:S519–22.

[22] Conlan JW, Ferris S, Clarke IN, Ward ME. Isolation of recombinantfragments of the major outer-membrane protein ofChlamydiatrachomatis: their potential as subunit vaccines. J Gen Microbiol1990;136:2013–20.

[23] Pal S, Barnhart KM, Wei Q, Abai AM, Peterson EM, de laMaza LM. Vaccination of mice with DNA plasmids coding forthe Chlamydia trachomatismajor outer membrane protein elicitsan immune response but fails to protect against genital challenge.Vaccine 1999;17(5):459–65.

[24] Hayes LJ, Conlan JW, Everson JS, Ward ME, Clarke IN.Chlamydiatrachomatismajor outer membrane protein epitopes expressed asfusions with LamB in an attenuated aroa strain ofSalmonellatyphimurium: their application as potential immunogens. J GenMicrobiol 1991;137:1557–64.

[25] Murdin AD, Su H, Manning DS, Klein MH, Parnell MJ, CaldwellHD. A poliovirus hybrid expressing a neutralization epitope from themajor outer membrane protein ofChlamydia trachomatisis highlyimmunogenic. Infect Immun 1993;61:4404–14.

[26] Szostak MP, Hensel A, Eko FO, et al. Bacterial ghosts: non-livingcandidate vaccines. J Biotechnol 1996;44(1–3):161–70.

[27] Eko FO, Witte A, Huter V, et al. New strategies for combinationvaccines based on the extended recombinant bacterial ghost system.Vaccine 1999;17:1643–9.

[28] Witte A, Wanner G, Sulzner M, Lubitz W. Dynamics of PhiX 174 protein E mediated lysis ofEscherichia coli. Arch Microbiol1992;157:381–8.

[29] Ramsey KH, Soderberg LSF, Rank RG. Resolution of chlamydialgenital infection in B-cell-deficient mice and immunity to reinfection.Infect Immun 1988;56:1320–5.

[30] Szostak M, Lubitz W. Recombinant bacterial ghosts as multivaccinevehicles. In: Chanock REA, editor. Vaccine 91: modern approachesto new vaccines including prevention of AIDS. Cold Spring Harbor:Cold Spring Harbor Laboratory Press; 1991. p. 409–14.

[31] Eko FO, Mayr UB, Attridge SR, Lubitz W. Characterizationand immunogenicity of Vibrio cholerae ghosts expressingtoxin-coregulated pili. J Biotechnol 2000;83:115123.

[32] Mermod N, Ramos J, Lehrbach P, Timmis K. Vector for regulatedexpression of cloned genes in a wide range of gram-negative bacteria.J Bacteriol 1986;167:447–54.

[33] Eko FO, Szostak MP, Wanner G, Lubitz W. Production ofVibriocholeraeghosts (VCG) by expression of a cloned phage lysis gene:potential for vaccine development. Vaccine 1994;12:1231–7.

[34] Igietseme JU, Murdin A. Induction of protective immunity againstChlamydia trachomatisgenital infection by a vaccine based on majorouter membrane protein lipophilic immune response-stimulatingcomplexes. Infect Immun 2000;68(12):6798–806.

[35] Igietseme JU, Uriri IM, Kumar SN, et al. Route of infection thatinduces a high intensity of gamma interferon-secreting T cells inthe genital tract produces optimal protection againstChlamydiatrachomatisinfection in mice. Infect Immun 1998;66(9):4030–5.

[36] Igietseme JU, Rank RG. Susceptibility to reinfection after aprimary chlamydial genital infection is associated with a decreaseof antigen-specific T cells in the genital tract. Infect Immun1991;59:1346–51.

[37] Igietseme JU, Ramsey KH, Magee DM, Williams DM, Kincy TJ,Rank RG. Resolution of murine chlamydial genital infection by theadoptive transfer of a biovarspecific, Thl lymphocyte clone. RegionImmunol 1993;5:317–24.

[38] Igietseme JU, Ananaba GA, Bolier J. The intercellular adhesionmolecule type I is required for rapid activation of T helper typeI (Th1) lymphocytes that control early acute phase of genitalchlamydial infection in mice. Immunology 1999;98(4):510–9.

[39] Igietseme JU, Black CM, Caldwell HD.Chlamydia vaccines:strategies and status. Biol Drugs 2002;16(1):19–35.

[40] Murdin AD, Su H, Klein MH, Caldwell HD. Poliovirus hybridsexpressing neutralization epitopes from variable domains I and IV ofthe major outer membrane protein ofChlamydia trachomatiselicitbroadly cross-reactiveC. trachomatisneutralizing antibodies. InfectImmun 1995;63(3):1116–21.

[41] Zhang DJ, Yang X, Berry J, Shen C, McClarty G, Brunham RC.DNA vaccination with the outer membrane protein gene inducesacquired immunity toChlamydia trachomatis(mouse pneumonitis)infection. J Infect Dis 1997;176:1035–40.

[42] Dong-Ji Z, Yang X, Shen C, Lu H, Murdin A, Brunham RC. Primingwith Chlamydia trachomatismajor outer membrane protein (MOMP)DNA followed by MOMP-ISCOM boosting enhances protection and

F.O. Eko et al. / Vaccine 21 (2003) 1694–1703 1703

is associated with increased immunoglobulin A and Th I cellularimmune responses. Infect Immun 2000;68(6):3074–8.

[43] Zhang DJ, Yang X, Shen C, Branham RC. Characterization ofimmune responses following intramuscular DNA immunization withthe MOMP gene ofChlamydia trachomatismouse pneumonitisstrain. Immunology 1999;96(2):314–21.

[44] De la Maza MA, De la Maza LM. A new computer model forestimating the impact of vaccination protocols and its applicationto the study ofChlamydia trachomatisgenital infections. Vaccine1995;13(1):119–27.

[45] Pal S, Theodor I, Peterson EM, de la Maza L. Immunization with theChlamydia trachomatismouse pneumonitis major outer membraneprotein can elicit a protective immune response against a genitalchallenge. Infect Immun 2001;69(10):6240–7.

[46] Rank RG. Models of immunity. In: RS Stephens, editor.Chlamydia:intracellular biology, pathogenesis and immunity. Washington, DC:ASM Press; 1999. p. 239–95.

[47] Murdin AD, Dunn P, Sodoyer R, et al. Use of a mouse lung challengemodel to identify antigens protective againstChlamydia pneumoniaelung infection. J Infect Dis 2000;181:S544.

[48] Whittum-Hudson JA, Ann LL, Saltzman WM, Prendergast RA,MacDonald AB. Oral immunization with an anti-idiotypic antibodyto the exoglycolipid antigen protects against experimentalChlamydiatrachomatisinfection. Nat Med 1996;2(10):1116–21.

[49] Eko FO, Lubitz W, Igietseme JU. Immunogenicity of anovel recombinant subunit candidate vaccine againstChlamydiatrachomatis. In: Proceedings of the 101st General Meeting of ASM,vol. Ab E-58, Orlando, FL; 2001. p. 341.