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Vaccine 25 (2007) 1581–1592

Vaccine potential for inactivated shigellae

Manuel Osorio, Mechelle D. Bray, Richard I. Walker ∗

Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research,Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852-1448, United States

Received 10 August 2006; received in revised form 1 November 2006; accepted 6 November 2006Available online 20 November 2006

bstract

We used human monocyte-derived dendritic cells (DC) and Balb/c mice as models to establish the immunogenic and protective potentialf formalin-inactivated Shigella spp. Incubation of DC with inactivated or live bacteria induced DC maturation and cytokine release. Micemmunized orally or intranasally with killed S. flexneri, S. sonnei, or S. dysenteriae developed IgG and fecal IgA titers to the homologousPS. Following respiratory challenge with the live homologous organisms, 80–100% survival was seen in all vaccinated groups compared toegligible survival in mice given PBS. Oral or intranasal immunization with an inactivated S. flexneri 2a strain (CVD1203) expressing the

FA/I and CS3 antigens of enterotoxigenic Escherichia coli induced IgG responses to both heterologous antigens. These in vivo and in vitroata indicate that inactivated shigellae retain the ability to interact effectively with key antigen presenting cells and induce protective immuneesponses in mice.

2006 Elsevier Ltd. All rights reserved.

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eywords: Inactivated bacteria; Enteric vaccines; Dendritic cells

. Introduction

Shigella continues to be a major infectious disease threat.ach year in the developing world, Shigella species cause

llness in over 150 million individuals and death in overne million [1]. Children under the age of 5 are mosteverely affected. In developing countries, Shigella flexnerind Shigella sonnei are the most prevalent species, caus-ng about 60% and 15% of episodes, respectively. Shigellaysenteriae occurs less frequently but produces a more severeisease, including pandemics with high attack and mortalityates. In the developed world, S. sonnei causes about 75% ofpisodes and is particularly important among children in day-are centers, travelers, and deployed military personnel [1].

The enormous global burden of Shigella, augmented byhe growing rate of anti-microbial resistance, makes devel-

pment of an effective vaccine essential. At present there iso vaccine against Shigella, although a variety of live andubunit vaccines which elicit an anti-LPS mucosal response

∗ Corresponding author. Tel.: +1 301 496 1014; fax: +1 301 402 2776.E-mail address: Richard.Walker@fda.hhs.gov (R.I. Walker).

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264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2006.11.012

ave been shown to confer protection in experimental mod-ls of shigellosis [2–4]. Live, attenuated Shigella strains haveeen the dominant approach to shigella vaccine developmentince a 1966 report [5] showing that monkeys were protectedgainst subsequent disease if previously administered atten-ated organisms, but not by an acetone-inactivated vaccine.ive vaccines can protect against Shigella in field studies [6]nd in volunteer challenge studies [7]. For live vaccines, how-ver, reactogenicity continues to be a major problem [7–9].istorically, the adequacy of attenuation has remained prob-

ematic in healthy adults and this problem may be even greatern infants or children in less developed countries or in otherersons whose defenses have been compromised by HIV oralaria. A potentially safer subunit, conjugate vaccine was

lso protective in a field trial [10]. Subunit vaccines, however,re expensive to prepare and are usually parenterally admin-stered. Protection associated with these vaccines may be dueo a boosting effect in individuals who have been previously

xposed to the organism.

If inactivated shigella whole cells (SWC) alone or carryingntigens of other enteric pathogens could be used for immu-ization, it would facilitate development of enteric vaccines

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ith the possibility of less reactogenicity for the recipient.he current study was undertaken to better characterize the

easibility of using mucosally-delivered inactivated wholeell vaccines for the major species of Shigella. This rel-tively simple approach would present the LPS and otherccessible antigens of Shigella in a particulate matrix which,iven mucosally, should be safe as well as effective. Mucosalaccination with inactivated whole cells has already shownromise against toxigenic enteric diseases caused by entero-oxigenic Escherichia coli and Vibrio cholerae [11,12].

Our hypothesis is that invasive enteric organisms suchs Shigella can also be vaccinated against with mucosally-elivered inactivated whole cells. Further, we evaluated theossibility that inactivated shigellae are not only immuno-enic for the pathogen, but may serve as safe and effectiveectors for heterologous antigens.

. Materials and methods

.1. Bacterial strains

For each experiment, a stock vial of S. flexneri, S. sonnei,r S. dysenteriae) was sub-cultured on Typtic Soy agar (TSA)Becton, Dickenson and Co., Sparks, MD) plates containing.065% Congo red (Sigma, St. Louis, MO). The plates wereultured overnight for approximately 12 h at 37 ◦C. Isolateded colonies were chosen for use in the respective experi-ents. The original stock vials of S. flexneri 2a (2457T) and S.

onnei (53G) were kindly provided by Dr. Jim Jackson, Antexiologics Inc. (Gaithersburg, MD). CVD 1203 [13,14], a S.exneri 2a vaccine strain attenuated by deletions in aroAnd virG, and engineered to express the CFA/I and CS3ntigens of entertoxigenic E. coli (ETEC) was kindly pro-ided by Dr. Eileen Barry at the University of the Marylandenter for Vaccine Development. Dr. Malabi Venkatesan,alter Reed Army Institute of Research, kindly provided

tock vials of S. dysenteriae 1 (1617) and WRSd1, a toxinegative strain of S. dysenteriae 1 [15]. A CFA/I-expressingnterotoxigenic E. coli (H10407) was obtained from Dr.red Cassels, Walter Reed Army Institute of Research. Dr.atricia Guerry, Naval Medical Research Center, suppliedials of Campylobacter jejuni strain 81–176 and V. choleraeATCC 14035).

.2. Vaccine preparation

Frozen vials from each of the respective Shigella stocktrains were used to inoculate Congo red TSA plates. Thelates were cultured overnight at 37 ◦C. Approximately fouro five red colonies were picked to inoculate a 2 L Erlen-

eyer flask containing 1 L of Brain Heart Infusion (BHI,

ecton, Dickenson and Company, Sparks, MD). The liq-id culture was incubated for about 12–14 h at 37 ◦C with00-rpm agitation. Next, 70 mL of the overnight cultureere transferred into a 2.8 L Fernbach flask containing

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(2007) 1581–1592

00 mL BHI broth. The flask was incubated at 37 ◦C for.5 h with 100-rpm agitation. Cells were harvested by cen-rifugation at 4000 rpm in a Beckman GS-6KR centrifugeor 30 min. After centrifugation, the supernatant was dis-arded. The bacterial pellet was resuspended in 70 mL ofank’s Balanced Salt Solution and inactivated by the addi-

ion of 0.7 mL Mallinckrodt formaldehyde solution (37.4%CHO). After 72 h at room temperature (RT) with mild

tirring with a magnetic stir-bar, the formaldehyde-fixedells were collected and centrifuged at 4000 rpm. The cellsere resuspended in phosphate buffered saline (PBS) forfinal optical density at 625 nm of 23 ± 2. A Petroff-

auser counting chamber was used to determine the celloncentration (∼2–3 × 1010 cells/mL). The vaccine prepara-ions were then aliquoted into 5 mL serum vials for storaget 4 ◦C.

.3. Preparation of human monocyte-derived dendriticells

Mononuclear cells were obtained by apheresis of normalolunteer donors as performed by the Blood Services Sec-ion in the Department of Transfusion Medicine (DTM) athe National Institutes of Health Warren G. Magnuson Clin-cal Center (Bethesda, MD). The mononuclear cells wereurther enriched for monocytes by centrifugal elutriation aserformed by the Cell Processing Section in the DTM. Thelutriated monocytes were then cultured (1 × 106 per well)n six-well tissue-culture plates with 3 mL/well of Roswellark Memorial Institute 1640 (RPMI) medium (Mediat-ch, Inc., Herndon, VA) containing 5% human AB (huAB)erum (Nabi, Miami, FL), 800 U/mL human granulocyteacrophage colony-stimulating factor (GM-CSF; Peprotech,ocky Hill, NJ), and 500 U/mL human Interleukin-4 (IL-; Peprotech). The non-adherent and loosely adherent cellsere harvested after incubating the plates for 5 days at7 ◦C in 5% CO2. The cells were centrifuged for 10 mint 1200 rpm in a Beckman GS-6KR centrifuge, then plated5 × 104 cells/well; 100 �L/well) in fresh RPMI containing% huAB serum (Nabi) and 800 U/mL GM-CSF (Peprotech)n a 96-well flat bottom plate. The culture process resultedn the generation of immature monocyte-derived dendriticells (DC) that were positive for CD11c, but negativeor CD14.

.4. Performance of the cytotoxicity test

The CytoTox 96® non-radioactive cytotoxicity assay kitPromega, Madison, WI) was used to determine the cytotoxicffect of bacterial preparations on DC. This assay quali-atively measures lactate dehydrogenase, a stable cytosolicnzyme that is released upon cell lysis. The assay and

espective calculations were performed according to the man-facturer’s instructions. The DC cell number was determinedo be optimal at 50,000 cells/well. The bacteria to DC ratiossed for the experiments were 20:1, 10:1, and 1:1.

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.5. Infection of DC

Cell suspensions of Shigella, ETEC, C. jejuni, or V.holera, or similar volumes of phosphate buffered salinePBS) (Invitrogen Corporation, Grand Island, NY) weredded into the cell culture wells at different multiplicitiesf infection (MOI 20:1, 10:1, or 1:1). Infected cultures wereentrifuged at 1000 rpm in a Beckman GS-6KR centrifuge formin, and incubated at 37 ◦C in 5% CO2. After 1 h, gentam-

cin (50 �g/mL) was added, and the incubation was continuedor a specified time period (i.e., 30 min to 48 h) as requiredy the respective assay.

.6. Flow cytometry analysis of S. sonnei-infected DC

Flow cytometry analysis was performed to examine theell surface expression of CD11c, CD14, CD80, CD86,nd HLA-DR in S. sonnei-infected DC and to compare thenduced level of maturation to the LPS control. Dendritic cellsere infected as described as above. The maturation state ofC was determined at 24 and 48 h after infection with S.

onnei (MOI 1:1) by measuring the expression of the surfacearkers by flow cytometry. Cell samples (0.5 × 106 cells)ere incubated (20 min, 4 ◦C) with 4% human AB serum

Nabi) in PBS (Invitrogen Corporation) to block the non-pecific binding sites and Fc receptors. The cells wereashed via centrifugation (1200 rpm; 10 min) with FACsuffer containing 0.2% bovine serum albumin (Sigma) and.1% sodium azide (Sigma) in PBS (Invitrogen Corporation),hen incubated (45 min, 4 ◦C) with CD11c (S-HCL-3), CD80L307.4), CD86 [2331(FUN-1)]FITC-labeled (1 �L; Bectonickinson, San Jose, CA), or CD14 (M�P9)-PE-labeled or

heir respective isotype-matched controls [1 �L IgG1-FITC×40) or IgG2a-PE (×39)] antibodies (Becton Dickinson).fter washing again, the cells were resuspended in cold PBS

Invitrogen Corporation) with 0.1% sodium azide (Sigma)upplemented with 0.5% paraformaldehyde (Sigma). Theell-associated immunofluorescence was measured with

FACS CaliburTM flow cytometer (Becton Dickinsonmmunocytometry Systems, San Jose, CA) and analyzedsing CellQuestTM software. For each determination, 10,000vents were acquired. Three experiments were performed,nd the results of a representative experiment are shown.

.7. Determination of cytokine responses

Quantitation of accumulated cytokines in cell cultureupernatants following co-culture of DC with bacteria waserformed using a multiplexed sandwich immunoassay basedn the Luminex® xMAP technology. A panel of 10 cytokinesas analyzed using a Human Cytokine 10-plex kit foruminex® (BioSource, Camarillo, CA) and the IL-12 p70

ntibody kit in culture supernatants harvested at 4, 24, or8 h from co-cultures of DC and bacteria. Cytokines exam-ned included interleukin-1 beta (IL-1�), IL-2, IL-4, IL-5,L-6, IL-8, IL-10, IL-12, interferon gamma, tumor necrosis

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(2007) 1581–1592 1583

actor alpha and granulocyte macrophage colony stimulat-ng factor (GM-CSF). Cytokine data analysis was performedsing the MasterPlex QT Quantitation Software (MiraiBio,lameda, CA).

.8. Preparation of lipopolysaccharides

A lipopolysaccharide (LPS) extraction kit (Intron Biotech-ology, Kyungki-Do, Korea) was used according to theanufacturer’s instructions to isolate LPS from S. flexneri

nd S. sonnei. The LPS preparations were air dried, weighed,nd resuspended in distilled water at 5 mg/mL. The LPS wastored at −20 ◦C, and thawed prior to use.

.9. Measurement of antibody titers

Serum samples were tested for specific antibody responsesy a capture enzyme-linked immunosorbent assay (ELISA)sing standard procedures. Blood samples (∼200 �L) wereollected from the lateral tail veins of all immunized andontrol mice on day 0, 14 and 28, and 2 weeks after the lastmmunization dose. Serum was assayed for Shigella species-pecific total IgG antibody responses in mice. Immunolon® 2at bottom microtiter plates (ThermoLabsystems, Franklin,A) were coated with 1 �g/mL of the respective antigen

nd incubated overnight at 4 ◦C. Whole cell and LPS anti-ens were prepared as described above. CFA/I and CS3 wererovided by Dr. Fred Cassels at WRAIR. All washing stepsere carried out with PBS containing 0.05% Tween-20. Thelates were blocked with 5% bovine serum albumin in PBSor 1 h at 37 ◦C. Each serum sample was serially diluted:3 in blocking buffer typically from 1:100 to 1:300,000.he samples were incubated for 1 h at 37 ◦C. Total IgGntibody titers were detected by using an affinity purifiederoxidase-labeled goat anti-mouse IgG (KPL, Gaithers-urg, MD) diluted 1:1000 in blocking buffer and incubatedor 30 min at RT. The plates were developed with 100 �Lf 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) sub-trate (ABTS; KPL). After incubating 15 min at RT, theeaction was stopped by adding 100 �L of ABTS Peroxi-ase Stop Solution (KPL). Absorbance at 405 nm (A405) waseasured using a spectrophotometer. Fecal IgA titers wereeasured as described by Grewal et al. [16]. All samplesere assayed in triplicate. End-point antibody titers were

xpressed as the maximum dilution of sample giving anbsorbance of greater than 0.2 A405. The results are presenteds the reciprocal of the dilution multiplied by the absorbancealue. The precision of antibody titer determination was esti-ated by calculating the coefficient of variation %CV.%CV was calculated with the formula ‘(S.D./mean)100’. Antibody titers against S. flexneri 2a LPS for a poly-

lonal rabbit serum raised against S. flexneri types 1–6 were

etermined in triplicate and in three separate occasions. Theithin (triplicate) assay variability was calculated to have aCV of 5.13, 3.0, and 1.7 for each one of three independent

ssays. The between (inter-plate) variability was calculated

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sing the geometric means of each one of three experimentsnd found to have a %CV of 2.07.

.10. Mice and immunizations

Balb/cAnN mice, purchased from the National Cancernstitute-Division of Cancer Treatment (Frederick, MD)r Taconic (Germantown, NY), were maintained in themall Animal Facility of the Food and Drug Administra-

ion (Bethesda, MD) and used with the approval of theood and Drug Administration’s Institutional Animal Carend Use Committee. For immunizations, inactivated Shigellahole cell (SWC) vaccine or Shigella vaccine vectors wereiven either orally (1 × 109–1 × 1010 cells), intranasally (i.n.)1 × 107–1 × 109 cells) or intraperitoneally (i.p.) (1 × 109

ells). The oral dose (0.2 mL) was administered using a.5 in., 20 gauge feeding needle. The intranasally immunizedroups received anesthesia (0.1 mL i.p. containing 80 mg/kgetamine and 15 mg/kg xylazine) followed by a dose of inac-ivated bacteria diluted in saline (20 �L of bacteria total or0 �L/nare) and administered with a pipette into the naresf the mice. The i.p. groups were immunized with bacte-ial suspension at a volume of either 0.25 or 0.5 mL deliveredith a 25 gauge, 5/8 in. needle. Each group received the sameaccine dose of either 3 times at 2-week intervals (i.n.) or 4imes (oral) at 24 or 48 h intervals, and in some experiments,ne booster dose 28 or 70 days later. Lateral tail vein bleeds0.2 mL) were performed on the mice on days 0, 14, and 28fter the first vaccine dose in the series, and then finally onay 14 after the final vaccine boost. Mice were rested fort least 72 h after bleeding, prior to bacterial challenge. The.n. challenge was conducted about 2–3 weeks after the lastmmunization using exactly the same technique as used forhe i.n. immunization. The immunized mice were challengedith a dose of 8.5 × 106–5.0 × 107 cells of the respectiveild-type bacteria.

.11. Statistical analyses

Statistical analyses were performed by means of annpaired t test using GraphPad Prism 4. A p-value of ≤0.05two-tailed) was considered to be statistically significant.

. Results

.1. Cytotoxicity to DC

The cytotoxic effect of live and inactivated shigellae onuman monocyte-derived DC was evaluated by measuringhe release of lactate dehydrogenase into the culture medium.acteria were incubated together with DC at ratios of up to

0:1 (bacteria to DC). As seen in Fig. 1, inactivated S. flexneriells were minimally cytotoxic, even after 4 h of incubation.ive S. flexneri, in contrast, were progressively cytotoxic,ith cytotoxicity after 4 h of incubation reaching 60% with

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ilutions of live or inactivated S. flexneri or live ETEC. All data points rep-esent the mean of triplicate cultures ± S.D. and are expressed as percentagef maximal LDH release.

20:1 ratio and around 25% using a 1:1 ratio of bacteria toC. Additionally, bacteria to DC ratios of 10:1 or 20:1 of. flexneri induced very similar DC mortality by 4 h. Live S.onnei, however, was less cytotoxic for DC than the otherhigella spp examined, with only about 25% mortality at 4 hith a 20:1 ratio (data not shown). S. dysenteriae was as cyto-

oxic as S. flexneri (data not shown). Enterotoxigenic E. colias used as a non-invasive control organism in these studies.ive ETEC behaved as did the inactivated shigellae and didot kill DC even at the 20:1 bacteria to DC ratio. Attenuatedtrains of S. flexneri (CVD 1203) and S. dysenteriae (WRSd-) were as cytotoxic as their parent strains (data not shown).ased on these results, subsequent studies were designed toxamine the DC immunological response elicited by live bac-erial cell preparations at a 1:1 ratio. Inactivated bacteria wereenerally added at a 10:1 ratio in comparative studies withive cells, although we found that responses to 1:1 and 10:1atios of inactivated cells were not significantly different.

.2. Cytokine induction and maturation in DC incubatedith live or inactivated Shigella spp.

DC surface marker expression profiles, following stimula-ion with either live or inactivated S. sonnei, were determinedn an attempt to further detect similarities or differencesn the two types of bacterial preparations. Both live andnactivated cells induced comparable levels of cell surface

olecule expression and this was generally similar to DC

esponses to LPS (data not shown). Live and inactivated. sonnei were incubated with DC (in the ratios describedbove) using DC derived from different donors each time.oth types of bacterial preparations induced increases in

M. Osorio et al. / Vaccine 25 (2007) 1581–1592 1585

Fig. 2. Production of proinflammatory cytokines following infection of DC with live and inactivated S. sonnei. With the exception of IL-8, stimulation of DCwtt

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ith live or inactivated resulted in significant levels of cytokine secretion (p < 0.05).han killed bacteria (p < 0.05). IL-8 secretion was not significantly different in theriplicate cultures ± S.D. The results shown are an example of one representative do

ig. 3. Cytokine responses by human DC incubated 24 h with killed species of shigp � 0.05). Levels of IL-12 secretion induced by S. sonnei were lower than those indhe mean of triplicate cultures ± S.D.

IL-1� production was significantly higher in DC incubated with live rathertwo experimental groups (p = 0.24). All data points represent the mean ofnor from three different experiments.

ellae. All bacterial stimuli induced significant levels of secreted cytokinesuced by either S. dysenteriae or WRSd1 (p < 0.05) All data points represent

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ytokine production over controls in cells from each of theonors. Although slight variations were observed in the lev-ls of cytokines secreted by DC from different donors, theelative levels of secretion induced by the different stim-li remained very similar. The results showed that, with thexception of IL-1� and IL-12, there was generally little oro difference in the responsiveness of DC to live or deadacterial cells (Fig. 2). Interleukin-1� and IL-12 productionncreased more following incubation with live cells than withilled, possibly due to the ability of the live bacteria to invadehe DC. Specifically, the Th-1 associated cytokine, IL-12,as induced by live bacteria at approximately twice the level

hat resulted from incubation of DC with the killed prepara-ion. Secretion of other pro-inflammatory cytokines such asL-6, IL-8, and TNF-� was similarly induced by both livend killed bacteria, as was the anti-inflammatory cytokineL-10.

We compared the ability of other inactivated Shigella spp.o modulate DC cytokine production. Generally, all inac-ivated Shigella preparations induced increases in cytokineroduction when samples were taken after 24 h of incubationith DC (Fig. 3). Killed S. sonnei generally induced similar

evels of proinflammatory cytokines, IL-6, IL-8, and TNF-�,hile resulting in lower levels of IL-12 than S. flexneri or. dysenteriae. S. flexneri induced the highest levels of IL-2 amongst all the species tested, and correspondingly, the

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(2007) 1581–1592

owest levels of IL-10. Finally, S. dysenteriae induced muchower TNF-� levels than S. sonnei or S. flexneri.

Tests were conducted to determine if the DC cytokineesponses seen following incubation with shigellae mighte similar to those following incubation with other inacti-ated enteric pathogens (Fig. 4). Compared to cultures tohich no bacteria were added, V. cholerae induced significant

esponses in most cytokines tested, but was quantitativelyess effective than other bacterial species tested. Highestesponses in this series were always associated with S. flexnerind ETEC which were comparable except for IL-10 and IL-2. S. flexneri induced higher levels of IL-12 while ETECnduced more IL-10. C. jejuni-infected DC usually resultedn the induction of slightly lower cytokine levels than ETECr S. flexneri, but was higher quantitatively than V. choleraen most cases.

.3. Immunologic potency of inactivated Shigellae inice

Humoral immune responses were determined followingP inoculation of two doses 1 × 109 cells of each of the

nactivated Shigella preparations given at an interval of 3eeks, and then bled at week 5 after the first inoculation.ll three Shigella preparations induced strong IgG responses

o their respective whole cell antigen (Fig. 5). Although the

arious enteric pathogens. DC were incubated with inactivated whole cellsted. V. cholera induced the production of most cytokines, IL-12 being theicate cultures ± S.D.

M. Osorio et al. / Vaccine 25 (2007) 1581–1592 1587

Fig. 5. Immunologic potency of inactivated shigella species to their respec-ti

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iters were quite high following systemic immunization, nonef the animals subsequently challenged intranasally withpproximately 5 × 107 live cells of the homologous speciesere protected (data not shown).

.4. Immune responses and protection followingntranasal immunization

Mice were inoculated intranasally with 20 �L containing× 109 inactivated shigellae. Mice were bled 2 weeks after

he third dose and challenged a week thereafter. Titers ofgG against WC antigen following immunization with thenactivated bacteria ranged from 10,000 to 20,000 (Fig. 6).eaths following challenge occurred in control animalsostly between days 4 and 7 post-challenge. Whereas all

ontrol animals following all three challenges died, 70–100%urvival was obtained in the vaccinated groups (Fig. 7). Micemmunized with live or inactivated S. sonnei were protectedqually.

.5. Vector potential for inactivated S. flexneri 2a

Because Shigella spp. are immunogenic and protective forhemselves, further tests were conducted to see if heterolo-ous antigens expressed on the bacterial surface would alsoe immunogenic. A S. flexneri 2a construct expressing the

FA/I and CS3 of ETEC was inactivated with formalin andsed to immunize mice intranasally as before with 1 × 109

ells. Groups of mice were also given either PBS or 1 × 107

ive CFA/I-expressing ETEC. Two weeks after the last dose

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amples were collected 2 weeks after the last immunization and titered inriplicate by ELISA.

f cells, mice had mean titers of 4000 to the Shigella LPSFig. 8). CFA1 titers were 125,000 and 25,000 for the inac-ivated Shigella and live ETEC, respectively. Anti-CS3 titersere 85,000 in mice given the inactivated Shigella vector, butere non-existent in mice given the CFA/I-expressing ETEC

train.The immunogenicity of the inactivated CVD 1203 vec-

or was also studied following oral immunization of mice.n this experiment, mice were given four doses of 1 × 1010

ive or inactivated cells at 48 h intervals, followed by a boost0 days after the initial dose of vaccine. Because the bacte-ial preparations were given orally, we examined the effectsn immune responses when the live vaccine was adminis-ered in buffered (5% bicarbonate) or unbuffered suspension.accination of mice induced anti-Shigella LPS IgG titers of0,000 to 20,000 in all preparations and the presence of theuffer did not affect the titers obtained (Fig. 9). Fecal IgAiters against S. flexneri LPS was also determined with similaresults. Again, the highest titers were obtained in mice immu-ized with the live preparations, with lower titers obtainedollowing immunization with the killed cells. Anti-CFA/IgG titers were 6000 for mice given live cells, but droppedo 2000 following immunization with formalin-treated cells.nti-CS3 titers were also 6000 for live cells, but were only000 with formalin-fixed cells. When challenged intranasally

ith 2 × 107 live S. flexneri 2a two weeks after the final immu-ization boost, all mice given live or killed CVD1203 weretrongly (70–90%) protected from death, while 80% of theBS treated mice died (Fig. 10).

1588 M. Osorio et al. / Vaccine 25 (2007) 1581–1592

Fig. 7. Protection to subsequent challenge following immunization withivd

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nactivated vaccine prepared from different shigella species. Percent sur-ival for any given day was determined by the number of mice remainingivided by the original total, and multiplied by 100.

. Discussion

These data support the concept that inactivated shigellaeepresenting the major species of clinical importance can besed safely to confer protection against these pathogens. Notnly do they induce protective immune responses to homolo-

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ig. 8. IgG titers following intranasal immunization with killed CVD 1203r live ETEC. Serum samples were titered in triplicate with ELISA.

ous challenge, but they can also induce significant immune

esponses to heterologous antigens expressed on their sur-ace, in this case the CFA1 and CS3 antigens of ETEC.he immunogenicity seen following mucosal immunizationf mice is consistent with the experience of Chakrabarti in

M. Osorio et al. / Vaccine 25

Fig. 9. IgG titers to ETEC CFA/1 and CS3, and S. flexneri 2a LPS follow-ing oral immunization with live or inactivated CVD 1203. Serum sampleswere collected at 2 weeks after the final immunization boost and analyzedin triplicate by ELISA. Compared to unimmunized control mice, all othergroups elicited significant CFA/I-specific antibody titers (symbol ‘*’ indi-cates a p-value of <0.05). There was no significant difference in the anti-CS3antibody titers elicited by inactivated CCVD1203 and those in the PBS con-trol mice (p = 0.13). Both live and inactivated bacterial preparations inducedsignificant anti-Shigella LPS antibody titers (p � 0.05).

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abbits [17,18] and Hartman in mice [19] immunized withilled shigellae. The immunogenicity obtained with inac-ivated whole cell shigella vaccines can be obtained withotentially greater safety than is possible with attenuatedaccines now being tested. This concept is supported by thebservation that, in contrast to the two attenuated shigellaesed in the present studies, the killed cells had no cyto-oxic activity. The safety of inactivated ETEC given to 6–18

onths old children [20] and inactivated cholera vaccine inIV patients [21] provides further evidence of the safety of

nactivated vaccines.DC play a major role in the initial uptake of microor-

anisms or antigens at mucosal surfaces and regulate thennate and adaptive immune responses [22–24]. Recentlydgeworth et al. [25] found that S. flexneri kills DC by an

paB-dependent mechanism involving caspase-1 and others yet unidentified proteases. These findings suggest that S.exneri infection of DC could have adverse consequencesor induction of immune responses to shigellosis. HumanC were used to demonstrate that in contrast to live cells

he inactivated shigellae were not cytotoxic. These particlesehaved in much the same way as live ETEC in that cytotoxicffects were not seen even at a 20:1 ratio of bacteria to DC.

hile this may not be true for all attenuated Shigella strains,he two attenuated Shigella strains used in this study weres cytotoxic as the parent strains. It is also of interest thatn other studies (unpublished data) we have shown that wildype Salmonella enterica serovar Typhi is as cytotoxic foruman DC as the live shigellae, but the attenuated typhoidaccine Ty21a was without cytotoxic effects. It is possiblehat this may relate to the relative safety of this vaccine. DCamage associated with live shigellae could contribute to theeactogenicity of the vaccine, possibly through the greaterL-1� production by dendritic cells exposed to live bacterias. killed cells. This cytokine is known to be associated withnflammatory responses in the host [26]. These observationsuggest that the cytotoxicity of DC associated with live cellsould interfere with generation of adaptive immunity [25],nd also contribute to some of the residual reactogenicityften associated with attenuated shigellae.

The interaction of bacteria with DC can be species spe-ific. For example, Pulendran et al. [27] found that LPS from. coli and Porphyromonas gingivalis activate DC subsets

o produce different cytokines and induce distinct types ofdaptive immunity in vivo. Yersinia enterocolitica down reg-lates certain functions of DC, and this may contribute tovasion of adaptive immune responses by this pathogen [28].imilarly Helicobacter pylori influences the activation andaturation of DC to favor a Th1 T-cell acquired immune

esponse [29,30]. The relative amounts of IL-10 and IL-12nduced by different Salmonella varied dramatically amongtrains [31]. All of these reports suggest that the cytokine

nduction by shigellae could be important to the host responsend indicate a need to establish that the profile associated withive organisms is not lost upon fixation. Except in the cases ofL-1� and IL-12 we found that there was little or no difference

1590 M. Osorio et al. / Vaccine 25 (2007) 1581–1592

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ig. 10. Oral immunization with live or inactivated CVD 1203 protected mics the number of animals remaining/total × 100, on any particular day after

etween most cytokine responses of DC exposed to killed orive cells. Likewise, both live and fixed preparations of shigel-ae were studied for mediation of DC maturation with similaresults obtained for both preparations. Taken together, theseata would indicate that similar immune responses could bebtainable with either live or killed shigella preparations.

Shigella was at least as effective as other inactivatednteric pathogens with regard to cytokine induction. Fur-her, ETEC, Campylobacter, and V. cholerae were all capablef inducing cytokine responses, suggesting the possibilityor their inclusion in future inactivated whole cell entericaccines.

In a potency assessment with mice immunized via thentraperitoneal route, the formalin-inactivated cells of allhree Shigella species induced high antibody titers to theathogen. While IgG titers were very high following two.p. injections, none of these animals were protected against

ortality in the respiratory challenge model. Since these ani-als had not previously been exposed to Shigella, it is quite

ossible that no local immunity occurred and hence no pro-ection. In contrast, mucosal immunization, which inducedignificant Shigella-specific IgG responses as well as fecalgA, was associated with clear protection against challenge.

The immunogenicity of inactivated shigellae led to fur-her studies to determine the potential these cells may haveo serve as vectors for heterologous antigen as well as beaccines. While there has been considerable interest in usingttenuated Shigella or other live enteric organisms as vaccineectors [32] little attention has been focused on non-livingectors. Studies in mice with Salmonella dublin expressinghe B subunit of the E. coli heat-labile toxin showed thathe immunogenicity of inactivated bacterial vectors could be

ore a factor of the fixation method than whether the bac-

erium was living or not [33]. With this in mind, future worksing shigellae as vectors of protein antigens might benefitrom use of less destructive inactivation methods, such as these of bacterial whole cell ghosts [34].

lfl[m

nged with 2 × 107 live S. flexneri bacteria. Percent survival was determinedbacterial challenge.

Several attenuated Shigella vaccines have been developedo express ETEC antigens which could be protective in aombined agent enteric vaccine [13,35,36]. We took the S.exneri CVD 1203 which was described by Noriega et al.13] and evaluated its potential as an inactivated whole cellector. Immune responses to both the CFA1 and CS3 anti-ens expressed by this organism were obtained followingither oral or nasal administrations. While the titers to shigellaere similar following either route of administration, titers

o the ETEC antigens were much greater following the nasalegimen. This may indicate that the nasal route avoids degra-ation of the ETEC antigens which could occur in the mouseastrointestinal tract and is more sensitive to antigen. It islso possible that the shigellae have an adjuvant effect onntigens when given nasally. Inactivated pertussis can haven adjuvant effect on intranasal coadministered viral antigenhich is poorly immunogenic when administered alone [37].The protective immunity afforded by S. flexneri 2a (CVD

203) to challenge with Shigella as was seen by Noriega etl. [13] in guinea pigs was confirmed by our studies in mice.n ETEC model is needed to determine whether immune

esponses to the subunits expressed on Shigella are sufficientor protection against this pathogen, too. It is not knownhether colonization antigens alone will provide sufficientrotection against challenge with ETEC, or whether otherell-associated antigens will also be required [38,39]. Recentuccess in producing a mouse nasal challenge model forTEC [40] may lead to a model capable of determining thetility of colonization antigens in protection.

A polyvalent vaccine against Shigella is needed becauserotective immunity is serotype-specific. There are 6erotypes and 15 subtypes of S. flexneri and one clinicallyelevant serotype of each S. dysenteriae and S. sonnei. Sero-

ogic cross-reactivity suggests that a smaller number of S.exneri strains could be represented in an effective vaccine41]. Thus, as few as five inactivated Shigella componentsay be sufficient for a vaccine, a goal that becomes more

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eadily obtainable due to the relative ease of preparationf inactivated vaccines. If some of these Shigella compo-ents express antigens of other enteric pathogens, such asas demonstrated in this report, a vaccine with broad cover-

ge becomes much more practical. The likelihood that the initro and animal studies reported here may predict success inumans seems strong. It is hoped that these preclinical find-ngs will help lead to challenge trials to establish both theafety and efficacy of killed shigellae in humans.

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