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Vaccine 29 (2011) 4771– 4777

Contents lists available at ScienceDirect

Vaccine

jou rn al h om epa ge: www.elsev ier .com/ locate /vacc ine

ecombinant (F1 + V) vaccine protects cynomolgus macaques againstneumonic plague

.D. Williamsona,∗, P.J. Packera, E.L. Watersa, A.J. Simpsona, D. Dyerb, J. Hartingsb,. Twenhafelb, M.L.M. Pittb

Dstl PortonDown, Salisbury, Wilts SP4 0JQ, UKUSAMRIID Fort Detrick, Frederick, MD, USA

r t i c l e i n f o

rticle history:eceived 5 January 2011eceived in revised form 14 April 2011ccepted 21 April 2011vailable online 12 May 2011

eywords:neumonic plagueaccine

mmune correlatesacaques

a b s t r a c t

Cynomolgus macaques, immunised at the 80 �g dose level with an rF1 + rV vaccine (two doses, threeweeks apart), were fully protected against pneumonic plague following inhalational exposure to a clinicalisolate of Yersinia pestis (strain CO92) at week 8 of the schedule. At this time, all the immunised animalshad developed specific IgG titres to rF1 and rV with geometric mean titres of 96.83 ± 20.93 �g/ml and78.59 ± 12.07 �g/ml, respectively, for the 40 �g dose group; by comparison, the 80 �g dose group haddeveloped titres of 114.4 ± 22.1 and 90.8 ± 15.8 �g/ml to rF1 and rV, respectively, by week 8. For all theimmunised animals, sera drawn at week 8 competed with the neutralising and protective Mab7.3 forbinding to rV antigen in a competitive ELISA, indicating that a functional antibody response to rV hadbeen induced. All but one of the group immunised at the lower 40 �g dose-level were protected againstinfection; the single animal which succumbed had significantly reduced antibody responses to both the

fficacy rF1 and rV antigens. Although a functional titre to rV antigen was detected for this animal, this wasinsufficient for protection, indicating that there may have been a deficiency in the functional titre to rF1and underlining the need for immunity to both vaccine antigens to achieve protective efficacy againstplague. This candidate vaccine, which has been evaluated as safe and immunogenic in clinical studies, hasnow been demonstrated to protect cynomolgus macaques, immunised in the clinical regimen, againstpneumonic plague.

. Introduction

Plague is caused by the Gram-negative organism Yersinia pestisnd although an ancient disease associated with epidemics fromhe Middle Ages onwards, it is still endemic in parts of the worldoday [1]. Bubonic plague is primarily a disease of small rodentsnd mammals that is spread by fleas in endemic areas, to establishnzootic foci which occasionally erupt as an epizootic outbreak,articularly after major geological disturbance such as earthquake2]. Humans can be infected either by flea bite or by inhala-ional exposure through a secondary host, for example wild rabbit,rairie dog or domestic cat and this leads to several thousandHO-reported cases of plague per annum globally [1,3]. The con-

equences of infection in man are serious and the infection needso be detected and treated promptly to prevent serious morbidity

eading to death. Transmission to man, by feeding fleas, leads tohe characteristic swelling of the draining lymph nodes, to formuboes, which may develop into a septicaemic illness or secondary

∗ Corresponding author.E-mail address: dewilliamson@dstl.gov.uk (E.D. Williamson).

264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2011.04.084

© 2011 Elsevier Ltd. All rights reserved.

pneumonic plague. However, plague is also highly transmissibleby the coughing of patients with bubonic or septicaemic plaguewho have developed pulmonary lesions [4,5]. An outbreak of pneu-monic plague at a diamond mine in Northern Congo in 2005 caused54 deaths and was limited only by the dispersal of miners flee-ing from the mine in panic [1]. Pneumonic plague would also bethe most likely form of disease if Y. pestis were to be used as abiowarfare agent; it is both the most serious and most feared man-ifestation of this disease, protection against which is the paramountrequirement to prevent epidemic spread.

There is accumulated epidemiological and experimental evi-dence that existing vaccine formulations, comprising sterilesuspensions of killed whole bacteria, provide little protectionagainst the pneumonic form of the disease caused by exposure towild type Y. pestis [6,7] and this is strongly supported by experimen-tal observation in animal models [8–10]. Additionally, whilst killedwhole cell vaccines (KWCVs) have been demonstrated to protectmice against bubonic plague arising from exposure to F1+ Y. pestis,

they do not protect against an F1− Y. pestis strain [11,12]. Renewedresearch effort in the last two decades has led to the developmentof a recombinant vaccine comprising the two protein antigens,rF1 and rV [10,13,14]. In combination, these antigens are potently

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mmunogenic in mouse, guinea-pig, macaque and human [15] ando date they have been demonstrated to induce protective immu-ity against plague in the mouse and guinea-pig models, leading tohe identification of potential immune correlates of protection [15].nlike KWCV, the experimental evidence indicates that the rF1 + rV

ormulation can induce protective immunity against pneumoniclague in the mouse model [10,16]. Furthermore these proteinsave been expressed as a genetic fusion to yield a single recombi-ant protein (rF1–V) in which the N-terminus of the V antigen is

used to the C-terminus of the F1 antigen [17] and which has simi-ar protective efficacy against pneumonic plague in the mouse [13]nd macaque [18,19] as do the combined antigens.

In previous studies in the cynomolgus macaque, we haveemonstrated that the rF1 + rV vaccine formulated by adsorption tolhydrogel in the dose range 5–40 �g of each sub-unit and used inhe same dosing regimen as used in a Phase 1 clinical trial [14], wasighly immunogenic [15]. Immune macaque sera from this study

nhibited the cytotoxic effect of Yersinia-delivered V antigen onacrophages in culture, competed with the protective monoclonal

ntibody Mab7.3 for binding to rV in vitro and conferred protectiongainst plague in mice by passive transfer [15]. The current studyas designed to extend the immunising dose range to 80 �g each

f rF1 and rV and to determine whether the functional serologicalssays used previously represent immune correlates of protec-ion by challenging the immunised macaques with aerosolised Y.estis. The identification of immune correlates of protection will bessential for the licensure of such a vaccine according to the FDA’snimal Rule, and equivalent guidance from the European Medicinesgency [20,21].

. Materials and methods

.1. Animals

This study was conducted in compliance with the U.S. Animalelfare Act and other Federal statutes and regulations relating to

nimals and it adhered to the principles stated in the Guide forhe Care and Use of Laboratory Animals, National Research Council996.

Twenty-two adult cynomolgus macaques (males and females)f bodyweight range 3–8 kg were used in this study. Telemetryevices (TA10DA-T70) were used to measure body temperaturend were implanted sub-cutaneously (s.c.) in all cynomolgusacaques with appropriate sedation and analgesia, approximately

0 days prior to the study start. These transponders were placedn the dorsum of the animals, between the scapulae. The animalsere offered environmental enrichment throughout the study andad access to food and water ad libitum.

.2. Immunisation

A batch (03D11601-04A) of formulated vaccine comprising20 �g rF1 + 120 �g rV in 0.5 ml 0.26% (w/v) alhydrogel (Brenntag,redericksaand, Denmark) was received from Avecia Billingham,K (now Pharmathene Annapolis, USA). Each recombinant antigenad been separately expressed from E. coli and purified as previ-usly described [14]. On receipt, the formulated vaccine was dilutedith alhydrogel in 0.9% saline to achieve dose-levels of either 40 �g

f each protein (group 1) or 80 �g (group 2) of each protein in a.5 ml final volume comprising 0.26% (w/v) alhydrogel, per animal.

Groups of 10 cynomolgus macaques were immunised at either

ose-level. Immunisation was carried out intra-muscularly (i.m.)

n 0.5 ml on two occasions, separated by 3 weeks. An additionalwo animals were administered a 0.5 ml placebo dose of 0.26%w/v) alhydrogel in 0.9% saline in the same dosing regimen. Two

e 29 (2011) 4771– 4777

placebo control animals were included with the objective of con-firming that the level of Y. pestis challenge delivered was indeedcapable of causing pneumonic plague as determined by lethalityand histopathology, rather than with the objective of achieving astatistically valid group size. Baseline blood samples were collectedfrom each animal immediately prior to vaccination, and these wereused to check that there was no pre-existing titre to either antigen.

2.3. Blood sampling and immunoassay

The animals were bled immediately prior to immunisation andthen approximately every week up to week 8. Serum antibody lev-els were measured by a conventional ELISA, as previously described[15] and using peroxidase-conjugated sheep anti-human IgG (TheBinding Site Ltd., Birmingham, UK) as the secondary reagent. TheIgG sub-class profiles at week 6 were also determined exactly aspreviously described [15] using human IgG standards from SigmaAldrich (Poole, Dorset), and peroxidise-conjugated anti-human iso-type reagents (The Binding Site Ltd., Birmingham, UK).

2.4. Competitive ELISA on week 8 serum samples

The detection of antibody which competed for binding to rVantigen in vitro with a murine monoclonal antibody (Mab7.3) wasdetermined as previously described [14] for individual serum sam-ples from macaques. Briefly, rV antigen was coated (5 �g/ml) tosolid phase prior to the binding of 80 ng Mab7.3. Subsequently, themacaque test sera, or negative control sera (from placebo animals99–310 and 49–470), or positive reference serum, were added induplicate in the dilution range 1:10 to 1: 80 in 1% (w/v) skimmedmilk powder in PBS. The positive reference serum was derivedas previously described [14] by pooling polyclonal sera collectedfrom cynomolgus macaques hyperimmune to rF1 + rV. The assaywas developed with HRP-goat anti-mouse IgG (Serotec; 1:2000 inPBS) followed by incubation (37 ◦C, 1 h). Plates were washed priorto addition of ABTS substrate (Sigma) with subsequent reading ofthe absorbance at 414 nm. The OD414nm was determined for eachtest and reference serum.

2.5. Challenge

The Y. pestis CO92 clinical isolate was prepared as describedpreviously [22] to challenge immunised animals by the inhala-tional route. Immunised macaques were anaesthetised with Telazol(6 mg/kg i.m.) prior to aerosol challenge with Y. pestis CO92 atweeks 8–9 of the schedule. The 40 �g and 80 �g rF1 + rV dosegroups were challenged on consecutive days, with 1 unvaccinatedcontrol in each cohort. Respiratory minute volumes were mea-sured by whole body plethysmography using a Buxco Biosystem XA(Buxco Electronics, Sharon, CT, USA) immediately before challenge.The anaesthetised macaques were then immediately exposed tothe bacterial aerosol, head-only, in a dynamic aerosol chambercontrolled using the Automated Bioaerosol Exposure System. Theaerosol (mass median aerosol diameter 1.2 �m) was generated bya three-jet Collision nebuliser and sampled continuously by anall-glass impinger (AGI-30; Ace Glass Inc., Vineland, NJ). For eachanimal, the aerosol concentration of Y. pestis organisms was calcu-lated by plating out dilutions of a sample from the AGI onto bloodagar plates (Remel). The inhaled doses were then determined.

2.6. Post-challenge monitoring

Post-infection (p.i.), the macaques were returned to their homecages where they were continuously monitored for body tem-perature, with data collected on an hourly basis up to 14 dayspost-challenge, using Dataquest A.R.T.2.3 software. Animals were

accine 29 (2011) 4771– 4777 4773

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E.D. Williamson et al. / V

losely monitored for clinical signs. Any animal which became crit-cally ill was promptly euthanized by deep anaesthesia with Telazol6–9 mg/kg, i.m.) followed by an intra-venous overdose of sodiumentobarbital.

.7. Pathology

Complete post mortem examinations were performed on ani-als that succumbed to disease in a biosafety level 3 necropsy

acility. Trachea, lungs, heart, thyroid gland, thymus, adrenallands, mandibular lymph node, mesenteric lymph node, liver, gall-ladder, spleen, kidneys, and brain were collected at necropsy foristopathology and were immersed in 10% neutral buffered forma-

in and held in biocontainment for 21 days. Organ weights wereecorded for the thymus (if present), thyroid gland, lungs withronchi, spleen, liver, adrenal glands, kidneys and brain. Tissuesor histopathology were embedded in paraffin, sectioned, and weretained with hematoxylin and eosin.

.8. Statistical analysis

The data were analysed using Graph Pad Prism version 5. Toompare geometric mean titres to rF1 and rV, a two-tailed paired t-est was used. Additionally, the mean titres to F1 and V, determinedor individual animals were compared with the GMT by unpaired-test. A correlation coefficient for the pairing of F1 and V titres forhe same animals was determined to assess the effectiveness of theairing. Statistical significance was assumed when p < 0.05.

. Results

.1. Antibody development with time

The serum antibody response to immunisation with rF1 + rV wasonitored in individual macaques in each of the dose-level groups

40 �g and 80 �g) on a weekly basis up to week 8. Generally, IgGitres to either antigen peaked in response to the booster immu-isation administered at 3 weeks, during the secondary responsehase at 4–7 weeks of the schedule (Fig. 1a–d). Animal 49 unexpect-dly died at week 7 from acute cardiac decompensation followingnaesthesia secondary to severe chronic heart disease, the etiol-gy of which could not be related to vaccination; therefore, the IgGitres for this animal were monitored only to week 7 (Fig. 1a and).

The specific serum IgG titres at the time of challenge (week) for each animal are shown in Table 1a and b, together withhe geometric mean titres (GMT) to rF1 and to rV, for the 40 and0 �g dose groups. The difference in GMT between dose-groupsas not statistically significant for either antigen. For individual

nimals, increases in titre to F1 correlated with increases in titre to, determined as a correlation coefficient for the 40 �g and 80 �gose-groups, respectively of r = 0.75, p < 0.006; and r = 0.68, p < 0.02.

.2. IgG sub-class profiles in the secondary response

The IgG response to rF1 and rV was further characterised byssay of IgG sub-classes at week 6 for individual animals in eachf the dose-level groups (Fig. 2a and b). IgG1 and IgG2 sub-classesponses to each antigen were detected, but no IgG3 or IgG4esponses. This was in agreement with our previous analysis ofmmune macaque sera [15]. However, the relative significancef these isotypes in the macaque, measured with anti-human

eagents, is still unclear. There was variability between individ-al animals in the isotype of response to F1 and V at each dose

evel. Calculation of GMT ± standard error of the mean valuesor IgG2 to F1 at the 40 and 80 �g dose-levels (123.6 ± 29.8 and

Fig. 1. Development of IgG titres to F1 and V with time in individual macaquesimmunised with either 40 mcg rF1 + rV (a and b) or with 80 mcg rF1 + rV (c and d).

269.8 ± 53) and comparison with the equivalent GMT values forIgG1 to F1 (105.8 ± 28 and 225.6 ± 46) showed no significant differ-ences between the sub-class response to rF1 at either dose-level.The GMT IgG1 response to rV significantly exceeded the GMTIgG2 response at both the 40 and 80 �g dose-levels (p < 0.05 and

p < 0.001, respectively) based on GMT values for IgG1 of 212.3 ± 31and 215.1 ± 31 and for IgG2 of 68.3 ± 35.7 and 39.4 ± 12, at thesedose-levels, respectively.

4774 E.D. Williamson et al. / Vaccine 29 (2011) 4771– 4777

Table 1Immune response and survival.

Animal identifier IgG titre in �g/ml at week 8 Survived/died at challenge TTD (d)

rF1 rV

(a) 40 �g rF1 + 40 �g rV group41–384 7.57 34.06 Died 5.349–516 46.86 61.15 SurvivedC964 47.22 36.95 SurvivedC512 37.23 68.43 Survived94984 121.96 86.76 SurvivedC440 138.3 139.98 Survived59–25 199.82 129.29 Survived79–27 172.07 58.58 Survived49–544 145.22 117.66 Survived49 52.08 (day 50) 53.06 (day 50) Died before challengea

GMT (sem) 96.83 (20.93) 78.59 (12.07)99–310 (negative control) 0 0 Died 3.6

(b) 80 �g rF1 + 80 �g rV groupc470 53.88 71.35 Survived49–458 95.55 115 Survived59–213 131.34 73.26 Survived41–318 70.17 49.7 SurvivedC616 203.34 198.85 Survived41–363 140.01 79.33 Survived30–318 18.97 38.11 Survived39–38 127.26 39.98 Survived78–143 243.69 138.82 Survived107–720 60.13 103.28 SurvivedGMT (sem) 114.4 (22.1)

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a Animal 49 died before challenge of vaccine-unrelated causes. The week 7 titres

.3. Competitive ELISA

All week 8 sera competed with Mab7.3 for binding to the rVntigen, in the CE assay, suggesting that the immunised macaques

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ig. 2. IgG isotype profile at week 6 for (a) 40 �g dose group and (b) 80 �g doseroup.

Died 3.7

is animal are included in the GMT calculation.

recognised the same protective epitope in rV (Fig. 3a and b). In thisassay, the further to the left the curves, the greater is the compe-tition with Mab7.3. All the test sera, apart from sample 41–363 inthe 80 �g dose-group, performed better than the positive referenceserum in competing with Mab7.3 for binding to rV antigen. How-ever, there was no discernible difference overall between the 40and 80 �g dose groups in the CE assay. Further, sera from the singleimmunised macaque (41–384) in the 40 �g dose group which diedp.i., competed well with Mab7.3 in this assay, illustrating the limi-tation of assaying CE titre to rV antigen only as a surrogate markerof protection.

3.4. Survival

The immunised macaques were challenged by the aerosol routewith Y. pestis CO92 at week 8 of the schedule with a target doseof 5 × 105 cfu (equivalent to 126 ± 36 LD50) and received 4.3 × 104

(standard deviation 1.2 × 104) cfu. All animals survived for theobservation period of 6 weeks p.i., except for one animal (41-384)in the 40 �g dose group. This animal died at 5.3 days p.i., havingdeveloped a fever at 3 days and becoming bacteremic by day 4.This animal had significantly reduced (p < 0.01) anti-rF1 and anti-rVresponses at week 8, compared with the GMT values for the 40 �grF1 + rV dose group (Table 1a). Another animal in the same dosegroup (C964) which had a similar response to the rV antigen buta 6-fold greater response to rF1 than 41–384, survived, indicatingthe important contribution of each antigen to protective immu-nity. The control animals in each of the dose groups, which receivedthe placebo formulation, died at 3.6 and 3.7 days p.i., respectivelyand both of these animals were bacteremic in the 24 h prior todeath. These animals displayed a regular circadian rhythm in body

temperature, with cycling between 36 ◦C and 37 ◦C up to approxi-mately day 2 p.i., after which the baseline drifted upwards peakingat 38–39 ◦C by day 3 (data not shown). A similar pattern, althoughmore protracted, was observed for the single immunised macaque

E.D. Williamson et al. / Vaccine 29 (2011) 4771– 4777 4775

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hich died p.i. (41–384); in this case, a circadian rhythm in bodyemperature was maintained until day 3 p.i., when the baselinencreased to 38 ◦C and peaked at 39 ◦C at day 4 p.i., after which ittarted to decline steadily until the animal died.

.5. Histopathology

The lesions observed in the immunised macaque 41–384 and thewo placebo-treated controls were similar and all were consistentith pneumonic plague, as described previously in the macaque

23]. The lungs were severely affected in these animals and con-isted of necrosuppurative bronchopneumonia with intralesionalacteria consistent with Y. pestis (Fig. 4a–d). Ninety percent ofhe small airways and alveoli contained a mixture of viable andegenerate neutrophils, necrotic debris, macrophages, edema, fib-in, hemorrhage, and often myriad extracellular bacteria (Fig. 4c).ungs also contained multifocal fibrinous pleuritis (Fig. 4b) andecrotizing vasculitis (Fig. 4d).

Other findings included necrotizing and suppurative lym-hadenitis of the tracheobronchial lymph node with numerousacilli, necrotizing tracheitis, pleural edema, and pleural effusion.mall numbers of bacteria were found in blood vessels of multi-le organs including the liver, indicating bacteremia was presentt death. The cause of death in these animals is cardiorespiratoryailure and sepsis due to pneumonic plague.

.6. Discussion

Previous studies indicated that the rF1 + rV vaccine formulations immunogenic in macaques and the passive transfer of immune

acaque serum into mice gave indirect evidence that the vaccineould protect macaques against live organism challenge [15]. In

C470

tal IgG titres of week 8 sera from macaques in 40 �g (a) and 80 �g (b) dose groups.

a previous study in which cynomolgus macaques were immunisedon 3 occasions with 50 �g each of rF1 and rV adsorbed to alhydrogelin combination, or with individual sub-units, 2 of 2 animals pergroup survived for 14 days post aerosol infection with >104 cfu Y.pestis CO92 [24].

The present study provides direct evidence that this vaccineis highly protective in immunised macaques for at least 6 weeksp.i. with >104 cfu Y. pestis CO92, using an immunisation regimenalready used in a Phase I clinical trial [14]. Here, the dose-range ofvaccine used has been extended to 80 �g of each antigen, whichafforded full protection. The importance of inducing an adequateresponse to each of the antigens is illustrated by the death p.i. ofa single immunised macaque (41–384) from the 40 �g dose group.This animal had a significantly depressed response to rF1, comparedwith other members of the same dose group. In contrast, macaqueC964 in the same dose group, which had a similar response to therV antigen but a 6-fold greater response to rF1 than 41-384, sur-vived. These observations are upheld by our early observations [14]that immunity to the rF1 and rV antigens is additive, presumablydue to the fact that two separate virulence mechanisms in whichF1 or V is pivotal, are being neutralised. The survival data gainedhere in immunised macaques justify the use of a similar dose rangecentering on 80 �g of rF1 + rV, in clinical studies.

The identification and assay of immune correlates of protectionis fundamental to the ability to extrapolate between non-clinicaland clinical data generated with this vaccine, since formal efficacytrials in man are neither feasible nor ethical.

In this context, assays of the inhibition of the V antigen are

attractive due to the inhibition of a defined cytotoxic endpointby Mab7.3; the latter has been well-characterised with respect torecognition of one protective B-cell epitope within the V antigen[25,26]. Mab7.3 has been demonstrated to protect cells in vitro

4776 E.D. Williamson et al. / Vaccine 29 (2011) 4771– 4777

Fig. 4. (a) Lung, placebo control. Severe pneumonic plague. Few alveolar spaces are air-filled (arrowhead) and the remaining air spaces are consolidated with cellular infiltrate,hemorrhage, and edema (asterisk). The surface of the lung, or pleura, appears only slightly thickened (arrow). Hematoxylin and eosin 2×. (b) Lung, placebo control. Severepneumonic plague. The lung is completely consolidated in the section (asterisk). The pleura is markedly expanded to 20 times normal (arrow). Hematoxylin and eosin2 ), viabH splaysw erate n

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×. (c) Lung, placebo control. Alveolar spaces are filled with edema fluid (asteriskematoxylin and eosin 40×. (d) Lung, placebo control. A pulmonary blood vessel diithin the vessel wall (arrow). The blood vessel is surrounded by viable and degen

rom the cytotoxicity of V antigen expressed from Y. pseudotu-erculosis, to passively protect naïve mice from challenge with Y.estis [25] and to compete in ELISA with polyclonal anti-rF1 + anti-

macaque serum for binding to the rV antigen [15]. However, thereay be more than one protective B-cell epitope on the V anti-

en [26] and it has been suggested that this may undermine theE approach involving Mab7.3 as a reliable inter-laboratory corre-

ate of protection [27,28]. Furthermore, it cannot be assumed that functional CE titre to V, in the absence of an adequate titre to1, will translate into protection, as illustrated here by the exam-le of the single immunised macaque from the 40 �g dose group,hich died. An alternative correlate of protection which we and

thers have reported is the passive transfer of immune serum into second animal model followed by challenge [14,29] which, relyingn anti-F1 and anti-V activity as it does, should become a reliableurrogate marker of protection for this bivalent vaccine. Whilsthe inhibition of cytotoxicity of V secreted from a Yersinia strain,

easured either qualitatively [15,26] or in a quantitative assay foraspase-3 activity [30] may be useful as an assay of the functional-

ty of the anti-V response, it may be of limited value on its own as surrogate marker of protection. In addition to serological assays,ssays of cell-mediated immunity may also yield surrogate markersf protection.

The protection demonstrated here for the rF1 + rV vaccinegainst pneumonic plague is a significant advance comparedo KWCV formulations, which have not been demonstrated toully protect against this form of the disease. Other studies haveemonstrated that African Green monkeys as well as cynomolgusacaques responded with a strong anti-F1 and anti-V response

hen immunised on two occasions with rF1–V conjugated to flag-

llin [19] whilst a Nicotiana benthamiana plant-derived LcrV-F1usion protein also induced high titres of serum IgG to both F1nd V antigens in cynomolgus macaques [18]. This LcrV-F1 fusion

le and degenerate neutrophils (N), fibrin (arrowhead), and myriad bacilli (arrow). necrotizing vasculitis characterized by disrupted endothelium and necrotic debriseutrophils admixed with fibrin and cellular debris (N).

was adjuvanted with alhydrogel and Quil A and 200 �g doses wereused to immunise macaques on three occasions prior to aerosolchallenge with 100LD50 Y. pestis CO92. Immunised macaques, chal-lenged 12 days after the final immunisation were fully protected[18]. Indirect evidence of efficacy of the E. coli-expressed rF1–Vfusion in cynomolgus macaques was gained in a further study inwhich mice were protected against Y. pestis by the passive transferof immune serum from immunised macaques or humans [29]. Thusvarious presentations of the recombinant F1 and V antigens havebeen demonstrated to be protective in the cynomolgus macaquemodel of pneumonic plague. It is therefore now feasible to protectagainst deliberate aerosolisation of Y. pestis with vaccine candidatescomprising F1 and V proteins, and this has significant implicationsfor both public health and military force protection.

Alternative formulations and delivery routes of the recombinantF1 and V proteins are being explored. With a view to oral adminis-tration of the vaccine, which would be convenient on a large scale,the rF1–V fusion has been expressed in transgenic tomato fruitand was demonstrated to be immunogenic in orally dosed mice[3] and in transgenic carrots with subsequent formulation for sub-cutaneous delivery [31]. Alternative routes of delivery of the rF1and rV proteins for needle-free delivery have been explored. The rF1and rV proteins, encapsulated in biodegradable polymeric micro-and nano-spheres, have been demonstrated to be protective whendelivered by the i.m. or intra-nasal (i.n.) routes [32] and after onlya single immunisation [33]. Similarly the rF1–V fusion, formulatedin polyanhydride nanoparticles and delivered i.n. protected miceagainst subsequent i.n. challenge with Y. pestis [34] or in a proteo-some adjuvant, protected them against aerosol challenge [35]. The

rF1 and rV proteins [36] or the rF1–V fusion [37] have protectedmice after transcutaneous delivery. Oral administration of the pro-teins expressed from attenuated Salmonella strains has protectedmice from bubonic plague [38,39]. Finally, the oral immunisation of

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ice with rF1 + rV formulated in an amphipathic oily emulsion [40],r in cationic liposome–nucleic acid complexes (CLDC) combinedith F1 antigen protected mice partially or fully against pneumoniclague [41]. The development and licensing of vaccines which pro-ect fully against all forms of plague, has the potential to eradicatehis ancient disease and the associated fear it engenders.

cknowledgements

The authors particularly wish to thank Dr. M.J. Duchars and Dr. P.peakman of Avecia, Billingham, UK (now Pharmathene, Annapolis,SA) for manufacture and supply of the vaccine and also Col. Neilmith, Dr. M.S. Lever and Craig Varney for their assistance with thistudy and the many technical personnel, involved in the carryingut of this study.

The research described herein was sponsored by the Defensehreat Reduction Agency JSTO-CBD as #/Medical Research/Materialommand Research Plan. Opinions, interpretations, conclusions,nd recommendations are those of the authors and are not nec-ssarily endorsed by the U.S. Army.

Research was conducted in compliance with the Animal Welfarect and other federal statutes and regulations relating to animalsnd experiments involving animals and adheres to the principlestated in the Guide for the Care and Use of Laboratory Animals,ational Research Council, 1996. The facility where this researchas conducted is fully accredited by the Association for Assessment

nd Accreditation of Laboratory Animal Care International.

eferences

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of an oil-based delivery formulation for both oral and parenteral vaccination.Open Drug Deliv J 2008;2:52–60.

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