Characterization of a Cynomolgus Macaque Model of PneumonicPlague for Evaluation of Vaccine Efficacy

Patricia Fellows,a Jessica Price,a* Shannon Martin,a* Karen Metcalfe,a Robert Krile,b Roy Barnewall,b Mary Kate Hart,a Hank Lockmanb

DynPort Vaccine Company LLC, a CSC Company, Frederick, Maryland, USAa; Battelle, Columbus, Ohio, USAb

The efficacy of a recombinant plague vaccine (rF1V) was evaluated in cynomolgus macaques (CMs) to establish the relationshipamong vaccine doses, antibody titers, and survival following an aerosol challenge with a lethal dose of Yersinia pestis strain Col-orado 92. CMs were vaccinated with a range of rF1V doses on a three-dose schedule (days 0, 56, and 121) to provide a range ofsurvival outcomes. The humoral immune response following vaccination was evaluated with anti-rF1, anti-rV, and anti-rF1Vbridge enzyme-linked immunosorbent assays (ELISAs). Animals were challenged via aerosol exposure on day 149. Vaccine dosesand antibody responses were each significantly associated with the probability of CM survival (P < 0.0001). Vaccination alsodecreased signs of pneumonic plague in a dose-dependent manner. There were statistically significant correlations between thevaccine dose and the time to onset of fever (P < 0.0001), the time from onset of fever to death (P < 0.0001), the time to onset ofelevated respiratory rate (P � 0.0003), and the time to onset of decreased activity (P � 0.0251) postinfection in animals exhibit-ing these clinical signs. Delays in the onset of these clinical signs of disease were associated with larger doses of rF1V. Immuniza-tion with >12 �g of rF1V resulted in 100% CM survival. Since both the vaccine dose and anti-rF1V antibody titers correlate withsurvival, rF1V bridge ELISA titers can be used as a correlate of protection.

Plague is caused by the Gram-negative bacterium Yersinia pes-tis. While natural outbreaks of disease occur in wildlife popu-

lations, humans are most often incidental victims in the life cycleof the bacterium in rodents. Human disease may also result fromcontact with blood or tissues of infected animals or exposure toaerosolized droplets containing bacteria (1, 2). Three forms of thedisease exist and are believed to be dependent upon whether thebacteria enter the lymph nodes (bubonic), the bloodstream (sep-ticemic), or the lungs (pneumonic). Primary pneumonic plague iscaused by inhalation of Y. pestis. It is the least common form of thedisease (2%); however, it is the most serious form because of thehigh fatality rate and the potential for direct human-to-humantransmission via the spread of respiratory droplets (3). Because ofthe potential person-to-person transmission of pneumonicplague, the Centers for Disease Control and Prevention (CDC)considers this disease to be a serious potential threat and has clas-sified Y. pestis as a category A (tier 1) bioterrorism agent (4).

Aerosol dissemination represents the most likely modern-dayscenario for the use of Y. pestis as a biological weapon, with abattlefield scenario or terrorist attack likely resulting in a signifi-cant number of pneumonic plague fatalities. In 1970, the WorldHealth Organization modeled an attack scenario that predictedthat the airborne release of 50 kg of Y. pestis over a city of 5 millionwould result in 150,000 cases of plague, 36,000 of which would befatal (5).

No licensed vaccines against plague are available for humanuse in the United States. The previously available U.S. Pharmaco-peia (USP) vaccine was a killed, whole-cell (KWC) vaccine. Al-though its efficacy was never confirmed in controlled clinical stud-ies, observations of vaccinated humans and a number of animalstudies suggested that the USP vaccine was effective against bu-bonic plague but had limited efficacy against pneumonic plague(6–10). Further, this vaccine produced a number of moderate-to-severe side effects, ranging from mild headache to severe malaiseand fever (9, 10). Live-attenuated vaccines have been used in sev-eral countries but were not licensed in the United States because of

their reactogenicity (11). To overcome the limited efficacy andreactogenicity associated with KWC and live-attenuated plaguevaccines, respectively, the Y. pestis F1 and V antigens were identi-fied as promising components of a new generation of recombi-nant protein vaccines.

Early proof-of-concept studies investigated the immunogenic-ity and efficacy afforded by vaccination with the individual F1 andV antigens and found that each provides some level of protectionfrom a challenge. However, the combined use of the F1 and Vantigens was found to have an additive protective effect and wasmore effective than single-antigen vaccines in mouse models ofpneumonic plague (7, 8, 12).

A recombinant plague vaccine (rF1V) is currently in advanceddevelopment by the U.S. Department of Defense (DoD) to pro-vide preexposure prophylaxis to military personnel 18 to 55 yearsold against battlefield exposure to aerosolized Y. pestis (13). Thevaccine contains both the F1 and V antigens fused into a singleprotein that is adsorbed to aluminum hydroxide adjuvant to en-hance the immunogenicity of the rF1V protein. The efficacy ofrF1V cannot be determined directly in humans because of the

Received 14 May 2015 Returned for modification 10 June 2015Accepted 20 July 2015

Accepted manuscript posted online 29 July 2015

Citation Fellows P, Price J, Martin S, Metcalfe K, Krile R, Barnewall R, Hart MK,Lockman H. 2015. Characterization of a cynomolgus macaque model ofpneumonic plague for evaluation of vaccine efficacy. Clin Vaccine Immunol22:1070 –1078. doi:10.1128/CVI.00290-15.

Editor: D. W. Pascual

Address correspondence to Patricia Fellows, pfellows@csc.com.

* Present address: Jessica Price, Medimmune, Gaithersburg, Maryland, USA;Shannon Martin, United States Army Medical Research Institute of InfectiousDiseases, Frederick, Maryland, USA.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00290-15

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ethical implications of conducting Y. pestis inhalational challengestudies. In addition, the incidence of pneumonic plague in thegeneral population is extremely low, making field studies imprac-tical. Therefore, licensure of rF1V will rely upon immunogenicityand efficacy data obtained in nonclinical studies, immunogenicityand safety data from clinical studies, and adherence to the require-ments of the Animal Rule (14). The CM was chosen as a suitablenonhuman primate model of pneumonic plague on the basis ofprevious studies that demonstrated that CMs exhibit a clinicalcourse of disease similar to that described for humans (15). Fur-ther, proof-of-concept model development studies indicated thatCMs responded to plague vaccines and were protected from dis-ease following a lethal aerosol challenge with Y. pestis (15, 16).

We initially developed and characterized the CM model ofpneumonic plague (17) by following the recommendations in ref-erence 18. The results of this initial model development studyshowed that a postexposure rise in temperature, loss of the tem-perature diurnal rhythm, bacteremia, and increased heart and res-piration rates, followed by a decrease in activity, strongly corre-lated with a lethal outcome. The pathology in the lungs of all CMsthat died included pneumonia, pulmonary congestion, fibrinouspleuritis, and neutrophil infiltration. This is consistent with thepathology described for human pneumonic plague (16).

The vaccination and challenge studies described here were de-signed to further develop the CM model. These studies were de-signed to establish the relationships among rF1V vaccine doses,bridge enzyme-linked immunosorbent assay (ELISA) titers, andthe survival of CMs with the goal of assessing the utility of thebridge ELISA as a correlate of protection, which is an importantcomponent of bridging clinical and nonclinical data to predictclinical benefit. Additionally, the effect of the rF1V vaccine doseon disease progression following an aerosol challenge was evalu-ated. These studies complete the planned development studiescharacterizing the CM model of pneumonic plague. The resultsindicate the CM is an appropriate model for demonstrating theefficacy of rF1V according to the Animal Rule.

MATERIALS AND METHODSAnimals. Indonesian-origin CMs, 80 male and 41 female, were procuredfrom a licensed USDA-approved vendor and weighed at least 2 kg at thetime of vaccination. All of the animals tested negative for the presence ofpreexisting titers of antibody to the rF1V vaccine antigen. They were alsotuberculin test negative and seronegative for simian T-cell leukemia virus,simian immunodeficiency virus, simian retroviruses (SRV-1, SRV-2,SRV-3, and SRV-5 via PCR) and herpes simian B virus. This study wasapproved by the Battelle Institutional Animal Care and Use Committeeand the Animal Care and Use Review Office of the U.S. Army MedicalResearch and Materiel Command and conducted according to the prin-ciples set forth in reference 19.

Test and control articles. Current good manufacturing practicesgrade rF1V drug product (160 �g/ml), formulated in 5 mM Na/K phos-phate buffer containing 150 mM NaCl and 1.3% (wt/vol) Alhydrogel, wasdiluted in sterile phosphate-buffered saline (PBS) without additional ad-juvant to prepare vaccine doses ranging from 0.016 to 20 �g per CM.Vaccine dilutions were prepared on each day of vaccination. Control an-imals received sterile PBS.

Study design. Three CM studies were planned and conducted in astagewise approach such that results from one study allowed for the designand selection of vaccine doses for subsequent studies. The first two studieswere dose-ranging studies (n � 21 and 44) that were designed to generatea range of survival outcomes to evaluate the relationships among thevaccine dose, the antibody response, and survival. The third study

(n � 56), an expanded efficacy study, was conducted to further evaluatethese relationships with the goal of increasing statistical confidence in thestudy data. Group sizes and vaccine doses were selected for each study onthe basis of statistical analysis of the cumulative results and on the basis ofa probabilistic simulation of the most likely subsequent outcomes. For allstudies, CMs were randomized into study groups according to bodyweight and age, with males and females randomized separately. On days 0,56, and 121, CMs were administered vaccine or saline in a 0.5-ml volumeby the intramuscular route. To control for bias, vaccine and control ma-terials were labeled to identify each individual CM and included the vac-cination day and date but not vaccine dose or study group information.Technicians performing CM vaccinations, observations, sample collec-tions, and gross necropsies were blind to the vaccination status of theCMs. Blood samples were collected to evaluate antibody titers on days 0,14, 28, 56, 70, 84, 98, 121, 135, and 149. Blood samples drawn on the dayof vaccination and the day of challenge were collected before those activ-ities were conducted. A target challenge dose of 229 50% lethal doses(LD50) of Y. pestis Colorado 92 (CO92) (5.5 � 103 CFU) was selected forthese studies on the basis of an earlier model development study thatestablished an LD50 of 24 CFU (17). Animals were challenged by aerosolinhalation on day 149 and observed for 21 days postchallenge.

Telemetry. Animals in the two dose-ranging studies (n � 65) wereimplanted with telemetry transmitter units (D70-PCT; Data Sciences In-ternational) for collection of body temperature, activity, and respirationdata. The cardiac lead was not used and was trimmed and tied off accord-ing to the manufacturer’s instructions. Implantation surgery was per-formed as previously described (17). An approximately 5-week recoveryperiod was allowed before the animals were placed in the study. To con-serve battery life, telemetry units were not turned on during the vaccina-tion period. Animals in the expanded efficacy study were not implantedwith telemetry units.

Immune response. Bridge ELISAs were performed as previously de-scribed (20) to determine the levels of anti-rV, anti-rF1, and anti-rF1Vantibodies elicited by vaccination. The bridge ELISA uses an assay formatthat allows direct comparison across clinical and nonclinical samples andis therefore considered species neutral. Antibody binding to the rF1Vantigen coating the plate is detected by a biotinylated rF1V antigen ratherthan a biotinylated species-specific antibody. The assay is consideredsemiquantitative because the standard curve is generated with chicken IgYantibodies to allow measurement of polyclonal antibodies to Y. pestis an-tigens in serum. Results are calculated by using a four-parameter logisticcurve fit. The assay’s limits of detection are 5 and 320 U/ml, with limits ofquantitation between 10 and 160 U/ml.

Challenge material. Four days before a challenge, a vial of CO92Working Cell Bank was thawed and streaked for isolation on Congo redagar (CRA). The plate was incubated at 26 � 2°C for 36 to 72 h. SeveralCRA-pigmented colonies were selected and suspended in sterile heartinfusion broth (HIB) until the optical density at 600 nm (OD600) of thesuspension was 0.200 � 0.05. The suspension was diluted 1:100 into ster-ile HIB, and the culture was incubated at 26 � 2°C with shaking forapproximately 24 h. Bacteria were collected by centrifugation, washed,and resuspended in PBS containing 0.05% (wt/vol) gelatin and 9.7% (wt/vol) �-�-trehalose (PBSGT) to an OD600 of 2.56 � 0.2, which corre-sponds to a reference concentration of approximately 2.05 � 109 CFU/ml.The bacteria were further diluted in PBSGT to the appropriate nebulizerconcentration required to achieve the target aerosol challenge.

Challenge material was characterized for titer, phenotype, purity, andGram staining. Titer and phenotype were determined by plating dilutionsof the challenge material in triplicate on tryptic soy agar (TSA) and CRA,respectively. The plates were incubated at 28 � 2°C for 36 to 96 h. Colonycounts were performed to determine titer and percent pigmentation. Pu-rity was evaluated by streaking a sample for single-colony isolation on thefollowing medium types: TSA with 5% sheep blood (TSAB), MacConkeyagar (MAC), phenylethyl alcohol agar (PEA), and cefsulodin-Irgasan-novobiocin (CIN) agar. A set of plates consisting of each medium type

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were incubated at three different temperatures, 28 � 2°C, 37 � 2°C, and23 � 2°C. Colony morphology on plates incubated at the three tempera-tures was recorded daily for 3 days (72 � 2 h), with the exception of theMAC plates, which were discarded after 48 h. After 72 h, the sets of platesincubated at 28 � 2°C and 37 � 2°C were also discarded. The remainingset of TSAB, PEA, and CIN plates was incubated at 23 � 2°C for anadditional 11 days, and the colony morphology was recorded at 7 and 14days. Gram staining was performed by using routine procedures.

Aerosol challenge. CMs were challenged on study day 149 by head-only aerosol exposure. Before exposure, each CM was anesthetized withtiletamine HCl and zolazepam HCl (Telazol; 2 to 6 mg/kg), moved into aclass III biological safety cabinet, and placed in a plethysmograph. Bodyplethysmography of each CM was performed in real time during aerosolexposure to measure tidal volume, total accumulated tidal volume, andminute volume.

Exposure doses were determined on the basis of the calculated aerosolconcentration of Y. pestis CO92 in the test atmosphere of the exposurechamber and the total volume of test atmosphere air inhaled by the CMsmeasured via real-time plethysmography with Buxco BioSystems soft-ware (Buxco Research Systems, Wilmington, NC). The aerosol concen-tration of Y. pestis CO92 in the exposure chamber was determined byviable plate counts performed with aerosol samples collected with a glassimpinger (model 7541; Ace Glass, Inc., Vineland, NJ). Samples were di-luted as necessary and plated in triplicate on TSA plates. The aerosolparticle size was determined during each exposure with an aerodynamicparticle sizer spectrometer (model 3321; TSI Inc., Shoreview, MN).

The preparation and use of Y. pestis challenge material, CM challenges,and all postchallenge activities were conducted under biosafety level 3(BSL3)/animal BSL3 conditions in a facility registered with the CDC Se-lect Agent Program. The facility also meets requirements of the DoDBiological Select Agent Transfer program.

Clinical observations. Trained technical personnel observed the CMsthroughout the study. CMs were observed twice daily throughout thevaccination period. Following a challenge, the CMs were observed forclinical signs of disease three times a day, at approximately 8-h intervals,throughout the 21-day postchallenge observation period. The time to theonset of clinical signs, as measured by telemetry or signs of disease re-corded by technical personnel, was documented in the study records.

Bacteria in blood and tissues. Approximately 2 ml of blood was col-lected from either the femoral artery or a vein of each CM for bacterio-logical culture. In the first dose-ranging study, samples were collectedapproximately 6 h following the onset of fever and continued daily for 7days, whereas samples in the second study were collected on postchallengedays 2 through 9, regardless of the onset of fever. In the expanded efficacystudy, blood was collected from the control CMs at the time of death oreuthanasia and from survivors at the end of the postchallenge observationperiod (day 21). Blood was collected in tubes containing sodium polya-nethol sulfonate as an anticoagulant and mixed thoroughly. Within 4 h ofcollection, the entire sample volume was inoculated into 40-ml Bactecblood culture bottles (Becton Dickinson Peds Plus/F). The bottles wereincubated at 28 � 2°C for 24 to 48 h. Samples from the blood culturebottles were streaked onto CRA and CIN agar and incubated at 28 � 2°Cfor 36 to 96 h. Following incubation, the agar plates were visually in-spected for colony morphology consistent with Y. pestis.

To determine bacterial burdens in tissues, samples of spleen, liver,kidney, and lung tissues were collected at the time of necropsy and beforetissue fixation in all studies. The samples were homogenized, plated onCIN agar, and incubated at 28 � 2°C for 36 to 96 h to determine theconcentration of Y. pestis bacteria per gram of tissue.

Necropsy. Gross necropsies were performed on all CMs that suc-cumbed or were found moribund and euthanized following aerosol ex-posure. Necropsies were also performed on CMs that survived to the endof the 21-day postchallenge observation period. The tissues collected in-cluded the liver, lungs, spleen, intrathoracic lymph nodes, and any grosslesions. Sections of lung, intrathoracic lymph nodes, and any gross lesions

were examined by histopathology. Tissues were processed, and approxi-mately 5-�m sections were made and stained with hematoxylin and eosinfor microscopic examination.

Statistical analysis. Data collected from all three studies were com-bined for statistical analysis with Stata Statistical Software, release 12.Logistic-regression models were used to evaluate the relationship betweenbridge ELISA titers and survival, as well as between vaccine doses andsurvival. One-sided Fisher exact tests were performed to compare thesurvival proportions in the vaccinated and control groups. Time-to-deathdata combined with survival data were analyzed to determine if there weredifferences in protection between the study groups based on a time-to-death model.

RESULTSChallenge material. Characterization of the challenge materialfor all three studies indicated that the average concentration of theovernight cultures was 1.65 � 109 CFU (range, 8.83 � 108 to5.53 � 109 CFU). Further, the cultures were Gram-negative, purecultures of Y. pestis with �99% pigmented colonies.

Aerosol exposures. The mean estimated inhaled dose of Y.pestis across all three studies was 5.54 � 103 CFU (range, 3.97 �102 to 2 � 104 CFU) which represents 231 LD50 (range, 17 to 833LD50). The mass median aerodynamic diameter of the particlesacross all aerosol exposures ranged from 1.65 to 2.87 �m. Aerosolswith these particle sizes are capable of reaching the alveoli of thelung, which was the target area for deposition in the lung (21).

Antibody responses. Data from all three studies were com-bined to evaluate the antibody responses following vaccination.Since the kinetics of the anti-rF1, anti-rV, and anti-rF1V antibodyresponses following vaccination were similar, the results pre-sented here focus on the anti-rF1V antibody response as the rep-resentative response (Fig. 1). Anti-rF1V antibody titers were mea-sured in CMs vaccinated with doses of �0.4 �g beginning 2 weeksfollowing the first vaccination. The geometric mean concentra-tion (GMC) of anti-rF1V antibody decreased before administra-tion of the second dose on day 56 but was above the baseline level.A robust antibody response was observed in all groups that re-ceived �0.4 �g of rF1V 2 weeks (day 70) following the secondvaccination, with approximately 2-log increases in the GMCs ofanti-rF1V antibody compared to those at day 56. A decrease fromthe peak antibody titers was observed by day 84; however, anti-body titers did not decline to the levels observed just before thesecond vaccination. Administration of the third vaccination onday 121 made the GMCs of anti-rF1V antibody slightly higherthan the second vaccination. Anti-rF1V antibodies were not de-tected in CMs vaccinated with rF1V doses of �0.08 �g or in sham-vaccinated control CMs.

Survival. A summary of vaccine dose, percent survival, meantime to death, and GMCs of anti-rF1V antibody at the time ofchallenge is shown in Table 1. Administration of the rF1V vaccineto CMs on a three-dose schedule with doses ranging from 0.0016to 20 �g demonstrated antibody-dependent survival and elicited arange of survival outcomes following a lethal aerosol challenge.A significant association between vaccine doses and survival(P � 0.0001) was observed, with higher vaccine doses associatedwith higher probabilities of survival. The proportion of CM sur-vival in groups vaccinated with �4 �g was significantly greaterthan that of the control group, in which 2 of 20 CMs survived thechallenge. Further, a statistically significant (P � 0.0001) vaccinedose-dependent response was observed in all three bridge ELISAs(rFl, rV, and rF1V). Similarly, the association between bridge

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ELISA titers (anti-rF1, anti-rV, and anti-rF1V) on day 149 andsurvival was statistically significant (all P values of �0.0001). Lo-gistic-regression curves illustrating these relationships, with anti-rF1V as the representative response, are shown in Fig. 2. Pairwisecomparisons assessing the time to death and overall survival of thegroups show significantly later deaths in groups vaccinated with�4 �g than in the controls (Table 2).

Clinical observations. Clinical signs associated with pneu-monic plague, such as fever, coughing, changes in respiration,lethargy, and hunched posture, were observed during the post-challenge period. There was a statistically significant differencebetween survivors and nonsurvivors (P � 0.05) in the frequencyof clinical signs, as determined by Fisher’s exact test (Table 3).There was a trend toward an increased incidence of the clinicalsigns hunched posture and lethargy with smaller doses of rF1V(data not shown).

Telemetry. An increase in body temperature was the first te-lemetry parameter exhibiting a change following a challenge. Fe-ver was identified as an increase in body temperature of �1.5°Cabove the animal’s baseline for �2 h. Mean temperature changesfrom the baseline plotted over time postchallenge are presented

for all vaccine dose groups in Fig. 3a. Of the 65 CMs in the dose-ranging studies, 33 (25 vaccinated and 8 control) exhibited feverand 29 (21 vaccinated and 8 control) succumbed to the challenge.The mean time to onset of fever was 63 h postchallenge (range,46.2 to 155.3 h), with a mean time to death following the onset offever of 55.7 h (range, 33.3 to 87.8 h). Statistically significant cor-relations between the vaccine dose and time to onset of fever(P � 0.0001) and the time from onset of fever to death (P �0.0001) were observed. None of the CMs vaccinated with 16 or 20�g of rF1V developed fever. Beginning 49 h postchallenge, groupmean temperatures of control CMs and those vaccinated with�0.4 �g of rF1V were elevated �2°C. A similar increase in tem-perature was delayed 24 h in CMs vaccinated with 2 �g of rF1V.Four vaccinated CMs developed transient fever postchallenge.Two of the CMs received vaccine doses of 0.016 and 0.4 �g, andonset of fever occurred at 54.5 and 47.4 h, respectively, postchal-lenge. The other two CMs were vaccinated with 12 �g and devel-oped fever at 82.3 and 155.3 h postchallenge. The body tempera-tures of three of the four CMs returned to the baseline by day 6postchallenge. The fever of the fourth CM (155.3 h) resolved to the

FIG 1 Group GMC rF1V bridge ELISA titers showing the kinetics of the immune responses following vaccination on days 0, 56, and 121 and just before achallenge on day 149. The vaccine doses used are shown on the right. Detectable antibody responses were not observed in CMs vaccinated with �0.08 �g, anddata at or below this level overlap in the graph. The error bars represent the standard error of the mean.

TABLE 1 Summary of GMCs determined by rF1V bridge ELISA and associated survival

GroupVaccine dose(�g)

GMC (U/ml) of rF1V(SD)a

Mean time todeath (h)

% Survival (no. ofsurvivors/total)

P value (survivorsvs controls)

1 20 11,880 (1.168) NAb 100 (4/4) 0.0071c

2 16 8,090 (1.306) NA 100 (6/6) 0.0009c

3 12 5,262 (1.552) NA 100 (9/9) �0.0001c

4 8 4,156 (2.060) 81.3 88 (22/25) �0.0001c

5 4 2,027 (2.264) 102.8 63 (15/24) 0.0023c

6 2 886 (2.542) 125.2 29 (6/21) 0.53757 0.4 50 (9.164) 111.1 20 (1/5) 1.00008 0.08 �10 (NA) 118.8 0 (0/3) 1.00009 0.016 �10 (NA) 102.5 25 (1/4) 1.000010 Control �10 (NA) 88.7 10 (2/20) NAa Anti-rF1V antibody titers determined by bridge ELISA.b NA, not applicable.c Survival is significantly greater than that of the control group, as determined by Bonferroni-Holm adjusted one-sided Fisher exact test (P value of �0.05).

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baseline by day 9. All four of these CMs survived to the end of thepostchallenge observation period.

Statistically significant dose-response relationships were ob-served between the vaccine dose and the onset of respiratory

changes such as an increased respiratory rate (P � 0.0003). As thevaccine dosage increased, the time to the onset of a respiratory rateincrease became longer (Fig. 3b).

A statistically significant relationship between increasing vac-cine doses and a delay in decreased activity was also observed (P �0.0251). Activity levels in the CMs postchallenge were generallylower than the baseline levels in the controls and groups vacci-nated with �4 �g of rF1V (Fig. 3c). The reduction in activityrelative to the baseline was especially evident in the 0.08-�g vac-cine dosage group. Group mean activity levels generally returnedto prechallenge baseline levels by day 6 postchallenge, althoughsome changes in activity continued in surviving CMs until the endof the postchallenge observation period.

Bacteremia and tissue bacterial burdens. All CMs that suc-cumbed to the challenge had positive blood cultures (data notshown). Two vaccinated CMs (0.4 and 0.016 �g) had positiveblood cultures for 1 day (day 3) but survived. The two survivingcontrols were not bacteremic. Increasing doses of the rF1V vac-cine reduced the numbers of CMs that were bacteremic and de-layed the onset of bacteremia in the vaccinated CMs that suc-cumbed. The proportions of CMs vaccinated with 4 �g (2/9), 8 �g(0/10), 12 �g (0/9), or 16 �g (0/6) of rF1V that became bacteremic

FIG 2 Logistic-regression curves showing the relationships between anti-rF1V bridge ELISA titers and the probability of survival (a) and between vac-cine doses and the probability of survival (b). Both were highly statisticallysignificant predictors of protection (P � 0.0001).

TABLE 2 Bonferroni-Holm adjusted P values from pairwise log rank tests comparing times to death and overall survival of groups

Vaccine dose (�g)

P value for vaccine dose (�g) of:

0.08 0.4 2 4 8 12 16 20 0 (control)

0.016 1.0000 1.0000 1.0000 0.8089 0.0019a 0.0755 0.3212 0.8089 1.00000.08 1.0000 1.0000 0.0864 0.0001a 0.0059a 0.0621 0.2717 1.00000.4 1.0000 0.5937 0.0002a 0.0361a 0.2054 0.6294 1.00002 0.7044 0.0009a 0.0411a 0.2262 0.6757 1.00004 0.8089 0.8089 1.0000 1.0000 0.0010a

8 1.0000 1.0000 1.0000 �0.0001a

12 1.0000 1.0000 0.0014a

16 1.0000 0.0221a

20 0.1421a The times to death and overall survival of the groups were significantly different at the Bonferroni-Holm adjusted 0.05 level of significance. The survival rates of groups that receivedvaccine doses of 4, 8, 12, and 16 �g were all significantly different from that of the control group. The survival rate of the 8-�g dosage group was significantly different from those of the �2-�g vaccine dosage groups. Similarly, the survival rate of the 12-�g dosage group was significantly different from those of the �2-�g vaccine dosage groups, except for the 0.0016-�g group,where the P value is close to significance. The survival rates in the 16- and 20-�g vaccine dosage groups, despite being 100%, with two exceptions (20-�g and control groups), were notstatistically significantly different from those of the �2-�g vaccine dosage groups. This appears to be the result of smaller sample sizes in the highest vaccine dosage groups.

TABLE 3 Incidence of clinical signs

Clinical sign

% (no./total) of CMs

Survivors(n � 66)

Nonsurvivors (n � 55)

Vaccinated(n � 37)

Controla

(n � 18)

Hunched posture 23 (15/66) 62 (23/37) 100 (18/18)Feverb 11 (4/35)c 97 (29/30)d 100 (18/18)Lethargy 2 (1/66) 78 (29/37) 72 (13/18)Changed respiratione 0 (0/66) 30 (11/30) 17 (3/18)Cough 5 (3/66) 30 (11/30) 6 (1/18)a Two control animals survived a challenge and had no clinical signs of disease.b Fever was monitored in the two dosage-ranging studies only.c This represents 4 of 35 CMs with transient fever that resolved.d Onset of fever was not determined in one vaccinated CM because of a malfunction inthe telemetry transmitter.e Changes in respiration included wheezing, labored breathing, and changes inrespiratory rate.

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by day 3 postchallenge and succumbed were statistically signifi-cantly lower than the proportion of control CMs (8/8) with posi-tive blood cultures (all P values of �0.05). Blood cultures collectedfrom survivors at the end of the 21-day postchallenge observationperiod were all negative.

All CMs that succumbed to the challenge had �103 CFU/g inlung, spleen, liver, and kidney tissue samples. The lungs consis-tently exhibited the largest quantities of bacteria, followed by thespleen, liver, and kidneys (Table 4). The differences in tissue bac-terial burdens between vaccinated and control CMs that suc-

cumbed were not statistically significant, as determined by t test(P � 0.8612).

Necropsy and histopathology. Gross lesions in animals thatdied following an aerosol challenge included mottled lung discol-oration and/or discolored intrathoracic lymph nodes and werepresent regardless of treatment (vaccinated or control). These le-sions correlated with microscopic observations of suppurative in-flammation, fibrin exudation, hemorrhage, necrosis, and edemain the airways and interstitium of the lungs and within the in-trathoracic lymph nodes, as well as intralesional rod-shaped bac-

FIG 3 Telemetry data for body temperature (a), respiratory rate (b), and physical activity (c) are shown as group mean values over 8-h intervals. Each plot showsthe mean change from the baseline; the zero ordinate represents no change from normal baseline values.

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teria with bipolar staining, all consistent with Y. pestis. There was atrend in the frequency of gross lung lesions and vaccine dosages.The majority of lesions were found in the lungs of CMs vaccinatedwith doses of �8 �g (Table 5). Among the CMs that succumbed,there were no major differences in lesion incidence and/or severitybetween the vaccinated and control groups. Lesions were not ob-served in CMs that survived to the end of the postchallenge obser-vation period.

DISCUSSION

The rF1V efficacy studies described here were conducted to fur-ther develop the CM model for use in testing the efficacy of rF1Vaccording to the requirements of the FDA Animal Rule. Vaccina-tion with rF1V elicited an immune response that provided protec-tion from a lethal aerosol challenge with Y. pestis. Our study resultsare similar to those reported by other groups conducting proof-of-concept studies showing that rF1- and rV-based vaccinesagainst plague elicit protective immune responses in CMs follow-ing an aerosol challenge (15, 22, 23). However, in contrast to theseproof-of-concept studies, which most often evaluated only onevaccine dose, our studies evaluated a broad range of vaccine doses.This provided the opportunity to evaluate the relationship be-tween vaccine dose and bridge ELISA titers across a range of sur-vival outcomes and allowed us to identify statistically significantcorrelations among vaccine doses, anti-rF1V antibody titers, andthe probability of survival. Combining all of our study data pro-vided a statistically precise estimate of these relationships, sup-porting the use of rF1V bridge ELISA antibody titers as a correlateof protection.

Our earlier model development study (17) identified fever, asmeasured by telemetry, as the first clinical sign of disease followingan aerosol challenge with CO92. Following the onset of fever,increased respiration and a decreased activity level (as measuredby telemetry), a hunched posture, and bacteremia were observedin CMs that succumbed following a challenge. Therefore, in thepresent studies, we sought to determine whether rF1V vaccination

delayed the onset or ameliorated signs of pneumonic plaguein CMs.

Four vaccinated CMs in the dosage-ranging studies developedtransient fever but survived a challenge. Two of these CMs re-ceived small vaccine doses (0.0016 and 0.4 �g) and were bactere-mic on day 3 but tested negative on all subsequent days. At thetime of the challenge, the CM vaccinated with 0.0016 �g of rF1Vhad no detectable anti-rF1V antibodies, whereas the CM vacci-nated with 0.4 �g of rF1V had an anti-rF1V antibody titer of 127U/ml. Given the low anti-rF1V antibody titer in one CM and thelack of antibodies in the other, the survival of these two CMs isbelieved to be associated with individual animal variability. Theother two surviving CMs were vaccinated with 12 �g of rF1V andnever developed bacteremia. The anti-rF1V antibody titers inthese two CMs were 3,584 and 7,290 U/ml at the time of challenge,which suggests the immune response elicited by rF1V limited theinfection and thus prevented bacteremia.

Postchallenge clinical observations were normal in two of thefour CMs with transient fever, whereas the other two CMs exhib-ited diarrhea and/or occasional hunched posture. In the CM vac-cinated with 0.4 �g, hunched posture was coincident with a tran-sient fever. The occurrence of diarrhea in the other CM wasobserved periodically during the vaccination period, as well as thepostchallenge observation period, and therefore not consideredstudy related. This CM also occasionally exhibited a hunched pos-ture postchallenge, but it was not associated with transient fever.The relevance of these clinical observations in the four survivingCMs is not known. Hunched posture, most often observed justbefore death, was accompanied by other clinical signs such as re-spiratory distress, lethargy, cough, or not eating and was consis-tent with observations in our model development study (23).Dose-dependent vaccine responses were associated with delays inother parameters measured by telemetry in the dosage-rangingstudies, such as an elevated respiratory rate and decreased activity.This provides further support that rF1V vaccination can provideprotection against pneumonic plague.

TABLE 4 Concentrations of Y. pestis in tissuesa

Tissue

Mean bacterial burden (CFU/g) in group given vaccine dosage (�g/ml) of:

0 (control) 0.016 0.08 0.4 2 4 8

Spleen 5.15 � 107 6.46 � 107 6.76 � 106 3.03 � 107 3.74 � 106 3.58 � 106 1.41 � 106

Liver 1.79 � 107 3.56 � 107 5.86 � 106 9.98 � 106 1.42 � 107 1.89 � 107 3.04 � 106

Kidney 2.37 � 106 8.73 � 106 1.6 � 105 1.68 � 106 1.25 � 106 6.86 � 106 6.25 � 105

Lung 5.4 � 108 1.69 � 107 1.49 � 108 3.18 � 107 9.64 � 109 1.03 � 108 9.46 � 108

a Mean concentrations of Y. pestis in tissues were determined at the time of necropsy.

TABLE 5 Incidence of microscopic lesions in lungs and lymph nodes

Tissue

No. of CMs with lesions/total (%) in group given rF1V dosage (�g)a of:

0 (control) 0.016 0.08 0.4 2 4 8

Lung 18/20 (100) 3/4 (75) 3/3 (100) 4/5 (80) 15/21 (71) 9/24 (38) 3/25 (12)

Lymph nodesBronchial 11/20 (55) 1/4 (25) 1/3 (33) 1/5 (20) 12/21 (57) 5/24 (21) 1/25 (4)Mediastinal 12/20 (60) 0/4 (0) 0/3 (0) 0/5 (0) 8/21 (38) 5/24 (21) 1/25 (4)

a Animals vaccinated with �12 �g of rF1V survived a challenge, and lesions were not observed at the time of necropsy (end of the postchallenge observation period). Lesions werenot observed in the two surviving controls.

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All of the CMs in the dosage-ranging and expanded efficacystudies that became bacteremic, with the exception of the twoanimals discussed above, succumbed to a challenge. There was nosignificant difference in the proportions of animals having posi-tive blood cultures between the small-dose vaccine groups (�2�g) and the control group, suggesting that these small vaccinedosages provided little benefit in ameliorating disease. Further,the concentrations of Y. pestis in the lungs, livers, spleens, andkidneys of vaccinated and control CMs that succumbed to a chal-lenge were not different between these groups. This suggests thatonce the bacteria reach the bloodstream and disseminate to theseorgans, low-level vaccine dosages do not induce an immune re-sponse sufficient to combat infection.

The observed survival of 2 of 12 control CMs in the expandedefficacy study was surprising, given the estimated dosages of 272LD50 (6.52 � 103 CFU) and 301 LD50 (7.22 � 103 CFU) inhaled bythese animals and that survival of control animals was not ob-served in the two dosage-ranging studies. An examination of theaerosol exposure system parameters indicated that the system op-erated as expected during the challenge of these CMs. Neitheranimal had anti-rF1V titers at the baseline or at any of the collec-tion points throughout the vaccination period. However, bridgeELISA titers measured at the end of the 21-day postchallenge ob-servation period in these CMs yielded anti-rF1V antibody titers of376 and 1,480 U/ml, indicating that the animals were exposed toY. pestis and mounted an immune response following the chal-lenge. Other groups have also reported observations of controlsurvival during vaccine efficacy studies conducted with CMs. In astudy by Cornelius et al. (22), one of four control CMs survived achallenge with an inhaled dose of 1.45 � 104 CFU (216 LD50) ofCO92, while three animals that succumbed to the challenge re-ceived inhaled doses ranging from 1.27 � 104 to 2.3 � 104 CFU(189 to 343 LD50). Quenee et al. (24) reported 25 and 50% survivalof control CMs following exposure to 250 and 50 LD50, respec-tively. Individual challenge data and assessments of rF1V antibodytiters pre- and postchallenge were not provided for all of the con-trol animals in these studies, making interpretation of the datadifficult. On the basis of these observations, designers of efficacystudies with CMs should consider low-level control survival indetermining group size to ensure that the studies are appropriatelypowered to determine survival differences between vaccinatedand control groups.

Our studies describe the further development of the CM modelto evaluate rF1V vaccine efficacy. Data generated in these studiesdetermined that bridge ELISA titers and vaccine dose were highlystatistically significant predictors of protection (P � 0.0001) andsupport the use of anti-rF1V bridge ELISA titers as a correlate ofprotection. Vaccination with rF1V was shown to ameliorate dis-ease in a dose-dependent manner. The data support the use of theCM model and provide important information that will be used todesign future pivotal efficacy studies that will play an importantrole in vaccine licensure based on the FDA Animal Rule.

ACKNOWLEDGMENTS

This study was supported by Medical Countermeasure Systems-JointVaccine Acquisition Program (MCS/JVAP) contract DAMD 17-98-C8024. The current development effort for the rF1V vaccine is supportedby an international partnering arrangement that includes the UnitedKingdom and Canada. The facilities performing these model develop-

ment studies are fully accredited by the Association for the Assessmentand Accreditation of Laboratory Animal Care International.

We thank the Battelle technical staff for excellent assistance during thisstudy. We also thank Stefanie Hone and Abraham Kallarakal for criticalreview of the manuscript.

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