DTIC ADA480405: Vaccination with F1-V Fusion Protein Protects Black-Footed Ferrets (Mustela Nigripes) Against Plague Upon Oral Challenge with Yersinia Pestis

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JournalofWildlifeDiseases,44(1),2008,pp.1–7
#WildlifeDiseaseAssociation2008
VACCINATION WITH F1-V FUSION PROTEIN PROTECTS
BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) AGAINST
PLAGUE UPON ORAL CHALLENGE WITH YERSINIA PESTIS
Tonie E. Rocke,1,5SusanSmith,1 PaulMarinari,2JulieKreeger,3JeffreyT. Enama,4 and
Bradford S.Powell4
1USGeologicalSurvey,NationalWildlifeHealthCenter,6006SchroederRoad,Madison,Wisconsin53711,USA
2USFishandWildlifeService,NationalBlack-FootedFerretConservationCenter,P.O.Box190,Wellington,Colorado
80549,USA
3USFishandWildlifeService,NationalBlack-FootedFerretConservationCenter,2362Highway34,Wheatland,
Wyoming82201,USA
4USArmyMedicalResearchInstituteofInfectiousDiseases,BacteriologyDivision,FortDetrick,Frederick,Maryland
21702,USA
5Correspondingauthor(email:[email protected])
ABSTRACT: Previous studies have established that vaccination of black-footed ferrets (Mustela
nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against
plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V
antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected
mouse,aprobableroutefornaturalinfection.Eightblack-footedferretkitswerevaccinatedwith
F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was
administered 3 mo later via SC injection. Four additional ferret kits received placebos. The
animalswerechallenged6 wkaftertheboostbyfeedingeachoneaY.pestis-infectedmouse.All
eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days
afterexposure.Todeterminethedurationofantibodypostvaccination,18additionalblack-footed
ferretkitswerevaccinatedandboostedwithF1-VbySCinjectionat60and120 days-of-age.High
titerstobothF1andV(meanreciprocaltitersof18,552and99,862,respectively)werefoundinall
vaccinatesupto2 yrpostvaccination,whereassevencontrolanimalsremainedantibodynegative
throughoutthesametimeperiod.
Key words: Black-footed ferrets, F1-V protein, sylvatic plague, vaccine, Yersinia pestis.
INTRODUCTION (Williams et al., 1994; Castle et al., 2001;
Rocke et al., 2006).
Plague, caused by the gram-negative
In recent studies conducted at the US
bacterium, Yersinia pestis, is a natural
Geological Survey, National Wildlife
threat to black-footed ferrets (Mustela
Health Center (NWHC), we demonstrat-
nigripes), and it has severely hindered ed that a majority of ferrets could be
recent efforts to restore this endangered protected against subcutaneous (SC) Y.
speciestoitshistoricrange(Barnes,1993). pestis challenge by vaccination with the
The disease occurs regularly in rodents F1-V protein (Rocke et al., 2004, 2006),
throughout the western USA and is a recombinant fusion protein that consists
particularly devastating for prairie dogs of two known protective antigens ex-
(Cynomys spp.). Ferrets, which feed pressed by Y. pestis (Powell et al., 2005).
exclusively on prairie dogs, are likely Vaccinated animals that survived an initial
exposed to Y. pestis by direct and indirect SC challenge with Y. pestis were com-
association with their prey. They may be pletely resistant to a secondary exposure
bitten by Y. pestis-infected fleas as they via consumption of a Y. pestis-infected
huntandkillprairiedogsintheirburrows. mouse (Rocke et al., 2006), and a few
Alternatively, and probably more impor- vaccinated animals even survived primary
tantly,ferretsmaycontractplaguethrough oral challenge (Rocke, unpubl. data).
direct contact or inhalation of infectious Although these results were considered
aerosols as they feed on ill or dead prey promising, both of these studies were
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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
01 JAN 2008 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Vaccination with F1-V fusion protein protects black-footed ferrets
5b. GRANT NUMBER
(Mustela nigripes) against plague upon oral challenge with Yersinia
pestis. Journal of Wildlife Diseases 44:1-7 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Rocke, TE Smith, S Marinari, P Kreeger, J Enama, J Powell, BS
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United States Army Medical Research Institute of Infectious Diseases, REPORT NUMBER
TR-07-083
Fort Detrick, MD
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
Earlier studies established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion
protein by subcutaneous (SC) injection protected animals against plague upon injection of the bacterium
Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague
contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight
black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60
days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits
received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y.
pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague
within 3 days after exposure. To determine the duration of antibody post-vaccination, 18 additional
black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age.
High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862 respectively) were found in all
vaccinates up to 2-yr post-vaccination, whereas seven control animals remained antibody negative
throughout the same time period.
15. SUBJECT TERMS
Yersina pestis, plague, F1-V vaccine, black-footed ferrets, efficacy, challenge studies, wildlife biology
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON

a. REPORT b. ABSTRACT c. THIS PAGE SAR 7
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Prescribed by ANSI Std Z39-18
2 JOURNALOFWILDLIFEDISEASES,VOL.44,NO.1,JANUARY2008
conducted in older animals that repre- NWHC, the animals were treated prophylac-
tically for coccidiosis and housed individually
sented less-than-ideal candidates for vac-
in stainless-steel cages as described previously
cination (Rocke et al., 2006) because they
(Rocke et al., 2004). The animals were fed
were4-to6-yr-oldspent breedersnearing
a diet of raw horse meat (Toronto Small
the end of their normal life span. Ferret CarnivoreDiet,MeatProducts,3347Kennedy
kits that are bred in captivity for field Road #1, Scarborough, Ontario M1V 3P1,
release are typically handled only twice: Canada),periodicallysupplementedwithmice
(NWHC) and prairie dog meat (NBFFCC).
whentheyareplaced inconditioning pens
This study was reviewed and approved by
at60 days-of-ageandjustbeforereleaseat
NWHC’s Animal Care and Use Committee
approximately 120 days-of-age. This tim- and Biosafety Committee. All personnel han-
ing may align with a practical two-dose dling Y. pestis-infected animals or carcasses
schedule for immunization, if vaccines were required to wear high efficiency partic-
ulate air-filtered respirators with full-face
administered at those age and time inter-
shields, rubber aprons and boots, and double
vals were shown to elicit protective
surgical gloves. Research was conducted in
immunity. Moreover, because ingestion compliance with the Animal Welfare Act and
and/or inhalation are probably important other federal statutes and regulations relating
transmission routes for Y. pestis in ferrets, to animals and experiments involving animals.
an effective vaccine for field use must
Experimentaldesign:Vaccineefficacytrial
demonstrate protection against plague
after consumption of infected prey. At approximately 60 days-of-age,eachof 12
ferretkitswasanesthetizedatNBFCC,andup
Here, we establish that vaccination of
to a 2 ml blood sample was drawn using
ferret kits with F1-V protein confers
methods described previously in Rocke et al.
protection against primary oral challenge
(2004). Eight of these ferrets received 0.5-ml
via consumption of a Y. pestis-infected F1-V vaccine-adjuvant preparation (100 mg of
mouse. Furthermore, we demonstrate the antigen)bySCinjectionbetweenthescapulae.
longevity of antigen-specific antibody ti- The F1-V fusion protein and our methods of
preparingthevaccine-adjuvantmixtureforSC
ters in immunized ferrets, which remain
injection have been described previously
high up to 2 yr postvaccination (PV).
(Rocke et al., 2004; Powell et al., 2005).
Briefly, the antigen was diluted in Modified
MATERIALSANDMETHODS Dulbecco’s medium (Sigma, PO Box 14508,
St. Louis, Missouri 63178, USA), mixed 1:1
Experimentalanimals with 0.2% Alhydrogel (United Vaccines, 7819
Thirty-seven black-footed ferret kits were AirportRd.,Madison,Wisconsin53562,USA),
selected for this study at the US Fish and and rocked gently overnight at 4 C. Four
Wildlife Service, National Black-Footed Fer- controlanimalsreceivedaplaceboof0.5 mlof
ret Conservation Center (NBFFCC), Wheat- the adjuvant (without protein antigen) sus-
land, Wyoming. One group of 12 kits (all pended in Dulbecco’s medium.
males) was designated for the vaccine efficacy Two mo later, all 12 animals were trans-
trial. The second group of 25 kits (mixed sex) portedtoNWHC.Afteranacclimationperiod
was used to determine the duration of of several wk, the animals were anesthetized,
antibody after vaccination. All animals were a2-mlbloodsamplewasdrawn,andabooster
60 days-of-age and were marked individually injection of F1-V was administered. About
with SC embedded microchips (AVID, 78294 6 wk after the booster dose, another blood
Oak Ridge Road, Folsom, Louisiana 70437, samplewasdrawn,andtheanimalswereorally
USA). At NBFCC, the kits were housed with challenged by feeding each a single Y. pestis-
dam and littermates until they reached infectedmouse.Six-wk-oldoutbredSwissICR
120 days-of-age. At NWHC, the animals were mice(HarlanSpragueDawley,POBox29176,
housed individually as described previously Indianapolis, Indiana 46229, USA) were in-
(Rocke et al., 2004). After the initial vaccina- oculatedwith.4,000cfu(in0.1 ml)wildtype
tion (described later), the group of 12 animals Y.pestisstrainCO92(,20050%lethaldoses)
was transported to the NWHC where they by intradermal injection. Upon death 3 days
were placed in a biosafety level-three animal- after challenge, one mouse was placed in the
holding facility; the other group of 25 cage of each ferret. Any carcasses or parts of
remained at the NBFFCC. Upon arrival at carcassesnotingestedbyferretswithin3–4 hr
ROCKEETAL.—VACCINATIONPROTECTSBLACK-FOOTEDFERRETSAGAINSTPLAGUE 3
wereremovedanddiscarded.Forthoseferrets
that didnotfullyconsume theirmice, another
Y.pestis-infectedmousewasgiventothemthe
following day. Ferrets were monitored daily
forsignsofillness,anddayofdeathwasnoted;
severely debilitated animals were euthanized
by CO asphyxiation.
2
Any ferrets that survived challenge were
bled to determine antibody titers after 3 wk
and then euthanized by intracardiac injection
of Euthasol (Delmarva Laboratories, Inc.,
1500 Huguenot Road, Suite 106, Midlothian,
Virginia 23113, USA). In both experiments, FIGURE 1. Geometric mean titers for anti-F1
antibody and anti-V antibody in black-footed ferret
dead or euthanized ferrets were immediately
kits that were vaccinated with F1-V fusion protein.
necropsied.Selectedtissueswerecollectedfor
Animals were vaccinated via subcutaneous (SC)
bacterial isolation (Rocke et al., 2004) and
injection on 17 August 2004, were boosted similarly
histologic examination.
on 15 November 2004, and challenged with wild-
type(CO92)Yersiniapestison1December2004.
Experimentaldesign:Durationof
antibodyresponse
The second group of 25 ferrets was vacci- anti-Vlogtiterfromthelogtiterofeachofthat
natedviaSCinjectionatapproximately60and same animal’s subsequent blood samples.
120 days-of-age as already described; 18 ani- Combined (anti-F1 + anti-V) log titers were
mals received the F1-V vaccine-adjuvant calculatedbyaddingthelogtitersofindividual
mixture, and seven animals received the animals for each antigen (Williamson et al.,
placebo. All of these animals remained at 1999). All statistical analyses were performed
NBFFCC throughout the duration of the using SAS software (SAS Institute, Cary,
study, and blood samples were drawn pre- NorthCarolina,27513USA).Statisticaldiffer-
vaccination, preboost, and ,3 mo, 6 mo, and ences in change of titer between groups were
1 yr postvaccination (PV) for all 25 animals, tested separately at each blood sampling
and 2 yr PV for 20 animals. period using a one-tailed Wilcoxon test. For
the vaccine efficacy trial, the difference in
Antibodydetermination survivorship between groups was analyzed
using the Fisher two-tailed exact test.
All blood samples were collected in sterile
glass serum separator tubes. After centrifuga-
tion of blood samples, the serum was trans- RESULTS
ferred to 2-ml polypropylene tubes and frozen
Vaccineefficacy
at220 Cforfutureanalyses.Antibodiesagainst
F1 and V antigens were measured using an All eight vaccinated ferrets developed
enzyme-linked immunosorbent assay (ELISA)
significantantibodytiterstobothF1andV
as previously described (Rocke et al., 2004),
after their first vaccine dose as compared
withslightmodificationsasfollows.AllELISA’s
were conducted at NWHC. Coating of ELISA to both their prevaccination titers
plates with V antigen was performed with the (P,0.0001)andthetitersofcontrol-group
same concentration ofprotein(1 mg/ml) but in animals (P,0.005). After the second
a 100-ml volume instead of 50 ml. Serum
vaccine dose, all eight vaccinates had
samples were serially diluted fourfold starting
significantly increased (Fig. 1) anti-F1
at1:640. The highest dilution that was positive
(definedasexceedingthemeanoffournegative and anti-V titers (at least fourfold and as
control samples by three standard deviations) much as 64-fold; P,0.0001). In contrast,
was considered to be the end point, and its the titers of control animals remained
reciprocalvaluewasrecordedasthetiter.
negative throughout the experiment.
All eight vaccinates survived oral chal-
Statisticalanalysis
lenge with a Y. pestis-infected mouse with
Antibody titers were transformed by calcu-
no signs of illness, whereas the four
latingthelog ofthetiter.Changeinlogtiter
10
was then calculated by subtracting an in- control animals that died succumbed to
dividual animal’s pre-inoculation anti-F1 or plague within 3 days of ingesting a Y.
4 JOURNALOFWILDLIFEDISEASES,VOL.44,NO.1,JANUARY2008
FIGURE 3. Geometricmeantitersofanti-F1and
FIGURE 2. Survival of black-footed ferrets vacci- anti-Vantibodiesinblack-footedferretkitsvaccinat-
nated with F1-V protein and control animals that ed with F1-V protein at 0, 3mo, 6mo, and 1yr
received a placebo after challenge via consumption (n518) and 2yr (n514) postvaccination. Animals
ofaY.pestis-infectedmouse. received the first vaccine dose in August 2004 and
receivedasimilarboostinOctober2004.
pestis-infected mouse (Fig. 2). Gross and
negative throughout the 2-yr duration of
histologic lesions consistent with plague
the study.
were observed in all dead control animals,
including marked congestion and hemor-
DISCUSSION
rhage in the liver, spleen, lymph nodes,
and intestines. Yersinia pestis was isolated In this study, we found that black-
from nasal swabs and liver in every case. footed ferret kits vaccinated and boosted
Antigen-specific titers of all survivors SC with F1-V protein were protected
rose significantly after challenge (Fig. 1). against plague contracted via ingestion of
Meananti-F1andanti-Vantibodytitersof a Y. pestis-infected mouse. All vaccinated
surviving vaccinates were significantly animals developed high antibody titers
higher (P50.0187 and 0.0368, respective- againstF1andVantigens,survivedoralY.
ly) than their mean prechallenge titers. pestis challenge, and showed a significant
Upon euthanasia and necropsy of surviv- increase in antigen-specific titers after
ing vaccinates at the end of the experi- challenge. Together with our earlier stud-
ment, no obvious lesions characteristic of ies(Rockeetal.,2004,2006),theseresults
plague were observed at the organ or demonstrated that the F1-V protein is an
tissue level, and no Y. pestis was isolated effective vaccine against plague for black-
from tested tissue samples. footed ferrets, eliciting protective immu-
nity against both an oral challenge (con-
Durationofantibody
sumption of infected prey) and an SC
All18vaccinatesdevelopedasignificant injection of the bacteria (simulated flea-
increase in antibody titers to F1 and V bite exposure). Our results are consistent
antigens (Fig. 3) after the initial vaccine with numerous other studies that have
dose (P,0.0001) at 60 days-of-age and an demonstrated the protective efficacy of
even higher increase in titer after the F1-V fusion protein against Y. pestis
booster injection (P,0.0001) at 120 days- infection in mice (Anderson et al., 1998;
of-age. Antibody titers remained high for Williamson etal.,1999;Glynnetal.,2005;
at least 2 yr PV (Fig. 2), and there was no Powelletal.,2005)andreaffirmtheutility
difference in combined log titers (anti-F1 of F1-V protein as a plague vaccine.
and anti-V) between males and females The antibodies elicited against Y. pestis
(P50.3584). Controls remained antibody antigens after captive ferret kits were
ROCKEETAL.—VACCINATIONPROTECTSBLACK-FOOTEDFERRETSAGAINSTPLAGUE 5
vaccinated with F1-V protein appear to
lastatleast2 yr(thelasttimepointtested)
and may possibly last longer, since they
showed little drop in titer. Although we
did not challenge these animals, we
believe their titers continued to be pro-
tective against plague. Anderson et al.
(1998)demonstratedthatF1-V-vaccinated
mice retained antibodies protective
against pneumonic plague for as long as
1 yr, the last time point tested in their
study. Furthermore, other investigators
have shown that the combined anti-F1
and anti-V log titer correlates better with
protection against plague in mice than do
FIGURE 4. Combined anti-F1 and anti-V anti-
either of the individual log-transformed body titers of black-footed ferrets immunized with
anti-F1 or anti-V titers (Williamson et al., F1-Vfusionproteinthateitherdiedorsurvivedupon
1999; J. Adamovicz, pers. comm.). By Y. pestis challenge. Data are combined from this
study and previous studies (Rocke, et al., 2006,
analyzing titer data (Fig. 4) generated
unpubl. data) and include animals that received
from vaccinated black-footed ferrets in
either1or2subcutaneous(SC)injectionsofvaccine
this study, a previous study (Rocke et al., at a dose of 100mg F1-V protein per animal and
2006), and unpublished data (Rocke), we animalschallengedeitherbySCinjectionofYersinia
determined that the mean combined anti- pestis or via consumption of a Y. pestis-infected
mouse. The horizontal lines indicate the combined
F1 and anti-V log titer of vaccinates that
anti-F1andanti-Vlogtitersatwhich50%(7.97)and
survived Y. pestis challenge in our studies
90% (9.05) of the animals would survive Y. pestis
(9.1461.05, n521) was significantly challengeasdeterminedbyprobitanalysis(with95%
higher (P,0.0009) than the combined confidence intervals of 6.91 to 8.57 and 8.50 to
log titer of vaccinated ferrets that died 13.26,respectively).
upon challenge (7.6960.68, n511). We
used probit analysis (SAS) to examine this risk, prairie dogs used for ferret feeding
relationship further and found that an are quarantined for at least 21 days.
animal with a combined log titer of 9.05 Vaccination of captive ferrets with F1-V
(confidence interval of 8.50 to 13.26) had protein should reduce the risk of Y. pestis
a 90% chance of surviving Y. pestis exposure even further. This vaccine could
exposure. The combined log titers of also be used to immunize ferret kits that
vaccinated ferrets 1 yr PV ranged from are released as part of reintroduction
8.62 to 10.43, with a mean of 9.5660.64. programs and whenever wild ferrets are
These values suggest that the majority of trappedformonitoringprograms.In2004,
these animals were probably protected a study was begun to measure survival
against plague 1 yr PV. At 2 yr PV, rates of both field-born and field-released
combined log titers ranged from 8.02 to ferret kits vaccinated with F1-V in com-
11.03, with a mean of 9.22. Probit analysis parison to animals not vaccinated. Al-
suggests that all the vaccinated animals though survival data are not available yet,
had a 50% chance of surviving Y. pestis six field-released vaccinates trapped ap-
exposure (Fig. 4) and most (8/14) had proximately 1 yr later had significant
a greater than 90% chance of survival. antibody titers to F1 and V (combined
At NBFFCC, zoos, and other facilities log titer range of 7.42 to 9.83).
where prairie dog meat is fed to captive Finally, although trapping and vaccina-
ferrets, there is a minimal but continual tion is a labor-intensive prospect, the
risk of Y. pestis exposure. To mitigate this vaccine might also be useful when plague
6 JOURNALOFWILDLIFEDISEASES,VOL.44,NO.1,JANUARY2008
threatens important ferret populations and long-term efficacy of single-dose subunit
that have been reestablished in their vaccines against Yersinia pestis in mice. Amer-
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58:793–799.
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outbreak occurred just 48 km south of the black-footed ferret. In Proceedings of the
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of black-footed ferrets resides (about 250
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5505.
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oral baits laden with a different plague BAETEN. 2004. Recombinant F1-V fusion pro-
tein protects black-footed ferrets (Mustela ni-
vaccine (Mencher et al., 2004).
gripes)againstvirulentYersiniapestisinfection.
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ACKNOWLEDGMENTS
146.
We thank T. Hoffman, S. Hwa, C. Pauly, ———, P. NOL, P. MARINARI, J. KREEGER, S. SMITH,
and P. Vertz for technical assistance, D. G. P. ANDREWS, AND A. M. FRIEDLANDER. 2006.
Heiseyforstatisticaladvice,andJ.Adamovicz, Vaccination as a potential means to prevent
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