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Biologicals 41 (2013) 279e294

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Biologicals

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Meeting report

Report on the international workshop on alternative methods forLeptospira vaccine potency testing: State of the science and the wayforwardq

William Stokes a,1, Geetha Srinivas b, Richard McFarland c, Jodie Kulpa-Eddy d,Warren Casey a,*, Angela Walker b, Hans Draayer e, Randy Sebring f, Karen Brown g,Elisabeth Balks h, Catrina Stirling i, Eric Klaasen j, Richard Hill b, Byron Rippke b,Kevin Ruby b, David Alt k, Suman Mukhopadhyay l, Hajime Kojimam, Nelson Johnson n,Lori Rinckel n, Vivian Doelling n, Brett Jones n

aNational Toxicology Program Interagency Center for The Evaluation of Alternative Toxicological Methods (NICEATM), Division of The National ToxicologyProgram, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, MD:K2-16, Research Triangle Park, NC 27709, USAbCenter for Veterinary Biologics, U.S. Department of Agriculture (USDA), P.O Box 844, Ames, IA 50010, USAcCenter for Biologics Evaluation and Research, U.S. Food and Drug Administration, 1401 Rockville Pike, HFM-700, Rockville, MD 20852, USAdUSDA Animal and Plant Health Inspection Service, 4700 River Road, Unit 2, Riverdale, MD 20737, USAeGourdneck View Consulting, LLC, Portage, MI 49002, USAfColorado Serum Company, 4950 York Street, P.O. Box 16428, Denver, CO 80216, USAg Pair O’Docs Consultants, 5501 N.W. Foxhill Road, Parkville, MO 64152, USAh Paul-Ehrlich-Institut, Paul-Ehrlich-Institut-Strasse 51-59, 63225 Langen, Germanyi Pfizer Ltd., Ramsgate Road, Sandwich, Kent CT13 9NJ, UKjMSD Animal Health, P.O. Box 31, 5830 AA Boxmeer, The NetherlandskUSDA Agricultural Research Service, 1920 Dayton Ave, Ames, IA 50010, USAlMicrobiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, 6610 Rockledge Drive, Bethesda, MD 20892, USAm Japanese Center for The Validation of Alternative Methods, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, 158-5801 Tokyo, Japann Integrated Laboratory Systems Inc., 630 Davis Drive, Suite 160, Morrisville, NC 27560, USA

a r t i c l e i n f o

Article history:Received 11 June 2013Accepted 22 June 2013

Keywords:Leptospira vaccinesAlternative methodsPotencyReplacementRefinementReduction

q This article may be the work product of an emploHealth (NIH), European Commission, or other organizaopinions or conclusions of NIEHS, NIH, the United Scomparative purposes only and does not constitute e* Corresponding author. Tel.: þ1 919 316 4729; fax

E-mail address: warren.casey@nih.gov (W. Casey).1 Present affiliation: Kelly Services, Inc., NIEHS, Res

1045-1056/$36.00http://dx.doi.org/10.1016/j.biologicals.2013.06.013

a b s t r a c t

Routine potency testing of Leptospira vaccines is mostly conducted using a vaccinationechallenge test thatinvolves large numbers of hamsters and unrelieved pain and distress. NICEATM, ICCVAM, and their inter-national partners organized aworkshop to review the state of the science of alternative methods that mightreplace, reduce, and refine the use of animals for veterinary Leptospiravaccine potency testing and to identifyways to advance improved alternative methods. Vaccine manufacturers were encouraged to initiate orcontinue product-specific validation using in vitro enzyme-linked immunosorbent assays as replacementsfor potency testing of four common Leptospira serogroups. Participants discussed the potential for elimi-nating the back-titrationprocedure in the hamster challenge assay,which could reduce animal useby 50% foreach individual potency test. Further animal reduction may also be possible by using cryopreserved Lep-tospira stock to replace continual passaging through hamsters. Serology assays were identified as a way tofurther reduce and refine animal use but should be considered only after attempting in vitro assays. Work-shop participants encouraged consideration of analgesics and use of earlier humane endpoints when thehamster vaccinationechallenge potency assay is used. International harmonization of alternative potencymethods was recommended to avoid duplicative potency testing to meet regionally different requirements.

.

yee or group of employees of the National Institute of Environmental Health Sciences (NIEHS), National Institutes oftions; however, the statements, opinions or conclusions contained therein do not necessarily represent the statements,tates government, the European Commission, or other organizations. The use of commercial product names is forndorsement by any of the authors, organizations or agencies.: þ1 919 541 0947.

earch Triangle Park, NC 27709, USA.

W. Stokes et al. / Biologicals 41 (2013) 279e294280

1. Introduction - The current availability and validation status of earlier hu-mane endpoints and other strategies to reduce or avoid pain

Leptospirosis is a widespread bacterial infection causing diseasein humans, livestock, pets, and wildlife. The infectious agents arespirochetes belonging to hundreds of different serovars of thegenus Leptospira. Vaccines have been used for many years to protectlivestock and companion animal species, and human vaccines areincreasingly being developed in response to widespread outbreaksand endemic disease [1e5]. Prior to release of each production lot(also referred to as “batch” or “serial”) of Leptospira vaccine, regu-latory authorities require the lot to be tested to ensure that it issufficiently potent to produce protective immunity. This requiredLeptospira vaccine testing consequently involves large numbers oflaboratory animals, and accounts for the largest single use of U.S.Animal Welfare Act regulated animals in regulatory testing thatexperience unrelieved pain and distress [6].

Several in vitromethods have been developed and validated thatcould potentially replace most of the animals used for Leptospiravaccine potency testing [6,7]. Implementation of these in vitromethods would avoid the pain and distress experienced by un-protected animals in the current vaccinationechallenge procedure.However, despite the availability of the methods, vaccine manu-facturers have not readily embraced the in vitro methods, as evi-denced by the large numbers of animals still used in traditionalpotency studies.

The Interagency Coordinating Committee on the Validation ofAlternative Methods (ICCVAM) is a permanent Federal interagencycommittee charged by law with promoting alternative methodsthat can replace, reduce, and refine (enhance animal well-beingand lessen or avoid pain and distress) the use of animals fortesting [8]. In order to effectively address this statutory re-sponsibility, ICCVAM agreed to convene an international workshopto address how to advance and implement the use of scientificallyvalid alternatives for Leptospira vaccine testing.

ICCVAM worked with the National Toxicology Program Inter-agency Center for the Evaluation of Alternative ToxicologicalMethods (NICEATM) to organize the “International Workshop onAlternative Methods for Leptospira Vaccine Potency Testing: Stateof the Science and the Way Forward”, that was held on September19e21, 2012, at the National Centers for Animal Health in Ames,Iowa, USA. Cosponsors included the Center for Veterinary Bi-ologics of the U.S. Department of Agriculture (USDA), the AnimalHealth Institute, the International Alliance for Biological Stan-dardization, and the European Union Reference Laboratory forAlternatives to Animal Testing (EURL ECVAM). The workshop wasorganized in collaboration with partner organizations in the In-ternational Cooperation on Alternative Test Methods, includingEURL ECVAM, the Japanese Center for the Validation of AlternativeMethods (JaCVAM), the Korean Center for the Validation ofAlternative Methods, and Health Canada. The organizing com-mittee included scientists from government, industry, academia,and the international community.

The objectives of the workshop were to:

� Identify and review the animal health and public health needsfor Leptospira vaccines, and the regulatory requirements forensuring the potency of each production lot of vaccine.

� Review the state of the science of currently available alterna-tive methods that may replace, reduce, and refine (enhanceanimal well-being and lessen or avoid pain and distress) (the3Rs) the use of animals for Leptospira vaccine potency testing,including:- The current validation and implementation status ofenzyme-linked immunosorbent assays (ELISAs) and sero-logical methods

and distress in the in vivo potency challenge test� Identify any unresolved knowledge/data gaps that must beaddressed to advance the research, development, and valida-tion of alternative 3Rs methods for Leptospira vaccine potencytesting

� Identify best practices for current and future integrated ap-proaches to Leptospira vaccine potency testing

� Develop an implementation strategy to achieve global regula-tory acceptance of alternative methods

More than 80 scientists from 10 different countries attended theworkshop. Participants represented government, industry,academia, and public interest groups. The workshop began with anintroductory session followed by plenary sessions on (1) publichealth needs, regulatory requirements, and research initiativesrelevant to Leptospira vaccines; (2) development, validation, andimplementation of in vitro replacement alternatives for potencytesting of Leptospira vaccines; and (3) development, validation, andimplementation of reduction and refinement alternatives for po-tency testing of Leptospira vaccines. The plenary sessions included17 lectures by invited experts and a roundtable discussion on in-ternational regulatory requirements for Leptospira vaccine potencytesting. Two breakout sessions, each with two breakout discussiongroups, provided the opportunity for participants to discuss issuesand questions prepared by the organizing committee. The firstbreakout session addressed available in vitro replacement methodsfor potency testing of Leptospira vaccines, including validation sta-tus, data gaps, implementation strategies, and expansion to includeadditional serovars. The second breakout session addressed animalreduction and refinement alternatives applicable to the currentin vivo Leptospira vaccine potency vaccinationechallenge test.

This workshop report briefly highlights the plenary speakerpresentations and summarizes workshop discussions, conclusions,and recommendations. Additional details can be found in indi-vidual speaker manuscripts that follow in this workshopproceeding.

1.1. ICCVAM, NICEATM, the International Cooperation onAlternative Test Methods, and workshop background

Dr. William Stokes (National Institute of Environmental HealthSciences [NIEHS], National Institutes of Health [NIH], USA) openedthe workshop by describing how the role and mandate of ICCVAMwas one of the key drivers for the workshop. ICCVAM is a perma-nent Federal interagency committee established by law to promotethe scientific validation, regulatory acceptance, and national andinternational harmonization of alternative testing methods thatcan replace, reduce, and refine animal use in testing, while main-taining the protection of human and animal health and the envi-ronment [8]. ICCVAM is composed of 15 U.S. Federal regulatory andresearch agencies that require, use, generate, or distribute toxicity,safety, and other types of testing information relevant to ensuringhuman and animal health and environmental protection. Memberagencies include the USDA, which regulates veterinary biologics inthe United States, and the U.S. Food and Drug Administration,which regulates human biologics and human and veterinary drugsin the United States. ICCVAM’s purposes and duties include:

� Reducing, refining, and replacing animal use in testing� Sharing experiences between agencies and avoiding duplica-tive efforts

� Advising on test method development and validation� Ensuring test methods are validated to meet agency needs

W. Stokes et al. / Biologicals 41 (2013) 279e294 281

� Increasing the efficiency and effectiveness of test methodreview

� Conducting technical reviews of new testing methods� Transmitting recommendations to Federal agencies for adop-tion decisions

� Fostering national and international harmonization

The ICCVAM Authorization Act of 2000 established ICCVAMunder NICEATM, a Center within the NIEHS at the NIH. NICEATMprovides scientific and operational support for ICCVAM-relatedactivities, and organizes, conducts, and participates in interna-tional validation studies on promising new safety testing methods.The NIH is charged by lawwith conducting and supporting researchfor methods of medical research and experimentation that do notuse animals, that reduce the number of animals used in suchresearch, and that produce less pain and distress in animals used inresearch and experimentation [9]. NIEHS is further charged by lawwith developing and validating alternative methods that canreduce or eliminate animal use in safety testing.

Since 1998, NICEATM, ICCVAM, and ICCVAM member agencieshave contributed to the national and international regulatoryacceptance of 63 alternative safety testing methods, including 36non-animalmethods and 27methods that significantly reduce and/or refine animal use [10]. More recently, the United States hasjoined together with Canada, Japan, Korea, and the European Unionto form the International Cooperation on Alternative Test Methods(ICATM) [11]. ICATM seeks to expedite the international adoption ofnew alternative methods through enhanced cooperation on vali-dation study designs, test method peer reviews, and developmentof harmonized test method recommendations.

Dr. Stokes noted that this workshop on Leptospira vaccines is thesecond in a series organized to address the highest priorities foralternative methods for vaccine potency and safety testing identi-fied at a 2010 NICEATMeICCVAM international workshop [7,12,13].Vaccine testing is one of the four highest priorities for NICEATMand ICCVAM because large numbers of animals are used for suchtesting and because many of the animals likely experience unre-lieved pain and distress [14].

In the most recent report on animal use for experimental andother scientific purposes in the European Union, vaccine testingaccounted for nearly two-thirds of all the animals reported as usedfor regulatory testing purposes [15]. Vaccine testing also accountedfor at least 59% of the 97,123 animals reported to the USDA in 2010that experienced unrelieved pain and distress [16]. Of the 59,437animals used for vaccine testing and reported in the category ofpain and distress without pain-relieving drugs, 38,118 were ham-sters. The reports provided by research and testing facilities for the2010 USDA report suggest that most if not all of these hamsterswere used for Leptospira vaccine potency testing. A review of datafor the past 10 years indicate that this number has not changedsignificantly, suggesting that further implementation of availablealternative methods for Leptospira potency testing could have asignificant beneficial impact on animal welfare.

1.2. USDA Center for Veterinary Biologics

Dr. Richard Hill (Center for Veterinary Biologics, USDA) providedan overview of the USDA National Centers for Animal Health andthe USDA Center for Veterinary Biologics (CVB), which hosted theworkshop. The National Centers for Animal Health consists of threekey organizations within the USDA. These include the CVB and theNational Veterinary Services Laboratories, which are componentsof Veterinary Services within the Animal and Plant Health Inspec-tion Service, and the National Animal Disease Center, a componentof the Agriculture Research Service. All of the organizations work

collaboratively to support animal health through research, di-agnostics, and review and approval of new biologics. The mission ofthe Veterinary Biologics Program is to implement the provisions ofthe Virus-Serum-Toxin Act to ensure that the veterinary biologicsavailable for the diagnosis, prevention, and treatment of animaldiseases are pure, safe, potent, and effective. Dr. Hill emphasizedthat CVB strategic goals include reducing animal use for testing andmaximizing the use of new technologies. He concluded by sum-marizing the many guidance documents that have been issued bythe CVB that contribute to the 3Rs, including the guidance for usingin vitro methods to quantify the potency of Leptospira vaccines fordogs, cattle, and swine.

1.3. Leptospira e public health and animal health perspectives

Dr. Marta Guerra (Centers for Disease Control and Prevention,USA) described leptospirosis as the most widespread zoonosis inthe world with incidence rates estimated at 10e100 per 100,000people in tropical regions [17]. The global burden of humanleptospirosis is significant, with more than 400,000 recorded casesof leptospirosis occurring annually and a fatality rate ranging from5% to as high as 20% or more in some tropical regions [1]. Lepto-spirosis in humans is largely a zoonotic disease, with most in-fections associated with direct contact with urine from infectedanimals or indirect contact with water or moist soil contaminatedwith infected urine [4,18]. Leptospirosis is considered both aneglected tropical disease and a neglected zoonotic disease [19e21]. The development of effective human vaccines is a globalresearch and public health priority [19e21].

Currently, more than 200 Leptospira serovars and 23 pathogenicserogroups have been identified [4,5]. Factors associated withendemic leptospirosis include tropical climates, stagnant waters,poor levels of sanitation, occupational or recreational exposure, andproximity of potential mammalian reservoirs to human pop-ulations [4,17,18]. In humans, the disease can vary in severitydepending on the infecting serovar of Leptospira and the age,health, and immunological competence of the patient. Severityranges from a mild, influenza-like illness to a severe infection withrenal and hepatic failure, pulmonary distress, and death (the classicWeil’s disease) [4,17]. In June 2012, the U.S. Council of State andTerritorial Epidemiologists approved reinstatement of leptospirosisas a Nationally Notifiable Condition. Reinstatement of nationalsurveillance will facilitate assessment of the incidence, geographicdistribution, trends, and risk factors associated with human casesand the identification of outbreaks and potential new animal res-ervoirs [17].

Dr. Ronald Schultz (University ofWisconsin, USA) explained thatLeptospira infects multiple animal species, including livestock(cattle and swine), companion animals (cats, dogs, horses), andwildlife. Leptospira infection causes significant disease and eco-nomic loss, especially in cattle and swine, and to a lesser extent ingoats and sheep. The Leptospira serogroups most often associatedwith disease in companion and food animals are canicola, grippo-typhosa, icterohaemorrhagiae, and pomona. Diagnosis is typicallyperformed using the microscopic agglutination test, which detectsagglutinating antibodies in the serum of infected animals. In ani-mals, leptospirosis is a systemic disease that can cause fever, renaland hepatic insufficiency, pulmonary manifestations, and repro-ductive failure [4]. Typical leptospirosis in dogs may result in fever,jaundice, vomiting, diarrhea, disseminated intravascular coagula-tion, uremia caused by renal failure, hemorrhages, and death[22,23]. In cattle and pigs, leptospirosis may cause infertility,abortion, stillbirths, fetal mummification, weak piglets or calves,and decreased milk production [24]. The disease can have a majoreconomic impact when it causes significant decreases in the

W. Stokes et al. / Biologicals 41 (2013) 279e294282

reproduction of food-producing animals. A chronic manifestationof leptospirosis that commonly occurs in horses is recurrent uveitis,which is not unique to this species and may also occur occasionallyin humans [25]. Animals recovering from leptospirosis may becomeasymptomatic carriers harboring virulent leptospires in the renaltubules for extended periods and shedding infectious leptospiresinto the environment [26]. Once excreted in the urine, leptospirescan survive in moist environments for months to years [4].

Inactivated Leptospira vaccines have been developed for mostsusceptible livestock and domestic pet species and are widely usedin the United States and other developed countries [2,3,5]. Forexample, 4-way multivalent veterinary vaccines are currentlyavailable for dogs, cattle, and swine, conferring protection againstLeptospira interrogans serogroups canicola, grippotyphosa, icter-ohaemorrhagiae, and pomona. In addition to these four serogroups,cattle and swine vaccines may also contain serovar hardjo in a 5-way multivalent vaccine, and selected swine vaccines containserovar bratislava in a 6-way multivalent vaccine. Human inacti-vated Leptospira vaccines that protect against regionally specificserovars are also available for workers in high-risk professions inselected countries, although none is currently approved for use inthe United States.

1.4. Human Leptospira vaccines and research efforts

Dr. Albert Ko (Yale University, USA) outlined the changing epide-miological patterns for leptospirosis outbreaks, current vaccinesavailable for human use, and newapproaches and current challengesfor next-generation Leptospira human vaccine development.Currently, three commercial or nationally produced leptospirosisvaccines are available for human use: (1) Spirolet (IMAXIO, France), amonovalent, whole-cell vaccine conferring protection against L.interrogans serovar icterohaemorrhagiae; (2) a Shanghai Institute ofBiological Products bivalent outer membrane vesicle-based vaccineprotective against Leptospira serovars icterohaemorrhagiae and heb-domadis; and (3) Vax-Spiral� (Finlay Institute, Cuba), a trivalentwhole-cell vaccine protective against Leptospira serovars canicola,mozdok, and copenhageni. Development of human leptospirosis vac-cines currently focuses on utilizing leptospiral outer-membraneproteins (i.e., Leptospira immunoglobulin-like proteins) as targets insubunit recombinant vaccine candidates [27e32]. However, all can-didates are still in the proof-of-concept stage.

Dr. Joseph Breen (NIAID, USA) described the current Leptospiraresearch portfolio supported by the National Institutes of Healththrough the National Institute of Allergy and Infectious Diseases(NIAID). The NIAID Division of Microbiology and Infectious Diseasessupports extramural basic and applied research to control andprevent diseases caused by human infectious agents, with ongoingresearch on over 293 different pathogens. The Leptospira researchprogram focuses on understanding (1) the basic mechanisms ofpathogenesis; (2) the role of pathogen-expressed immunogenicdeterminates in establishing an infection; (3) the role of host im-munity; (4) leptospirosis pulmonary hemorrhagic syndrome; (5)identifying determinates of clinical outcome; (6) improved di-agnostics; and (7) the development of improved interventional andprevention procedures including vaccines. Although no licensedhuman vaccines are currently available for leptospirosis in theUnited States, the NIH is actively supporting advances and in-novations in science and technology to further understand,develop, and overcome the public health gaps that must beaddressed to combat this pervasive disease. This research includes:

� Whole genome sequencing of over 250 globally diverse Lep-tospira serovars, including those that cause human morbidityand mortality.

� A clinical proteomics initiative that includes a reference arrayof over 3667 Leptospira-encoded proteins that can be used toscreen serum antibodies for evidence of exposure to Leptospirainfection. This research may aid in the development of diag-nostic markers and vaccine candidates.

� Cooperative agreements that have generated prototypes ofpoint-of-care diagnostics to differentiate the early symptoms ofleptospirosis from other fever-causing agents (e.g., malaria,dengue).

� Investigation of the critical elements of hostepathogen inter-action and identification and development of potential vaccinecandidates using animal models of infection.

� Preclinical and clinical services to support product develop-ment of diagnostics and vaccines.

� Fellowships to provide U.S. medical doctors valuable experi-ence identifying and treating the disease.

Further information on NIAID microbiology and infectious dis-ease resources and funding opportunities can be found at http://www3.niaid.nih.gov/LabsAndResources/resources/dmid/.

2. Leptospira veterinary vaccine potency testing e U.S. andinternational regulatory requirements

The routine lot release of most veterinary Leptospira vaccinestraditionally requires a hamster potency challenge test using viru-lent Leptospira bacteria to ensure that the final product has suffi-cient potency to produce an effective immune response invaccinated animals. This in vivo test accounts for the majority ofhamsters reported to the USDA as experiencing unrelieved pain anddistress in research in the United States [6]. Specific potency testingrequirements vary slightly among regulatory authorities (Table 1).Regulatory authorities have also issued guidance on serological andin vitro alternativemethods that can be used in place of the hamsterpotency challenge test (Table 1).

2.1. U.S. regulatory requirements

Dr. Geetha Srinivas (USDA, USA) reviewed the current guidelinesand practices for potency testing of veterinary Leptospira vaccinesin the United States [44]. Historically, potency testing of leptospiralvaccines licensed by the USDA was conducted using only thehamster vaccinationechallenge (i.e., serogroups pomona, icter-ohaemorrhagiae, canicola, and grippotyphosa) [45e48]. Eachserogroup test generally requires a total of 40 hamsters. Ten ham-sters are vaccinated with a specified dilution of the final vaccineproduct. After 14e18 days, the 10 vaccinated hamsters and 10nonvaccinated controls are challenged with a virulent suspensionof Leptospira. A back-titration is conducted typically using 20hamsters to estimate the LD50 (range of 10e10,000 LD50) of thechallenge inoculum, using four groups of five hamsters eachinjected with serial dilutions of the inoculum. A valid test requiresthat 8 or more of the 10 nonvaccinated hamsters must die after avirulent challenge of 10e10,000 LD50. Moribund animals exhibitingclinical signs consistent with the expected disease pathogenesismay be humanely euthanized and considered as deaths. The chal-lenge inoculum must also be determined to contain 10e10,000LD50 of the virulent Leptospira serovar. A serial is consideredsatisfactory if 80% or more of vaccinated hamsters are protectedfrom the challenge dose.

Due to the high cost of the Codified hamster vaccinationechallenge tests (i.e., monetary expense, animal welfare, time), theCVB developed an in vitro ELISA for the four Leptospira fractionsmost commonly used in vaccines. Briefly, the ELISAs utilize apolyclonal capture and monoclonal detecting antibody to

Table 1United States and European regulatory requirements for veterinary Leptospira vaccine potency testing.

Leptospira Reference alternativemethod

Alternative test method Referencetraditional test

Traditional test Species

Leptospira interrogansserogroup pomona

USDA SAM 624 [33] Sandwich ELISA (rabbit polyclonal capturewith mouse mAb-detecting antibody

USDA SAM 608;9 CFR 113.101 [34]

Hamstervaccinationechallenge

Swine, cattle,dogs

Leptospira interrogansserogroup canicola

USDA SAM 625 [35] Sandwich ELISA (rabbit polyclonal capturewith mouse mAb-detecting antibody

USDA SAM 609;9 CFR 113.103 [36]

Hamstervaccinationechallenge

Swine, cattle,dogs

Leptospira kirschneriserogroup grippotyphosa

USDA SAM 626 [37] Sandwich ELISA (rabbit polyclonal capturewith mouse mAb-detecting antibody

USDA SAM 617;9 CFR 113.104 [38]

Hamstervaccinationechallenge

Swine, cattle,dogs

Leptospira interrogansserogroupicterohaemorrhagiae

USDA SAM 627 [39] Sandwich ELISA (rabbit polyclonal capturewith mouse mAb-detecting antibody

USDA SAM 610;9 CFR 113.102 [40]

Hamstervaccinationechallenge

Swine, cattle,dogs

Leptospira interrogansserovar hardjo

N/A N/A 9 CFR 113.105 [41] Test defined in theoutline of production(not specified)

Cattle

Canine leptospirosis vaccine(inactivated) e can include oneor more strains of one of moreLeptospira interrogans serovarcanicola, icterohaemorrhagiae,or any other epidemiologicallyappropriate serovar

Ph. Eur., monograph447 [42]

1) Vaccines with/without adjuvants:a) Hamster vaccinationechallengeb) Hamster immunization and serology(in vitro method to detectantibodies e methodnot defined in Ph. Eur.)2) Vaccine without adjuvants:Validated in vitro method for antigeniccomponents

Hamstervaccinationechallenge

Dogs

Bovine leptospirosis vaccine(inactivated) e can includeone or more suitable strains ofLeptospira borgpeterseniiserovar hardjo, L. interrogansserovar hardjo, or otherL. interrogans serovars

Ph. Eur., monograph1939 [43]

Guinea pig vaccination and serology;use a suitable validated method suchas micro-agglutination test

Cattle

W. Stokes et al. / Biologicals 41 (2013) 279e294 283

determine the relative potency of the antigen compared to that of aqualified National Reference Standard [33,35,37,39]. As stated inUSDA CVB Notices No. 07-02, 07-12, and 09-16, licensees and per-mittees may request an exemption to the existing Codified hamsterpotency tests to implement the ELISA alternatives [49]. The CVBrequires that firms conducting the ELISA use either a NationalReference Standard or a product-matched reference that has beenqualified in a host animal vaccinationechallenge study or hamster-model potency test. Furthermore, the CVB can supply interestedvaccine manufacturers with the Leptospira-specific antibodies andNational Reference Standards necessary to conduct the in vitroELISAs, at least until further notification. The CVB’s goal is tofacilitate the global acceptance of the validated ELISAs by sharinginformation on these alternatives with regulatory agencies in othercountries.

The CVB will continue to review potency testing of other Lep-tospira serovars that do not have Codified requirements (e.g., serovarhardjo) on a case-by-case basis. Any in vitro ELISA developed forthese remaining serovars must undergo appropriate validation andcomply with specified U.S. regulatory requirements [50]. Finally, theCVB promotes the use of humane endpoints for the existing hamsterchallenge assay and actively encourages their implementation byvaccine manufacturers following appropriate validation [51].

2 http://www.edqm.eu/en/EDQM-Downloads-527.html.

2.2. EU regulatory requirements

Dr. Lukas Bruckner (Institute of Virology and Immunoprophy-laxis, Switzerland) reviewed the current regulatory framework andpractices for veterinary Leptospira vaccines in Europe [52] (Table 1).The hamster vaccinationechallenge test is used for lot release po-tency testing of canine vaccines, commonly referred to as batchrelease testing in Europe [42]. Specifically, European Pharmaco-poeia (Ph. Eur.) Monograph 0447 states that, for potency testing ofinactivated canine Leptospira vaccines, the potency of each serovarcontained in the vaccine must be established by vaccinating 5

hamsters with 1/40th of the dose for the target species, thenchallenging the vaccinates 15e20 days later with virulent Lep-tospira of the same serovar in the vaccine. Five unvaccinated con-trols are also challenged at this time. At least 4 vaccinates mustsurvive the test, and 4 nonvaccinated challenge controls should dieand exhibit typical signs of Leptospira infection. There is norequirement for estimating the LD50 of the challenge inoculum.Alternatively, a serological test or suitably validated in vitro test (fornonadjuvanted vaccines only) may be used to determine the con-tent of one or more antigenic components as an indicator of pro-tection [42]. Leptospira vaccines for use in cattle can be tested forpotency using a serological assay in guinea pigs [43].

At the January 2012 European Directorate for the Quality ofMedicines and HealthCare workshop “Alternatives to the Lepto-spirosis Batch Potency Test”, participants unanimously agreed thatcomplete in vitro testing for Leptospira vaccine potency testing ispossible and should be promoted.2 Furthermore, participants alsostated that consistency of production is necessary to corroborateand support reliable in vitro test results. They agreed that furthereffort must be put on defining appropriate parameters for startingmaterials and the entire production process in order to support thereplacement of the current in vivo batch potency test.

2.3. International regulatory requirements

An international regulatory roundtable chaired by Mr. HansDraayer (Gourdneck View Consulting, LLC, USA) discussed thespecific requirements for importing Leptospira vaccines in theUnited States, Europe, and Mexico and the opportunities forreducing duplicative or similar retesting [53]. Leptospira vaccines,as with many animal health vaccines, are manufactured anddistributed both on a regional basis and internationally via large

W. Stokes et al. / Biologicals 41 (2013) 279e294284

multinational firms. Currently, three general scenarios exist for theinternational distribution of veterinary vaccines, including Lep-tospira vaccines:

� The importing country recognizes the country of origin’stesting and batch release data with no additional testingrequired.

� The importing country requires that a specific potency assay beconducted by the exporting firm based on the importingmarket’s current regulations (in addition to the current serialrelease assay in the country of origin).

� The importing country requires in-country retesting of theproduct following importation by a local laboratory and/or alocal government testing laboratory prior to distribution [53].

The European Union (EU) requires vaccine production to be incompliance with Good Manufacturing Practices and the use ofGood Laboratory Practices for demonstrating safety of new vaccineproducts. The EU requirements for Leptospira vaccine testing alsoutilize fewer animals per test group (i.e., 5 versus 10 hamsters).However, participants noted that retests are a significant problemwhen using fewer animals and that this can result in a greateroverall number of animals being used.

Dr. de la Peña-Moctezuma (Universidad Nacional Autónoma deMéxico, México) reported that Mexican regulatory authorities donot require additional potency testing for imported Leptospiravaccines. Similarly, Canada does not retest imported Leptospiravaccines. Speakers from the United States and internationalagencies agreed that the acceptance of internationally recognizedtests and/or standards should be a primary objective for futureefforts after this workshop. Other proposals from the internationalroundtable discussion included:

� For Leptospira vaccines, the goal should be global imple-mentation of a mutually accepted standard in vitro assay withglobally available international reference standards and re-agents, with provisions for appropriate product-specific vari-ations as justified.

� When relative potency testing against national or internationalreferences is required, the impact of the method ofmanufacturing on the antigenic mass of references and vaccinesamples should be considered (e.g., the inactivation agent usedfor the antigen source) [54].

� A limited number of valid assays (e.g., 4e5) should be avail-able for use by vaccine manufacturers following carefulexpert review and recommendation by appropriate scientificreview organizations and approval by major regulatoryagencies.

� A widely available database of in vitro reagents would be avaluable resource toward implementing internationallyrecognized in vitro potency assay(s).

� An international collaborative study should be considered togenerate confidence among interested stakeholders (e.g.,manufacturers, trade organizations) in the alternative in vitropotency assays. Such a collaborative effort would leverage re-sources and improve the likelihood of international acceptanceof the methods and data.

� Manufacturers should consider implementing a consistency-of-production approach to facilitate international acceptance,especially in those countries where Good ManufacturingPractices are required for veterinary vaccine production.

� Testing and importation requirements should be clearlydifferentiated and defined for well-established products with aproven quality history versus newly developed productswithout such a history.

3. Leptospira veterinary vaccine potency testing: in vitroELISAs as replacement alternatives

3.1. State of the science

Dr. Kevin Ruby (USDA, USA) reviewed the USDA development ofELISAs for measuring relative potencies of leptospiral vaccinescontaining serogroups canicola, grippotyphosa, icterohaemor-rhagiae, and pomona [55e59]. Briefly, the sandwich ELISAs utilizeserogroup-specific polyclonal antibodies to capture antigen in thevaccine product, followed by incubation with serogroup-specificmurine monoclonal antibodies (mAbs) and goat anti-mouseimmunoglobulin horseradish peroxidase-labeled antibody conju-gate. The specificity of the mAbs was confirmed via the microscopicagglutination test, passive protection studies in hamsters, andWestern blot analyses. The mAbs, which are directed against aserogroup-specific lipopolysaccharide-like moiety, were shown tobe agglutinating and protective against a homologous challenge butfailed to protect against a heterologous challenge.

Hundreds of randomly selected serials of licensed leptospiralbacterins, which included combination products and representedat least eleven licensed establishments, were evaluated using theELISAs [55]. The reference utilized was a monovalent bacterinprepared by a biologics manufacturer and titrated to the minimumprotective dose in the hamster. A test bacterin was consideredsatisfactory by ELISA if a relative potency of 1.0 or greater was ob-tained when compared to the reference bacterin. All licensedproducts examined successfully passed the ELISAs. In cases wherethe product did not pass the initial ELISA, all of these subsequentlypassed the ELISA after adjuvant elution [55]. All products retestedin the hamster challenge test also passed.

Veterinary biologics industry stakeholders also evaluated theELISAs using a coded panel of bacterins representing low, me-dium, and high concentrations of each serogroup, as well asnegative controls. Samples were tested on three different days bythree different laboratory technicians. All participating manufac-turers reported positive results and consistent standard de-viations of relative potency. Greater interlaboratory, rather thanintralaboratory, variation was observed. Therefore, the dataindicate that all four ELISAs are serogroup specific and sensitivefor their respective serogroup. Accordingly, the CVB encouragesproduct-specific validation, implementation, and use of in vitroELISAs as viable alternatives to the existing in vivo hamster po-tency assay.

Dr. Carole Bolin (Michigan State University, USA) summarizedthe conduct and evaluation of target host efficacy trials (i.e., dogs,pigs) used to validate the potency of monovalent leptospiralreference bacterins against serogroups canicola, grippotyphosa,icterohaemorrhagiae, and pomona. The studies evaluated the effi-cacy of nonadjuvanted reference bacterins in dogs and pigs. Thestudies were designed to correlate protection in the host animalswith protection in the hamster challenge test. They did not attemptto determine a minimal protective dose in the host animals. Mix-tures of two challenge strain isolates for each of the four Leptospiraserogroups were evaluated. Briefly, dogs (10 per group) werevaccinated at 8 and 11 weeks of age subcutaneously (SC) or intra-muscularly (IM) with bacterin. A challenge dose was administeredvia conjunctival instillation three weeks after the second vaccina-tion, and animals were observed twice daily for clinical outcomes.Meloxicamwas available for analgesia. Necropsies were performedtwo weeks postchallenge or sooner if required. All four referencebacterins, administered either SC or IM, induced protection fromsignificant Leptospira challenge in vaccinated dogs and pigs. Inaddition, animals with significantly low postvaccinal titers werealso protected. Outcomes measured to define protection include:

W. Stokes et al. / Biologicals 41 (2013) 279e294 285

� Daily body temperature measurements� Clinical chemistry panel on days 1, 7, and 14� Blood cultures on days 4, 7, 10, and 12� Postmortem examinations with cultures of liver, kidney, andurine on day 14 or earlier

� Antibody titers measured prior to study and postmortem

The majority of nonvaccinated animals exhibited clear evidenceof infection. The host efficacy trials clearly demonstrated thatprotection of dogs and pigs correlated with that observed inhamsters and with results from the CVB ELISA potency tests.

Mr. Brett Webster (Animal Health Institute, USA) provided anindustry perspective of vaccine manufacturers with the product-specific validation of the USDA ELISAs for product release potencytesting. Manufacturers that have or are attempting to convert fromin vivo testing to the in vitro ELISAs have reported:

� Failure of primary antibodies from the CVB Supplemental AssayMethods (SAMs) to detect the in-house leptospiral serovarsused by some firms

� Difficulties in the elution/extraction of adjuvant from product� High background response� Unsatisfactory reproducibility and threshold of detection� Concerns regarding the continued availability of assay reagents� The need for increased antigen input when utilizing the CVBnonadjuvanted reference standards

� Inability to meet the validation requirements of VeterinaryServices (VS) Memo 800.112

Concern was expressed regarding the testing of adjuvantedproduct and comparing this to CVB-provided nonadjuvantedreference standards. Testing bulk vaccine prior to adjuvanting andblending was proposed as an alternative approach. Another optionto consider would be for manufacturers to validate their own vac-cine reference standards using a challenge test. Manufacturersmust also determine if antigen extraction from the final product isnecessary to avoid effects of the adjuvant and whether this willaffect parallelism of the assay. Currently, U.S. veterinary vaccinemanufacturers are at varying stages of product-specific validationusing the ELISAs, and some firms have already successfully imple-mented ELISAs for product release. Mr. Webster concluded byemphasizing that product specific validation and implementationof alternative ELISAs require vaccine manufacturers to investconsiderable resources.

Dr. Jeffrey Galvin (Pfizer Animal Health, USA) summarized thesuccessful development, optimization, product-specific validation,and EU regulatory approval of an in-house in vitro ELISA potencytest for a bovine Leptospira vaccine (Spirovac� [Pfizer AnimalHealth, UK]). He highlighted the major issues encountered and keymilestones achieved during the development and approval of theassay. Briefly, Spirovac� is a second-generation leptospirosis vac-cine containing inactivated Leptospira borgpetersenii serovar hardjotype hardjobovis adjuvanted with aluminum hydroxide. The vac-cine is suitable for use in healthy cattle 4 weeks of age or older,including pregnant and/or lactating cows and heifers, for pre-venting infection caused by type hardjobovis and urinary sheddingfor a period of 12 months. Due to the nature of the bovine strain ofinterest, and in an effort to refine the existing hamster potencymodel in accordance with 3Rs initiatives, a bacterial kidney colo-nization endpoint (instead of lethality) was previously imple-mented. More recently, during product development of newcombination vaccines, specific reagents (i.e., monoclonal antibody)were generated for the bovine strain. Subsequently, a quantitativein-process ELISAwas successfully developed to evaluate the qualityand quantity of Leptospira antigen in fluids prior to formulation into

final vaccine. Compared to previously utilized antigen-quantification methods, this ELISA demonstrated significantlystronger correlation to the hamster kidney colonization potencytest. Furthermore, during a 3-year period in which this ELISA wasconducted (1) potency failure rates decreased 6�, (2) fewer retestswere required, and (3) a 50% reduction in numbers of hamstersrequired to release product was observed. Following additionaldevelopment, optimization, and validation, the ELISA format wasapplied to final product testing as a complete replacement alter-native. The ELISA is currently a critical final product test for thequality control release of the vaccine in the European Union but notyet in the United States. Finally, this ELISA confers significantbenefits over the existing in vivo hamster potency test in terms ofrelease time (1e2 days versus w4 months) and cost ($500 versus$1000).

Dr. Eric Klaasen (MSD Animal Health, The Netherlands)described a successful manufacturers’ initiative to develop anin vitro ELISA potency test that could replace the hamster vacci-nationechallenge test for an inactivated canine leptospirosis vac-cine: Nobivac� Lepto (MSD Animal Health) [60]. Briefly, Nobivac�

Lepto is a nonadjuvanted vaccine containing inactivatedwhole cellsof L. interrogans serovar canicola type portlandvere and L. inter-rogans serovar icterohaemorrhagiae type copenhageni. Dr. Klaasendetailed the development and validation of an antigenic mass ELISAbatch potency test against lipopolysaccharide epitopes [60]. ThisELISA was approved in the European Union as a suitable replace-ment for the current in vivo hamster potency test based on (1) theresults of assay validation, (2) an examination of the relationship ofantigenic mass with efficacy in dogs, and (3) the protective effectsof the monoclonal antibodies. Encouragingly, preliminary ELISAtesting showed no adjuvant interference using either analuminum-adjuvanted Leptospira/rabies combination vaccine or asaponin-adjuvanted Leptospira vaccine [60].

Dr. David Alt (USDA, USA) summarized recent research on thepotential expansion of in vitro potency assays for L. borgpeterseniiserovar hardjo [61]. Data from target host animal efficacy studiesusing whole-cell bacterins, and issues involved in extrapolatingthese studies to either hamster-based or other potency testingmethods were discussed. Leptospira serovar hardjo infection incattle results in significant production and reproductive lossesworldwide. Overt clinical signs of disease are rarely observed incattle outside of pregnancy, during which late-term abortions,stillbirths, and birth of weak calves have been reported. Due tothese observed differences in infectionwith other serovars, a directcomparison to a lethal hamster potency assay may not provide anappropriate, accurate assessment of vaccine efficacy [61]. Thedevelopment of a chronic hamster model of infection, more closelymimicking infection in cattle, in conjunction with a lethal hamstermodel, may prove valuable in the evaluation of suitable vaccinecandidates and future development of appropriate in vitromethodsfor potency testing [61].

3.2. Workshop discussion

Workshop participants discussed how to facilitate product-specific validation of the four validated in vitro antigen quantifi-cation methods as replacements for the current hamster vaccina-tionechallenge Leptospira potency test. Associated data gaps,implementation challenges, and expansion of suitable in vitromethods to other serovars were also discussed. The discussionswere limited to potency testing required for serial/batch releasetesting of approved vaccines and did not attempt to address testingto demonstrate product efficacy. The goal of serial/batch releasetesting is to ensure that each serial/batch is consistent with thosepreviously shown to be safe and effective in target species.

W. Stokes et al. / Biologicals 41 (2013) 279e294286

As stated previously, Leptospira serogroups canicola, grippoty-phosa, icterohaemorrhagiae, and pomona currently have validatedELISAs available for serial release potency testing. In addition, theUSDA CVB commented that it had successfully evaluated potencyutilizing these ELISAs with a representative sample (i.e., 35%) ofmarketed Leptospira vaccine products. Workshop participantsencouraged the USDA to share these data with respective manu-facturers to assist them with identifying their vaccine products forwhich the ELISAs have the greatest potential for successful product-specific validation.

A key technical challenge for vaccine manufacturers in devel-oping these ELISAs for product-specific validation is the qualifica-tion of a reference bacterin for individual products. CVB Notices 07-02, 07-12, and 09-16 detail the options for leptospiral referencebacterin qualification and include information on

� The availability and use of a National Reference StandardBacterin (i.e., nonadjuvanted), currently available from the CVB

� The production of a product-matched reference bacterin andits subsequent qualification via the Codified hamster potencytest

� The production of a product-matched reference bacterin andits subsequent qualification via a host-animal vaccinationechallenge study

Regulatory authorities noted that a product-matched referencequalified by the current hamster potency test or host-animalvaccinationechallenge study may be required for several prod-ucts currently released in the United States. Because the qualifiedCVB references are nonadjuvanted, they may exhibit a differentdoseeresponse curve compared to some adjuvanted serials testedfor release, and thus may be inappropriate for those products.Several international workshop participants questioned the use-fulness of correlating any proposed ELISA to the current hamsterpotency test due to their experience with inconsistent linearity ofthe hamster test, and proposed that any alternative in vitro po-tency assay for Leptospira vaccines should only be correlated withefficacy in the host animal. However, the USDA and most U.S.based vaccine manufacturers commented that they had notexperienced these linearity issues, and suggested that differencesin the design of the hamster assay across countries may beaffecting the outcome.

Workshop participants considered achievement of parallelismrequirements specified in VSMemorandum 800.112 to be themajorissue facing vaccine manufacturers in the product-specific valida-tion of the USDA ELISAs. The memorandum states that anyapproved ELISA must “be relevant, reliable, reproducible, andscientifically sound”, with sensitivity and specificity requirementsalso inherent in these guidelines. Test methods used for potencytesting of multivalent vaccine formulations must show that thedoseeresponse curve of each formulation is similar to the referenceor standard doseeresponse curve [50]. Vaccine manufacturersexpressed concern about the ability to achieve the required paral-lelism across the entire doseeresponse curve, particularly at theupper and lower asymptotes. Due to the noted variability in ELISAperformance with different formulations, workshop participantsproposed assessing parallelism only in the critical linear range, ascurrently accepted in the European Union. U.S. regulatory author-ities expressed a willingness to share ELISA testing data with themanufacturers whose products were examined, contingent onavailability of staff resources. Workshop participants recom-mended that manufacturers that receive data from regulatory au-thorities also share their results with regulators, thus allowing atwo-way dialog to address potential differences and provideconstructive suggestions.

Workshop participants noted that the parallelism specificationsmight not be achieved when using a nonadjuvanted reference totest adjuvanted product. The measurement of bulk antigen usingthe ELISAs (preformulation antigen content, preadjuvant addition)was proposed as an alternative to avoid nonparallelism hurdles,thereby increasing the utility of in vitro potency assays. U.S. regu-latory authorities stated that current policy requires potencytesting on the final product; however, they encouraged manufac-turers to propose viable alternatives for consideration and to sub-mit potency test data on both bulk and final product. Theimplementation of current good manufacturing practice pro-cedures for the mixing and addition of specific adjuvant to finalproduct was proposed to address the concerns of regulatory au-thorities. Prior to approval, sufficient data are required by regula-tory authorities to demonstrate that the antigen does not change inbulk and final product. Finally, workshop participants briefly dis-cussed the difficulties with eluting antigen from adjuvant. Regu-latory authorities and vaccine manufacturers were encouraged toshare information and testing experiences on antigen elution toprevent the repetition of failed initiatives. Participants encouragedorganization of a follow up workshop to explore elution techniquesnecessary to advance ELISA implementation.

Workshop participants also reviewed the requirement for anELISA to identify substandard (i.e., subpotent) serials. In addition, anassay must be able to differentiate between the fraction of interestand cross-reactive substances. U.S. regulatory authorities indicatedthat an ELISA must discriminate between products that meet therelease value (100%) and those with an antigen content of 80% and90% of the release value. U.S. regulatory authorities reported anability to differentiate antigen content of 80%, 90%, and 100% inproducts using the validated ELISAs. However, a concern wasexpressed that this levelofprecision isextremelyhighandnot readilyachievable for vaccines based on inactivated whole bacterial cells.

While the ability to determine subpotent lots is also required inthe European Union, it was mentioned that EU regulatory author-ities will accept tolerance intervals that are calculated based on theresults of “n” standard batches (i.e., batches formulated to thetarget antigenic mass according to the manufacturer’s license/dossier). Such a procedure has been accepted recently for centralregistration of a canine leptospirosis vaccine by the Committee forMedicinal Products for Veterinary Use at the European MedicinesAgency. Depending on the variation in potency results (e.g., anti-genic mass results) of these “n” batches, the lower and upperrelease limit may even be <80% and >120%, respectively, of thetarget antigen concentration in the vaccine. This may be accepted if(1) the margin between the lower batch release limit and theminimum protective dose in the target animal is considered largeenough and (2) consistency of production of antigens and vaccine isconsidered satisfactory.

Workshop participants discussed the usefulness and validity ofthe consistency approach in the product-specific validation of theUSDA ELISAs. The consistency approach for the routine release ofvaccines is based upon the principle that the quality of each vaccineserial is a result of a quality system that ensures consistent pro-duction of serials with characteristics similar to those previouslyshown to be safe and effective in humans or target species [62]. U.S.regulatory authorities, while amenable to the consistency approachand awell-characterized final product, stated that high-quality datamust be submitted to support any proposal. In addition, a morespecific and detailed Outline of Production is also required.Potentially relevant in-process parameters that could be measuredand contribute to ensuring consistency of the final product include:

� Medium sterility� Master and working seed quality (purity, cell count, etc.)

W. Stokes et al. / Biologicals 41 (2013) 279e294 287

� Optical density of precultures and main culture� Purity of precultures� Monitoring of parameters/settings in main culture (e.g., pH,temperature)

� Purity of antigen prior to inactivation� Inactivation control (with validated test)� Antigen sterility� Antigen quantification

U.S. regulatory authorities communicated their willingness toprovide reagents, particularly monoclonal antibodies, necessary forvaccine manufacturers to develop in vitro ELISA tests and to qualifyreference bacterins for their products (i.e., serogroups canicola,grippotyphosa, icterohaemorrhagiae, and pomona). Accordingly,these reagents are available from the CVB upon request, with cur-rent supplies forecast to last at least 15 years. In addition, lepto-spiral antibodies and references are also available to internationalregulatory agencies to achieve global implementation of the vali-dated ELISAs. Workshop participants concurred that assurance of areadily available reagent supply is essential for the implementationof these methods, as well as assurance of the stability of thereference bacterins supplied by the CVB. In the United States,reference standards should be qualified via the Codified hamsterpotency test or via a host-animal vaccinationechallenge study. Inthe European Union, requalification in the target species is notusually required. The CVB does not have reagents available forLeptospira fractions other than those identified above.

Finally, other Leptospira serovars (e.g., hardjo or region-specificserovars) do not currently have USDA-developed ELISAs or Codi-fied potency tests approved in the United States. In the EuropeanUnion, approved ELISAs are available for an adjuvanted hardjobovisvaccine (cattle) and a nonadjuvanted tetravalent canine vaccine(antigens from serogroups canicola, icterohaemorrhagiae, bratislava,and grippotyphosa). Workshop participants noted that achievingthe specified validation requirements outlined in VS Memorandum800.112 might preclude the approval of these ELISAs in the UnitedStates.

3.3. Recommendations

� Vaccine manufacturers are encouraged to continue product-specific validation of the USDA in vitro ELISA tests for potencytesting of Leptospira vaccines containing serogroups canicola,grippotyphosa, icterohaemorrhagiae, and pomona. Specificmonoclonal antibodies and National Reference Standards areavailable from the CVB upon request to facilitate product-specific validation.

� Workshop participants encouraged the CVB to share itsarchival information on ELISA testing of marketed Leptospiravaccine products with respective manufacturers to assist withidentification of products for which the ELISAs have thegreatest likelihood for successful product-specific validation.The ELISA potency test appeared useful for a representativesample (i.e., 35%) of marketed Leptospira vaccines tested withand without elution or further assay optimization duringproof-of-concept testing at the USDA.

� Vaccine manufacturers are encouraged to further investigatethe identification and use of parameters during the vaccinemanufacturing process that can be used to indicate and ensureproduct consistency, which may help overcome obstaclesencountered during product-specific validation of Leptospiravaccines.

� Meeting the requirements for parallelism, as outlined in VSMemorandum 800.112, may be problematic when using anonadjuvanted reference vaccine with an adjuvanted product.

The following guidelines should be considered by regulatoryauthorities and manufacturers to address this issue:- Consider assessing parallelism only in the critical linear range- Summarize and communicate the most appropriate methodsfor recovering relevant antigen from adjuvanted vaccines

- Conduct potency tests on both the combined antigens (“bulkproduct”) prior to addition of adjuvant as well as the adju-vanted final product. While testing of the final product is aregulatory requirement, regulatory authorities may considerreviewing ELISA data comparing bulk and final product as away to demonstrate consistency and potency of the finalproduct

- Consider providing guidance on issues that may arise due todifferences in antigen manufacturing methods between ref-erences and products with regard to adequate assessment ofthe in vitro potency of products.

4. Leptospira veterinary vaccine potency testing: serologicalassays as refinement alternatives

4.1. State of the science

Dr. Elisabeth Balks (Paul-Ehrlich-Institut, Germany) provided anoverview of a current serological potency test utilized for licensedveterinary Leptospira vaccines in Europe [63]. Briefly, Ph. Eur.Monograph 01/2008:0447 (Canine Leptospirosis Vaccine [Inacti-vated]) and Monograph 01/2008:1939 (Bovine Leptospirosis Vac-cine [Inactivated]) describe the use of a serological immuneresponse test to assess batch potency [42,43]. The relative strengthsand weaknesses of the microscopic agglutination test, which esti-mates agglutination (50%) as an endpoint titer in serially dilutedsamples (i.e., diluted serum þ equal volume of leptospirosis), werediscussed. In addition, specific validation criteria (e.g., specificity,accuracy, precision, linearity) for the routine quality control ofvaccines were reviewed. The two main strategies to assess batchpotency and their specific demands were also addressed [63].

Dr. Catrina Stirling (Pfizer Animal Health, UK) summarized theprinciples, benefits, and limitations of a successfully implementedserological potency test for a multicomponent adjuvanted canineLeptospira vaccine currently released in the European Union [64].Versican� (Pfizer Animal Health, UK) is a multicomponent andmultiple Leptospira serovar canine vaccine protective againsticterohaemorrhagiae, canicola, and grippotyphosa. The alternativeassay is a measure of batch potency relative to the potency of anefficacious reference batch. The assay assesses the ability of com-mercial batches to stimulate an immune response in rabbits that iscomparable to the immune response in rabbits from a referencevaccine previously shown to be efficacious in dogs [64]. The assaydoes notmeasure the protective antibody titer in target species, norprovide any direct correlation to target species efficacy. Theserology assay, originally developed by Bioveta A.S. and listed as anoption in the Czechoslovakian Pharmacopoeia since 1971, requiredadditional development and validation studies to achieve Europeanproduct approval [64]. The associated benefits conferred by thevalidated alternative serology assay include:

� No in vivo vaccinationechallenge test or challenge strainmaintenance required

� Ability to be used with adjuvanted vaccines� Is serovar-specific can be used on multicomponent vaccines� Higher sensitivity than the existing in vivo hamster potencytest

� Good stability indicator� Reduced costs and release testing timelines� Better product consistency

W. Stokes et al. / Biologicals 41 (2013) 279e294288

4.2. Workshop discussion

Workshop participants recognized that the ideal alternativeLeptospira vaccine potency test is one that completely replaces theuse of animals, such as antigen quantification using ELISA as dis-cussed earlier. However, during the transition phase when in vitrotests are not available or are still in development, serologicalmethods can offer advantages in terms of animal use and animalwelfare. For example, while serological methods require thevaccination of animals, they do not require challenging animalswith a virulent dose that typically results in the death of unpro-tected vaccinates, nonvaccinated controls, and the majority of an-imals used in the back-titration procedure. Thus, serologicalmethods provide for refinement in that no significant pain ordistress is involved. They also provide for reduction in that they usefewer animals compared to a challenge test.

Workshop participants noted that the current hamster vacci-nationechallenge lethality test may not be suitable for all Lep-tospira serovars. For example, 99% of globally available serovarhardjo strains (e.g., hardjobovis and hardjoprajitno) are not lethal tohamsters.

Validated serological potency tests (i.e., rabbit) are currentlyavailable for assessing the potency of an adjuvanted monovalentbovine serovar hardjo vaccine and trivalent canine Leptospira vac-cine in the European Union, and several Leptospira products (pre-sumably serovar hardjo) in the United States. However, some U.S.participants expressed concern that current data do not support theuse of serology for potency testing of Leptospira. Before U.S. regu-latory authorities accept a serology assay, solid scientific datawould have to be established showing that host protection can bedirectly correlated to a specific antibody titer for a specific Lep-tospira strain. Furthermore, the data would have to be generatedusing a statistically valid number of animals and would validateserology for only the specific Leptospira serovar strain utilized in thestudy.

Serology is a highly beneficial diagnostic tool for determiningincreases in antibody titers of animals potentially exposed to Lep-tospira; however, an antibody titer that results in host protectionhas not yet been clearly defined [65e67]. U.S. vaccine manufac-turers stated that it would be necessary to demonstrate that anti-bodies are protective against challenge (i.e., correlating serology toprotection) to achieve regulatory approval and product release. Inaddition, validation studies correlating the level of antibody inlaboratory animals to host-animal efficacy would also be a regu-latory requirement. Currently, no vaccines for the four Leptospiraserogroups with a Codified test utilize a validated serology releaseassay in the United States (USDA, personal communication). Reg-ulatory agency representatives support the development andimplementation of alternative in vitro assays (i.e., SAMs 624e627)in preference to serology release tests. However, the developmentand introduction of serology assays may represent a more cost-effective alternative for vaccine manufacturers with existingproducts.

One vaccine manufacturer discussed a validated, approvedserology assay (i.e., in rabbits) for canine Leptospira vaccines in theEuropean Union. Specifically, the assay can differentiate only be-tween 75% and 100% formulated batches, thus a product incorrectlyformulated within this range would not be detected. As a conse-quence, the manufacturer is required to prove efficacy in targetspecies at 75% commercial minimum input or to increase the an-tigen input accordingly. As previously stated, EU regulatory au-thorities do not necessarily require a direct correlation of protectiveantibody titers in rabbits to target species efficacy, but rather thedemonstration of potency relative to an efficacious batch (i.e.,ability of commercial batches to stimulate an immune response in

rabbits comparable to the immune response for a batch previouslyshown to be efficacious in dogs) [64].

Workshop participants recognized that a serological potencytest for multicomponent Leptospira vaccines must be sufficientlysensitive and antigen specific. Correlation of the serological po-tency test to vaccine efficacy in the target species must bedemonstrated. In addition, any proposed serology assay must beable to detect substandard (i.e., incorrectly formulated) vaccinebatches. Regulatory agency representatives requested an opendialog with vaccine manufacturers early and often during thedevelopment of any alternative serology assay in order to providequalified feedback.

4.3. Recommendations

� The use of serological potency test methods (i.e., a single-injection vaccination and measurement of specific, immuno-logically relevant antibodies) is recommended instead ofvaccinationechallenge tests to avoid animal pain and distress,to reduce animal use, and to avoid worker safety issues asso-ciated with using live pathogenic Leptospira bacteria andhandling infected animals.

� U.S. regulatory authorities encourage vaccine manufacturers toprogress directly to product-specific validation and imple-mentation of ELISAs that are currently available for four com-mon Leptospira serogroups. However, when in vitro assays arefound to be inappropriate, the use of serology methods shouldbe considered. The following should be considered during thedevelopment of serological assays:- In the United States, none of the four Leptospira serogroupswith a current Codified test has a serology release test. Forany alternative serological assay, U.S. regulatory authoritiesrequire manufacturers to submit data correlating antibodytiter in the surrogate species to host-animal efficacy.

- EU regulatory authorities do not necessarily require a directcorrelation of protective titers in the surrogate species totarget species efficacy but rather the demonstration of po-tency in the surrogate species relative to an efficacious batch(i.e., ability of commercial batches to stimulate an immuneresponse in the surrogate species comparable to titers in thesurrogate species for batches previously shown to be effica-cious in the target species).

- Regulatory authorities encourage manufacturers to seekguidance and provide preliminary data early in the devel-opment process of alternative serological potency assays forthose serovars for which no in vitro assay is available.

- A serological potency assay for multicomponent Leptospiravaccines must be sufficiently sensitive and antigen specific;and a protective dose must be demonstrated. Serologicalassays must be able to detect substandard (i.e., incorrectlyformulated) vaccine batches.

5. Leptospira veterinary vaccine potency testing: humaneendpoints, moribund euthanasia, and analgesics asrefinement alternatives

5.1. State of the science

Dr. Elsio Wunder (Yale University, USA) reviewed the challengesinherent in identifying earlier humane endpoints when conductingthe current hamster Leptospira vaccinationechallenge test. Specificconcerns included:

� Leptospires rapidly disseminate in the hamster, and death canoccur within a few hours of overt signs of disease.

W. Stokes et al. / Biologicals 41 (2013) 279e294 289

� Within the same pathogenic species (e.g., L. interrogans),different serovars and routes of infection cannot be compareddue to large differences in virulence and progression of infec-tion, making identification of general cross-serovar humaneendpoints difficult.

� The intraperitoneal route of injection does not mimic naturalinfection.

� The LD50 of individual serovars and the associated median timeto death is highly variable across serovars.

Furthermore, weight loss, decrease in rectal temperature, andgeneral clinical signs have not been found to be sufficientlyconsistent and predictive that they could be considered as routineearlier surrogate endpoints for death. Nonetheless, based on Dr.Wunder’s experience, the following practices and earlier humaneendpoints may help alleviate animal pain and distress during thecurrent hamster Leptospira vaccinationechallenge test:

� Observe animals at least twice a day during light cycle� Check movement of animals in response to tactile stimuli:- Normal awake animal: responds by moving more than 30 cmin the cage

- Moribund animal: responds by moving less than 30 cm.These animals should be euthanized.

� Immediately euthanize animals exhibiting the followingsymptoms:- Seizures, dyspnea, bleeding from any orifice, and ruffled furcoat

Dr. Wunder explained that an ultimate goal of his studies is toprovide an improved hamster model for leptospirosis, including:

� Awell-standardized animal model (i.e., location, animal, agent,dose of infection, and route of infection)

� Awell-established timeframe, taking into account the differentdoses of infection and route of infection used

� Improved (i.e., quantifiable) descriptions of parameters toidentify symptomatic animals

� Complete and efficient tracking spreadsheet to collect infor-mation on animal monitoring

� Identification and evaluation of new surrogates for deter-mining infection

Finally, Dr. Wunder presented recent data demonstrating thathamster death can be correlated with the quantity of Leptospiraorganisms in specific tissues (e.g., kidney, blood, liver, spleen), andthat tissue quantification might in the future serve as a more sen-sitive endpoint than death.

Dr. Karen Brown (Pair O’Docs Consultants, USA) provided areview of humane endpoints that have been evaluated for Lep-tospira infections and discussed opportunities and strategies tofurther refine animal use for the hamster Leptospira vaccinationechallenge test, including the potential use of postchallenge anal-gesics. Clinical signs such as lethargy, hunched posture, and dullsunken eyes have been associated with impending death. Thepresence of blood on the nose and paws of hamsters as well as inurine has also been found to be indicative of infection and even-tual death.

Dr. Brown stressed the importance of defining the “CriticalPhase” for each serovar and vaccine testing facility by collectingdata on clinical signs from ongoing hamster potency testing pro-grams. The Critical Phase is when clinical signs and deaths begin toappear, which for most serovars is between days 7 and 10 post-challenge. The Critical Phase for a specific serovar and facility canbe defined by a retrospective review of available data, or a

prospective study where there is collection of detailed data onclinical signs that occur in animals involved in ongoing studies.After identifying the onset time for the Critical Phase, morefrequent observations should occur during this period through theend of the study. The following measures should be considered tofacilitate the collection and/or recognition of clinical signs duringthe hamster Leptospira vaccinationechallenge test:

� Reverse lighting cycle (i.e., lights off during the day) should beconsidered to allow hamsters to be observed during their mostactive period since they are nocturnal; red lighting can be usedby personnel to observe animals during the dark phase.

� White or light-colored bedding material can be used to detectbleeding and/or bloody urine.

� Appropriate training for laboratory animal technicians isessential to facilitate detection of clinical signs and imple-mentation of earlier humane endpoints.

The use of analgesics to avoid or minimize animal pain anddistress during the Critical Phase of the in vivo challenge test wasalso reviewed. The relative properties of ketoprofen, meperidine,butorphanol, and buprenorphine were highlighted. Sustained-release analgesic formulations of buprenorphine have been shownto effectively manage pain in rodents for up to 72 h [68]. Further-more, buprenorphine effectively relieves pain and distress in micechallenged with Toxoplasma gondii without interfering with testoutcomes, including the time to death in control animals [69]. Toavoid animal handling and minimize worker exposure to infectedanimals, analgesics may be delivered via drinking water or gelatincubes. However, clinically ill animals may eat and drink less, so thiswill need to be considered in dosing calculations. Comparativestudies should be conducted to determine the effect of analgesics onthe achievement of test objectives prior to implementation.

5.2. Workshop discussion

5.2.1. Refinement e earlier humane endpointsWorkshop participants recognized that the ultimate goal for

Leptospira vaccine potency testing is to completely eliminate ani-mal testing. However, the current vaccinationechallenge test willcontinue to be required until suitably validated serology and/orin vitro assays that can reduce, refine, or replace animal use arewidely accepted by regulatory authorities. Therefore, during thistransition period when it is necessary to conduct the hamstervaccinationechallenge test, the primary goals are to:

� Conduct the test in the most humane manner possible in orderto minimize or avoid pain and distress

� Use the fewest number of animals necessary to achievescientifically valid data while still ensuring potency of the testvaccine serials

� Minimize experimental variation and test failures

Participants agreed that data to support the implementationand use of earlier humane endpoints for the current hamsterLeptospira vaccinationechallenge model are limited. U.S. regula-tory authorities noted that hamsters sometimes proceed from “noclinical signs of illness” to “moribund” in fewer than 3 h and canprogress from moribund to death in less than 1 h. In accordancewith CVB Notice No. 12-12, any hamsters observed in a moribundstate that exhibit “clinical signs consistent with the expecteddisease pathogenesis that are unable to rise or move under theirown power should be humanely euthanized and considered asdeaths” [51]. Furthermore, if humane endpoints prior to the onsetof the moribund condition could be defined for hamsters that

W. Stokes et al. / Biologicals 41 (2013) 279e294290

receive one serovar, the same endpoint may not be useful for theother serovars. U.S. regulatory authorities require sufficient veri-fication that earlier humane endpoints, in addition to thosecurrently described in CVB Notice No. 12-12 and 9 CFR 117.4, areappropriate for each product tested with a challenge inoculum.Specifically, verification requires a statistically valid number ofanimals vaccinated with a defined vaccine product and infectedwith a quality-controlled Leptospira challenge strain, exhibitingdefined clinical sign(s) and progressing to moribund condition (ordeath).

U.S. Codified regulations specify that a valid test requires thatthe challenge inoculum induce death (or moribund condition) in 8of 10 nonvaccinated hamsters. Therefore, a concern is that the useof inappropriate earlier humane endpoints may mask a weakchallenge dose and inadvertently allow a substandard serial to beconsidered satisfactory for release. Furthermore, a loss of virulencein challenge cultures is of particular concern for leptospirosis, as thespirochetes are known to routinely undergo loss of virulence whenmaintained in in vitro cultures [70]. The dynamic nature of lepto-spiral virulence, combined with the lack of uniformity betweenstrains, reinforces the importance of quality control of Leptospirachallenge cultures if earlier humane endpoints are utilized.

The CVB is actively investigating the use of ataxia, bloody urine,and bleeding from nostrils as definitive earlier humane endpointsfor nonvaccinated animals receiving one of four CVB-approvedchallenge cultures, via observations of hamsters currently utilizedfor challenge maintenance and routinely performed potency as-says. One manufacturer stated that the presence of blood on thenostrils and/or paws of hamsters is already used at their facility asan earlier humane endpoint during Leptospira vaccine potencytesting. U.S. regulatory authorities requested that manufacturersreport such data for further review and consideration. In addition,manufacturers were requested to review in-house data and collectprospective data on the potential use of decreased responses tostimuli (e.g., movement of awake animals less than 30 cm whentouched) as an earlier humane endpoint and to forward their datato regulatory authorities for consideration if appropriate.

Participants recommended immediate training of laboratoryanimal technicians to recognize clinical signs that may be appro-priate as humane endpoints, and increasing the frequency of ani-mal monitoring during the “Critical Phase” of their challengemodel. Presently, at least two observations per day are recom-mended and performed at most facilities during the Critical Phase.In addition, participants discussed the use of reverse lighting (i.e.,switching light/dark cycles) to facilitate the detection of clinicalsigns during the period of nocturnal activity. Several participantsstated that the usefulness and validity of reverse lighting are beinginvestigated at their facilities and may be implemented if it doesnot interfere with other activities. Workshop participants notedthat increased manipulation and handling of animals infected withknown zoonotic agents present a risk to laboratory staff and maycause additional stress to the animal. One solution might be theapplication and use of remote observation and telemetry devices toidentify moribund animals; however, this was considered ascurrently cost-prohibitive.

Workshop participants noted that a further limitation to intro-ducing earlier humane endpoints for the in vivo challenge test is theexistence of Leptospira serovar- and strain-specific differencesresulting in varying clinical expressions of disease. For example,hamsters infected with serovar hardjo typically present no symp-toms, while serovars pomona and grippotyphosa generate distinctclinical symptoms. Thus, each strain requires identification of itsrespective earlier humane endpoint(s). Prior to implementation,additional data on this issue are needed to ensure that productpotency is not jeopardized as a result of premature euthanasia.

5.2.2. Refinement e use of analgesicsAnimals are routinely infected with virulent Leptospira to

maintain virulent challenge cultures and to conduct the CodifiedLeptospira potency vaccinationechallenge test. With regard tomaintenance of challenge cultures in animals, workshop partici-pants considered that the opportunities for pain relief (i.e., anal-gesics) in hamsters infected with leptospirosis were minimal. Aspreviously stated, the disease progression for leptospirosis is rapidfollowing the initial appearance of clinical signs. Therefore, anal-gesics (e.g., buprenorphine) are not likely to have sufficient time toact prior to animal death unless administered prior to the onset ofclinical signs. Furthermore, U.S. regulatory authorities stated thatcurrent transfer conditions (i.e., dose and interval between pas-sages) are optimized to facilitate transfer of Leptospira at the firstindication of clinical disease, thereby limiting the impact of anyprophylactic analgesia.

Some workshop participants considered the use of analgesicsduring the potency testing of Leptospira vaccines as impractical andof limited benefit. This was based on what they viewed as variabletime of onset of clinical signs and the resulting difficulty inselecting the most appropriate timepoint for administering anal-gesia. In addition, they noted that nonvaccinated animals used forLD50 determination rapidly succumb to the disease once clinicalsigns appear but appear completely healthy without clinical signsof pain or distress prior to the onset of clinical signs.

For other challenge strains that confer a slower disease pro-gression, the use of analgesics may be more useful in reducing oravoiding pain and distress. Again, comparative studies should beconducted to verify that the analgesia does not alter the number ofhamsters that succumb to the disease and that it does not other-wise interfere with the objectives of the potency test. Regulatorssuggested that three valid (as defined by Codified regulations) ex-periments in which similar LD50 values were demonstrated inhamsters with and without analgesia before challenge would berequired to support analgesic use for a particular product anddefined challenge.

For future Leptospira potency tests, workshop participants sug-gested the use of simple husbandry refinements such as (1)providing water sources and gel packs on cage floors to facilitateimproved access to ill animals and (2) use of white bedding ma-terial to more easily detect the presence of blood.

5.3. Recommendations

� When it is still necessary to conduct the hamster vaccinationechallenge test, the following actions are proposed:- Where possible, analgesics (e.g., buprenorphine) should beprovided to avoid or minimize pain and distress associatedwith the hamster Leptospira vaccinationechallenge test. Priorto implementation, comparative studies should be conductedto determine that the use of analgesics is consistent withtesting objectives. To avoid animal handling and minimizeworker exposure to infected animals, consideration shouldbe given to delivering analgesics via drinking water or gelatincubes.

- Manufacturers should further investigate potential humaneendpoints such as ataxia, bloody urine, presence of blood onthe nose or feet, and reduced responsiveness and lethargy fortheir usefulness as the basis for earlier humane euthanasia toavoid or minimize spontaneous deaths. White bedding ma-terial should be considered to facilitate the detection of thepresence of blood.

- The short time from onset of clinical signs to death should betaken into account when considering the use of humaneendpoints in a test with a defined observation period. In all

W. Stokes et al. / Biologicals 41 (2013) 279e294 291

cases, the frequency of monitoring should be increasedappropriately to facilitate moribund euthanasia and tofurther reduce spontaneous deaths.

- Vaccine producers and regulatory authorities conducting thechallenge test should consider reverse animal-room lightingto facilitate observation of clinical signs during the nocturnalperiod of activity. These observations may be useful as thebasis for humane euthanasia and therefore further reducespontaneous deaths.

6. Leptospira veterinary vaccine potency testingdreductionalternatives

6.1. State of the science

Dr. Angela Walker (USDA, USA) reviewed ongoing USDA activ-ities for reducing animal use for the current hamster vaccinationechallenge test and for maintaining Leptospira challenge cultures[70]. Hamsters are infected with virulent Leptospira for two pur-poses in the regulation of Leptospira biologics: (1) to conduct theregulatory potency test, and (2) to maintain virulent Leptospiracultures. The most efficient way to achieve an immediate reductionin the number of hamsters used for potency determination is tocombine challenge control and back-titration groups [70]. Thismeasure could effectively reduce hamster use by at least 50%. Inaddition, the CVB is evaluating the current requirement for back-titration and will publish their recommendations upon completion.

The second target area for potentially reducing animal use forLeptospira vaccine potency testing is during the maintenance ofvirulent Leptospira challenge cultures. To date, a completely in vitroapproach is not feasible due to the associated loss of virulence thatis observed with multiple passages in media. Previous studies haveshown that many variables affect virulence in cultured strains,including:

� pH� Osmolarity� Temperature� Bovine serum albumin source

In vivo passaging of Leptospira serovars in hamsters is requiredto maintain virulence; however, significant variation is observedamong the more than 250 pathogenic serovars. To reduce animaluse, the CVB is investigating the feasibility of maintaining virulentLeptospira using cryopreserved Leptospira stock for direct infectionof the host animal or hamster. This alternative strategy wouldenable intermittent pauses in the in vivo passaging of virulentLeptospira through hamsters. However, such cultures mustdemonstrate consistent virulence, stability in the challenge char-acteristics, rapid recovery, and ease of propagation. The cry-opreserved challenge cultures would not be considered MasterSeed, but many of the same benefits associated with the limitedpassage numbers of Master Seeds would be possible with a cry-opreserved challenge material. The potential impact of this mea-sure on Leptospira virulence, time to initiate testing, and continuoussupply of challenge strains is being considered [70]. Additionalwork is required to validate this alternative reduction strategy.

6.2. Workshop discussion

Workshop participants discussed opportunities to reduce thenumbers of animals used in the Codified regulatory requirementsfor the current hamster Leptospira vaccinationechallenge test.Specifically, participants considered potential opportunities forreducing the number of hamsters used (1) within the vaccinated

group, (2) within the nonvaccinated control group, (3) in routineback-titration to assess the virulence of the challenge inoculum, (4)through simultaneous batch testing to share controls, and (5) forculture maintenance.

U.S. and EU regulatory requirements currently differ with regardto the number of hamsters used for each test group in the vaccina-tionechallenge test. The Codified USDA test requires 10 vaccinatesand 10 nonvaccinates, while the EU test requires 5 hamsters in eachgroup. Due to the significant variability associated with the currentin vivo test (and subsequent need for statistical validity), U.S. regu-latory representatives did not encourage reducing the number ofanimals below current requirements in the near term. Participantsnoted that reducing the number of hamsters to 5 each in the test andcontrol groups may actually increase overall animal usage due toincreased repeat testing that can potentially use more animalsoverall than if the higher number had been initially used [71].

Workshop participants discussed the possibility of reducinganimal numbers in the hamster vaccinationechallenge test byeliminating the routine use of 20 hamsters to perform a back-titration to estimate the LD50 of the challenge inoculum.Currently, U.S. Codified regulations for Leptospira potency testingspecify that the challenge inoculum must contain 10e10,000 LD50equivalents. Because the minimum LD50 requirement is achievedwhen at least 8 of 10 control animals die (or are considered mori-bund) during regulatory testing, the additional information pro-vided by the back-titration procedure only addresses if thehamsters are overchallenged. U.S. regulatory representatives statedthat studies are underway to investigate the necessity of ascer-taining the precise lethality of the challenge inoculum using theback-titration procedure. Routine back-titrations would be unnec-essary if no impact on the vaccinated group is observed due tooverchallenge, thus only 10 control animals would be required toensure that the minimum LD50 requirement is met. Implementingthis reduction alternative could reduce animal use for potencytesting of Leptospira vaccines by at least 50%. U.S. regulatory rep-resentatives stated that a minimum of three independent testsmust be conducted in hamsters vaccinated with a potent vaccineand a subpotent vaccine(s), and each vaccinate group challengedwith at least two doses (i.e., challenge titration and overchallengetitration) to support the removal of routine back-titrations. The CVBis currently conducting such studies. In the European Union, noback-titration step is required; however, at least 4 out of 5 controlhamsters must die within 14 days post-challenge.

Workshop participants stated that it is common practice toconduct simultaneous testing of serials, which reduces animal useby sharing common control and back-titration groups. However,vaccine manufacturers noted that when using this approach, theoccurrence of an invalid control or back-titration group wouldresult in testing delays for all of the serials involved and wouldrequire repeat testing using additional animals.

Workshop participants also discussed potential ways to reducethe number of animals used to maintain Leptospira challenge cul-tures necessary for potency testing procedures. Presently, Lep-tospira must be transferred via a live host (i.e., hamsters) tomaintain virulence. To date, only one study has reported success inmaintaining virulence in any leptospiral strain beyond approxi-mately 10 in vitro passages [72]. A reliable method of maintainingvirulent cultures in vitro has not been identified for the four frac-tions with Codified hamster vaccinationechallenge potency assaysin the United States [70]. The other limitation to the in vitroapproach is that in vitro propagation of Leptospira culturesmay takea significant period of time (i.e., greater than one month) to achieveappropriate levels for hamster challenge. Therefore, rapid testingfor serial release may not be possible if any issues arise with cultureconditions and stocks must be regrown.

W. Stokes et al. / Biologicals 41 (2013) 279e294292

The only viable mechanism at this time for reducing hamsteruse for maintaining Leptospira challenge cultures is to use cry-opreserved Leptospira for infection of a host animal or hamster. Inthis manner, the continuous transfer of Leptospira through ham-sters could be paused for extended periods. A primary concern isthat no reliable method is currently available to directly inoculatehamsters from cryopreserved Leptospira stock without growth insemisolid culture. U.S. regulatory authorities noted that, beforethese methods for maintaining Leptospira cultures can be accepted,scientific data must demonstrate that hamsters are infected andexhibit clinical signs of disease following different periods in thecryopreserved state and viamultiple infected tissue collections. TheCVB is currently investigating procedures for direct challenge ofhamsters from cryopreserved Leptospira stock to allow pauses inin vivo passaging through hamsters without reducing the virulenceof challenge strains.

In the European Union, regulatory authorities do not require thecontinuous serial passaging of Leptospira challenge strains inhamsters. Rather, manufacturers pass challenge strains via a singlehamster every 2e3 years. A trend analysis of challenge results incontrol groups is performed to monitor the virulence of the chal-lenge strains.

6.3. Recommendations

� The CVB is encouraged to complete its evaluation of the ne-cessity to perform back-titrations to calculate the LD50 of thechallenge inoculum in each hamster vaccinationechallengetest. Elimination of the routine back-titration requirementcould reduce the number of animals required for potencytesting of a single serial from 40 to 20 for each serovar,providing for up to a 50% reduction in animal use. This wouldalso avoid significant pain and distress that occurs in the ma-jority of the animals used for the back-titration procedure.

� Regional differences currently exist in the regulatory re-quirements for the number of animals per control and testgroup (e.g., 10 versus 5). However, reducing the animal numbermay result in increased repeat testing that ultimately requiresmore animals. Therefore, vaccine manufacturers and regula-tory authorities are encouraged to investigate and share in-formation on which approach uses the lowest overall numberof animals for individual serovars and challenge strains.

� To further reduce animal use, manufacturers are encouraged tocontinue performing simultaneous testing of multiple serialsso that the nonvaccinated control and back-titration groupsmay be shared.

� The CVB is strongly encouraged to continue investigating andstandardizing procedures for the use of cryopreserved Lep-tospira challenge inoculum to directly inoculate hamsters. Thisprocedure would allow pauses in the continuous in vivopassaging of challenge strains, thereby significantly reducingthe number of animals required to maintain virulent Leptospiracultures and that otherwise would experience pain anddistress.

7. Workshop conclusions

This international workshop provided a comprehensive reviewof the current state of the science for alternative methods that canreplace, reduce, and refine animal use for veterinary Leptospiravaccine potency testing. Participants considered this informationand developed recommendations on how to implement andfurther advance alternative methods and approaches that couldpotentially eliminate animals for most Leptospira potency testing.Where animals must still be used, recommendations weremade on

how to further reduce the numbers of animals required per test andfurther minimize the duration and severity of pain and distress.

The active participation of U.S. and international academic, in-dustry, and regulatory scientists sharing their combined experi-ences and insights contributed to new ideas on the best wayforward to advance alternatives for potency testing of Leptospiravaccines. Although the workshop focused primarily on veterinaryvaccines, the participation by experts working to develop humanLeptospira vaccines provided the opportunity to exchange infor-mation and ideas on their current approaches as well as next-generation vaccines. Continuing the productive interactions andcooperation established at the workshop will aid in the imple-mentation of workshop recommendations and potential fruitfulcollaborations that will advance new methods and approaches forLeptospira vaccine testing.

One of the most important aspects of the workshop was dis-cussion of how manufacturers could move forward with product-specific validation studies using the USDA ELISA tests for theirLeptospira vaccine products. Most importantly, the USDA/CVBaffirmed that they will continue to provide the monoclonal anti-bodies and other critical reagents necessary to perform thesein vitro tests. Workshop participants discussed critical issues anddeveloped recommendations for product-specific validation of theELISAs, including appropriate methods for recovering relevant an-tigen from adjuvanted vaccines and achieving the parallelism re-quirements for the reference and test vaccine. The CVB agreed toshare archival information on ELISA testing of commercial Lep-tospira vaccine products with respective manufacturers to assistwith identifying products for which the assays have the greatestlikelihood for successful product-specific validation. Finally, furtheridentification of parameters that can be used to assure in-processconsistency of vaccines was encouraged, along with explorationof how such consistency parameters can contribute to facilitatingproduct-specific validation.

The workshop participants discussed how serological methodsfor potency testing could avoid pain and distress and worker safetyissues by eliminating the need to use live Leptospira pathogenicbacteria in animals. At least two Leptospira vaccine products arecurrently being released using serological potency tests in Europe.However, workshop participants recommended that serologicalmethods should be considered in the future only after firstattempting to use in vitro assays.

Other important outcomes of the workshop were discussionsand recommendations on how to further reduce and refine animaluse where animals must still be used for Leptospira vaccinationechallenge testing. Most importantly, workshop participantsencouraged the CVB to complete its evaluation of the necessity forconducting a back-titration to calculate the LD50 of the challengeinoculum in each hamster vaccinationechallenge test. If the non-vaccinated control animals in the potency assay could be usedinstead to indicate the adequacy of the Leptospira challenge inoc-ulum, this could reduce the number of animals required for eachvaccine potency test by 50% (20 versus 40).

Another area for significant potential reduction of animals is theuse of hamsters to maintain Leptospira challenge cultures used inpotency testing procedures. Workshop participants stronglyencouraged the CVB to complete their research on the use of cry-opreserved Leptospira stock to directly inoculate hamsters, which, ifsuccessful, will allow for extended pauses in the continuous in vivopassaging of challenge strains.

Workshop participants proposed several immediate actions thatcould further minimize or avoid pain and distress in the currenthamster Leptospira potency challenge test. These included consid-eration and use of analgesics, identification and use of earlier hu-mane endpoints that could serve as the basis for humane

W. Stokes et al. / Biologicals 41 (2013) 279e294 293

euthanasia, and increased frequency of observation to avoid orminimize spontaneous deaths. The immediate use of reverseanimal-room lighting to facilitate the observation of clinical signswas also encouraged.

Finally, workshop participants supported international recog-nition of established potency tests and harmonization of newalternative methods to avoid duplicative potency testing currentlyconducted to meet regionally different requirements. Such recog-nition and harmonization could significantly reduce animal testingand vaccine testing expenses. The international availability ofcritical reagents such as monoclonal antibodies and referencestandards is considered essential to implementation of this effort.

The workshop highlighted how the application of new scienceand innovative technologies is providing opportunities for moreefficient and effective vaccine testing. The open public discussionsamong a wide range of interested stakeholders identifiednumerous ways to further advance the development, validation,and use of improved alternativemethods. The timely and successfulimplementation of the workshop recommendations is expected toadvance alternative methods for Leptospira vaccine potency testingthat will significantly benefit animal welfare while continuing toensure safe and effective vaccines.

Acknowledgments

The authors extend their sincere appreciation to all participantsin the international workshop for their contributions leading to theworkshop conclusions and recommendations. The members of theLeptospira Workshop Organizing Committee, ICCVAM InteragencyBiologics Working Group, and NICEATM staff are acknowledged fortheir contributions to the planning of the workshop, and all theinvited experts are acknowledged for their presentations andcontributions to breakout group discussions and workshop pro-ceedings. Participating national/international validation organiza-tions from the International Cooperation on Alternative TestMethods, including NICEATM, ICCVAM, EURL ECVAM, and JaCVAM,are also gratefully acknowledged for their scientific contributionsand financial support. Finally, the authors gratefully acknowledgeDr. Raymond Tice and Dr. John Bucher from the National Institute ofEnvironmental Health Sciences, National Institutes of Health, USAfor their review of the manuscript.

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