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Development and validation of a serological potency test for the release of Leptospira vaccines – Requirements in the European Union
Biologicals 41 (2013) 325e329
Contents lists available at SciVerse ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Short paper
Development and validation of a serological potency test for the release of Leptospira vaccines e Requirements in the European Union Elisabeth Balks*, Heike Gyra, Babett Kobe, Klaus Cussler, Esther Werner Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Veterinary Department, Section Bacterial Vaccines and Immune Sera, Paul-Ehrlich-Institut-Strasse 51-59, 63225 Langen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2013 Received in revised form 19 June 2013 Accepted 22 June 2013
Both European Pharmacopoeia Monograph 01/2008:0447 “Canine Leptospirosis vaccine (inactivated)” and the more recent Monograph 01/2008:1939 “Bovine Leptospirosis vaccine (inactivated)” explicitly allow for a sero-response test to assess batch potency. Test setup and requirements for in vivo and in vitro validation are described. Furthermore, the two main strategies to assess batch potency and their specific demands are addressed. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Leptospira Vaccine Batch potency Alternative method 3R Serology
1. Control of leptospirosis Vaccination against leptospirosis constitutes an important preventive health measure in veterinary medicine. Canine vaccines have been available in Europe since 1961 [1]. In Germany, vaccines against leptospirosis in dogs are considered to be core vaccines [2]. Currently, products from four manufacturers are licensed. So far, most vaccines are bivalent, comprising Leptospira (L.) interrogans serogroups Icterohaemorrhagiae and Canicola. Changes in the epidemiology of canine leptospirosis led recently to the inclusion of L. interrogans serogroup Australis and L. kirschneri serogroup Grippotyphosa. Whereas exposure to most of these serogroups is consistently observed across Europe, the relative importance of serogroups Grippotyphosa and also Sejroe, which is currently not included in vaccines available in Germany, differs according to region. This is due to the fact that infections by serogroups Grippotyphosa and Sejroe result from incidental exposure to strains maintained by rodent hosts, whose distribution and concentration varies across Europe. Infections by serogroup Pomona, which are important in North America, are rare in Europe [1]. Canine vaccines currently available are based on inactivated whole bacteria. All but one vaccine are non-adjuvanted. Leptospires
* Corresponding author. Tel.: þ49 6103 777402. E-mail address: [email protected] (E. Balks).
are grown in media containing rabbit serum and/or bovine serum albumin or serum-free media, inactivated by heat, formalin, thiomersal, or bepropriolactone. In some cases, additional washing and concentration steps by centrifugation are performed. In 2008, a L. borgpetersenii serovar Hardjo (type Hardjobovis) vaccine for cattle was licensed in Germany. This is an adjuvanted whole-cell bacterin.
2. Immunity and protection Immunity to acute leptospirosis is thought to be primarily humoral. In contrast, immunity against L. borgpetersenii serovar Hardjo in bovine reservoirs correlates with T helper 1 cell response. High levels of agglutinating antibodies do not appear to be protective [3]. Nevertheless, several studies could show a clear dosee response of antibody titers in vaccinated guinea pigs [4e6]. The response was related to the degree of protection achieved in calves given graded vaccine doses [4]. In consequence, current alternatives to the hamster challenge test for potency testing of Leptospira vaccines are based on humoral immunity. Due to their potential ability to stimulate heterologous immunity, surface-exposed, constitutively expressed outer membrane proteins specific to pathogenic serovars represent an important focus of current leptospiral research [7,8]. However, findings concerning protectivity evoked by protein antigens appear to be
1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2013.06.011
326
E. Balks et al. / Biologicals 41 (2013) 325e329
Table 1 Batch potency tests outlined in Ph. Eur. monographs on leptospirosis. Monograph 01/2008:0447 Canine Leptospirosis Vaccine (inactivated)
Monograph 01/2008:1939 Bovine Leptospirosis vaccine (inactivated)
Vaccines with or without adjuvants (I): 10 Hamsters for each serovar s.c. vaccination of 5 animals with 1/40 of the dose for dogs, 5 hamsters maintained as controls i.p. challenge 15e20 d p. vacc. Vaccine complies if at least 4 control hamsters die within 14 d of receiving challenge suspension and at least 4 vaccinees remain in good health for 14 d after the death of the controls. Vaccines with or without adjuvants (II): Suitable validated sero-response test for each serovar In-vitro evaluation of individual blood samples to determine the antibody-response to one or more antigenic components which are indicators of protection and which are specific for that serovar Criteria for acceptance set with reference to a batch that has given satisfactory results in dogs Vaccines without adjuvants: Suitable validated in-vitro test for each serovar to determine the content of one or more antigenic components which are indicators of protection and which are specific for that serovar Criteria for acceptance set with reference to a batch that has given satisfactory results in dogs
ambiguous. From studies with single purified whole proteins, combinations thereof, and combinations of recombinant (fusion) proteins, it has been concluded that protective effects are synergistic, reflecting genetic redundancy of leptospires [8,9]. At the same time, there is convincing evidence for the causative role of lipopolysaccharides (LPS) in pathogenicity and their key role in immunity. Studies with monoclonal antibodies have shown that LPS is the target for agglutinating and opsonizing antibodies. Differences in the polysaccharide component, which is the outermost domain of the LPS molecule, account for more than 260 serovars of Leptospira. Consequently, LPS-mediated immunity is mainly serovar specific. It correlates with levels of agglutinating LPS-specific antibodies in transferred sera. LPS-specific monoclonal antibodies passively protect naive animals from leptospirosis [9,10]. Purified LPS can stimulate active immunity [9]. 3. Serological approaches to assess batch potency In the chapter “General principles” of the introduction, the European Pharmacopoeia (Ph. Eur.) states commitment to the reduction of animal use wherever possible. Chapter I, “General notices,” specifies that “. alternative methods of analysis may be used for control purposes, provided that the methods used enable an unequivocal decision to be made as to whether compliance with the
Not fewer than 12 guinea pigs for each serovar 10 Vaccinees, not fewer than 2 controls Blood collection 19e23 d after vaccination Evaluation of antibody titers of individual samples by a suitable validated method such as the MAT Vaccine complies with the test if antibody levels are equal to or greater than those obtained with a batch that has given satisfactory results in cattle.
standards of the monograph would be achieved, if the official methods were used.” In addition, both Ph. Eur. monograph “Canine Leptospirosis vaccine (inactivated)” [11] and the more recent monograph “Bovine Leptospirosis vaccine (inactivated)” [12] explicitly allow for a seroresponse test to assess batch potency (Table 1). Currently available alternatives to the hamster challenge test comprise various ELISA formats aiming at antigen quantification by the use of serovar-specific, LPS-directed monoclonal antibodies. Furthermore, a serological approach is in use, where antibody titers are evaluated by the microscopic agglutination test (MAT). Due to its good specificity, the MAT, which became available as early as 1918 [13], is still regarded as the gold standard for serological diagnosis of leptospirosis [14]. It includes evaluation of paired serum samples and their ability to agglutinate live leptospires. The endpoint is defined as that dilution of serum that shows 50% agglutination, leaving 50% free cells compared with a control culture diluted in phosphate-buffered saline (Fig. 1) [14]. There have been several studies to evaluate the suitability of the MAT for batch potency testing of Leptospira serovar Hardjo vaccines using guinea pigs. Goddard et al., 1986 [4] compared challenge results in calves to guinea pig serology. Cattle and guinea pig serology evaluated by the MAT were re-addressed in an ECVAM research project [5,6]. Important technical findings from these studies were
Fig. 1. Microagglutination of live Leptospira (dark field microscopy, 400) A e no agglutination, B e 50% agglutination, C e 100% agglutination.
E. Balks et al. / Biologicals 41 (2013) 325e329
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Table 2 Strengths and weaknesses of the MAT as a tool to assess batch potency.
Fig. 2. Procedure to demonstrate accuracy A e validation successful, B e validation not successful x e mean of first set of data, x0 e mean of second set of data, s e standard deviation Accuracy is usually demonstrated in a two-step procedure. In a first step, several trials are performed to fix the specification of the test signal (e.g., mean 2 s). In a second step, several (usually 9) tests are run to demonstrate whether the specification is still met. Validation is successful if the mean value of the results obtained in the second step, including a safety margin (usually the 95% confidence interval), fall completely within the specifications set beforehand. This could be shown in the upper graph (blue interval) but not in the lower graph (red interval), where the mean value but not the complete confidence interval fullfils the validation criteria.
that titers as low as 1:2 can be significant when an animal has been previously negative, and the titer can be attributed to a known input of antigen [4]. Younger guinea pigs (350 g) exhibited a better antibody response than older animals (500 g). Furthermore, old
Strengths
Weaknesses
Good specificity (detection of group- or serovar-specific antibodies, where the serovars have been well described) [14] Evidence for the detection of protective antibodies provided [21] Titers reflect reaction to entire vaccine including immunomodulatory effects of product components; no further vaccine processing required [22] Titers reflect vaccine dose/ vaccination scheme Suitable for nonlethal strains (as compared to protection tests) [4] and for stability testing
Requires animal testing (ethics, costs, time, extrapolation of test data between target and surrogate species) Standardization and transfer difficult (cultivation of strains, read out semiquantitative and subjective) Requires maintenance of live reference strains (risk of cross-contamination, contamination with non-Leptospira spp. or rapid-growing saprophytic leptospires, mislabeling, switching of strains, infection of personnel) [23] No differentiation of IgM and IgG (as compared to ELISA) Might not be suitable for all vaccines. (A multivalent and a multivalent combined vaccine did not induce measurable antibody titers [6]. In this study, 8 vaccines representative for products used in Europe, North America, and Australia/New Zealand were included)
animals appear to include a higher number of nonresponders [6]. General conclusions with regard to the MAT as a tool for batch potency testing of Leptospira Hardjo vaccines are listed in Table 2. As an outcome of their studies, both groups proposed a potency test based on the MAT response of guinea pigs [4,5]. This led to the inclusion of serology in the Ph. Eur. monographs on leptospirosis. At present, the manufacturer of a canine vaccine successfully established a batch potency test utilizing the MAT. The test measures the antibody responses to each of the Leptospira serovars following vaccination of rabbits. 4. Assay validation Current expectations for assay validation are outlined in two complementary International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines [15,16] and also in the Technical Guide for the elaboration of monographs [17]. Specific aspects on the validation of alternative methods have been addressed by Hendriksen et al., in 1998 [18]. Table 3 lists those validation
Table 3 Validation/test validity criteria for routine quality control (Technical guide for the elaboration of monographs, 6th ed. 2011, Chapter III, Analytical validation, modified). Parameter
Fig. 3. Relevance of accuracy and precision A e accuracy, P e precision, “þ” e acceptable, ““ e not acceptable Poor accuracy is characterized by marked deviations of the measured values from the target value. Deviations are of the same nature and occur in the same direction. Precision expresses the closeness of agreement between a series of measurements. Accuracy and precision have not only to be addressed during validation but also need to be surveyed with every test result obtained thereafter.
Specificity Accuracy Precision Linearity Detection limit Quantitation limit Range
Type of test Identity
Purity Quantitative Qualitative
Potency
þ
þ þ þ þ þ þ
þ þ þ þ þ
þ þ
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E. Balks et al. / Biologicals 41 (2013) 325e329
Table 4 Validation of relevant parameters for potency testing of veterinary vaccines. Parameter
Terminology and validation
Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of other antigenic components, excipients, residuals, or degradants [17]. This is especially important for veterinary vaccines, which are not purified in most cases. For multivalent vaccines, it is essential to test the specificity of the response for each component in the vaccine. In case of a direct correlate to protection, specificity may be shown by passive protection or vaccination/challenge experiments or by sero-response studies. Alternatively, reactivity of vaccinates to immunorelevant or immunodominant epitopes is demonstrated. Mostly, ELISA assays e sometimes in combination with Western blot, agglutination tests or in-vitro growth inhibition tests are used. In the light of the consistency approach, there might be a change of paradigm with regard to the understanding of specificity. Once consistency of production has been shown, testing is then based on in vitro tests which do not have to provide the same information as in vivo tests [24,25]. Accuracy expresses the closeness of agreement between the conventional true value or an accepted reference value and the value found [17]. Examining the accuracy of a method serves to recognize and/or eliminate systematic errors. Accuracy should be assessed by using a minimum of 9 determinations [17]. Accuracy is usually demonstrated in a two-step process by confirming a specification set beforehand (Fig. 2). Less commonly, a validated alternative procedure is used to demonstrate accuracy. Precision expresses the closeness of agreement between a series of measurements obtained from multiple testing of the same homogeneous sample. The rationale of such tests is to recognize and avoid random errors. It is usually expressed as variance, standard deviation, or coefficient of variation. A minimum of 6 determinations at 100% of test concentration is recommended. Data obtained for accuracy may be included [17]. Precision may be considered at 3 levels:
Accuracy (Figs. 2 and 3)
Precision (Fig. 3)
characteristics regarded as the most important for the validation of different types of analytical procedures. Table 4 further details technical aspects of the validation of potency assays. Parameters should be regarded as typical for the analytical procedures, but occasional exceptions should be dealt with on a case-by-case basis [17]. 5. Assessment of potency The principle applied whenever possible throughout the bioassays prescribed in the Ph. Eur. is the comparison of a standard preparation with the batch under test. Bioassays included in the Ph. Eur. have been conceived as dilution assays whenever possible. The batch under test is supposed to contain the same active principle as the standard preparation but in a different ratio of active and inert components [19]. To ensure this requirement is met, it is essential to compare the doseeresponse relationships of the standard and the batch under test. The standard is either a vaccine shown to be efficacious in the target species or a pool serum derived thereof. The latter may have some advantage in terms of the 3Rs because it further reduces the number of animals needed.
p Repeatability/intra-assay precision (same operation conditions over a short interval of time, i.e., results from the same day, same technician) p Intermediate/inter-assay precision (expresses intralaboratory precision, i.e., results from different days, different analysts, different equipment) p Reproducibility (includes comparison of results obtained by different laboratories; usually applied to standardization of methodology [17]) Linearity
Range
Linearity ensures that the test results within a given range are directly proportional to the amount of analyte. Determination of linearity requires measuring the analytes at different concentrations. International guidelines recommend measuring 5 concentrations of the analyte [5]. Other approaches should be justified. To ensure this condition, the batch release value (OD, antigen content, titer) must fall within the linear part of the titration curve. Testing for linearity requires duplicate testing. Ph. Eur. requires regression analysis to demonstrate linearity. In some cases data may need to be subjected to mathematical transformation [19]. The range denotes the interval between the upper and lower concentration of analyte in the sample. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy, and precision when applied to samples containing an amount of analyte within or at the extremes of the analytical procedure [17]. The range is determined by application of the procedure to a series of samples having analyte concentrations spanning the claimed range.
Fig. 4. A. Assessment of batch potency (I). Relative potency: similarity testing of dosee response curves (parallel line assay and 4-parameter logistic curve model) x-axis e dose, y-axis e response. Horizontal shift of curves along the x-axis describes the relative potency of the batch under test (green line) as compared to standard (blue line). The relevant parameter for the “parallel line assay” is parallelism in the linear section of doseeresponse curves (red parallels). Relevant parameters for the “4parameter logistic curve model” are minimal and maximal response, slope at the steepest increase (descent), and the ED50 value. B. Assessment of batch potency (II) Calculation of fixed acceptance criteria x-axis e batches tested, y-axis e parameter scale (example) Acceptance criteria for batch release can be based on the calculation of a fixed cutoff value or by calculation of a reference interval (e.g., mean 2 (3) standard deviations) that covers 95.4% (99.7%) of the population. Another approach is to use tolerance intervals. Tolerance intervals can either cover a certain percentage of the population (as is the case with reference intervals) or cover percentiles of the population with a certain probability [26]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
E. Balks et al. / Biologicals 41 (2013) 325e329
Similarity testing of curves is mostly based on a “parallel line assay” evaluating parallelism of the linear portion of the curves or the “4-parameter logistic curve model,” which includes the complete doseeresponse curve (Fig. 4A). Alternatively, reference or tolerance intervals can be calculated based on pilot batches. This approach results in a fixed threshold or a lower and upper acceptance limit for batch release (Fig. 4B). In contrast to the relative potency assay, the use of fixed acceptance limits also requires demonstration that subformulated batches will be reliably detected. Ideally, such substandard batches have been shown to be efficacious in the target animal species but will not pass the batch potency test. In general, there should not be an overlap between the release range and the substandard range. Otherwise, the probability of releasing substandard batches might increase. The difference in the amount of antigen that can be detected depends on the type of vaccine and also on the type of test used to assess potency. Sero-agglutination tests often lead to high variability of test results. This will affect the discriminative power of a test. Thus, suitable dilution factors should be applied to research and development batches when the doseeresponse relationship is investigated. Serovar-specific adjustment of antigen input or additional challenge studies in the target species may be required. 6. Conclusions Direct measurement of vaccine antigens in inactivated vaccines is often difficult because of interference and immunomodulation by the vaccine matrix. Keeping in mind that “potency is a measure of some parameter which has been shown to be related directly or indirectly with efficacy” [18], specific serological methods can be suitable interim solutions for routine batch potency testing of Leptospira vaccines to replace challenge procedures. Several changes have recently been implemented in relevant regulatory documents including the Ph. Eur. [20] to further reduce the number and severity of animal tests. Along with adequate product-specific validation, this will facilitate regulatory acceptance of in vitro assays for new and long-established vaccines. References [1] Ellis WA. Control of canine leptospirosis in Europe time for a change? Vet Rec 2010;167:602e5. [2] Duchow K, Hartmann K, Horzinek M, Lutz H, Straubinger R, Truyen U. Leitlinie zur Impfung von Kleintieren. Available from: http://www.bundestieraerztekammer. de/downloads/btk/leitlinien/Impfleitlinie-Kleintier_2009.pdf; 2009. [3] Dellagostin OA, Grassmann AA, Hartwig DD, Félix SR, da Silva EF, McBride AJA. Recombinant vaccines against leptospirosis. Hum Vaccin 2011;7:1215e24. [4] Goddard RD, Hopkins IG, Thornton DH. The development of a potency test for Leptospira hardjo vaccines: a comparison of protection in calves and serology in guinea pigs. J Biol Stand 1986;14:337e44. [5] Ebert E. Guinea pig serology as an alternative to the hamster challenge test for potency testing of Leptospira hardjo vaccines. Pharmeur Bio 1999;99-2: 102e10.
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[6] Ebert E, Hartinger J, Balks E, Cussler K. Use of the micro agglutination test for replacing the challenge of hamster stipulated for the potency testing of Leptospira hardjo vaccines. ECVAM project, contract no. 12992-97-06 F1ED ISP D, Study 2 2000. [7] Gamberini M, Gómez RM, Atzingen MV, Martins EAL, Vasconcelllos SA, Romero EC, et al. Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis. FEMS Microbiol Lett 2005;244:305e13. [8] Cao Y, Faisal SM, Yan W, Chang YC, McDonough SP, Zhang N, et al. Evaluation of novel fusion proteins derived from extracellular matrix binding domains of LigB as vaccine candidates against leptospirosis in a hamster model. Vaccine 2011;29:7379e86. [9] Zuerner R, Haake D, Adler B, Seegers R. Technological advances in the molecular biology of Leptospira. J Mol Microbiol Biotechnol 2000;2(4):455e62. [10] Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 2009;7:736e47. [11] Monograph 01/2008 0447. Canine leptospirosis vaccine (inactivated). In: European Pharmacopoeia. 7th ed. Strasbourg: Council of Europe; 2012. [12] Monograph 01/2008:1939. Bovine leptospirosis vaccine (inactivated). In: European Pharmacopoeia. 7th ed. Strasbourg: Council of Europe; 2012. [13] Mulla S, Chakraborty T, Patel M, Pandya HP, Dadhaniya V, Vaghela G. Diagnosis of leptospirosis and comparison of ELISA and MAT techniques. Indian J Pathol Microbiol 2006;49(3):468e70. [14] Leptospirosis, chapter 2.1.9. In: OIE terrestrial manual. Available from: http:// www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.09_LEPTO. pdf; 2008. [15] VICH Guideline 1. Validation of analytical procedures: definition and terminology. Available from: http://www.vichsec.org/pdf/gl01_st7.pdf; 1998. [16] VICH Guideline 2. Validation: methodology. Available from: http://www. vichsec.org/pdf/gl02_st7.pdf; 1998. [17] Analytical validation, chapter III. In: Technical guide for the elaboration of monographs. 6th ed. Strasbourg: Council of Europe. Available from: http:// www.edqm.eu/medias/fichiers/technical_guide_for_the_elaboration_of_mon ographs_.pdf; 2011. [18] Hendriksen C, Spieser JM, Akkermans A, Balls M, Bruckner L, Cussler K, et al. Validation of alternative methods for the potency testing of vaccines. Altern Lab Anim 1998;26:747e61. Available from: http://staging-ecvam.jrc.it/ publication/WorkshopReport31.pdf. [19] 01/2008:50300, Chapter 5.3 Statistical analysis of results of biological assays and tests. In: European Pharmacopoeia. 7th ed. Strasbourg: Council of Europe; 2012. [20] Woodland R. European regulatory requirements for veterinary vaccine safety and potency testing and recent progress towards reducing animal use. Procedia Vaccinol 2011;5:151e5. [21] Challa S, Nally JE, Jones C, Sheoran AS. Passive immunization with Leptospira LPS-specific agglutinating but not non-agglutinating monoclonal antibodies protect guinea pigs from fatal pulmonary hemorrhages induced by serovar Copenhageni challenge. Vaccine 2011;29(27):4431e4. [22] EMA/CVMP/IWP/582970/2009. Reflection paper on control of the active substance in the finished product for immunological veterinary medicinal products (IVMPs). Available from: http://www.ema.europa.eu/docs/en_GB/document_ library/Scientific_guideline/2010/03/WC500076617.pdf; 15 March 2010. [23] Chappel RJ, Goris M, Palmer MF, Hartskeerl RA. Impact of proficiency testing on results of the microscopic agglutination test for diagnosis of leptospirosis. J Clin Microbiol 2004;42:5484e8. [24] De Mattia F, Chapsal JM, Descamps J, Halder M, Jarrett N, Kross I, et al. The consistency approach for quality control of vaccines e a strategy to improve quality control and implement 3Rs. Biologicals 2011;39(1):59e65. [25] Flash report on EPAA Workshop, Brussels, April 7th 2011. Available from: http://ec.europa.eu/enterprise/epaa/3_events/3_3_workshops/flash-reportvaccines-workshop-april-2011.pdf. [26] Hanschmann KM. Statistical considerations in setting acceptance criteria. In: EMA Expert workshop on setting specifications for biotechnological products. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/ Presentation/2011/10/WC500115820.pdf; 2011.
Contents lists available at SciVerse ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Short paper
Development and validation of a serological potency test for the release of Leptospira vaccines e Requirements in the European Union Elisabeth Balks*, Heike Gyra, Babett Kobe, Klaus Cussler, Esther Werner Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Veterinary Department, Section Bacterial Vaccines and Immune Sera, Paul-Ehrlich-Institut-Strasse 51-59, 63225 Langen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2013 Received in revised form 19 June 2013 Accepted 22 June 2013
Both European Pharmacopoeia Monograph 01/2008:0447 “Canine Leptospirosis vaccine (inactivated)” and the more recent Monograph 01/2008:1939 “Bovine Leptospirosis vaccine (inactivated)” explicitly allow for a sero-response test to assess batch potency. Test setup and requirements for in vivo and in vitro validation are described. Furthermore, the two main strategies to assess batch potency and their specific demands are addressed. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Leptospira Vaccine Batch potency Alternative method 3R Serology
1. Control of leptospirosis Vaccination against leptospirosis constitutes an important preventive health measure in veterinary medicine. Canine vaccines have been available in Europe since 1961 [1]. In Germany, vaccines against leptospirosis in dogs are considered to be core vaccines [2]. Currently, products from four manufacturers are licensed. So far, most vaccines are bivalent, comprising Leptospira (L.) interrogans serogroups Icterohaemorrhagiae and Canicola. Changes in the epidemiology of canine leptospirosis led recently to the inclusion of L. interrogans serogroup Australis and L. kirschneri serogroup Grippotyphosa. Whereas exposure to most of these serogroups is consistently observed across Europe, the relative importance of serogroups Grippotyphosa and also Sejroe, which is currently not included in vaccines available in Germany, differs according to region. This is due to the fact that infections by serogroups Grippotyphosa and Sejroe result from incidental exposure to strains maintained by rodent hosts, whose distribution and concentration varies across Europe. Infections by serogroup Pomona, which are important in North America, are rare in Europe [1]. Canine vaccines currently available are based on inactivated whole bacteria. All but one vaccine are non-adjuvanted. Leptospires
* Corresponding author. Tel.: þ49 6103 777402. E-mail address: [email protected] (E. Balks).
are grown in media containing rabbit serum and/or bovine serum albumin or serum-free media, inactivated by heat, formalin, thiomersal, or bepropriolactone. In some cases, additional washing and concentration steps by centrifugation are performed. In 2008, a L. borgpetersenii serovar Hardjo (type Hardjobovis) vaccine for cattle was licensed in Germany. This is an adjuvanted whole-cell bacterin.
2. Immunity and protection Immunity to acute leptospirosis is thought to be primarily humoral. In contrast, immunity against L. borgpetersenii serovar Hardjo in bovine reservoirs correlates with T helper 1 cell response. High levels of agglutinating antibodies do not appear to be protective [3]. Nevertheless, several studies could show a clear dosee response of antibody titers in vaccinated guinea pigs [4e6]. The response was related to the degree of protection achieved in calves given graded vaccine doses [4]. In consequence, current alternatives to the hamster challenge test for potency testing of Leptospira vaccines are based on humoral immunity. Due to their potential ability to stimulate heterologous immunity, surface-exposed, constitutively expressed outer membrane proteins specific to pathogenic serovars represent an important focus of current leptospiral research [7,8]. However, findings concerning protectivity evoked by protein antigens appear to be
1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2013.06.011
326
E. Balks et al. / Biologicals 41 (2013) 325e329
Table 1 Batch potency tests outlined in Ph. Eur. monographs on leptospirosis. Monograph 01/2008:0447 Canine Leptospirosis Vaccine (inactivated)
Monograph 01/2008:1939 Bovine Leptospirosis vaccine (inactivated)
Vaccines with or without adjuvants (I): 10 Hamsters for each serovar s.c. vaccination of 5 animals with 1/40 of the dose for dogs, 5 hamsters maintained as controls i.p. challenge 15e20 d p. vacc. Vaccine complies if at least 4 control hamsters die within 14 d of receiving challenge suspension and at least 4 vaccinees remain in good health for 14 d after the death of the controls. Vaccines with or without adjuvants (II): Suitable validated sero-response test for each serovar In-vitro evaluation of individual blood samples to determine the antibody-response to one or more antigenic components which are indicators of protection and which are specific for that serovar Criteria for acceptance set with reference to a batch that has given satisfactory results in dogs Vaccines without adjuvants: Suitable validated in-vitro test for each serovar to determine the content of one or more antigenic components which are indicators of protection and which are specific for that serovar Criteria for acceptance set with reference to a batch that has given satisfactory results in dogs
ambiguous. From studies with single purified whole proteins, combinations thereof, and combinations of recombinant (fusion) proteins, it has been concluded that protective effects are synergistic, reflecting genetic redundancy of leptospires [8,9]. At the same time, there is convincing evidence for the causative role of lipopolysaccharides (LPS) in pathogenicity and their key role in immunity. Studies with monoclonal antibodies have shown that LPS is the target for agglutinating and opsonizing antibodies. Differences in the polysaccharide component, which is the outermost domain of the LPS molecule, account for more than 260 serovars of Leptospira. Consequently, LPS-mediated immunity is mainly serovar specific. It correlates with levels of agglutinating LPS-specific antibodies in transferred sera. LPS-specific monoclonal antibodies passively protect naive animals from leptospirosis [9,10]. Purified LPS can stimulate active immunity [9]. 3. Serological approaches to assess batch potency In the chapter “General principles” of the introduction, the European Pharmacopoeia (Ph. Eur.) states commitment to the reduction of animal use wherever possible. Chapter I, “General notices,” specifies that “. alternative methods of analysis may be used for control purposes, provided that the methods used enable an unequivocal decision to be made as to whether compliance with the
Not fewer than 12 guinea pigs for each serovar 10 Vaccinees, not fewer than 2 controls Blood collection 19e23 d after vaccination Evaluation of antibody titers of individual samples by a suitable validated method such as the MAT Vaccine complies with the test if antibody levels are equal to or greater than those obtained with a batch that has given satisfactory results in cattle.
standards of the monograph would be achieved, if the official methods were used.” In addition, both Ph. Eur. monograph “Canine Leptospirosis vaccine (inactivated)” [11] and the more recent monograph “Bovine Leptospirosis vaccine (inactivated)” [12] explicitly allow for a seroresponse test to assess batch potency (Table 1). Currently available alternatives to the hamster challenge test comprise various ELISA formats aiming at antigen quantification by the use of serovar-specific, LPS-directed monoclonal antibodies. Furthermore, a serological approach is in use, where antibody titers are evaluated by the microscopic agglutination test (MAT). Due to its good specificity, the MAT, which became available as early as 1918 [13], is still regarded as the gold standard for serological diagnosis of leptospirosis [14]. It includes evaluation of paired serum samples and their ability to agglutinate live leptospires. The endpoint is defined as that dilution of serum that shows 50% agglutination, leaving 50% free cells compared with a control culture diluted in phosphate-buffered saline (Fig. 1) [14]. There have been several studies to evaluate the suitability of the MAT for batch potency testing of Leptospira serovar Hardjo vaccines using guinea pigs. Goddard et al., 1986 [4] compared challenge results in calves to guinea pig serology. Cattle and guinea pig serology evaluated by the MAT were re-addressed in an ECVAM research project [5,6]. Important technical findings from these studies were
Fig. 1. Microagglutination of live Leptospira (dark field microscopy, 400) A e no agglutination, B e 50% agglutination, C e 100% agglutination.
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Table 2 Strengths and weaknesses of the MAT as a tool to assess batch potency.
Fig. 2. Procedure to demonstrate accuracy A e validation successful, B e validation not successful x e mean of first set of data, x0 e mean of second set of data, s e standard deviation Accuracy is usually demonstrated in a two-step procedure. In a first step, several trials are performed to fix the specification of the test signal (e.g., mean 2 s). In a second step, several (usually 9) tests are run to demonstrate whether the specification is still met. Validation is successful if the mean value of the results obtained in the second step, including a safety margin (usually the 95% confidence interval), fall completely within the specifications set beforehand. This could be shown in the upper graph (blue interval) but not in the lower graph (red interval), where the mean value but not the complete confidence interval fullfils the validation criteria.
that titers as low as 1:2 can be significant when an animal has been previously negative, and the titer can be attributed to a known input of antigen [4]. Younger guinea pigs (350 g) exhibited a better antibody response than older animals (500 g). Furthermore, old
Strengths
Weaknesses
Good specificity (detection of group- or serovar-specific antibodies, where the serovars have been well described) [14] Evidence for the detection of protective antibodies provided [21] Titers reflect reaction to entire vaccine including immunomodulatory effects of product components; no further vaccine processing required [22] Titers reflect vaccine dose/ vaccination scheme Suitable for nonlethal strains (as compared to protection tests) [4] and for stability testing
Requires animal testing (ethics, costs, time, extrapolation of test data between target and surrogate species) Standardization and transfer difficult (cultivation of strains, read out semiquantitative and subjective) Requires maintenance of live reference strains (risk of cross-contamination, contamination with non-Leptospira spp. or rapid-growing saprophytic leptospires, mislabeling, switching of strains, infection of personnel) [23] No differentiation of IgM and IgG (as compared to ELISA) Might not be suitable for all vaccines. (A multivalent and a multivalent combined vaccine did not induce measurable antibody titers [6]. In this study, 8 vaccines representative for products used in Europe, North America, and Australia/New Zealand were included)
animals appear to include a higher number of nonresponders [6]. General conclusions with regard to the MAT as a tool for batch potency testing of Leptospira Hardjo vaccines are listed in Table 2. As an outcome of their studies, both groups proposed a potency test based on the MAT response of guinea pigs [4,5]. This led to the inclusion of serology in the Ph. Eur. monographs on leptospirosis. At present, the manufacturer of a canine vaccine successfully established a batch potency test utilizing the MAT. The test measures the antibody responses to each of the Leptospira serovars following vaccination of rabbits. 4. Assay validation Current expectations for assay validation are outlined in two complementary International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines [15,16] and also in the Technical Guide for the elaboration of monographs [17]. Specific aspects on the validation of alternative methods have been addressed by Hendriksen et al., in 1998 [18]. Table 3 lists those validation
Table 3 Validation/test validity criteria for routine quality control (Technical guide for the elaboration of monographs, 6th ed. 2011, Chapter III, Analytical validation, modified). Parameter
Fig. 3. Relevance of accuracy and precision A e accuracy, P e precision, “þ” e acceptable, ““ e not acceptable Poor accuracy is characterized by marked deviations of the measured values from the target value. Deviations are of the same nature and occur in the same direction. Precision expresses the closeness of agreement between a series of measurements. Accuracy and precision have not only to be addressed during validation but also need to be surveyed with every test result obtained thereafter.
Specificity Accuracy Precision Linearity Detection limit Quantitation limit Range
Type of test Identity
Purity Quantitative Qualitative
Potency
þ
þ þ þ þ þ þ
þ þ þ þ þ
þ þ
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Table 4 Validation of relevant parameters for potency testing of veterinary vaccines. Parameter
Terminology and validation
Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of other antigenic components, excipients, residuals, or degradants [17]. This is especially important for veterinary vaccines, which are not purified in most cases. For multivalent vaccines, it is essential to test the specificity of the response for each component in the vaccine. In case of a direct correlate to protection, specificity may be shown by passive protection or vaccination/challenge experiments or by sero-response studies. Alternatively, reactivity of vaccinates to immunorelevant or immunodominant epitopes is demonstrated. Mostly, ELISA assays e sometimes in combination with Western blot, agglutination tests or in-vitro growth inhibition tests are used. In the light of the consistency approach, there might be a change of paradigm with regard to the understanding of specificity. Once consistency of production has been shown, testing is then based on in vitro tests which do not have to provide the same information as in vivo tests [24,25]. Accuracy expresses the closeness of agreement between the conventional true value or an accepted reference value and the value found [17]. Examining the accuracy of a method serves to recognize and/or eliminate systematic errors. Accuracy should be assessed by using a minimum of 9 determinations [17]. Accuracy is usually demonstrated in a two-step process by confirming a specification set beforehand (Fig. 2). Less commonly, a validated alternative procedure is used to demonstrate accuracy. Precision expresses the closeness of agreement between a series of measurements obtained from multiple testing of the same homogeneous sample. The rationale of such tests is to recognize and avoid random errors. It is usually expressed as variance, standard deviation, or coefficient of variation. A minimum of 6 determinations at 100% of test concentration is recommended. Data obtained for accuracy may be included [17]. Precision may be considered at 3 levels:
Accuracy (Figs. 2 and 3)
Precision (Fig. 3)
characteristics regarded as the most important for the validation of different types of analytical procedures. Table 4 further details technical aspects of the validation of potency assays. Parameters should be regarded as typical for the analytical procedures, but occasional exceptions should be dealt with on a case-by-case basis [17]. 5. Assessment of potency The principle applied whenever possible throughout the bioassays prescribed in the Ph. Eur. is the comparison of a standard preparation with the batch under test. Bioassays included in the Ph. Eur. have been conceived as dilution assays whenever possible. The batch under test is supposed to contain the same active principle as the standard preparation but in a different ratio of active and inert components [19]. To ensure this requirement is met, it is essential to compare the doseeresponse relationships of the standard and the batch under test. The standard is either a vaccine shown to be efficacious in the target species or a pool serum derived thereof. The latter may have some advantage in terms of the 3Rs because it further reduces the number of animals needed.
p Repeatability/intra-assay precision (same operation conditions over a short interval of time, i.e., results from the same day, same technician) p Intermediate/inter-assay precision (expresses intralaboratory precision, i.e., results from different days, different analysts, different equipment) p Reproducibility (includes comparison of results obtained by different laboratories; usually applied to standardization of methodology [17]) Linearity
Range
Linearity ensures that the test results within a given range are directly proportional to the amount of analyte. Determination of linearity requires measuring the analytes at different concentrations. International guidelines recommend measuring 5 concentrations of the analyte [5]. Other approaches should be justified. To ensure this condition, the batch release value (OD, antigen content, titer) must fall within the linear part of the titration curve. Testing for linearity requires duplicate testing. Ph. Eur. requires regression analysis to demonstrate linearity. In some cases data may need to be subjected to mathematical transformation [19]. The range denotes the interval between the upper and lower concentration of analyte in the sample. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy, and precision when applied to samples containing an amount of analyte within or at the extremes of the analytical procedure [17]. The range is determined by application of the procedure to a series of samples having analyte concentrations spanning the claimed range.
Fig. 4. A. Assessment of batch potency (I). Relative potency: similarity testing of dosee response curves (parallel line assay and 4-parameter logistic curve model) x-axis e dose, y-axis e response. Horizontal shift of curves along the x-axis describes the relative potency of the batch under test (green line) as compared to standard (blue line). The relevant parameter for the “parallel line assay” is parallelism in the linear section of doseeresponse curves (red parallels). Relevant parameters for the “4parameter logistic curve model” are minimal and maximal response, slope at the steepest increase (descent), and the ED50 value. B. Assessment of batch potency (II) Calculation of fixed acceptance criteria x-axis e batches tested, y-axis e parameter scale (example) Acceptance criteria for batch release can be based on the calculation of a fixed cutoff value or by calculation of a reference interval (e.g., mean 2 (3) standard deviations) that covers 95.4% (99.7%) of the population. Another approach is to use tolerance intervals. Tolerance intervals can either cover a certain percentage of the population (as is the case with reference intervals) or cover percentiles of the population with a certain probability [26]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Similarity testing of curves is mostly based on a “parallel line assay” evaluating parallelism of the linear portion of the curves or the “4-parameter logistic curve model,” which includes the complete doseeresponse curve (Fig. 4A). Alternatively, reference or tolerance intervals can be calculated based on pilot batches. This approach results in a fixed threshold or a lower and upper acceptance limit for batch release (Fig. 4B). In contrast to the relative potency assay, the use of fixed acceptance limits also requires demonstration that subformulated batches will be reliably detected. Ideally, such substandard batches have been shown to be efficacious in the target animal species but will not pass the batch potency test. In general, there should not be an overlap between the release range and the substandard range. Otherwise, the probability of releasing substandard batches might increase. The difference in the amount of antigen that can be detected depends on the type of vaccine and also on the type of test used to assess potency. Sero-agglutination tests often lead to high variability of test results. This will affect the discriminative power of a test. Thus, suitable dilution factors should be applied to research and development batches when the doseeresponse relationship is investigated. Serovar-specific adjustment of antigen input or additional challenge studies in the target species may be required. 6. Conclusions Direct measurement of vaccine antigens in inactivated vaccines is often difficult because of interference and immunomodulation by the vaccine matrix. Keeping in mind that “potency is a measure of some parameter which has been shown to be related directly or indirectly with efficacy” [18], specific serological methods can be suitable interim solutions for routine batch potency testing of Leptospira vaccines to replace challenge procedures. Several changes have recently been implemented in relevant regulatory documents including the Ph. Eur. [20] to further reduce the number and severity of animal tests. Along with adequate product-specific validation, this will facilitate regulatory acceptance of in vitro assays for new and long-established vaccines. References [1] Ellis WA. Control of canine leptospirosis in Europe time for a change? Vet Rec 2010;167:602e5. [2] Duchow K, Hartmann K, Horzinek M, Lutz H, Straubinger R, Truyen U. Leitlinie zur Impfung von Kleintieren. Available from: http://www.bundestieraerztekammer. de/downloads/btk/leitlinien/Impfleitlinie-Kleintier_2009.pdf; 2009. [3] Dellagostin OA, Grassmann AA, Hartwig DD, Félix SR, da Silva EF, McBride AJA. Recombinant vaccines against leptospirosis. Hum Vaccin 2011;7:1215e24. [4] Goddard RD, Hopkins IG, Thornton DH. The development of a potency test for Leptospira hardjo vaccines: a comparison of protection in calves and serology in guinea pigs. J Biol Stand 1986;14:337e44. [5] Ebert E. Guinea pig serology as an alternative to the hamster challenge test for potency testing of Leptospira hardjo vaccines. Pharmeur Bio 1999;99-2: 102e10.
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