SPECIAL FOCUS y Pertussis
Review
Whole-cell pertussis vaccine potency assays: the Kendrick test and alternative assays Expert Review of Vaccines Downloaded from informahealthcare.com by Cornell University on 12/15/14 For personal use only.
Expert Rev. Vaccines 13(10), 1175–1182 (2014)
Dorothy Xing, Kevin Markey, Rose Gaines Das and Ian Feavers* Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG, UK *Author for correspondence: Tel.: +44 1707 641450 Fax: +44 1707 641540 [email protected]
Whole-cell pertussis vaccines are still widely used across the globe and have been shown to produce longer lasting immunity against pertussis infection than acellular pertussis vaccines. Therefore, whole-cell vaccines are likely to continue to be used for the foreseeable future. The intracerebral mouse protection test (Kendrick test) is effective for determining the potency of whole-cell pertussis vaccines and is the only test that has shown a correlation with protection in children. Here we review the Kendrick test in terms of international requirements for vaccine potency and critical technical points to be considered for a successful test including test validity, in-house references and statistical analysis. There are objections to the Kendrick test on animal welfare and technical grounds. Respiratory challenge assays, nitric oxide induction assay and serological assays have been developed and have been proposed as possible methods which might provide alternatives to the Kendrick test. These methods and their limitations are also briefly discussed. Establishment of validated in vitro correlates of protection has yet to be achieved. New technical developments, such as genome sequence and the use of gene microarrays to screen responses triggered by vaccine components may also provide leads to alternative assays to the Kendrick test by identifying biomarkers of protection. KEYWORDS: intracerebral mouse protection test • Kendrick test • pertussis • potency assay • whole-cell pertussis vaccine
Whole-cell pertussis vaccines
Whooping cough, caused by the gram-negative bacterium Bordetella pertussis, is an important cause of infant death worldwide and continues to be a public health concern even in countries with high vaccination coverage. Whole-cell pertussis (wP) vaccines have been recommended and widely used for routine vaccination of children and have been used worldwide as part of combined diphtheria, tetanus, pertussis (DTP) vaccine in national childhood immunization programs for decades [1–4]. Although concerns about possible adverse events following their administration led to the adoption of acellular pertussis (aP) vaccines in some countries, wP vaccines are still widely produced and used globally [5]. The wP vaccine is more cost– effective than the aP vaccine which requires extensive purification processes. Moreover, a recent increase in pertussis incidence in countries where aP coverage is high and related
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10.1586/14760584.2014.939636
reports has indicated that wP vaccines provide better and longer-lasting immunity than aP vaccines. Thus, wP vaccines may continue to be used for the foreseeable future [6–9]. wP vaccines are suspensions of killed B. pertussis organisms which are prepared by growing selected strains of B. pertussis under conditions that favor the expression of the virulent Phase I phenotype. Synthetic or semisynthetic culture media have been used in production for many years [10]. Strains are selected to cover the agglutinogen serotypes 1, 2 and 3, now referred to as serotype 1, 2 and 3 fimbriae (Fim). In some cases, a single strain of serotype 1, 2 and 3 is employed but often one or more strains each of serotypes 1, 2 and 1, 3 are used. As strains have not been shown to express Fim 2 or 3 without also expressing 1, it is only necessary to monitor the presence of Fim 2 and Fim 3. These are considered to be equivalent to Fims 2 and 3 and monoclonal antibodies are available for their detection [11,12].
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Various combinations of vaccinations with animal infection models have been used for pertussis vaccine research and development or quality assessment. A comprehensive review on these animal models has recently been published by van der Ark et al. [13]. The routine batch release control test required by regulatory bodies can be different from the assays used for research and development in a number of ways. For example, quantitative results comparing a test vaccine against a reference by statistical calculation to define clear pass/fail end points is essential; the method should be robust, and if not all, at least the majority of regulatory laboratories and vaccine manufacturers should be able to perform the test; it needs to be standardized worldwide and so on. In this article, we focus only on the intracerebral mouse protection (Kendrick) model, which is used as potency test in the routine control and batch release of wP or wP-based combination vaccines. Intracerebral mouse protection (Kendrick) test
wP vaccine prepared from bacteria harvested from cultures on Bordet–Gengou agar or similar media and killed by exposure to heat or various chemical agents have been shown to vary widely in both toxicity and protective efficacy. The results of clinical trials performed in the 1920s and 1930s produced largely inconclusive results in terms of efficacy and demonstrated the need for standardization of the properties of pertussis vaccines [14–16]. A major stumbling block in the development and assessment of pertussis vaccines was the lack of a suitable means, other than clinical trials, for assessing the potency of the vaccine [16]. Leslie and Gardner first demonstrated that use of Phase I B. pertussis is crucial for vaccine efficacy [17], and the molecular mechanism for the loss of virulence factors in later phase B. pertussis was described by Weiss and Falkow [18]. The standardization and control of wP was addressed by Kendrick and Eldering [19–21], who regulated the bacterial cell content in vaccine by measuring opacity and developed a mouse protection assay involving intracerebral challenge (Kendrick test) to control potency. Subsequently, the evidence was published in the 1950s that vaccines shown to protect mice against intracerebral challenge also protected immunized children against whooping cough when they were exposed to the disease at home by an infected sibling. This correlation was the basis for the establishment of the current potency test [22–26], and the Intracerebral Mouse Protection Test became the official potency assay [5,27]. According to the WHO requirements, wP vaccine should have a potency of at least 4.0 International Unit (IU) per single human dose, with lower fiducial limit of at least 2.0 IU as determined in the Kendrick test [28]. The Kendrick test has been in use for many years by control laboratories and manufacturers [5,23,25,29]. The test involves immunizing mice with serial dilutions of the reference vaccine and the test vaccine. At 14–17 days after the immunization, mice are challenged intracerebrally with a dose of B. pertussis (18.323) suspension prepared from a 20–24 h culture grown on Bordet–Gengou agar or other suitable medium. Simultaneously, to obtain an estimate of the LD50 of the challenge suspension dose, dilutions of the challenge dose are injected into control mice by the 1176
intracerebral route. All mice are observed for lethal effects over the next 14 days. The potency of the test vaccine is estimated in terms of IUs of the vaccine standard by parallel line assay after the LD50 of the challenge dose has been determined [5]. Although the potency test has a long record of use, it has often been criticized on the grounds of its reproducibility and the use of a challenge route unrelated to the natural model of infection. However, a WHO proficiency study involving 13 laboratories in 12 countries confirmed that the Kendrick test was effective in distinguishing potent and sub-potent batches of vaccine and gave consistent results both between repeat tests and between different laboratories [30]. The intracerebral challenge route involves localization of bacteria on ciliated cells of the ependymal lining of ventricles, and therefore the model involves ciliated cells like those of the respiratory tract – but at a different location [31,32]. It has been reported that for a vaccine to be protective against intracerebral challenge with B. pertussis, pertussis toxin (PT) is required to enhance the protective activities of other pertussis antigens [33,34]. The similar enhancement effect of PT was also found in an international collaborative study with the modified Kendrick test for aP vaccines [35]. The mechanism of this enhancement was thought to be due to the ability of PT to increase the vascular permeability of the blood–brain barrier which allows the immune response to develop at the site of infection [33,34]. It was also observed that a low concentration of PT acting as a co-factor in combination with other B. pertussis antigens can potentiate the activation of macrophages and this was associated with increased protection not only against the intracerebral challenge but also by the aerosol challenge route of infection [36]. The enhanced protective effect conferred by PT against the intranasal challenge was also observed in another study involving aP [35]. These studies indicate that the effect of PT on increasing the vascular permeability of the blood–brain barrier may not be the only mechanism for the enhanced protection observed. It is well known that in both humans and animals, wP induces different types of immune responses from aP [37,38], and complete bacterial clearance requires cellular immunity mediated by Th1 and Th17 cells [39]. Studies in murine models have shown that innate immune mechanisms involving dendritic cells, macrophages, neutrophils, natural killer cells and antimicrobial peptides help to control the infection. The protective immunity generated by wP appears to be mediated largely by Th1 cells and cell-mediated immune response are likely to play a significant role in protection, whereas alum-adjuvanted aP vaccines induce strong antibody Th2 and Th17 responses [39]. This may be a reason why the original intracerebral mouse protection test is not suitable for aP vaccines. In Japan, China and Korea, aP potency is assessed in a modified intracerebral challenge test which extends the challenge time from 2 to 3 weeks post-immunization to allow antibody development [40–42]. The efficacy of wP vaccine was questioned when one particular vaccine showed lower-than-expected efficacy in two clinical trials in 1990s [43,44]. However, other trials reported higher efficacy for the European wP vaccine [45–47]. Since all these wP vaccines were claimed to have passed the Kendrick test, these Expert Rev. Vaccines 13(10), (2014)
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wP potency assays
reports could be considered a warning sign of differences in both the quality of wP vaccines and the potency tests performed on them. It has been noted that specifications for wP vaccines to pass the intracerebral mouse protection test are not identical worldwide. The vaccines that showed higher efficacy in the clinical trials were produced in Europe and satisfied the WHO/EP requirements with a relative potency estimate of no less than 4.0 IU per single human dose, with a lower 95% fiducial limit of no less than 2.0 IU per single human dose [5,48]. However, the vaccine lots with lower efficacy were produced in North America at that time and presumably passed the US requirements ‘shall have a potency of 12 units per total human immunizing dose’ based on ‘a single test estimate of no less than 8 units’ [49]. It is thus possible that a vaccine that passed the US requirement on the basis of a single test may not pass the WHO/EP test. A similar situation has been described for other vaccines such as tetanus and diphtheria [50]. Furthermore, large variations in almost all animal-based tests are expected. Thus in carrying out this type of bioassay, it is essential to keep the reagents and procedure as consistent as possible and to use mice obtained from the same closely bred stock. Despite such ‘standardization,’ the main difficulties of the Kendrick test are the day-to-day variation in the infective potency of the challenge suspension; the relatively restricted range of dosage of vaccine that can be given; the large day-today variation in ED50 of immunizing dose of the same vaccine and the large number of mice necessary to get a precise potency estimate. The original Medical Research Council trial highlighted several points that should be addressed when carrying out the Kendrick test [23,51]. Results from several more recent international collaborative studies using the Kendrick test have confirmed that the test can be carried out effectively but further emphasize the factors that affect it [30,52–54]. Assay conditions: (immunization doses ED50 , mouse strain & preparation of challenge strains [LD50])
The Kendrick test depends on both the efficacy of the vaccine and the virulence of the challenge strain. The strain 18.323 first used by Kendrick as challenge strain in the test is still in use today because of its exceptionally low intracerebral LD50. The virulence of the challenge strain depends on the growth conditions, for example, age of culture and the culture media. The virulence depends on the number of viable organisms; the proportion of viable organisms at 72 h can be as low as 14% of the value at 24 h [55]. It is also essential to use Phase I virulence bacteria for the challenge [17,18]. The LD50 of the challenge strain varies substantially between assays in the same laboratory and between laboratories and thus requires assessment in each assay. The virulence of the challenge strain may affect the ability of the assay to distinguish between vaccines of different potency [30,54]. Requirements for the strength of the challenge strain are set in WHO recommendations and pharmacopoeial monographs at 100–1000 LD50 [5,48]. Vaccine efficacy in response to challenge can only be determined if the vaccinated mice give a measurable response, that informahealthcare.com
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is, greater than 0% and less than 100% of mice respond to challenge dose, and thus the effective vaccine dose range is limited. Moreover, the ED50 (i.e., vaccine dose at which 50% of mice respond) should be within the vaccine dose range used. The effective vaccine doses are more consistent between assays in the same laboratory, provided the mice are broadly consistent between assays. Nevertheless, the effective vaccine doses may depend on mouse age, weight, sex, strain and other conditions and must be determined in the individual laboratories. These assays are designed to minimize animal use and the effective vaccine doses are typically at fivefold intervals. In-house reference
The between-assay variability of both the LD50 of the challenge dose and the ED50 of the vaccine necessitates the use of a reference standard, in terms of which vaccine potency can be expressed. An International Standard is available, but are not sufficient for use as working reference in all assays; therefore, secondary standards or in-house references are required. The nature and calibration of the secondary standards or in-house references can be a major source of inter-laboratory differences in potency estimation of the same vaccines [30,54]. The calibration of secondary or in-house reference preparations is an important subject and has been described in detail elsewhere [56]. Statistical analysis
Estimation of vaccine potency from the results of these assays requires rigorous statistical analysis. The most widely used method is the parallel line analysis of probit responses of the vaccinated mice [57]. To ensure that, as far as possible, this analysis is appropriate, assays for regulatory purposes are required to meet certain conditions, namely, the ED50 of both the test vaccine and the reference vaccine must be between the largest and smallest vaccine doses; the two dose response lines must be parallel (significant deviations from parallelism less than 5%). If more than one test is carried out, the geometric mean result combined for all valid tests will meet the previous limits [5,48]. Combination vaccine issue
Diphtheria-tetanus-wP (DTwP)-based combined vaccines vary in the amounts of each antigen and the range of antigens according to the intended age range for use. Possible interference between antigens may affect the potency of wP in the combination vaccine and/or affect the performance of the Kendrick test. One example is the observation of suppression of in vivo wP potency in the presence of an inactivated polio vaccine component in the combination vaccine [58]. Important issues for Kendrick assay of wP in combined vaccines include the suitability of the test conditions originally established for monocomponent wP vaccines and the suitability of mono-component wP reference material for use with combined vaccines. There is currently no International Standard or International Reference Preparation specifically designed for such combined vaccines. The use of stable wP international, regional and national reference materials, which have been calibrated against the 1177
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International Standard for assaying the potency of DTwP-based combined vaccines, serve as the starting point for assay standardization. It has practical advantages and should be used whenever possible [59]. However, the qualitative differences in antigen and/ or excipient composition between a monovalent reference preparation and a combined vaccine under test could potentially affect the potency test. A careful evaluation of the slopes of the dose–response lines and/or variability within and between assays should be carried out. The most complex vaccines currently approved in some countries include DTwP-inactivated polio vaccine, DTwP–Hib, DTwP+Hib, DTwP–HepB, DTwP– HepB–Hib and DTwP–HepB+Hib.
the Kendrick test, attempts have been made to develop more acceptable alternatives. However, the development of a reliable simpler alternative to the Kendrick test is still a challenge. Considerable investigations have been carried out on the B. pertussis organism, its toxins, molecular biology and pathogenicity. Human and animal studies have provided more data on immunity, including the role of cell-mediated mechanisms [69–73], which may lead to improved assays or the development of alternatives. Several attempts to develop alternatives are described below. However, a consequence of the decreasing demand for wP for national immunization programs is that progress in this area has been slow.
Issues with Kendrick test
Respiratory challenge test
As mentioned above, the challenge strain 18.323 first used by Kendrick is still in use today in the test and this strain has recently been analyzed by whole-genome sequencing [60]. With the observed increase of pertussis in various vaccinating countries in recent years, antigenic mutations were reported in circulating B. pertussis strains [61–64]. Global transmission of new strains is very rapid and the worldwide population of B. pertussis is evolving in response to vaccine introduction [65]. Data from a comparative genomics study suggested that B. pertussis is evolving in response to vaccination pressure, resulting in expansion of clones carrying new variants of genes encoding immunogenicity and pathogenicity associated antigens [66]. The continued use of an antigenic mismatched or an unrepresentative strain as the challenge strain in the Kendrick test has been questioned. Since antigenic mismatches between pertussis vaccine strains and circulating vaccine-evasive B. pertussis strains may affect the effectiveness of vaccines through escape from immune recognition, the adaptation may also affect strain virulence properties or the biological impact of antigens. Therefore, it would be more desirable to choose a more recently isolated strain as the challenge strain in the protection test as this may reflect the protective effect of the test vaccine against currently circulating strains. Strain 18.323 was chosen by Kendrick at that time because of its exceptionally low intracerebral LD50. To change the 18.323 challenge strain to a more recent isolate in the Kendrick test for use in regulatory batch release control needs careful consideration such as the choice of the new challenge strain, the time of isolation, LD50 and stability and availability of worldwide supply, etc. Extensive evaluation and standardization in an international setting would be required. The Kendrick test has been heavily criticized on the grounds of animal welfare and for being technically demanding [67]. There was a strong recommendation from the European Directorate for the Quality of Medicines/European Centre for the Validation of Alternative Methods consultation in 2005 to use validated humane end points in the Kendrick test [68].
The production of a respiratory tract infection with B. pertussis in mice, using an intranasal or aerosol challenge, has considerable potential for evaluating the protective activity of B. pertussis antigens [74–77]. The murine respiratory challenge model simulates natural B. pertussis infections in humans in several ways including the route of infection, the localization of the bacteria, age dependency of severity of the disease, duration of changes such as leukocytosis, hyperinsulinemia, hypoglycemia and acquired immunity to re-infection. Both the intranasal and aerosol challenge models have been shown to be able to differentiate between highly efficacious and less effective pertussis vaccines [74,75,78,79]. Therefore, it has considerable value for evaluating the protective activity of B. pertussis antigens and has been used extensively for research purposes [75,80,81]. A WHO collaborative study showed that a harmonized method of intranasal challenge could be successfully transferred between laboratories. The results of the study also showed that both the intranasal challenge model and the aerosol challenge model can discriminate between vaccines with different protective capacities. The dose–response relationship for the vaccine tested indicated that, if optimized, these methods would permit the estimation of relative potency [82]. However, at the moment, the intranasal challenge model has the disadvantage that it cannot be used to determine the quantitative relationships between vaccines. This limits the comparisons between laboratories as well as between this and other methods, including the Kendrick test. Further optimization of the intranasal challenge systems to allow the estimation of relative potency is recommended. Although attempts to develop the aerosol challenge model as alternative to the intracerebral mouse protection test for potency assay of wP have showed promising results [37], the limited availability of specialized aerosol equipment may restrict the adoption of the aerosol challenge method [83]. Because of this, the method has not yet been validated by international collaborative study for further vaccines and reference preparations to investigate possible differences in slopes of dose–response lines related to different products [67]. More recently, a baboon model has been developed that offers many advantages; chiefly the ability to investigate pertussis pathogenesis, transmission and host immune responses to infection and vaccination in a primate species that is >96% genetically similar to humans [84–86]. However, while from a
Other novel assays/alternative tests
For the reasons outlined above, it is recognized that a better potency assay is needed and, in addition to modifications of 1178
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wP potency assays
research perspective this model is already providing interesting insights into pertussis disease and vaccination, it is not suitable for the development of a routine potency test for practical reasons, for example, accessibility, cost and ethical acceptability for extensive use.
Review
test for wP vaccines. However, the correlation between the serological response to agglutinins in mice and protection in children, demonstrated as early as the Medical Research Council trials in the 1950s, should be further explored as a potential alternative or a complementary test to the currently recommended potency test [68].
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Nitric oxide & hydrogen superoxide assays
There is now convincing evidence to suggest that B. pertussis can be taken up and survive within macrophages in the lungs and that cell-mediated immunity plays a role in protection. It has been found that murine macrophages can be activated by immunization with wP vaccines to induce nitric oxide (NO) production. An alternative in vitro assay based on the determination of reactive nitrogen intermediates produced as a result of macrophage activation has been reported as an indicator of the whole-cell vaccine protective effect [87,88]. The production of NO by macrophages from mice immunized with wP in response to in vitro re-stimulation with the bacterial antigen was immunization dose-dependent and correlated with protective immunity in vivo as determined by Kendrick test. The results suggested that NO production may serve as a marker of macrophage activation in mice immunized with whole-cell vaccine and could form the basis of a potential protective assay. However, whether the duration of the production of protective levels of NO coincides with that of the protection itself remains to be established. This correlation would strongly support the case for NO as an indicator of protection. Furthermore, wP has been reported to induce a strong Th1/Th17 response [39]. If those relevant markers are proved to be immunization dose dependent, it would warrant further investigation. Serological potency assay
Serological potency tests for wP vaccines in mice and guinea pigs have been reported in a number of publications [89–93]. Such assays have the potential to reduce the overall severity of animal procedures used in assessing the potency of wP vaccines, but, at the moment, there remain a number of unresolved issues. Among these issues, understanding the immunological mechanisms of protection in humans afforded by wP vaccines is particularly important including the value of neutralizing antibodies, the nature of the critical antigens and the role of cell-mediated immunity. There is little evidence for a correlation between serum antibody responses and the protective activity of wP vaccines [5]. Furthermore, as the antigenic composition of each batch of coating bacteria can vary, neither the identity of the antigens to which responses are being measured nor their relevance to protection is known. The serological approach was discussed extensively at an European Directorate for the Quality of Medicines/European Centre for the Validation of Alternative Methods consultation in 2005 where the issue of the relevance of simple antibody measurements to human clinical protection was considered. It was concluded that such tests cannot yet be considered as validated alternatives to the mouse protection potency informahealthcare.com
Expert commentary & five-year view
wP vaccines still remain the only option for a major part of the global immunization programs. The recent increase in rates of pertussis infection in countries with high aP vaccine coverage indicates that the immunity induced by this type of vaccines is not as long lasting as wP vaccine and may encourage more use of whole-cell vaccines. Thus, whole-cell vaccines will continue to be used for the foreseeable future. Further research into the underlying principles of the Kendrick test or the use of information from the modified intracerebral challenge assay for acellular vaccine may ultimately result in the development of a less severe alternative requiring fewer animals. Less severe alternatives to the Kendrick test, such as respiratory challenge assay, are available, but need further optimization and validation. Progress in the development of in vivo and in vitro alternatives is slow because most of the developed countries with resources to engage in bioassay development have switched to aP and therefore lack the incentive to develop new tests for wP. The serological assay has the critical limitation that it measures antibody-binding activity but not functional activity and the relevance of simple antibody binding measurements to human clinical protection is unknown. The intranasal challenge model has gone through a first-phase international collaborative study and can be successfully transferred between laboratories. Also, the ability to distinguish mice vaccinated with an untreated aP and a denatured vaccine has been demonstrated. However, as it stands at the moment, the intranasal challenge model is based on clearance curves over a time period after challenge and is not designed for calculating relative potency to a reference vaccine [82]. Further work on evaluating a reference vaccine, for example, the 4th International Standard for whole cell pertussis vaccine, and immunization dose–responses are needed. This could be done through the next phase of the international collaborative study. The identification of early biomarkers predicting the outcome of adaptive immune mechanisms would be valuable. System immunology data generated by genomic and proteomic studies may point the way to alternative assays in the future [94,95]. Several studies using gene microarrays have already indicated a wide range of responses triggered by exposure to B. pertussis cells [96,97]. Further analysis of the human and animal immune responses to whole-cell vaccination using gene expression arrays or high throughput RNA sequencing together with other techniques, for example, advances in systems biology [98,99], will undoubtedly result in a better understanding of immunity to the whole-cell vaccine, which in turn may lead to the identification of biomarkers for protection and hence the development of alternatives to the Kendrick test. 1179
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Financial & competing interests disclosure
This work was supported by NIBSC. The authors have no other relevant affiliations or financial involvement with any organization or entity with
a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues • A potency assay (that correlates with vaccine efficacy) is essential for quality assurance of whole-cell pertussis (wP) vaccine. The Kendrick test is the only internationally agreed official potency test for batch release control of wP vaccines or wP-based combination vaccines. • wP vaccines shown to meet strictly the WHO requirements for intracerebral challenge assay were efficacious in clinical trials. Suitable standards are critical for this assay. • Assay validation is essential and requires a good appreciation of the assay conditions, especially the choice of mouse strain, the ED50 of Expert Review of Vaccines Downloaded from informahealthcare.com by Cornell University on 12/15/14 For personal use only.
immunization doses and the preparation of challenge strains (LD50). It is important to consider the suitability of the reference materials and the assay conditions originally established for mono-component wP vaccines when applying the Kendrick assay to the evaluation of wP in combined vaccines. • The Kendrick test has proved to be an effective potency test for wP vaccines, but, on the grounds of animal welfare and ease of performance, suitable alternative tests are urgently needed.
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Describes the development of alternatives to Kendrick test and challenges. Mascart F, Verscheure V, Malfroot A, et al. Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses. J Immunol 2003;170(3):1504-9 Giammanco A, Taormina S, Chiarini A, et al. Analogous IgG subclass response to pertussis toxin in vaccinated children, healthy or affected by whooping cough. Vaccine 2003;21(17-18):1924-31 Guiso N, Njamkepo E, Vie le Sage F, et al. Long-term humoral and cell-mediated immunity after acellular pertussis vaccination compares favourably with whole-cell vaccines 6 years after booster vaccination in the second year of life. Vaccine 2007;25(8):1390-7 Schure RM, Hendrikx LH, de Rond LG, et al. T-cell responses before and after the fifth consecutive acellular pertussis vaccination in 4-year-old Dutch children. Clin Vaccine Immunol 2012;19(11):1879-86
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mouse spleen cells via gamma interferon. Infect Immun 1996;64(4):1309-13 89.
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Expert Rev. Vaccines 13(10), (2014)
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Expert Rev. Vaccines 13(10), 1175–1182 (2014)
Dorothy Xing, Kevin Markey, Rose Gaines Das and Ian Feavers* Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG, UK *Author for correspondence: Tel.: +44 1707 641450 Fax: +44 1707 641540 [email protected]
Whole-cell pertussis vaccines are still widely used across the globe and have been shown to produce longer lasting immunity against pertussis infection than acellular pertussis vaccines. Therefore, whole-cell vaccines are likely to continue to be used for the foreseeable future. The intracerebral mouse protection test (Kendrick test) is effective for determining the potency of whole-cell pertussis vaccines and is the only test that has shown a correlation with protection in children. Here we review the Kendrick test in terms of international requirements for vaccine potency and critical technical points to be considered for a successful test including test validity, in-house references and statistical analysis. There are objections to the Kendrick test on animal welfare and technical grounds. Respiratory challenge assays, nitric oxide induction assay and serological assays have been developed and have been proposed as possible methods which might provide alternatives to the Kendrick test. These methods and their limitations are also briefly discussed. Establishment of validated in vitro correlates of protection has yet to be achieved. New technical developments, such as genome sequence and the use of gene microarrays to screen responses triggered by vaccine components may also provide leads to alternative assays to the Kendrick test by identifying biomarkers of protection. KEYWORDS: intracerebral mouse protection test • Kendrick test • pertussis • potency assay • whole-cell pertussis vaccine
Whole-cell pertussis vaccines
Whooping cough, caused by the gram-negative bacterium Bordetella pertussis, is an important cause of infant death worldwide and continues to be a public health concern even in countries with high vaccination coverage. Whole-cell pertussis (wP) vaccines have been recommended and widely used for routine vaccination of children and have been used worldwide as part of combined diphtheria, tetanus, pertussis (DTP) vaccine in national childhood immunization programs for decades [1–4]. Although concerns about possible adverse events following their administration led to the adoption of acellular pertussis (aP) vaccines in some countries, wP vaccines are still widely produced and used globally [5]. The wP vaccine is more cost– effective than the aP vaccine which requires extensive purification processes. Moreover, a recent increase in pertussis incidence in countries where aP coverage is high and related
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reports has indicated that wP vaccines provide better and longer-lasting immunity than aP vaccines. Thus, wP vaccines may continue to be used for the foreseeable future [6–9]. wP vaccines are suspensions of killed B. pertussis organisms which are prepared by growing selected strains of B. pertussis under conditions that favor the expression of the virulent Phase I phenotype. Synthetic or semisynthetic culture media have been used in production for many years [10]. Strains are selected to cover the agglutinogen serotypes 1, 2 and 3, now referred to as serotype 1, 2 and 3 fimbriae (Fim). In some cases, a single strain of serotype 1, 2 and 3 is employed but often one or more strains each of serotypes 1, 2 and 1, 3 are used. As strains have not been shown to express Fim 2 or 3 without also expressing 1, it is only necessary to monitor the presence of Fim 2 and Fim 3. These are considered to be equivalent to Fims 2 and 3 and monoclonal antibodies are available for their detection [11,12].
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Various combinations of vaccinations with animal infection models have been used for pertussis vaccine research and development or quality assessment. A comprehensive review on these animal models has recently been published by van der Ark et al. [13]. The routine batch release control test required by regulatory bodies can be different from the assays used for research and development in a number of ways. For example, quantitative results comparing a test vaccine against a reference by statistical calculation to define clear pass/fail end points is essential; the method should be robust, and if not all, at least the majority of regulatory laboratories and vaccine manufacturers should be able to perform the test; it needs to be standardized worldwide and so on. In this article, we focus only on the intracerebral mouse protection (Kendrick) model, which is used as potency test in the routine control and batch release of wP or wP-based combination vaccines. Intracerebral mouse protection (Kendrick) test
wP vaccine prepared from bacteria harvested from cultures on Bordet–Gengou agar or similar media and killed by exposure to heat or various chemical agents have been shown to vary widely in both toxicity and protective efficacy. The results of clinical trials performed in the 1920s and 1930s produced largely inconclusive results in terms of efficacy and demonstrated the need for standardization of the properties of pertussis vaccines [14–16]. A major stumbling block in the development and assessment of pertussis vaccines was the lack of a suitable means, other than clinical trials, for assessing the potency of the vaccine [16]. Leslie and Gardner first demonstrated that use of Phase I B. pertussis is crucial for vaccine efficacy [17], and the molecular mechanism for the loss of virulence factors in later phase B. pertussis was described by Weiss and Falkow [18]. The standardization and control of wP was addressed by Kendrick and Eldering [19–21], who regulated the bacterial cell content in vaccine by measuring opacity and developed a mouse protection assay involving intracerebral challenge (Kendrick test) to control potency. Subsequently, the evidence was published in the 1950s that vaccines shown to protect mice against intracerebral challenge also protected immunized children against whooping cough when they were exposed to the disease at home by an infected sibling. This correlation was the basis for the establishment of the current potency test [22–26], and the Intracerebral Mouse Protection Test became the official potency assay [5,27]. According to the WHO requirements, wP vaccine should have a potency of at least 4.0 International Unit (IU) per single human dose, with lower fiducial limit of at least 2.0 IU as determined in the Kendrick test [28]. The Kendrick test has been in use for many years by control laboratories and manufacturers [5,23,25,29]. The test involves immunizing mice with serial dilutions of the reference vaccine and the test vaccine. At 14–17 days after the immunization, mice are challenged intracerebrally with a dose of B. pertussis (18.323) suspension prepared from a 20–24 h culture grown on Bordet–Gengou agar or other suitable medium. Simultaneously, to obtain an estimate of the LD50 of the challenge suspension dose, dilutions of the challenge dose are injected into control mice by the 1176
intracerebral route. All mice are observed for lethal effects over the next 14 days. The potency of the test vaccine is estimated in terms of IUs of the vaccine standard by parallel line assay after the LD50 of the challenge dose has been determined [5]. Although the potency test has a long record of use, it has often been criticized on the grounds of its reproducibility and the use of a challenge route unrelated to the natural model of infection. However, a WHO proficiency study involving 13 laboratories in 12 countries confirmed that the Kendrick test was effective in distinguishing potent and sub-potent batches of vaccine and gave consistent results both between repeat tests and between different laboratories [30]. The intracerebral challenge route involves localization of bacteria on ciliated cells of the ependymal lining of ventricles, and therefore the model involves ciliated cells like those of the respiratory tract – but at a different location [31,32]. It has been reported that for a vaccine to be protective against intracerebral challenge with B. pertussis, pertussis toxin (PT) is required to enhance the protective activities of other pertussis antigens [33,34]. The similar enhancement effect of PT was also found in an international collaborative study with the modified Kendrick test for aP vaccines [35]. The mechanism of this enhancement was thought to be due to the ability of PT to increase the vascular permeability of the blood–brain barrier which allows the immune response to develop at the site of infection [33,34]. It was also observed that a low concentration of PT acting as a co-factor in combination with other B. pertussis antigens can potentiate the activation of macrophages and this was associated with increased protection not only against the intracerebral challenge but also by the aerosol challenge route of infection [36]. The enhanced protective effect conferred by PT against the intranasal challenge was also observed in another study involving aP [35]. These studies indicate that the effect of PT on increasing the vascular permeability of the blood–brain barrier may not be the only mechanism for the enhanced protection observed. It is well known that in both humans and animals, wP induces different types of immune responses from aP [37,38], and complete bacterial clearance requires cellular immunity mediated by Th1 and Th17 cells [39]. Studies in murine models have shown that innate immune mechanisms involving dendritic cells, macrophages, neutrophils, natural killer cells and antimicrobial peptides help to control the infection. The protective immunity generated by wP appears to be mediated largely by Th1 cells and cell-mediated immune response are likely to play a significant role in protection, whereas alum-adjuvanted aP vaccines induce strong antibody Th2 and Th17 responses [39]. This may be a reason why the original intracerebral mouse protection test is not suitable for aP vaccines. In Japan, China and Korea, aP potency is assessed in a modified intracerebral challenge test which extends the challenge time from 2 to 3 weeks post-immunization to allow antibody development [40–42]. The efficacy of wP vaccine was questioned when one particular vaccine showed lower-than-expected efficacy in two clinical trials in 1990s [43,44]. However, other trials reported higher efficacy for the European wP vaccine [45–47]. Since all these wP vaccines were claimed to have passed the Kendrick test, these Expert Rev. Vaccines 13(10), (2014)
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wP potency assays
reports could be considered a warning sign of differences in both the quality of wP vaccines and the potency tests performed on them. It has been noted that specifications for wP vaccines to pass the intracerebral mouse protection test are not identical worldwide. The vaccines that showed higher efficacy in the clinical trials were produced in Europe and satisfied the WHO/EP requirements with a relative potency estimate of no less than 4.0 IU per single human dose, with a lower 95% fiducial limit of no less than 2.0 IU per single human dose [5,48]. However, the vaccine lots with lower efficacy were produced in North America at that time and presumably passed the US requirements ‘shall have a potency of 12 units per total human immunizing dose’ based on ‘a single test estimate of no less than 8 units’ [49]. It is thus possible that a vaccine that passed the US requirement on the basis of a single test may not pass the WHO/EP test. A similar situation has been described for other vaccines such as tetanus and diphtheria [50]. Furthermore, large variations in almost all animal-based tests are expected. Thus in carrying out this type of bioassay, it is essential to keep the reagents and procedure as consistent as possible and to use mice obtained from the same closely bred stock. Despite such ‘standardization,’ the main difficulties of the Kendrick test are the day-to-day variation in the infective potency of the challenge suspension; the relatively restricted range of dosage of vaccine that can be given; the large day-today variation in ED50 of immunizing dose of the same vaccine and the large number of mice necessary to get a precise potency estimate. The original Medical Research Council trial highlighted several points that should be addressed when carrying out the Kendrick test [23,51]. Results from several more recent international collaborative studies using the Kendrick test have confirmed that the test can be carried out effectively but further emphasize the factors that affect it [30,52–54]. Assay conditions: (immunization doses ED50 , mouse strain & preparation of challenge strains [LD50])
The Kendrick test depends on both the efficacy of the vaccine and the virulence of the challenge strain. The strain 18.323 first used by Kendrick as challenge strain in the test is still in use today because of its exceptionally low intracerebral LD50. The virulence of the challenge strain depends on the growth conditions, for example, age of culture and the culture media. The virulence depends on the number of viable organisms; the proportion of viable organisms at 72 h can be as low as 14% of the value at 24 h [55]. It is also essential to use Phase I virulence bacteria for the challenge [17,18]. The LD50 of the challenge strain varies substantially between assays in the same laboratory and between laboratories and thus requires assessment in each assay. The virulence of the challenge strain may affect the ability of the assay to distinguish between vaccines of different potency [30,54]. Requirements for the strength of the challenge strain are set in WHO recommendations and pharmacopoeial monographs at 100–1000 LD50 [5,48]. Vaccine efficacy in response to challenge can only be determined if the vaccinated mice give a measurable response, that informahealthcare.com
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is, greater than 0% and less than 100% of mice respond to challenge dose, and thus the effective vaccine dose range is limited. Moreover, the ED50 (i.e., vaccine dose at which 50% of mice respond) should be within the vaccine dose range used. The effective vaccine doses are more consistent between assays in the same laboratory, provided the mice are broadly consistent between assays. Nevertheless, the effective vaccine doses may depend on mouse age, weight, sex, strain and other conditions and must be determined in the individual laboratories. These assays are designed to minimize animal use and the effective vaccine doses are typically at fivefold intervals. In-house reference
The between-assay variability of both the LD50 of the challenge dose and the ED50 of the vaccine necessitates the use of a reference standard, in terms of which vaccine potency can be expressed. An International Standard is available, but are not sufficient for use as working reference in all assays; therefore, secondary standards or in-house references are required. The nature and calibration of the secondary standards or in-house references can be a major source of inter-laboratory differences in potency estimation of the same vaccines [30,54]. The calibration of secondary or in-house reference preparations is an important subject and has been described in detail elsewhere [56]. Statistical analysis
Estimation of vaccine potency from the results of these assays requires rigorous statistical analysis. The most widely used method is the parallel line analysis of probit responses of the vaccinated mice [57]. To ensure that, as far as possible, this analysis is appropriate, assays for regulatory purposes are required to meet certain conditions, namely, the ED50 of both the test vaccine and the reference vaccine must be between the largest and smallest vaccine doses; the two dose response lines must be parallel (significant deviations from parallelism less than 5%). If more than one test is carried out, the geometric mean result combined for all valid tests will meet the previous limits [5,48]. Combination vaccine issue
Diphtheria-tetanus-wP (DTwP)-based combined vaccines vary in the amounts of each antigen and the range of antigens according to the intended age range for use. Possible interference between antigens may affect the potency of wP in the combination vaccine and/or affect the performance of the Kendrick test. One example is the observation of suppression of in vivo wP potency in the presence of an inactivated polio vaccine component in the combination vaccine [58]. Important issues for Kendrick assay of wP in combined vaccines include the suitability of the test conditions originally established for monocomponent wP vaccines and the suitability of mono-component wP reference material for use with combined vaccines. There is currently no International Standard or International Reference Preparation specifically designed for such combined vaccines. The use of stable wP international, regional and national reference materials, which have been calibrated against the 1177
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International Standard for assaying the potency of DTwP-based combined vaccines, serve as the starting point for assay standardization. It has practical advantages and should be used whenever possible [59]. However, the qualitative differences in antigen and/ or excipient composition between a monovalent reference preparation and a combined vaccine under test could potentially affect the potency test. A careful evaluation of the slopes of the dose–response lines and/or variability within and between assays should be carried out. The most complex vaccines currently approved in some countries include DTwP-inactivated polio vaccine, DTwP–Hib, DTwP+Hib, DTwP–HepB, DTwP– HepB–Hib and DTwP–HepB+Hib.
the Kendrick test, attempts have been made to develop more acceptable alternatives. However, the development of a reliable simpler alternative to the Kendrick test is still a challenge. Considerable investigations have been carried out on the B. pertussis organism, its toxins, molecular biology and pathogenicity. Human and animal studies have provided more data on immunity, including the role of cell-mediated mechanisms [69–73], which may lead to improved assays or the development of alternatives. Several attempts to develop alternatives are described below. However, a consequence of the decreasing demand for wP for national immunization programs is that progress in this area has been slow.
Issues with Kendrick test
Respiratory challenge test
As mentioned above, the challenge strain 18.323 first used by Kendrick is still in use today in the test and this strain has recently been analyzed by whole-genome sequencing [60]. With the observed increase of pertussis in various vaccinating countries in recent years, antigenic mutations were reported in circulating B. pertussis strains [61–64]. Global transmission of new strains is very rapid and the worldwide population of B. pertussis is evolving in response to vaccine introduction [65]. Data from a comparative genomics study suggested that B. pertussis is evolving in response to vaccination pressure, resulting in expansion of clones carrying new variants of genes encoding immunogenicity and pathogenicity associated antigens [66]. The continued use of an antigenic mismatched or an unrepresentative strain as the challenge strain in the Kendrick test has been questioned. Since antigenic mismatches between pertussis vaccine strains and circulating vaccine-evasive B. pertussis strains may affect the effectiveness of vaccines through escape from immune recognition, the adaptation may also affect strain virulence properties or the biological impact of antigens. Therefore, it would be more desirable to choose a more recently isolated strain as the challenge strain in the protection test as this may reflect the protective effect of the test vaccine against currently circulating strains. Strain 18.323 was chosen by Kendrick at that time because of its exceptionally low intracerebral LD50. To change the 18.323 challenge strain to a more recent isolate in the Kendrick test for use in regulatory batch release control needs careful consideration such as the choice of the new challenge strain, the time of isolation, LD50 and stability and availability of worldwide supply, etc. Extensive evaluation and standardization in an international setting would be required. The Kendrick test has been heavily criticized on the grounds of animal welfare and for being technically demanding [67]. There was a strong recommendation from the European Directorate for the Quality of Medicines/European Centre for the Validation of Alternative Methods consultation in 2005 to use validated humane end points in the Kendrick test [68].
The production of a respiratory tract infection with B. pertussis in mice, using an intranasal or aerosol challenge, has considerable potential for evaluating the protective activity of B. pertussis antigens [74–77]. The murine respiratory challenge model simulates natural B. pertussis infections in humans in several ways including the route of infection, the localization of the bacteria, age dependency of severity of the disease, duration of changes such as leukocytosis, hyperinsulinemia, hypoglycemia and acquired immunity to re-infection. Both the intranasal and aerosol challenge models have been shown to be able to differentiate between highly efficacious and less effective pertussis vaccines [74,75,78,79]. Therefore, it has considerable value for evaluating the protective activity of B. pertussis antigens and has been used extensively for research purposes [75,80,81]. A WHO collaborative study showed that a harmonized method of intranasal challenge could be successfully transferred between laboratories. The results of the study also showed that both the intranasal challenge model and the aerosol challenge model can discriminate between vaccines with different protective capacities. The dose–response relationship for the vaccine tested indicated that, if optimized, these methods would permit the estimation of relative potency [82]. However, at the moment, the intranasal challenge model has the disadvantage that it cannot be used to determine the quantitative relationships between vaccines. This limits the comparisons between laboratories as well as between this and other methods, including the Kendrick test. Further optimization of the intranasal challenge systems to allow the estimation of relative potency is recommended. Although attempts to develop the aerosol challenge model as alternative to the intracerebral mouse protection test for potency assay of wP have showed promising results [37], the limited availability of specialized aerosol equipment may restrict the adoption of the aerosol challenge method [83]. Because of this, the method has not yet been validated by international collaborative study for further vaccines and reference preparations to investigate possible differences in slopes of dose–response lines related to different products [67]. More recently, a baboon model has been developed that offers many advantages; chiefly the ability to investigate pertussis pathogenesis, transmission and host immune responses to infection and vaccination in a primate species that is >96% genetically similar to humans [84–86]. However, while from a
Other novel assays/alternative tests
For the reasons outlined above, it is recognized that a better potency assay is needed and, in addition to modifications of 1178
Expert Rev. Vaccines 13(10), (2014)
wP potency assays
research perspective this model is already providing interesting insights into pertussis disease and vaccination, it is not suitable for the development of a routine potency test for practical reasons, for example, accessibility, cost and ethical acceptability for extensive use.
Review
test for wP vaccines. However, the correlation between the serological response to agglutinins in mice and protection in children, demonstrated as early as the Medical Research Council trials in the 1950s, should be further explored as a potential alternative or a complementary test to the currently recommended potency test [68].
Expert Review of Vaccines Downloaded from informahealthcare.com by Cornell University on 12/15/14 For personal use only.
Nitric oxide & hydrogen superoxide assays
There is now convincing evidence to suggest that B. pertussis can be taken up and survive within macrophages in the lungs and that cell-mediated immunity plays a role in protection. It has been found that murine macrophages can be activated by immunization with wP vaccines to induce nitric oxide (NO) production. An alternative in vitro assay based on the determination of reactive nitrogen intermediates produced as a result of macrophage activation has been reported as an indicator of the whole-cell vaccine protective effect [87,88]. The production of NO by macrophages from mice immunized with wP in response to in vitro re-stimulation with the bacterial antigen was immunization dose-dependent and correlated with protective immunity in vivo as determined by Kendrick test. The results suggested that NO production may serve as a marker of macrophage activation in mice immunized with whole-cell vaccine and could form the basis of a potential protective assay. However, whether the duration of the production of protective levels of NO coincides with that of the protection itself remains to be established. This correlation would strongly support the case for NO as an indicator of protection. Furthermore, wP has been reported to induce a strong Th1/Th17 response [39]. If those relevant markers are proved to be immunization dose dependent, it would warrant further investigation. Serological potency assay
Serological potency tests for wP vaccines in mice and guinea pigs have been reported in a number of publications [89–93]. Such assays have the potential to reduce the overall severity of animal procedures used in assessing the potency of wP vaccines, but, at the moment, there remain a number of unresolved issues. Among these issues, understanding the immunological mechanisms of protection in humans afforded by wP vaccines is particularly important including the value of neutralizing antibodies, the nature of the critical antigens and the role of cell-mediated immunity. There is little evidence for a correlation between serum antibody responses and the protective activity of wP vaccines [5]. Furthermore, as the antigenic composition of each batch of coating bacteria can vary, neither the identity of the antigens to which responses are being measured nor their relevance to protection is known. The serological approach was discussed extensively at an European Directorate for the Quality of Medicines/European Centre for the Validation of Alternative Methods consultation in 2005 where the issue of the relevance of simple antibody measurements to human clinical protection was considered. It was concluded that such tests cannot yet be considered as validated alternatives to the mouse protection potency informahealthcare.com
Expert commentary & five-year view
wP vaccines still remain the only option for a major part of the global immunization programs. The recent increase in rates of pertussis infection in countries with high aP vaccine coverage indicates that the immunity induced by this type of vaccines is not as long lasting as wP vaccine and may encourage more use of whole-cell vaccines. Thus, whole-cell vaccines will continue to be used for the foreseeable future. Further research into the underlying principles of the Kendrick test or the use of information from the modified intracerebral challenge assay for acellular vaccine may ultimately result in the development of a less severe alternative requiring fewer animals. Less severe alternatives to the Kendrick test, such as respiratory challenge assay, are available, but need further optimization and validation. Progress in the development of in vivo and in vitro alternatives is slow because most of the developed countries with resources to engage in bioassay development have switched to aP and therefore lack the incentive to develop new tests for wP. The serological assay has the critical limitation that it measures antibody-binding activity but not functional activity and the relevance of simple antibody binding measurements to human clinical protection is unknown. The intranasal challenge model has gone through a first-phase international collaborative study and can be successfully transferred between laboratories. Also, the ability to distinguish mice vaccinated with an untreated aP and a denatured vaccine has been demonstrated. However, as it stands at the moment, the intranasal challenge model is based on clearance curves over a time period after challenge and is not designed for calculating relative potency to a reference vaccine [82]. Further work on evaluating a reference vaccine, for example, the 4th International Standard for whole cell pertussis vaccine, and immunization dose–responses are needed. This could be done through the next phase of the international collaborative study. The identification of early biomarkers predicting the outcome of adaptive immune mechanisms would be valuable. System immunology data generated by genomic and proteomic studies may point the way to alternative assays in the future [94,95]. Several studies using gene microarrays have already indicated a wide range of responses triggered by exposure to B. pertussis cells [96,97]. Further analysis of the human and animal immune responses to whole-cell vaccination using gene expression arrays or high throughput RNA sequencing together with other techniques, for example, advances in systems biology [98,99], will undoubtedly result in a better understanding of immunity to the whole-cell vaccine, which in turn may lead to the identification of biomarkers for protection and hence the development of alternatives to the Kendrick test. 1179
Review
Xing, Markey, Das & Feavers
Financial & competing interests disclosure
This work was supported by NIBSC. The authors have no other relevant affiliations or financial involvement with any organization or entity with
a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues • A potency assay (that correlates with vaccine efficacy) is essential for quality assurance of whole-cell pertussis (wP) vaccine. The Kendrick test is the only internationally agreed official potency test for batch release control of wP vaccines or wP-based combination vaccines. • wP vaccines shown to meet strictly the WHO requirements for intracerebral challenge assay were efficacious in clinical trials. Suitable standards are critical for this assay. • Assay validation is essential and requires a good appreciation of the assay conditions, especially the choice of mouse strain, the ED50 of Expert Review of Vaccines Downloaded from informahealthcare.com by Cornell University on 12/15/14 For personal use only.
immunization doses and the preparation of challenge strains (LD50). It is important to consider the suitability of the reference materials and the assay conditions originally established for mono-component wP vaccines when applying the Kendrick assay to the evaluation of wP in combined vaccines. • The Kendrick test has proved to be an effective potency test for wP vaccines, but, on the grounds of animal welfare and ease of performance, suitable alternative tests are urgently needed.
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