Laboratory Tests for Live Attenuated Poliovirus Vaccines

827674—BIOL 25/1 (ISSUE)—MS 0060

Biologicals (1997) 25, 3–15

Laboratory Tests for Live Attenuated Poliovirus Vaccines D. J. Wood and A. J. Macadam NIBSC, Blanche Lane, South Mimms, Potters Bar, EN6 5QG, U.K.

Abstract. A new generation of tests to control live attenuated poliovirus vaccines are under development based on major advances in our understanding of the molecular basis of attenuation and reversion to virulence of polioviruses. These include an alternative in vivo neurovirulence test in transgenic mice that express the human poliovirus receptor and a new in vitro test, the MAPREC (mutant analysis by polymerose chain reaction and restriction enzyme cleavage assay, that assesses consistency of production at a molecular level. Excellent progress is being made with both methods but neither is sufficiently developed yet for regulatory use. Critical review of existing control tests shows that the WHO neurovirulence test is well standardized and contributes significantly to the assessment of each batch. On the other hand, the current rct40 test is neither standardized nor particularly informative, though improvements could be made in both areas. The continued relevance of other marker tests such as the d or antigenic marker is doubtful. Potency, identity and thermal stability tests are crucial for control of the final trivalent vaccine. = 1997 The International Association of Biological Standardization

Introduction Worldwide experience has shown that the live attenuated strains of poliovirus developed by Dr Albert Sabin have yielded vaccines that are both immunogenic and safe when orally administered to susceptible children and adults. Although these strains are attenuated, they are potentially capable of reversion to virulence and production of safe and efficacious vaccines has depended in part on consistency of the manufacturing process and in part on laboratory tests of working seed viruses and vaccine lots derived from them. The aim of this review is to examine critically the role of established laboratory tests in assuring the quality of live attenuated poliovirus vaccines. In addition, major advances in our understanding of the molecular basis of attenuation and reversion of polioviruses have resulted in the development of a new generation of tests which are also reviewed. The paper will focus on assays for poliovirus and will not consider the wide range of additional laboratory tests that are applied to each batch of poliovirus vaccine to ensure, for example, freedom from extraneous agents. Polioviruses Polioviruses occur in three serotypes designated types 1, 2 and 3 and are the type members of the 1045–1056/97/010003 + 13 $25.00/0/bg970055

Enterovirus genus in the Picornaviridae family. They are non-lipid containing viruses of about 28 nm diameter with a positive sense RNA genome (Fig. 1).1 Some molecular biological features of the virus relevant to laboratory tests are reviewed later. Cell cultures derived from Old World primates or humans are naturally susceptible to infection with polioviruses. Cell cultures from other species can be made susceptible by transformation with DNA that codes for the human or primate cellular receptor.2,3,4 Humans are believed to be the only natural reservoir of the virus although chimpanzees and many old world primates including rhesus and cynomolgous monkeys can be experimentally infected by a variety of routes. Production of live attenuated poliovirus vaccines The World Health Organization (WHO) defines international standards for the production and control of live attenuated poliovirus vaccines.5 The production procedures are designed to minimize reversion to virulence during manufacture and have been developed from experience gained over many years. A fundamental requirement is that vaccine must be produced using a seed lot system from virus strains shown to be immunogenic and safe in susceptible children and adults. Most 7 1997 The International Association of Biological Standardization

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VPg P3B P3A

VP4 5' non-coding VP2

VPg 1000

VP3 2000

VP1

P2A P2B

3000

Protease Polymerase P3C P3D

P2C

4000

3' non-coding

5000

6000

AAAAAA

7000

Figure 1. Organization of the poliovirus RNA genome which encodes a single polyprotein. Individual products result from cleavage by viral proteases. Serotype-specific immune responses are directed against determinants in the capsid proteins VP1-4. Attenuating determinants have been found in the 5' non-coding, capsid protein and non-structural P3 coding regions (see text).

manufacturers use the Sabin virus strains6 although an alternative type 3 strain, termed Chung III2 , has been used in parts of China.7 The number of passages from master virus seed to vaccine is strictly limited and most type 1 and type 2 vaccines are at the SO + 3 (Sabin original plus three passages) level. Two types of seed are used for type 3 vaccines for historical reasons. Vaccine may be derived from SO seed virus but since some higher passage level vaccine lots had unacceptable neurovirulence levels, they may alternatively be derived from a plaque-purified seed that is inherently less neurovirulent in monkeys.8 This plaque was obtained following transfection of SO + 2 viral RNA, undertaken in order to circumvent potential SV40 contamination, so the master seed is designated as RSO1 and vaccine as RSO3. Three types of cell substrate may be used for production of live attenuated poliovirus vaccines,5 namely primary monkey kidney, human diploid cells (MRC-5 or WI38) and a continuous cell line, Vero, derived from African Green Monkey kidney cells. Crucial production parameters are temperature and duration of incubation. Cultures must be incubated at a constant temperature in the range of 33–35°C and virus must be harvested no later than 96 h after infection. Other important production parameters are cell density and age of cells at inoculation, multiplicity of infection, composition and pH of the culture medium. A virus suspension prepared in a single production run is termed a single harvest. Virus grown in primary monkey kidney or human diploid cells is clarified by filtration to remove cellular debris. Virus grown in Vero cells undergoes additional purification to remove unwanted cellular material.9 Usually, several single harvests of the same poliovirus type are pooled to form a monovalent bulk. The final stage in the manufacturing process is preparation of a trivalent blend using satisfactory

monovalent bulks of each virus type. These are mixed together with the addition of a thermal stabilizer which is most often 1 m magnesium chloride10 but alternative thermal stabilizers such as sucrose or sorbitol are also used. The recommended minimum titres for the Sabin strains are 6.0 log10 TCID50 , 5.0 log10 TCID50 and 5.5 log10 TCID50 per human dose for types 1, 2 and 3, respectively,5 but some countries or agencies require higher titres, especially for the type 3 component. The final vaccine is distributed into vials or plastic tubes with single-dose or, for large vaccination campaigns, multidose presentations. Laboratory tests are applied to both the monovalent bulk and trivalent vaccine. Currently the monovalent bulk is tested for identity, potency, neurovirulence in monkeys, and in vitro consistency, whereas the final trivalent vaccine is tested for identity, potency and thermal stability.

The WHO neurovirulence test Neurovirulence is a complex phenotypic marker and can be experimentally increased or decreased. Tests of neurovirulence by direct inoculation of poliovirus into the central nervous system of susceptible primates were crucial in development of the vaccine strains since they enabled Sabin to compare accurately the neurovirulence of candidate vaccine preparations. Results provided by these tests were critical in the selection of the strains that are now used for vaccine production.11 Certain manufacturing practices are now known to be associated with unacceptable rates of neurovirulence test failures. For example, a significant increase in neurovirulence was observed with high virus passage levels for Sabin type 3 strains12 and some Sabin type 3 seed preparations were found to be more neurovirulent than others.13 Current

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international requirements for poliovirus vaccine production reflect these findings.5 The monkey neurovirulence test is used nowadays to establish that newly produced batches of monovalent bulk show suitably low levels of neurovirulence compared to a reference lot. Historically, a variety of neurovirulence test methods were used but, as this heterogeneity culminated in different decisions being made on the acceptability of some type 3 monovalent bulks in different countries,14 a standardized method was developed by WHO.15 The standard WHO neurovirulence test5 was first introduced in 1982 and requires a new monovalent bulk to be tested concurrently with a homotypic reference preparation. For each preparation a single dose is intraspinally inoculated into monkeys. Even with attenuated strains this causes lesions in target areas of the central nervous system which are histologically scored using a standard grading scale. A lesion score is calculated for each animal and a mean lesion score (mls) is obtained for each preparation. A monovalent bulk passes the test if the difference between mls for the test preparation and reference is not greater than a constant value calculated from pooled within-test variance for each laboratory. There must also be no significant difference in clinical pathogenicity between the preparations. Originally, reference preparations from field trials were used, but these are now exhausted. The WHO Biologicals Unit and the U.S. and Japanese control authorities currently supply different reference preparations to different parts of the world. The WHO neurovirulence test is thus an extremely complex assay. Nevertheless, experience shows that good within-laboratory reproducibility can be achieved as there is little change in acceptance–rejection criteria, which as noted above are based on pooled within-test variance, with time.16 Also, although the mls assigned to preparations by different histopathologists can vary up to two-fold, the between-laboratory differences when two preparations are assessed are considerably less as readers tend to score consistently high or low.17 Probability curves show that there is a 99% chance of rejecting a monovalent bulk with just a two-fold increase in virulence compared to the reference,16 whereas there is a 1% chance of rejecting a batch that is not significantly different from the reference.5 There are some technical problems with the assay. Occasional tests give an unusually high incidence

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of clinical paralysis. This has been ascribed to intercurrent infection for example with measles in the animals during the test,18 which, if undetected, would cause difficulties in evaluation of results. Another difficulty is with batches that give inconsistent results. Repeat tests are allowed in some circumstances,5 yet for Sabin type 1 there are, as yet, no batches that consistently fail. Batches with inconsistent test results are not usually used in the final vaccine but it is debatable, for example, whether they constitute a break in manufacturing consistency. These problems are rare and results from three national control authorities show that certain manufacturers can consistently produce vaccines that pass the WHO neurovirulence test. Thus, over a 12-year period 81/81 type 1 and 34/34 type 2 batches passed the test.14 Some type 3 batches (4/89) failed but manufacturers have progressively switched to a RSO seed that is less neurovirulent in monkeys.13 No RSO-derived type 3 vaccine failed the WHO neurovirulence test in this series. As the incidence of vaccine-associated poliomyelitis in recipients in the three countries that participated in this study is low14 it can be concluded that when consecutive lots of monovalent bulks from a manufacturer consistently pass the neurovirulence test, there is a high level of assurance that final vaccines derived from such lots will be safe.5 However, there is not necessarily a direct correlation between monkey neurovirulence after intraspinal inoculation and human neurovirulence after oral feeding. Indeed, it would be surprising if there were, given that virulence for one host may be different from virulence in another host especially when different routes of inoculation are used.19 Thus, the vaccineassociated cases of poliomyelitis that have occurred in recipients are hardly ever linked to the Sabin type 1 component20,21 yet Sabin 1 is the most virulent serotype in the WHO neurovirulence test as judged by mls14 or paralytogenic activity (DJW, unpublished). Understanding the virological properties assayed by the WHO neurovirulence test and defining more precisely the relationship with human safety are particularly pertinent as new approaches to neurovirulence testing are developed.22 Molecular studies of attenuation and reversion of the Sabin strains have defined some virological factors measured in this assay. High quality epidemiological studies are now required to define better the correlation between monkey and human safety. For example the impact of the change in the type 3

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component from SO-derived to RSO-derived vaccine on rates of vaccine associated poliomyelitis should be studied in several countries. Genetic basis of attenuation and reversion The immunogenicity and safety of Sabin’s live attenuated vaccine strains depend on genetically determined properties such as ability to replicate in the human gut and low neurovirulence. Attenuation of neurovirulence was achieved by multiple passage in primate tissues and cells and attenuated strains were selected largely through tests in animal models of poliomyelitis on the assumption that virulence in monkeys inoculated by the intraspinal route was an exaggerated test of virulence in humans infected by the oral route. The demonstrable safety of Sabin’s strains in humans supports this assumption which also lies at the heart of the current WHO neurovirulence test. The correlation is further supported by the increased neurovirulence for monkeys of revertant strains isolated from the rare cases of vaccine-associated paralysis. Thus, genetic analysis of the attenuation phenotypes of the vaccine strains can be used to identify the determinants of attenuation that are measured in the current neurovirulence test, and possibly the determinants that are important in human vaccinations. The genetic basis of the attenuated phenotype of each vaccine strain has been investigated extensively over the last 15 years. The nucleotide sequences of the three vaccine strains have been determined23 as well as those of neurovirulent progenitor and revertant strains: the type 3 strain has 11 mutations compared to the virulent strain from which it was derived (P3/Leon) and differs from a virulent revertant strain (P3/119) at nine nucleotide positions;24 the type 2 strain differs from a neurovirulent revertant (P2/117) at 23 positions25 and the type 1 vaccine strain differs from its neurovirulent parent (P1/Mahoney) by 55 point mutations.26 Some or all of the genetic differences between attenuated and virulent strains must be responsible for the phenotypic differences. The identification of the principal genetic determinants of attenuation of the Sabin strains has involved a common approach which relies on the infectivity of cloned cDNA copies of the poliovirus genome27 and RNA transcripts derived from them in vitro.28 Hybrid genomes containing parts from attenuated and virulent strains are constructed

using recombinant DNA techniques and viruses are recovered by transfection of permissive cells. These recombinant viruses are assayed for neurovirulence using animal models of poliomyelitis and viruses whose neurovirulence is lower than that of the virulent parental strain are assumed to contain parts of the vaccine strain genome that include determinants of attenuation. Determinants can be narrowed down further by construction of sitedirected mutants. Using this approach, significant determinants of attenuation were found in the 5' non-coding and capsid protein coding regions of the type 3 vaccine strain. In one study, employing recombinants between the Sabin 3 vaccine, P3/Leon parent and P3/119 revertant strains, it was concluded that the attenuated phenotype of Sabin 3 was almost entirely due to a U at base 472 in the 5' non-coding region (ncr) and a U at nucleotide 2034 (resulting in a phenylalanine residue at position 91 of the capsid protein VP3).24 Another study identified a third attenuating residue in the type 3 vaccine strain, namely a C at nucleotide 2493 (giving a threonine at residue 6 of VP1).29 Recombinants between Sabin 2 and either a virulent revertant strain, P2/117, or the mousevirulent strain P2/Lansing were used to identify regions of the vaccine strain genome that attenuated the virulence of P2/117 in monkeys and P2/Lansing in mice.30,31 These studies both concluded that the major attenuating determinants in the type 2 vaccine strain are an A at nucleotide 481 in the 5' ncr and an A at nucleotide 2908 (residue 143 of VP1 is thus isoleucine). The genetic basis of attenuation of Sabin 1 is less clear cut than that of the other two strains, probably because the genome contains a greater number of attenuating mutations, some of which do not influence virulence sufficiently to be detected in all animal models. A study of recombinants between Sabin 1 and P1/Mahoney in primates showed that all genomic regions of Sabin 1, when recombined into a P1/Mahoney strain, contained attenuating determinants, the strongest of which was in the 5' 1122 nucleotides.26 The principal attenuating determinant in this region was identified as a G at nucleotide 480 in the 5' ncr.32 Studies in transgenic mice expressing the human poliovirus receptor (TgPVR mice) using the same constructs again identified determinants of attenuation in all genomic regions of Sabin 1 the strongest of which again appeared to be in the 5' 1122 nucleotides.33 However, these results suggested that the contri-

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bution of nucleotide 480 to neurovirulence may have been overestimated by the previous work. In a study using a different mouse model of poliomyelitis an attenuating determinant was identified at nucleotide 6203 (residue 73 in the polymerase gene 3D) in addition to the G at 480 in the 5' ncr.34 Nucleotide 6203 had previously been implicated in the attenuation of Sabin 1 by a study of nontemperature-sensitive variants in primates35 but subsequent evidence from site-directed mutants of Sabin 1 and Mahoney questioned the significance of the attenuating effect of a C at 6203.36 In this work, which also used the primate model, mutation of the G at 480 in Sabin 1 to the P1/Mahoney-like A resulted in only a slight increase in virulence. Recently, in a study using a different strain of TgPVRmice to that above, no evidence was found of attenuating mutations outside the capsid protein coding region (the contribution of the 5' ncr was not assessed).37 The major determinants in the capsid region were identified as nucleotides 935 (VP4-65), 2438 (VP3-225) and 2795 (VP1-106). In summary, the evidence concerning attenuating determinants in the non-structural protein coding regions of Sabin 1 is contradictory, whereas conclusions about mutations in non-coding regions differ mainly in the degree of attenuation conferred. It is well established that the Sabin strains increase in virulence during replication in the human host38,39,40 and, while these vaccine strains have an excellent safety record, reversion to virulence occasionally results in cases of poliomyelitis in vaccinees or contacts. These occur at an estimated frequency of 1 per 500 000 first doses41 and are mostly due to type 2 and type 3 strains.20 Isolates from these cases, as well as some isolates from healthy vaccinees and tissue-culture variants selected for growth at elevated temperatures, are virulent in animal models of poliomyelitis.30,32,42,43,44 Sequence analysis of viruses from these sources has shed light on the genetic basis of reversion to virulence. Clearly, direct back mutation to the sequence present in the neurovirulent vaccine precursor is sufficient to achieve reversion of attenuating determinants and, indeed, has frequently been observed in revertant strains. In some cases, however, reversion to virulence has occurred in strains that have retained the original attenuating mutations. Phenotypic suppression by secondsite mutation has been observed for three different attenuating determinants so far. This phenomenon was first observed in type 3 isolates that had lost (at least partially) their

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attenuated phenotypes while retaining the Phe at residue 91 of VP3 (U at nucleotide 2034). Several substitutions in the capsid proteins of these strains were identified45 and shown by site-directed mutagenesis to be capable of suppressing the attenuating effect of VP3-91 Phe.42 This residue is also the major determinant of temperature sensitivity of Sabin 345 and loss of the ts phenotype was an inevitable consequence of these second-site mutations.42 In the light of structural data46 and in vitro analysis of the ts defect,47 these observations can be explained by a model in which the Phe at VP3-91 inhibits an early virion assembly step by disrupting subunit interactions and the second-site changes act either by restoring these interactions or improving the efficiency of later assembly steps. Suppression of the G at nucleotide 480 in Sabin 1 can be achieved by a U–C mutation at nucleotide 525.35 This mechanism can be understood in terms of the local RNA secondary structure of the 5' ncr (Fig. 2) which is well characterized.48 In wild-type strains the A at 480 and the U at 525 form a Watson–Crick A–U base pair and in the vaccine strain the G at 480 results in a weakened G–U base pair. The U–C mutation at 525 observed in some revertant strains then acts to restore the basepair strength through formation of a G–C pair. Attenuation of virulence by weakening of this secondary-structure domain (domain V) has been demonstrated by site-directed mutagenesis.48 In some cell lines, weakening of the secondary structure of this domain results in a ts phenotype, the severity of which correlates with the extent of disruption.49 These mutations also decrease in vitro translation efficiency,50 probably as a result of reduced affinity for a translation factor. Neurovirulent revertants of Sabin 2 always lose the Ile residue at VP1-143, having instead one of four different amino acid residues (Val, Thr, Ser, Asn) resulting from mutations at nucleotides 2908 and 2909.30 The valine and threonine residues, at least, result in loss of attenuation in both monkey and TgPVR mouse models of poliomyelitis.30,31 Molecular methods: theoretical considerations The knowledge gained from the work described above has raised the question whether molecular characterization can play a useful, or even central, role in the laboratory testing of poliovaccines. In order to answer this question it is important to decide exactly what are the desirable molecular properties of vaccine lots which, as a consequence

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C C C A

A

G A C· C · G ·A U ·G C ·C C G A A C · G G · C 480G(1) G · C 481A(2) A ·U C · G U G A A C 472U(3) U C C A U G G A G · · · · · · · C· U G G U G C C U C· G C A G A A U· G A C A A C G CG A 525C(1) Figure 2. RNA secondary structure of domain V of the 5' non-coding region of P3/Leon. The structure and, to a lesser extent, the sequence are conserved between serotypes. Arrows show attenuating nucleotide changes found in the three Sabin strains (attenuating bases are in bold followed by the relevant serotype in parentheses; nucleotide positions refer to the sequence of the relevant strain). The U–C mutation at 525 occurs in some revertants of Sabin 1 that retain the G at 480.

of the high error rate of RNA polymerases, are inevitably mixtures of sequence variants. The sequence composition of any population of viruses will depend on the polymerase error rate and the prevalent selection pressures. For poliovirus, the error rate has been estimated at 10−4 to 10−3 per nucleotide51 so in the absence of selection every genome will differ from the consensus sequence at between 0.74 and 7.4 positions, and the minimum level of variation at each position will be between 0.01% and 0.1%. Positive or negative selection would tend to increase or decrease, respectively, these proportions. The basis of any laboratory test would be the monitoring of this sequence variation and the criteria for judging vaccine lots would

include the level of sequence variation at some or all nucleotide positions. This approach has been pioneered by Chumakov and colleagues at CBER in the USA whose assay (MAPREC) is described below. The questions that arise are: which nucleotide positions should be monitored and how much sequence variation can be tolerated? In answer to the latter it is reasonable to propose that levels of variation should not exceed those in vaccine preparations now in use. Methods need to be sufficiently sensitive to detect differences between batches that passed and those that failed the current neurovirulence test. It is not practical to monitor sequence variation at every nucleotide position

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in each genome. Alternative strategies would be to concentrate on the positions at which attenuated and virulent strains differ, or simply those positions implicated in attenuation phenotypes of vaccines by the studies described above. A complete knowledge of the genetic basis of attenuation would seem to be a prerequisite for the last approach, or rather, a complete knowledge of the genetic basis of reversion to virulence. For instance, type 3 strains with capsid mutations that suppress attenuation due to 2034 (VP3-91 Phe) would not be detected if only nucleotides 472, 2034 and 2493 were monitored. Two such strains, constructed or selected in vitro (T7/S3/1001 and SLR1ts+),42 would have failed the monkey neurovirulence test while retaining vaccine-like residues at these three positions. In practice, such strains are unlikely to arise in vaccine batches produced at 34°C since VP3-91 Phe–Ser mutations52 and all suppressor mutations tested (AJM, unpublished) are selected against at this temperature. Indeed, it is has been suggested that it may be sufficient to define the selection pressures in cell culture and then only monitor sequence variation at positions that influence attenuation and are subject to selection. Selection pressures have been studied by extensive passaging, sequencing and MAPREC analysis of vaccine strains.52,53,54 Selection was found to be influenced by incubation temperature, cell substrate and, in some cases, confluence of monolayers, multiplicity of infection (moi) and even other nucleotide positions (the rate of reversion of 472U–C was faster for SO-derived than RSO-derived type 3 vaccines; ref. 55). The mutations selected included some but not all attenuation determinants as well as others such as non-coding changes which were unexpected. The same mutations were sometimes found in different experiments but when multiple replica passages were conducted the rate of selection was variable.55 Greater variability in results was reported for low moi passages. The results of these experiments are informative and help explain correlations between variation at particular positions and monkey neurovirulence (see below). However, it is questionable whether selection pressures can be defined confidently enough to predict variation in view of potential differences or changes in vaccine production conditions. MAPREC assay Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) is a molecu-

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lar biological assay for quantitation of the trace amount of revertant sequence(s) in monovalent bulks.56 The development of this method demonstrates the feasibility of using a new generation of molecular biological assays for the control of live attenuated poliovirus vaccines. MAPREC assays for all three poliovirus serotypes are described.53,54,56 The assay involves extraction of RNA from virions, reverse transcription, amplification of a target stretch of cDNA by PCR, restriction enzyme digestion of the amplified products with enzymes that cut either the attenuated or virulent sequence and quantitation of cut and uncut bands after polyacryclamide gel electrophoresis (usually mutations need to be introduced via the PCR primer in order to generate enzyme recognition sequences). The attenuated phenotype of Sabin 3 involves mutation at nucleotides 472, 2034 and 2493 as described above. MAPREC assays for reversions at each of these bases have been devised. For nucleotide 472, for example, a MboI restriction site is generated in sequences with the reverted 472-C base and a HinFI restriction site in sequences with the attenuated 472-U. The ratio of cut to uncut fragments after MboI digestion gives the proportion of reverted sequences present in the sample.56 Analysis of sequence heterogeneity by MAPREC for poliovirus type 3 preparations, which included commercial monovalent bulks that either passed or failed the monkey neurovirulence test, showed that there was good correlation between MAPREC results at position 472 and the neurovirulence test.57 In one laboratory, all monovalent bulks with less than 0.8% 472-C passed the neurovirulence test while most with greater than 1.0% 472-C failed. No sequence heterogeneity was observed at nucleotide 2034, which suggested that direct back mutation at this position does not contribute to increased neurovirulence of commercial monovalent bulks. Variation at positions of second-site suppressors of 2034 was not measured but the good correlation between 472-C content and neurovirulence test results suggests that reversion by this mechanism does not contribute either. This is consistent with observed selection against these mutations in tissue culture at vaccine production temperatures (AJM, unpublished). The mutation at 2493 was found to be most variable but not to be directly associated with neurovirulence test results in monkeys. Monovalent bulks prepared from the RSO seed contained about 1% U at this position, whereas bulks prepared from the SO seed contained from 60 to 99% U, depending on passage level, (Leon, the wild-type progenitor of

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the Sabin 3 vaccine strain, contains U at position 2493). Levels of 2493-C are thus a good indication of the seed used for vaccine production. Taken together these results suggest that the MAPREC test for 472-C is a very good predictor of the monkey neurovirulence test for Sabin type 3 and suggest that the monkey neurovirulence test may simply measure differences in 472-C content of type 3 vaccines, something more accurately determined by an in vitro assay such as MAPREC. However, it is important to remember that this is only the case because reversion to virulence at other positions such as 2034 does not appear to happen during vaccine production. In view of these very encouraging results the WHO initiated an international collaborative study of MAPREC for poliovirus type 3 to transfer the method from the originating laboratory to other national control laboratories and manufacturers, to establish international reference materials for the assay and to determine whether samples that passed or failed the monkey neurovirulence test could be discriminated by MAPREC in a large group of laboratories. The results of the study were recently discussed at a WHO meeting58 and showed that all objectives were met. Therefore, this method, albeit a complex molecular biological assay, was successfully implemented in many laboratories. The imperative now is to establish for each manufacturer a database of a MAPREC profile of their working seeds, individual harvests and monovalent bulks as a means to monitor consistency of production. With this information it is envisaged that MAPREC could be used as a preliminary test before the monkey neurovirulence test to identify individual harvests or monovalent bulks with excessive amounts of 472-C revertants and so reduce the use of monkeys. MAPREC also offers a tool to monitor the genetic stability of the vaccine at key nucleotides if important production conditions are changed. Further work is however required to develop a standardized assay that is suitable for regulatory decisions and the present state of knowledge does not permit replacement of the neurovirulence test for type 3 poliovirus by MAPREC. The molecular basis of attenuation of Sabin 1 is less well defined than Sabin 3. Nevertheless, MAPREC assays are described to quantitate variation at nucleotides 480 and 525 that form a base pair in the 5' non-coding region as described above53 (Fig. 2). Sequence heterogeneity for the combined positions was found to be in a range of 1 to 3% for

commercially produced monovalent bulks and did not correlate with monkey neurovirulence. Quantitation of reversions at these positions may contribute towards monitoring of manufacturing consistency for Sabin 1 but the value of such results to predict the outcome of the neurovirulence test is less clear. Again, it will be necessary to establish specific mutation profiles for each manufacturer to enable meaningful conclusions to be drawn about manufacturing consistency. Commercially produced monovalent bulks of Sabin type 2 were found to contain 481-G mutations in the range 0.4 to 1.1%54 with no clear correlation between 481-G content and mean lesion score. Viruses passaged under experimental conditions failed the monkey neurovirulence test when the 481-G mutation was present at over 4%. The pattern of accumulation of mutations with cell culture passage differed in primary AGMK and Vero cells which emphasises that application of the methodology to monitor production consistency will depend on comparison with an historic database for each manufacturer. The MAPREC assay therefore offers the opportunity to control live attenuated poliovirus vaccine through monitoring molecular consistency of production. This technique should find considerable application not only for the final monovalent bulks but also for in process testing of single harvests. The method has limitations however in that it assays for direct back mutation at specific nucleotides and, since it may not be possible to predict all possible ways for the Sabin strains to revert to virulence, there will be a continued need for in vivo assays. Transgenic mice that express the human cellular receptor for poliovirus Until recently, primates were the only animal host susceptible to all three poliovirus types. The host restriction is due to the absence of a cellular receptor found on human and primate cells. The genes for both human and primate poliovirus receptors (PVRs) have been identified and isolated.2,3,4 This in turn allowed the establishment of transgenic mouse lines that express the human receptor (TgPVR mice).59,60 Unlike non-transgenic mice, TgPVR mice can be infected with each of the poliovirus serotypes by various routes and develop clinical signs and morphological lesions in the central nervous system similar to those observed in primates. TgPVR mice therefore offer a potential

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new in vivo model for testing poliovirus vaccines.61 A variety of TgPVR mouse lines have been established that differ in genetic background, copy number, insertion site and expression of the transgene in the central nervous system. These factors, particularly expression levels, can influence the sensitivity of TgPVR mice to poliovirus.62 To standardize assay development the WHO initiated an international collaborative study to assess one TgPVR strain, designated TgPVR 21, as a model for neurovirulence testing. This strain was selected as it is the first TgPVR strain to become available as a laboratory animal in sufficient quantities and of standardized genetic and microbiological quality.63 Furthermore, initial WHO studies were restricted to poliovirus type 3 which historically is the most problematic strain to control. The strategy was to develop a test system in TgPVR 21 mice that would distinguish monovalent bulks that passed or failed the monkey neurovirulence test. Intraspinal inoculation of TgPVR 21 mice is necessary to detect the subtle differences between passed and failed batches.64 A standardized intraspinal inoculation technique was tested in the WHO collaborative Study. Results presented at a recent meeting58 showed that the inoculation method was successfully transferred to several laboratories. Furthermore, the majority of mice, unlike monkeys, develop flaccid paralysis and so a clinical dose response occurs.44 Various clinical scoring models, such as severity of disease and time to onset of flaccid paralysis, showed promising results in the first round of the WHO collaborative study and are to be evaluated further. The histological distribution of lesions differs between TgPVR 21 mice and monkeys61,64 since there is less spread in the mice from the inoculation site in the lumbar cord. Nevertheless, it is possible using a modified histological scoring system to distinguish a failed monovalent bulk from a reference virus.58 Again this model is to be evaluated in further rounds of the WHO collaborative study. The TgPVR mouse model is thus at an early but promising stage of development for testing poliovirus type 3. Preliminary results presented at the WHO meeting suggest TgPVR21 mice may also be useful for poliovirus type 2 but less so for poliovirus type 1.58 The key assumption with TgPVR mice is that the factor(s) that increase neurovirulence in primates also increase neurovirulence in TgPVR mice. Evidence is available that mutations that increase virulence of poliovirus type 2 in TgPVR mice31 also do so in primates.30 This may

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also be the case for poliovirus type 344 but requires further rigorous testing. There is evidence for type 1 that mutations selected in TgPVR mice can be different from those selected in primates.37 Full assessment of the validity of the transgenic mouse model therefore requires extensive testing of monovalent bulks that have been previously, or are concurrently, tested in primates. rct40 and other genetic marker tests For many years it has been apparent that a wide variety of in vitro markers will distinguish virulent from attenuated polioviruses. The earliest international requirements for poliovirus vaccine production and control65 specified that each monovalent bulk suspension should be tested for reproductive capacity at supra-optimal temperature (the rct 40 marker)38,66 and at least one other genetic marker. Tests of the antigenic character, d-marker and MS-marker were suggested as suitable additional marker tests. The most recent international requirements5 retain the test for rct40 and suggest that either a test for antigenic character or the d-marker are suitable additional marker tests. The rct40 test is also a pharmacopoeial test of monovalent bulks. The rct40 test quantifies virus replication in cells incubated at 36 2 0.1°C and 40 2 0.1°C. Attenuated viruses replicate poorly at the higher temperature and a monovalent bulk passes the test if the titre at 36°C is at least 5.0 log10 greater than that determined at 40°C. The d(=delayed) marker assay67,68 assesses sensitivity to inhibition by an acid–agar overlay. Attenuated strains give lower titres (by around 3.0–6.0 log10 ) when grown under an agar overlay with a low bicarbonate concentration, which produces an acid overlay, compared to an agar overlay with a normal bicarbonate concentration. A variety of antigenic marker tests exist but none are specified by the requirements. All of these tests can differentiate virulent strains such as the type 1 vaccine progenitor Mahoney from attenuated vaccine strains.38,39,66 As such they can provide a test for identity. With the advent of improved antigenic tests and molecular-based assays that can serve as identity tests the continued relevance of the tests for this purpose must be questionable. They also can provide information on the consistency of manufacture since reversion to virulence of vaccine strains after propagation in vivo or in vitro is accompanied by loss of the rct40 and d markers,38,39 suggesting a common genetic basis for attenuation

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and these markers. This has indeed been found to be true to some extent (see below). However, even from the early days of vaccine production it was recognized they do not necessarily predict neurovirulence for monkeys.38,66 Neither the rct40 nor the d marker test are internationally standardized. There are no international reference reagents, descriptions of the test methodologies provide only outline details and there have been no international collaborative studies of application of the methods to vaccine batches. An international collaborative study of the rct marker test to differentiate vaccine from wild-type diagnostic isolates found that between laboratory variation was a major problem and concluded that the method was not suitable for intratypic differentiation of poliovirus strains.69 Taken together, the lack of predictive power and lack of standardization suggests that continued use of the currently configured tests in poliovirus vaccine control is questionable. Genetic basis of rct40 and d markers Sensitivity of viral growth to elevated temperatures, measured in the rct40 test, is common to all three Sabin vaccine strains, consistent with their derivation at low temperatures. The genetic basis of each phenotype has been studied using recombinant strains and site-directed mutants similar to those used for the analysis of attenuation phenotypes. The major determinant of temperature sensitivity of the type 3 vaccine strain is the 2034-U (VP3-91 Phe).45 Other mutations contribute to the ts phenotype of Sabin 3 including 472-U49 and 2493-C (VP1-6 Thr) (AJM, unpublished observation). Thus, these three positions influence both ts and attenuation phenotypes, though the relative contributions to each are not equal. With respect to VP3-91 Phe suppression of temperature sensitivity by second site mutation also results in reversion to virulence. The U at 472 affects ts in a cell-specific way (see below). The major determinant of temperature-sensitivity of Sabin 2 is the A at 480‘ms70 (Fig. 2). As with 472-U, the precise effect of the 480A varies, depending on the cell line used for assays (see below). Other mutations in the 5' ncr and at unknown positions elsewhere in the genome make minor contributions to the ts phenotype.56 Since 480-A is also a major determinant of attenuation the rct40 test (or a more precise temperature-sensitivity assay) can be a good correlate of attenuation though the influence

of particular cell lines on ts as well as secondsite suppression (see below) must be considered. Furthermore, temperature sensitivity is not influenced by reversion of the attenuating determinant at VP1-143. The type 1 Sabin strain has multiple determinants of both ts and attenuation only some of which are the same. Major contributions to the ts phenotype are made by the attenuating mutation at VP4-6537 and sequences in the 3D pol/3' non-coding regions.34 The latter is due to synergistic effects of at least three positions71 only one of which (3Dpo173) influences attenuation. Other positions contributing to the temperature-sensitivity of Sabin 1 are 480,36 VP3-225 and VP1-134.37 Mutations in 5' ncrs of vaccine strains that weaken the secondary structure of domain V (such as those at 480, 481 and 472 in Sabin 1, 2 and 3, respectively; Fig. 2) influence temperature sensitivity to a different extent in different cell lines.49,70 When such a mutation is the major determinant of temperature sensitivity, as is the case for Sabin 2, the rct40 of the vaccine itself can depend on the cell line used for assay.49 Furthermore, in some cell lines the ts phenotype associated with the 481-A can be suppressed by mutations in the 2A protease gene.72 However, in others, such as L20B cells [mouse L cells expressing the human PVR73 ts assays can detect 481 A : G revertants at levels of approximately 1% (AJM, unpublished). The rct40 is a relatively crude, poorly standardized test. A temperature of 40°C is high, even for some neurovirulent strains, so that vaccines can pass the rct40 test and fail the monkey neurovirulence assay. Some virulent type 2 revertants would pass the rct40 test (AJM, unpublished). However, as described, the majority of attenuation determinants do influence temperature sensitivity in some cells. With this knowledge it should be possible to design assays that are more informative than the current rct40 test. The genetic basis of the d marker has not been studied to such an extent. In Sabin I this genetic marker maps to the genomic region from 1123–366426 which encodes a large part of the capsid proteins and 2A (Fig. 1) and is known to contain some determinants of attenuation. Potency assays and thermal stability The titre of poliovirus is determined in each monovalent bulk to determine the dilution required for the neurovirulence test and for preparing the

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final bulk. The titre of each individual serotype in the final trivalent vaccine is determined to ensure that a minimum infectious dose is present in the vaccine. For the assay of the individual serotypes in the trivalent vaccine it is necessary to use highly specific neutralizing antisera to neutralize two of the three components. The thermal stability test compares the titre of an unheated sample of the trivalent blend in the absence of antibody neutralization (the total virus content) with the total virus titre of a sample heated at 37°C for 2 days. A vaccine batch passes this test if the loss of titre on exposure is not greater than 0.5 log10 TCID50 per human dose.5 The potency of live attenuated poliovirus is determined in an in vitro assay using Hep2C cells. Replicate wells are inoculated with suitable dilutions of virus which results in specific cytopathic effect that can either be detected microscopically or, after suitable staining, macroscopically. The dose response (number of wells with cytopathic effect/ number inoculated) can be statistically treated to yield a tissue culture infective dose (TCID50 ). The assay is apparently simple but is in fact a complex biological reaction between virus, cells, antisera and culture media. International collaborative study has shown that when different assay methods are used between-laboratory variation for identical preparations can be in excess of 10-fold.74 For this reason standard methods are described for titration of monovalent bulks5 and trivalent final vaccine.75 In addition international reference reagents are established for titration of monovalent bulks74 and in 1995 an international reagent, 85/659, was established for trivalent final vaccine (E. Griffiths, personal communication). In an effort to improve further the standardization of this assay, working standard materials are also available. For example, in 1995 the European Pharmacopoeia Commission established a working standard for oral poliovirus vaccine. This reagent can be included in every assay in both national control and manufacturers laboratories. The reagent is intended for validation of assays and is expected to improve between-laboratory agreement. The thermal stability test for trivalent poliovirus vaccine was introduced because of concerns about the relatively low, compared with other vaccines, thermal stability of the vaccine under actual conditions of use.5 The recommended storage temperature for live attenuated poliovirus vaccine is 2–8°C so the test at 37°C is an accelerated degradation test. Some manufacturers have had

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to modify their thermal stabilizer formulations to produce vaccines that consistently pass this test. Therefore it may be expected that the quality of vaccine administered in the clinic will be improved through implementation of this test. References 1. Minor PD. Polioviruses. In Encyclopedia of Virology ISBN 0-12-226960-8, London: Academic Press Ltd, 1994. 2. Mendelsohn CL, Wimmer E and Racaniello VR. Cellular receptor for poliovirus: molecular cloning, nucleotide sequence and expression of a new member of the immunoglobulins superfamily. Cell 1989; 56: 855–865. 3. Koike S, Horie H, Ise I et al. The poliovirus receptor protein is produced both as membrane-bound and secreted forms. EMBO J 1990; 9: 3217–3224. 4. Koike S, Ise I, Sato Y et al. A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain. J Virol 1992; 66: 7059–7066. 5. World Health Organization. Requirements for poliomyelitis vaccine (oral), Technical Report Series 1990; 800: 30–86. 6. Sabin AB and Boulger LR. History of Sabin attenuated poliovirus oral live vaccine strains. J Biol Stand 1973; 1: 115–118. 7. Cooperative research group of poliotype III vaccine. Studies on new attenuated strains of type III live poliomyelitis vaccine. I. The development of a new type III2 attenuated poliovirus. Acta Microbiologia Sinica 1976; 16: 41–47 (In Chinese). 8. Stones PB, MacDonld CR, McDougall JK et al. Preparation and properties of a derivative of Sabin’s type 3 poliovirus strain Leon 12a, b. 10th Symposium of the European Association against poliomyelitis, Warsaw, 1964, pp 390–397. 9. Montagnon BJ. Polio and rabies vaccines produced in continuous cell lines: reality for Vero cell line. Dev Biol Stand 1989; 70: 27–48. 10. Melnick JL, Ashkenazi A, Midulla UC et al. J Am Med Assoc 1963; 185, 406–408. 11. Minor PD. Use of animals in the development and control of viral vaccines. Dev Biol Stand 1996; 86: 113–120. 12. Boulger LR, Marsden SA, Magrath DI et al. Comparative monkey neurovirulence of Sabin type III poliovirus vaccines. J Biol Stand 1979; 7: 97–111. 13. Boulger LR, Magrath DI. Differing neurovirulence of three Sabin attenuated type 3 vaccine seed pools and their progeny. J Biol Stand 1973; 1: 139–147. 14. Furesz J, Contreras G. Some aspects of the monkey neurovirulence test used for the assessment of oral poliovirus vaccines. Dev Biol Stand 1993; 78: 61–70. 15. Magrath DI, Reeve P. On the role of the World Health Organization in the development of Sabin vaccines. Biologicals 1993; 21: 345–348. 16. Contreras G, Furesz J, Karpinski K et al. Experience in Canada with the new revised monkey neuroviru-

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62. Koike S, Taya C, Aoki J et al. Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene—influence of the transgene expression of pathogenesis. Arch Virol 1994; 139: 351–363. 63. Nomura T and Hioki K. Systems for development of transgenic animals as laboratory animals. Scand J Lab Animal Sci, in press. 64. Abe S, Ota Y, Koike S et al. Neurovirulence test for oral live poliovaccines using polio-sensitive transgenic mice. Virology 1995; 206: 1075–1083. 65. World Health Organization. Requirements for poliovirus vaccine (oral). Technical Report Series 1962; 237: 23. 66. Sabin AB, Lwoff A. Relation between reproductive capacity of polioviruses at different temperatures in tissue culture and neurovirulence. Science 1959; 129: 1287–1288. 67. Vogt M, Dulbecco R, Wenner HA. Mutants of poliomyelitis viruses with reduced efficiency of plating in acid medium and reduced neuropathologenicity. Virology 1957; 4: 141–155. 68. Hsuing GD, Melnick JL. Effect of sodium bicarbonate concentration on plaque formation of virulent and attenuated polioviruses. J Immunol 1958; 80: 282–293. 69. Anonymous. Markers of poliovirus strains isolated from cases temporally associated with the use of live poliovirus vaccine: report on a WHO Collaborative study. J Biol Stand 1981; 9: 163–184. 70. Macadam AJ, Pollard SR, Ferguson G et al. The 5' non-coding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature-sensitivity. Virology 1991; 181: 451–458. 71. Georgescu M-M, Tardy-Panit M, Guillot S et al. Mapping of mutations contributing to the temperature sensitivity of the Sabin vaccine strain of poliovirus. J Virol 1995; 69: 5278–5286. 72. Macadam AJ Ferguson G, Fleming T et al. Role for poliovirus protease 2A in cap-independent translation. EMBO J 1994; 13: 924–927. 73. Pipkin PA, Wood DJ, Racaniello VR et al. Characterization of L cells expressing the human poliovirus receptor for the specific detection of polioviurses in vitro. J Virol Methods 1993; 41: 333–340. 74. Magrath DI and Seagroat V. The standardization of infectivity titrations of poliovaccines—a WHO Collaborative study. J Biol Stand 1985; 13: 159–166. 75. World Health Organization. Manual of laboratory methods for testing the potency of final vaccines used in the WHO Expanded Programme on Immunization. 1995; WHO/BLG/95.1.