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Office of Vaccines Research and Review, Office of Blood Research and Review
Office of the Director, Center for Biologics Evaluation and Research, Food and Drug Adminstration, Bethesda, Maryland
Division of Virology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts, United Kingdom
Because of the highly neurotropic and neurovirulent properties of wild-type mumps viruses, most national regulatory organizations require neurovirulence testing of virus seeds used in the production of mumps vaccines. Such testing has historically been performed in monkeys; however, some data suggest that testing in monkeys does not necessarily discriminate among the relative neurovirulent risks of mumps virus strains. To address this problem, a collaborative study was initiated by the National Institute for Biological Standards and Control in the United Kingdom and the Food and Drug Administration in the United States, to test a novel rat-based mumps virus neurovirulence safety test. Results indicate that the assay correctly assesses the neurovirulence potential of mumps viruses in humans and is robust and reproducible.
Mumps virus is a negative-strand RNA virus in the Paramyxoviridae family that causes a communicable disease characterized by a number of acute inflammatory reactions, including aseptic meningitis. The highly neurotropic nature of mumps virus was established in a landmark study by Bang and Bang in 1943 that demonstrated evidence of invasion of the central nervous system (CNS) by the virus in 65% of cases [1]. Mumps virus remains a leading cause of virus-induced aseptic meningitis and encephalitis in unvaccinated populations [2, 3].
Because of the neurotropic and neurovirulent properties of wild-type mumps viruses, testing of live mumps vaccines for neurovirulence is required by most national regulatory organizations. Such testing has historically been performed in monkeys [4, 5], but results obtained from these tests have raised questions as to whether this assay can reliably discriminate neurovirulent from nonneurovirulent mumps virus strains [69]. The difficulty in evaluating the neurovirulence potential of mumps viruses by use of the monkey-based assay is suggested further by reports of causal links between mumps virus CNS infections and immunization with selected mumps virus vaccines [1014]. This has resulted in the withdrawal from the market of some mumps vaccines, public resistance to vaccination, and, in some countries, complete cessation of national vaccination programs. Thus, as new strains of mumps vaccine continue to be licensed for clinical use, the development of a validated mumps virus neurovirulence safety test with more relevance to human disease risk remains an important international public health objective.
To address this issue, a rat-based neurovirulence safety test was developed at the Center for Biologics Evaluation and Research at the Food and Drug Administration (FDA), and preliminary testing suggested that the model was capable of assessing the neurovirulence potential of mumps virus in humans [15]. To better evaluate the performance of the rat-based neurovirulence safety test, an interlaboratory collaborative study was initiated between the FDA and the National Institute for Biological Standards and Control (NIBSC) in the United Kingdom. For the present study, 12 mumps virus preparations of varying neurovirulence potential in humans were blinded and distributed, along with a standard operating procedure for performing the rat-based neurovirulence safety test, to each laboratory. At the conclusion of the study, the predictive value of the test for the assessment of the neurovirulence potential of these mumps viruses in humans was measured, and statistical analyses were performed to evaluate the comparability of the results between the 2 laboratories.
MATERIALS AND METHODS
Mumps viruses.
Twelve mumps virus preparations were used in the present study and are described in table 1. All were divided into 0.5-mL aliquots, kept at -80°C, and shipped on dry ice. The identity of the virus strains was blinded throughout the experimental phase of the study and was revealed at the conclusion of the study, for statistical analysis.
Virus content determination.
Virus concentrations were determined by plaque assay. Briefly, 1 aliquot of each virus was thawed and diluted in serial 10-fold increments, from 10-2 to 10-6, by use of MEM that contained 2% fetal bovine serum (FBS; Quality Biologicals) as the diluent. Each dilution was incubated in duplicate in 12-well plates that contained 12-dayold confluent Vero cell monolayers. Plates were incubated at 37°C and in 5% CO2 for 1 h. Viral inocula were removed, and wells were overlaid with a 42°C 1-mL mixture of equal parts Agar Nobel Gel 1.5% (Quality Biologicals) and 2× MEM that contained 10% FBS. Plates were incubated at 37°C and in 5% CO2 for 5 days. Plates were stained following the addition of a second layer of agar overlay mixture containing 0.02% neutral red (Sigma). Plaques were counted the following day. The assays at the NIBSC were not performed under the agar overlay mixture; instead, they were performed with an overlay medium containing 0.8% carboxymethyl cellulose (NBS Biologicals). The cell sheet was then stained with methyl violet after 7 days of incubation. The virus titer was determined by calculation of the mean number of plaques for each pair of duplicate wells and multiplication by the inverse of the dilution factor and volume plated. Observed titers are shown in table 2. Each laboratory determined the inoculum dose on the basis of observed titers.
Inoculation of rats.
Pregnant Lewis rats were purchased from either Harlan Sprague Dawley (FDA) or Bicester (NIBSC). Two to 3 litters of 1-day-old rats were inoculated with 100 pfu of virus in a 10-L volume of MEM by use of a 27-gauge needle. Each litter consisted of 810 pups. The inoculation site was in the left parietal area of the skull, 2 mm left of midline and midway between the bregma and lambda. On day 25 after inoculation, all rats were euthanized by CO2 asphyxiation. Brains were removed and were fixed in 10% neutral buffered formalin for 1 week. Institutional guidelines for the care and use of laboratory animals were strictly followed.
Brain processing.
Fixed brains were cut in half in the sagittal plane along the anatomical midline. To each brain hemisphere, a second cut was made in the same plane 35 mm from the first cut. The resulting 35-mmthick sections of brain tissue from each rat were placed in a cassette, were dehydrated by use of a graded series of ethanol baths (70%, 85%, 95%, 100%, 100%, and 100%) in which they were placed for 1 h in each bath, and were immersed in xylene for 2 h. Dehydrated tissues were then immersed in paraffin at 60°C for 2 h and were then mounted on a microtome with the side closest to anatomical midline facing the knife. A single 10-m-thick section was obtained from each hemisphere at a depth of 0.51.0 mm from the surface. Paraffin-embedded tissue sections were placed on glass slides, warmed overnight, rehydrated, stained with hematoxylin-eosin (H-E), and dehydrated, and cover slips were attached with Permount (Fisher Scientific).
Statistical analysis.
After all testing was completed, data were unblinded. The raw neurovirulence scores, being percentages, were subjected to arcsin transformation, for analysis of values and rank order. A comparison between laboratories was done by the Tukey honestly significant difference (HSD) test, and P = .05 was considered to be significant; rank was evaluated by use of the Wilcoxon 2-sample test. An analysis of variance (ANOVA) was then used to evaluate the ability of each laboratory to discriminate among 3 groups of virus strains: attenuated vaccine virus strains, partially attenuated vaccine virus strains, and wild-type virus strains. Members of these 3 groups are listed in table 1. In the first analysis, members of the attenuated and partially attenuated groups were market products only, and therefore, for the purposes of the analysis, were considered to be the "conservative" group. In the second analysis, members of the attenuated and partially attenuated groups included market products, as well as laboratory-passaged vaccine virus strains and clinical isolates (vaccine virus strains isolated from patients with mumps vaccineassociated meningitis after vaccination with Pluserix or Trivirex Urabe-AM9containing vaccines) and were considered to be the "expanded" group. A nested model was used in which specimens were nested within a group.
RESULTS
An ANOVA of the neurovirulence scores represented by these 3 conservative groups demonstrated that, at both laboratories, attenuated strains could be distinguished from partially attenuated strains, and partially attenuated strains could be distinguished from wild-type strains (P < .001). Group comparisons were then performed by use of the Tukey HSD test (P = .05). For each laboratory, all 3 pairwise comparisons were significant (table 3).
In the second analysis, members of the attenuated and partially attenuated groups consisted of market products, as well as laboratory-passaged vaccine virus strains and clinical isolates (the expanded group in table 1). When these groups were used in the analysis, the results were similar to those for the conservative group, in that the ANOVA of the neurovirulence scores represented by these groups demonstrated that, at both laboratories, attenuated strains could be distinguished from partially attenuated strains, and partially attenuated strains could be distinguished from wild-type strains (P < .001). Group comparisons performed by use of the Tukey HSD test (P = .05) also found that, for each laboratory, all 3 pairwise comparisons were significant (table 4).
DISCUSSION
The standard practice for assessment of the neurovirulence risk of mumps virus vaccines in humans is to test either the virus seed stock or the first few production lots of vaccine in monkeys. Data from 2 recent independent studies showed that mumps virus neurovirulence testing as currently performed in monkeys did not discern with statistical certainty the relative neurovirulence risk of many mumps virus strains in humans [6, 8]. However, preliminary test results of a novel rat-based neurovirulence safety test demonstrated correct identification of the relative neurovirulence risk of various mumps virus strains in humans [15]. These results suggested that the rat-based neurovirulence safety test might be a more predictive assay than the currently recommended monkey-based neurovirulence safety test.
Results of the present study show that, although the neurovirulence scores differed by laboratory, the overall rank order of the scores was the same. Further, in both laboratories, wild-type viruses could be differentiated with statistical certainty from vaccine viruses, and attenuated vaccine viruses could be differentiated with statistical certainty from partially attenuated vaccine viruses.
That the neurovirulence scores measured for each virus differed by laboratory was not unexpected. Because the scores were determined by use of a biological assay, the absolute neurovirulence scores for any given virus preparation tested in the rat-based neurovirulence safety test will differ from study site to study site, even if all procedures are executed in identical fashion using identical reagents. However, it should be noted that there were some differences in execution between the 2 laboratoriesnamely, use of different virus doses, rat breeding colonies, and brain area measurement methods. Of these, virus dose is likely to be the major contributor to the observed interlaboratory differences in neurovirulence scores, as is indicated by results of a study published elsewhere [15]. The use of different virus doses at the 2 laboratories was a result of virus potency being determined at each test site (see table 2). Fixed virus titers were not preassigned for common use, because product manufacturers and regulatory agencies that use this test will likely prepare material for inoculation according to their own potency determinations. However, in an effort to reduce interlaboratory differences in potency determinations, in future assays we plan to include a standard preparation of a positive control sample that would be assigned a defined infectivity titer. In this way, potency values obtained at each study site can be adjusted up or down, according to the performance of the standard in the potency test.
Both brain area measurement methods used in the present study were found to yield comparable results, according to an interim analysis of blinded samples (data not shown), and were therefore not likely to affect test outcome. Notably, the standard operating procedure for the assay does not specify the method to measure area; any reasonable approach to the quantitation of the ratio of the cross-sectional area of the lateral ventricle to the cross-sectional area of the entire brain should be acceptable. Whether the use of different breeding colonies of Lewis rats played a significant role in the different neurovirulence scores obtained at the 2 laboratories is not known. Although inbred rat strains differ in their susceptibility and reaction to infection by some viruses, depending on their breeding histories [16, 17], preliminary data from testing different breeding lines of Lewis rats (from Harlan Sprague Dawley and Charles River Laboratories) with the same mumps virus stocks indicated no differences in neurovirulence scores (data not shown).
To assure better control for intraassay and interassay variability, reference viruses are needed for use in the assay. Thus, for future validation studies, distribution of a "low" virulence reference virus and a "high" virulence reference virus will be needed. An acceptable range in neurovirulence scores for these references will be established for use in the validation of assay performance. In addition, the neurovirulence of a test virus should not be determined according to its neurovirulence score per se, but, rather, according to its performance relative to that of the low virulence reference virus, by use of an appropriate confidence interval.
In summary, on the basis of these initial validation studies, the rat-based neurovirulence safety test for mumps virus vaccine appears to be a robust, reproducible, and predictive test. Additional modifications are needed, such as the establishment of reference viruses and testing of other mumps vaccinessuch as the Leningrad-3based vaccine strains, which have been associated with reports of meningitis in vaccine recipients [10]. Further validation of the rat-based neurovirulence safety test through larger studies by additional independent laboratories are required to provide a basis for the adoption of this assay by regulatory authorities and vaccine manufacturers as a replacement for the monkey-based test for the assessment of the neurovirulence potential of candidate mumps vaccine virus strains in humans.
References
1. Bang HO, Bang J. Involvement of the central nervous system in mumps. Acta Med Scand 1943; 113:487505. First citation in article
2. Aygun AD, Kabakus N, Celik I, et al. Long-term neurological outcome of acute encephalitis. J Trop Pediatr 2001; 47:2437. First citation in article
3. Johnson RT. Viral infections of the nervous system. 2nd ed. Philadelphia: Lippincott-Raven, 1998. First citation in article
4. Mumps virus vaccine live. [CFR 21], parts 600799. Washington, DC: Office of the Federal Register, National Archives and Records Administration. Code of Federal Regulations, 1996; 63050, 1067. First citation in article
5. World Health Organization. Requirements for mumps vaccine. World Health Organ Tech Rep Ser 1994; 840:12136. First citation in article
6. Afzal MA, Marsden S, Hull RM, Pipkin PA, Bently ML, Minor PD. Evaluation of the neurovirulence test for mumps vaccines. Biologicals 1999; 27:439. First citation in article
7. Buynak EB, Hilleman MR. Live attenuated mumps virus vaccine. 1. Vaccine development. Proc Soc Exp Biol Med 1966; 123:76875. First citation in article
8. Rubin SA, Snoy P, Wright KE, et al. The mumps virus neurovirulence safety test in rhesus monkeys: a comparison of mumps virus strains. J Infect Dis 1999; 180:5215. First citation in article
9. Sassani A, Mirchamsy H, Ahourai SP, et al. Development of a new live attenuated mumps virus vaccine in human diploid cells. Biologicals 1991; 19:20311. First citation in article
10. Arruda WO, Kondageski C. Aseptic meningitis in a large MMR vaccine campaign (590,609 people) in Curitiba, Parana, Brazil, 1998. Rev Inst Med Trop Sao Paulo 2001; 43:3012. First citation in article
11. Balraj V, Miller E. Complications of mumps vaccines. Rev Med Virol 1995; 5:21927. First citation in article
12. Brown EG, Furesz J, Dimock K, Yarosh W, Contreras G. Nucleotide sequence analysis of Urabe mumps vaccine strain that caused meningitis in vaccine recipients. Vaccine 1991; 9:8402. First citation in article
13. Ki M, Park T, Yi SG, Oh JK, Choi B. Risk analysis of aseptic meningitis after measles-mumps-rubella vaccination in Korean children by using a case-crossover design. Am J Epidemiol 2003; 157:15865. First citation in article
14. Odisseev H, Gacheva N. Vaccinoprophylaxis of mumps using mumps vaccine, strain Sofia 6, in Bulgaria. Vaccine 1994; 12:12514. First citation in article
15. Rubin SA, Pletnikov M, Taffs R, et al. Evaluation of a neonatal rat model for prediction of mumps virus neurovirulence in humans. J Virol 2000; 74:53824. First citation in article
16. Bender K, Balogh P, Bertrand MF, et al. Genetic characterization of inbred strains of the rat (Rattus norvegicus). J Exp Anim Sci 1994; 36:15165. First citation in article
17. Ritter M, Bouloy M, Vialat P, Janzen C, Haller O, Frese M. Resistance to Rift Valley fever virus in Rattus norvegicus: genetic variability within certain "inbred" strains. J Gen Virol 2000; 81:26838. First citation in article
18. Hilleman MR, Buynak EB, Weibel RE, Stokes J. Live, attenuated mumps-virus vaccine. N Engl J Med 1968; 278:22732. First citation in article
19. Usonis V, Bakasenas V, Kaufhold A, Chitour K, Clemens R. Reactogenicity and immunogenicity of a new live attenuated combined measles, mumps and rubella vaccine in healthy children. Pediatr Infect Dis J 1999; 18:428. First citation in article
20. Afzal MA, Pickford AR, Forsey T, Heath AB, Minor PD. The Jeryl Lynn vaccine strain of mumps virus is a mixture of two distinct isolates. J Gen Virol 1993; 74:91720. First citation in article
21. Miller E, Goldacre M, Pugh S, et al. Risk of aseptic meningitis after measles, mumps and rubella vaccine in UK children. Lancet 1993; 341:97982. First citation in article
22. Afzal MA, Yates PJ, Minor PD. Nucleotide sequence at position 1081 of the hemagglutinin-neuraminidase gene in the mumps Urabe vaccine strain. J Infect Dis 1998; 177:2656. First citation in article
23. Brown EG, Dimock K, Wright KE. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J Infect Dis 1996; 174:61922. First citation in article
24. Amexis G, Rubin S, Chatterjee N, Carbone K, Chumakov K. Identification of a new genotype H wild-type mumps virus strain and its molecular relatedness to other virulent and attenuated strains. J Med Virol 2003; 70:2846. First citation in article
25. Afzal MA, Buchanan J, Heath AB, Minor PD. Clustering of mumps virus isolates by SH gene sequence only partially reflects geographic origin. Arch Virol 1997; 142:22738. First citation in article