Neurovirulence safety testing of mumps vaccines—Historical perspective and

Vaccine29 (2011) 2850–2855Contents lists available at ScienceDirectVaccinej o u r n a l h o m e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/v a c c i neReviewNeurovirulence safety testing of mumps vaccines—Historical perspective and current statusS.A.Rubin a,∗,M.A.Afzal b,1a Center for Biologics Evaluation and Research,United States Food and Drug Administration,Bethesda,MD20892,USAb NIBSC,South Mimms,Potters Bar,Hertfordshire,EN63QG,UKa r t i c l e i n f oArticle history:Received22December2010Received in revised form2February2011 Accepted3February2011Available online 18 February 2011Keywords:MumpsVaccineNeurovirulenceSafetyMonkeys a b s t r a c tMany live,attenuated viral vaccines are derived from wild type viruses with known neurovirulent properties.To assure the absence of residual neurotoxicity,pre-clinical neurovirulence safety testing of candidate vaccines is performed.For mumps virus,a highly neurotropic virus,neurovirulence safety testing is performed in monkeys.However,laboratory studies suggest an inability of this test to correctly discern among virus strains of varying neurovirulence potential in man,and,further,some vaccines found to be neuroattenuated in monkeys were later found to be neurovirulent in humans when administered in large numbers.Over the past decade,concerted efforts have been made to replace monkey-based neurovirulence safety testing with more informative,alternative methods.This review summarizes the current status of mumps vaccine neurovirulence safety testing and insights into models currently approved and those under development.Published by Elsevier Ltd.Contents1.Introduction (2850)2.Neurovirulence safety testing in monkeys (2851)3.Neurovirulence safety testing in small animal models (2851)4.Neurovirulence safety testing in marmosets (2851)5.Non-animal based assessment of mumps virus neurovirulence (2853)6.Perspectives and future directions (2853)Acknowledgements (2853)References (2854)1.IntroductionMumps is an acute communicable respiratory viral infection transmitted by oropharyngeal secretions.Symptoms are usually the result of secondary infection of tissues and organs following viremia,resulting in a wide array of acute inflammatory reactions. While most symptoms such as parotitis and orchitis are relatively benign,more serious reactions,such as meningitis and encephali-tis can occur[1,2].The high tropism of mumps virus for the central nervous system(CNS)was suggested by a study of a mumps epi-demic in Copenhagen in the winter of1941/42.Of255individuals presenting with uncomplicated parotitis on whom routine lum-∗Corresponding author.Tel.:+13018271974;fax:+13014805679.E-mail address:steven.rubin@(S.A.Rubin).1Current address:265-Mutton Lane,Potters Bar,Hertfordshire,EN62AT,UK.bar puncture was performed,cerebrospinalfluid pleocytosis was identified in129(51%)[3].Although symptomatic CNS infection is less frequent,occurring in approximately1–10%of cases[4,5], in the pre-vaccine era mumps virus was the leading cause of virus-induced aseptic meningitis and encephalitis in developed countries [6],a statistic maintained in countries lacking strict mumps vacci-nation programs[7–10].Given the neurotropic and neurovirulent properties of mumps virus,it is critical that candidate live-attenuated vaccine strains be assessed for neuroattenuation.The only approved method for such an assessment is based on intracerebral inoculation of mon-keys[11,12];however,the reliability of this method has been questioned following the occurrence of aseptic meningitis in recip-ients of vaccines passing monkey neurovirulence safety testing [13,14].Such a problem places public confidence in all mumps vaccines at risk,as indicated by the experience in Japan where national mumps vaccination programs were discontinued in19930264-410X/$–see front matter.Published by Elsevier Ltd. doi:10.1016/j.vaccine.2011.02.005S.A.Rubin,M.A.Afzal/Vaccine29 (2011) 2850–28552851following established links to aseptic meningitis;consequently, more than a million new mumps cases occur annually in that country[15,16].The recent resurgence of mumps outbreaks world-wide highlights the need for maintenance of robust immunization programs,and,hence,the importance of assuring the safety of mumps vaccines[17–26].In this article we review the avail-able data on mumps virus vaccine neurovirulence safety testing, including promising alternatives to the current monkey-based model.2.Neurovirulence safety testing in monkeysThe use of a monkey model is presumably based on the notion that non-human primates,due to their close relatedness to humans, would be appropriate hosts for mumps virus replication and patho-genesis.Indeed,monkey neurovirulence safety testing of some vaccines,including poliovirus and yellow fever virus,has been suc-cessful[27,28];however,this is not the case with mumps virus.The major criteria by which mumps virus neurovirulence is assessed in monkeys includes inflammation within and damage to the ven-tricular system,specifically the choroid plexus,ependymal cell lining and cells in the subependymal zone following inoculation of the CNS[29,30].In regions of extensive inflammation and nearby areas(e.g.,the hippocampal cortex and corpus collosum),dys-trophic neurons can occasionally be seen and are also considered in the analysis[29,31,32].The array of histopathological changes in monkeys following inoculation of different mumps viruses by several routes is provided in Table1,indicating that virus-induced neuropathology in monkeys appears to be poorly predictive of the attenuation phenotype of mumps viruses for humans.For example,the Jeryl Lynn vaccine strain,which has an excellent safety record and has not been causally associated with aseptic meningitis in humans[33–35]is histopathogically indistinguish-able in monkeys from wild type virus strains or vaccines known to produce aseptic meningitis in children.It is therefore perhaps not entirely surprising that certain mumps vaccines presumably passing the monkey neurovirulence safety test have produced aseptic meningitis in vaccinees,including the widely used Urabe-AM9[16,33,36–38]and Leningrad-Zagreb strains[39–42],and the less widely used Leningrad-3[43],Hoshino[35],Torii[44], and Sofia-6[45]strains.However,it is important to point out that all“wild type”viruses tested to date have beenfirst pas-saged in vitro,a process potentially leading to partial attenuation. Thus it is quite possible that the monkey model could distinguish true wild type viruses from attenuated viruses,but the model seemingly cannot reliably distinguish sufficiently vs.insufficiently attenuated viruses.This may in part be due to the limited num-ber of monkeys used in such analyses and perhaps with larger group sizes,the distinction between varying levels of attenua-tion may become apparent;however,for ethical and logistical reasons,testing of large numbers of non-human primates is not feasible.Despite this apparent shortfall,in the absence of an alternative methodology,the monkey neurovirulence safety test continues to be recommended by national regulatory authorities for the pre-clinical assessment of the neurological safety of candidate vac-cine strains.Notably,although the incidence of aseptic meningitis associated with administration of some mumps vaccines may be as high as1in1000doses,this is but a fraction of the incidence of aseptic meningitis following natural infection,suggesting a benefit of testing candidate vaccines in the monkey neurovirulence safety test.Indeed,many vaccines known to cause aseptic meningitis are still used today in several countries based on the benefit of protection outweighing the small risk of non-life threatening adverse events[46].3.Neurovirulence safety testing in small animal modelsThe best characterized small animal model for the study of mumps virus pathogenesis is the hamster;however,like the monkey model,neuropathology following infection by virulent versus attenuated strains are largely indistinguishable[47–50]. Mice were also explored as model system;however,virus repli-cation in this species is abortive[51–54].Early studies in rats indicated that productive infection required host adaptation[55], a phenomenon that would preclude the use of rats as means of assessing vaccine virus safety.However,it was later determined that non-adapted mumps viruses can productively replicate in rats if inoculated intracerebrally within thefirst24h of birth[56].The major neuropathological outcome in neonatally inoculated rats is hydrocephalus of the lateral and third ventricles,the magnitude of which was found to be a correlate of the neurovirulence potential of the virus for humans[57].The greater predictive value of the rat based assay versus the monkey based assay is clearly apparent when comparing neurovirulence scores measured in the two assays (Fig.1).Further support for the ability of the rat assay to correctly assess neuroattenuation is indicated by results of inoculation of newborn rats with passage levels8,13and17of the Jeryl Lynn mumps virus strain(kindly provided by Merck and Co).Although passage levels8and13were not tested in humans,during vac-cine development passage level12was found to be insufficiently attenuated in children whereas passage level17was of low reac-togenicity in children[58].From these data it can be inferred that virus at passage level8is the least attenuated whereas virus at pas-sage level17is the most attenuated,with passage level13being of intermediate reactogenicity in the clinic.This trend is clearly demonstrated in testing of these passage levels in rats(Fig.2).To pursue the possible use of the newborn rat-based test as a valid alternative to the monkey-based test,robustness and repro-ducibility of the former was examined in a collaborative study involving two laboratories,one at the U.S.Food and Drug Adminis-tration(FDA)and the other at the National Institute for Biological Standardization and Control(NIBSC)in the U.K.Twelve mumps virus preparations of varying human neurovirulence potential were blinded and distributed to each laboratory along with a standard operating procedure for performing the assay.Although the abso-lute measured neurovirulence scores differed between the two laboratories,the overall rank order of the scores was maintained at both study sites wherein wild-type viruses could be differentiated with statistical certainty from vaccine viruses and attenuated vac-cine viruses could be differentiated with statistical certainty from partially attenuated vaccine strains[59].The newborn rat neurovirulence safety test is currently the sub-ject of a World Health Organization sponsored validation study that will include vaccines produced by many of the current product manufacturers.4.Neurovirulence safety testing in marmosetsAs an alternative to the Cercopithecidae monkey-based test, marmosets(Callithrix jacchus)were investigated as a possible model for testing of the human neurovirulence potential of mumps viruses.Marmosets inoculated intraspinally with the wild type Odate strain or the insufficiently attenuated NK-M46or Urabe vac-cine strains developed extensive encephalitis and meningitis and virus could be readily recovered from brain tissue.In contrast,ani-mals inoculated with the Jeryl Lynn vaccine strain manifested only minor histopathological changes and virus could not be recov-ered from brain or other tissues including kidney,pancreas and parotid gland[60].In a head-to-head comparison of reactivity in the marmoset model versus the newborn rat model,the Jeryl Lynn2852S.A.Rubin,M.A.Afzal/Vaccine29 (2011) 2850–2855Table1Synopsis of the literature describing histopathological changes in monkeys following inoculation of different mumps viruses by various routes.JL:Jeryl Lynn;L-3:Leningrad-3; wt:wild type;p.attn:partially attenuated;vac.isolate:vaccine isolate from cerebrospinalfluid of patient with vaccine-induced aseptic meningitis;HAU50:heamaglutining units;TCID50:tissue culture infection dose units;PFU:plaque forming units;IT:intrathalamic;IC:intracisternal;IS:intraspinal;IM:intramuscular;IP:intraparotid.Virus strains Biological phenotype Species(dose range)Injection site Histologicalfindings and conclusions ReferenceJL p17Vaccine a Rhesus macaques(103.7−5.0HAU50)IT/IS/IM(combined)Slight cellular infiltration of choroids plexus(cp)and ependymal cells with all viruses;noqualitative or quantitative histologicaldifferences between viruses[58]JL p12p.attn JL p7wt ABC wt Farina wtL-3p21Vaccine b Rhesus macaques(104.3−4.5HAU50)IT/IS/IC(combined)Cellular infiltration of cp and periventricular(pv)areas with all viruses;neuronal dystrophywith L-3p6and Sophia6,but not L-3p21[31]L-3p6wt Sophia-6Vaccine bL-3p22Vaccine b Rhesus macaques(105.5HAU50)IT/IC(combined)Neuronal dystrophy and cellular infiltration ofcp,pv and meninges with both viruses;histological changes more extensive with L-3p22vs.p19[29]L-3p19p.attnJL p17Vaccine a Rhesus macaques and Greenmonkeys(105.0−5.8HAU50)IP;IM(separately)IP:cellular infiltration of cp and pv with allviruses following;encephalitis and neuronaldystrophy with L-3p7and Sofia-40.IM:astrogliosis and cellular infiltration of pv withall viruses[69]L-3p22Vaccine b L-3p7wtSofia-6Vaccine bJL p17Vaccine a Rhesus macaques and Greenmonkeys(104.4−4.5HAU50)IT Cellular infiltration of cp and pv with allviruses;neuronal dystrophy with Berlin andSofia-6,L-3p7,and to a lesser extent JL p17and L-3p22[32]L-3p22Vaccine b L3p7wtSofia-6vaccine b Berlin wtHoshino L-32Vaccine b Cynomolgus Macaques(105.0TCID50)IP;IM;IT;IC(separately)No histological changes in the CNS in monkeysinoculated with any of the viruses[70]Sasazaki wtS-12wtS-12p14Vaccine cJL p17Vaccine a Rhesus macaques(104.5pfu)IT Cellular infiltration of cp and pv with allviruses;no significant differences betweenviruses[71]RIT-4385vaccine aKilham wtLo1wt87-1004vac.isolateJL p17Vaccine1Cynomolgus macaques(104.4−5.0pfu)IT Cellular infiltration of cp and pv with allviruses;no significant differences betweenviruses[72]Urabe-AM9Vaccine b Lo1(wt)wtNt-5vac.isolate MJ14Vaccine aHoshinoL-32Vaccine b Green monkeys(105.0TCID50)IT/IC.(combined)Cellular infiltration of cp,pv and meningeswith Hoshino and Hoshino CA-35;nohistological changes with Hoshino L-32[73]Hoshino wtHoshino CA-35p.attna Not causally associate with meningitis.b Causally associate with meningitis.c Association with meningitis unknown.vaccine strain was found to be attenuated and the Odate and NK-M46strains were found to be neurovirulent,as expected.However, whereas the Urabe strain was found to be neurovirulent in mar-mosets,it was found to be attenuated when tested in newborn rats by Saika et al.[61].This contrasts withfindings by Rubin et al., who reported the Urabe vaccine strain to be neurovirulent in new-born rats[59].Reasons for this discrepancy are unclear;however, manufacturer-to-manufacturer as well as lot-to-lot differences in rate of Urabe vaccine virus associated aseptic meningitis have been reported,suggesting the potential for biological characteris-tics to differ between different vaccine lots[62,63].Additionally, the Urabe vaccine preparation used by Rubin et al.was unpas-saged material obtained as marketed product whereas the vaccine preparation used by Saika et al.was passaged once more in chicken embryo cells to generate a stock for testing,and,thus,mutations could have arisen in the passaged virus that could have affected itsS.A.Rubin,M.A.Afzal /Vaccine 29 (2011) 2850–28552853J L R I T (v a c )J L (v a c )L o 1 (w t )88-1961 (w t )N .a .N .J L -R I T (v a c )J L (v a c )U r -A M 9 (v a c )L o 1 (w t )M e a n L e s i o n S c o r e0.00.51.01.52.02.53.03.54.0J L -R I T (v a c )J L (v a c )L -3 (v a c )U r -A M 9-1 (v a c )U r -A M 9-2 (v a c )T e n n (w t )L o 1 (w t )N Y (w t )P e t r e n k o (w t )88-1961 (w t )N e u r o v i r u l e n c e S c o r e2468101214161820parison of neurovirulence scores measured in the rat based assay versus the monkey based assay.Neurovirulence scores determined in the rats (left panel)and monkeys (right panel).Viruses are listed in order of increasing human neurovirulence from left to right.Attenuated vaccines are shown as white bars,insufficiently attenuated vaccines as light grey bars,and wild type viruses as dark grey bars.In rats,the Jeryl Lynn-based attenuated vaccines (JL and JL-RIT)could be distinguished with statistical certainty from the wild type viruses as well as the insufficiently attenuated Leningrad-3vaccine (L-3)and Urabe-AM9vaccine (Ur-AM9-1and Ur-AM9-2,representing two different lots from the same manufacturer).In contrast,in the monkey assay,no statistically significant differences were seen between any of the viruses tested either at the FDA or at the NIBSC (left and right halves of graph,respectively)in two independent studies.Neurovirulence and mean lesion scores were determined as described previously [57,71,72].Passage LevelP17P13P8N e u r o v i r u l e n c e S c o r e123456789Fig.2.Results of inoculation of newborn rats with passage levels 8,13and 17of the Jeryl Lynn mumps virus strain showing the ability of the rat assay to correctly assess neuroattenuation.Neurovirulence scores measured in adult rats inoculated as new-borns with 100pfu of increasing passage levels of the Jeryl Lynn strain.Error bars represent the standard error of the mean.Neurovirulence scores were determined as described previously [57].Differences between each group were statistically sig-nificant (all p <0.001,Mann–Whitney rank sum test).phenotype.Although data from the marmoset model are promising,this system has not been rigorously tested.5.Non-animal based assessment of mumps virus neurovirulenceEthical concerns surrounding the use of animals in product testing,coupled with questions over the predictive value of such testing,have resulted in a concerted effort by the scientific com-munity to replace animal-based neurovirulence safety testing with more meaningful,validated alternative methods.To date,no such alternatives have been developed for mumps virus.In vitro characteristics such as the relative extent of virus-induced cyto-pathic effects (e.g.,cell-to-cell fusion,cell lysis,etc.),virus plaque morphology,or replication kinetics do not appear to reliably distin-guish neurovirulent from non-neurovirulent mumps virus strains [64–68].However,the observation that compared to attenuated mumps virus strains,non-attenuated strains possess a replication advantage in neuronal cells suggests that cell line-specific permis-siveness to mumps virus infection may be a parameter worthy of further pursuit [56].Nonetheless,such an in vitro -based test,even if validated,would more likely be approved as a screening tool,rather than as a complete replacement for animal-based testing.6.Perspectives and future directionsThe studies reviewed in this article indicate the potential for the newborn rat-based and marmoset-based models as alternatives to the monkey-based model for pre-clinical neurovirulence safety testing of mumps virus vaccines.Both alternative methods require further validation.Despite some promise for use of in vitro -based markers to assess the neurovirulence potential of mumps viruses,it is unlikely that mumps virus neurovirulence safety testing could be wholly accomplished outside of animal testing given the risk of in vitro systems not fully reflecting the complexity of the pathogen-esis of neurotropism and neurovirulence.Nonetheless,meaningful data obtained from non-animal based testing would be of immense value as a screening tool in development of vaccine candidates prior to pre-clinical animal testing,and,thus,this area deserves further investigation.AcknowledgementsThis work was supported in part by a grant from the National Vaccine Program Office and in part by an appointment to the Research Participation Program at the Center for Biologics Eval-uation and Research administered by the Oak Ridge Institute for Science and 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