Biologicals (1992) 20, 27-33

Possible Influence of Measles Virus Infection of Cynomolgus Monkeys on the Outcome of the

Neurovirulence Test for Oral Poliovirus Vaccine

G. Contreras and J. Furesz* Bureau of Biologics, Drugs Directorate, Health Protection Branch, Health and Welfare, Canada

Abstract. Macaque monkeys are susceptible to measles infection which triggers temporary immuno- depression similar to the well known phenomenon in humans. It is known that feral monkeys become infected with measles virus when they are exposed to humans. Since Macaca mulata and M. fascicu- laris are species used to assay the neurovirulence of oral poliovirus vaccine, the immunodepression caused by measles infection of the test monkeys could significantly alter the results of the neuroviru- lence test. The serum titers of measles-neutralizing antibodies were studied in over 1500 monkeys used for neurovirulence tests. A high proportion of the feral monkeys had measles antibodies (51-100%); in contrast, none of 493 M. fascicularis monkeys which had been bred in a primate colony under strict isolation measures was found positive for measles antibodies. An increase in the preva- lence of measles in the population of Ontario and Quebec provinces was accompanied with an increase in the proportion of measles-positive monkeys and their serum antibody titers were found higher. It was observed that monkeys used in tests that had been performed during high measles prevalence presented with a poliomyelitis of more pronounced severity clinically and histologically. The analysis of 29 tests conducted on type 1 vaccines over several years showed a positive correla- tion (correlation coefficient = 0.5141, P < 0.0022) between severity of poliomyelitis and the presence of measles serum antibodies in test monkeys (some animals seroconverted during the test). A similar observation, when type 3 Sabin vaccines were tested in M. fascicularis, was recently reported from another laboratory in Ontario.

Introduction

The neurovirulence test (NVT) for oral poliovirus vac- cine (OPV) is based on the evaluation of specific histopathology, detected in the central nervous sys- tem of monkeys 17-21 days after inoculation of a large number of infectious vaccine virus particles, in the grey mat ter of the lumbar cord of cynomolgus (Macaca fascicularis) or rhesus monkeys (M. mulatta).' During the test most animals lose weight, over 25% develop some degree of leg flaccid paraly- sis, occasionally tetraplegia, rarely severe enough to justify euthanasia. The proper evaluation of the NVT requires that all test animals be in good health when inoculated. For this purpose the feral monkeys are quarantined, tested for infectious agents and

Corresponding author: J. Furesz, Bureau of Biologics, Virus Building, Tunney's Pasture, Ottawa, Ontario, Canada K1A OL2.

1045-1056/92/010027+07 $03.00/0

antibodies and clinically monitored for at least 6 weeks before the test. During the last 6 years, cynomolgus monkeys bred in a colony (Health Protection Branch, Ottawa) have become available for this test.

Measles virus infection is known to be immuno- compromising in humans. The virus induces mostly subclinical infections in both species of monkeys which are free of measles in nature but become infected upon contact with human populations. 2 The infection may go unnoticed during transit or quar- antine but the virus will induce antibodies,'-' and for a few weeks will disrupt the animal's normal immune responsiveness. 3 The disruption of the normal immune responsiveness induced by measles in humans and in monkeys may increase the suscepti- bility to other viral infections. Two observations in humans lend support to this concept; one refers to a particularly severe poliomyelitis outbreak in a small

© 1992 The International Association of Biological Standardization

28 G. Contreras and J. Furesz

community which followed within weeks a measles epidemic. 4 The other observation is the increased sus- ceptibility to herpes simplex virus infections in chil- dren with acute measles. 5 When investigating immunosuppression during a natural measles epi- demic in a primate research centre, it was found that there was a high case/fatality ratio associated with opportunistic infections of rhesus monkeys. 6

Subclinical measles infections in cynomolgus mon- keys may also increase susceptibility to other viruses. This paper presents the results of neutralizing anti- body determinations in over 1000 non-human pri- mates used in the laboratory, and attempts are made to correlate the frequency and size of measles anti- body titers with histological lesion scores in monkeys used in various tests that were performed during recent years.

Materials and methods

Viruses

Monotypic reference OPV prepared in this labora- tory and monotypic lots of one commercial source were employed in the NVTs.

Low passage of virulent measles virus Edmonston strain was received from Dr J. F. Enders in 1961 and subcultured five times (E5) in our laboratory. Live measles virus vaccine (Attenuvax; Merck, Sharp and Dohme) was used in one experiment.

Monkeys

Feral non-human primates (M. mulatta, M. fasci- cularis and Cercopithecus aethiops) were quaran- tined in our laboratory for at least 2 months, in addition to the 6-week quarantine performed by the supplier. Feral animals were clinically examined, tuberculin tested, treated for endo- and ectoparasites and bled for antibody determinations and for usual blood tests. Feral and colony-bred animals were bled upon entry and at termination of the NVT.

M. fascicularis were bred in the primate colony of our Department in Ottawa under strict isolation mea- sures.

Neurovirulence test

The NVT designed for the testing of OPV was per- formed following WHO guidelines, t Briefly, 0.1 ml of a test vaccine containing 10s6-106'6 plaque-forming units of virus was inoculated intraspinally between the first and second lumbar vertebrae. Animals that became moribund or severely paralysed were eutha- nized during the test; all other monkeys were eutha- nized at the end of the observation period (17-21

days). The following number of histological sections were examined from each animal: lumbar cord-12; cervical cord-12; lower and upper medulla oblongata- 1 each; mesencephalon-1; pons-1; and tha lamus-1 (giving a total of 29 sections). The scoring was done on a scale from 0-4:0 = no lesion; 1 = minimal lesion (only a few focal cellular infiltrates--no neuronal damage present); 2 = mild lesions of a few neurons in the motor areas; 3 = moderately severe lesions (most neurons damaged in the motor areas); and 4 = severe lesions (all neurons damaged in the motor areas of cord and brainstem). Because the specific poliovirus lesions were often quite different on the two sides of the section, more accurate readings were obtained by scoring them by hemisections as previ- ously described. 7 Individual monkey lesion scores (LS) were calculated as follows:

[sum of lumbar score] [sum of cervical score] L S = +

No. of hemisections No. of hemisections

[sum of brain score] + - ' 3

No. of hemisections

The LS for all positive monkeys were used to calcu- late a mean lesion score (MLS) for the virus being tested. Positive monkeys were those showing at least one specific neuronal lesion.

Measles neutralizing antibody assay. This was per- formed as previously described. ~

Experimental measles infection of monkeys. This was performed with the low passage virulent measles Edmonston strain (E5). Two groups of 10 cynomolgus monkeys received, intravenously, 3 ml of virus with a titer of 106 pfu ml-t. Four C. aethiops were inocu- lated subcutaneously with one dose of live measles vaccine (Attenuvax).

Results

Measles antibodies in feral and colony-bred monkeys

The presence of measles antibodies varied among the three monkey species (Table 1). Antibodies were found in 975 of 1105 feral cynomolgus (88%) and in 30 of 59 rhesus monkeys tested (51%). None of the 493 colony-bred cynomolgus monkeys had measles antibodies, confirming that the strict isolation mea- sures taken by the breeding colony prevented infec- tions from human pathogens and kept the animals in conditions similar to their natural habitat. It is of interest that none of 26 feral cerco monkeys tested was positive, although they had been in contact with

Measles virus inf luence on pol iovirus vaccine test 29

Table 1. Measles serum neutralization antibody titers in feral and colony-bred monkeys

Species Source

Measles antibodies No. positive

No. tested (%) GMT*

Macaca fascicularis Macaca fascicularis Macaca mulatta Cercopithecus aethiops

Feral 975/1105 (88) 1:11.4 Colony bred 0/493 (0) NAt

Feral 30/59 (51) 1:6.6 Feral 0/26 (0) NA

* GMT = geometric mean antibody titer. t NA = not applicable.

humans for some time; when four of these cercos were inoculated with a live measles vaccine, all developed low titer antibodies, GMT 1:8, but no clinical signs were observed. It should be noted that cerco monkeys were used only as kidney or blood donors, never included in NVTs.

The feral cynomolgus monkeys positive for measles could have become infected while in contact with humans when in transi t or in the laboratory, since they seem to be free of measles when colony-bred with the required isolation. This finding is similar to the observation that rhesus monkeys are negative for measles in nature. 2

The chances of infection of the monkeys would increase at the time of measles outbreaks. In 1979-1980, Canada experienced a large measles epi- demic with close to 20 000 cases, many of them in Ontario and Quebec; another smaller epidemic took place in 1984 following a quiet 3-year period (as shown in Figure 1 copied from the Canada Disease Weekly Report)2 During this 6- year period, 82 NVTs

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1980 1981 1982 1983 1984

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Figure 1. Measles--reported cases in 4-week periods, Canada, 1980-1984.

were performed and in most tests the monkeys were tested for measles antibodies. The proportion of measles-positive monkeys included in NVTs during the two epidemic periods, 1979-1980 and 1984, is higher than the proportion of measles positives in the non-epidemic period, 1981-1983 (Table 2). Since it is not proper to combine the 1979, 1980 and 1984 monkeys together in order to compare them with the combined 1981-1983 group, we compared the mon- keys in pairs, i.e. each group of monkeys used yearly in 1979, 1980 and 1984 was compared with groups of monkeys tested in 1981, 1982 and 1983. This statis- tical procedure showed that all the pairwise compar- isons were significant (P < 0.001) (Table 2). The geo- metric mean antibody, titers (GMT) were also higher for the groups of 1979, 1980 and 1984 compared with the other years. These results suggest that in those NVTs in which there was a very high proportion of cynomolgus monkeys with measles antibodies, a few with high titers, some animals could have been ex- periencing a subclinical measles infection before, or at the time, they were included in the NVT.

Subclinical measles infection could alter the mon- keys' immunological reaction to poliovirus attenu- ated vaccine strains in the NVT. When measles prevalence was low in Ontario and Quebec 32 NVTs performed on poliovirus type 1 vaccines (18 reference and 14 test vaccines) demonstrated that five of 524 (1%) monkeys died of poliomyelitis during the test (Table 3). This monkey mortality increased more than 12-fold (12 of 95 monkeys died, P < 0-0001)when NVTs were performed during a period of high measles prevalence in Ontario and Quebec (Table 3). Sixty- nine of these 95 monkeys were tested at the begin- ning of the NVTs for measles antibodies. All were positive; their GMT increased from 1:20 to 1:39 dur- ing the test, and two seronegative monkeys serocon- verted at the end of the test (Table 3). The MLS for these 95 monkeys was 1.52, higher and different

30 G. Contreras and J. Furesz

Tab le 2. Annual measles prevalence in Ontario and Quebec and measles antibody prevalence in monkeys used in neurovirulence tests of oral poliovirus vaccine

Measles antibodies

Annual measles monkeys prevalence No. positive

Year No. of Cases No. tested (%) GMT

1979 8099 132/132 (100)* 1:20.3 1980 11401 217/227 (96)* 1:17.3 1981 974 258/318 (80) 1:9-8 1982 559 173/210 (82) 1:14.2 1983 544 135/158 (85) 1:15.1 1984 2760 60/60 (100)* 1:25-3

* Higher proportion of positive cynomolgus monkeys in years 1979, 1980 and 1984, com- pared pairwise with the lower proportion years 1981, 1982 and 1983, as explained in the text (P < 0.001).

(P < 0.001) from the MLS of 1.22 observed in the 524 monkeys tested during the low measles prevalence period. A similar level of measles antibodies (GMT 1:39) was observed following intravenous inoculation of a 'wild' measles strain E5 (See Materials and methods) into 20 seronegative cynomolgus mon- keys. Two groups of 10 monkeys were infected with the measles E5 virus; one group was inoculated 5 days later and the other group 21 days later, both groups with a poliovirus type 1 reference vaccine in a regular neurovirulence test. One of the 10 monkeys inoculated 21 days previously with measles virus died presenting complete flaccid paralysis of the four limbs. The mean lesion score for the 20 monkeys was 1-57 higher and different (P < 0.001/from the mean of 1.22 observed in the 524 monkeys (Table 3). The data presented so far suggest a correlation between measles infection of monkeys used in NVTs and a higher MLS score with a higher incidence of fatal poliomyelitis.

In order to establish a correlation between lesion

scores and measles antibody status of monkeys, 29 NVTs were selected from the 36 NVTs described above for fur ther statistical analysis. The 29 NVTs (15 and 14 tests with reference and test vaccines, respectively) included 492 monkeys, and were grouped from the lowest to the highest MLSs (Table 4). The bottom nine NVTs (Nos. 21-29) are the high lesion score group; in this group there were 12 of 141 monkeys dying from paralytic poliomyelitis, v s 2 of 351 monkeys with medium and lower lesion scores (P < 0.0001). The proportion of monkeys with measles antibodies in the high lesion score group was 119 out of 120 (99%) v s 271 out of 321 (87%) (P < 0.01). Two monkeys seroconverted among the 119 of the high lesion score group as did three among the 271 of the low lesion score group. It is note- worthy that one vaccine lot tested in NVT No. 53, in the group with high lesion scores, was retested 45 days later when the measles epidemic was waning, in NVT No. 56 (in the group with low lesion scores); the mean lesion scores were 1.49 and 1.16, respec-

Tab le 3. Correlation between poliovirus type 1 neurovirulence severity and measles antibody prevalence

Neurovirulence test Measles antibodies

No. neuro- No. dying polio Lesion score No. positive Measles virulence prevalence* tests No. inocul. (%) Mean SD No. tested (%) GMTt

<100 32 5/524 (1.0)$ 1.22 -+0.45 371/414 (89.6) 1:18.6 >1000 4 12/95 (12.6)$ 1.52 -+0.58 69/69 (100) ÷ 1:32.2

* Measles prevalence expressed as the number of monthly reported cases in Ontario and Quebec at the time the NVTs were performed.

t GMT = Geometric mean titer. ,2 Difference proportions of animals dying from poliomyelitis, 5/524 v s 12/95 (P < 0.0001). " Two monkeys seroconverted to measles during the NVT.

Measles virus influence on poliovirus vaccine test 31

Tab l e 4. Neurovirulence test lesion scores and measles neutralizing anti- body titers in test monkeys: poliovirus type 1 0 P V strains

Observation Neurovirulence test Measles antibodies

Test Lesion No. dying No. positives

No. No. score No. tested GMT No. tested

1 61 0.79 0/30 1:14.3 25/25 2 63 1.01 0/24 13.4 18/23 3 110 1.03 0/12 28.5 11/12 4 101 1.04 0/12 17.0 12/12 5 64 1.05 1/20 5-7 9/18 6 89 1.08 0/12 12.7 12/12 7 100 1.09 0/12 20.2* 12/12 8 60 1.14 0/15 17.8 13/13 9 111 1.15 0/12 19.3" 11/12

10 75 1.15 0/20 8.3 15/20 11 88 1.15 0/20 32.0* 12/12 12 56 1.16 0/23 13.7 21/23 13 76 1.17 0/20 15.4 18/19 14 62 1.22 1/24 5.4 16/23 15 74 1.23 0/20 13.3 16/19 16 54 1.27 0/24 14.7 23/23 17 44 1.29 0/11 21.5 11/11 18 108 1.30 0/12 21.4 12/12 19 65 1.30 0/10 4.9 4/10 20 49 1.31 0/18 25-4 17/18

21 112 1.35 0/12 42.7 12/12 22 90 1.41 0/10 22.6 10/10 23 107 1.41 0/12 26.5 11/11 24 55 1.44 5/24 35-1" 14/14 25 109 1.48 0/12 17.0 11/12 26 53 1.49 5/24 43.3* 17/17 27 105 1.58 1/12 28.2 12/12 28 42 1.69 1/23 36.2 20/20 29 106 1.96 0/12 28.5 12/12

Mean 1.27 (X) 14/492 Mean 1:20.8 (Y) 407/449 Correlation coefficient 0.5141 (P < 0.0022)

* One monkey seroconverted while on test.

tively; five monkeys died in the first NVT, none in the second. The GMTs were 43.3 and 13.7 respectively. One monkey seroconverted in the first test, none in the second. The correlation coefficient between the two variables: the lesion scores and the GMT of measles antibodies is 0.5141 (P < 0.0022). If the log- arithm of the GMT is used, the correlation coefficient is 0.4430 (P = 0.008).

D i s c u s s i o n

The susceptibility of macaque monkeys to measles infection has been known about since 1911,1° but the

fact tha t they are measles-free in the wild and become infected upon contact with human populations was demonstrated only in 1962. 2 Data presented in this paper showed fur ther tha t macaques become infected with the human virus spreading in populations dur- ing measles epidemics. Although reports on the high infectivity of measles virus for monkeys are unequiv- ocal, clinical measles of different degrees of severity has been described by several investigators. In macaques, clinical measles appears to be a mild dis- ease, with subtle skin rash that often goes unnoticed in the furred animals,ll but occasional epizootics with greater severity have been described. ~2 In contrast,

32 G. Contreras and J. Furesz

measles can be highly pathogenic for marmosets, which appear to be the most susceptible primate species to measles infection. '3

Several facts indicate that the measles antibodies detected in monkeys presented in this paper were the result of active immunization by subclinical infec- tions with measles virus acquired shortly before or while in our laboratory. These facts are: (i) some mon- keys seroconverted for measles antibodies while.on test or in quarantine; (ii) there were groups in which every single animal showed measles antibodies (with high antibody titers in some); (iii) not a single decrease in antibody titer was detected in 670 cynomolgus monkeys followed for periods of over 6 weeks; this would suggest that the antibodies were not transferred passively by administration of serum immune globulin; H and (iv) all the monkeys inocu- lated with measles E5 virus developed a subclinical infection, as shown by the antibody response in every test animal. The antibody titers in the experimen- tally inoculated cynomolgus monkeys were similar to those found in naturally infected monkeys.

Measles infection in humans causes a depression in skin test reactivity 14 and a depression of human lymphocyte responsiveness, 6,'5 as well as in macaque lymphocytes2 The alteration of the immune respon- siveness, as well as other pathological changes induced by measles virus infection, may increase the susceptibility to other viral diseases. Bloch and Massry reported in 1963 that a severe poliomyelitis epidemic occurred within weeks following a severe measles epidemic in a small community, with an inci- dence of 7-6% for children under 4 years of age2 The authors analysed all aspects of the outbreak and dis- cussed possible mechanisms to explain the predis- posing role of measles. One mechanism could be the immunocompromise caused by measles infection; another could be the vascular changes produced by the measles virus, allowing a greater penetration of the central nervous tissue by poliovirus. 4 In another epidemic children with acute measles showed greater susceptibility to oral infections of herpes simplex virus and their lymphocyte response to mitogens was decreased. 5 The authors also considered the role of malnutrit ion as a contributing factor together with measles, but could not arrive at a conclusion. 5

The finding that 12 of 95 monkeys presented with very severe poliomyelitis when four type 1 vaccines were assessed for neurovirulence during two con- secutive springs of 1979 and 1980 (Table 3), led to a search for an explanation for this unusual severity. As a result of our investigation, it was found that all monkeys showed measles neutralizing antibodies

with high titers and two seroconverted; epidemiolog- ical surveillance revealed tha t measles epidemics were developing in the area during those two springs .9

Results obtained for 29 NVTs for type 1 poliovac- cines suggest a correlation between the severity of poliomyelitis and the presence of measles antibodies in test monkeys (Table 4). There is a statistically significant correlation between the mean lesion score for an NVT and the number of monkeys experiencing very severe poliomyelitis with high antibody levels to measles virus. Similar observations were reported to us from another laboratory when four cynomolgus monkeys used in three Type 3 NVTs showed severe neurological symptoms and high lesion scores. Of the 98 monkeys employed in these tests there were 27 monkeys which had four-fold or higher increases in measles antibodies titers during the test (unpub- lished data). These findings lend support to the hypothesis that measles infection of test monkeys may increase the severity of clinical signs and the lesion scores caused by OPV.

No vaccine failed an NVT due to possible measles infection of monkeys. Since the NVT requires the simultaneous inoculation of test monkeys with a ref- erence and a test vaccine, one may presume tha t a measles infection would equally affect both groups of monkeys. In fact, the lesion scores in both groups of monkeys were not significantly different.

In a recent review of immunosuppression caused by measles, McChesney and Oldstone analysed the mechanisms through which measles infection exerts this well-known effect; the authors had an oppor- tunity to extend their observations on this subject when a natural measles epidemic developed in rhe- sus monkeys at a primate research centre. During the epidemic there was a high case/fatality ratio associ- ated with opportunistic infections of the rhesus mon- keys. 6

It is a matter of concern that testing a poliovirus vaccine in monkeys experiencing or recovering from measles subclinical infection could yield an unac- ceptably high neurovirulence for an otherwise satis- factory vaccine and such a vaccine could be rejected. Moreover, in view of recent measles outbreaks in 1989 and 1990 in Canada, the source of virus infection will be around for some considerable time into the future. One possible solution for this problem already pro- posed I' could be to routinely immunize monkeys pass- ively with immune serum globulin against measles at suitable times before or during quarantine. Another solution could be to use monkeys which have been bred in colonies under strict isolation pro-

Measles virus influence on poliovirus vaccine test 33

cedures , since these m o n k e y s will cons is ten t ly t e s t nega t ive for m e a s l e s ant ibodies .

Acknowledgements

The au tho r s t h a n k Dr K. K a r p i n s k i for his valu- able profess ional advice on the s ta t i s t ica l ana lys i s of the data . They also t h a n k the la te Mrs J o a n Anderson, Mrs F r a n c e s Kane , and Mess r s G. Label le and R. A. J. Lapo in te for the i r va luab le technical ass is tance .

R e f e r e n c e s

1. WHO requirements for poliomyelitis vaccine (oral). WHO Tech Rep Ser 1983; 687.

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4. Bloch A, Massry S. Poliomyelitis following recent measles. Pediatrics 1963; 31: 929-935.

5. Orren A, Kipps A, Moodie JW, Beatty DW, Dowdle EB, McIntyre JP. Increased susceptibility to herpes sim- plex virus infections in children with acute measles. Infect Immun 1981; 31: 1-6.

6. McChesney MB, Oldstone MBA. Virus-induced immunosuppression: Infections with measles virus

and human immunodeficiency virus. Adv Immunol 1989; 45: 335-360.

7. Contreras G, Furesz J, Karpinski K, Grinwich K, Gardell C. Experience in Canada with the new revised monkey neurovirulence test for oral poliovirus vaccine. J Biol Stand 1988; 16: 195-205.

8. Moreau P, Furesz J. A rapid micro tissue culture assay in BS-C1 cells for the titration and neutralization of measles virus. Can J Microbiol 1967; 13: 313-319.

9. Varughese P, Measles in Canada--Update. Can Dis Weekly Rep 1984; 10: 193-194.

10. Goldberg J, Anderson JF. An experimental demon- stration of the presence of the virus of measles in mixed buccal and nasal secretions. JAMA 1911; 57: 476-478.

11. Barsky D, Palmer AE, London WT, Kerber WT. Use of immune serum globulin (human) to reduce mortality in newly imported rhesus monkeys (Macaca mulatta). J Med Primatol 1976; 5: 150-159.

12. Remfry J. A measles epizootic with 5 deaths in newly impor ted rhesus monkeys (Macaca mulatta). Laboratory animals 1976; 10: 49-57.

13. Albrecht P, Lorenz D, Klutch MJ, Vickers JH, Ennis FA. Fatal measles infection in marmosets pathogene- sis and prophylaxis. Infect Immun 1980; 27: 969-978.

14. von Pirquet C. Das Verhalten der kutanin Tuberculin- Reaktion w~ihrend der Masern. Dtsh Med Wochenschr 1908; 34: 1297-1300.

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Received for publication 28 August 1991; accepted 14 November 1991