Rabies neutralizing antibody in serum of children compared to adults following post-exposure prophylaxis

Biologicals (1992) 20, 283-287

Rabies Neutralizing Antibody in Serum of Children Compared to Adults Following Post-exposure Prophylaxis Fred Y. Aoki,* Margaret E. Rubint and Margaret V. FastS§ *Departments of Medicine, Medical Microbiology, Pharmacology and Therapeutics, ¢Department of Medical Microbiology, University of Manitoba and $Communicable Disease Control, Province of Manitoba, Winnipeg, Manitoba, Canada Abstract. Rabies neutralizing antibody concentrations in serum (SRNA) elicited by experimental postexposure prophylaxis (PEP) regimens are used to assess their potential clinical efficacy. Although PEP with human rabies immunoglobulin (HRIG) plus human diploid-cell culture vaccine (HDCV) is almost completely protective, the limited availability of these components has stimulated the search for alternative regimens. Since 33% to over 60% of PEP is administered to children, a need exists for data on the SRNA response to HRIG plus HDCV in children, to be used to assess the potential value of alternative PEP treatments. We measured SRNA by the rapid fluorescent focus inhibition test on days 0, 1, 3, 7, 14, 28, 56 and 90 after initiation of PEP with HRIG (20 IU/kg) and HDCV (1.0 ml on day 0, 3, 7, 14, 28) in 10 children [8 _ 4 (mean _+ SD) years of age] and 32 adult control patients (38 _+ 16 years of age). Early, uniform appearance of SRNA was observed with no differences between groups. These data are consistent with the demonstrated efficacy of PEP with HRIG plus HDCV in children as in adults and provide results that can be used for initial evaluation of experimental pediatric PEP regimens in the future.

Introduction The efficacy of post-exposure prophylaxis (PEP) against rabies when vigorous cleaning of the bite site is combined with injection of h u m a n rabies immune globulin (HRIG) and h u m a n diploid-cell culture vaccine (HDCV) initiated promptly after exposure, approaches 100%.~ Although rare failures have been reported," it is probably not unreasonable to consider this regimen as a standard for PEP. However, the high cost and limited availability of HRIG and HDCV relative to the magnitude of the need for PEP in many countries of the developing world have stimulated interest in alternative regimens, such as one in which a purified Vero cell culture vaccine (PCVR) is substituted for HDCV? One approach to developing suitable alternative regimens of PEP for evaluation in the field is to compare the serum rabies neutralizing antibody (SRNA) §Current address: Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada. Correspondence to: Dr Fred Aoki, Department of Medical Microbiology, Rm 510, Basic Medical Sciences Building, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 0W3. 1045-1056/92/040283 +05 $08.00/0

responses to the experimental regimen, with those observed with HRIG plus HDCV? Since one-third 4 to over 60% '~ of PEP is administered to children, there is a compelling case to confirm that experimental regimens administered to children produce SRNA profiles comparable to those of effective treatments, such as HRIG plus HDCV. Few data are published specifically on the SRNA response of children given HRIG plus HDCV. 6 Therefore, we undertook the following study to compare SRNA in children given HRIG plus HDCV for PEP, with results in an adult control group studied concurrently. We confirmed thereby t h a t the SRNA profiles in children and adults were not different and describe data that may be useful in developing alternative PEP regimens for children in the future.

Materials and Methods

Subjects All persons in the province of Manitoba (population approximately one million), who required PEP from March 1987 to March 1988 were invited to participate in this study which had been approved by © 1992 The International Associationof BiologicalStandardization

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the Faculty Committee on the Use of H u m a n Subjects in Research, University of Manitoba. We defined a child as a patient who was 12 years old or less. During the 12 months of the study, 78 individuals received P E P (7-8 per 100 000 population). Forty-two individuals participated including 10 children (3 girls), 8 +- 4 (mean _+ SD) years of age and 32 adults (13 women), 38 +_ 16 years of age. Of the 10 children who were immunized, 3 had been exposed to, or bitten by, pets whose brain tissue was positive by fluorescent antibody (FA) testing (1 dog bit 2 children; 1 cat). The remaining 7 were given P EP after contact with animals t h a t escaped (4 dogs, 1 rodent) or whose brain tissue was FA-negative (2 dogs). Of the 32 adults, 18 had been exposed to 11 animals with FA-positive brain tissue [7 cows (14 adults exposed), 2 horses, 1 dog and 1 cat]; 13 had been exposed to 13 animals t h a t escaped (7 cats, 2 dogs, 2 coyotes, 2 others) and one to a cat whose brain tissue was FA-negative. Thirty-six individuals were not included in the study: 19 veterinarians or members of their staff had previously been immunized against rabies and required only HDCV while 17 others who received both HDCV and HRIG declined to be included in the study: 9 children (3 girls), 6 -+ 5 years of age and 8 men, 37 _ 16 years of age.

Analysis Ages of men and women and their SRNA at each test time were compared by the non-parametric M a n n - W h i t n e y U test, as were SRNA between the children and adults. Proportions of adults and children with SRNA on days 1, 3 and 7 were compared by the Fisher Exact Probability test. Differences with P ~< 0-05 for two-tailed tests were considered significant. Results

SRNA concentrations over 90 days after initiation of P E P for both groups are shown in Fig. 1 and Tables 1 and 2. One m an had SRNA detectable in preimmunization (day 0) serum. His subsequent SRNA concentration profile was not different t h a n t h a t of those who had no pre-existing SRNA before initiation of P E P and his SRNA data were therefore not excluded from the analysis. Of the 42 study subjects, four did not receive a complete course of immunization: one child's P E P (subject num ber 2, Table 1) was discontinued after three doses of vaccine because the FA test on the

50.0 [

Vaccine HDCV (L'Institut Merieux, Lyons, France) was stored, prepared and injected as recommended by the manufacturer. A dose of 1.0 ml was injected intramuscularly (i.m.) in the deltoid region (adults and older children) or quadriceps muscle (small children) on days 0, 3, 7, 14 and 28.

l(}-Org ",~,

8 8 C, e.-,

Antiserum HRIG (Hyperrab R, Cutter Laboratories, Berkeley, CA, USA) was stored and injected as recommended by the manufacturer. A dose of 20 IU/kg was administered on day 0. Up to one-half was infiltrated around the bite site where practical and the remainder injected i.m. in one or more sites aforementioned where no vaccine was being administered t h a t day.

SRNA SRNA was measured using the rapid fluorescent focus inhibition test (RFFIT) s in venous blood samples obtained on days 0, 1, 3, 7, 14, 28, 56 and 90. Inclusion of a standard antiserum enabled all results to be expressed as IU/ml.

/

l.o L

I

0 I! I

/ I

i

E

.= (I.Ol 0

7 14 28 5O 90 Days after initiation of post-exposure prophylaxis

Figure 1. Serum rabies neutralizing antibody concentration (mean +_ SD) in 10 children (O--O) and 32 adults (Q--O) given human rabies immune globulin plus human diploid-cell culture rabies vaccine for post-exposure prophylaxis.

Rabies neutralizing antibody in serum of children

285

Table 1. R a b i e s n e u t r a l i z i n g a n t i b o d y in s e r u m of children given p o s t - e x p o s u r e p r o p h y l a x i s with H R I G a n d H D C V according to the s c h e d u l e described in M a t e r i a l s a n d M e t h o d s Serum rabies neutralizing antibody concentration (mean _+ SD; IU/ml) on day Volunteer number

Sex

Age (yearsl

0

1

3

7

1 2 3

F F F

1 3 11

<0.10

0-11

0.40 0.11

0.50 . 0-13

4 5 6 7 8 9 10

M M M M M M M

2 7 8 11 11 12 12

<0.10 <0.10 <0.10 <0.10 <0.10 <0-10 <0.10

0.11 0.11 0.11 0.11 0.08 0.09

0.13 0.33 0.11 0.21 0.29 0-11 0.33

8 4

<0.10

0.10 0.01

0.20 0.10

Mean SD

14

28

12.00

14,00 . 11.00

0.54 2.10 0-11 0.13 0.33 0-16 0-42

7-00 14.00 0.56 2-50 2.00 2.10 9.00

0.49 0-63

6-10 5.10

.

.

56

90

12.00

-

. 5.40

2.90

9.00 14.00 2.70 2.80 4.80 12.00 14.00

14.00 14.00 2.80 2.80 2.80 2.80 10.00

3.60 14-00 2.70 2-80 2.80 1.80 2.70

9.40 4-80

7.40 5.00

4.20 4.00

Table 2. R a b i e s n e u t r a l i z i n g a n t i b o d y in s e r u m of a d u l t s given p o s t - e x p o s u r e p r o p h y l a x i s with H R I G a n d H D C V according to the schedule described in M a t e r i a l s a n d M e t h o d s Serum rabies neutralizing antibody concentration (mean _+ SD; IU/ml) on day N

Age

0

1

3

7

14

28

56

90

Females

13

34-+12 <0.10

0.07---0.50

0.11-+0.06

0.52-+1.01

4.15-+3.48

Males

19

40_+17 <0-10 0.10_+0.02

0-21_+0.13

0.31-+0.16

7-58_+5-33 10.39-+4-94 8.95_+4.85

7.36_+5.72

Overall

32

38_+16 <0.10

0.17_+0-12

0.38_+0.59

6.50_+5-00

9.30_+5-00 7.90_+4.60

6.70_+5.20

0.09_+0.03

b r a i n of the dog t h a t h a d bitten her, was negative. Two adults w i t h d r e w from the s t u d y a f t e r receiving H R I G and t h r e e doses of vaccine a n d one after receiving four doses of vaccine. One adult from the group withdrew on e n t e r i n g the hospital for elective hip s u r g e r y while the reason(s) for w i t h d r a w a l in the other two instances are not known. None of the 38 subjects who completed the s t u d y complained of side effects but a d v e r s e s y m p t o m s were not s y s t e m a t i c a l l y sought. No subject developed r a b i e s d u r i n g n e a r l y five y e a r s of o b s e r v a t i o n (59 -+ 3 m o n t h s in children a n d 57 _+ 3 m o n t h s in adults). T h e r e w a s no difference b e t w e e n the a g e s of the 13 w o m e n or 19 m e n (Table 2) n o r b e t w e e n t h e i r S R N A c o n c e n t r a t i o n s on a n y day. Moreover, the r e s u l t s w e r e not significantly different w i t h i n each g e n d e r g r o u p or b e t w e e n t h e m , if d a t a fl'om the one subject w i t h p r e - e x i s t i n g S R N A or the t h r e e who did not c o m p l e t e P E P t r e a t m e n t , were excluded. Accordingly, t h e s e d a t a w e r e r e t a i n e d a n d d a t a for all a d u l t s pooled for c o m p a r i s o n w i t h those of the chil-

7.48_+4.79 6.03 -+3.88 5.60 _+4.46

dren. T h e r e w e r e no differences in S R N A b e t w e e n the children a n d the adults at a n y day. Excluding the d a t a of the one child and the t h r e e adults, with incomplete courses of PEP, did not affect the conclusion. At day 90, SRNA in children was lower t h a n in adults but the small n u m b e r of children studied m a y h a v e precluded d e m o n s t r a t i o n of a difference at this time. S R N A w a s detected in all 7 children t e s t e d a t d a y 1 a n d all 9 t e s t e d at d a y s 3 a n d 7. In the adults, S R N A was detected in 24 of 27 individuals t e s t e d at d a y 1, 26 of 28 t e s t e d at d a y 3 a n d all 27 t e s t e d at d a y 7. No differences in p r o p o r t i o n b e t w e e n adults a n d children on a n y day were significant. All 25 a d u l t s a n d 8 children t e s t e d on day 14 h a d S R N A c o n c e n t r a t i o n s 1>0.5 IU/ml. Discussion

T h e n o t e w o r t h y findings of this s t u d y are the detailed description of the S R N A c o n c e n t r a t i o n s in children following P E P with H R I G a n d H D C V a n d

286

F.Y. Aoki et aL

their similarity to those in adults given the same regimen and studied concurrently. Such data have not previously been reported. SRNA is the best measure of the protective i m m u n i t y provided by P E P against rabies infection although the minimum protective concentration is not known. P E P with HRIG and HDCV appears to be uniformly effective in both children and adults.' The data from our study suggest strongly t h a t these uniformly good results in children and adults are associated with similar SRNA concentrations over the first 90 days following initiation of PEP. Following initiation of simulated P E P with HRIG plus HDCV in adults 9''° SRNA concentrations have been similar to those observed in our adult patient control population, with uniform, early appearance of neutralizing antibody in serum. In one study, SRNA was detected by RFFIT in all 6 adult subjects at day 1,9 while in the other, Nicholson and Turner, observed SRNA by mouse neutralization test (MNT) in 7 of 15 veterinary students at day 1 and 13 of 15 at day 3. '0 In the current study, the proportions of adults with SRNA at days 1 and 3 were 24 of 27 and 26 of 28, respectively. Thus, SRNA results observed in the adult control group in this study were likely a valid representation of those in other adult patients given HRIG plus HDCV for PEP. Therefore, in developing alternatives to HRIG and HDCV for PE P in children, doses and schedules should be selected t h a t yield SRNA concentrations over time t h a t are similar to those described in this study. In this regard, it seems appropriate to emphasize the importance of early a t t a i n m e n t of detectable SRNA by day 19.'' or at least day 3 '°'r-' or day 7 '~ as evidenced by data from the aforementioned studies of simulated P E P with HRIG and HDCV 9.'° as well as data from two clinical studies. ''.'2 In one study of 17 patients severely bitten by a rabid wolf, Habel and Koprowski measured SRNA by MNT after P E P with either heterologous (mule) rabies antiserum plus vaccine, or vaccine alone." The higher survival rate in those given combined passive-active immunization compared to vaccine alone was associated with earlier appearance of SRNA in the former group, in which 10 of 11 had SRNA detected at day 1 whereas no antibody was detected until day 19 in the latter group. In another study, SRNA was measured by MNT in 45 patients given P E P with HDCV plus mule antirabies serum, following bites inflicted by rabid dogs or wolves.'" All survived. SRNA was detected (mean concentration 0.74 IU/ml) in 23 of 24 adults tested for the first time at day 3 following initiation of PEP. Finally, Anderson and colleagues

measured SRNA by RFFIT in 90 patients given HDCV and HRIG for PEP. '3 The m ean SRNA concentration was 0.20 IU/ml in 83 individuals at first testing on day 7. All were well 10-18 months after exposure. Taken together, these SRNA data from simulated P E P t r e a t m e n t s plus those from field studies, strongly support the idea t h a t SRNA is detectable early (days I to 7) after initiation of effective P E P regimens in adults and t h a t this should be the goal in developing new P E P regimens for children also. The potential value of HDCV plus HRIG for children was initially suggested by limited, uncontrolled data from two children given 20 IU/kg i.m. of HRIG and 5 doses of HDCV on days 0, 3, 7, 14 and 28 or 302 However, SRNA m e a s u r e d by RFFIT was not detectable on days 0 or 7 although concentrations were 0.4 and 13.5 IU/ml on day 14, 27 IU/ml on day 28 or 30, and 30 and 35 IU/ml on days 42 and 90, respectively. The reasons for differences in SRNA compared to those in our study are unclear. Subsequently, SRNA in children and adults given HDCV and HRIG were reported '3.'4 but the data in the children could not be identified specifically. In one of these studies, '3 it was noted t h a t all 84 recipients tested at day 42 (including 35 children) had neutralizing antibody equal to, or greater than, 0.5 IU/ml, which was purported to be the m a x i m u m concentration attained with HRIG plus 23 doses of duck embryo vaccine, one of the vaccines used widely before HDCV became available. By contrast, 59% had SRNA t>0.5 IU/ml at day 14. In our study, all subjects tested, both adult and pediatric, had neutralizing antibody concentrations >t0.5 IU/ml at day 14. SRNA responses to other P E P regimens in children have been described. ,2.,5 In 19 children 12 years old or younger successfully t r e a t e d with HDCV and a mule antirabies serum, antibody concentrations m easured by MNT were 0.98 _+ 0.20, 1.68 +_ 1.37, 16.69 _ 17.39, 46.02 _+ 37-78 and 69.83 _ 58-65 IU/ml on days 3, 7, 14, 30 and 90, respectively. '2 As in adults successfully treated, early uniform appearance of SRNA was observed. '2 In 7 children, 2 to 14 years of age, SRNA was used to assess the potential efficacy of HRIG combined with PCVR. '5 SRNA measured by the MNT was not detectable at day 0; the concentration was next m easured at day 14 (4.68 _ 2-0 IU/ml); at day 30 it was 20-8 _+ 12.1 IU/ml; and on day 90, 4.1 ± 0.9 IU/ml. The variation in these SRNA results and the fact t h a t SRNA measured by MNT may be 1.4 to 9-6 times higher t h a n by RFFIT, 'G may explain in large part the differences in

Rabies neutralizing antibody in serum of children

t h e s e r e s u l t s f r o m o u r d a t a in children given H R I G a n d HDCV. T h e s e r e s u l t s with P C V R a n d H R I G w e r e d e e m e d to be sufficient to w a r r a n t u s e of this c o m b i n e d r e g i m e n for P E P for children. O u r d a t a a n d t h o s e p r e v i o u s l y p u b l i s h e d ~2 s u p p o r t t h a t conclusion which would be e v e n m o r e r o b u s t if S R N A h a d b e e n d e m o n s t r a t e d in t h e first d a y s a f t e r initiation of PEP. In conclusion, d a t a f r o m t h e c u r r e n t s t u d y indicate t h a t H R I G plus H D C V a d m i n i s t e r e d as curr e n t l y r e c o m m e n d e d for P E P in children, is a s s o c i a t e d w i t h e a r l y u n i f o r m a p p e a r a n c e of S R N A w i t h p e r s i s t e n c e o v e r 90 d a y s at levels c o m p a r a b l e to those o b s e r v e d in a d u l t s given the s a m e r e g i m e n . T h e S R N A d a t a m a y be useful to aid initial selection of a l t e r n a t i v e P E P r e g i m e n s for children in the future.

Acknowledgements We t h a n k o u r v o l u n t e e r s a n d t h e i r p h y s i c i a n s for t h e i r co-operation, t h e s t a f f of t h e C a d h a m Provincial L a b o r a t o r y for f a c i l i t a t i n g the collection of s p e c i m e n s , Ms K a t h y Tolli a n d Ms A n g e l a Nelson for t y p i n g the m a n u s c r i p t , a n d D r T i s s a V i t a r a n a for critical r e v i e w of t h e m a n u s c r i p t .

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Received for publicatio17 23 June 1992; accepted 5 December 1992.