R e v i s t a d a S o c ie d a d e B ra s ile ir a d e M e d ic in a T ro p ic a l 2 2 (1 ): 2 5 -2 9 , ja n - m a r , 1 9 8 9
PATTERN OF AC Q U ISITIO N OF ROTAVIRUS ANTIBO D Y IN CH ILDREN
FOLLOW ED U P FROM BIRTH TO THE A G E OF THREE YEARS
Alexandre C. Linhares1, Valéria R. M elo 2 , Joana D’Arc P. M ascarenhas1,
Yvone B. G abbay1 and Ronaldo B. de Freitas'1.
N in e h u n d re d a n d fo r ty - e ig h t ser u m s a m p le s f r o m 8 3 ch ildr en liv in g in B e le m , B r a z il, co lle c te d 'w ith in th e ir f i r s t th re e y e a r s o f life, w ere te s te d f o r th e p r e s e n c e o f gro u p
-s p e c ific r o ta v ir u -s -a n tib o d y b y a n e n z y m e -lin k e d im m u n o -s o r b e n t a -s -s a y ( E L I S A ) blo ck in g -tes t. P a s s iv e ly tr a n sfe rr e d m a te r n a l a n tib o d y la s te d a b o u t tw o a n d h a l f
m on th s; su b se q u e n te ly , lo w le v e ls o f ro ta v iru s a n tib o d y s ta r te d to a p p e a r a t sev en m on th s, re a ch in g a p e a k a t ele ven m o n th s o f age. F ro m on e y e a r o n w a r d s p o s it iv i ty g r a d u a lly in cre ased, re a ch in g h ig h e st v a lu e s a t 3 4 m o n th s o f life. I n d iv id u a l res pon se s w er e e x a m in e d in s e r a f r o m 61 c h ild re n w ho w ere f o l lo w e d u p sin c e birth to th re e y e a r s o f
ag e: 3 8 ( 6 2 ,3 % ) o fth e m d e v e lo p e d a lon g-te rm im m u n ity f o llo w in g f i r s t in fection ; eleven ( 1 8 .0 % ) ch ildr en d e v e lo p e d a sh o rt- te r m im m u n ity a fte r f i r s t in fec tion b y ro ta viru s;
se v e n ( 1 1 .5 % ) h a d no a n tib o d y re s p o n s e w ith in th e ir f i r s t th re e y e a r s o f life; a n d 5 ( 8 . 2 % ) s h o w e d p o s it iv e a n tib o d y re sp o n se f r o m birth to th ree y e a r s old.
Key-words: Rotavirus. Antibody. Children.
The worldwide occurrence o f rotaviruses as major enteropathogens, has been described by several investigators during the past decade.2 714 17 21 They constitute the single m ost important cause o f diarrhoea requiring admission to hospital and attack rates for severe rotavirus diarrhoea in young children appear to be similar in both developed and developing countries1. In the latter regions, where malnutrition represents a common finding, rotavirus diarrhoea certainly accounts for a high fatality-rate among children less than 3 years o f age. In Brazil, following the first findings in the North region, many authors have assessed the public health importance o f rotavirus diarrhoea throughout the country4 5 9 19.
M ost o f the studies carried out to date on rotavirus infection, have dealt with detection o f this agent in faeces from diarrhoeic children, and the concomitant evaluation o f their immune response.
The precise role o f immune response with respect to the pathogenesis o f rotavirus infection remains to be fully elucidated. It is believed that local immunity in the gut plays a more important role in protecting against rotavirus diarrhoea than circula
1. Instituto Evandro Chagas, Fundação Serviços de Saúde Pública - 66050 - Belém, P ará - Brasil.
2. Universidade Federal do Pará, Centro de Ciências Bioló gicas - 66000 - Belém, P ará - Brasil.
This work was supported by the World Health Organization, Control of Diarrhoeal Diseases Programme and Fundação Serviços de Saúde Pública, Ministério da Saúde.
Recebido para publicação em 28/10/88.
ting antibodies2*^. The latter humoral response, howe ver, may reflect the immunological status o f the small intestine, at least in terms o f immunoglobulin A^.
Seroepidemiological studies conducted in both temperate6 and tropical1*} 1 ' countries have clearly demonstrated that most ( about 80%) o f children by the age o f three years have antibodies to rotavirus.
Results presented here emerged from a three-year community-based longitudinal investigation and basically concern the kinetics o f acquisition o f humo ral rotavirus antibodies within the first three years o f life.
M A T E R IA L S A N D M E T H O D S
Our community-based investigation was carried out in the periphery ofB elem , Para state, NorthBrazil, involving 83 children who lived under poor hygienic conditions. B etw een N ovem b erl982 and March 1986 they were followed up (since birth), through regular fortnightly visits, with the main purpose o f studying both clinical and epidemiological aspects o f rotavirus infection. Apart from faecal specimens, which were collected fortnightly (or whenever symptoms o f diarrhoea were present), serum samples were taken in the following occasions: at birth, throughout every diarrhoeal episode and, regularly, each six months.
L i n h a r e s A C , M e lo V R , M a s c a r e n h a s J D P , G a b b a y Y B , F r e ita s R B . P a tte rn o f a c q u is itio n o f ro ta v iru s a n tib o d y in c h ild re n f o l l o w e d u p f r o m b ir th to th e a g e o f th re e y e a rs. R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 2 : 2 5 - 2 9 , ja n - m a r ,
1 98 9.
polystyrene microtitration plate (N U N C 239454, G ibco Biocult Ltda., Roskilde, Denmark) and incu bate 4°C overnight or at 37°C for two hours. Plates were then washed six times in phosphate-buffered saline (pH 7.3) which contained Tween 20 (polysor-bate) at a final concentration o f 0.1% v/v (P B S/T ). Fifty microliters o f P B S /T containing 0.01 M E D T A (pH 7.3) (P B S /T /E D T A ) were added to the precoa ted wells; 25 ul o f human serum samples (including four negative and two positive controls), diluted 1 /2 0 in P B S /T /E D T A , were added to duplicate wells; and 25 ul o f an optimal dilution o f a clarified rotavirus-positive human stool in P B S /T /E D T A were added to each well. The plates were incubated at 4°C overnight and then washed six times in P B S/T . A 100 ul sam ple o f a hyperimmune guinea-pig anti-human rotavirus serum, diluted 1 /1 0 ,0 0 0 in P B S /T containing bovine serum albumin (B S A ) at a final concentration o f 1 per cent v/v (P B S /T /B S A ), was added to all wells. Plates were incubated at 37°C for two and one half hours and then washed six times in P B S/T . Then, 100 ul o f Kirkegaard and Perry (goat) anti-guinea-pig immunoglobulin G , conjugated with alkaline phosphatase, diluted 1 /2 0 0 in P B S /T /B S A , were added to all wells. Plates were incubated at 37°C for on and a half hours, washed again six times in P B S/T , and 100 ul o f p-nitrophenylphosphate substra te (Sigm a 104-105, 1 mg/ml) in 10 per cent v/v diethanolamine buffer (pH 9.8) were added. After a 20-minute incubation at 37>.C, the reaction was stop
ped by adding 50 ul o f 3M sodium hydroxide. Readings o f optical density (O D ) were performed with a F low E L ISA reader (Multiskan), using a 405 nm filter. The percentage o f blocking value o f each specimen was determined as follows: per cent blocking = (m ean O D o f four negatives - O D o f sample) I
(m ean o f O D four negatives - mean o f O D o f two positives) x 100. A ll samples that yielded a per cent blocking value o f greater than 25 were regarded as positive.
R E SU L T S
N in e hundred and forty-eight sera from 83 children were tested for the presence o f group-specific rotavirus-antibody, with the purpose o f determining the pattern o f acquisition o f immunity within the first three years o f age. Figure 1 shows mean values o f percentages o f blocking o f these sera in relation to the age in months and number o f children o f whom specim ens were collected. Cord blood samples yielded relatively high levels o f rotavirus antibody (percentage o f blocking greater than 50), which gradually declined to negative values at 2-3 months o f age. Low levels o f rotavirus antibody start to appear at seven months, reaching a peak at eleven months o f age. From one year onwards, children had progressively increasing E L IS A antibody rates with increasing ages; the highest percentage o f rotavirus antibody was reached at 34 months o f age.
A 8 E I N M O N T H S
NUMBER OF — ^ 6 3 >8 IJ 16 3 8 9 « 2 6 2 7 3 0 S3 2 8 6 « 33 2 0 17 2 0 4 0 4 7 I f 21 2 0 14 17 5 4 13 2 0 14 8 3 3 3 3 13 I I 13 12 2 2 37 SERA TESTE0
• PO SI TI V E FOR RO TAVIRUS-ANTIBO DY O NEGATIVE FOR ROTAVIRUS-ANTIBODY
Figure 1 - Rotavirus antibody in 948 sera from 83 children, measured as mean values of percentages of blocking, according to age
L in h a r e s A C , M e lo VR, M a s c a r e n h a s J D P , G a b b a y YB, F r e ita s R B . P a tte r n o f a c q u is itio n o f r o ta v ir u s a n tib o d y in ch ild ren f o llo w e d u p f r o m b ir th to th e a ge o f th ree ye ar s. R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 2 : 2 5 - 2 9 , ja n -m a r ,
198 9.
Figure 2 shows the immune response of ten present patterns of short-term immnnp responses different patterns of immune response to rotavirus yielded by eleven (18,0% ) children; seven (11.5%)
infection,
as evaluated in 61 children who were individuals had no antibody response within their first followed up from birth to three years of age. Diagrams three years of life, as shown in diagrams 2F and G; and A and B show the pattern o f immune response of 38 5 (8.2% ) had rotavirus antibody positivity since birth (62.3% ) children who developed a long-term immu- to three years of life (Figure 2E).nity following first infection; figures 2C, D, H, I and J
( ) NUMBER OF CHI LD REN
L i n h a r e s A C , M e l o V R , M a s c a r e n h a s J D P , G a b b a y Y B , F r e ita s R B . P a tte rn o f a c q u is itio n o f ro ta v iru s a n tib o d y in c h ild r e n f o l l o w e d u p f r o m b ir th to th e a g e o f th re e y e a rs. R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T ro p ic a l 2 2 : 2 5 - 2 9 , j a n - m a r ,
198 9.
D IS C U S S IO N
Previous studies conducted in both temperate3 and tropical 13 regions, have shown that early rotavirus infection (i.e. infections which occur within the first 3-4 months o f life) are more likely to be asymptomatic. The reason for this is not yet well understood. It is possible that circulating antibodies, which may reflect the status o f intestinal immunity, can play a role in this context Our longitudinal investigation has shown that passively transferred maternal antibodies in gene ral disappear after 3 months o f age. The children would therefore become susceptible to rotavirus diarrhoea. W e also believe that, in the light o f our findings, administration o f a possible rotavirus vaccine should not be carried out within the first month o f life, because o f interference which might occur in the presence o f high levels of maternal antibody in blood serum.
After six months, children had progressively increasing E L ISA antibody rates with increasing age, reaching a peak at eleven months of age. This finding strongly suggests that the rate o f rotavirus infection in our region is greatestin children aged 6-24 months. Other authors in both temperate and tropical countries have yielded remarkably similar results with respect to the incidence rate o f rotavirus disease among children less than one year of age1.
The interval between the complete decline o f passively transferred maternal rotavirus antibody and the emergence o f naturally acquired rotavirus is o f 2-3 months. This immunological “ window” may reflect the absence of rotavirus infection in m ost o f the studied children, from their third to five months o f life. It is possible that maternal milk, containing anti-rotavirus secretory IgA (and other non-immune factors), could play a role in protecting them from rotavirus infection22. In our region breast feeding is observed in general from birth to six months o f age, particularly among children belonging to the low socio-econom ic level.
The evaluation o f individual immune responses among 61 children who were followed up from birth to three years o f age yielded ten different patterns, which can be divided into three groups: a) that involving children who apparently developed a long-term immunity; b) those who had a short-term immunity; and c) the children who surprisingly did not show any serological evidence o f rotavirus infection.
Immune responses o f either short or long dura tion have already been described with respect to gastroenteritis caused by Norwalk agent18. In our investigation, the apparent long-term immunity aga inst rotavirus could perhaps be a result o f repeated infections involving different serotypes. Previous stu dies by us I2 indicate that all four human rotavirus serotypes circulate in our region; serotype 1 is the most prevalent within the first year o f life, whereas serotype
2 is mainly detected during the second and third years o f life; serotype 4 occurs throughout the first three years o f life and serotype 3 is the less prevalent, accounting for a relatively low number o f cases o f rotavirus infection.
The occurrence o f short-term immunity may indicate that following first infection by one rotavirus serotype, a child can further becom e susceptible to the same serotype. Studies on this particular subject are required in order to determine whether neutralizing antibodies (not detected by our E L IS A assay) would also be o f limited duration. Another possibility is that our blocking test used to detect group-specific rotavi rus antibody is not sufficiently sensitive to demonstrate low levels o f serum antibodies.
With regards to the seven children who did not develop antibody response throughout the three-year longitudinal study, we could postulate the following: a) they were immunocompromised, probably by severe malnutrition; b) they in fact had no rotavirus infection during their first three years o f life and therefore did not develop serum antibody; and c) their immune response was not detected by our E L IS A blocking test, proba bly they had too low levels o f serum antibodies.
Further and broader studies on the immune response to rotavirus infection are necessary, parti cularly with respect to the serotype-specific immunity. A better understanding o f this subject is o f importance regarding further strategies o f administration o f a suitable vaccine.
SU M Á R IO
A m o s tr a s d e soro, em n ú m ero d e 94 8, fo r a m c o le ta d a s de 8 3 c rian ça s, a o lon g o d e se u s tr ês
p r im e ir o s a n o s d e vid a, e t e s ta d a s q u a n to à p r e s e n ç a d e a n tic o r p o s g ru p o -e s p e c ific o s p a r a r o ta v iru s p o r
u m te ste d e blo q u eio , u tiliz a n d o s e _o en s a io im u n o -e n z im á tic o (E L I S A ) . O b s-e rv o u -s -e q u -e a n tic o r p o s tr a n s fe r id o s p a s s iv a m e n te p e l a m ã e p e r s is tir a m p o r
ce rc a d e d o is e m e io m ese s; su b se qü e n te m en te , b a ix o s n ív e is d e a n tic o r p o s p a r a ro ta v ir u s c o m e ç a r a m a
a p a r e c e r a o s se te m e se s d e id ade , a tin g in d o u m p ic o p o r v o lta d o s on ze. A p a r t i r d e um a n o a p o s it iv i d a d e
a u m e n to u g ra d u a lm e n te , a lc a n ç a n d o o s m a is a lto s v a lo r e s a o s 3 4 m e se s d e vida . R e s p o s ta s im u n e s in d iv id u a is fo r a m a v a lia d a s em 61 c ria n ç a s a c o m
p a n h a d a s d e s d e o seu n a s c im e n to a té o s três a n o s d e ida de , o b te n d o -s e o s se g u in te s re su lta d o s: 3 8 ( 6 2 ,3 % ) d e la s d e s e n v o lv e r a m im u n id a d e d e lon g a d u raç ão , a p ó s a p rim o -in fe c ç ã o ; 11 ( 1 8 ,0 % ) in d iv í
d u o s a p r e s e n ta r a m a n tic o r p o s es p e c ífic o s q u e se
m a n tiv e r a m p o r u m in te r v a lo d e te m p o re la tiv a m e n te curto; 0 7 ( 1 1 ,5 % ) n ã o e x ib ir a m q u a lq u e r r e s p o sta im u n e a o a g e n te em q u e stã o , a o lon go d o s s e u s três p r im e ir o s a n o s d e vida ; e 05 ( 8 , 2 % ) m o s tr a r a m -s e
L i n h a r e s A C , M e lo V R , M a s c a r e n h a s J D P , G a b b a y Y B , F r e ita s R B . P a tte rn o f a c q u is itio n o f ro ta v iru s a n tib o d y in c h ild re n f o l l o w e d u p f r o m b ir th to th e a g e o f th re e y e a rs. R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T ro p ic a l 2 2 : 2 5 - 2 9 , ja n - m a r ,
1 9 8 9 .
p o s it iv o s q u a n to à p r e s e n ç a d e a n tic o r p o s p a r a A C K N O W L E D G E M E N T S
ro ta viru s, d e s d e o n a s c im e n to a té o s trê s a n o s d e
id a d e . W e thank Mrs. Margarete Figueiredo Garcia
Palavras-chaves: Rotavirus. Anticorpos. Crianças. for typing the manuscript
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