CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 1994, p. 89-941071-412X/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Neutralizing Antibody Immune Response in Children withPrimary and Secondary Rotavirus Infections

CARLOS F. ARIAS,'* SUSANA LOPEZ,' JOANA D'ARC P. MASCARENHAS,2 PEDRO ROMERO.'PAUL CANO,' YVONE B. GABBAY,2 RONALDO B. DE FREITAS,2 AND

ALEXANDRE C. LINHARES2Departamento de Biologia Molecular, Instituto de Biotecnologia, Universidad Nacional Aut6noma de Mexico,

Apartado Postal 510-3, Colonia Miraval, Cuernavaca, Morelos 62271, Mexico,1 and Se,ao de Virologia,Instituto Evandro Chagas, Fundaqdo Servicos de Saude Publica, 492, 66.050 Belem, Pard, Brazil2

Received 10 May 1993/Returned for modification 7 September 1993/Accepted 23 September 1993

We have characterized the neutralizing antibody immune response to six human rotavirus serotypes (GI toG4, G8, and G9) in Brazilian children with primary and secondary rotavirus infections and correlated theresponse with the G serotype of the infecting rotavirus strain. Twenty-five children were studied: 17 had a

single rotavirus infection, 4 were reinfected once, and 4 experienced three infections. Two of the reinfectionswere by non-group A rotaviruses. Among the 25 primary infections, we observed homotypic as well as

heterotypic responses; the serotype Gl viruses, which accounted for 13 of these infections, induced mostly a

homotypic response, while infections by serotype G2 and G4 viruses induced, in addition to the homotypic, a

heterotypic response directed primarily to serotype Gl. Two of the primary infections induced heterotypicantibodies to 69M, a serotype G8 virus that by RNA electrophoresis analysis was found not to circulate in thepopulation during the time of the study. The specificity of the neutralizing antibody immune response inducedby a virus of a given serotype was the same in primary as well as secondary infections. These results indicatethat the heterotypic immune response induced in a primary rotavirus infection is an intrinsic property of thevirus strain, and although there seem to be general patterns of serotype-specific seroconversion, these may vary

from serotype to serotype and from strain to strain within a serotype.

Group A rotaviruses are the leading cause of severe dehy-drating gastroenteritis in children under 3 years of age, andthere is considerable interest in developing an effective vaccine(22). The surface of group A rotaviruses is formed by twoproteins, VP4 and VP7. VP4 forms spikes that extend from thesurface of the virus particles and is involved in a variety of viralfunctions, including virulence, agglutination of erythrocytes,and trypsin-enhanced infectivity (13). VP7 is a glycoproteinthat, in addition to VP4, has been proposed to be responsiblefor the initial attachment of rotaviruses to the target cells (13).The antibody response to these proteins has the ability toneutralize the infectivity of the virus in vitro as well as in vivo(28, 32, 36), and the specificity of these antibodies in neutral-izing different rotavirus strains has been used to classifyrotaviruses in various serotypes. Since VP7 and VP4 bothinduce neutralizing antibodies, the viruses can be classified byeither of these two surface proteins (13, 14, 18, 21).Based on VP7, 14 different G serotypes (G for glycoprotein)

have been identified among group A rotaviruses so far (2-4, 13,16, 37). Nine of these serotypes (Gl to G4, G6, G8 to G10, andG12) infect humans, although four of them (Gl to G4) appearto account for the majority of isolates (23). The G serotypeshave been defined by cross-neutralization assays with hyper-immune animal sera to the complete virus particles (21). Basedon VP4, at least 11 P types (P for protease sensitivity) havebeen found by antigenic reactivity (18) and genomic (hybrid-ization and amino acid sequence) analysis (13, 20, 35). Four ofthe genomic P types have been isolated from humans (13, 14),corresponding to serotypes PlA, PlB, P2, and P3, defined byneutralization with hyperimmune sera directed to the VP4protein (18). Recently, the human rotavirus strain 69M (sero-

* Corresponding author. Phone: (5273) 11-4900. Fax: (5273) 17-2388.

type G8) was reported to be a new genomic P type (35) thatmight represent a fifth human P serotype, but this has not beenconfirmed serologically.Both homotypic and heterotypic neutralizing antibody

(NtAb) serological responses have been observed in rotavirusinfections of seronegative children (8, 10, 11, 15, 34, 40).However, there is very limited information which correlatesthe serotype of the infecting virus with the specificity of theneutralizing immune response in primary rotavirus infections(5, 17). Determination of the specificity of the neutralizingimmune response induced by the various rotavirus serotypesand study of the role of this response in protection againstsubsequent reinfections or disease would be most useful fordesigning rotavirus vaccines.

In this work, we characterized the neutralizing immuneresponse to rotavirus serotypes Gl to G4, G8, and G9 inchildren with serologically defined primary infections andreinfections and correlated this immune response with the Gserotype of the infecting virus.

MATERIALS AND METHODS

Patients and specimens. We studied the immune responseto rotavirus infection in 25 children who were part of a

longitudinal study carried out in Belem, Brazil (24). In thatstudy, the children were monitored from birth to 3 years of age.While they were in the hospital, feces were collected daily fromthe newborn children; when the child went home, fecal sam-

ples were obtained on alternate days up to day 14, and fecessamples were collected during any diarrheal episode. After day14, feces samples were collected fortnightly or whenever signsof diarrhea were present. Serum samples were routinely col-lected every 6 months, and acute- and convalescent-phaseserum samples were collected during any diarrheal episode. At

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the time of this study, about one-half of the acute- andconvalescent-phase sera had been used up; therefore, in thosecases, the routinely collected sera were used as preinfectionand postinfection sera to study the immune response. Theacute-phase serum samples were collected 1 to 2 days afteronset of symptoms, and convalescent-phase serum was ob-tained 2 to 3 weeks later. The time that the preinfection serumsamples were collected ranged from 4 days to 2.5 months(mean, 29 days) before the onset of symptoms, while thepostinfection serum samples were collected from 1 to 6 months(mean, 3.2 months) after infection. Most children were be-tween 6 and 24 months old (mean, 13 months) at the time ofprimary infection.IgM and IgG ELISA. Immulon II (Dynatech Laboratories,

Inc.) microtiter plates were coated with a 1:5,000 dilution ofgoat anti-human rotavirus strain D (kindly provided by HarryB. Greenberg, Stanford University) in phosphate-bufferedsaline (PBS) for the enzyme-linked immunosorbent assay(ELISA). After overnight incubation at 4°C, the plates werewashed twice with PBS and blocked with 3% fetal bovineserum in PBS for 1 h at 37°C. The plates were then washedtwice with PBS and incubated for 2 h at 37°C with a Freon-extracted MA104 cell lysate that had been infected withrotavirus SA114fM or mock infected. After the plates werewashed four times, serial dilutions of the children's sera wereadded to duplicate wells and incubated for 1 h at 37°C. Theplates were then washed four times and incubated with a1:1,000 dilution in PBS of goat anti-human immunoglobulin G(IgG) or IgM conjugated to peroxidase (Kirkegaard & PerryLaboratories) and incubated for 1 h at 37°C. The plates werewashed four times, and the presence of peroxidase activity wasdetected by incubation fot 30 min at room temperature witho-phenylenediamine hydrochloride (Sigma Chemical Co.) asthe substrate. The reaction was stopped with 4 N H2SO4, andthe optical density was read at 492 nm. The IgG and IgMantibody titers were defined as the highest serum dilution thatgave an optical density of .0.2 and greater than twice thenegative control value obtained when mock-infected cells wereused as the antigen.

Serotyping ELISA. The serotyping ELISA was done asdescribed previously (33).NtAb assay. NtAb titers in the children's sera were mea-

sured by an immunochemical focus reduction neutralizationtest (1). The titer of NtAb in a serum sample was defined as thehighest serum dilution at which a reduction of at least 60% inthe number of infected cells was observed compared withcontrols for which PBS had been used instead of serum.

RESULTS

ELISA antibody response. The aim of this study was todetermine the specificity of the NtAb immune response ofchildren who had experienced primary and secondary infec-tions with rotavirus and associate this response with the Gserotype of the infecting rotavirus strain. Since the childrenincluded in this work were followed up from birth in a

longitudinal study, it is reasonable to assume that the firstrotavirus infections detected represented primary infections.To support this assumption, we analyzed the immunoglobulinclass specificity of the children's serum antibody response,

since it has been shown that the presence of virus-specific IgMis a reliable marker for primary rotavirus infections (17, 19). Ofthe 25 children studied, 17 had a single rotavirus infection and8 were reinfected once or twice with rotavirus.As mentioned in Materials and Methods, some of the acute-

and convalescent-phase serum samples collected from the

TABLE 1. ELISA IgM and IgG antibody response for serumsamples from 25 children with presumed primary rotavirus infections

No. of positive samples/totalSerum' (geometric mean titer)

IgM IgG

Acute phase 8/14 (218) 2/14 (400)Preinfection 0/9 0/9

Convalescent phase 9/10 (216) 8/10 (566)Postinfection 0/15 12/15 (599)

Total 15/25 20/25U The acute-phase or preinfection sera were not available for two of the

children.

infected children included in this study were no longer avail-able. In such cases, the preinfection and postinfection serumsamples that had been obtained routinely were used instead. In15 of the 25 (60%) presumed primary infections, we detectedIgM in either the acute- or convalescent-phase serum or inboth serum samples, suggesting that these were indeed primaryinfections. Eight of 14 acute-phase serum samples had detect-able virus-specific IgM levels (titer, 1:100 to 1:400), while noneof the 9 preinfection serum samples did (Table 1). In addition,9 of 10 convalescent-phase serum samples had IgM (titer,1:100 to 1:400), while none of the 15 postinfection serumsamples had any detectable IgM. Only two of the children hadIgM in both the acute- and convalescent-phase serum samples.We had an acute-phase serum sample for only 3 of the 10IgM-negative children; for the other 7, we had only thepreinfection serum. Furthermore, for all 10 IgM-negativecases, we had postinfection rather than convalescent-phaseserum. Therefore, it is not surprising that we could not findIgM in these children, since this antibody class appears earlierthan IgG and apparently disappears after 2 to 3 weeks (17). NoIgM response was observed in the reinfections.We also analyzed the IgG response; 2 of the 14 acute-phase

serum samples had detectable IgG (titer, 1:200 and 1:800), andin both samples, were also detected IgM. None of the 9children whose preinfection sera were analyzed had preexistingIgG. For the convalescent-phase and postinfection serumsamples, 8 of 10 and 12 of 15, respectively, had detectable IgGlevels, giving a total of 20 of 25 children (80%) with detectableIgG levels (titer, 1:200 to 1:3,200 in both types of sera). In 18of these cases, there was seroconversion (fourfold increases inthe titer), and in 2 cases, the IgG titer did not change. Thus, theabsence of rotavirus-specific IgG in the preinfection sera andthe presence of IgM antibody in 90% of the convalescent-phase sera from the children studied seem to indicate that thefirst rotavirus infections detected were primary infections.NtAb response in primary infections. The G serotype of 21

of the 25 rotavirus strains isolated from the primary infectionshad been reported previously (25). In this work, we determinedthe G serotype of two of the strains that could not be typed inthe previous study (patients 24.075 and 24.145), both of whichwere shown to have serotype G3 specificity. Overall, 13 of therotavirus strains isolated from primary infections were sero-type Gl, 4 were serotype G2, 2 were serotype G3, and 4 wereserotype G4. Two rotavirus strains were untypeable but hadbeen shown before to belong to subgroup II (25).We analyzed the specificity of the NtAb response induced by

these rotavirus strains; the acute-phase or preinfection seraand the convalescent-phase or postinfection sera were testedfor their ability to neutralize the infectivity of human rotavi-

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TABLE 2. NtAb seroconversion in children witirotavirus infections

Patientno.'

23.979*24.004*24.013*24.05924.09324.108*24.17524.19124.376*24.378*24.38324.384*24.415

23.996*24.072*24.169*24.195*

24.145*24.075*

23.943*24.00224.16624.333

23.98324.053*

Infecting virusG serotype

1111111111111

2222

Seroconversionbto G serotype

111111,211,811111

1,21, 2, 81, 21, 2

33

4444

? (II)y? (II)

1,41,41, 3, 4d1, 4d

1,31

a *, IgM detected in either the acute- or convalescentb Seroconversion is defined as a fourfold increase in tl

of the children had any detectable NtAb in the preimmunsample.

A, asymptomatic; S, symptomatic rotavirus infectiond Twofold increase in NtAb titer but no seroconversioThe subgroup specificity is indicated in parenthe

serotype could not be determined.

presumed primary rotavirus strains, one seroconverted to serotype Gl, and theother one seroconverted to serotypes Gi and G3. It is remark-

Age able that all but the two serotype G3 viruses induced a(mo) SymptomsC neutralizing response to rotavirus strain Wa (serotype GI).

The heterotypic NtAb response induced by serotype G2 and15 S G4 viruses to the serotype Gi virus Wa was strong, since it was15 S equal to the homotypic response when the infecting virus was

11 S1 A serotype G2 and even two- to fourfold higher than the

12 S homotypic response in serotype G4 infections.6 S NtAb response in secondary infections. Of the 25 children6 A analyzed in this study, 8 had more than one rotavirus infection:10 S 4 had two and 4 had three infections. Five of the eight11 S reinfected children were initially infected by a serotype Gl3 A virus, and four of these five children were reinfected by a7 S serotype G2 virus. As observed in the primary infections, the19 S four secondary infections with serotype G2 strains induced10 A homotypic antibodies (except in patient 24.059; see Table 3)20 S and increased the titer of already existing NtAb to the serotype30 S GI strain Wa. The one secondary infection caused by a18 A serotype G3 virus induced homotypic antibodies as well as a23 S heterotypic response to rotavirus strain Wa. The other three

secondary infections were caused by subgroup II viruses that10 A could not be typed; one of these infections induced NtAb to22 S the serotype G8 virus 69M. None of the five serotyped

24 S rotavirus strains isolated from secondary infections was of15 S serotype Gl.26 A Of the four strains isolated from children who had already10 S experienced two rotavirus infections, two were non-group A

rotaviruses, one was serotype G2, and one was serotype G3.4 S The infection by the two non-group A rotaviruses did not7 S modify the preexisting levels of NtAb to the group A viruses

tested, as expected, since these two groups of rotaviruses are

he titer of NtAb. None not antigenically related (30). The tertiary infection by thee or preinfection serum serotype G2 virus induced homotypic antibodies and increased

the level of preexisting antibodies (already present after theprimary infection) to serotypes Gi and G3 (patient 23.983).

nses for strains whose The serotype G3 tertiary infection induced homotypic antibod-ies and increased the level of preexisting NtAb to serotype Gi(patient 24.059).

ruses belonging to G serotypes 1 (Wa), 2 (S2), 3 (P), 4 (ST3),8 (69M), and 9 (WI61). The VP4-based P serotype has beenreported to be 1A for rotavirus strains Wa, P, and WI61; PIBfor strain S2; and P2 for strain ST3 (18). Rotavirus strain 69Mmay represent a new human P serotype (35). Since the Pserotype of the infecting strains was not determined, wheneverwe refer to a heterotypic response, we are doing so only withregard to VP7, but it should be kept in mind that the responsecould be not heterotypic with regard to VP4.

All children infected with serotype GI strains seroconvertedto the serotype Gi virus Wa, and two seroconverted to eitherserotype G2 or G8 in addition to serotype Gl (Table 2). Allchildren infected with serotype G2 viruses seroconverted toboth serotypes Gi and G2, and one of these children alsoseroconverted to serotype G8. Neither of the two serotypeG3-infected children had detectable neutralizing activity to anyof the six strains tested. It is interesting that these two childrenhad no detectable IgG in the postinfection sera, and one ofthese two infections was asymptomatic; however, in both cases,IgM was detectable in the acute-phase serum. The fourserotype G4-infected children seroconverted to serotype Gl,and all four also had an increase in the level of NtAb to theserotype G4 strain ST3, but only two of them seroconverted;one serotype G4-infected child also seroconverted to serotypeG3. Finally, of the two children infected with the untypeable

DISCUSSION

We have analyzed the serotype specificity of the NtAbresponse of children with primary and secondary rotavirusinfections. The serological analysis supports the idea that thefirst rotavirus detected in the children studied represented aprimary infection; the children did not have preexisting class-specific or neutralizing rotavirus antibodies in the preinfectionserum samples, and 9 of 10 of the available convalescent-phasesera had detectable IgM, which has been shown to be a specificmarker for primary rotavirus infections (17, 19). The child thatdid not have detectable IgM in the convalescent-phase seruminstead had IgM on the acute-phase serum.

In the first studies that characterized the NtAb serologicalresponse of children infected with rotavirus, there were con-flicting results regarding the ability of a primary rotavirusinfection to induce a heterotypic antibody response in humans.Some observed a homotypic response (40), while others foundit to be heterotypic (10, 11, 15, 34); however, in most of thesestudies, the nature of the infection (primary or secondary) wasnot defined or the serotype of the infecting virus was notdetermined, complicating the interpretation of the heterotypicresponses observed and the understanding of the relationshipbetween such responses and the serotype of the infecting virus.Heterotypic antibody responses induced by single rotavirusinfections have also been suggested from seroepidemiological

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TABLE 3. NtAb immune response in children with secondary rotavirus infections

Infecting NtAb titer in acute-phase or preinfection/convalescent-phase or postinfection sera to indicatedPatient Age virus G rotavirus strain (G scrotype) Symptoms"no. (mo) viuGserotype' Wa (1)" S2 (2) P (3) ST3 (4) 69M (8) WI161 (9)

23.983 4 ? (II) --"/400 -1<50 -/200 -1<50 -1<50 -1<50 S20 ? (II) 400/200 <501<50 200/200 <501<50 <50/400 501<50 S27 2 100/400 <50/200 200/400 <501<50 100/<50 <50/<50 A

24.004 15 1 <50/800 <501<50 <501<50 <501<50 <501<50 <501<50 S26 2 100/400 <50/100 <501<50 501<50 <501<50 <501<50 S

24.053 7 ? (II) 50/200 <501<50 <501<50 <501<50 <501<50 <501<50 S20 3 200/800 <501<50 <50/1600 <501<50 <50/<50 <501<50 S35 Non-A 800/200 <501<50 1,600/200 <501<50 <501<50 <501<50 S

24.059 1 1 -/400 -1<50 -1<50 -1<50 -1<50 -1<50 A20 2 100/400 <501<50 <501<50 <501<50 <501<50 <501<50 S32 3 400/800 <501<50 50/200 <501<50 <501<50 <501<50 S

24.108 6 1 50/800 <50/100 <501<50 <501<50 <501<50 <501<50 S18 2 100/400 100/200 501<50 <501<50 <501<50 <501<50 S

24.145 10 3 <50/<50 <501<50 <501<50 <501<50 <501<50 <501<50 A30 ? (II) <50/<50 <501<50 50/200 <501<50 <501<50 <501<50 A33 Non-A <50/<50 <501<50 200/<50 <501<50 <501<50 <501<50 S

24.378 3 1 <50/200 <501<50 <501<50 <501<50 <501<50 <501<50 A16 2 200/400 50/200 <501<50 <501<50 <501<50 501<50 S

24.384 9 1 <50/100 <50/<50 <501<50 <501<50 <501<50 <501<50 S17 ? (?) 100/NDe <501<50 <50/100 <501<50 <501<50 <501<50 S

aWhen the serotype of the infecting strain could not be determined, the subgroup specificity is indicated in parentheses./In this column, acute- and convalescent-phase sera are underlined and pre- and postinfection sera are not underlined.'See Table 2, footnote c."-, neither the acute nor the preinfection sera were available.ND, not determined.

studies (7, 8, 39) and have also been found in an epitope-blocking assay designed to detect the antibody response toindividual epitopes (27, 28, 38). However, with the epitope-blocking assay, heterotypic responses have been observedmostly in individuals with preexisting rotavirus antibodies intheir sera, suggesting that it represents a broadening of theresponse to heterotypic epitopes after multiple infections.

Recent studies have correlated the known infecting rotavirusG serotype with the specificity of the NtAb immune responsein serologically defined primary rotavirus infections (5, 17, 31).In one study (17), the pattern of response was characteristic ofthe infecting serotype: infections with serotype G1 strainsinduced antibodies to serotypes G1 and G4; serotype G2viruses induced NtAb to serotypes G1, G2, and G4; andserotype G4 strains induced antibodies to serotypes GI andG4. Similarly, Briussow et al. (5) found primary heterotypicresponses to serotypes G3, G4, and G9 but not to serotypes G2and G8 after serotype G1 infections. In the study by Offit et al.(31), primary rotavirus infections induced a heterotypic NtAbimmune response that was primarily directed at VP4.

In this study, we observed homotypic as well as heterotypicpatterns of response in primary infections. The serotype G1viruses induced mostly a homotypic response (85%), whileinfections by serotype G2 and G4 viruses induced, in additionto the homotypic response, a heterotypic response directedprimarily to serotype G1. The induction by serotype G1 strainsof preferentially homotypic responses has also been observedby others (15, 33). Overall, 92% of the 25 children developedNtAb to the serotype G1 strain Wa in the primary infection,

regardless of the serotype of the infecting virus. Our results,compared with those of Gerna et al. (17) and Brussow et al.(5), indicate that although some virus serotypes may be moreprone to induce heterotypic responses and there may be apattern of NtAb response associated with a specific serotype ina particular time and geographical location, the specificity ofthe NtAb immune response will ultimately depend on theparticular immunogenic characteristics of the infecting rotavi-rus strain, regardless of its serotype.As recognized by others, the induction of heterotypic anti-

bodies in primary infections is most likely due to the presence ofheterotypic domains on VP4 and VP7. In this regard, it isinteresting that only a few of the children's sera tested neutral-ized strain P (serotype G3) and none of the sera neutralizedstrain W161 (serotype G9), rotavirus strains that have beenshown to have the same VP4 type (serotype PIA) as theserotype G1 strain Wa. These results suggest that VP7 is theprotein that induced the cross-reactive NtAb; however, theseobservations may also be explained by antigenic differenceswithin specific G or P serotypes among the strains isolated fromthe patients and the reference strains used in the neutralizationassay, as suggested before (31), or by the interaction of partic-ular types of VP7 and VP4 (9). Elucidation of the molecularbasis of the heterotypic response and of the interactions be-tween VP4 and VP7 that influence such a response is of obviousrelevance for the design of rotavirus vaccines.Two of the primary infections by serotypes G1 and G2

induced heterotypic antibodies to the serotype G8 virus 69M,which has a supershort RNA pattern (29). Since this conspic-

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uous RNA pattern was not detected during the longitudinalstudy (24), it is very unlikely that serotype G8 viruses circulatedin the study population at that particular time, suggesting thatprimary infections can indeed induce heterotypic NtAb. In thisregard, in a study conducted in Ecuador, NtAb to serotype G8viruses was detected in the serum of 23% of 870 children,although only 12 of these children showed neutralizing activityexclusively to serotype G8 and not to serotypes G1 to G4,which were also tested (6). In addition, the absence of NtAb toserotype G9 viruses in Brazilian children from the area ofBelem, Para, is in contrast to the high prevalence reported forserotype G9 antibody in sera from Ecuadorian (5) and Amer-ican (12) children.

In secondary infections, the pattern of heterotypic NtAbinduced by a given rotavirus serotype was the same as thatinduced by that strain in a primary infection. For instance,primary infections with a serotype G2 strain induce NtAb toserotypes G1 and G2, while a secondary infection by a serotypeG2 virus, when the primary infecting strain was of serotype Gl,induced homotypic NtAb and boosted the response of NtAb toserotype G1. These results suggest that the broadening of theNtAb immune response in secondary infections will depend onthe particular infecting strain and that the second rotavirusinfection with a given serotype will not induce a broaderimmune response than that induced by the same virus in aprimary infection, as has been suggested (7). The immuneresponse induced by rotavirus serotypes G2 and G3 in tertiaryinfections was also characterized by seroconversion to theinfecting serotype and a boost to the preexisting heterotypicNtAb; no further broadening of the NtAb response wasobserved. We found no difference in the level or specificity ofthe NtAb response in children with asymptomatic and symp-tomatic rotavirus infections, as was observed by Losonsky andReymann (26).We have shown here that primary rotavirus infections can

induce a heterotypic NtAb immune response and that this is anintrinsic property of the virus strain. Although there seem to begeneral patterns of serotype-specific seroconversion, these mayvary from serotype to serotype and from strain to strain withina serotype.

ACKNOWLEDGMENTS

We are grateful to A. Ruiz for excellent technical assistance and toL. Padilla-Noriega for critical reading of the manuscript.

This work was partially supported by grants 75191-527101 from theHoward Hughes Medical Institute and RF89088#66 from the Rock-efeller Foundation.

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