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Rotaviruses are recognized as the major cause of severe acute infantile gastroenteritis. Based on the viral outer capsid proteins VP4 and VP7, which independently elicit virus-neutralizing, protective antibodies, a dual serotyping system for rotavirus has been adopted. VP4 genotypes, which were identified on the basis of sequence differences and which, when tested, correlate with serotypes, have also been designated. Human rotaviruses contain at least eight VP4 (P) genotypes and at least nine VP7 (G) serotypes, the most common of which are P[4], [6], and [8] and G1 to 4.

Immunity to rotavirus infection in children has been shown to correlate with serum (15) and intestinal or stool antibodies (5) to rotavirus, but titers of serotype-specific, heterotypic, and neutralizing serum antibodies and isotype-specific antibodies in serum and intestine or stools cannot be used reliably as markers of protection against subsequent illness (15). The contribution of neutralizing coproantibodies (fecal antibodies) to immunity in children requires more study, particularly as serological immune correlates of protection have not been identified for design and evaluation of effective rotavirus vaccines, and intestinal antibody responses have not yet been measured during vaccine trials (16).

Intestinal immunoglobulin A (IgA) to rotavirus has been shown to be the most-sensitive marker of rotavirus infection (6), and fecal antirotaviral IgA levels can be used to predict the presence of duodenal IgA (14). Fecal IgA coproconversions correlate with fecal rotavirus-neutralizing antibody conversions (8). Coproconversions in rotavirus-neutralizing IgA are more-sensitive indicators of rotavirus infection and reinfection than seroconversion in IgG, IgM, IgA, or neutralizing antibodies, and persistent elevations in stool rotavirus-neutralizing IgA (termed coproIgA plateaus) correlate with protection against reinfection and symptomatic illness in young children (5). In a small number of children, the serotype specificity of the stool rotavirus-neutralizing IgA responses has been studied (6, 8). However, it is not known whether the P or G serotype specificity of these responses parallels the specificity of the rotavirus-neutralizing responses in serum following severe rotavirus gastroenteritis and rotavirus reinfection. The duration of neutralizing coproantibody excretion in stools following rotavirus infection is not known either.

The aim of this study was to compare the nature and duration of rotavirus-neutralizing antibody responses in sera and stools of children during the acute and convalescent phases of severe rotavirus gastroenteritis and during at least 5 months of longitudinal monitoring thereafter. The children studied were admitted to the infectious diseases ward of the Royal Children's Hospital, Melbourne, Australia, between April 1984 and September 1985 with acute rotavirus gastroenteritis diagnosed on clinical grounds and in the laboratory by the presence of rotavirus by electron microscopic examination of stool extracts and/or by the presence of viral antigen in stools detected by enzyme immunoassay (EIA). The 15 children studied, 2 to 39 months old at recruitment, were a subset of the 44 children recruited at this time for longitudinal study of rotavirus infection and immune responses. This subset was selected from the first 24 children from whom complete sets of samples were obtained and was chosen to contain similar numbers of children infected with G1 and G4 rotavirus. The clinical, demographic, and laboratory findings for these 44 children have already been described (5, 6, 14). Prior to enrollment, parents were provided with a detailed explanation of the study (including the need to obtain blood samples from the infants), and they gave their signed consent. The study was approved by the Human Ethics Committee of the Royal Children's Hospital.

Titers of neutralizing antibody were measured in sera collected in the acute and convalescent phases and at 4-month intervals post-onset of diarrhea, in fecal specimens collected daily while the child was in the hospital, and in stools collected at 7- to 10-day intervals for 219 to 721 days from the onset of severe rotavirus gastroenteritis. Stools collected by parents at home were stored frozen at 4°C for up to 1 month before transport to the Royal Children's Hospital (14). Feces and sera were stored at 70°C until tested. Rotavirus-neutralizing antibodies were measured by fluorescent focus reduction neutralization assay (FFN) with MA104 cells as described previously (6, 8). Samples were titrated against cell culture-adapted human rotavirus strains RV-4, Wa and Ku (P[8], G1), RV-5 (P[4], G2), RV-3 (P[6], G3), ST-3 (P[6], G4), and VA70 (P[8], G4). RV-4 and RV-5 were isolated from stools of Melbourne children with rotavirus gastroenteritis, whereas RV-3 was obtained from an asymptomatically infected Melbourne neonate (RV-3). Strains Wa, Ku, and VA70 were obtained from children hospitalized with gastroenteritis in the United States, Japan, and Italy, respectively. ST-3 was isolated from an asymptomatically infected neonate in the United Kingdom. The origins and sources of these rotaviruses have been reported previously (3, 7, 9). All virus strains were propagated in MA104 cells in the presence of trypsin (9). Fourfold dilutions of sera, starting at 1:100, were incubated with each trypsin-activated, cultivated rotavirus strain. The neutralization titer of each sample was expressed as the reciprocal dilution giving 50% reduction in the number of fluorescing cells. Fecal extracts (n = 40) containing no antirotaviral IgA, IgM, or IgG by EIA all gave reciprocal titers of <200 by FFN. Stool samples with titers of <200 were therefore considered to be negative for neutralizing antibody to the rotavirus strain tested. An immune conversion in neutralizing antibody in sera or stools was considered to be a fourfold increase in reciprocal titer to at least 400 (6).

Stool rotavirus antigen, VP7 serotypes, and VP7 monotypes (classified on the basis of antigenic variation within a VP7 serotype) were determined with rotavirus-specific monoclonal antibodies in antigen-capture EIAs as described previously (4, 10). For a few patients, sufficient stool sample was available for determination of VP4 genotype by reverse transcription-PCR amplification of viral RNA with nested primers (11). VP4 (P) genotypes were determined to confirm that they corresponded to the genotype expected of G1 and G4 rotaviruses causing gastroenteritis in children, i.e., P[8]. All genotypes were P[8] and are listed in Table 1.

All children infected with either G1 or G4 rotavirus showed serum neutralizing antibody responses to G1 and G4 rotaviruses (Table 1). Compared with serum responses, neutralizing antibody was found less often in stools, and fewer children showed antibody conversions to fewer rotavirus strains. All of the children with any detectable stool antibody response (80%) produced neutralizing antibody to G1 rotavirus RV-4. A broadening of these responses to include additional virus serotypes and strains was observed with increased age of the children at the time of illness. For example, only one child aged 12 months or less had serum neutralizing antibody to the P[4], G2 virus RV-5, and none had antibodies neutralizing the P[6], G3 virus RV-3. Nine of the ten children aged 16 to 39 months had serum antibody to at least one of these two heterotypic viruses. Most children seroconverted and coproconverted (or showed positive coproantibody levels) to P[8], G1 rotavirus(es). Many showed seroconversion to P[6] or [8], G4 rotaviruses (80%) and coproconversion to P[6], G3 virus (67%) and P[6] or P[8], G4 viruses (47%). Fewer children seroconverted to the P[4], G2 and P[6], G3 strains (33%) or coproconverted to P[4], G2 virus (13%).

As neutralizing antibody titers of children infected with G1a and G1c virus strains were similar, comparisons of the geometric mean titers (GMT) of serum and fecal neutralizing antibody in children infected with G1 or G4 rotavirus could be made (Table 2). The GMT to G1 and G4 viruses in convalescent-phase sera were at least twice the GMT to RV-5 and RV-3 viruses. Titers to G1 viruses Wa and Ku were significantly higher in acute-phase sera in G1 than G4 rotavirus infections, whereas G4 infections stimulated significantly higher levels of neutralizing antibody to ST-3 than did G1 infections. The highest GMT of serum neutralizing antibody were directed to local P[8], G1 virus RV-4, followed by P[8], G1 viruses Ku and Wa, in the case of P[8], G1-infected children, and by P[8], G4 virus in the case of P[8], G4 virus-infected children. The children infected with G4 rotavirus showed slightly higher GMT of neutralizing coproantibodies to all strains tested than did the G1 virus-infected children, although these differences were not significant. The highest titers observed were to the local P[8], G1 virus RV-4 and the local P[6], G3 virus, RV-3, irrespective of the infecting rotavirus serotype. These results show that during the acute and convalescent phases of severe primary G1 or G4 rotavirus gastroenteritis, neutralizing antibody responses were directed to viruses of the infecting G type in both serum and stools. However, significant responses were also directed to G1 viruses, in the case of G4-infected children, and G4 viruses, in the case of G1-infected children, as has been reported previously for serum responses alone (12). Thus, the responses in serum and stools during and following primary P[8], G1 and P[8], G4 rotavirus gastroenteritis were heterotypic for P and G serotypes and G monotypes and similar between sites.

The duration of excretion of virus-neutralizing coproantibodies after severe rotavirus gastroenteritis was determined for the first time in this study. As shown in Fig. 1, stools collected between 5 and 8 days after the onset of diarrhea were the most likely to contain neutralizing coproantibodies (70%). In contrast, only 31 (56%) of those collected on days 9 to 16 contained neutralizing coproantibody and 6 (26%) of stools collected on days 17 to 28 contained this antibody. Thus, in vaccine trials and further studies of natural infection, more limited acute- and convalescent-phase stool sampling than was done here is still likely to be informative.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Frequency of detection of rotavirus-neutralizing antibodies in stools relative to the timing of stool collection after the onset of rotavirus gastroenteritis.

The FFN coproantibody and serum antibody profiles of the children followed from 120 days to at least 391 days post-onset of severe rotavirus gastroenteritis are presented in Tables 3 and 4. As assessed by seroconversion in antirotaviral IgG, IgA, and IgM and neutralizing antibodies, coproconversion in IgA and neutralizing antibodies, and rotaviral antigen excretion in stools, these children had one to four rotavirus reinfections during this period (5, 6). All sera collected >120 days after onset contained neutralizing antibodies to P[8], G1 virus Wa, and most contained antibodies able to neutralize P[4], G2, P[6], G3, and P[6], G4 rotaviruses (Table 4). In the children originally infected with G1 rotavirus, a higher percentage of sera (78%) than stools (10%) contained G4-neutralizing antibody. Children with P[8], G1 or P[8], G4 virus as the cause of their severe gastroenteritis showed similar percentages of stools containing P[8], G1, P[4], G2, and P[6], G3-neutralizing antibodies. However, children with severe gastroenteritis caused by P[8], G4 rotavirus showed a significantly higher proportion of stools (74%) containing P[6], G4 (ST-3)-neutralizing antibody (P < 0.01) than did children initially enrolled with P[8], G1 virus-associated gastroenteritis (10%). The prevailing rotavirus serotypes during this period in this population were G1 (common) and G4 (rare) in the first year and G1 in the second year (2). Thus, although it was not possible to type the reinfecting virus, the children monitored longitudinally were likely to have been reinfected with G1 rotavirus. This suggests that the concept of "original antigenic sin," whereby prior exposure to one influenza virus strain is able to divert the antibody response to a second challenging virus strain to focus on the shared (cross-reactive) epitopes (13), may apply to the rotavirus-neutralizing coproantibody responses of these children. Further studies of these antibodies during natural infection and in vaccine trials are needed to resolve this question.

As was seen in stools collected within 28 days of the onset of severe gastroenteritis (Table 1), stools collected at least 120 days after the onset of severe rotavirus diarrhea contained neutralizing antibody to the local neonatal G3P[6] rotavirus RV-3 as often as antibody directed to the homologous serotype (G1 or G4) (Table 3). In contrast, the most frequently detected antibodies in sera collected at least 120 days after the onset of severe rotavirus diarrhea were directed to virus of the infecting serotype (Table 4). In a previous study of children in this population, we showed nonspecific neutralization of RV-3 by a maximum of 7% of stool extracts tested (8), so it is likely that at least 93% of responses to RV-3 in this study were specific. These children may have been infected with this virus as neonates (2), made no lasting serum rotavirus-neutralizing antibody response to RV-3, and remained susceptible to severe disease. Neonatal infection with RV-3-like rotaviruses has been shown to be protective against later severe rotavirus gastroenteritis but not against reinfection (1). However, neutralizing antirotaviral antibodies were not measured in that study. Alternatively, the neutralizing coproantibody responses in these children may have been directed to epitopes on VP7 or VP4 which are shared with RV-3 (9), whereas serum FFN responses might not include these epitopes. Differences like this in FFN responses to rotavirus between stools and sera may help explain serotype-specific immunity in the absence of type-specific neutralizing antibody in serum (16) and need further study.

FFN responses in sera and stools to the local virus RV-4 were of a higher level and were more frequent than those to the U.S. strain, Wa, and the Japanese isolate, Ku, irrespective of monotype of infecting or test virus. Similarly, as discussed above, stool FFN responses to local virus RV-3 were frequent and of a high level. Thus, local rotavirus isolates may be needed to give the most-sensitive measures of rotavirus-neutralizing antibodies in both sera and secretions. Overall, this study shows that serum neutralizing antibody responses appear to be a more useful measure of severely symptomatic rotavirus infection than stool responses, but these serum responses did not correlate with immunity to rotavirus in previous studies with nonlocal virus strains. Use of local strains in neutralization assays may help identify immune correlates of protection in vaccine trials.