This was the third time that this set of paired sera from the adult volunteers challenged with virulent D strain (G1P1A,NSP[B]) HRV were analyzed [14
]. In the original study[14
], virus neutralizing (VN) antibody titers were measured with plaque formation inhibition assay or inhibition of infectivity in monolayer tube cultures using HRV strain Wa (G1P1A,NSP4[B), DS-1xUK reassortant (G2P7,NSP4[A]), bovine rotavirus strains NCDV (G6P6,NSP4[A]) and UK (G6P7,NSP4[A]). Serum antibody titers were also measured with indirect immunoflurescence (IF) assay using the D strain as detector antigen, and with immune adherence HA (IAHA) assay and ELISA using 10% stool suspension of gnotobiotic calves infected with the D or DS-1 strain as detector antigens. The presence of IF antibody to the homologous D and relatively high levels of VN antibody to the DS-1xUK reassortant (≥1:60) or Wa virus (≥1:100) were associated with resistance to diarrhea; and IF and IAHA antibody to the D, and VN antibodies to the Wa, DS-1xUK reassortant and UK were associated with resistance to virus shedding[14
]. Thus, antibodies associated with resistance to virus shedding or diarrhea identified by all the assays performed in this original study were those directed against the VP7 or VP4 or both of the homotypic G1P1A HRV. Antibodies to DS-1 reassortant were associated with protection by VN but not other assays.
In the second study of these volunteer sera[20
], selected neutralizing monoclonal antibodies (mAb) directed to type-specific epitopes on G1, G2, G3 or G4 VP7 and on RRV VP4 (P5B[3
]) and to a cross-reactive epitope shared by VP4 of most HRV strains were used in a competitive epitope-blocking immunoassay in an attempt to dissect the specificity of the antibodies associated with protection against the D challenge. Certain levels of antibodies that blocked the binding of mAb 2C9 and 954/159, G1 and G3 VP7 specific, respectively, both mapping to antigenic site A correlated with resistance to infection or diarrhea. Thus the consensus of the two previous studies was that protection against the virulent D infection or diarrhea was associated with certain levels of serum antibody to the homotypic G1 VP7.
In the present study, serum IgG and IgA antibody responses to a total of 16 individual rotaviral proteins were determined, including all major serotypes/genotypes of HRV VP4, VP7 and human and animal RV NSP4. Statistical analysis showed that the presence of high levels of prechallenge IgG antibody to the G1 and G3 VP7, the P1A and P2A VP4, and the DS-1 [A] NSP4 correlated with resistance to the D infection. Prechallenge IgA antibody to the G1 VP7 and SA11 [A] NSP4 were also correlated with resistance to the D infection. However, among them, antibodies to G1 VP7 and P1A VP4 were most strongly associated with protection, as indicated by the smallest likelihood ratio p values. Thus, although the immunoassays as well as statistical analyses used in the current and two previous studies[14
] were different, the same conclusion (i.e., protective immunity was most closely associated with the presence of certain levels of prechallenge homotypic VP7 and VP4 antibody) was reached. This conclusion supports the development of a multivalent vaccine that would provide antigenic coverage to VP7 and VP4 types of global epidemiologic importance (e.g. multivalent human-animal reassortant rotavirus vaccines)[36
]. In an early study, logistic regression analysis of antibody titers in adult volunteers challenged with virulent HRV CJN strain also showed correlation between prechallenge serum IgG antibody titers to the homotypic G1P1A Wa (whole virus was used as detector antigen) and the probability of resistance to the HRV infection[26
]. In the current study, slightly less but similar levels of associations were also observed between IgG titers to G3 VP7 and P VP4 and resistance indicating that heterotypic antibodies also provided protection. This may explain why a monovalent Rotarix vaccine is effective against some heterotypic strains in clinical trials.
Our results indicate an important role of homotypic IgG VP7 antibodies to D (G1) as well as heterotypic IgG VP7 antibodies in protection. The heterotypic antibodies were likely induced by cross-reactive epitopes shared among VP7s[39
]. It is interesting to note that in both groups, there were individuals (albeit small in number) that IgG developed a seroresponse against G1, G3, and G9 viruses after D challenge whereas no IgG seroresponse was observed against G2 and G4 viruses (except for one individual in non-infected group against G2). This may indicate that G1 VP7 shares some cross-reactive neutralization epitopes with G3 and G9 VP7s, which may explain an observed protective role of certain levels of G3- and G9-specific IgG antibodies against G1 infection. VP7-specific neutralizing mAbs that displayed cross-reactivity between G1 and G3 viruses and between G3 and G9 viruses, respectively, have been generated[39
The prechallenge GMTs of IgG antibodies to DS-1[A] NSP4 were the highest among NSP4 antibody titers in the non-infected group which were significantly higher than those in the infected group and correlated with protection. As noted earlier, neutralizing antibodies to DS-1xUK in the initial study also correlated with protection. In contrast, prechallenge GMTs of IgG antibody to the homotypic Wa [B] NSP4 did not differ significantly between the groups and did not significantly correlate with protection, although the GMT was higher in the non-infected group than in the infected group. Our previous studies of experimentally infected gnotobiotic pigs showed that antibody responses to the homologous-host homotypic NSP4 did not confer protection against rotavirus infection or diarrhea[30
]. Broad and heterotypic antibody responses to NSP4 were reported also in children naturally infected with rotaviruses[27
The IgG antibody GMTs to P1A and P2A VP4 in the infected group increased the most after challenge with D (G1P1A). These observations are in accord with previous findings in which repeated infection/vaccination enhanced and broadened antibody responses to heterotypic VP4s [13
]. These findings also suggest the presence of cross-reactive epitopes on P1A and P2A VP4. In a recent study, porcine rotavirus strain Gottfried (P2B) which is closely related serologically to human P2A induced VN antibodies to VP4 with P1A or P1B specificity in guinea pigs[40
]. The GMTs of IgA antibody to all VP7 and VP4 genotypes (except for DS-1 VP4) in the infected group decreased postchallenge. The reason for the decrease in IgA antibody titers in contrast to the increased IgG antibody titers, albeit slightly, is unclear; it may suggest different dynamics of serum IgA and IgG VP7 antibody anamnestic responses to rotavirus infections in adult humans.
Growing evidence indicates that viremia occurs commonly during the acute phase of rotavirus infection in humans and other animal species[42
]. The systemic nature of the acute phase of rotavirus infection implies that rotavirus-specific IgG and IgA antibodies in the serum may play a direct role in protective immunity by directly neutralizing infectious viruses in the blood. Such neutralization will be most effectively mediated by antibodies to homotypic VP4 and VP7. In conclusion, the present study, which was the latest and most comprehensive analysis of the set of sera from adult volunteers challenged with the virulent D HRV, (i) provided important information on the homotypic and heterotypic viral protein-specific antibody responses and (ii) improved our understanding of the immunogenicity of the major viral proteins VP4, VP7 and NSP4 and their roles in homotypic and heterotypic protective immunity against rotavirus infection and diseases.