Viral proteins VP2, VP6, and NSP2 are strongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection

Rotavirus-specific IgA has been correlated with immune protection against rotavirus reinfection and symptomatic disease. Systemic and mucosal antibody responses were determined by an enzyme-linked immunosorbent assay in 11 infants with severe rotavirus gastroenteritis. Geometric mean titers of antirotavirus serum IgG and IgA antibodies were significantly higher during the convalescence of the disease (P < 0.001 vs. acute-phase titers). Rotavirus-specific fecal sIgA antibodies increased 4 times during the convalescence in 9 (81.8%) children (P < 0.001). The serum IgG and IgA antibody and fecal sIgA antibody responses to individual rotavirus polypeptides were characterized by radioimmunoprecipitation assay (RIPA) using Staphylococcus aureus protein A and the lectin jacalin to precipitate IgG- and IgA-immune complexes, respectively. The main IgG response was directed toward the structural viral proteins VP2, VP4, and VP6 and toward the nonstructural protein NSP2. Serum IgA reactivity was detected by RIPA in all serum samples, with major responses to VP2, VP6, and NSP2. Interestingly, fecal sIgA in convalescent samples reacted strongly toward NSP2 and VP6. These data reinforce the antigenic importance of rotaviral proteins other than VP4 and VP7, such as VP2, VP6, and NSP2, as main targets in the immune response to rotavirus. J. Med. Virol. 56:58–65, 1998. © 1998 Wiley-Liss, Inc.     

Serotype Specificity of the Neutralizing-Antibody Response Induced by the Individual Surface Proteins of Rotavirus in Natural Infections of Young Children

The relative contribution of the rotavirus surface proteins, VP4 and VP7, to the induction of homotypic as well as heterotypic neutralizing antibodies (NtAbs) in natural infections was studied. The NtAb titers of paired sera from 70 infants with serologically defined primary rotavirus infections were determined with a panel of rotavirus reassortants having one surface protein from a human rotavirus (serotypes G1 to G4 for VP7 and P1A and P1B for VP4) and the other surface protein from a heterologous animal rotavirus strain. A subset of 37 children were evaluated for epitope-specific antibodies to the two proteins by an epitope-blocking assay. The infants were found to seroconvert more frequently to VP4 than to VP7 by both methods, although the titers of the seroconverters were higher to VP7 than to VP4. Both proteins induced homotypic as well as heterotypic NtAbs. G1 VP7 frequently induced a response to both G1 and G3 VP7s, while G3 VP7 and P1A VP4 induced mostly homotypic responses.     

Analysis of homotypic and heterotypic serum immune responses to rotavirus proteins following primary rotavirus infection by using the radioimmunoprecipitation technique.

Three sequential serum samples collected from each of 20 young children with a naturally acquired primary rotavirus infection were assayed by the radioimmunoprecipitation technique for immunoglobulin G antibodies to rotavirus structural and nonstructural proteins of the four major human rotavirus serotypes G1, P1A; G2, P1B; G3, P2; and G4, P2. Fourteen children were infected with a serotype G1 rotavirus strain and six children were infected with a serotype G4 rotavirus strain. Sera were collected from each child in the acute and convalescent periods postinfection and also approximately 4 months later. Serum immune responses to rotavirus core antigens VP2 and VP3, to the major inner capsid antigen VP6, to nonstructural proteins NS35, NS28, and NS26, and to the outer capsid neutralization antigen VP4 of all test strains were detected in the majority of patients. These responses do not appear to be influenced by the G type or P type of the rotavirus strain used in the reactions. Homologous responses to the main neutralization antigen VP7 were detected in 93% of patients with serotype G1 infections and 50% of patients with serotype G4 infections. Heterologous VP7 responses were less frequently detected and were restricted to G1, G3, and G4 serotype rotavirus strains. No responses to VP7 of the serotype G2 rotavirus strain were detected in any patients. Heterotypic immune responses to the neutralization antigens, at least following serotype G1 and G4 infections, therefore appear to consist primarily of responses to VP4 rather than to VP7.     

Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection.

The outer capsid proteins VP4 and VP7 of group A rotaviruses are both targets of neutralizing antibody produced following natural infection in humans. Of interest is the relative importance and immunodominance of each protein in the generation of a protective immune response. In order to measure neutralizing antibody responses to VP4 and VP7 separately, reassortants bearing VP4 of each of the major human rotavirus P types with VP7 of SA11 origin were successfully produced by neutralizing monoclonal antibody selection. The resulting reassortants, together with reassortants representing each of the major VP7 types, were antigenically characterized with serotype-specific neutralizing monoclonal antibodies and hyperimmune sera. The neutralization proteins of human rotavirus origin were found to be unaffected antigenically by reassortment. The abilities of these reassortants to discriminate between VP4 and VP7 immune responses were evaluated with postinfection sera collected from three patients infected with either a P1A[8],G1, a P1B[4],G2, or a P1A[8],G4 rotavirus strain. The reassortants were shown to be capable of separating the neutralizing antibody responses to VP4 and VP7, with each patient showing a different immune response with respect to VP4 or VP7 immunodominance. These reassortants can now be applied to analyses of individual immune responses to VP4 and VP7 proteins after primary rotavirus infections and reinfections in humans.     

Quantification of systemic and local immune responses to individual rotavirus proteins during rotavirus infection in mice.

The purpose of the present study was to develop a quantitative assay that could be used to measure the local and systemic immune responses to specific rotavirus proteins following rotavirus infection of adult mice. To measure these responses, we used an immunocytochemical staining assay of Spodoptera frugiperda (Sf-9) cells which were infected with recombinant baculovirus expressing selected rotavirus proteins. The specificity of the assay was documented by using a series of monoclonal antibodies to individual rotavirus proteins. We observed that the assay had high levels of sensitivity and specificity for a series of VP7- and VP4-specific neutralizing monoclonal antibodies which recognized conformation-dependent epitopes on their target proteins. We also studied immunoglobulin G (IgG) immune responses in serum and IgA immune responses in the stools of mice infected with wild-type murine rotavirus strain EHPw. In both sera and stools, the most immunogenic proteins were VP6 and VP4. VP2 was less immunogenic than VP6 or VP4, and the immune responses to VP7, NSP2, and NSP4 were very low in serum and undetectable in stools.     

Homotypic serum antibody responses to rotavirus proteins following primary infection of young children with serotype 1 rotavirus

The class-specific antibody responses to serotype 1 rotavirus structural proteins were examined by immunoblotting with sera obtained from young children hospitalized with acute rotavirus diarrhea caused by serotype 1. All were believed to be primary infections. Three consecutive samples were obtained from 16 patients during the acute and convalescent phases of the disease and then approximately 4 months later. Immunoglobulin G (IgG)-class antibody responses to two inner capsid proteins (VP2 and VP6) and to the major homologous outer capsid protein (VP7) were detected in all patients. Antibody responses to VP6 were rapid, increased in intensity during 20 to 40 days after the onset of symptoms, and persisted for more than 4 months. Responses to VP2 and VP7 were more delayed, were maximal in convalescent-phase sera, and decreased markedly in intensity 4 months after the onset of symptoms in the majority of children. Two patients with evidence of mixed infection showed persisting high levels of antibody to VP7. Responses to the outer capsid protein VP4 were detected in 67% of patients, peaked at 20 to 40 days after the onset of symptoms, and were no longer detected at 4 months in the majority of patients. It is likely that the immunoblotting technique underestimated responses to VP4. Acute- and convalescent-phase sera (known to contain antirotavirus IgM or IgA measured by enzyme immunoassay) were also examined by immunoblotting. IgM- and IgA-class antibody responses to viral proteins VP2, VP4, and VP7 appeared to be qualitatively identical to those observed for IgG in the same serum samples.     

Humoral immunity patterns based on antibody reactivity to rotavirus antigens in Brazilian children under 5 years of age

The age distribution of antibody to simian rotavirus (SA-11) was studied in serum specimens obtained from 399 children aged to 5 years and living in the city of Recife (PE), located in the north eastern region of Brazil. Sera were examined for group-specific rotavirus antibody using a blocking enzyme immunoassay (bELISA) and a hemagglutination inhibition antibody (HIA) test, and for anti-VP2, anti-VP4, anti-VP6, and anti-VP7 antibodies using an immunoblotting assay (IBA). Antibody prevalence was similar in all bELISA and HIA assays, showing a steep rise in the 6- to 17-month-old age groups. The results indicate early acquisition of antibody to rotavirus. The majority of children aged 2 to 4 years had bELISA (50% to 60%) and HIA (70% to 81%) antibodies. There was an association in prevalence data obtained by HIA and bELISA with immunoblotting (IBA), revealing four serologic profiles. Children with profiles I and II (60%) respectively had HAI and ELISA antibody or HAI antibody alone and all had immunoprotective antibodies to VP4 and/or VP7. These children were regarded as “immune,” resembling convalescent patients with a rotavirus infection. Children with profile III (4%) had no HIA antibody and only non-protective anti-VP6 and/or VP7 antibody, and were considered to be “partially immune.” Children with profile IV (36%) had no detectable antibody and were classified as “nonimmune.” These children should be considered to be susceptible to rotavirus infection, with the risk of developing clinically severe diarrhea. © 1996 Wiley-Liss, Inc.     

轮状病毒VP7 DNA免疫研究

目的 研究促使基因分泌或膜锚定表达的基因修饰对轮状病毒VP7 DNA疫苗所诱导抗体水平的影响。方法 把携带野生型（VP7wt）、分泌型（VP7s）和膜锚定型（VP7tm）3种VP7基因的重组pcDNA3质粒分别免疫小鼠,用大肠杆菌表达的重组VP7抗原检测VP7特异的抗体反应,统计分析。结果 3种DNA疫苗均能诱导轮状病毒VP7特异的IgG抗体反应,但其抗体水平之间无显著性差异。结论 把轮状病毒VP7基因导入分泌或膜锚定表达途径不能增强其DNA疫苗抗体应答。     

Heterogeneity of VP4 neutralization epitopes among serotype P1A human rotavirus strains.

We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains.     

Preparation and characterization of a neutralizing monoclonal antibody directed to VP4 of rotavirus strain K8 which has unique VP4 neutralization epitopes

Summary For selecting the neutralizing monoclonal antibodies (N-MAbs) directed to VP4 of rotavirus strain K8, which has unique VP4 neutralization epitopes, we prepared several reassortant viruses by mixed infection of two different strains K8 (serotype 1) and P (serotype 3) in vitro: three reassortant clones having VP4 of K8 and VP7 of P and four clones having VP4 of P and VP7 of K8. By using these reassortants in screening hybridomas, a N-MAb (K8-2C12) directed to strain K8-specific VP4 was obtained. The MAb K8-2C12 neutralized only K8 when tested against numerous strains of different serotypes, while in enzyme-linked immunosorbent assay this MAb reacted also with simian rotavirus SA11 (serotype 3), bovine rotavirus NCDV (serotype 6), and human rotavirus (HRV) strain 69M (serotype 8). Neutralization-resistant mutants of K8 were selected by the K8-2C12 antibody and VP4 amino acid sequences of the mutants were determined. Single amino acid substitution was detected in the three mutant clones at position 394, which is included in the major cross-reactive neutralization region identified in other rotaviruses.     

轮状病毒组特异性抗原VP6在重组腺病毒中的表达及其免疫学效果的研究

目的 构建可表达A组轮状病毒组特异性抗原VP6的非复制型重组腺病毒载体，并对其生物学和免疫学性质进行研究。方法 将人轮状病毒VP6基因插入腺病毒载体pShuttle-CMV中，通过细胞内同源重组方法获得重组腺病毒DNA，将其转染293细胞获得重组腺病毒。用聚合酶链反应（PCR）及Southern blot方法，检测目的基因在重组腺病毒中的整合，用Western blot检测VP6的表达。通过灌胃和滴鼻两种途径对小鼠进行免疫，并对免疫后小鼠体液和粘膜免疫进行分析。结果 得到了重组腺病毒rvAd-VP6,VP6基因整合于腺病毒基因且中，在293细胞中可稳定表达。2次免疫后灌胃和滴鼻两组小鼠均产生明显免疫应答，血清IgG抗体滴度分别为1：1000和1：10000－1：100000。除了血清IgG外，小鼠还产生了较强的针对轮状病毒的血清IgA，滴度为1：10－1：100。滴鼻组在肺灌洗液和肠匀浆液中均可检测到分泌型IgA(sIgA)，灌胃组仅在肠道检测到sIgA。滴鼻组的免疫学效果明显优于灌胃组。结论 人轮状病毒VP6基因重组腺病毒载体的成功构建及所取得的良好免疫学效果，为我国具有自主知识产权的新型轮状病毒基因工程疫苗的研制奠定了基础。     

Antibody prevalence and specificity to group C rotavirus in Swedish sera

Of 160 sera collected from different age groups throughout Sweden, 38% were found to be antibody positive for group C rotavirus. The highest antibody prevalence rate was found in individuals aged 11-30 years (45%). An immunoprecipitation assay revealed that the antibodies were directed against VP2, VP4, VP6, VP7, and NSP2, with VP6 being the most immunogenic protein. Neutralising antibodies against a cultivable porcine group C rotavirus (strain AmC-1/Cowden) were detected in 16/19 individuals at titres from 160 to 5,120. The results indicate that group C rotavirus infections are relatively common in older Swedish children and adults but appear to be less common in children younger than 5 years of age. It is concluded that porcine and human group C rotaviruses share epitopes critical for stimulation of neutralising antibodies.     

Antibody response to serotype-specific and cross-reactive neutralization epitopes on VP4 and VP7 after rotavirus infection or vaccination.

By using a competitive solid-phase immunoassay with serotype-specific and cross-reactive neutralizing monoclonal antibodies directed at VP4 and VP7, we tested the antibody responses to some neutralization epitopes on VP4 and VP7 in individuals infected or vaccinated with rotavirus. Antibody responses to VP7 epitopes of the infecting serotype of virus were found at a high frequency in both infants and children. In contrast, antibody responses to VP4 and heterotypic VP7 were observed only when the individuals possessed antibodies to any serotype of rotavirus in their acute-phase or prevaccination sera.     

Evidence of conserved epitopes in variable region of VP8* subunit of VP4 protein of rotaviruses of P[8]-1 and P[8]-3 lineages

Although antibody responses to the human rotavirus VP4 protein have been reported, few studies have analyzed the specificity of these responses to the VP8* subunit. This study investigated antibody responses generated against the variable region of the VP4 protein (VP8* subunit) in children infected with rotavirus genotype P[8]. Recombinant VP8* subunit (rVP8*) and truncations corresponding aa 1-102
(peptide A) and 84-180 (peptide B) of rotavirus strains P[8]-1 and P[8]-3 lineages were expressed in Escherichia coli and examined for antibody reactivity using ELISA and Western blot assays. Sera from infected children had IgG antibodies that reacted with full-length rVP8*, peptide A and B of both lineages, with stronger reactivity observed against peptide B. In addition, anti-strain Wa (P[8]-1) and anti-rVP8* (P[8]-3) rabbit polyclonal antiserum reacted against peptide B sequences of both lineages. These data indicate that the VP8* variable region of rotavirus belonging to P[8]-1 and P[8]-3 lineages have conserved epitopes recognized by antibodies elicited during natural infections.     

Identification of two subtypes of serotype 4 human rotavirus by using VP7-specific neutralizing monoclonal antibodies

Two distinct subtypes of human rotavirus serotype 4 were identified by using neutralizing monoclonal antibodies directed to the major outer capsid glycoprotein, VP7, of strains ST3 (subtype 4A) and VA70 (subtype 4B). Specimens containing serotype 4 rotavirus, obtained from different countries, were examined for subtyping by using solid-phase immune electron microscopy, enzyme-linked immunosorbent assay, and, for cell culture-adapted strains, neutralization assay. All 59 human rotavirus strains identified as serotype 4 by using animal antisera were classified into either subtype by monoclonal antibodies. This suggests that the antigenic difference between the two subtypes is a consequence of critical variations within the immunodominant serotype 4-specific neutralization site of rotavirus VP7. Subtype 4A (ST3-like) strains were predominant and were detected in stools from patients with gastroenteritis, as well as from healthy infants and young children.     

Protection against rotavirus shedding after intranasal immunization of mice with a chimeric VP6 protein does not require intestinal IgA

Intranasal immunization of mice with chimeric VP6 and the adjuvant LT(R192G) consistently elicits >95% reductions in fecal rotavirus shedding following challenge. To determine the association between mucosal antibody and protection, we immunized BALB/c wt and J chain knockout (Jch-/-) mice with VP6 and either LT(R192G) or cholera toxin (CT). Both strains developed nearly equal levels of serum rotavirus IgG, but Jch-/- mice, which cannot transport dimeric IgA across epithelial cell surfaces, developed >4-fold higher levels of serum rotavirus IgA. Stool rotavirus IgA was present in wt but undetectable in Jch-/- mice. When challenged with rotavirus strain EDIM, reductions in rotavirus shedding were nearly identical in VP6-immunized wt and Jch-/- mice (i.e., 97% and 92%, respectively; P > 0.01). Th1 CD4 T cell responses were also detected in VP6-immunized animals based on high levels of IFN-gamma and IL-2 found after in vitro VP6 stimulation of spleen cells. Therefore, protection induced by intranasal immunization of mice with VP6 and adjuvant does not depend on intestinal rotavirus IgA antibody but appears to be associated with CD4 T cells.     

Longitudinal Studies of Neutralizing Antibody Responses to Rotavirus in Stools and Sera of Children following Severe Rotavirus Gastroenteritis

Rotavirus-neutralizing antibody responses in sera and stools of children hospitalized with rotavirus gastroenteritis and then monitored longitudinally were optimally detected by using local rotavirus strains. Stool responses were highest on days 5 to 8 after the onset of diarrhea. Longitudinal monitoring suggested that serum neutralizing antibody responses were a more useful measure of severely symptomatic rotavirus infection than stool responses but that stool antibody responses may be a useful measure of rotavirus immunity.     

Class and subclass antibodies to Haemophilus influenzae type b capsule: comparison of invasive disease and natural exposure

Recently, the role of immunoglobulin G (IgG) subclasses in the immune responses to organisms with polysaccharide capsules, particularly Haemophilus influenzae type b, has been of interest. We developed assays to measure IgG2- and IgG4-specific antibodies to the polyribosylribitol phosphate (PRP) polysaccharide antigen of H. influenzae type b and demonstrated that these assays were subclass specific. Relative levels of subclass-specific antibody were assayed in serum from 30 Alaskan Eskimo children who had invasive H. influenzae type b disease and 30 healthy controls that were matched for age and village of residence. We also measured total PRP antibody and total serum IgG4. The group with invasive H. influenzae type b disease had a significantly higher mean level of IgG4-specific PRP antibody than did the controls (P = 0.0006). However, we found no significant difference between cases and controls for IgG2-specific PRP antibody, total IgG4, or total PRP antibody. The data suggest that IgG4-specific PRP antibody is elicited by invasive H. influenzae type b disease, independent of age. The IgG4 subclass thus may be a critical determinant of the immune response to invasive infection caused by H. influenzae type b, especially for young infants who generally have a weak immune response to this organism.     

Evaluation of serum antibody responses against the rotavirus nonstructural protein NSP4 in children after rhesus rotavirus tetravalent vaccination or natural infection

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of < or = 200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.     

A longitudinal cohort study in calves evaluated for rotavirus infections from 1 to 12 months of age by sequential serological assays

Using an immunocytochemical staining assay involving six different recombinant baculoviruses with each expressing one of the major bovine rotavirus VP7 (G6, G8 and G10) and VP4 (P6[1], P7[5] and P8[11]) serotypes, we analyzed IgG antibody responses to individual proteins in archival serum samples collected from 31 calves monthly from 1 to 12 months of age during 1974–1975 in Higley, Arizona. Seroresponses to VP7 and VP4, as determined by a fourfold or greater antibody response, were not always elicited concurrently following infection: in some calves, (1) seroresponses to VP7 were detected earlier than to VP4 or vice versa; and (2) a subsequent second seroresponse was detected for VP7 or VP4 only. In addition, a second infection was more likely to be caused by different G and/or P types. Analyses of serum samples showed that the most frequent G–P combination was G8P6[1], followed by G8P7[5], G8P8[11] and G6P6[1]. Francis R. Abinanti: Deceased.