Non-structural protein NSP2 induces heterotypic antibody responses during primary rotavirus infection and reinfection in children

Rotaviruses are the single most important causes of severe acute diarrhoea in children worldwide. Despite success in developing vaccines, there is still a lack of knowledge about many components of the immune response, particularly those to non-structural proteins. This study established radioimmunoprecipitation (RIP) assays using labeled G1P[8], G2P[4], and G4P[6] human rotaviruses to examine the spectrum and duration of rotavirus antibodies in sera collected sequentially for 18-36 months from 27 children after hospitalization for primary rotavirus gastroenteritis. Five children experienced rotavirus re-infections. Primary responses detected to non-structural protein NSP2 declined to baseline after 100-150 days. Responses were heterotypic between NSP2 of G1P[8] and G4P[8] rotaviruses. Re-infections after 465-786 days boosted antibody levels to NSP2of both serotypes, together with the appearance of anti-NSP2 to G2P[4], even though there was no evidence of infection with this serotype. We developed an enzyme-immunoassay to measure sequential levels of anti-NSP2 IgG and IgA, using recombinant (heterotypic) NSP2 derived from SA11 (G3P[2]). Anti-NSP2 IgG and IgA were detected in sera from 23/23 (100%) and 18/24 (75%) of children after primary infection, declined to baseline after 100-150 days, were boosted after rotavirus re-infections, and again declined to baseline 150 days later. Anti-NSP2 IgA was also detected after primary infection, in duodenal juice from 14/16 (87%), and faecal extract from 11/19 (57%) of children. Sequential estimation of anti-NSP2 EIA levels in sera could be a sensitive index of rotavirus infection and re-infection. The potential of anti-NSP2 to limit viral replication after re-infection deserves further study.     

Evaluation of IFN-gamma response to rotavirus and non-structural protein NSP4 of rotavirus in children following severe rotavirus diarrhea

BACKGROUND: Rotavirus (RV) is the commonest cause of severe gastroenteritis in young children worldwide. However the natural immune mechanisms controlling and preventing rotavirus disease in humans are not fully understood. OBJECTIVE: To examine cellular immune responses to whole rotavirus (vaccine strain, 116E) and non-structural protein-4 (116E-NSP4) in children during natural rotavirus-infection. STUDY DESIGN: Gamma-interferon (IFN-gamma) responses were evaluated by enzyme-linked immunospot assay in peripheral blood mononuclear cells from children with RV (n=26) or non-RV (n=10) gastroenteritis and from RV-exposed adults (n=10). Additionally, IL-4 responses were assessed in 5 of the 10 adults and 6 of 26 RV-infected children. RESULTS: IFN-gamma secreting cells specific to whole RV were detected in 68% of RV-positive children and to NSP4 in 43% of these children between 4 and 30 days of illness onset. IFN-gamma responses were transient and were found higher in RV-exposed adults than in children (P<0.05). Within the RV-positive group, IFN-gamma responses in children with prior RV-exposure were higher than children without prior exposure (P<0.05). The response to whole RV and NSP4 were positively correlated (P<0.01, r(s)=0.66). CONCLUSIONS: Significant IFN-gamma responses to rotavirus candidate vaccine strain 116E were detected in children during natural RV-infection and in RV-exposed adults. Significant IFN-gamma responses to NSP4 were also observed in these study groups.     

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.     

我国各血清型轮状病毒非结构蛋白NSP4基因特征的初步分析

目的 对我国血清学调查中发现的四型（G型）轮状病毒NSP4序列进行测定和分析,了解NSP4的基因类型和变异状况。方法 利用银染测离方法对G1、G2、G3、G9型毒株NSP4 cDNA序列进行测定。结果 四个血清型毒株NSP4的核苷酸和氨基酸序列的同源性分别为78.8%,93.5%;83.0%,95.5%。G1、G3、G9型标本多属Wa组;G2型标本多属KUN组。NSP4的变异主要位于VP4结合域内的135－145位氨基酸。结论 Wa组和KUN组是我国主要的两种NSP4基因型。     

Characterization of serum antibody responses to natural rotavirus infections in children by VP7-specific epitope-blocking assays.

Knowledge of the immune response to rotavirus is crucial for vaccine development. We compared an epitope-blocking assay (EBA) that uses VP7-specific monoclonal antibodies with neutralization assays (NAs) with polyclonal antisera for detecting serum antibody responses after natural rotavirus infection in children. Twenty-six serum pairs from children living in an orphanage with and without symptoms during two rotavirus outbreaks were evaluated for VP7 type 1-, 2-, 3-, and 4-specific antibody responses. In the first outbreak, which was caused by a VP7 type 3 strain, homotypic antibody responses were detected in 11 of 11 symptomatic children by NA and in 10 of 11 symptomatic children by EBA. Heterotypic antibody responses were detected more frequently (12 of 15 children) by NA than by EBA, and the heterotypic epitope-blocking antibody responses occurred in children older than 14 months of age. Antibody responses in asymptomatic children were more commonly detected by EBA than by NA. EBA results from the sera of children in the second outbreak indicated that it was caused by VP7 type 4, whereas NA results suggested it was caused by VP7 type 3. Our results confirm that EBA is a sensitive and specific method for determining VP7 type-specific immune responses after natural rotavirus infections.     

人轮状病毒NSP4蛋白131和133位氨基酸变异对毒力影响的研究

目的研究两株A组人轮状病毒NSP4蛋白131位和133位氨基酸位点的变异对毒力的影响。方法从昆明地区2002年和2005年秋冬季婴幼儿腹泻流行期不同程度腹泻患儿中分离得到两株轮状病毒流行株02k38和05k44,RT-PCR扩增NSP4全长基因,进行cDNA序列测序。通过pGEX-5X-1载体,转化E.coliBL21以谷胱苷肽S-转移酶融合蛋白的形式表达两株病毒的NSP4蛋白C-端86～170位氨基酸（GST-NSP486-170）;并酶切融合头纯化得到两种NSP486-170蛋白,在ICR乳鼠中比较这两种蛋白致小鼠腹泻的差异。结果两种NSP486-170蛋白毒性无差异。结论两株轮状病毒131位和133位氨基酸位点的变异不影响毒力的改变,其致腹泻活性没有明显差异（P〉0.05）。     

Homotypic and heterotypic serum neutralizing antibody response to rotavirus proteins following natural primary infection and reinfection in children

Worldwide trials of rotavirus vaccines are currently in progress, but the basis of cross-reactive immunity between rotavirus serotypes is yet to be elucidated. The involvement of the outer capsid proteins, VP7 and VP4, in the production of cross-reactive neutralizing antibody (N-Ab) is unclear, and may be important for the success of animal rotavirus-based candidate vaccines that lack a VP4 of human rotavirus origin. In this study, VP7- and VP4-specific N-Ab was assayed in sera from children experiencing primary (27 children) and/or secondary (14 children) rotavirus infections using human-animal reassortant strains. These reassortants contained genes encoding the major G- and P-types found in human infection, including G1, 2, 3, and 4; or P1A[8], 1B[4], and 2[6]. After primary infection, the N-Ab response to VP7 was generally serotype-specific, whereas the response to VP4 was heterotypic. After reinfection (with the same or different serotypes) there was a significant increase (P = 0.0313) in the number of VP7 serotypes seroconverted against with no broadening of cross-reactivity to VP4. Increases in homotypic N-Ab titer, following both primary and secondary infection, were greater against VP7 than VP4, with the seroconversion against VP7 being significantly greater upon reinfection than following primary infection (P = 0.0280). In summary, heterotypic N-Ab produced following primary infection appears to be primarily against VP4. However, upon reinfection, VP7 becomes increasingly immunodominant both in terms of cross-reactive N-Ab production and increases in N-Ab titer. J. Med. Virol. 57:204–211, 1999. © 1999 Wiley-Liss, Inc.     

Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pig model of human rotavirus disease

Antibody responses to the Wa human rotavirus (HRV) nonstructural protein NSP4, a viral enterotoxin, were evaluated in neonatal gnotobiotic (Gn) pigs. Gn pigs were inoculated orally with one dose of 10(5) fluorescent focus units (FFU) of virulent Wa HRV (HRV-V), to mimic natural infection, or with three doses of 5 x 10(7) FFU attenuated Wa HRV (HRV-A) at 10-day intervals, to mimic oral attenuated rotavirus vaccines, or they were mock inoculated (mock). Subsets of pigs were challenged with 10(6) FFU of virulent Wa HRV at post-inoculation day 28 (PID 28). Post-challenge, the HRV-V pigs were completely protected against diarrhea and virus shedding, whereas the HRV-A pigs had a 50% protection rate against diarrhea and a 67% protection rate against virus shedding. All mock-inoculated pigs shed virus and had diarrhea post-challenge. Isotype antibody titers to NSP4 were compared in serum and intestinal contents, at post-inoculation day (PID) 28 and at post-challenge day 7 (PCD 7/PID 35) by indirect ELISA, using purified recombinant NH2-6xHis-tagged NSP4 of virulent Wa HRV. Pre-challenge, both the HRV-V and HRV-A-inoculated pigs had similar moderate titers of serum IgG antibodies to NSP4. However, only the HRV-V-inoculated pigs developed detectable serum and intestinal IgA antibody titers to NSP4 pre-challenge, compared with the HRV-A-inoculated pigs. The mock-inoculated pigs had no IgM, IgA, or IgG antibodies to NSP4 pre-challenge. All Wa HRV-inoculated pigs developed low to moderate titers of serum IgM, IgG, and IgA antibodies to NSP4 post-challenge, but the mock-inoculated pigs had only IgM antibodies post-challenge. Both Wa HRV-inoculated groups developed low titers of IgA antibody to NSP4 in the small intestinal contents post-challenge, but titers were 5.8-fold higher in the HRV-V pigs. Our results concur with findings that both rotavirus vaccinated and naturally infected children seroconvert with modest IgG antibodies to NSP4 [Johansen et al. (1999) J Med Virol 59:369-367]. These data suggest that Gn pigs could be a useful model to evaluate serum and intestinal IgA antibodies to NSP4 and their role in protection against HRV infection. Further experiments may clarify whether (1) the NSP4 antibodies detected pre-challenge in the HRV-V pigs contribute to the higher protection rates observed, or (2) the reduced or delayed NSP4 antibody responses of the HRV-A pigs are associated with the lower protection rates in these pigs.     

Species-specific but not genotype-specific primary and secondary isotype-specific NSP4 antibody responses in gnotobiotic calves and piglets infected with homologous host bovine (NSP4[A]) or porcine (NSP4[B]) rotavirus

Using recombinant baculoviruses expressing rotavirus NSP4 [A], [B], [C], and [D] genotypes of bovine, porcine, human, simian, or murine origin, we analyzed serum antibody responses to NSP4s in gnotobiotic calves and piglets infected by the oral/alimentary or intraamniotic route with bovine (NSP4[A]) (Wyatt, R.G., Mebus, C.A., Yolken, R.H., Kalica, A.R., James, H.D., Jr., Kapikian, A.Z., Chanock, R.M., 1979. Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus. Science 203(4380), 548-550) or porcine (NSP4[B]) (Hoshino, Y., Saif, L.J., Sereno, M.M., Chanock, R.M., Kapikian, A.Z., 1988. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 62(3), 744-748) rotaviruses. Following primary infection and challenge with virulent rotaviruses, the animals developed higher or significantly higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous host heterotypic NSP4s, indicating that antibody responses were species specific rather than genotype specific. Antibody responses to NSP4s corresponded closely with the phylogenetic relationships of NSP4s within a species-specific region of amino acids (aa) 131-141. In contrast, NSP4 genotypes determined by amino acid full-length sequence identity predicted poorly their "serotypes". In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, in an approach to immunization with a live oral rotavirus vaccine, the NSP4 protein does not appear to play an important role in protection against rotavirus disease and infection.     

Development of serum and intestinal antibody response to rotavirus after naturally acquired rotavirus infection in man

The temporal characteristics of the response of rotavirus specific IgM, IgG, IgA in serum and secretory antibody in feces to rotavirus were studied in 77 hospitalized patients with rotavirus induced gastroenteritis. The response in serum was characterized by the sequential appearance of rotavirus specific IgM, IgG, and IgA antibody. The IgM antibody appeared to be higher in the acute phase of the disease and was subsequently replaced by the IgG and IgA antibodies. However, the titers of IgG rotavirus antibody in convalescent specimens of serum were found to be statistically significantly lower in patients with severe or prolonged rotavirus infection than in specimens from subjects with mild or moderate disease. Most fecal specimens collected during both the acute and convalescent phase of illness contained virus specific secretory IgA. Higher concentrations of antibody were measured in convalescent samples from patients with prolonged diarrhea and virus shedding. These observations suggest a possible relationship between the severity of rotavirus infection and the nature of systemic and secretory antibody response.     

Serologic responses by immunoblot following natural infection with rotavirus serotypes G1 and G4 in children

Serologic responses to proteins of rotavirus serotypes G1, P1A[8]; G2, P1B[4]; G3, P1A[8]; and G4, P2A[6] were evaluated by immunoblotting paired sera from 17 children with primary rotavirus infection. Ten children were infected with G1, P1A[8]; five with G4, P1A[8]; and two with G4, P2A[6] viruses. Anti-VP6 and anti-VP2 were seen in most responses. Homotypic anti-VP7 developed following G1 and G4 infections in 8 (80%) and 6 (86%) cases, respectively. Homotypic anti-VP4 developed in 9 (60%) cases following P1A[8] infection and in 0 of 2 cases following P2A[6] infection. Heterotypic anti-VP7 appeared against G4 (20%) and G3 (20%) following the 10 G1 infections, and against G3 (86%) and G1 (57%) following the 7 G4 infections. Heterotypic anti-VP4 occurred in only 3 (18%) children. The data show the antigenic predominance of internal proteins VP6 and VP2. Homotypic antibodies developed against VP7 but not against VP4 in most cases, while heterotypic antibodies were infrequent. J. Med. Virol. 56:52–57, 1998. © 1998 Wiley-Liss, Inc.     

Human immune response to meningococcal outer membrane protein epitopes after natural infection or vaccination

Antibody levels in 41 sets of human acute- and convalescent-phase meningococcal sera were compared with those in 23 sets of human prevaccination and 2-week postvaccination sera. We used a modification of a solid-phase radioimmunoassay (SPRIA) technique to test each of the human serum samples as inhibitors of monoclonal antibodies (MAbs) that bind (HIMSPRIA) to the outer membrane complex from a 2a:P1.2:P5.1 strain. We used three murine MAbs specific for the 2a, P1.2, and P5.1 epitopes on meningococcal class 1, 2, and 5 proteins, respectively, to detect antibodies with similar specificities in human sera. Each of 40 available matching strains from patients were also screened with the three MAbs in a nitrocellulose spot blot assay. A total of 37 (92%) were positive for the 2a epitope, 36 (90%) were positive for the P1.2 epitope, and 16 (40%) were positive for the P5.1 epitope. Of 38 available convalescent-phase sera, 27 (71%) matched with these strains and had detectable inhibiting antibody for each of the MAb-defined protein epitopes of the infecting strain. Three convalescent-phase sera had no HIMSPRIA activity for MAb-defined epitopes that were present on the infecting strain; others had activity for one or two of the epitopes. The results were similar for pre- and postvaccination sera. The average level of HIMSPRIA activity for the P1.2 epitope was greater than fivefold higher in postvaccination sera compared with that in convalescent-phase sera. Sera with distinct patterns of HIMSPRIA activity also were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis and showed a correlation between the HIMSPRIA activity for particular epitopes and the level of antibody binding to the immunoblotted proteins possessing those epitopes. A comparison of the HIMSPRIA and the bactericidal activity of selected postvaccination sera indicated a possible correlation between HIMSPRIA and bactericidal activity, but it also suggested the presence of bactericidal antibodies with specificities other than those defined by the MAbs.     

Structure of the extended diarrhea-inducing domain of rotavirus enterotoxigenic protein NSP4

Rotavirus nonstructural protein 4 (NSP4) is a multidomainal and multifunctional protein and is recognized as the first virus-encoded enterotoxin. Extensive efforts to crystallize the complete cytoplasmic tail (CT), which exhibits all the known biological functions, have been unsuccessful, and to date, the structure of only a synthetic peptide corresponding to amino acids (aa) 95-137 has been reported. Recent studies indicate that the interspecies-variable domain (ISVD) from aa 135 to 141 as well as the extreme C-terminus are critical determinants of virus virulence and the diarrhea-inducing ability of the protein. Among the five NSP4 genotypes identified, those belonging to genotypes A1, B and C possess either a proline at position 138 or a glycine at 140, while those of A2, D and E lack these residues in the ISVD, suggesting conformational differences in this region among different NSP4s. Here, we examined the crystallization properties of several deletion mutants and report the structure of a recombinant mutant, NSP4:95-146, lacking the N-terminal 94 and C-terminal 29 aa, from SA11 (A1) and I321 (A2) at 1.67 and 2.7 A, respectively. In spite of the high resolution of one of the structures, electron density for the C-terminal 9 residues could not be seen for either of the mutants, and the crystal packing resulted in the creation of a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as the crystal packing owing to the additional space required to accommodate the un structured virulence-determining region. The crystal structure and secondary structure prediction of the NSP4:95-146 mutants from different genotypes suggest that the region C-terminal to aa 137 in all the NSP4 proteins is likely to be unstructured, and this might be of structural and biological functional significance.     

Saliva, breast milk, and serum antibody responses as indirect measures of intestinal immunity after oral cholera vaccination or natural disease.

The possibility that antibody responses in serum, saliva, or breast milk samples to oral vaccines or enteric infections may reflect the intestinal immune response was evaluated in Bangladeshi volunteers orally immunized with a cholera B subunit-whole-cell vaccine (B + WCV) and in patients convalescing from enterotoxin-induced diarrheal disease. Two peroral doses of B + WCV induced antitoxin and antibacterial antibody responses in the intestinal fluids of 76 and 92%, respectively, of the volunteers and in serum samples in 90 and 69% of those tested. These responses were comparable to those obtained after cholera or enterotoxigenic Escherichia coli disease. Whereas immunoglobulin A (IgA) antitoxin titer increases in saliva (44%) and breast milk (29%) specimens after vaccination were less frequent than in intestinal fluid (76%), antitoxin responses in saliva and breast milk occurred in 80 to 90% of the patients after disease. Also, antilipopolysaccharide (anti-LPS) titer increases in extraintestinal body fluids were found more frequently after disease than after vaccination. A comparison of the frequency and magnitude of antibody response in different body fluids with those in intestinal lavage fluid revealed no extraintestinal antibody that directly reflected the intestinal immunity. However, comparison of vibriocidal and IgG antitoxin antibodies in serum specimens with antitoxin and anti-LPS IgA responses in intestinal fluids after the vaccination of volunteers showed a sensitivity of 70 to 90% and a predictive accuracy of about 80% for the serum analyses reflecting the intestinal immune responses. Furthermore, antitoxin and anti-LPS antibody responses in saliva and breast milk samples seemed to be useful proxy indicators of a gut mucosal response of these antibodies after enterotoxin-induced diarrheal disease showing sensitivity vales of 70 to 90% and predictive accuracy vales of 70 to 100%.     

Humoral and cell-mediated immune responses in humans to the NSP4 enterotoxin of rotavirus.

Rotavirus nonstructural protein NSP4 has recently been suggested to function as a viral enterotoxin and play a role in the pathophysiological mechanism whereby rotaviruses induce diarrhea. The ability of rotavirus NSP4 to stimulate a humoral immune response was examined in naturally infected children and adults, rotavirus vaccinated children, as well as a cellular immune response in adults. In this study, 10 of 10 naturally infected children and 9 of 10 rotavirus-vaccinated children showed a weak humoral IgG immune response to recombinant NSP4 (rNSP4) and/or a synthetic peptide corresponding to residues 114-134 of NSP4. Modest serum IgG antibody responses were detected in 20 of 20 adults. A cellular immune response to rNSP4 and/or NSP4(114-134) were detected in 8 of 10 adults measured either as a T-cell proliferative response (7 of 10), an increased production of IL-2 (6 of 10), or increased production of interferon-gamma (8 of 10). These results indicate that NSP4 induces a humoral immune response in humans and show for the first time that NSP4 stimulates a cellular immune response, possibly including cytolytic T-cells.     

Comparison of serum and mucosal antibody responses following severe acute rotavirus gastroenteritis in young children.

The development of mucosal immunity is presumed to be the most important marker of rotavirus infection. The practical difficulties of obtaining small-bowel secretions stimulated this study of the antibody response to acute rotavirus infection at other sites. Forty-four infants admitted to the hospital with rotavirus gastroenteritis had serum, saliva, and feces collected at the acute phase (median, 5.5 days), during convalescence (median, 33.5 days), and 4 months later (median, 12.2 weeks). A subgroup of 19 children also had duodenal juice collected in parallel. Rotavirus-specific immunoglobulin G (IgG), IgA, secretory immunoglobulin, and IgM were measured and compared in all samples. The results showed that the estimation of antirotavirus serum IgM, serum IgG, duodenal juice IgA, and duodenal juice IgM by an enzyme immunoassay indicated an immune response to severe primary rotavirus infection in all children. Four months later, the levels of serum IgG and IgA served as the most sensitive markers of the preceding rotavirus infection. The predictive accuracies of immune responses at different sites in relation to a positive IgA immune response in the duodenum were calculated. Fecal IgA predicted duodenal IgA rotavirus antibodies with accuracies of 86% at 1 month and 92% at 4 months. The high sensitivity of serum IgM and IgG in detecting rotavirus infection and the high predictive accuracy of fecal IgA as an indicator of duodenal IgA abrogates the need for duodenal intubation to detect (or monitor) an immune response to rotavirus infection. This finding has important practical implications for epidemiological studies of acute diarrhea in children and in rotavirus vaccine trials.     

Antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus

The antibody response of pregnant swine to transmissible gastroenteritis (TGE) virus was studied, with special reference to the titers and the immunoglobulin (Ig) class of TGE neutralizing antibodies in colostrum and milk. Animals vaccinated twice intramuscularly or intramammarily with live attenuated TGE virus developed high levels of antibodies in serum and colostrum, but the levels in milk declined markedly within a few days post-farrowing. In contrast, animals naturally or experimentally infected with virulent virus generally developed lower levels of antibodies in serum and colostrum but maintained higher levels in milk, as compared to the vaccinated animals. Gel filtration studies indicated that antibodies in milk from vaccinated animals were primarily of the IgG class, whereas those from the naturally or experimentally infected animals were primarily of the IgA class. The ability of sows to transmit a high degree of passive immunity to their suckling progeny was more closely associated with TGE antibodies of the IgA than the IgG class. Present evidence suggests that high levels of TGE antibodies of the IgA class occur in milk as a result of an infection of the intestinal tract. Probable reasons for this are discussed.     

Enterotoxin-specific immunoglobulin E responses in humans after infection or vaccination with diarrhea-causing enteropathogens

Cholera toxin (CT)-specific antibody responses of the immunoglobulin E (IgE) isotype in the sera of adult patients suffering from infection with either Vibrio cholerae O1, V. cholerae O139, or enterotoxigenic Escherichia coli (ETEC) were analyzed and compared with those in the sera of volunteers immunized with a bivalent B subunit O1/O139 whole-cell cholera vaccine. A significant IgE response to CT was observed in 90% of the patients with V. cholerae O1 infection (18 of 20; P = <0.001) and 95% of the patients with V. cholerae O139 infection (19 of 20; P = <0.001). Similarly, the majority of the patients with ETEC diarrhea (83%; 13 of 15) showed a positive IgE response to CT. Eight of 10 North American volunteers (80%) orally challenged with V. cholerae O1 showed CT-specific IgE responses (P = 0.004). In contrast, Swedish volunteers immunized with the oral cholera vaccine showed no IgE responses to CT (P value not significant). During the study period, total IgE levels in the sera of the diarrheal patients, the North American volunteers, and the Swedish cholera vaccinees alike remained unchanged. However, the total IgE levels in the sera of patients and healthy Bangladeshi controls were on average 89-fold higher than those in the sera of the healthy Swedish volunteers and 34-fold higher than those in the sera of the North American volunteers.     

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 ≤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.