Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Development of a method for detection of human rotavirus in water and sewage

The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.     

Development of a method for detection of human rotavirus in water and sewage.

The simian rotavirus SA11 was used to develop a simple, reliable, and efficient method to concentrate rotavirus from tap water, treated sewage, and raw sewage by absorption to and elution from Filterite fiberglass-epoxy filters. SA11 adsorbed optimally to Filterite filters from water containing 0.5 mM AlCl3 at pH 3.5. Filter-bound virus was eluted with 0.05 M glycine-NaOH supplemented with 10% tryptose phosphate broth at pH 10. SA11 was quantitated by plaque assay, whereas human rotavirus was detected by immunofluorescence. The method was applied to detect rotavirus in raw and treated sewage at two Houston, Tex., sewage treatment plants. The sewage isolates were identified as rotavirus, probably a human strain, based on several criteria. The sewage isolates were detectable by an immunofluorescence test, using anti-SA11 serum which would detect the simian, human bovine, and porcine rotaviruses. No reaction was noted by immunofluorescence with the reoviruses or several common enteroviruses. The sewage isolates were neutralized by convalescent sera from a human adult and infant who had been infected by rotavirus as well as by a hyperimmune serum prepared in guinea pigs against purified human rotavirus. Preimmune or preillness sera did not react with the isolates by neutralization or immunofluorescence. The natural isolates were sensitive to pH 11 and other inactivating agents, similar to SA11. The buoyant density of the sewage isolates in CsCl gradients was 1.36 g/cm3, which is the value usually reported for complete, infectious rotavirus particles. The double-shelled particle diameter was 67.1 +/- 2.4 nm. Finally, electron micrographs of cell lysates inoculated with the sewage isolate showed particles displaying characteristic rotavirus morphology.     

Demonstration of an immunodominant neutralization site by analysis of antigenic variants of SA11 rotavirus

Serotype-specific monoclonal antibodies were used to select mutants of SA11 rotavirus that were resistant to neutralization. The antigenic characteristics of these mutants were studied with with a panel of monoclonal antibodies. We isolated one type of mutant which showed a dramatic increase (greater than 10-fold) in resistance to neutralization by hyperimmune antiserum, and this together with other data indicates the presence on the rotavirus major outer shell glycoprotein of an immunodominant antigenic site involved in virus neutralization. The mutants were also useful in classifying neutralizing monoclonal antibodies.     

Rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein.

We investigated properties of the rotavirus genome segment 11 protein. A rotavirus SA11 genome segment 11 cDNA which contains the entire coding region was sequenced and inserted into the baculovirus transfer vector pVL941. Recombinants containing gene 11 cDNA were selected, and the gene 11 product expressed in Spodoptera frugiperda cells infected with these recombinants was inoculated into guinea pigs to produce hyperimmune antiserum. Characterization of the antiserum showed that it recognized a primary translation product with a molecular weight of 26,000 (26K protein) in recombinant-infected insect cells, in SA11-infected monkey kidney cells, and in cell-free translation reactions programmed with SA11 mRNA. A modified 28K product was also detected but only in SA11-infected monkey kidney cells. The 26K 28K proteins were shown to be phosphorylated in infected monkey kidney cells, and the 26K protein was phosphorylated in insect cells. We were unable to identify what type of modification caused the molecular weight shift to 28,000 in infected monkey kidney cells. Large amounts of the gene 11 product were detected by immunofluorescence in discrete foci in the cytoplasm of infected monkey kidney cells. Viruses of all known serotypes were also detected by immunofluorescence by using hyperimmune antiserum to the SA11 gene 11 product. The antiserum reacted with particle-depleted cytosol fractions but did not react with purified virus particles by immunoprecipitation or immunoblotting; it also did not neutralize virus infectivity in plaque reduction neutralization assays. Therefore, we conclude that the primary gene 11 product is a nonstructural phosphoprotein which we designated NS26.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum?

A panel of single and double neutralization-resistant escape mutants of serotype G11 porcine rotavirus strains A253 and YM, selected with G11 monotype- and serotype-specific neutralizing monoclonal antibodies (MAbs) to VP7, was tested in neutralization assays with hyperimmune sera raised against rotavirus strains of different serotypes. Escape mutants with an amino acid substitution in antigenic region A (amino acids [aa] 87 to 101) resulting in a residue identical or chemically similar to those present at the same positions in serotype G3 strains, at positions 87 for strain A253 and 96 for strain YM, were significantly more sensitive than the parental strains to neutralization with sera against some serotype G3 strains. Also, one YM antigenic variant (YM-5E6.1) acquired reactivity by enzyme-linked immunosorbent assay with MAbs 159, 57/8, and YO-1E2, which react with G3 strains, but not with the serotype G11 parental strain YM. Cross-adsorption studies suggested that the observed cross-neutralization by the G3-specific sera was due to the sera containing antibodies reactive with the parental strain plus antibodies reactive with the epitope(s) on the antigenic variant that mimick the serotype G3 specific one(s). Moreover, antibodies reactive with antigenic region F (aa 235 to 242) of VP7 might also be involved since cross-reactivity to serotype G3 was decreased in double mutants carrying an additional mutation, which creates a potential glycosylation site at position 238. Thus, single point mutations can affect the serotype reactivity of G11 porcine rotavirus strains with both monoclonal and polyclonal antibodies and may explain the origin of rotavirus strains with dual serotype specificity based on sequence divergence of VP7.     

A synthetic peptide corresponding to the cleavage region of VP3 from rotavirus SA11 induces neutralizing antibodies.

Antibodies were elicited in rabbits by immunization with the synthetic tetradecapeptide Gln-Asn-Thr-Arg-Asn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3. Protein specificity of the antipeptide serum is demonstrated. The antipeptide serum revealed neutralizing activity directed against SA11 in a neutralization assay. Human rotavirus strains Wa, S2, and Hochi and bovine strains NCDV and UK were not neutralized, demonstrating the strain-specific neutralizing activity of the raised antipeptide serum. Upon immune electron microscopy, aggregation of SA11 particles was observed.     

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.     

Generation of recombinant rotavirus with an antigenic mosaic of cross-reactive neutralization epitopes on VP4

Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.     

Primary virus envelope cross-reactivity of the broadening neutralizing antibody response during early chronic human immunodeficiency virus type 1 infection

To test the hypothesis that changing neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1) during chronic infection were a response to emergence of neutralization escape mutants, we cloned expressed and characterized envelope clones from patients in the Multicenter AIDS Cohort Study (MACS). Pseudotyped HIV-1 envelope clones obtained from differing time points were assessed for sensitivity to neutralization by using sera from different times from the same and different patients. Clones from early and late time points during chronic infection had similar neutralization sensitivity, and neutralizing antibody responses cross-reacted with early, late, and heterologous envelopes. The potential for broadly effective HIV-1 immunization is supported.     

Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus.

Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.     

Antigenic relationships among five reovirus-like (RVL) agents by complement fixation (CF) and development of new substitute CF antigens for the human RVL agent of infantile gastroenteritis.

The human reovirus-like (HRVL) agent, Nebraska calf diarrhea virus (NCDV), epizootic diarrhea of infant mice (EDIM) virus, simian agent (SA)-11, and the "O" (offal) agent were found to be similar, if not identical, in reciprocal complement fixation (CF) tests employing hyperimmune animal sera. In addition, in CF tests with paired sera from 35 diarrhea patients who shed the HRVL agent, 74% developed serologic evidence of infection with the HRVL antigen, 43% with NCDV, 51% with EDIM virus, 57% with SA-11, and 71% with the "O" agent. Thus, in addition to the NCDV, which had previously been described as a suitable substitute CF antigen for the HRVL agent, the SA-11, "O", and EDIM viruses may also be utilized as substitute antigens for the HRVL agent. However, the "O" agent appears to be the most efficient of the four substitute CF antigens and thus should be used preferentially when the HRVL agent is not available. The "O" agent was about as efficient as the HRVL agent and significantly more efficient than the NCDV for detecting seroresponses. The greatest efficiency for detecting infection with the HRVL agent resulted when sera were tested with both the HRVL and "O" agents as 31 (89%) of the patients developed serologic evidence of infection with one or both antigens. The finding of additional substitute CF antigens for the HRVL agent may have implications in the immunoprophylaxis against human disease.     

Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins.

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.     

Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C Infection

We previously showed that HIV-1 subtype C viruses elicit potent but highly type-specific neutralizing antibodies (nAb) within the first year of infection. In order to determine the specificity and evolution of these autologous nAbs, we examined neutralization escape in four individuals whose responses against the earliest envelope differed in magnitude and potency. Neutralization escape occurred in all participants, with later viruses showing decreased sensitivity to contemporaneous sera, although they retained sensitivity to new nAb responses. Early nAb responses were very restricted, occurring sequentially and targeting only two regions of the envelope. In V1V2, limited amino acid changes often involving indels or glycans, mediated partial or complete escape, with nAbs targeting the V1V2 region directly in 2 cases. The alpha-2 helix of C3 was also a nAb target, with neutralization escape associated with changes to positively charged residues. In one individual, relatively high titers of anti-C3 nAbs were required to drive genetic escape, taking up to 7 weeks for the resistant variant to predominate. Thereafter titers waned but were still measurable. Development of this single anti-C3 nAb specificity was associated with a 7-fold drop in HIV-1 viral load and a 4-fold rebound as the escape mutation emerged. Overall, our data suggest the development of a very limited number of neutralizing antibody specificities during the early stages of HIV-1 subtype C infection, with temporal fluctuations in specificities as escape occurs. While the mechanism of neutralization escape appears to vary between individuals, the involvement of limited regions suggests there might be common vulnerabilities in the HIV-1 subtype C transmitted envelope.     

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

Priming for rotavirus neutralizing antibodies by a VP4 protein-derived synthetic peptide.

In the rotavirus SA11 surface protein VP4, the trypsin cleavage sites associated with the enhancement of infectivity are flanked by two amino acid regions that are highly conserved among different rotaviruses. We have tested the ability of synthetic peptides that mimic these two regions to induce and prime for a rotavirus neutralizing antibody response in mice. After the peptide immunization schedule, both peptides induced peptide antibodies, but neither was able to induce virus antibodies, as measured by an enzyme-linked immunosorbent assay or a neutralization assay. However, when the peptide-inoculated mice were subsequently injected with intact SA11 virus, a rapid and high neutralizing antibody response was observed in mice that had previously received the peptide comprising amino acids 220 to 233 of the VP4 protein. This neutralizing activity was serotype specific; however, this peptide was also able to efficiently prime the immune system of mice for a neutralizing antibody response to the heterotypic rotavirus ST3 when the ST3 virus was used for the secondary inoculation.     

Attachment of Trypanosoma brucei to rabbit peritoneal exudate cells

Attachment of trypanosomes to cultured rabbit peritoneal cells was enhanced in the presence of hyperimmune Trypanosoma brucei antiserum and sera from infected rabbits. During infection attachment rose rapidly from control levels reaching a maximum value after two or three weeks; this was maintained until the death of the animal. The initial rise in activity was preceded by an increase in the serum titres of trypanosome agglutinating antibody. Attachment did not appear to be mediated by variant specific antibodies, no association being found between adherence and the appearance of successive variant subpopulations. Fractionation of hyperimmune and immune sera indicated that the majority of activity was present in the gamma globulin fraction with less activity in the macroglobulin fraction, despite its elevation during infection. Increased activity obtained with partially-purified immunoglobulin G prepared from hyperimmune serum was reduced following absorption with either disrupted or live trypanosomes.     

Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11.

An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.