Douglas R. Knowlton

Development of an improved method for measuring neutralizing antibody to rotavirus.

An improved assay was developed for measuring neutralizing antibody titers against rotaviruses. This procedure used the same initial steps as performed to determine antibody titers by the focus reduction neutralization (FRN) assay. However, instead of counting infected cells after staining with fluorescein, reductions in virus infectivity by neutralizing antibody were determined by quantitation of viral antigen production using an ELISA. A linear relationship was found between ELISA absorbance values and focus forming units for each of four prototype rotaviruses, representative of serotypes 1-4. Thus, the serum dilution that resulted in neutralization of 60% of infectious virus (i.e. the neutralizing antibody titer) could be readily determined from absorbance values. Titers found by this method were similar to and as reproducible as those found by the FRN assay. Because this method is less laborious and the results are obtained by objective rather than subjective methods, it represents an improvement over the FRN assay.

Serologic Correlates of Immunity in a Tetravalent Reassortant Rotavirus Vaccine Trial

The correlation of antibody responses (serum rotavirus IgA and neutralizing antibody to serotype G1-G4 human rotaviruses and rhesus rotavirus [RRV]) in a reassortant rotavirus vaccine trial with protection against rotavirus infection or disease was investigated. Most subjects administered 4 x 10(5) pfu of either the serotype G1 monovalent or serotype G1-G4 tetravalent vaccine seroconverted for at least one of the six antibodies (85% and 91%, respectively). However, fewer than one-third seroconverted to any prototype G1-G4 human rotavirus. Analyses of covariance indicated that higher prevaccination neutralizing antibody titers negatively affected postvaccination titers. Significant relationships were found between several postvaccination rotavirus antibody titers and protection, and serotype-specific correlates of protection were identified between anti-Wa titers and G1 illnesses (P = .03) and between anti-RRV titers and G3 illnesses (P < .001). Overall, however, serotype-specific immunity was no more significant than heterotypic immunity, and no specific titer of any antibody analyzed was a reliable indicator of protection.

Comparison of seven kits for detection of rotavirus in fecal specimens with a sensitive, specific enzyme immunoassay

A sensitive, specific enzyme immunoassay (SSEIA) was compared to four commercial, enzyme-linked immunosorbent assay (ELISA) kits and three latex agglutination assay (LAA) kits: (1) Rotavirus EIA, International Diagnostic Laboratories (IDL), (2) Pathfinder, Kallestadt (KAL), (3) Rotavirus Bio-EnzaBead, Litton (LIT), (4) Rotazyme II, Abbott (RTZII), (5) Slidex Rota-Kit, bioMerieux (SRK), (6) Meritec-Rotavirus, Meridian (MER), and (7) Rotalex, Medical Technology Corporation (RLX). The SSEIA was chosen as the reference method due to its greater sensitivity in comparison to immunoelectron microscopy and polyacrylamide gel electrophoreses of viral RNA segments. Upon evaluation of 136 specimens (of which 44 were positive by SSEIA), the ELISA kits (LIT, KAL, IDL, and RTZII) had sensitivities of 80%, 98%, 91% and 84%; specificities of 95%, 78%, 100%, and 88%; positive predictive values (PPV) of 88%, 68%, 100%, and 77%; and negative predictive values (NPV) of 91%, 99%, 96%, and 92%. When compared with SSEIA, the three LAA tests (SRK, MER, and RLX) had sensitivities of 73%, 75%, and 62%; specificities of 99%, 93%, and 95%; PPVs of 97%, 85%, and 84%; and NPVs of 88%, 89%, and 84%. LAA test results appeared to be reliable, if positive, but the sensitivities of these tests were less than those of the ELISA tests. The ELISA tests that employed specimen specific negative controls were superior in minimizing false positive reactions.

Effect of wastewater spray irrigation on rotavirus infection rates in an exposed population

The Lubbock Infection Surveillance Study was conducted between June 1980 and October 1983 to detect potential increases in enteric infection and disease incidence in a community surrounding a wastewater spray irrigation site. This report concerns the incidence of rotavirus infections in study participates during that period. Rotavirus infection was defined as a > 2-fold increase in rotavirus serum antibody between blood collections which occurred approximately every 6 months. Antibody was detected by an enzyme-linked immunosorbent assay (ELISA). Of the 368 participants who provided ⩾ 2 blood specimens, 67 seroconversions to rotavirus were detected, an average annual rate of 6.8 infections/100 subjects. One subject was infected twice. Seroconversions were observed in 32.7% (3298) of children ⩽ 16 years of age and in 12.7% (33260) of adults (⩾ 17 years old) with a single infection. Thus, significantly more (P < 0.0001) rotavirus infections occurred in children. Baseline rotavirus serum antibody titers were found to be significantly lower (P = 0.047) in subjects who seroconverted. However, many children and adults with high titers were also infected. More seroconversions were observed between June and December (January) than between December (January) and June. Wastewater spray irrigation had no detectable effect on the incidence of rotavirus infection.

Reassortant Formation and Selection Following Coinfection of Cultured Cells with Subgroup 2 Human Rotaviruses

Reassortant formation following coinfection has been suggested as a mechanism of evolution of rotaviruses. This study was designed to examine the selection of reassortants following coinfection of cultured cells with pairs of subgroup 2 human rotaviruses. The three pairs studied (Wa x P, CJN x 31, 62 x 69) were chosen to maximize the number of RNA segments that could be electrophoretically distinguished. After coinfection and multiple passages, reproducible selection of reassortants was observed with each pair. Although more segments were selected from the virus of a pair that grew to higher titre, certain segments were selected independently of the relative growth properties or multiplicities of infection of the coinfecting viruses; selection of other segments was dependent on both. In determining the time and cause of selection it was found that no selection of genomic RNA segments was detectable prior to or during viral particle assembly in coinfected cells. However, selection was evident within the infectious progeny population after a single cycle of replication. Therefore, selection of specific reassortants following coinfection was apparently due to differences in the infectivities of progency viruses and not in their assembly. This implies that these infectivities were a function of the parental origin of specific genomic segments.

Genotypic Selection Following Coinfection of Cultured Cells with Subgroup 1 and Subgroup 2 Human Rotaviruses

The purpose of this study was to determine why identifiable reassortants between subgroup 1 and subgroup 2 rotaviruses have been so rarely isolated from human specimens. Cultured cells were coinfected with pairs of subgroup 1 and 2 human rotaviruses and passaged multiple times to simulate natural reassortant formation and selection in vivo. After coinfection of MA-104 cells with subgroup 1 (DS-1) and subgroup 2 (either Wa or P) strains, approximately 14% of the plaque-picked progeny were shown to be reassortants. During multiple passages of these coinfected cultures, however, complete (Wa virus coinfection) or nearly complete (P virus coinfection) loss of detectable DS-1 segments from progeny was observed. Thus, when all segments of the subgroup 2 viruses were present in coinfected cultures, these segments dominated in the selected progeny. Coinfection with subgroup 1-subgroup 2 rotavirus reassortants and the DS-1 strain followed by multiple passages, however, resulted in complete loss of some segments from the subgroup 2 strains originally present in the reassortants. Therefore, segments from the parental subgroup 2 viruses appeared to be selected in toto during multiple passages because they were dominant as a group, not because individual segments of these viruses were consistently favoured over their subgroup 1 virus counterparts.

Effect of mutation in immunodominant neutralization epitopes on the antigenicity of rotavirus SA-11.

Exposure of rotavirus SA-11 to polyclonal neutralizing antibody from hyperimmunized guinea-pigs permitted selection of variants which were poorly neutralized by antisera against the parental virus. In one-way cross-neutralization experiments, at least 22 of 24 plaque-purified variants could be classified as belonging to a serotype different from that of the parent. Most antisera generated against the variants, however, readily neutralized the parental virus. This indicates that immunodominant neutralization epitopes in the parent differed from those in the variants. Changes in immunodominant epitopes caused the serotypic relationships between the variants and other strains of rotavirus to differ from those of the parental SA-11. The serotypic relatedness of human strain P (human serotype 3) was reduced while, in contrast to results found with the parental SA-11, several of the antisera against the variants recognized the bovine rotavirus NCDV as the same serotype. Causes for these changes are discussed.

Sharing of identical idiotypic determinants among the monoclonal IgG, IgA and IgM in a single individual.

The monoclonal IgM-κ and IgA-κ proteins from the serum of patient CM were initially shown to share identical idiotypic determinants (Fair et al., 1976). It was also shown that a portion of the IgG fraction from this patients serum also contained the reacting idiotypic component. In this report we describe the purification to homogeneity of the IgG monoclonal protein from a later serum sample and show that this isolated IgG monoclonal protein shares identical idiotypic determinants with both the monoclonal IgM and IgA proteins. Our conclusions were based on four separate double antibody competitive equilibrium radioimmunoassays. Sequential changes in the levels of both IgG and IgA over a period of 75 months indicated that these two paraproteins fluctuated in parallel to one another during this period of time and were not significantly influenced by Chlorambucil therapy. Previous immunofluorescent data and the establishment of the structural identities of IgG to IgM and IgA suggest that both pathways of immunoglobulin differentiation (i.e. IgM → IgA and IgM → IgG) may be occurring in patient CM. The differences in the expression of these classes of immunoglobulin observed over the 75-month period may reflect the gradual maturation of these developmental pathways of antibody forming cells.

Shared idiotypic determinants among three electrophoretic components in the IgG-K immunoglobulins from a single individual

Abstract The serum from patient GT was originally shown to contain an IgG1 (κ) + IgG2 (κ) combination of biclonal proteins. Subsequently, we showed that there were three electrophoretic components in this patients serum that appear to be monoclonal proteins: IgG1 (κ) + IgG2 (κ) + IgG2 (κ). Rabbit antisera generated to the three different IgG species were rendered specific for idiotypic (id) determinants. The three anti-idiotypic antisera reacted with determinants on all three of the 125I-labeled proteins to varying degrees in a double antibody radioimmunoassay (RIA). Further, the two IgG2 molecules inhibited the reaction between labeled protein and anti-idiotype serum generated to either of the two proteins to the same extent, indicating that both immunoglobulins shared identical idiotypic determinants. The IgG1 component did not compete in the reaction between either labeled IgG2 and the heterologous anti-IgG2 id antisera. However, the two IgG2 components did compete in the reaction between labeled IgG1 and anti-IgG2 id antiserum or labeled IgG1 and anti-IgG1 id antiserum. These data suggest that the IgG1 molecules share a limited number of idiotypic sites with both of the IgG2 molecules.