Characterization of a monoclonal antibody against a nuclear antigen associated with serotype-1 Marek's disease virus-infected and transformed cells

A monoclonal antibody (MAb), 2BN90, was characterized on a panel of Marek's disease virus (MDV) isolates representing three antigenically related serotypes and lymphoblastoid cell lines established from Marek's disease (MD) tumors. It was reactive with all serotype 1 isolates, including CV1988, in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) test. The MAb immunoprecipitated and immunoblotted a 40-kilodalton protein, which was found to be phosphorylated by metabolic labeling with 32P inorganic phosphate, and localized to the cell nucleus using the IF test. It reacted with MDV-transformed lymphoblastoid cell lines, and the percentage of reactive cells was enhanced after treatment with iododeoxyuridine. MAb 2BN90 may be useful as a type-specific antibody of choice for serotyping MDV serotype 1 strains and as a probe for studying the role of pp40 in MDV infection and transformation.     

Strategy for cross-protection among Shigella flexneri serotypes

Based upon the lipopolysaccharide (LPS) structure and antigenicity of Shigella group B, a strategy for broad cross-protection against 14 Shigella flexneri serotypes was designed. This strategy involves the use of two S. flexneri serotypes (2a and 3a), which together bear the all of the major antigenic group factors of this group. The novel attenuated strains used in these studies were S. flexneri 2a strain CVD 1207 (DeltaguaB-A DeltavirG Deltaset1 Deltasen) and S. flexneri 3a strain CVD 1211 (DeltaguaB-A DeltavirG Deltasen). Guinea pigs were immunized with an equal mixture of these strains and later challenged (Sereny test) with a wild-type S. flexneri serotype 1a, 1b, 2b, 4b, 5b, Y, or 6 strain of demonstrated virulence in the same model. Guinea pigs that were immunized with these two vaccine strains produced serum and mucosal antibodies that cross-reacted with all the S. flexneri serotypes tested (except of S. flexneri serotype 6) as assessed by enzyme-linked immunosorbent assay, immunoblotting, and slide agglutination. Furthermore, the combination vaccine conferred significant protection against challenge with S. flexneri serotypes 1b, 2b, 5b, and Y but not with serotypes 1a, 4b, or (as predicted) 6.     

Genetic and antigenic analysis of human rotavirus prevalent in Al-Taif, Saudi Arabia

The subgroup, serotype and electropherotype diversity of human rotavirus strains was investigated in Al-Taif, Saudi Arabia. Out of 349 faecal samples collected from diarrhoeic children, 150 (43 percent) tested rotavirus positive by a group-A specific enzyme-linked immunosorbent assay (ELISA). The majority (87 percent) of the infected children were below 2 years of age. Subgrouping and serotyping of rotaviruses with specific monoclonal antibodies showed that of the 150 rotavirus positive specimens, 17 percent belonged to subgroup I, 59 per cent belonged to subgroup II, and 24 percent were neither subgroup I nor subgroup II. The specimens were typed, as serotype 1 (43 percent), serotype 2 (5 percent), serotype 3 (11 percent), serotype 4 (10 percent) or mixed serotypes (3 percent). The remaining 41 (27 percent) specimens were untypeable. None of the serotypes showed association with a particular age group. An electrophoretic analysis of viral RNA revealed 11 distinct patterns (six long and five short). The majority, 78 percent were long patterns and 22 percent were short patterns. Analysis of the specimens for which subgroups, serotypes and electropherotypes were available indicated that a given RNA pattern does not correspond to a particular subgroup or serotype.     

Immune response to three doses of quadrivalent rotavirus vaccine: 1-year follow-up

Twenty-eight children who received three doses of the quadrivalent rotavirus vaccine with 4 x 10(5) plaque-forming units (p.f.u.) were followed during a year after vaccination. Serum samples were obtained and evaluated for rotavirus IgA and neutralizing antibodies against vaccine and human rotavirus strains. At the end of the study, up to 61% of the children showed an increase in circulating IgA antibody levels. Nearly all of the vaccinated children increased their neutralizing antibody titres against the vaccine strains, and 25-54% against human rotavirus serotypes. After comparing the vaccinees with a population of children naturally infected with serotype G1 in the same study area, we conclude that three doses of 4 x 10(5) p.f.u. of the quadrivalent vaccine should prepare the child against future severe rotavirus diarrhea.     

Rotavirus

Rotavirus, the most common diarrheal pathogen in children worldwide, causes approximately one third of diarrhea-associated hospitalizations and 800,000 deaths per year. Because natural infection reduces the incidence and severity of subsequent episodes, rotavirus diarrhea might be controlled through vaccination. Serotypespecific immunity may play a role in protection from disease. Tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) (which contains a rhesus rotavirus with serotype G3 specificity and reassortant rhesus-human rotaviruses with G1, G2, and G4 specificity) provides coverage against the four common serotypes of human rotavirus. In clinical trials in industrialized countries, RRV-TV conferred 49% to 68% protection against any rotavirus diarrhea and 61% to 100% protection against severe disease. This vaccine was licensed by the U.S. Food and Drug Administration on August 31, 1998, and should be cost-effective in reducing diarrheal diseases in industrialized countries. The vaccine's efficacy and cost-effectiveness in developing countries should be evaluated.     

Detection of an Actinobacillus pleuropneumoniae serotype 2 lipopolysaccharide (LPS) variant

Until now 12 serotypes of Actinobacillus pleuropneumoniae have been recognized. The specificity of the serotypes reside in the carbohydrate composition of the capsular polysaccharides and lipopolysaccharides (LPS). The LPS of A. pleuropneumoniae serotype 2 is a smooth type LPS with O-chains of linear repeating pentasaccharide units with an O-acetyl group linked to a glucose unit. A monoclonal antibody (MAb 102-G02) directed against A. pleuropneumoniae serotype 2 was characterized in enzyme linked immunosorbent assay (ELISA) and in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The MAb 102-G02 was directed against an epitope on the O-chain of the LPS and was used to define a new. LPS variant of A. pleuropneumoniae serotype 2 (referred to as A. pleuropneumoniae serotype 2X). Investigation of the reactivity of the MAb 102-G02 against an A. pleuropneumoniae serotype 2X, field isolate (9008) and the Danish App-2 strain 4226 in electron microscopy, confirmed the different binding patterns.     

Evaluation of a rapid immunochromatographic test for diagnosis of dengue virus infection

A rapid (<7-min) immunochromatographic test for immunoglobulin M (IgM) and IgG antibodies to dengue viruses was evaluated by using hospital admission and discharge sera from 124 patients. The reference laboratory diagnosis was based on the results of virus isolation, hemagglutination-inhibition assay (HAI), and enzyme immunoassay (EIA). By the standard assays, patients experienced primary dengue virus infection (n = 30), secondary dengue virus infection (n = 48), Japanese encephalitis (JE) virus infection (n = 20), or no flavivirus infection (n = 26). The rapid test demonstrated 100% sensitivity in the diagnosis of dengue virus infection and was able to distinguish between primary and secondary dengue virus infections through the separate determinations of IgM and IgG. For all patients with primary dengue virus infection a positive test for IgM to dengue virus and a negative test for IgG to dengue virus were obtained, whereas for 46 of 48 patients (96%) with secondary dengue virus infection, a positive test for IgG to dengue virus with or without a positive test for IgM to dengue virus was obtained. The remaining two patients with secondary dengue virus infection had positive IgM test results and negative IgG test results. Furthermore, the rapid test was positive for patients confirmed to be infected with different dengue virus serotypes (12 infected with dengue virus serotype 1, 4 infected with dengue virus serotype 2, 3 infected with dengue virus serotype 3, and 2 infected with dengue virus serotype 4). The specificity of the test for nonflavivirus infections was 88% (3 of 26 positive), while for JE virus infections the specificity of the test was only 50% (10 of 20). However, most patients with secondary dengue virus infection were positive for both IgM and IgG antibodies to dengue virus, while no patients with JE virus infection had this profile, so cross-reactivity was only a concern for a small proportion of patients with secondary dengue infections. The rapid test demonstrated a good correlation with the reference EIA and HAI and should be useful for the rapid diagnosis of dengue virus infections.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Managing pain after coronary artery bypass surgery

From June 1992 to May 1993, rotaviruses were detected by an immunoenzymatic assay in 159 (49.5%) of 321 children admitted to the hospital with acute diarrhea. Of the 159 cases ELISA positive, 80 samples were chosen at random to investigate subgroups and serotypes of group A human rotavirus. By the ELISA test 9 (11.3%) of the strains were subgroup I, 46 (57.5%) were subgroup II, and 25 (31.3%) could not be grouped. The serotype G1 was identified in 52 cases (65%), G2 in 11 cases (13.8%), G3 in 1 case (1.2%), and 7 cases (8.8%) showed more than one serotype. By electrophoretic analysis of viral RNA, 137 (42.7%) of the samples exhibited an RNA pattern. The long pattern (59.1%) prevailed over the short pattern (35.8%), and by coelectrophoresis 8 different electropherotypes were found throughout the period of study. These results illustrate the great variety of rotavirus strains in this region of the country.     

Protection against rotavirus disease after natural rotavirus infection. US Rotavirus Vaccine Efficacy Group

Determination of protective efficacy after natural rotavirus infection is important as a basis for evaluating rotavirus vaccines. Therefore, placebo recipients in a large 2-year rotavirus vaccine trial conducted across the United States were followed to determine the protection afforded by natural rotavirus infection. Serotype 1 rotaviruses predominated (93% year 1, 66% year 2), but isolates of all four major human rotavirus serotypes circulated during both years. Of the 45 placebo recipients with documented rotavirus illnesses in year 1, 1 developed rotavirus disease in year 2 compared with 29 of the other 235 placebo recipients (P = .03). Serologic data were available for 171 placebo recipients, and 37 of 140 without rotavirus illnesses in year 1 had evidence of asymptomatic rotavirus infection. None of these 37 experienced rotavirus disease in year 2 compared with 22 of the remaining 103 (P < .001). Overall efficacy after natural rotavirus infection was 93% (95% confidence interval, 50%-99%).     

Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus

Three groups of 10 pigs were vaccinated with an American serotype porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and three groups of 10 pigs were vaccinated with a European serotype PRRSV vaccine. A control group of 12 pigs was left unvaccinated. Four weeks after vaccination the PRRSV-specific antibody titres were determined and each group was challenged with either a Spanish, German or Dutch PRRSV wild-type strain. The serological responses four weeks after vaccination confirmed that the two vaccines were of different serotypes. Vaccination with the American serotype vaccine hardly reduced the level of viraemia after challenge with the European PRRSV wild-type strains, and only after challenge with the Spanish PRRSV strain was a moderate, statistically significant reduction in viraemia observed. In contrast, after vaccination with the European serotype vaccine, viraemia was completely suppressed after challenge with the German PRRSV isolate and almost completely suppressed after challenge with the Spanish and Dutch PRRSV isolates.     

Unstable expression and thermal instability of a species-specific cell surface epitope associated with a 66-kilodalton antigen recognized by monoclonal antibody EM-7G1 within serotypes of Listeria monocytogenes grown in nonselective and selective broths

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective Listeria enrichment broth (LEB). When cells were grown in Listeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used Listeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other Listeria spp. (Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100 degrees C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.     

Serologic correlates of immunity in a tetravalent reassortant rotavirus vaccine trial. US Rotavirus Vaccine Efficacy Group

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.     

Pneumococcal vaccination in HIV-1-infected adults in Uganda: humoral response and two vaccine failures

OBJECTIVES: To assess the feasibility of establishing a pneumococcal vaccine trial among HIV-1-infected adults in Uganda and to characterize their responses to 23-valent pneumococcal polysaccharide vaccine. DESIGN: An open-label pilot trial to assess recruitment and compliance of HIV-1-infected adults in Uganda to vaccination and to determine the immunogenicity of the vaccine. SETTING: A community clinic for HIV-1-infected adults in Entebbe, Uganda. METHODS: Levels of capsule-specific IgG to four common vaccine capsular serotypes were measured before vaccination and 1 month after vaccination. Subsequent rates of disease episodes and deaths, and immunologic responses in two vaccine failures, were followed. RESULTS: One month after-vaccination, both HIV-1-infected (n = 77) and seronegative control subjects (n = 10) demonstrated a significant rise in capsule-specific immunoglobulin G (IgG) for three of four serotypes tested, but levels were significantly lower among HIV-1-infected patients. In 149 patient-years of follow-up, two (2.6%) developed pneumococcal pneumonia, one bacteremic with serotype 1 and one non-bacteremic with serotype 13, a non-vaccine serotype; both patients showed inadequate killing of the organism in vitro. In this same follow-up period, 29 (38%) patients died. CONCLUSION: HIV-1-infected adults in Uganda are at high risk of pneumococcal disease and show a significant but suboptimal response to pneumococcal vaccine. Although reliable recruitment and follow-up of vaccinees is feasible, evaluation of vaccine efficacy may be compromised by limited responses to common vaccine serotypes, an unknown incidence of disease with non-vaccine serotypes, and a high rate of mortality unrelated to Streptococcus pneumoniae infection.     

Antigenic and genomic diversity of human rotavirus VP4 in two consecutive epidemic seasons in Mexico

In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.     

A minor prevalent strain in a severe outbreak of foal diarrhea associated with serotype 3 rotavirus

An epizootic of foal diarrhea due to serotype 3 rotavirus (RV) was observed in 89 of 168 cases (53%) during the period from March to July in 1987. A total of 51 strains of RV were isolated from the 62 diarrheal feces examined, and one isolate (CH-3) showed a unique electropherotype of viral RNA which differed from the others that widely prevailed on this farm. No positive reaction was observed between strain CH-3 and each of the antisera against serotypes 1 to 12 of human and animal RV in neutralization tests. However, dsRNAs of the CH-3 virus were hybridized with a probe prepared from a strain of equine serotype 3 RV.     

Disinfection of eyelid speculums for retinopathy of prematurity examination

Depending on the growth medium used for enrichment of bacterial cells prior to assay, the monoclonal antibody (MAb) EM-6E11 recognizing Listeria genus-specific epitope on 43 and 94 to 97 kDa cell-surface antigens (A. K. Bhunia and M. G. Johnson, Appl. Environ. Microbiol. 58:1924-1929, 1992) exhibited extensive variability in the detection of Listeria species. MAb EM-6E11 strongly detected live cells of all Listeria species and all serotypes of L. monocytogenes by ELISA when cells were grown in nonselective brain heart infusion (BHI) broth, in selective Listeria enrichment broth (LEB), or in Listeria repair broth (LRB). In contrast, EM-6E11 detected only four of the thirteen serotypes of L. monocytogenes (serotypes 1/2c, 3b, 4ab, and 7) when cells were grown in the UVM1 formulation of Listeria enrichment broth (UVM1) or Fraser broth (FRB). This MAb failed to react with live cells of four other Listeria species, including L. ivanovii, L. welshimeri, L. grayi, and L. murrayi cells grown in UVM1 or FRB. Heating of Listeria cells at 100 degrees C for 20 min, irrespective of the enrichment media used, led to large losses of MAb EM-6E11 reactivity in ELISA, suggesting that the specific cell-surface epitopes involved may not be heat stable. Our results confirm that MAb EM-6E11 is suitable for detection of live cells but not heat-killed cells of Listeria spp. and can be used in conjunction with an enrichment step in BHI, LEB, or LRB but not in UVM1 or FRB.     

Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain

The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1-4, and 6-11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype.