No abstract

Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ϳ92% of samples (3, 17, 19).

The Merck pneumococcal (Pn) enzyme-linked immunosorbent assays (ELISAs) for measuring antibodies to 12 serotypes (serotypes 1, 3, 4, 6B, 7F, 8, 9V, 12F, 14, 18C, 19F, and 23F) were validated in 1999. Merck Laboratories developed the Pn assays using 10 g/ml C polysaccharide, 100 g/ml Pn polysaccharide (PnPs) 25, and 100 g/ml PnPs 72 for preadsorption of samples, standards, and controls in order to improve the specificity to the Pn serotypes in the vaccine. The Pn assays utilize postimmunization sera obtained from subjects immunized with PNEUMOVAX 23 as standards for measuring immunoglobulin G concentrations in sera obtained from vaccine clinical trials with adults and infants. This material was calibrated to the Pn reference standard serum, 89SF, subjected to the Merck Pn ELISA adsorbants. Comparisons were made between the Merck Pn assay and the international Pn assay, showing moderate agreement between the two assay formats. This work describes the test procedures and operating characteristics of the Merck Pn assays and the results of experiments performed to compare the Merck Pn ELISAs to the international Pn ELISAs.PNEUMOVAX 23 (pneumococcal [Pn] polyvalent vaccine), which was licensed in 1983, is a sterile, liquid vaccine for intramuscular or subcutaneous injection. It consists of a mixture of highly purified capsular polysaccharides from 23 of the most prevalent or invasive serotypes of Streptococcus pneumoniae, including the six serotypes (serotypes 6B, 9V, 14, 19A, 19F, and 23F) that most frequently cause invasive drug-resistant Pn infections among children and adults in the United States (1). Bacterial capsular polysaccharides (Ps) induce antibodies primarily by T-cell-independent mechanisms (15); therefore, the antibody response to most Pn capsular polysaccharide types is generally poor or inconsistent in children Ͻ2 years of age, whose immune systems are immature and unable to respond effectively to the polysaccharide antigens.The immunogenicity of PNEUMOVAX 23 in early clinical trials was determined using a radioimmunoassay measuring antibodies to the Pn polysaccharides (PnPs) (14). However, due to the practicality of supporting large-scale testing, the enzyme-linked immunosorbent assay (ELISA) became the preferred method for estimating antibody concentrations. In this respect, the first-generation ELISA showed a poor correlation of antibody concentration with the efficacy of the vaccines. This observation was attributed to an overestimation of the serotype-specific PnPs antibody concentrations due to the presence of contaminating C Ps in the antigen preparations used in the ELISA (4, 6). Subsequently, a second-generation ELISA was developed by taking steps to neutralize C Ps antibodies in the serum prior to ELISA measurements. These Pn assays, utilizing the steps with preadsorption of test sera with C Ps, were compared in a multicenter study (11). The study compared results among 12 laboratories, with each laboratory testing the same series of 48 Pn paired adult serum specimens from 24 individuals (q...