Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies.

Control of measles and rubella through use of attenuated vaccines.

A summary report of the current status of morbidity and mortality associated with measles and rubella vis á vis immunization practices in the United States since these vaccines were licensed in 1963 (measles) and 1969 (rubella) is presented.

A comparison of the reactivity and immunogenicity of RA 27/3 strain rubella vaccine prepared in WI-38 or MRC5 human diploid cells.

The immunogenicity and clinical reactivity of rubella vaccine derived from WI-38 or MRC5 human diploid cells was compared in 125 seronegative adolescent females. Seroconversion rates, assessed by single radial haemolysis testing of paired pre- and post-vaccination samples exceeded 98% (56/57 and 68/68 vaccinees, respectively) for both vaccines. Quantitative assessment of rubella-specific antibodies in 53 post-vaccination sera by an ELISA technique also failed to reveal any difference in immunogenicity between the vaccines. Assessable calendar records documenting the occurrence of local and systemic signs and symptoms in the four weeks following vaccination were returned by 106 subjects. No important statistically significant difference in parameters of clinical reactivity between the vaccine groups was observed although the incidence of pain at the injection site was found to be significantly higher for vaccinees receiving WI-38 derived vaccine.

Mapping T-cell epitopes of rubella virus structural proteins E1, E2, and C recognized by T-cell lines and clones derived from infected and immunized populations.

To design a safe and effective synthetic peptide vaccine against rubella virus (RV) infection, it is necessary to identify immunodominant T-cell epitopes of RV structural proteins. To define such epitopes, 49 overlapping synthetic peptides (17-34 residues in length) corresponding to more than 95% of the amino acid sequence of RV virion proteins E1 (23 peptides) and C (11 peptides) and all of E2 (15 peptides) were synthesized and tested for their capacities to induce proliferative responses of rubella-specific T-cell lines and T-cell clones derived from 4 study groups (5 women infected with RV in pregnancy, 5 patients with congenital rubella syndrome, 5 seropositive healthy donors, and 5 RV vaccine recipients). The most frequently recognized epitopes were E1-21 (residues 358-377) with 11/20 responders, E2-4 (residues 54-74) with 6/20 responders, and C11 (residues 255-280) with 11/20 responders, respectively. E1-10 (residues 174-193), E1-16 (residues 272-291) and E1-18 (residues 307-326) were responded to strongly by corresponding T-cell clones, and were recognized by 4 or 5 T-cell lines. T-cell lines derived from three congenital rubella syndrome patients did not respond to any of the synthetic peptides. The results showed that more T-cell epitopes were present in E1 (19/23) and C (10/11) than in E2 (8/15). The identification of T cell sites recognized frequently by RV-infected or -immunized populations could provide the basis for selecting candidate T-cell epitopes for the development of an effective synthetic vaccine against rubella.

Detection and characterization of pestivirus contaminations in human live viral vaccines.

In view of the use of potentially contaminated foetal calf serum (FCS) in cell cultures pestiviruses may be present in live viral vaccines. Thirty-six lots of human live viral vaccines produced by three manufacturers were tested for the presence of pestiviruses. Bovine viral diarrhoea virus (BVDV) RNA was detected in 33% of the vaccine lots. All positive results were caused by the mumps component of a single manufacturer. Partial sequences of the 5' untranslated region of BVD viral RNA were determined. The sequences were closely related to that of the NADL strain of BVDV. The amount of BVDV RNA in the vaccines was determined by real-time RT-PCR using the LightCycler. Between 3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present in the vaccine samples.Additionally, culture tests were done with FCS and human diploid cells used in the vaccine production of the manufacturer whose vaccines were positive by PCR. All attempts to detect virus antigen in MRC-5 human diploid cells or to infect these cells with BVDV failed. This suggests that BVDV RNA detected in human live viral vaccines represents passive carry over of BVDV from contaminated FCS rather than active virus replication in human diploid cells. Our results indicate that contamination with BVDV of FCS used in vaccine production does not appear to be of immediate concern to human health. Furthermore, our results indicate that gamma-irradiation of FCS destroys BVDV particles and is also effective in preventing the presence of BVDV RNA in the vaccines.

Expression and characterization of a soluble rubella virus E1 envelope protein.

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1 delta Tm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1 delta Tm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1 delta Tm by the insect cells. The secreted E1 delta Tm protein was purified from serum-free medium by one-step immunochromatography. The purified E1 delta Tm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.

Vacunación antirrubéolica: la vacuna y las estrategias / Vaccination antimeasles: the vaccine and the strategies

Las primeras vacunas utilizadas para uso generalizado fueron las elaboradas con las cepas HPV77 y la Cendehill en 1969. A partir de entonces diversas vacunas han estado disponibles en el mercado, pero la de uso más generalizado es la preparada con la cepa RA27/3 cultivada en células diploides humanas que es más inmunogénica y estimula tanto la producción de anticuerpos humorales como secretorios, todo ello sin que se presente un incremento de los efectos colaterales indeseables. La vacuna antirrubéolica existe en tres presentaciones: sola o asociada con otras, la viral doble (rubéola-sarampión) y la viral triple (rubéola-sarampión-parotiditis). En las tres formas la dosis es de 0.5 mL, se prepara en forma liofilizada y debe guardarse en refrigeración (entre 2§C y 8§C) antes de su reconstrucción. Una vez reconstruida debe aplicarse antes de ocho horas. En relación con la vacunación antirrubéolica, existen varias posibles estrategias. Las más importantes son: 1.No incluir a la vacuna contra la rubéola en los programas nacionales de vacunación. 2.Vacunar a todos los suceptibles mayores de un año de edad con énfasis en niños, adolescentes y mujeres adultas. 3.Vacunar a todas las niñas de 11 a 14 años de edad. Y 4.vacunar a grupos específicos: mujeres adultas rubéola-seronegativas, mujeres en el post-parto y personal médico y paramédico principalmente