Neurovirulence safety testing of mumps vaccines--historical perspective and current status.

Many live, attenuated viral vaccines are derived from wild type viruses with known neurovirulent properties. To assure the absence of residual neurotoxicity, pre-clinical neurovirulence safety testing of candidate vaccines is performed. For mumps virus, a highly neurotropic virus, neurovirulence safety testing is performed in monkeys. However, laboratory studies suggest an inability of this test to correctly discern among virus strains of varying neurovirulence potential in man, and, further, some vaccines found to be neuroattenuated in monkeys were later found to be neurovirulent in humans when administered in large numbers. Over the past decade, concerted efforts have been made to replace monkey-based neurovirulence safety testing with more informative, alternative methods. This review summarizes the current status of mumps vaccine neurovirulence safety testing and insights into models currently approved and those under development.

Detection of a new mumps virus genotype during parotitis epidemic of 2006-2007 in the state of São Paulo, Brazil.

Nucleotide sequence analyses of the SH gene of 18 mumps virus isolates collected in the 2006-2007 parotitis epidemic in the state of São Paulo identified a new genotype, designated genotype M. This new designation fulfills all the parameters required to define a new mumps virus genotype. The parameters were established by an expert panel in collaboration with the World Health Organization (WHO) in 2005. This information will enhance the mumps virus surveillance program both at the national and global levels.

Absence of detectable measles virus genome sequence in blood of autistic children who have had their MMR vaccination during the routine childhood immunization schedule of UK.

Leukocyte preparations from children with documented evidence of MMR vaccination and confirmed diagnosis of autism were examined by several assays designed to target multiple regions of the measles virus genome sequence. No sample was found positive by any method. The assays applied were highly sensitive, specific and robust in nature, and were based on the amplification of measles virus RNA transcripts by real-time quantitative RT-PCR (QRT-PCR) as well as by conventional RT-PCR-nested PCR. The assays applied were potentially able to detect measles virus RNA down to single figure copy numbers per reaction. The amount of total nucleic acid extract of leukocytes subjected to various measles virus-specific investigations was several fold higher than minimally required of a sample where measles virus persistence is well documented. This study failed to substantiate reports of the persistence of measles virus in autistic children with development regression.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates.

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.

Comparative evaluation of measles virus-specific RT-PCR methods through an international collaborative study.

Comparison of RT-PCR assays established in house at various places revealed that laboratories could differ in sensitivity by as much as 1,000-fold in terms of the ability to detect measles virus sequences in clinical samples. The study indicates that PCR findings, positive or negative, are questionable if they are not supported by the associated data demonstrating the overall sensitivity of the assay applied. Measles virus-specific RT-PCR-based assays need to be validated using standard virus preparation or nucleic acid-based target templates. A correlation between real-time quantitative PCR and the conventional PCR for measles virus is highly desirable.

Mumps virus reinfection--clinical findings and serological vagaries.

Sera taken from two cases of mumps reinfection were tested against different strains of mumps virus in a plaque reduction neutralisation assay. Antibodies to mumps in the sera reacted with all strains, but the pattern of emergence of these antibodies differed with the strains tested. These findings raise the possibility of emergence of a mutant strain under the selective pressure of immunisation, with limited or no cross protection induced by the vaccine strain.

Safety evaluation of MMR vaccine during a primary school campaign in Saudi Arabia.

Monitoring of adverse events following the administration of MMR vaccine containing the Urabe mumps virus vaccine strain, to over 2 million schoolchildren (aged 6-13 years) revealed that the incidence of vaccine-associated aseptic meningitis was one case per 295 000 doses given. About 92 per cent of these children had had their primary immunization against MMR at 12 months of age and, therefore, were probably not immunologically naïve. It appears from our data that the use of the Urabe-based mumps vaccine in the booster-dose format induces much less adverse effects than usually observed following the primary immunization with it. Further studies are needed to prove this conclusively.

Further evidence of the absence of measles virus genome sequence in full thickness intestinal specimens from patients with Crohn's disease.

Specimens of macroscopically inflamed and normal intestine along with mesenteric lymph nodes were obtained at resection from patients with Crohn's disease. The samples were systematically examined by RT-PCR-nested PCR targeting N, M and H gene regions of the measles virus genome. None of the samples examined gave any evidence of the persistence of measles virus in the intestine of Crohn's disease patients. The study supports previous findings produced by this laboratory and others using highly sensitive measles virus specific PCR diagnostic technology.

Absence of chicken myelin basic protein residues in commercial formulations of MMR vaccine.

Several preparations of MMR vaccines and their progenitor monovalent vaccine bulks produced by two different manufacturers were examined serologically for the presence of chicken myelin basic protein (MBP) residues. The products were challenged against several commercial preparations of anti-hMBP antisera that reacted positively with the control MBP preparations of human and chicken origins. There was no evidence of the presence of MBP components in MMR vaccines or their progenitor vaccine bulks as shown by the reactivity profiles of the antibody preparations against control and test antigens.

Clinical safety issues of measles, mumps and rubella vaccines.

The clinical safety of measles and measles-mumps-rubella vaccines has been questioned in recent reports that propose a possible link between measles virus or measles vaccines and the occurrence of juvenile Crohn disease and autism. This article reviews the outcomes of several laboratory investigations which were carried out independently to identify the presence or absence of measles virus in the intestinal tissues derived from cases of inflammatory bowel disease. One research group reported the presence of measles virus particles and genomic RNA in inflammatory bowel disease tissues, but this could not be confirmed by other groups, despite use of techniques that are highly specific and sensitive for the detection of measles virus nucleic acid in clinical specimens down to the molecular level. Based on the published data reviewed here, it can be concluded that there is no direct association between measles virus or measles vaccines and the development of Crohn disease, a conclusion which is supported by most epidemiological findings.

Evaluation of the neurovirulence test for mumps vaccines.

Neurovirulence tests in Macaca fascicularis using commercial preparations of different vaccine bulks and a wild-type strain revealed that the test was unable to distinguish mixed from pure populations or a suitable vaccine from a related strain which has been shown to be associated with clinical meningitis. However, the test was able to distinguish a wild-type strain from the vaccine strains successfully. The ability of the test to discriminate between acceptable and unacceptable seeds requires further examination.

Assay of humoral immunity to mumps virus.

One hundred randomly chosen sera from blood donors from North London were assayed for antibodies to mumps virus by plaque reduction and microtitre neutralisation assay, haemagglutination inhibition and in-house ELISA. The assay reproducibility was determined, and there was reasonable agreement between antibody levels measured by the two neutralising methods. Neutralising antibody levels measured by either method were low but the strain used in the assay had a large effect on the antibody titres observed. Titres measured by neutralisation assay, HI assay and ELISA did not correlate well. Assessment of immunity to mumps virus remains problematical.

Wild type mumps viruses circulating in China establish a new genotype.

By analysing the nucleotide sequence of the SH genes of five mumps virus strains derived from the clinical specimens collected during the 1995/96 mumps epidemic in China a new genotype has been established. The circulating viruses showed divergence ranging from 0.8-4.5% at the nucleotide level and 3.5-12.3% at the amino acid level. In addition, a more rational approach has been taken in proposing genotype groupings to MuV strains.