Calibration and use of an in-house anti-measles IgG standard serum.

For the purpose of research a large quantity of anti-measles IgG working reference serum was needed. A pool of sera from five teenagers was prepared and named Alexandre Herculano (AH). In order to calibrate the AH serum, 18 EIA assays were performed testing in parallel AH and the 2nd International Standard 1990, Anti-Measles Antibody, 66/202 (IS) in a range of dilutions (from 1/50 to 1/25,600). A method which compared parallel lines resulting from the graphic representation of the results of laboratory tests was used to estimate the power of AH relative to IS. A computer programme written by one of the authors was used to analyze the data and make potency estimates. Another method of analysis was used, comparing logistic curves relating serum concentrations with optical density by EIA. For that purpose an existing computer programme (WRANL) was used. The potency of AH relative to IS, by either method, was estimated to be 2.4. As IS has 5000 milli international units (mIU) of anti-measles IgG per millilitre (ml), we concluded that AH has 12,000 mIU/ml.

Comparison of a commercial enzyme immunoassay with plaque reduction neutralization for maternal and infant measles antibody measurement.

The most practicable assay for measurement of measles IgG (mIgG) in large numbers of sera is an enzyme immunoassay (EIA). To assess how EIA results would agree with those by the gold standard method of plaque reduction neutralization (PRN) we compared the results from the two methods in 43 pairs of maternal and umbilical cord sera, and sera from the corresponding infants when aged 11-14 months. In maternal-cord sera, the differences between mean antibody levels by EIA or PRN were not statistically significant, though in individual sera, differences could be large. However, agreement was less good for infants sera, in which levels of mIgG were very low. The conclusions of a study of transplacental transport of mIgG would not be affected by the use of either technique. When studying waning immunity in infants, PRN should be the method of choice, while results from studies using EIA should be interpreted with caution.

An evaluation of nine commercial EIA kits for the detection of measles specific IgG.

Nine commercial EIAs for measles-specific IgG were compared with haemagglutination inhibition (HI) and plaque reduction neutralization (PRN). A total of 174 sera selected, to give approximately half of the sera without measles antibody by HI, were tested by all EIAs and HI. However, there was sufficient volume of only 101 samples for testing by PRN. A dilution curve of the British Standard measles antibody serum was also tested by each EIA. Assays were evaluated qualitatively against a consensus EIA result, HI and PRN: Gull, Melotest and Behring EIAs performed best. Quantitative evaluation was by assessment of the characteristics of the standard dilution curve, and by plotting differences with PRN against mean: Gull and Melotest EIAs were best.

A study of maternally derived measles antibody in infants born to naturally infected and vaccinated women.

Maternal, cord and infant measles antibody levels were measured and compared in a group of 411 vaccinated mothers and 240 unvaccinated mothers, and their babies, between 1983 and 1991. Maternal and cord sera were tested by haemagglutination inhibition and/or enzyme-linked immunosorbent assay, and plaque reduction neutralization tests were also used to test infant sera. Geometric mean titres were significantly higher in the unvaccinated than in the vaccinated mothers (P < 0.001). Infants born to mothers with a history of measles had higher antibody levels at birth than infants of vaccinated mothers and, although the difference narrowed over time, continued to have higher levels up to 30 weeks of age. Between 5 and 7 months of age significantly more of the children of vaccinated mothers had plaque reduction neutralization antibody levels below that which would interfere with vaccination. As the boosting effect of circulating natural measles disappears, earlier measles vaccination may need to be considered, perhaps as part of a two-dose policy.

Antibodies to measles, mumps and rubella in UK children 4 years after vaccination with different MMR vaccines.

Persistence of antibodies 4 years after vaccination with measles, mumps and rubella (MMR) vaccine from three different manufacturers was compared in 475 children who received a single injection of vaccine when aged 12-18 months. Antibodies to measles and mumps were measured using a plaque reduction neutralisation assay; rubella antibodies were measured by radial haemolysis and latex agglutination. Children given MMR vaccine containing the Urabe mumps strain were less likely to be antibody negative than those given the Jeryl Lynn mumps strain (39/266, 15% vs 39/204, 19%, p = 0.048). However, the relatively high proportions in both groups without detectable mumps neutralising antibody suggests the probable need for a second dose in order to achieve mumps elimination. No significant differences were found in the proportions with detectable antibody to measles between vaccines containing the Schwarz and the Enders-Edmonston strains. Overall, only 3% of vaccinees were without detectable measles antibody, although a further 28% had a level below 200 mille International Units, the putative protective level for clinical measles. Geometric mean titres (GMTs) to measles were twofold higher in girls vaccinated after than before 14 months of age; GMTs in boys were intermediate and showed no age effect. Over 99% of vaccinees were seropositive to rubella, confirming the excellent immunogenicity of the RA 27/3 rubella strain and the potential for elimination of rubella with a single dose strategy.

Fragmentation of therapeutic human immunoglobulin preparations.

Some commercial batches of human therapeutic immunoglobulins (Ig) have been found to show evidence of molecular fragmentation when examined by molecular sizing methodologies including sodium dodecyl sulphate polyacrylamide gel electrophoresis [SDS-PAGE] and size exclusion high performance liquid chromatography (SE-HPLC). These batches all demonstrated impaired immunobiological activity (efficacy) as assessed by Fc function measured using a rubella haemolytic assay and as such are likely to be subpotent for therapeutic use. Fragmented Igs were characterized by the presence of at least three protein bands and peaks additional to monomeric IgG. Incubation of Igs with blood enzymes (plasmin and kallikrein) reproduced the fragmentation patterns observed for intrinsically degraded batches, suggesting that fragmentation occurred by contamination with these proteases from the source material (human blood) during manufacture. Intravenous Igs (IVIG) were found to be more susceptible to proteolysis than intramuscular Igs, probably as a consequence of the post-fractionation processing that some IVIGs receive which may induce molecular alterations, allowing enzyme access and fragmentation. Two of the products examined were found to be relatively resistant to proteolysis and both were formulated by processes that limit enzyme activity. These processes were inclusion of an enzyme inhibitor, alpha 2-macroglobulin, and formulation at acidic pH. Enzyme carry-over into the final product is a likely cause of Ig fragmentation, and reduction in levels of such contamination should lead to improvements in product stability and efficacy.

Immunogenicity of high-titre AIK-C or Edmonston-Zagreb vaccines in 3.5-month-old infants, and of medium- or high-titre Edmonston-Zagreb vaccine in 6-month-old infants, in Kinshasa, Zaire.

The effect of measles vaccine potency was evaluated among 485 children aged 6 months, and the effect of vaccine strain was evaluated among 538 children aged 3.5 months, in Kinshasa, Zaire. Children aged 6 months were randomly assigned to receive either high-titre Edmonston-Zagreb (EZ-H), potency 5.7 log10/dose, or medium-titre EZ (EZ-M), potency 4.7 log10/dose, those aged 3.5 months were randomly assigned to receive either AIK-C, potency 5.5 log10/dose, or EZ-H, and were revaccinated with EZ-M vaccine at age 9.5 months. Measles antibodies were measured using the plaque reduction neutralization assay. Among children vaccinated at age 6 months, the seroresponse was significantly higher after EZ-H than EZ-M vaccine, with 92 and 83% seroconverting by 6 months postvaccination and 59 and 40% respectively having antibody titres > 200 mIU. Among children vaccinated at age 3.5 months, only 24% (AIK-C) and 22% (EZ-H) attained antibody titres > or = 200 mIU 6 months postvaccination. After revaccination at age 9.5 months, 81% of children in the AIK-C group and 73% in the EZ-H group had antibody levels > 200 mIU (p = 0.056). A retrospective survey was conducted in January 1993 to determine the mortality experience of vaccine groups, and information was obtained for 94% of the children. A total of 44 deaths (4%) were identified, with no significant differences between groups when stratified by age at vaccination.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between dose and response of standard measles vaccines.

A considerable number of field trials have been published over the last 30 years, showing measles vaccines to be highly effective. Due to inconsistencies in methodology, comparisons between these studies have often been difficult with regard to the potencies used and the sero-response of vaccinated children. This article reviews key studies which have measured the response of children to different potencies of vaccine. The review concludes that increasing the titre of vaccine above log10 3.0 does not appear to improve vaccine efficacy. It is recommended that standard titre measles vaccine should be administered with a minimum potency of log10 3.0 TCID50 or PFU per dose at 9 months of age or later. This recommendation is endorsed by the Global Advisory Group and the Research and Development Group of the Expanded Programme on Immunization.

A European collaborative study to assess the proficiency of laboratory estimates of potency of live measles, mumps and rubella tri-valent vaccines.

A collaborative study involving 10 European laboratories was undertaken to assess the variability of estimates of the potency of measles, mumps and rubella tri-valent vaccines. The precision of assays as demonstrated by tests on duplicate samples was good; differences averaged around 0.2 log10 steps. Similarly, assay to assay variation within laboratories was small with most achieving consistency around 5% over three assays. In contrast, overall median variations in potency between laboratories were around 1.0 log10 for measles, 3.0 log10 for mumps and 2.0 log10 for rubella. Unexpectedly, the variations in estimates for measles and rubella were not improved when potencies were expressed relative to reference preparations. However, for mumps variability was reduced by using a reference but only for the vaccines of the same strain as the reference. For these Urabe mumps vaccines the variation in relative potency was around 1.5 log10.

The Jeryl Lynn vaccine strain of mumps virus is a mixture of two distinct isolates.

Sequence analysis of the region of the mumps virus genome encoding the putative small hydrophobic protein gene confirms that it is a highly variable region. Jeryl Lynn, the mumps vaccine strain used in the U.K., is shown to be a mixture of two closely related viruses, both probably of American origin.

Identification of a new mumps virus lineage by nucleotide sequence analysis of the SH gene of ten different strains.

The SH gene and its flanking sequences have been analysed for 10 strains of mumps virus (MuV) and compared to 5 others. A new lineage has been identified among UK isolates. Changes in the transcription pattern could not be correlated with differences in the sequences of the F-SH and SH-HN intergenic regions of the genome.

A collaborative study to assess the proficiency of laboratory estimates of potency of live measles vaccines.

A collaborative study was undertaken to assess the variability in estimates of the potency of measles vaccines. Overall a median variation of 2.0 log10 between estimates was observed. This was reduced to a median of 1.0 log10 when the potencies were expressed relative to a reference vaccine. A difference in the sensitivity between plaque assays and TCID50 assays was also reduced when relative potencies were used. The benefit of including a common reference preparation in vaccine assays was therefore demonstrated. For the vaccines assayed in this study, it was not necessary to use a measles reference of the same strain as the vaccines tested. We therefore recommend that measles vaccines be assayed against a single international reference preparation.

Viral infection induces cytokine release by beta islet cells.

Viral infection has been suggested to play a triggering role in the pancreatic beta cell destruction which occurs in insulin-dependent diabetes (IDDM). However, the underlying mechanism of this phenomenon is unknown. In this study a human insulinoma cell line has been infected with measles, mumps and rubella viruses since a temporal association is reported between the clinical onset of IDDM and diseases caused by these viruses. The infection with measles and mumps viruses induced the release of interleukin-1 (IL-1) and interleukin-6 (IL-6) by the cell line as assessed by a bioassay and up-regulated the expression of human leucocyte antigen (HLA) class I and class II antigens as evaluated by cytofluorimetric analysis. Stimulation with rubella virus induced the release of IL-6 only and had no effect on HLA antigen expression. These data show for the first time that IL-1 and IL-6 secretion by an insulinoma cell line may occur after viral infection and suggest that cytokine release and increased expression of HLA molecules by beta cells may act to induce the immune response towards beta cells in IDDM.

The 1st International Standard for anti-measles serum.

The 1st International Standard for anti-measles serum has been established on the basis of a collaborative study. There were four participating laboratories in four countries and three types of assay used. This standard has been assigned a potency of 5 IU anti-measles antibody per ampoule.