The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

The standardization of inactivated equine influenza vaccines by single-radial immunodiffusion

Single-radial-immunodiffusion (SRD) assays were used for measuring the haemagglutinin (HA) antigen content of equine influenza vaccines containing the virus strains A/equine/Prague/56 (H7N7) and A/equine/Miami/63 (H3N8). Three bivalent aqueous vaccines and one bivalent adjuvanted vaccine were standardized by SRD to contain graded amounts of HA antigen activity. The SRD reaction was influenza subtype specific and was not influenced by the presence of adjuvant in vaccine.     

Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards

Each year the production of seasonal influenza vaccines requires antigen standards to be available for the potency assessment of vaccine batches. These are calibrated and assigned a value for haemagglutinin (HA) content. The calibration of an antigen standard is carried out in a collaborative study amongst a small number of national regulatory laboratories which are designated by WHO as Essential Regulatory Laboratories (ERLs) for the purposes of influenza vaccine standardisation. The calibration involves two steps; first the determination of HA protein in a primary liquid standard by measurement of total protein in a purified influenza virus preparation followed by determination of the proportion of HA as determined by PAGE analysis of the sample; and second, the calibration of the freeze-dried reference antigen against the primary standard by single radial immunodiffusion (SRD) assay. Here we describe a collaborative study to assess the effect of adding a deglycosylation step prior to the SDS-PAGE analysis for the assessment of relative HA content. We found that while the final agreed HA value of the samples tested was not significantly different with or without deglycosylation, the deglycosylation step greatly improved between-laboratory agreement.     

A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen

A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.     

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.     

Collaborative study to assess the suitability of a candidate International Standard for yellow fever vaccine

Yellow fever vaccines are routinely assayed by plaque assay. However, the results of these assays are then converted into mouse LD(50) using correlations/conversion factors which, in many cases, were established many years ago. The minimum required potency in WHO Recommendations is 10(3) LD(50)/dose. Thirteen participants from 8 countries participated in a collaborative study whose aim was to assess the suitability of two candidate preparations to serve as an International Standard for yellow fever vaccine. In addition, the study investigated the relationship between the mouse LD(50) test and plaque forming units with a view to updating the WHO recommendations. Plaque assays were more reproducible than mouse assays, as expected. Differences in sensitivities of plaque assays were observed between laboratories but these differences appear to be consistent within a laboratory for all samples and the expression of potency relative to the candidate standard vaccine improved the reproducibility of assays between laboratories. However, the use of potencies had little effect on the between laboratory variability in mouse LD(50) assays. There appears to be a consistent relationship between overall mean LD(50) and plaques titre for all study preparations other than sample E. The slope of the correlation curve is >1 and it would appear that 10(3) LD(50) is approximately equivalent to 10(4) plaque forming units (PFU), based on the overall means of all laboratory results. The First International Standard for yellow fever vaccine, NIBSC Code 99/616, has been established as the First International Standard for yellow fever vaccine by the Expert Committee of Biological Standards of the World Health Organisation. The International Standard has been arbitrarily assigned a potency of 10(4.5) International Units (IU) per ampoule. Manufacturers and National Control Laboratories are including the First International Standard for yellow fever vaccine in routine assays so that the minimum potency in IU of vaccines released for use and which meet the current minimum potency of 10(3) LD(50) in mouse assays, can be determined. These data will be analysed before a review of the WHO requirements, including the minimum potency per dose, is undertaken.     

Approaches to the control of acellular pertussis vaccines.

The quality control of acellular pertussis vaccines presents particular problems related to the differences in composition and method of detoxification used in the various type of preparation. These vaccines are not amenable to potency assay by the active mouse protection test used for whole-cell pertussis vaccines and assurance of protective activity is problematic.In contrast, monitoring of these vaccines for safety is relatively straightforward and is centred on assays for the lipooligosaccharide endotoxin, active pertussis toxin and absence of reversion to toxicity of detoxified product. The absence of heat-labile toxin, tracheal cytotoxin and adenyl cyclase toxin is assumed provided that adequate validation of the process has been performed.Confirmation of the antigenic content of the detoxified bulk components is difficult to achieve by conventional binding assays based on monoclonal antibodies because of changes in accessibility of reactive sites post-toxoiding. However, single radial diffusion assay using polyclonal antisera permits estimation of pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (P69). Dot blot immunoassay can be used for the fimbrial agglutinogens 2 and 3 (Fim 2 and 3) and potentially could also be used to check the composition of final filling lots for PT, FHA, P69 and Fim 2 and 3.Gel electrophoresis and immunoblotting can be applied to monitor purity of purified bulk components and the characteristics of these change after chemical detoxification. Electron microscopy provides a useful semi-quantitative supporting method for checking purity of bulk components. Physico-chemical examination, particularly CD and fluorescence spectroscopy, offer a means of monitoring the consistency of detoxified bulk components.No completely satisfactory method is available for monitoring potency. Immunogenicity assays may be useful for checking consistency but do not necessarily correlate with protection. At present, active protection against aerosol challenge offers the best prospect of a functional assay.     

Inactivated rabies vaccine control and release: use of an ELISA method.

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations.The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.     

Quantitative estimation of diphtheria and tetanus toxoids. 5. Comparative assays in mice and in guinea-pigs of two diphtheria toxoid preparations

Two freeze-dried international reference diphtheria toxoids of different origin were compared in biological assays in guinea-pigs and mice under different adjuvant conditions. When the antigenic content in the two toxoids was used as denominator for determination of relative potency, that is to say quantitation of immunogenic power per unit amount of antigen, the design of the animal assay proved to have a major influence. Similar observations have been made previously also for tetanus vaccines. It is concluded that diphtheria vaccines as well as tetanus vaccines can hardly be quantitated unambiguously using the currently recommended potency assays in animals. A new scheme for control of toxoid vaccine production is suggested, with more emphasis on the control of the bulk purified toxoid, which would make the release of final products more simple and rapid.     

The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens

Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.     

In vitro potency assay for yellow fever vaccines: comparison of three vero cell lines sources.

Quality control of Yellow Fever vaccines performed by Control Authorities prior to marketing vaccines batches requires in vitro potency assays. The two currently available methods are the plaque formation assay and the cytopathic effect assay based on the use of porcine kidney PS cells or monkey kidney Vero cells. Among several sources of variation in virus titration, the cell systems are considered as important issues and Quality Assurance strongly recommends working with cell banks from certified suppliers. The aim of our study was to compare the behaviour and the sensitivity of three Vero cell sources obtained from ATCC, WHO and EP used at different passage levels in a plaque formation test. The conclusion of this work was that the yellow fever live attenuated virus titration, adapted in Vero cell lines appeared as a reliable method applicable for routine in vitro potency assay. The comparison of Vero cell lines, originated from three different sources, showed that they could be equally used as substrates by laboratories having the basic facility of cell culture, without influence on the final viral titre.     

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.     

Rabies vaccine standardization: International collaborative study for the characterization of the fifth international standard for rabies vaccine

A collaborative study was carried out to establish a replacement for the International Standard for Rabies Vaccine, the stocks of which are exhausted. Three rabies vaccines for human use derived from different rabies virus strains and prepared on different cell culture substrates were compared with the International Standard for Rabies Vaccine using in vivo and in vitro assay methods in a collaborative study involving 14 participants. The proposed fifth International Standard (PISRAV) which was derived from the same virus strain as the present international standard preparation, the Pitman Moore (PM) strain, was found to be approximately twice as potent relative to the International Standard in immunogenicity assays as in antigenicity assays. On the other hand another vaccine, derived from the LEP strain, was considerably more potent in antigenicity assays than in immunogenicity assays. The glycoprotein of the proposed replacement standard measured in antigenicity assays appeared to be stable at +37 degrees C for 245 days, whereas the immunogenicity of the proposed replacement vaccine was sensitive to this heat treatment and the vaccine lost 66% of its immunogenic potency. The results of this study indicate that the NIH protection test should continue to form the primary basis for potency assay of rabies vaccine as glycoprotein content does not appear to correlate with immunogenic potency for different types of vaccine. The vaccine coded PISRAV has been established as the fifth International Standard for Rabies Vaccine and a potency of 16 International Units of Rabies Vaccine (based on the immunogenicity assays) assigned to the contents of each ampoule. Each ampoule has also been assigned a unitage of 10 IU of PM Rabies Virus Glycoprotein and 135 IU of PM Rabies Virus Ribonucleoprotein.     

Regulation and standardization of IPV and IPV combination vaccines.

Inactivated poliovirus vaccine (IPV) is not only increasingly used on a global basis but also is a component of many combination vaccines. Standardization and control of IPV continues to be a challenge for manufacturers and regulators. A rat immunogenicity assay is currently recommended by many authorities, including WHO, as the definitive in vivo potency. Alternative in vitro assays to determine D-antigen content have been developed and are routinely used in some countries to assess IPV potency assay. However, the other less reliable in vivo immunogenicity assays are also used (e.g. monkey, chick). We review some of the regulatory challenges facing current and future IPV assessment, with a focus on the relevance of in vivo and in vitro tests, considerations for Sabin derived IPV and discussion of future efforts for standardization.     

Report of a collaborative study for assessing the potency of hepatitis B vaccines

A collaborative study was carried out to examine the suitability of a hepatitis B vaccine derived from plasma as an immunogenicity reference for vaccines produced by recombinant DNA technology in yeast. The use of a plasma derived vaccine as reference appeared satisfactory, although the use of homologous reference improved agreement in potency estimates. The use of a recombinant standard did not however improve agreement for a recombinant vaccine produced by a different manufacturer. The variation in the dilution of vaccine required to induce antibodies in 50% of test animals and in potency estimates varied widely between laboratories (25-fold and 10-fold respectively). However this was similar to the variation found in a previous collaborative study.     

The minimum significant ratio: a statistical parameter to characterize the reproducibility of potency estimates from concentration-response assays and estimation by replicate-experiment studies

The authors show by illustration that procedures used to validate the reliability of single-concentration high-throughput screens such as the signal window and Z' factor do not ensure sufficient reliability in potency estimates from concentration response assays. They develop the minimum significant ratio as a statistical parameter to characterize the fold change between 2 compounds run in the same experiment that can be considered a real difference and use this parameter to characterize the reliability of the assay. They adapt methods described by Bland and Altman to develop a simple set of 2 experiments to estimate the minimum significant ratio and show that this protocol can identify assays that lack reproducibility. The methods are then extended to validate the equivalency of the same assay run by multiple laboratories.     

Sources of variability in foot and mouth disease vaccine potency estimates based on serum neutralizing antibody assay

Some vaccines can be assayed for potency by measuring the serum antibody response they produce in vaccinated test animals. Using data obtained from potency assays on batches of foot and mouth disease vaccine, the sources of variability in such a method were examined. A linear model is proposed for the analysis of replicate serum neutralizing antibody assays, which represents an improvement on the usual approach of working with only a mean serum assay value for each test animal. Components of variance were calculated, allowing the relative importance of the numbers of test animals, or the numbers of serum assays per test animal, to be estimated in terms of the variability of the overall group mean antibody response. A method is described for calculating fiducial intervals for the serological potency estimates, and it is shown that these intervals are no larger than, and are in fact probably smaller than, those obtained from quantal challenge tests. The results have important implications for the design and analysis of similar biological tests used for other products.     

The development and standardization of an ELISA for ovalbumin determination in influenza vaccines

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ovalbumin in influenza vaccines has been developed and standardized. Commercially available reagents were used. ELISA was compared to single radial immunodiffusion (SRD) and immunoelectro-osmophoresis (IEOP) techniques. The detection limit by ELISA was 0.5 ng/ml. This method was found to be at least 1000 times more sensitive than SRD and at least 200 times more sensitive than IEOP. It was concluded that ELISA is a specific, sensitive and reproducible method for the determination of the small amounts of ovalbumin found as an impurity in unconcentrated influenza vaccines.     

Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide

Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.     

Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine

Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15–CRM197, using capillary Western technology.