The establishment of a highly sensitive ELISA for detecting bovine serum albumin (BSA) based on a specific pair of monoclonal antibodies (mAb) and its application in vaccine quality control.

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying BSA was established, based on two mAbs that recognize different epitopes on a BSA molecule. Our ELISA system was used to detect BSA concentrations in several vaccines, such as the MMR (measles, mumps and rubella) vaccine, hepatitis A vaccine, and hepatitis B vaccine. Moreover, we compared the mAb ELISA and the present pAb ELISA by detecting BSA standards and bovine serum samples. The results showed that our ELISA system was in good accordance with the pAb ELISA system. A pair of mAbs (FMU-BSA NO.6 and FMU-BSA NO.11) from 11 murine hybridomas secreting BSA-specific mAbs was selected for the development of the sandwich ELISA. The detection limit of this quantitative assay reaches 0.38 µg/L, which is 10-fold more sensitive than those previously reported. The quantitative range of BSA concentration is from 0.5 to 40 µg/L, which is comparable to the currently used polyclonal antibody (pAb) ELISA. Intra-assay and inter-assay coefficient variations are both lower than 10% at the three concentrations used (10, 20, and 40 µg/L). Thus, the mAb sandwich ELISA developed herein may provide a stable, precise, and highly sensitive method for quantifying BSA, which is very useful in the quality control of some vaccines.

Establishment of a biological reference preparation for hepatitis A vaccine (inactivated, non-adsorbed).

A reference standard calibrated in International Units (IU) is needed for the in vitro potency assay of hepatitis A vaccines prepared by formalin-inactivation of purified hepatitis A virus grown in cell cultures. Thus, a project was launched by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish one or more non-adsorbed inactivated hepatitis A vaccine reference preparation(s) as working standard(s), calibrated against the 1st International Standard (IS), for the in vitro potency assay (ELISA) of all vaccines present on the European market. Four non-adsorbed liquid preparations of formalin-inactivated hepatitis A antigen with a known antigen content were obtained from 3 manufacturers as candidate Biological Reference Preparations (BRPs). Thirteen laboratories participated in the collaborative study. They were asked to use an in vitro ELISA method adapted from a commercially available kit for the detection of antibodies to hepatitis A virus. In-house validated assays were to be run in parallel, where available. Some participants also included commercially available hepatitis A vaccines in the assays, after appropriate desorption. During the collaborative study, several participants using the standard method were faced with problems with some of the most recent lots of the test kits. Due to these problems, the standard method did not perform satisfactorily and a high number of assays were invalid, whereas the in-house methods appeared to perform better. Despite this, the overall mean results of the valid assays using both methods were in agreement. Nonetheless, it was decided to base the assignment of the potency values on the in-house methods only. The results showed that all candidate BRPs were suitable for the intended purpose. However, based on availability of the material and on the results of end-product testing, 2 candidate reference preparations, Samples C and D, were selected. Both were from the same batch but filled on different days; no statistically significant difference in potency was observed. They were thus combined in 1 single batch. The candidate preparation (Sample C/D) was adopted at the June 2009 session of the European Pharmacopoeia (Ph. Eur.) Commission as the Ph. Eur. BRP batch 1 for hepatitis A vaccine (inactivated, non-adsorbed), with an assigned potency of 12 IU/ml for in vitro antigen content assays. Accelerated degradation studies have been initiated. The preliminary data show that the BRP is stable at the recommended storage temperature (< -50 degrees C). The BRP will be monitored at regular intervals throughout its lifetime.

Reduced-dose intradermal vaccination against hepatitis A with an aluminum-free vaccine is immunogenic and can lower costs.

A reduced dose (0.1 mL) of intradermal hepatitis A virus (HAV) vaccine could facilitate the control of hepatitis A in countries of endemicity. All study subjects receiving an aluminum-free HAV vaccine intradermally were seroprotected 28 days after vaccination (anti-HAV titer, > or =10 mIU/mL). Seroprotection rates decreased to 80.8% at 12 months but returned to 100%, with titers increasing 28-fold, after receipt of a booster vaccination.

Impact of hepatitis A vaccination of Indigenous children on notifications of hepatitis A in north Queensland.

OBJECTIVE: To describe the impact of a hepatitis A vaccination program for Indigenous children in north Queensland. DESIGN: Enhanced surveillance of all notified cases of hepatitis A in north Queensland from 1996 to 2003. SETTING: North Queensland; population, 596 500 people, including about 6900 Indigenous children aged under five years. INTERVENTIONS: Hepatitis A vaccine was provided to Indigenous children in north Queensland from February 1999; two doses were recommended (at 18 months and 2 years of age), as was catch-up vaccination up to the sixth birthday. RESULTS: In the 4 years 1996-1999, 787 cases of hepatitis A were notified in north Queensland, 237 (30%) of which were in Indigenous people. The average annual notification rates in Indigenous and non-Indigenous people during this period were 110 and 25 cases per 100 000 persons, respectively. In the first 4 years after introduction of the vaccination program (2000-2003), 66 cases of hepatitis A were notified. Only nine of the 66 (14%) were in Indigenous people. The average annual notification rates in Indigenous and non-Indigenous people in 2000-2003 were 4 and 2.5 cases per 100 000 persons, respectively. CONCLUSION: Hepatitis A seems to have been eradicated from Indigenous communities in north Queensland very soon after the vaccination program began. The rapid decline in notifications in non-Indigenous as well as Indigenous people suggests the program quickly interrupted chains of transmission from Indigenous children to the broader community. To our knowledge this is the first evidence that a hepatitis A vaccination program targeting a high-risk population within a community can reduce disease in the broader community. Hepatitis A vaccine should be provided to other high-risk Indigenous children elsewhere in Australia.

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines.

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.

Biophysical validation of Epaxal Berna, a hepatitis A vaccine adjuvanted with immunopotentiating reconstituted influenza virosomes (IRIV).

Inactivated vaccines usually contain an adjuvant which potentiates the immune response to the antigen. During the last 70 years aluminium salts have been the only adjuvant licensed for human use. The adjuvanting activity is based on their serving as an antigen depot and inducing a localized inflammatory response. Our efforts to develop a potent and well tolerated adjuvant has focussed on the use of immunopotentiating reconstituted influenza virosomes (IRIV). The IRIV base is a liposome with a mean diameter of 150 nm, comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These mammalian phospholipids are virtually non-immunogenic and have enjoyed a long history of use in human pharmaceutical preparations. The haemagglutinin (HA) and trace quantities of viral neuraminidase and phospholipids from the A/Singapore 6/86 virus strain are intercalated within the phospholipid bilayer. The presence of the HA is necessary to enhance the immunopotentiating effect to antigen associated with IRIV The excellent characteristics of IRIVs as adjuvants have been demonstrated in several systems. IRIVs as alternative adjuvant system for human use are registered by most European, Asian and American countries in commercial hepatitis A and influenza vaccines. IRIVs were first used in the manufacture of a hepatitis A vaccine. This contains formalin-inactivated and highly purified hepatitis A virus (HAV) of strain RG-SB, cultured in human diploid cells, which is coupled to the IRIV vesicle. For a new influenza vaccine the surface spikes (HA and NA) of three currently circulating influenza strains were jointly inserted into the vesicle membrane of the IRIVs and successfully tested clinically. In Epaxal Berna, the first commercially available liposomal vaccine is expected to be the inactivated hepatitis A virus adsorbed to IRIV particles. In the virosomal hepatitis A vaccine, the antigen is believed to be attached to the IRIV by interacting with phospholipids which are considered to correspond to its natural receptor on hepatocytes. The present investigation includes data based on light scattering measurements which show the binding of the virions to vesicles.