Dianna E. Wilkinson

Establishment of the 1st World Health Organization international standards for human papillomavirus type 16 DNA and type 18 DNA.

A World Health Organization collaborative study was conducted to evaluate candidate international standards for human papillomavirus (HPV) Type 16 DNA (NIBSC code 06/202) and HPV Type 18 DNA (NIBSC code 06/206) for use in the amplification and detection steps of nucleic acid‐based assays. The freeze‐dried candidate international standards were prepared from bulk preparations of cloned plasmid containing full‐length HPV‐16 or HPV‐18 genomic DNA. Nineteen laboratories from 13 countries participated in the study using a variety of commercial and in‐house quantitative and qualitative assays. The data presented here indicate that, upon freeze‐drying, there is no significant loss in potency for the candidate HPV‐18 DNA and a slight loss in potency for the candidate HPV‐16 DNA; although this is likely not scientifically relevant when assay precision is considered. In general, the individual laboratory mean estimates for each study sample were grouped ±∼2 log10 around the theoretical HPV DNA concentration of the reconstituted ampoule (1 × 107 HPV genome equivalents/mL). The agreement between laboratories is improved when potencies are made relative to the candidate international standards, demonstrating their utility in harmonizing amplification and detection steps of HPV‐16 and −18 DNA assays. Degradation studies indicate that the candidate international standards are extremely stable and suitable for long‐term use. Based on these findings, the candidate standards were established as the 1st WHO international standards for HPV‐16 DNA and HPV‐18 DNA, each with a potency of 5 × 106 international units (IU) per ampoule or 1 × 107 IU mL−1 when reconstituted as directed.

The first international standard for antibodies to HPV 16

Current HPV vaccines and vaccine candidates are based on recombinant virus capsid proteins, so called virus-like particles (VLPs). Standardisation of assays for HPV capsid antibody will assist with epidemiology studies and future vaccine development. A World Health Organization international collaborative study was undertaken to assess the suitability of a freeze-dried serum, obtained from women naturally infected with HPV 16 and reactive against HPV 16 only, to serve as the International Standard for antibodies to HPV 16 in immunoassays and pseudovirion neutralisation assays. Eleven laboratories from nine countries participated in the collaborative study in which the candidate (NIBSC code 05/134) was assayed alongside samples from both vaccinees and naturally infected individuals. 05/134 had titres which were comparable to those obtained with serum from a naturally infected individual. Overall the variation between laboratories is similar to that observed in the previous study for samples from naturally infected individuals although slightly wider for sera from vaccinees. 05/134 has been established by the WHO Expert Committee on Biological Standardization as the 1st International Standard for antibodies to HPV 16, human serum, with an assigned potency of 5IUper ampoule.

Human papillomavirus antibody reference reagents for use in postvaccination surveillance serology.

ABSTRACT Suitably controlled serosurveillance surveys are essential for evaluating human papillomavirus (HPV) immunization programs. A panel of plasma samples from 18-year-old females was assembled, the majority of the samples being from recipients of the bivalent HPV vaccine. Antibody specificities were evaluated by three independent laboratories, and 3 pools that displayed no antibodies to any HPV type tested or intermediate or high levels of antibody to HPV16, HPV18, HPV31, and HPV45 were created. These pools will be useful as control reagents for HPV serology.

Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins

Highlights • Comprehensive pre-clinical immunogenicity evaluation of HPV L1 major capsid protein.• Majority neutralizing antibody response was genotype-specific.• Reciprocal cross-neutralization between some Alpha-7 and Alpha-9 genotypes.• Tetravalent formulation (HPV16/18/39/58) induced broadly neutralizing antibodies.• These data improve our understanding of the antigenic diversity of the L1 protein.

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Characterization of the 3rd International Standard for hepatitis B virus surface antigen (HBsAg)

BACKGROUND HBsAg is the most important marker for laboratory diagnosis of HBV infection. Validation and quality control of HBsAg tests requires International Standards (IS). Recently the 2nd IS was replaced by the 3rd IS. Both IS are made from plasma-derived hepatitis B vaccines, but production and geographical origin are different. OBJECTIVE Characterization of the HBsAg in the source material (SM) for the 3rd IS and comparison with the 2nd IS and native HBsAg. STUDY DESIGN The SM was analyzed using solid-phase immunoassays, quantitative immune electrophoresis, ultracentrifugation, immunoblotting and HBV DNA sequencing. RESULTS The plasma-derived HBsAg of the SM originated from at least two different HBV strains, both of subgenotype (sgt) B4, typical for Vietnam. The HBsAg subtype was heterogeneous with ayw1 and adw2. The HBsAg concentration was 23,700 IU/ml as determined by solid-phase immunoassay; immune electrophoresis calibrated with sgt B2 revealed a concentration of 24,500 IU/ml while calibration with sgt D1 provided lower values. Proteins in the SM are heterogeneous in size containing only traces of preS. The protein subunits are partially cross-linked. CONCLUSIONS The antigenicity of the 3rd IS is suitable for HBsAg calibration in laboratory tests. In contrast to the 2nd IS, the 3rd IS is representative for a highly endemic region. Similar to the 2nd IS and different from native HBsAg, preS domains are depleted, protein subunits are partially cross-linked and the HBsAg particles are partially aggregated in the 3rd IS. The HBV subgenotype differences between the two IS may lead to variations in different quantitative assays.