WHO International Standards for antibodies to HPV6 HPV11 HPV31 HPV33 HPV45 HPV52 and HPV58

Abstract

Previously established World Health Organization (WHO) International Standards (IS) for anti-HPV16 and HPV18 antibodies are used to harmonize results across human papillomavirus (HPV) serology assays. Here, we present an international collaborative study to establish ISs for antibodies against HPV6 (NIBSC code 19/298), HPV11 (20/174), HPV31 (20/176), HPV33 (19/290), HPV45 (20/178), HPV52 (19/296) and HPV58 (19/300). The candidate standards were prepared using sera from naturally infected individuals. Each candidate was shown to be monospecific for reactivity against its indicated HPV type except for the HPV11 candidate, which was also reactive against other types. Expression of antibody levels relative to the relevant candidate IS reduced inter-laboratory variation allowing greater comparability between laboratories. Based on these results, the WHO Expert Committee on Biological Standardization established each of the 7 candidates as the 1st IS for antiserum to its indicated HPV type for use in the standardization of HPV pseudovirion-based neutralization and antibody-binding assays.

Fig. 1. Process flow diagram for testing, selection and formulation of donations from naturally infected women to produce the 7 candidate WHO International Standards for HPV antibodies.

Twenty anonymized donations obtained from women naturally infected with Human Papillomavirus (HPV) were provided for initial testing in HPV type-specific pseudovirion-based neutralization assays (PBNA) and antibody binding (Ab-binding) assays. Thirteen candidate samples shown to be seropositive for the target HPV types were selected for further development. Candidate samples for antibodies to HPV6, HPV31, HPV33, HPV45, HPV52 and HPV58 were selected for optimization of pooling ratios to obtain lowest possible cross-reactivities for non-target HPV types. The HPV11 candidate samples were pooled without optimization. The candidate samples were then filled into ampoules and freeze-dried in separate manufacturing procedures to produce the 7 candidate International Standards. Prior to their formal assessment in the multicenter international collaborative study, the candidate International Standards underwent validation testing in PBNA and Ab-Binding assays. The single asterisk indicates that seronegative serum was used for optimizing the reactivities of candidate samples for HPV31 and HPV45 antibodies. The double asterisk indicates that pooling of candidate samples for HPV11 antibodies was not optimized based on the exception criteria of “no type-cross-reactivity” due to difficulty in sourcing monospecific material. The triple asterisk indicates that the candidate International Standards for HPV31 and HPV45 antibodies were validated after the optimization procedure. The candidate International Standards for HPV6, HPV11, HPV33, HPV52 and HPV58 were validated after freeze-drying.

Fig. 2. Assessment of intra-assay variability of duplicate samples tested in PBNA.

Ratios of median absolute antibody concentrations (un-transformed) plotted for duplicate samples (I:P, A:J and B:K) tested for HPV16, 18, 31, 33, 45 and 52 antibodies. A ratio of 1 indicates that the duplicates had matching results. A greater than 20% difference in results for duplicate samples was selected as a guideline to identify greater variability (blue dashed lines mark the 0.8 and 1.2 ratio cut-offs). Data points plotted as 0 on the x axis indicates at that 1 sample (I or P) was reported below the assay cut-off. Ratios of potencies for HPV6, HPV11 and HPV58 for duplicates I and P were not determined as ≥ 50% of laboratories scored the samples negative.

Fig. 3. Assessment of intra-assay variability of duplicate samples tested in Ab-binding assays.

Ratios of median absolute antibody concentrations (un-transformed) plotted for duplicate samples (I:P, A:J and B:K) tested for HPV16, 18, 31, 33, 45, 52 and 58 antibodies. A ratio of 1 indicates that the duplicates had matching results. A greater than 20% difference in results for duplicate samples was selected as a guideline to identify greater variability (blue dashed lines mark the 0.8 and 1.2 ratio cut-offs). Data points plotted as 0 on the x-axis indicates at that 1 sample (I or P) was reported below the assay cut-off. Ratios for responses to HPV6 and HPV11 for duplicates I and P were not determined as ≥ 50% of laboratories scored the samples negative.

Fig. 4. Assessment of inter-assay (within-laboratory) variability for samples tested in PBNA.

Ratios of maximum and minimum laboratory estimates of HPV antibody concentration (un-transformed) for samples tested in at least 3 independent PBNA. A greater than 4-fold difference (blue dashed line cut-off) in sample antibody concentrations across assays was selected as a guideline to identify greater inter-assay variability.

Fig. 5. Assessment of inter-assay (within-laboratory) variability for samples tested in Ab-binding assays.

Ratios of maximum and minimum laboratory estimates of HPV antibody concentration (un-transformed) for samples tested in at least 3 independent Ab-binding assays. A greater than 4-fold difference (blue dashed line cut-off) in sample antibody concentrations across assays was selected as a guideline to identify greater inter-assay variability.