High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2) = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively

Robust in vitro and in vivo neutralization against multiple high-risk HPV types induced by a thermostable thioredoxin-l2 vaccine

Abstract Current prophylactic virus-like particle (VLP) human papillomavirus (HPV) vaccines are based on the L1 major capsid protein and provide robust but virus type-restricted protection. Moreover, VLP vaccines have a high production cost, require cold-chain storage, and are thus not readily implementable in developing countries, which endure 85% of the cervical cancer–related death burden worldwide. In contrast with L1, immunization with minor capsid protein L2 elicits broad cross-neutralization, and we previously showed that insertion of a peptide spanning amino acids 20–38 of L2 into bacterial thioredoxin (Trx) greatly enhances its immunogenicity. Building on this finding, we use, here, four different neutralization assays to demonstrate that low doses of a trivalent Trx-L2 vaccine, incorporating L2(20–38) epitopes from HPV16, HPV31 and HPV51, and formulated in a human-compatible adjuvant, induce broadly protective responses. Specifically, we show that this vaccine, which uses a far-divergent archaebacterial thioredoxin as scaffold and is amenable to an easy one-step thermal purification, induces robust cross-neutralization against 12 of the 13 known oncogenic HPV types. Immune performance measured with two different in vitro neutralization assays was corroborated by the results of mouse cervico-vaginal challenge and passive transfer experiments indicating robust cross-protection also in vivo. Altogether, our results attest to the potential of Trx-L2 as a thermostable second-generation HPV vaccine particularly well suited for low-resource countries. Cancer Prev Res; 8(10); 932–41. ©2015 AACR.</jats:p

Effect of PSV-serum premix incubation time on neutralization titer.

<p>A serum from a Gardasil® vaccinated individual was pre-incubated for different times with HPV 16 or HPV 18 PSVs before the neutralization assay was initiated by the addition of reporter cells. ED₅₀ values with 95% confidence intervals are shown.</p

HT-PBNA inter- and intra-run variability of neutralization titers.

<p>The ED₅₀ values for HPV 16 and HPV 18 PSV of the serum standard were determined in 58 repeats on seven assay days (runs) over a period of 2 months. For six of the 7 assay days, triplicates of the standard serum dilutions were assayed 8 times each, for one assay date the standard serum was assayed 10 times.</p

Influence of PSV concentration on HT-PBNA analytical sensitivity and robustness.

<p>Neutralization titers of a serum from a Gardasil® immunized individual expressed as ED₅₀ values (open circles and open squares) with the variability (bars indicating the 95% confidence intervals) were determined at different PSV concentrations. The maximal luminescence intensities (RLU) obtained without serum are shown as crosses. An arrow indicates the dilution of the PSV preparation that was used in subsequent neutralization assays.</p

Comparison of HPV 16 HT-PBNA (Gaussia) with manPBNA (SEAP and Gaussia) and GST-HPV 16 L1 antibody binding assay for natural and vaccine-induced HPV16-specific responses.

<p>HPV 16 HT-PBNA (A–D), manPBNA using SEAP reporter (A and B; titers, ED₅₀) and a bead-based GST-HPV 16 L1 antibody binding assay (C and D; median fluorescent intensity (MFI) at 1∶100 or 1∶2700 serum dilution) were used to determine reactivity of pre- (n = 35; A and C) and post-vaccination (n = 72; B and D) sera. Serum samples analyzed were from women with HPV 16 positive, high-grade cervical intraepithelial neoplasia (base-line sera of the chimeric HPV 16 L1-E7 vaccination trial). Cut off values used for positive/negative classification (broken lines in C) were an ED₅₀ of 80 for the HT-PBNA and 109 MFI at 1∶100 for the GST-L1 antibody binding assay.</p

Titration of vaccine sera in the HT-PBNA using PSV of HPV types 16, 18, 31, 45, 52, 58 and BPV-1. A

<p>) Standard serum from Gardasil® vaccinated person. <b>B</b>) Cervarix® vaccine serum. <b>C</b>) Serum from a mouse immunized with L2 epitopes (amino acids 17–36) from HPV16 inserted in the capsid of adeno-associated virus 2 particles.</p

Detection of neutralizing antibodies as result of natural infection by HT-PBNA.

<p>Thirty-five pre-vaccination sera from a study involving patients with CIN2+ lesions were tested for neutralizing antibodies against PSVs of HPV types 16, 18 and 31. The geometrical mean titer for each HPV type is indicated as a horizontal line and the cut off value (ED₅₀ = 80) as a dashed line.</p