Evaluation of Two Types of Sponges Used To Collect Cervical Secretions and Assessment of Antibody Extraction Protocols for Recovery of Neutralizing Anti-Human Papillomavirus Type 16 Antibodies

Abstract

Immunogenicity evaluations in human papillomavirus (HPV) vaccine trials have relied on serologicalsamples, yet cervical antibodies are likely to be most relevant for protection against infection. In order to assess functional antibody levels at the cervix, the secreted-alkaline-phosphatase neutralization assay (SEAPNA) was used to measure HPV-neutralizing activity. We assessed the variability of the SEAPNA with serum samples after vaccination with an HPV type 16 (HPV16) L1 virus-like particle vaccine and whether the SEAPNA can be used to monitor neutralizing activity at the cervix. The SEAPNA has an overall coefficient of variation of 29.3%. Recovery from ophthalmic sponges was assessed by spiking V5 (mouse anti-HPV16) antibody onto and extracting it from sterile Merocel and Ultracell sponges and sponges used to collect specimens from participants. V5 recovery from sterile Merocel sponges was complete, yet that from Ultracell sponges was null. The mean V5 recoveries from participant Ultracell and Merocel sponges were 61.2% and 93.5%, respectively, suggesting that Merocel sponges are more appropriate for specimen collection. The SEAPNA can be applied to determine the surrogates of protection and to examine the durability of protection at the cervix.Prophylactic human papillomavirus (HPV) L1-based viruslike particle (VLP) vaccines have been shown to be safe, immunogenic, and protective against cervical infection and the associated lesions by homologous HPV types (7-9, 11, 17, 18). In addition, HPV VLP vaccinations induce neutralizing antibodies not only in the periphery but also at the cervix (6, 12). Several assays for monitoring HPV type-specific antibody responses have been developed; however, validation efforts for these assays have focused on serum rather than the cervix, where protection is executed.HPV VLP enzyme-linked immunosorbent assays (ELISAs) have been used to determine serum antibody titers in epidemiological studies of HPV infection and in HPV vaccine trials (8,9,15,16,19). Enzyme-linked immunosorbent-based assays have the advantage of being applicable for large-scale studies. However, these assays lack the ability to discern between neutralizing and nonneutralizing antibodies and may have the disadvantage of detecting antibodies to yeast and baculovirusderived proteins, compromising their specificity. It is therefore still unclear whether ELISAs are optimal for the evaluation of the levels of protective antibodies at the cervix.Recently, a pseudovirion (PsV) neutralization assay (a secreted-alkaline-phosphatase neutralization assay [SEAPNA]) was developed by Pastrana et al. to evaluate the neutralizing potential against HPV (13). This assay has the advantage of specifically measuring the biological activity believed to be relevant for protection (i.e., neutralization).Here, we explored whether the SEAPNA could be used to monitor neutralizing antibody levels at the cervix by the use of two different ophthalmic sponges (Merocel and Ultracell) commonly used in large-scale studies for cervical mucus s...

Evaluation of a multiplex panel of immune‐related markers in cervical secretions: A methodologic study

“…25 First, the wet-weight of the sponge was recorded, and then each sponge was placed in a 2 ml Spin-X centrifuge filter tube (Corning Inc, Corning, NY) to which 300 μl of extraction buffer [PBS (Invitrogen, Grand Island, NY), 256 mM NaCl (Sigma–Aldrich, St. Louis, MO), and 100 μg/ml Aprotinin (Sigma–Aldrich, St. Louis, MO)] was slowly added. The sponges was incubated at 4 °C for 30 minutes and then centrifuged at 13,000 × g for 15 minutes at 4 °C.…”

Production of Furin‐Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1‐ or L2‐Specific Antibodies

“…In addition, Life Technologies also offers a kit (cat # N10578), and a relatively more recent product from Jaden Bioscience (cat# CM025) is also available. Clontech’s detection kit in the past has been used to measure SEAP in the original 293TT cell-based neutralization assay (now known as L1-PBNA) in our studies (Kemp et al, 2008; Kemp et al, 2011; Pastrana et al, 2005). This L1-PBNA system measures HPV anti-L1 antibody titers using PsV particles where little or no L2 proteins on the virion have been cleaved.…”

Immunogenicity Testing in Human Papillomavirus Virus‐Like‐Particle Vaccine Trials

“…In the most widely employed variation, the pseudovirus carries the gene for secreted alkaline phosphatase (SEAP), which enables infection to be quantitated by measuring the ability of culture supernatants to cleave a colorigenic substrate. The sensitivity of the SEAP neutralization assay (SEAP-NA), in terms of titers measured in response to natural infection or VLP vaccination, is similar to that of standard VLP-based IgG ELISAs [15, 17, 18]. The assay has been adapted to a high-throughput 96- well-plate format.…”

“…However, a strong correlation between individual ELISA and neutralizing-antibody titers was observed in trials of the GSK vaccine [17, 18]. In some situations, the neutralization assay may be less type specific than the cLIA.…”