Evaluation of HPV-16 and HPV-18 specific antibody measurements in saliva collected in oral rinses and merocel® sponges

Abstract

Background: Current Human papillomavirus (HPV) L1 VLP vaccines protect against HPV-16 and HPV-18-associated cancers, in females and males. Although correlates of protection have not been identified, HPV-specific antibodies at sites of infection are thought to be the main mechanism of protection afforded by vaccination. Oral sampling has gained increased attention as a potential alternative to serum in monitoring immunity to vaccination and understanding local immunity in oral cancers.

Methods: Serum was collected via venipuncture, and saliva was collected via oral rinses and Merocel® sponges from healthy volunteers: 16 unvaccinated females, 6 females (ages 24-41) and 6 mid-adult aged males (ages 27-45) recipients of three doses of the HPV-16/18/6/11 vaccine (Gardasil®). Mid-adult male vaccine trial participants were compared to female participants. Samples were tested for anti-HPV-16 and anti-HPV-18 immunoglobulin G levels by an L1 virus-like particle-based enzyme-linked immunosorbent assay (ELISA).

Results: All vaccinated participants had detectable serum anti-HPV-16 and anti-HPV-18 antibodies. Optimal standard concentration range and sample serial dilutions for oral rinses were determined. The standard curve was not affected by the type of solution examined. Reproducibility of HPV-16 and HPV-18 antibody titers in mouthwash (overall CV < 10%) or in Merocel® extraction buffer was robust (CV < 13%). Excellent assay linearity (R2 > 0.9) was observed for sera spiked controls in both solutions. HPV-16 and HPV-18 specific antibodies were detectable in saliva from vaccine recipients, both in mouthwash and in Merocel® sponges but levels were several logs lower than those in serum.

Conclusions: This study confirms the application of HPV-16 and HPV-18 ELISAs currently used in sero-epidemiological studies of immunogenicity of HPV vaccines for use with oral samples. Oral samples may be a useful resource for the detection of HPV-16 and HPV-18-specific antibodies in saliva following vaccination.

Keywords: HPV vaccine; Human papillomavirus; Mouthwash; Saliva.

Conflict of interest statement

Potential Conflicts of interest: ARG reports receiving grants from Merck during the conduct of the study and other grants from Merck outside the submitter work. All other authors reported no potential conflicts.

Copyright © 2018 Elsevier Ltd. All rights reserved.

Figures

Figure 1

Standard performance for HPV-16 or HPV-18 in different days (A, C), or in different buffer type (B, D). Mouthwash and extraction buffer were tested for anti-HPV-16 and anti-HPV-18 antibodies by ELISA. Testing over different days and matrices did not impact HPV-16 or HPV-18 assay standard performance.

Figure 2

Assay linearity in mouthwash (A, B) and in sponge extraction buffer (C, D) for HPV-16 and HPV-18. Mouthwash and Merocel® extraction buffers spiked with HPV-16 and HPV-18 antibody positive sera display robust linearity. R2 is representative of 1 sample serially diluted.

Figure 3

HPV-16 (A) and HPV-18 (B) antibodies can be detected by ELISA in saliva from vaccine recipients. ELISA cutoffs were determined by using mouthwash and sponge samples from 15 HPV-16 and HPV-18 seronegative females, as tested by ELISA. The antibody levels, expressed as ELISA Units (EU/ml), were calculated by interpolation of OD values from the standard curve by averaging the calculated concentrations from all dilutions that fall within the working range of the standard curve. The mean OD or EU/ml of these samples plus 3 standard deviations were set as the cutoff values for these assays (Table 2). Large variations exist in antibody levels as the collection of blood from male samples were part of a clinical trial, collected at 30 months post vaccination, while females were healthy volunteers from the Occupational Health Services that self-reported vaccination and were not part of a clinical trial.