A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions

Abstract

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

Fig. 1

Schematic representation of the placebo-controlled randomized trial and summary of immunological results. a Patients were recruited at colposcopy visit. After informed consent, HPV testing was performed by PCR and an extra biopsy was taken for the culture of HSIL-infiltrating lymphocytes. Patients with histological proven HPV16+ HSIL then consented to the vaccination study at which time blood was drawn for chemistry and base-line immunomonitoring. Patients in arm 1 received the vaccine at a dose of 300 μg per peptide twice with a 3-week interval; patients in arm 2 received a placebo (PBS). Seven weeks after the first vaccination, a LEEP excision was performed at which time an extra biopsy of the HSIL was taken and blood drawn for immunomonitoring. DTH skin test was performed 2–4 weeks after surgery at which time blood was drawn to measure the effect of the LEEP excision on the systemic immune response. b Immunological results of all the patients using PBMC from three different time points. Week 0 (prevaccination), week 7 (post-vaccination) and week 9–11 (after LEEP excision). Systemic HPV16-specific T-cell reactivity against six peptide pools (4 E6 and 2 E7 peptide pools) was determined by IFNγ-ELISPOT. The boxes in gray show the number of HPV-specific IFNγ-producing T cells per 100,000 cells. c HPV specificity determined by the proliferation assay (LST). The gray boxes indicate the (stimulation index) SI of the HPV-specific proliferative responses. The culture at week 0 of patient 3006 was not tested due to technical problems. To the right, the overall cytokine profile based on the outcome of tested supernatants of the LST by cytokine bead array (CBA) is indicated. A Th-0 response indicates weak cytokine production inconclusive for a Th-1 or Th-2 response. Patient 3008 was randomized, but never showed up for vaccination; NS, not started

Fig. 2

Example of the results from immunomonitoring. The results of patient 3007 who received two vaccinations and of whom blood was tested at week 0 (prevaccination; white), at week 7 (post-vaccination; black) and at week 9–11 (after LEEP excision; gray). The arrows indicate a preexisting response and the stars indicate a positive reaction during the course of the study. a Results of the IFNγ-ELISPOT assay. b Results of the proliferation assay showed no preexcising HPV-specific reactivity. c Cytokine bead array (CBA) was used to test the HPV16-specific production of the indicated cytokines measured in the culture supernatants of the proliferation assay. d DTH results showing clear redness and swelling of sites injected with E6.1, E6.2, E6.3, E6.4 and E7.2. e Overview of the IFNγ-ELISPOT results compared to the DTH skin test results