Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses

Abstract

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNgamma (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells (P = 0.005) and displayed a lower HPV16-specific IFNgamma/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells was predictive of clinical success. Foxp3(+) T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.

Fig. 1.

Strong and broad vaccine-induced T-cell immunity was seen. (A) HPV16 E6/E7-specific proliferation of PBMC in blood samples drawn before and after vaccination as determined in the LST assay. Each symbol represents a particular patient; black and red symbols indicate CR. (B) The strength of vaccine-induced proliferative T-cell response in time (median + interquartile range). The numbers above each time point indicate the ratio of the number of patients displaying a positive proliferative response to the number of patients evaluated before vaccination (pre-vac), after one (1-vac), two (2-vac), three (3-vac), and four (4-vac) vaccinations, and at 3-mo, 1-y, and 2-y follow-up (FUP).

Fig. 2.

Stronger HPV16-specific cytokine production by patients with small VIN3 lesions was observed. Patients are grouped according to clinical outcome (CR or non-CR) and lesion size (smaller or larger than ≤9.5 cm²) at study entry: The strength (median + interquartile range) of cytokine production (A–D) and proliferation at indicated time points (E) is depicted. *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001. (Exact P values are given in Tables S3 and S4.)

Fig. 3.

More CD4⁺CD25⁺Foxp3⁺ T-regulatory cells were seen in patients with large lesions. The median + interquartile range percentage of CD4⁺CD25⁺Foxp3⁻ and CD4⁺CD25⁺Foxp3⁺ T cells is depicted for patients grouped according to (A) lesion size at study entry and (B) clinical response.