Epitope specificity and longevity of a vaccine-induced human T cell response against HPV18

Abstract

Persistent human papillomavirus (HPV) type 16 and 18 infection can lead to pre-malignant and malignant diseases of the lower genital tract. Several lines of evidence suggest that T cell responses can control HPV infection. However, relative to other human viruses, strong effector memory T cell responses against HPV have been difficult to detect. We used an in vitro stimulation step prior to enzyme-linked immunospot assays to identify IFN-gamma-secreting T cells specific for HPV16 and 18 E6/E7 peptides. This allowed the detection of HPV-specific CD4+ T cells that were not evident in direct ex vivo assays. T cell responses against HPV16 or 18 peptides were detected in healthy volunteers (7/9) and patients with lower genital tract neoplasia (10/20). Importantly, this assay allowed tracking of vaccine-induced T cell responses in nine patients, following inoculation with a live recombinant vaccinia virus (HPV16 and 18 E6/E7, TA-HPV). Novel vaccine-induced T cell responses were demonstrated in five patients, but no clinical responses (lesion regressions) were seen. For one vaccinated patient, the T cell response was mapped to a single dominant HPV18 E7 epitope and this response was sustained for >3 years. Our data suggest that systemic memory T cells against HPV16 and 18, induced naturally or by TA-HPV vaccination, are relatively rare. Nevertheless, the assay system developed allowed estimation of magnitude, epitope specificity, and longevity of vaccine-induced CD4+ T cell responses. This will be useful for vaccine design and measurement of immunological endpoints in clinical trials.

Fig. 1.

T cell responses against HPV16 and 18 peptides in healthy volunteers. Representative responses are shown from (A) HV003 (HPV16 E6 response) and (B) HV007 (HPV18 E6 response). PBMC were cultured for 7 days with peptide pools from HPV18 E7, HPV18 E6, HPV16 E7, HPV16 E6 or the PPP. Cells were harvested and tested in ELISPOT assays against autologous PBMC plus the appropriate HPV peptide pool. The counts shown are for nothing (T cells only) or plus peptide [(T cells + PBMC + peptide) − (T cells + PBMC)].

Fig. 2.

Dissection of HPV16 peptide-specific T cell responses measured by ELISPOT. (A) Mapping an HPV16 E6 epitope in a healthy volunteer (HV003). PBMC were cultured with HPV16 E6 peptides before testing in ELISPOT assays against the whole HPV16 E6 pool (25 peptides), pool of four peptides (19, LPQLCTELQTTIHIDI; 25, RDLCIVYRDGNPYAV; 31, YGTTLEQQYNKPLCD and 37, DKKQRFHNIRGRWTG) or individual peptides. (B) Immunomagnetically enriched populations of either CD8⁺ (98.5% purity) or CD4⁺ T cells (96% purity) from HV003 were cultured with peptide 37, before testing in an ELISPOT assay.

Fig. 3.

T cell responses in patients vaccinated with TA-HPV. Responses against HPV peptide pools were measured pre- and post-vaccination (at least three time points) in nine patients with HPV-associated LGTN (VAC001–003, VAC005, VAC006 and VAC008–011). For each patient, cryopreserved PBMC from multiple time points were tested in the same experiment to minimise variability. Responses marked with asterisk represent significant post-vaccine T cell responses (>2SD of pre-vaccine response). Note that VAC002 produced a mean of 303 spots for day 61 and a mean of 340 spots for day 91. For the majority of patients, PPP responses exceeded 200 spots per well and were not precisely counted above this number.

Fig. 4.

Vaccine-induced T cell response against an immunodominant epitope of HPV18 E7. (A) Day 91 PBMC from patient VAC002 were cultured for 7 days with HPV18 E7 pool (25 peptides) before testing in ELISPOT assays against either the whole HPV18 E7 pool or smaller HPV18 E7 pools containing four individual peptides (C1–C4 pools) or mitogens. (B) Day 91 PBMC were cultured as above, before testing against entire HPV18 E7 pool, HPV18 E7 C2 pool (peptides 45, LLCHEQLSDSEEEND; 46, LSDSEEENDEIDGVN; 47, ENDEIDGVNHQHLPA and 48, GVNHQHLPARRAEPQ) or individual HPV18 E7 peptides (45, 46, 47 or 48). The response against mitogens in experiment B exceeded 500 spots.

Fig. 5.

Longevity of vaccine-induced HPV18-specific T cell responses. Day 901 PBMC from patient VAC002 were cultured with the whole HPV18 E7 peptide pool before testing in ELISPOT assays against entire HPV18 E7 pool, HPV18 E7 C2 pool (peptides 45–48) or individual HPV18 E7 peptides 45 and 48. The response against mitogens exceeded 500 spots.