Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia

Abstract

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n 5 11) and healthy individuals (n 5 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV161 patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD41 Th1/Th2 and CD81 T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity. ' 2008 Wiley-Liss, Inc.Key words: HPV16/delayed type hypersensitivity; cellular immunity; ELISPOT; skin testThe main cause of cervical carcinoma and cervical intraepithelial neoplasia (CIN) is a persistent infection with one of the highrisk, oncogenic human papilloma viruses.1,2 Anogenital infection with a high-risk HPV type is very common.3-5 The rate of incidence of infection is estimated to be 80-85% worldwide.6 Fortunately, the majority of infected subjects will clear the virus infection within a year 7,8 , and only a small proportion of women will become persistently infected and are at risk to develop HPVrelated malignancies.9,10 Cell-mediated immune responses play an important role in controlling HPV infections. 11,12Delayed Type IV hypersensitivity (DTH) reactions are used as a general measure of cell-mediated immunity in vivo.13 A local inflammatory reaction (induration and erythema), orchestrated by activated cytokine releasing CD41 memory T cells, usually appears within 24-72 hr after intradermal injection of antigen. [14][15][16] Delayed-type hypersensitivity reactions have been used to demonstrate an encounter with pathogens (e.g., Mantoux Test), vaccineinduced immune responses, [17][18][19][20] and in particular to show HPVspecific immune responses in various animal models.21-23 H€ opfl et al. were the first to study HPV16-specific cellular immunity by skin test in humans 24,25 and showed that a skin reaction, appearing within 2-6 days after intradermal injection with HPV16 E7, was correlated with regression of HPV-induced CIN lesions.We have previously studied the HPV16-specific T-cell responses in great detail in vitro, and the results of these studies suggested that the HPV16 E2, E6 and E7 specific Type 1 and Type 2 T-cell response ...

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

“…Infiltrating lymphocytes from a 3-mm biopsy of the VIN lesion before vaccination, from the last vaccination site 2 wk after the last vaccination, and from the VIN lesion 3 mo after the last vaccination were expanded as described previously (38,39). When sufficient cells were obtained, their HPV16-specific proliferative capacity was tested in triplicate in a 3-d proliferation assay.…”

Induction of p53-Specific Immunity by a p53 Synthetic Long Peptide Vaccine in Patients Treated for Metastatic Colorectal Cancer

“…T cells from PBMC or skin biopsies were isolated and cultured as described previously (39,43) and either directly used or cryopreserved. A set of six pools of long overlapping peptides, indicated by the first and last amino acids in the p53 protein, was used for the screening of T-cell responses: p53.1: 1-78; p53.2: 70-115; p53.3: 102-155; p53.4: 142-203; p53.5: 190-248; and p53.6: 241-393.…”

“…Detection of IFN-g, IL-2, and IL-5 production by p53-specific T cells using flow cytometry. PBMCs were either directly ex vivo used for intracellular cell staining or 10 days presensitized using the peptides in pools p53.2 to p53.5 (2.5 Ag/peptide/mL) as described previously (43). T cells from the biopsy were directly tested ex vivo.…”

Immunization with a P53 synthetic long peptide vaccine induces P53‐specific immune responses in ovarian cancer patients, a phase II trial

“…from skin biopsies taken at the fourth injection site (n 5 17) using a successful protocol to obtain antigen-specific T-cells, which is based on the induction of homeostatic proliferation and as such does not strongly affect the phenotype of the infiltrating lymphocytes, as published previously. 15,35,38 Only in 2 cases (P15 and P20) we were not able to obtain sufficient numbers of lymphocytes from the skin biopsy to analyze the p53-specific T-cell response by proliferation assay. The median yield of cells was 1.25 3 10 6 (range 0.06 3 10 6 -11.1 3 10 6 ) after 2-3 weeks of culture.…”