Memory T Cells Specific for Novel Human Papillomavirus Type 16 (HPV16) E6 Epitopes in Women Whose HPV16 Infection Has Become Undetectable

Abstract

Human papillomavirus (HPV)-specific T-cell response to the HPV type 16 (HPV16) E6 protein has been shown to be associated with successful viral clearance. The patterns of CD8 T-cell epitopes within HPV16 E6 protein were previously studied in two women with HPV16 clearance. The goal of this study was to characterize these epitopes in terms of their minimal and optimal amino acid sequences and the human leukocyte antigen (HLA) restriction molecules. The presence of the epitope-specific memory T cells after viral clearance was also examined. In subject A, the dominant epitope was characterized to be E6 75-83 (KFYSKISEY), restricted by the HLA-B62 molecule, while that of subject B was E6 133-142 (HNIRGRWTGR), restricted by the HLA-A6801 molecule. Homologous epitopes were identified in five other high-risk HPV types for both of these epitopes, but they were not recognized by respective T-cell clone cells. An enzyme-linked immunospot assay or tetramer analysis was performed on peripheral blood mononuclear cells from blood samples collected after viral clearance but prior to isolation of the T-cell clones. The presence of epitope-specific memory T cells was demonstrated. These data suggest that HPV-specific memory T cells were generated in vivo and that they may remain in circulation many months, if not years, after viral clearance. Our findings broaden the spectrum of the CD8 T-cell epitopes of the HPV16 E6 protein. The characterization of novel T-cell epitopes and long-lasting epitope-specific memory T cells may be useful for the development of a potential epitope-based vaccine.Cervical cancer is the second most common malignant cancer in women worldwide. High-risk human papillomaviruses (HPVs), most commonly HPV type 16 (HPV16), have been shown as the major cause of cervical cancer (29, 33). The transforming properties of high-risk HPV16 E6 and E7 proteins play a critical role in the initiation of oncogenic processes. The E6 protein can lead to the degradation of the p53 tumor suppressor protein (4, 28), while the E7 protein can interfere with the retinoblastoma tumor suppressor protein (28). These two proteins are foreign viral antigens, and their constitutive expression is required for the maintenance of the malignant phenotype of cervical cancer (32, 33). Therefore, HPV16 E6 and E7 proteins have been sought as ideal targets of antigenspecific immunotherapeutic strategies for the prevention and treatment of cervical cancer.Immune responses, especially HPV-specific T-cell immune responses, are thought to play an important role in successfully clearing HPV infection and controlling HPV-associated diseases. CD8-positive, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) are known to be responsible for recognizing and killing virus-infected host cells and virus-induced tumor cells (12). Immunohistochemical analyses of squamous intraepithelial lesion and cervical cancer specimens have demonstrated the presence of activated CTLs in lesions (2). A study using stimulated peripheral blood m...

Current Insight into Anti-HPV Immune Responses and Lessons for Prophylactic and Therapeutic Vaccines

“…In this fragment, the dominant epitope E6 29-37 is restricted by HLA-B48, E6 31-38 by HLA-B4002 and the subdominant epitope E6 52-61 by HLA-B35 (Nakagawa et al, 2007). The same group had also shown that the peptide E6 33-42 61 is recognized by CD8+ T lymphocytes in association with HLA-A68, peptide E6 52-61 in association with HLA-B57 and -B35, peptide E6 75-83 in association with HLA-B62, peptide E7 7-15 in association with HLA-B48 and peptide E7 79-87 in association with HLA-B60 (Nakagawa et al, 2004(Nakagawa et al, , 2007Wang et al, 2008). In addition, E7 7-15 is also able to bind HLA-A2 and -B8 to be recognized by CTL (Oerke et al, 2005;Ressing et al, 1995).…”

Cross-Reactivity, Epitope Spreading, andDe NovoImmune Stimulation Are Possible Mechanisms of Cross-Protection of Nonvaccine Human Papillomavirus (HPV) Types in Recipients of HPV Therapeutic Vaccines

“…A CD8 T-cell clone specific for the HPV-16 E6 aa-52-to-61 epitope restricted by the HLA class I B57 molecule has been shown to cross-recognize HPV-35 (␣9), -39 (␣7), -45 (␣7), -51 (␣5), and -73 (␣11) (35) (Table 4), while another CD8 T-cell clone specific for the same peptide but restricted by another HLA class I molecule, B58, has been shown to cross-recognize HPV-31 (␣9), -33 (␣9), -35 (␣9), -39 (␣7), -45 (␣7), -51 (␣5), -58 (␣9), and -73 (␣11) (36) ( Table 5). On the other hand, a CD8 T-cell clone recognizing the HPV-16 E6 aa-75-to-83 epitope restricted by the HLA class I B62 molecule (Table 6) and another clone recognizing the E6 aa-133-to-142 epitope restricted by the HLA class I A6801 molecule (Table 7) did not recognize any homologous peptides tested (37). Therefore, the presence of amino acid homology does not automatically lead to cross-recognition and it is not species specific when present.…”

“…Our group approached this question by isolating T-cell clones positive for an HPV-16 epitope and examining the recognition of homologous sequences from other high-risk HPV types using a gamma interferon (IFN-␥) enzyme-linked immunosorbent spot assay (ELISPOT) (35)(36)(37). If the number of spot-forming units for a homologous sequence from another HPV type was equal to or greater than 50% of the number for HPV-16, then that HPV type was considered to be cross-reactive.…”

Hepatitis B Surface Antigen-based Vaccines for Human Neoplastic Disease

“…During a natural infection, the growth of HPV-associated tumours is hindered and eradicated by E6-and E7-directed cytotoxic CTL responses (Coleman et al, 1994;Kadish et al, 2002;Wang et al, 2008). However preliminary studies using E6-and E7-targeting vaccines in humans has met with limited success (Borysiewicz et al, 1996;van Driel et al, 1999;Muderspach et al, 2000;Davidson et al, 2003;Frazer et al, 2004;Hallez et al, 2004;Kenter et al, 2008;Trimble et al, 2009) and there is currently no immunotherapeutic approach to treat HPV-associated carcinoma.…”

“…CTL responses targeting E7 and E6 are defined in mice (Feltkamp et al, 1993;Eiben et al, 2002;Peng et al, 2004) and are utilised for immunoprophylactic and immunotherapeutic anti-tumour strategies Greenstone et al, 1998;Chen et al, 1999;Chu et al, 2000;Schirmbeck et al, 2003a;Cassetti et al, 2004;Doan et al, 2005;Yan et al, 2007). CTL epitopes have also been defined in E6 and E7 in humans as potential targets for immunotherapeutic anti-tumour strategies (Kast et al, 1994;Peng et al, 2006b;Liu et al, 2007a;Nakagawa et al, 2007;Wang et al, 2008).…”