Yonsei Med J.  2017 Jan;58(1):43-50. 10.3349/ymj.2017.58.1.43.

Identificaiton of Novel Immunogenic Human Papillomavirus Type 16 E7-Specific Epitopes Restricted to HLA-A*33;03 for Cervical Cancer Immunotherapy

• Kim S
• Chung HW
• Kong HY
• Lim JB

• KMID: 2374187
• DOI: http://doi.org/10.3349/ymj.2017.58.1.43

Abstract

PURPOSE
To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer.
MATERIALS AND METHODS
We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells.
RESULTS
Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs.
CONCLUSION
We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.

Keyword

HLA-A*33;03; HPV 16 E7; immunotherapy; cervical cancer; epitopes

MeSH Terms

Amino Acid Sequence
CD8-Positive T-Lymphocytes/immunology/metabolism
Epitopes/*immunology/therapeutic use
Female
*HLA-A Antigens
Human papillomavirus 16/*immunology
Humans
*Immunotherapy
Interferon-gamma/analysis/*biosynthesis
Leukocytes, Mononuclear/immunology/metabolism
T-Lymphocytes, Cytotoxic/immunology/metabolism
Uterine Cervical Neoplasms/*therapy
Epitopes
HLA-A Antigens
Interferon-gamma

Figure

• 

  Fig. 1 Quantitation of IFN-γ production and a cytotoxicity assay of candidate 15-amino acid peptide-sensitized CTLs. (A) Flow cytometry analyses were performed to measure intracellular IFN-γ production of PBMCs sensitized with candidate peptides. E749-63 induced significantly higher IFN-γ production in PBMCs from HLA-A*33;03 subjects than negative controls (non-peptide). The data are representative of ten independent experiments using PBMCs from HLA-A*33;03 subjects. (B) The bar graph depicts the numbers of each peptide-specific INF-γ+CD8+ T cells per 7.5×104 PBMCs. Columns, mean (n=10); bars, SE. (C) ELISpot assays were performed to measure IFN-γ production from CD8+ CTL that were sensitized with fourteen candidate peptides. HPV 16 E749-63 induced greater number of IFN-γ+ spots from CD8+ CTLs of HLA-A*33;03 donors than other peptides as well as negative control. The bar graph depicts the numbers of SFU of INF-γ produced by CD8+ T cells. Columns, mean (n=6); bars, SE. (D) E749-63 (▵)-sensitized PBMCs lysed a significantly higher number of SNU1299 cells than negative control in a cytotoxic assay using 51Cr release. Point, mean (n=10); bars, SE. CMV A33 (○)-sensitized PBMCs was used as a positive control and non-peptide (•) or CMV A02 (▾)-sensitized PBMCs were used as negative controls matched with each donor's HLA type. All statistically significant differences between the test group and the control group (non-peptide) were determined using the Mann-Whitney U test. *p<0.05, **p<0.01. IFN-γ, interferon-γ; CTLs, cytotoxic T lymphocytes; PBMCs, peripheral blood mononuclear cells; SE, standard error; HPV, human papillomavirus.

• 

  Fig. 2 Quantitation of intracellular IFN-γ production and a cytotoxicity assay of candidate 9- or 10-amino acid peptide-sensitized CTLs. (A) E750-59 and E752-61 induced significantly higher IFN-γ production in PBMCs from HLA-A*33;03 subjects than negative controls (non-peptide). The data are representative of ten independent experiments using PBMCs from HLA-A*33;03 subjects. (B) The bar graph depicts the number of each peptide-specific INF-γ+CD8+ T cells per 7.5×104 PBMCs. Columns, mean (n=10); bars, SE. Statistically significant differences between the test group and the non-peptide group were determined using the Mann-Whitney U test. *p<0.05, **p<0.01. (C) E750-59 (▵)- and E752-61 (▪)-sensitized PBMC lysed a significantly higher number of SNU129 cells than negative control (non-peptide, •). CMV A33 (○)-sensitized PBMCs was used as a positive control and CMV A02 (▾)-sensitized PBMCs was used as a negative control. Point, mean (n=10); bars, SE. (D) E750-59 (▾)- and E752-61 (▵)-sensitized PBMCs lysed a significantly higher number of EBV-BLCs than negative control. CMV A33 (○)-sensitized PBMCs was used as a positive control and non-peptide sensitized-PBMCs (none, •) or non-peptide pulsed-EBV-BLCs (▪, □) were used as negative controls. Point, mean (n=10); bars, SE. (E) CD69 expression by CD8+ T cells was measured by flow cytometry to determine the activation of CD8+ CTLs after a 1-week sensitization with each candidate peptide, and E750-59 and E752-61 -sensitized CD8+ CTLs expressed higher quantities of CD69 than negative control. CMV A33-sensitized CD8+ CTLs were used as a positive control and non-peptide or CMV A02-sensitized CD8+ CTLs were used as negative controls. Data are representative of four independent experiments using PBMCs from HLA-A*3303 subjects. IFN-γ, interferon-γ; CTLs, cytotoxic T lymphocytes; PBMCs, peripheral blood mononuclear cells; SE, standard error; EBV-BLCs, Epstein-Barr virus-B lymphoblastoid cells.

• 

  Fig. 3 HPV 16 E7 peptide-specific cytotoxicity by the cytolytic effect of CD8+ CTLs in PBMCs (A) E749-63-sensitized- and isolated-CD8+ CTLs (▪) as well as E749-63-sensitized PBMCs (○) lysed significantly greater quantities of SNU1299 cells than the CD8+ T lymphocyte-depleted PBMCs (▵) and non-peptide sensitized-PBMCs (•) used as a negative control. Point, mean (n=7); bar, SE. (B) E750-59 (▾)- and E752-61 (▵)-sensitized and isolated-CD8+ CTLs also showed significantly higher cytotoxicity against SNU1299 cells than CD8+ T lymphocyte-depleted PBMCs (▪, □) as well as the negative control (non-peptide, •). Point, mean (n=7); bar, SE. HPV, human papillomavirus; PBMCs, peripheral blood mononuclear cells; SE, standard error; CTLs, cytotoxic T lymphocytes.

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