Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909

Abstract

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Figure 1

Rapid in vivo responses of Melan-A–specific T cells to vaccination (vacc) with low doses of CpG 7909, Melan-A peptide, and IFA. PBMCs were collected before, as well as 7–10 days after, vaccinations 2 and 4, and they were analyzed ex vivo by flow cytometry. (A) Dot plots from PBMCs of patient LAU 627, with percentage of Melan-A–specific cells among CD8⁺ T cells. (B) After 2 and 4 vaccinations, 6/8 and 8/8 patients, respectively, had significantly increased percentages (i.e., greater than 2-fold) of Melan-A–specific T cells. (C) A control group of 8 patients was similarly treated with Melan-A peptide and IFA but without CpG (23). After 2 vaccinations, none of the patients had more than 2-fold increased percentages. After 4 vaccinations, 4/8 patients had more than 2-fold increased frequencies, but percentages of Melan-A–specific T cells remained significantly (P < 0.01) lower as compared to those of CpG-vaccinated patients. (D) Fold increase of Melan-A–specific T cells before or after 4 vaccinations in patients vaccinated with or without CpG. Horizontal lines indicate mean values.

Figure 2

T cell responses to CpG vaccination (arrows) in patients that previously did not respond to vaccination with Melan-A peptide and immunological adjuvants MPL and QS21 (vertical lines). Percentages of multimer⁺ T cells were determined ex vivo in PBMCs collected over an observation period of 4 to 5 years. (A) Patient LAU 321; (B) Patient LAU 371.

Figure 3

Short-term kinetics of Melan-A–specific T cells from 2 representative patients with maximal (max.) responses on day 7 (patient LAU 818) and on day 10 (patient LAU 627) after recall vaccination with CpG 7909, Melan-A peptide, and IFA. PBMCs were collected immediately before, as well as 7, 10, and 14 days after, recall vaccination. Percentages of multimer⁺ T cells were determined ex vivo and calculated in percentages of maximally reached values (100%) per patient.

Figure 4

Expression of effector mediators by vaccine-activated Melan-A–specific T cells. Multiparameter cytometric analysis and sorting was performed with A2/Melan-A multimers and antibodies specific for CD8, CD45RA, and CCR7. (A) Expression of CD45RA and CCR7 is shown for A2/Melan-A multimer⁺ CD8⁺ gated cells. (B) RT-PCR gene expression analysis was performed (33) using primers specific for CD3, granzyme B, perforin, IFN-γ, TNF-α, and CD94, on sorted A2/Melan-A multimer⁺ T cells which were RA⁺CCR7⁺ (naive cells) or RA^–CCR7⁺ effector memory cells. Each band represents the RT-PCR product from RNA isolated from sorted 5-cell aliquots. Data in A and B (representative of 8 and 2 patients analyzed, respectively) are from PBMCs collected from patient LAU 371 before and after 2 and 4 vaccinations. +, positive control; –, negative control; N, naive T cells; EM, effector memory T cells.

Figure 5

T cell receptor fine-specificity and tumor cell recognition. (A) PBMCs from patient LAU 371 were tested ex vivo in IFN-γ Elispot assays before and after vaccination with HIV, tyrosinase, and Melan-A natural and analog peptides. (B) Melan-A–specific T cells were sorted by flow cytometry and cloned, and cytotoxicity was tested against T2 cells in the presence of the following titrated HLA-A2 binding peptides: Melan-A analog (squares), Melan-A natural decamer (circles), Melan-A natural nonamer (triangles), and influenza matrix protein GILGFVFTL (diamonds). (C) Cytotoxicity against HLA-A2⁺ melanoma cell lines Me 275 (Melan-A⁺; filled symbols) and NA8 (Melan-A^–; open symbols) in the presence (squares) or absence (circles) of synthetic Melan-A analog peptide. Data shown were generated with clone 6 derived from patient LAU 371 and are representative for 12 of 22 clones generated from patients LAU 371 and LAU 444.