A purified recombinant lipopeptide as adjuvant for cancer immunotherapy

Abstract

Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter) induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs), leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m) protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion). Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4). We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines.

Figure 1

The lipid structures of Pam3CSK4 and Triacyl-CysSQEAK (lipo-Nter). (a) Identification of purified lipo-Nter by mass spectrometry. Lipo-Nter was obtained after trypsinizing rlipo-D1E3 and was analyzed on a Waters MALDI micro MX mass spectrometer. The MALDI-TOF MS spectra revealed the existence of four peaks with m/z values of 1451.9, 1465.9, 1479.9, and 1493.9. (b) Pam3CSK4 contains an N-acyl-S-diacylglyceryl cysteine moiety, and the fatty acids in all of the groups are 16-carbon saturated fatty acids. (c) The lipid structure of lipo-Nter is also an N-acyl-S-diacylglyceryl cysteine structure. The R2 group of lipo-Nter contains unsaturated fatty acids with different chain lengths. (d) The masses of Pam3CSK4 and lipo-Nter were determined. We previously [23] identified the masses of the corresponding lipid structures.

Figure 2

The responses of BM-DCs after stimulation with lipo-Nter. BM-DCs were generated from wild-type (WT), TLR1^−/− (TLR1 KO), TLR2^−/− (TLR2 KO), or TLR6^−/− (TLR6 KO) mice. Different types of BM-DCs were stimulated with/without LPS (0.1 μg/mL) or lipo-Nter (100 nM) for 24 h. The levels of TNF- in the culture supernatant were measured by ELISA. LPS (TLR4 agonist) was used as positive control. The results are expressed as the means + S.D. of the amount of cytokine.

Figure 3

IgG isotype and T cell cytokine profile induced in mice following immunization with rE7m mixed with lipo-Nter. Wild-type C57BL/6 mice were subcutaneously administered rE7m (30 μg) mixed with/without lipo-Nter (30 μg/100 μL) or PBS twice at a 2-week interval. (a) After 2 weeks, serum samples were collected, and anti-rE7m antibody titers were determined by sandwich ELISA. (b) On day 7 after the second immunization, splenocytes from immunized mice were stimulated with rE7m (100 nM), and the levels of IL-2, IFN-γ, and IL-5 in the culture supernatant were measured by ELISA. The data are expressed as the means + S.D. of the samples.

Figure 4

CD8⁺ T cell response and antitumor effect elicited by lipo-Nter in a protein vaccine. (a) C57BL/6 mice were immunized twice by subcutaneous injection with PBS or rE7m (30 μg) mixed with/without lipo-Nter (30 μg/100 μL) at a 2-week interval. On day 7 after the second immunization, the mice were sacrificed, and splenocytes (2 × 10⁵ cells/well) were stimulated with or without 10 μg/mL RAHYNIVTF (RAH) peptide for 48 h in an anti-IFN-γ-coated 96-well ELISPOT plate. The IFN-γ-secreting spots were measured using an ELISPOT reader. (b) 2 × 10⁵ TC-1 cells were injected into the abdominal region of the mice. After 7 days, TC-1 tumor-bearing mice (6 animals/group) received a single injection with PBS or rE7m (30 μg) mixed with or without lipo-Nter (30 μg/100 μL). The tumor diameters are shown (cm³). The data are expressed as the means + SD of 6 animals per group.

Figure 5

CD8⁺ T cell response and antitumor effect elicited by lipo-Nter in a peptide vaccine. (a) Mice were subcutaneously administered twice at a 1-week interval with RAH and PADRE (30 μg) mixed with IFA. CD8⁺ T cells were purified from the spleens of the immunized mice. Purified CD8⁺ T cells were cocultured with/without BM-DCs and stimulated with PBS or 1 μM RAH mixed with/without 200 nM lipo-Nter for 72 h. Proliferation of T lymphocytes was measured by uptake of  [³H]thymidine. (b) TC-1 tumor-bearing mice (6 animals/group) received a single injection of PBS or 10 μg of RAH mixed with/without lipo-Nter (10 μg/100 μL). The tumor diameters are shown (cm³).

Figure 6

Anti-tumor effects of Pam3CSK4. TC-1 tumor-bearing mice (5 or 6 animals/group) received a single injection of PBS or 10 μg of RAH mixed with Pam3CSK4 (10 μg/100 μL). The tumor diameters are shown (cm³).