HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen

Abstract

There is a need for new adjuvants that will induce immune responses to subunit vaccines. We show that a short peptide, named Hp91, whose sequence corresponds to an area within the endogenous molecule high mobility group box (HMGB1) protein 1 potentiates cellular immune responses to peptide antigen and cellular and humoral immune responses to protein antigen in vivo. Hp91 promoted the in vivo production of the immunomodulatory cytokines, IFN-γ, TNF-α, IL-6, and IL-12 (p70), as well as antigen-specific activation of CD8+ T cells. These results demonstrate the ability of a short immunostimulatory peptide to serve as an adjuvant for subunit vaccines.

Fig. 1. Hp91 causes release of cytokines in vivo

Mice were injected i.v. into the tail vein with Hp91 (10 or 100 μg) or PBS (denoted as 0 μg Hp91). Blood was collected after 2 and 24 h and serum was analysed for IFN-γ, IL-6, IL-12 (p70), and TNF-α by ELISA. Data shown are mean (+/− SEM) from 3-4 mice per group.

Fig. 2. Cellular immune response in Hp91 immunised mice

(A-B) Mice were immunised with OVA peptides in PBS (denoted as “0”), Hp91 (250 and 500 μg), or IFA. One group of mice was immunised with OVA protein and Hp91 (500 μg). Freshly-isolated splenocytes from the immunised mice were cultured in the presence of (A) OVA-I (SIINFEKL) peptide (2.5 μg ml⁻¹) or (B) OVA-II (ISQAVHAAHAEINEAGR) (2.5 μg ml⁻¹) in an IFN-γ ELISPOT assay. The number of IFN-γ-secreting cells was determined 18 hours later. The data shown (IFN-γ spot-forming cells per million cells) are means (+/− SEM) for 10 mice/group, except the Hp91-OVA protein group which is n=5. Asterisk, p < 0.05 between groups; Student's t-test. Data are representative of at least 3 independent experiments. (C) Freshly isolated splenocytes from PBS/OVA-peptide and Hp91/OVA-peptide immunised mice were depleted of CD8+ or CD4+ T cells and stimulated overnight in the presence of 2.5 μg ml⁻¹ OVA-I (SIINFEKL) peptide in an IFN-γ ELISPOT assay as above. Data shown are means (+/− SEM) for 5 mice per group. (D) Mice were immunised twice with OVA-I (SIINFEKL) peptide (50ug) co-injected with 0, 30, 60, 125, or 250 μg Hp91 dissolved in PBS. Splenocytes from immunised mice were cultured in an OVA-I IFN-γ ELISPOT assay as above. Spleens were collected 6 days after the boost. Asterisk, p < 0.05 between groups; Student's t-test compared to PBS. (E) Mice were immunized with OVA peptides in PBS, Hp91 or Hp121 (250 μg). Mice received an additional boost s.c. into the contralateral flank one month after the first boost. Freshly-isolated or OVA-I-expanded splenocytes were cultured in an OVA-I IFN-γ ELISPOT assay as above. The number of IFN-γ spot-forming cells are shown as means (+/− SEM) for 4 mice per group. Asterisks, (*<0.05 and **<0.005); 2-way ANOVA.

Fig. 3. Cytokine secretion in Hp91 immunised mice

Mice were immunised with OVA peptides (50μg) co-injected with Hp91 (0, 250 or 500 μg dissolved in PBS) or IFA. One group of mice was immunised with OVA protein and Hp91 (500 μg) in PBS. Splenocytes from immunised mice were stimulated overnight with 2.5 μg ml⁻¹ of (A) OVA-I (SIINFEKL) peptide or (B) OVA-II (ISQAVHAAHAEINEAGR). Culture supernatants were collected and analysed for IL-2 secretion by ELISA. Data shown are mean (+/− SEM) for 5-10 mice per group. Asterisk, p < 0.05; Student's t-test.

Fig. 4. Antibody responses in Hp91 immunised mice

Serum was obtained from immunised mice (5 mice per group) 10 days after the final immunisation and analysed for antibody levels by ELISA. (A) A 1:100 dilution of the serum from immunised mice as indicated, (B) a 1:100 dilution of serum from mice immunised with OVA protein and Hp91 (0 or 250μg in PBS) or (C) a serial dilution of serum from mice immunised with OVA protein and Hp91 (500 μg) in PBS was added to the plates, followed by a peroxidase-conjugated anti-mouse IgG1 antibody. Plates were developed with TMB substrate and absorbance was analysed on a microplate reader. Asterisks, p < 0.001; Student's t-test.

Fig. 5. CTL Induction in immunised mice

Expanded splenocytes from immunised mice were cultured with E.G7-OVA cells at the indicated effector to target ratios for 6h. Cell culture supernatants were collected and cytotoxicity was quantified in a CytoTox96 non-radioactive cytotoxicity assay. Data from at least 3 mice per group are shown. Data were analysed by linear regression and slopes were compared for significance. Asterisk, p < 0.05; compared to PBS.