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Vaccine. Author manuscript; available in PMC 2011 Nov 3.
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PMCID: PMC2963688

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


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 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.

Keywords: HMGB-1 peptides, Adjuvants, Vaccine, Immunotherapy, Dendritic cells

1. Introduction

Vaccines traditionally have, and still consist of whole-inactivated or live-attenuated pathogens or toxins [1,2]. The usage of these modified pathogens is however unattractive for several reasons. Live attenuated pathogens can cause disease by reverting to a more virulent phenotype, especially in the non-developed immune system of newborns or immunodeficient patients, and whole inactivated pathogens contain reactogenic components that can cause undesirable vaccine side effects. Therefore, there is growing interest and research to develop a new generation of vaccines containing recombinant protein subunits, synthetic peptides, and plasmid DNA [1]. While these new modalities promise to be less toxic, many are poorly immunogenic when administered without an immune-stimulating adjuvant. As adjuvants are a crucial component of the new generation of vaccines, there is a great need for safer and more potent adjuvants [1-3].

The development of the appropriate type of immune response is essential for successful immunisation. Robust cell-mediated immunity, which is associated with a Th1 type immune response, is thought to be required for the control of intracellular pathogens [4], viruses [5] as well as cancer [6]. Humoral immunity, characterized by a Th2 type response is useful for vaccination against extracellular pathogens, such as bacteria. By choosing an appropriate adjuvant, the immune response can be selectively modulated to initiate a Th1 or Th2-type [7]. Aluminum salts (alum), which are the only vaccine adjuvants currently approved by the US Food and Drug Administration for use in humans [8,9] are not ideal adjuvants for certain pathogens, since they favor a Th2 response with weak or absent Th1 responses [10-14]. Although neutralizing antibodies from a Th2 response can be protective against many pathogens, the generation of Th1 and cytotoxic T lymphocyte (CTL) responses are important, playing crucial roles in the protection and recovery from viruses, intracellular bacteria, and cancer cells.

Pathogen associated molecular patterns (PAMPs) are small molecular sequences commonly associated with pathogens, such as CpG unmethylated bacterial DNA sequences, lipopolysaccharide (LPS), or poly(I:C) [15-18]. While many PAMPs have been investigated for their use as vaccine adjuvants, their development has been slowed for several reasons, including reactogenicity, toxicity, and ability to induce or exacerbate autoimmune diseases[19]. For instance, CpG oligodeoxynucleotides, which signal through TLR9, can activate antigen-presenting cells, induce a wide variety of cytokines, and generate a potent cellular Th1 immune response in mice, initially showed strong clinical promise [20-23]. However, clinical trials in humans utilizing CpG as a cancer immunotherapy adjuvant failed to produce the potent immune responses that were anticipated, and low TLR9 expression in human plasmacytoid DCs may be implicated [24]. Identification of new adjuvants demonstrating low-toxicity and the ability to stimulate a cellular Th1 response in humans would be a great advancement in the development of vaccines for infectious disease and cancer.

In contrast to PAMPs, endogenous molecules and proteins have been proposed and studied as adjuvants. Examples of such endogenous molecules, or danger-associated molecular patterns (DAMPs), include heat stock proteins, cytokines, and high mobility group box 1 (HMGB1) protein [25,26]. Originally identified as a nuclear protein, HMGB1 modulates the innate immune response when released into the extracellular compartment by necrotic and damaged cells [27,28]. HMGB1 is a potent pro-inflammatory cytokine, released by monocytes and macrophages following exposure to LPS, tumor necrosis factor (TNF)-α or IL-1β and as a result of tissue damage [27,29]. Extracellular HMGB1 promotes the maturation of myeloid and plasmacytoid DCs [30-32] and it has been shown to act as immune adjuvant by enhancing immunogenicity of apoptotic lymphoma cells and eliciting antibody responses to soluble ovalbumin protein [33].

We have previously identified a short peptide, named Hp91, within the B box domain of HMGB1 that induces activation of human and mouse DCs [25]. Hp91-activated DCs show increased secretion of pro-inflammatory cytokines and chemokines, including the Th1 cytokine, IL-12. In addition, DCs exposed to HMGB1-derived peptides induced proliferation of antigen-specific syngeneic T cells in vitro [25]. These immunostimulatory properties of Hp91 and the fact that peptides are easy to manufacture make it an attractive candidate as an adjuvant for vaccine development. Here we show that the immunostimulatory peptide (ISP) Hp91 acts as an adjuvant in vivo by enhancing immune responses to peptide and protein antigen.

2. Materials and methods

2.1 Reagents and cell lines

The OVA-transfected EL4 line, E.G7-OVA (ATCC, Manassas, VA, USA), was cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA, USA), 10 mM HEPES (Invitrogen), and penicillin (100 U ml−1) - streptomycin (100 μg ml−1) - L-glutamine (2 mM) (Invitrogen).

2.2 Peptides and protein

The peptides, including the ISP Hp91 (DPNAPKRPPSAFFLFCSE), Hp121 (SIGDVAKKLGEMWNNTAA), the MHC-Class I (H-2Kb)-restricted peptide epitope of ovalbumin (OVA-I: OVA 257-264 aa, SIINFEKL), and the MHC-Class II (I-Ab)-restricted peptide epitope of ovalbumin (OVA-II: OVA 323-339 aa, ISQAVHAAHAEINEAGR) were all purchased from GenScript Corp (Piscataway, NJ, USA) and CPC Scientific (San Jose, CA, USA). Hp91 and Hp121 peptides were synthesized with an N-terminal biotin. Peptides were routinely synthesized with greater than 95% purity. LPS-free chicken egg white ovalbumin protein was kindly provided by Dr. Thomas Moran (Department of Microbiology, Mount Sinai School of Medicine, New York, NY, USA). Unless otherwise stated, all peptides and proteins were dissolved in PBS in preparation for immunisation.

2.3 Mice and Immunisations

Female C57BL/6 mice 8-12 weeks of age were used for most experiments. All mice were purchased from Charles River Laboratories (Boston, MA, USA) and housed at the Moores UCSD Cancer Center animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee of UCSD and were performed in accordance with the institutional guidelines. For most experiments, mice were immunised s.c. with 50 μg of SIINFEKL (OVA-I) peptide and 50 μg of ISQAVHAAHAEINEAGR (OVA-II) peptide. The OVA peptide was co-administered with either Hp91 (30 to 500 μg) or PBS (negative control). For some experiments, a protein vaccine group was included, wherein 500 μg of Hp91 was co-administered with 100 μg of LPS-free OVA protein. As a positive control, mice were immunised s.c. with OVA peptide(s) or protein in Incomplete Freund's Adjuvant “IFA” (Sigma-Aldrich, St. Louis, MO, USA). If not otherwise indicated, mice were immunised and boosted two weeks later and spleens and blood were collected 10-14 days after the final immunisation.

2.4 Intravenous administration of ISP

C57BL/6 mice were injected i.v. with 0, 10 or 100 μg Hp91 dissolved in PBS into the tail vein. Blood was collected after 2h and 24h by retroorbital puncture. Blood was allowed to clot and serum was isolated after centrifugation. Serum was diluted and analysed for systemic cytokine and chemokine release by ELISA (eBioscience, San Diego, CA, USA).

2.5 Detection of antigen-specific antibody production by ELISA

Serum was obtained by retroorbital puncture or cardiac puncture from mice following immunisation. Blood was allowed to clot and serum was isolated after centrifugation. Microtiter plates were coated overnight with OVA protein (Sigma-Aldrich), blocked with BSA, and dilutions of serum were added to the plates for incubation. Plates were washed, incubated with anti-mouse IgG or IgM peroxidase conjugated antibodies (Roche, Basel, Switzerland), developed using Zymed TMB substrate (Invitrogen), and analysed using a microplate reader at 450 nm.

2.6 Spleen cell preparation

Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70 μm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, IN, USA) and the splenocytes were subsequently resuspended in complete medium (RPMI 1640 with 10% FBS, L-glutamine, penicillin, streptomycin, and HEPES). In some experiments, CD4+ and CD8+ cells were depleted from bulk splenocyte populations using anti-CD4 or anti-CD8α conjugated microbeads (Miltenyi-Biotec, Auburn, CA, USA) according to the manufacturer's instructions.

2.7 Enzyme-linked immunospot assay

Freshly isolated splenocytes were plated in duplicate to wells of a nitrocellulose bottom enzyme-linked immunospot (ELISPOT) plate (Millipore, Millerica, MA, USA) that had been previously coated overnight with 5 μg ml−1 monoclonal anti-mouse IFN-γ antibody (Mabtech, Stockholm, Sweden). Splenocytes were cultured overnight at 37°C with 2.5 μg ml−1 SIINFEKL (OVA-I) peptide, 2.5 μg ml−1 ISQAVHAAHAEINEAGR (OVA-II) peptide, or left unstimulated (medium only). After 18 h, culture supernatants were collected for cytokine analysis and ELISPOT plates were developed using 1 μg ml−1 biotinylated anti-mouse IFN-γ antibody (Mabtech), Streptavidin-HRP (Mabtech), and TMB Substrate (Mabtech). The plate was scanned and the spots were counted using an automated ELISpot Reader System (CTL ImmunoSpot, Shaker Heights, OH, USA).

2.8 Measurement of cytokines and chemokines

Splenocytes were cultured overnight with 2.5 μg ml−1 OVA-I peptide, 2.5 μg ml−1 OVA-II peptide, 5 μg ml−1 concanavalin A positive control (Sigma), or left unstimulated (media only). After 18 h, cell culture supernatants were collected and analysed for the presence of IL-2 and IL-4 by ELISA (eBioscience).

2.9 LDH Cytotoxicity Assay

Splenocytes were expanded in culture at 3 × 106 cells ml−1 in complete medium with mitomycin-C (Sigma-Aldrich)-treated E.G7-OVA at a 5:1 ratio in 6 well plates. Four days later, live cells were isolated on a lympholyte gradient (Cedarlane Laboratories Limited, Burlington, Ontario, Canada) and cultured in complete medium with 25 U ml−1 IL-2 (R&D Systems, Minneapolis, MN, USA) for two additional days. Cytotoxicity assays were performed using a CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, USA). 1 × 104 E.G7-OVA cells per well were plated as target cells. Expanded splenocyte effector cells were incubated with the target cells at effector to target ratios of 1:1, 3:1, 10:1, and 30:1. Cultures were incubated in phenol-red free RPMI (Invitrogen) with 5% FBS (Omega) for 6h at which point the cell culture supernatants were harvested. The lactate dehydrogenase (LDH) released from lysed cells was proportional to the resulting red formazan product, and was quantified using a microplate reader at 490 nm absorbance. The percentage of cytotoxicity was calculated according to the following equation: % Cytotoxicity = [(E – St – Se)/(M – St)] × 100. Abbreviations are as follow; E = LDH release by effector-target coculture, St = spontaneous release by target cells, Se = spontaneous release by effectors, and M = maximal release by target cells.

2.10 Statistical analysis

Data represented are mean ± SEM. Data were analysed for statistical significance using unpaired Student's t-test, 2-way ANOVA, or linear regression. Statistical analyses were done using GraphPad software version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). A p value <0.05 was considered statistically significant for these analyses.

3. Results

3.1 Hp91 induces cytokine release in vivo

We have previously shown that exposure of DCs to an immunostimulatory peptide (ISP) named Hp91 in vitro leads to secretion of inflammatory as well as Th1 skewing cytokines[25]. To examine the adjuvant properties of Hp91 in vivo, serum cytokine responses were measured after intravenous (i.v.) injection of Hp91 into mice. Increased secretion of the Th1 cytokines IFN-γ, IL-12 (p70), as well as TNF-α and IL-6, was observed within 2 h of injection, with levels generally rising further over 24 h (Fig.1).

Fig. 1
Hp91 causes release of cytokines in vivo

3.2 Hp91 enhances CD8 T cell responses to peptide antigen

Since the ISP Hp91 activates DCs and induces antigen-specific T cell responses in vitro[25], we tested whether Hp91 acts as a adjuvant to induce antigen-specific immune responses in vivo. The ISP Hp91 at both doses tested (250 μg and 500 μg) when co-administered with OVA peptides, caused a significant increase in the number of antigen-specific IFN-γ secreting T cells when splenocytes were restimulated with the CD8 epitope SIINFEKL (Fig. 2A), but not when restimulated with the CD4 epitope ISQAVHAAHAEINEAGR (Fig. 2B). Incomplete Freund's Adjuvant (IFA) a known stimulator of cell-mediated immune responses, elicited strong cellular immune responses when splenocytes were restimulated with either the CD8 or CD4 epitope (Fig. 2A,B).

Fig. 2
Cellular immune response in Hp91 immunised mice

The OVA-I peptide (SIINFEKL) is recognized in the context of H-2Kb MHC-Class I molecules and is specific for CD8+ T cells. To further confirm that the observed immune response is an OVA-specific CD8+ T cell response, CD4+ or CD8+ cells were depleted from the splenocytes prior to setup up the ELISPOT assay. While greater than 95% of CD4+ cells were depleted, CD8+ depletion was not as complete; 20% of CD8+ cells remained in the cultures as observed by flow cytometry (data not shown). As expected, the number of IFN-γ secreting cells was reduced to near background levels (OVA peptides/PBS) in the CD8+ depleted splenocyte populations (Fig. 2C). In contrast to the CD8+ depletion, the CD4+ depleted splenocytes from the Hp91-OVA immunised groups retained the ability to secrete IFN-γ in response to OVA-I peptide stimulation further supporting the involvement of CD8+ T cells following co-immunisation with the ISP Hp91 and OVA peptide, showing that the ISP Hp91 causes activation and proliferation of antigen-specific CD8+ T lymphocytes in vivo.

To test whether lower doses of Hp91 would suffice as adjuvant for immunization, titrated doses of Hp91 were co-injected with OVA peptide to determine the minimum injection dose required for a significant increase in antigen-specific IFN-γ secreting T cells. As expected, a dose response was observed (Fig. 2D). However, with only 5 mice per group, a significant increase in IFN-γ secreting T cells was observed by ELISPOT only in the group receiving an Hp91 dose of 250 μg, suggesting 250 μg is an optimal dose when immunising small groups of mice. Subsequent experiments showing 500 μg of Hp91 were conducted prior to the titration experiment.

3.3 The ISP adjuvant effect is sequence specific

To test if the in vivo adjuvant effect is related to the sequence of Hp91 or if any peptide will cause similar effects, we used a control peptide named Hp121. Hp121 is also derived from HMGB1 B-box and has the same length, a similar charge, and isoelectric point as Hp91. Hp121 does not cause activation of human DCs [25] or mouse BM-DCs in vitro (data not shown). Mice were immunised s.c. with OVA-I peptide co-injected with either Hp91, Hp121 or PBS control. Mice immunised with Hp91/OVA-I peptide showed a significantly increased number of INF-γ secreting cells as compared to the Hp121/OVA-I peptide and PBS/OVA-I peptide immunised mice using freshly isolated as well as expanded splenocytes (Fig. 2E). No significant increase was observed between the Hp121/OVA peptide and PBS/OVA peptide immunised groups.

3.4 Hp91 induces Th1-type immune response in vivo

Since IL-2 is critical for the activation, survival, and proliferation of T lymphocytes, we tested whether IL-2 secretion is increased in Hp91/OVA peptide immunised mice. Freshly isolated splenocytes were cultured overnight in presence of OVA peptide and culture supernatants were analysed for IL-2 (Figure 3) and IL-4, which may indicate the induction of a Th2 type immune response (data not shown) by ELISA. The highest IL-2 secretion was observed in OVA-I restimulated splenocytes from mice immunised with Hp91/OVA peptides (Fig. 3A), which showed higher IL-2 secretion compared to mice vaccinated with whole OVA protein/Hp91 or OVA peptide/IFA, both of which were also significantly increased as compared to the PBS/OVA control. After exposure to the MHC-Class II specific OVA-II peptide, splenocytes from Hp91/OVA peptide immunised mice showed a low but significant increase in IL-2 secretion as compared to PBS/OVA immunised mice (Figure 3B). IL-4 was not detected in the splenocytes cultures at any of the conditions, though it was detected in the ConA stimulated positive control (data not shown).

Fig. 3
Cytokine secretion in Hp91 immunised mice

3.5 Hp91 elicits antibody responses to soluble protein

Immunisation using OVA protein in context of Hp91 promoted an antibody response to OVA which was dominated by the IgG1 isotype (Fig. 4A, 4B). Two-fold serial dilutions of the Hp91/OVA protein group show similar serum titers for the group (Fig. 4C). Minimal increase in IgG2b and IgM OVA specific antibodies was detected (data not shown). As expected, immunisation using OVA peptides (OVA-I and OVA-II) together with Hp91 did not induce antibody responses (Fig. 4A and data not shown).

Fig. 4
Antibody responses in Hp91 immunised mice

3.6 Co-administration of Hp91 with antigen induces CTL responses

To further investigate whether OVA-specific cytotoxic T lymphocytes (CTL) responses were induced by immunisation with the ISP Hp91, splenocytes from immunised mice were assessed for their ability to lyse OVA-expressing E.G7-OVA cells. The strongest killing was observed using effector splenocytes from mice immunised with Hp91/OVA peptides (Fig. 5), which was even higher than of splenocytes from IFA/OVA peptide immunised mice. When data were analysed by linear regression and slopes were compared, the percent killing of target cells by effectors cells from mice immunized with Hp91/OVA peptides was significantly higher than the PBS control group.

Fig. 5
CTL Induction in immunised mice

4. Discussion

Although subunit vaccines promise to be less toxic, many are poorly immunogenic when administered without adjuvant. Alum, though FDA-approved, generates a weak Th1 response with a questionable safety profile. Thus, there is a great need for safer and more potent adjuvants [1-3].

We have previously shown that the 18 amino acid long ISP Hp91, is a potent stimulus for human DCs with the ability to generate a Th1-type immune response in vitro [25]. Here we demonstrate that Hp91 acts as adjuvant in vivo; inducing cellular immune responses to peptide and both cellular and humoral immune responses to protein antigen. The CD8 immune response was strong, since no in vitro expansion of splenocytes was needed to obtain a significant response as is commonly performed when testing vaccine responses. We show that the ISP Hp91 acts as an immune adjuvant to induce antigen-specific CD8 T cell responses in vivo. In addition, the immunostimulatory effects of Hp91 are related to its sequence, as the HMGB1 control peptide, Hp121, while matching Hp91 in length, isoelectric point, and charge, failed to induce cellular immune response.

The cytokine profile induced by an immune adjuvant plays an important role in the polarization of the immune response. The data show that co-immunisation with the ISP Hp91 and OVA peptides as well as OVA protein results in OVA-specific secretion of IL-2, suggesting that immunised mice are able to mount an adaptive immune response that activates T cells to synthesize and secrete IL-2 for in vivo proliferation of OVA-specific effector T cells. Interestingly, IL-4 levels were undetectable. Intravenous administration of Hp91 resulted in increased secretion of the Th1 cytokines IFN-γ and IL-12 (p70) associated with cell-mediated immunity. This together with the measured IFN-γ secretion by the T cells along with undetectable IL-4 suggests that Hp91 induces a Th1-type of immune response in vivo. We also show that immunisation with Hp91/OVA peptide elicited stronger CTL responses than IFA/OVA.

Although the main objective was to test the potency of Hp91 as adjuvant for peptide vaccines and induction of cellular immune responses, we show that immunisation using OVA protein mixed with Hp91 also induced humoral immune responses. This is very promising for future development of this novel adjuvant, as it could be used for prophylactic vaccination against infectious disease.

We have previously shown that Hp91 can activate DCs, but the exact mechanism of action is still under investigation. The Hp91 peptide sequence corresponds to a region within the B-Box domain of HMGB1 protein [25]. Toll-like receptors (TLRs) 2 and 4 have been shown to be involved in HMGB1 signaling in vitro [34-37] and in vivo data have shown TLR4 to be involved in HMGB1-induced inflammation [38]. We have previously shown that that the HMGB1 immunostimulatory peptides, including Hp91, activate mouse BM-DCs independent of TLRs 2, 4, 9, and MyD88 [25]. HMGB1 has been shown to contribute to LPS-mediated DC maturation via RAGE [39]. The C-terminal motif of HMGB1 (150–183 amino acids) is responsible for RAGE binding [40]. The HMGB1-Bx and the “active” peptides do not contain the C-terminal motif; therefore, it is unlikely that DC maturation induced by HMGB1-Bx or the peptides occurs through RAGE. We have recently shown that utilizing Hp91-loaded nanoparticles for endocytic delivery to DCs demonstrate enhanced potency of the ISP and may suggest that the receptor is not on the cell surface [41], but likely in an intracellular compartment such as an endosome. At this time we cannot exclude that Hp91 signals through a TLR3, MyD88 independent pathway or acts via other receptors. Studies are currently underway to identify the Hp91 receptor.

The ability of Hp91 to induce antigen-specific cell mediated, Th1 immune response may make Hp91 suitable as an adjuvant in cancer immunotherapies as well as vaccines against infectious diseases caused by intracellular bacteria [4] or viruses [42,43]. Some of the adjuvants that are being evaluated in clinical and preclinical settings like CD40L [44] and poly(I:C) synthetic double stranded RNA [45], act on myeloid DCs and have shown promising results for tumour immunotherapy [46], emphasizing the importance of activating myeloid DCs. As a peptide adjuvant that acts directly on myeloid DCs [25], Hp91 has several advantages. It can be made synthetically, is inexpensive, can be produced in high quantities at GMP quality, and it can also be genetically engineered to DC targeting molecules like DEC-205 which promotes strong immune responses when linked to a DC stimulatory molecule [47,48]. Since the tested doses of Hp91 have shown no adverse effects in mice to date, this current data suggest this endogenous peptide should be well-tolerated for use in vaccines.


We would like to thank D. Futalan, D. Seible, J. Steiner, J. Ahlqvist, and N. Ambren for excellent technical assistance. We would like to thank J.F. Fecteau for critical reading of the manuscript. This work is supported by 5 U54 CA119335 from the National Institutes of Health/NCI (to S.E. and D.M.) and the Swedish Research Council AI52731 and the Swedish International Development Cooperation Agency; SIDA and VINNMER (Vinnova) to (ML).


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