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.

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], [2], [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, characterised 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], [11], [12], [13], [14]. Although neutralising 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], [16], [17], [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], [21], [22], [23]. However, clinical trials in humans utilising 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, tumour 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], [31], [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.