Systemic immune responses in sheep, induced by a novel nano-bead adjuvant

Abstract

Although a number of adjuvants are currently approved for use in veterinary species, only alum has been widely used in humans. While it induces strong antibody responses, cell mediated responses are often low and inflammatory reactions at the site of injection are common. We investigated the immunological properties of a novel nano-bead adjuvant in a sheep large-animal model. In contrast to alum, antigen covalently coupled to nano-beads induced substantial cell mediated responses along with moderate humoral responses. No adverse reactions were seen at the site of immunisation in the sheep. Thus, nano-bead adjuvants in veterinary species may be useful for the induction of immunity to viral pathogens, where a cell mediated response is required. These findings also highlight the potential usefulness of nano-bead vaccines for intracellular pathogens in humans.

Introduction

Vaccines for large animals need to be cheap and easy to administer, with minimal side effects. Live or attenuated vaccines, administered by the subcutaneous (SC) or intramuscular (IM) route have been traditionally administered to livestock. These are usually based on attenuated live or heat killed pathogens and incorporate natural “danger” signals, which promote immunity, but can also cause inflammation systemically and at the site of injection [1]. There are however many diseases for which whole organism vaccines have proven ineffective or unsafe to administer. Thus, newer vaccines with clearly-defined, quality-controlled antigens, such as plasmid DNA-encoded polypeptides, proteins produced in vitro by fermentation and synthetic peptides, have been developed, but these require concurrent administration of adjuvants to provide the activating “danger” signals to trigger an immune response. A current challenge is to develop vaccine formulations which promote protective immunity, but do not have adverse inflammatory side-effects (e.g. [1], [2]).

Adjuvant development has a major role in vaccine technology [3]. Adjuvants are particularly important in eliciting responses to simple proteins delivered in solution. The lack of suitable adjuvants has often hampered the development of effective recombinant peptide and protein vaccines for many diseases. It has been postulated that the role of adjuvants is to act as an in vivo depot for antigen while at the same time stimulating the immune system by providing “danger” signals to antigen presenting dendritic cells (DC). DC act as sentinels in tissues (including the skin), by sampling the local immunological environment. “Danger” signals, such as necrotic cells, bacterial products and cytokines released from injured tissues activate DC, thus promoting migration of these antigen-carrying cells through the afferent lymph vessels to the draining lymph node (reviewed in [4]), where primary immune responses are initiated [5], [6]. The effectiveness of many adjuvants is directly linked to their ability to mimic the “danger” signals associated with the tissue damage and hence adjuvants may cause inflammation, both systemically and at the site of injection (i.e. reactogenicity). Thus, adjuvants should be carefully selected for their ability to maximise immunogenicity, while at the same time minimising reactogenicity.

A number of approaches, including soluble mediators that interact with surface molecules present on DC [7], particulate antigens which are taken up by antigen presenting cells, including DC, but not other cells [8], [9], [10], [11], [12], [13] and viral/bacterial vectors that infect antigen presenting cells [14], [15], are currently being developed as effective vaccine adjuvants. The activity of particulate adjuvants, such as ISCOMs [8], virus-like particles (VLP), non-ionic surfactant vesicles (NISV) and PLGA micro-particles (reviewed in [16], [17], [18]) is known to be dependent on the size of the particles. In mice, lipid vesicles with a size ≥225 nm induced predominantly Th1-type responses while vesicles with a size ≤155 nm induced predominantly Th2-type responses [19].

One way of achieving maximal immunogenicity with minimal reactogenicity is to use particulate antigens without adding or inducing inflammatory mediators. Recent studies by Fifis et al. [13], [20], have demonstrated in mice that antigen covalently linked to inert nano-beads with a size range of 40–50 nm are preferentially taken up by DC and induce high antibody titres, as well as cell mediated immune responses (including cytotoxic T cells), without significant side effects. Thus, in mice, the nano-bead based adjuvant met the requirements as an ideal adjuvant for maximising immunogenicity while minimising reactogenicity. Here, we explored the cellular and humoral immune responses induced in sheep by this novel nano-particle based adjuvant delivered via different routes.