Preventive (prophylactic) vaccines have greatly helped the global eradication of some infectious viral diseases, for example poliomyelitis or smallpox1. However, vaccines used against pathogens such as the HIV and the hepatitis C virus (HCV) have for the most part failed. One problem is that vaccines based on attenuated viruses, despite generating protective immunity2,3, are frequently too toxic for clinical use. On the other hand, non-viral vaccines, which typically have an excellent toxicity profile, are relatively ineffective at promoting immunity. It is therefore necessary to develop a vaccination strategy that can generate effective immunity and have low toxicity. As reported in Nature Materials, Irvine and colleagues have achieved an important step in this direction4: a non-viral vaccine carrier that provides immune responses comparable to viral vectors5.

The vaccine carriers are multilamellar vesicles (liposomes) with crosslinked bilayers that entrap protein antigens in the vesicle core, and immunostimulatory Toll-like receptor (TLR) ligands in between bilayers (TLR is a protein that recognizes structural patterns). Irvine and colleagues showed that the crosslinked liposomes act as a controlled-release reservoir of protein antigen and can also target dendritic cells in vivo. Dendritic-cell targeting is a key part of the multi-step process by which vaccines activate antigen-specific memory T cells6 — cells that recognize and rapidly clear pathogens that caused previous infections. In this process (Fig. 1), dendritic cells pick up the injected liposomes and present the delivered antigen and immunostimulatory molecules to cytotoxic T cells. The dendritic cells secrete cytokines — cell-signalling protein molecules — that help activate T cells against the pathogen-specific antigens. These cells then proliferate and circulate through the body, most of them dying off within a few days. However, a small subset of the population of cytotoxic (or killer) T cells survives in the long term (even for decades), giving rise to memory T cells. On infection by a pathogen with the known antigen, the antigen-specific memory T cells proliferate and differentiate into cytotoxic T cells, which clear the infection before it can gain a foothold. Hence, the presence of antigen-specific memory T cells greatly accelerates the timescale and magnitude of the immune response towards the infecting pathogen.

Figure 1: Crosslinked liposomes target and deliver antigen and immunostimulatory drugs to dendritic cells to trigger the generation of memory T cells.
figure 1

Activated dendritic cells generate cytotoxic T cells, which then divide and clear the infection. Some of the cytotoxic T cells give rise to memory T cells, which survive long term.

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Although the detailed mechanism by which antigen-specific memory T cells are generated is not completely understood, key steps seem to be the presentation of the antigen to T cells, the secretion of cytokines by dendritic cells, and the controlled release of antigen. To secrete the cytokines needed for T cell activation, dendritic cells need to be stimulated by TLR ligands. Thus, several polymeric microparticles have been developed that can deliver both antigen and TLR ligands7. However, because these particles have difficulties in reaching the cytosol of dendritic cells, they tend to be less effective as vaccine carriers.

As an alternative, the delivery of vaccines loaded into liposomes is a very attractive strategy not only because the cytosol can be accessed, but also because both antigen and TLR ligands can be easily encapsulated within the same particle, owing to the presence of both aqueous and hydrophobic components. Furthermore, the synthesis of liposomes is simple, it can be performed on a small scale in an aqueous environment, and it provides high encapsulation of proteins. Despite the advantages, however, liposomes have previously not been critically successful as vaccine-delivery vehicles owing to their variable stability and unpredictable release profile8. Indeed, liposomes can be destabilized under the shear forces present in biological environments, resulting in structural disintegration and aggregation or fusion, and also have problems with chemical stability because of hydrolysis, oxidation and enzymatic degradation.

Irvine and co-authors avoided the usual problems in the stability and release profile of the liposomes by crosslinking their bilayers with thioether linkages (Fig. 2). Additionally, polyethylene glycol was conjugated on the surface of the resulting interbilayer crosslinked multilamellar vesicles (ICMVs) to enhance their in vivo performance. The authors showed that ICMVs have excellent stability in physiological solutions but rapidly break down intracellularly as a result of the presence of lipases — lipid-hydrolysing enzymes — within the cells4. They also showed that ICMVs provide both sustained release and intracellular delivery of the entrapped antigen, thus allowing efficient activation of both T and B cells, and the generation of memory T cells. B cells — cells that produce antibodies against antigens — are important because they neutralize pathogens that are circulating in the blood, and it is believed that effective vaccines for pathogens such as HIV will require activation of both T and B cells.

Figure 2: Interbilayer-crosslinked multilamellar vesicles (ICMVs) encapsulating TLR ligands (not shown) and pathogen-specific antigens were synthesized4 from dried liposomes by Mg2+-induced fusion and by crosslinking lipid head groups from opposite lipid bilayers with bilayer-permeable dithiols.
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Thiol-terminated polyethylene glycol (PEG-SH) was then conjugated to the surface of the resulting ICMVs to enhance their performance in vivo.

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Although ICMVs have shown tremendous potential with the model antigen Ovalbumin, it needs to be seen if this approach shows similar levels of efficacy with protein antigens from pathogens such as HIV and HCV. Also, because the immune system of humans can be substantially different from that of a mouse, determining if ICMVs also have high efficacy in higher animal models is critical. All in all, the concept of crosslinking liposomes with dithiol linkages is a powerful way of delivering protein antigens and TLR ligands to immune cells, with the potential to greatly improve vaccine development and drug delivery.