Elsevier

Clinical Immunology

Volume 140, Issue 2, August 2011, Pages 142-151
Clinical Immunology

REVIEW
Clinical development of a novel CD1d-binding NKT cell ligand as a vaccine adjuvant

https://doi.org/10.1016/j.clim.2010.11.009Get rights and content

Abstract

Natural killer T (NKT) cells are known to play a role against certain microbial infections, including malaria and HIV, two major global infectious diseases. Strategies that can harness and amplify the immunotherapeutic potential of NKT cells can serve as powerful tools in the fight against such diseases. 7DW8-5, a novel glycolipid, may be one such tool. The interaction of 7DW8-5 with CD1d molecules induces activation of NKT cells, thereby activating various immune-competent cells including dendritic cells (DCs) to provide a significant adjuvant effect for several vaccines. This review discusses the discovery and characterization of 7DW8-5 and the practical considerations of its preclinical and clinical development as a potential glycolipid adjuvant for candidate malaria and HIV vaccines.

Introduction

Natural killer T cells (NKT) are distinct from conventional T cells and natural killer (NK) cells [1] as they are characterized by the expression of both a T cell antigen receptor (TCR) and NK1.1 (NKR-P1 or CD161c), a C-lectin type natural killer (NK) receptor. As the TCR is invariant for the majority of murine and human NKT cells, encoded by the Vα14 and Jα18 gene segments [2], or Vα24 and Jα18 segments [1], respectively, these NKT cells are called invariant NKT (iNKT) cells. The Vβ repertoire utilized by NKT cells is somewhat more diverse [2], [3], [4]. Murine NKT cells usually belong to either CD4CD8 (double negative) or CD4+CD8 sub-populations [2], [3], [4]. Human NKT cells are more heterogeneous and a significant percentage of human NKT cells are CD4CD8+, both in the blood and the liver [5].

The only characterized ligand of NKT cells to date is CD1d, an MHC class I-like molecule [2], [6], [7], [8]. While the CD1d protein is not encoded by the MHC gene locus [6], [7], this molecule associates non-covalently with β2-microglobulin and exhibits limited, yet significant, homology as well as overall structural similarity with MHC class I heavy chains [6], [7]. CD1d is constitutively expressed by many cells, including dendritic cells (DCs), macrophages, and B and T lymphocytes [9], [10]. Although all iNKT cells recognize and are restricted by CD1d molecules, some NKT cells having more diversified TCR also recognize CD1d [1]. There is a population of NKT cells that does not depend on the recognition of CD1d molecules and known as non-CD1d-restricted NKT cells [1].

A synthetic glycolipid designated α-galactosylceramide (α-GalCer), originally identified in an Okinawan marine sponge by a research group at the Kirin Brewery Co. in Japan [1], [11], was shown to bind to CD1d. This was documented by surface plasmon resonance using immobilized α-GalCer and soluble murine [12], [13] or human CD1d [12]. This glycolipid, when presented by the CD1d molecule, activates both murine and human NKT cells in vivo and in vitro [14], [15], [16], [17]. X-ray crystallography studies have revealed that the lipid portion of the glycolipid fits tightly into the CD1d binding groove, wherein the sphingosine chain associates with one pocket in the groove and the longer acyl chain anchors within a separate pocket [18], [19]. A more recent structural study using a tri-molecular complex consisting of CD1d-glycolipid and Vα24 TCR confirmed that the galactose ring extends above the surface of the lipid-binding groove, and thereby is exposed for recognition by the TCR of NKT cells [20].

Eliminating or exchanging this moiety with various sugars has been shown to either diminish or abrogate activity [14], indicating the importance of the galactose head group to α-GalCer function. We and others have shown that the α-anomeric conformation of the glycolipid as well as the equatorial configuration of the 2-hydroxyl group of the sugar moiety and the 3-hydroxyl group of the phytosphingosine are also crucial for α-GalCer to bind CD1d molecules and to activate NKT cells through their TCR [1], [13], [21], [22]. One study has shown that, albeit at lower potency than α-anomeric GalCer, β-anomeric GalCer can induce CD1d-dependent biological activities in mice [23]. Earlier research has shown that α-GalCer has been amendable to modification in the acyl tails of the molecule, since varying the hydrocarbon lengths and/or introducing unsaturation in the fatty acid chains [24], [25], as well as the truncation of the fatty acid chain from 24 to 2 carbons [21] did not significantly affect mouse NKT cell responses. Interestingly, a compound that has only 9 carbons at the sphingosine chain, OCH, is shown to skew the cytokine release profile towards Th-2 cytokines [26]. In contrast, we have previously found that a C-glycoside analog of α-GalCer, α-C-GalCer, preferentially stimulates a Th1-type response in mice [27], [28]. This analog, in comparison to α-GalCer, consistently stimulated prolonged production of IFN-γ, increased production of IL-12, and decreased production of the Th2 cytokine IL-4.

A number of studies have documented the protective role of α-GalCer-activated NKT cells in anti-tumor immunity [29], [30], autoimmune diseases [31], [32], [33], [34] and infectious diseases [35], [36], [37], [38], [39]. α-GalCer and α-C-GalCer have recently been shown to induce full maturation of DCs, as determined by an increased expression of co-stimulatory molecules, which include CD40, CD80 and CD86, as well as MHC class II molecules on DCs [40], [41]. The presence of NKT cells is required in order for the glycolipids to activate DCs. Although the precise mechanisms for this DC maturation process and for the functional consequence of the DC maturation are still unknown, it is thought to involve multi-factorial pathways in order to lead to full maturation/activation of DCs [42].

To date, several clinical trials have been conducted using α-GalCer primarily for the treatment of cancer, but also as a potential therapeutic agent against Hepatitis B and Hepatitis C [43], [44], [45], [46], [47], [48]. Direct intravenous (IV) administration of α-GalCer did not result in a noticeable clinical benefit against cancer, hepatitis B, or hepatitis C infection [43], [48]. In order to improve the activity in cancer patients, subsequent clinical trials conducted were based on the transfer of DCs pulsed with α-GalCer [45], [46]. These trials again failed to display any noticeable anti-tumor effect. In a recent Phase I clinical trial, however, APCs were pulsed with α-GalCer in the presence of autologous Vα24 NKT cells, before transferring them back to the cancer patients. In short, the combined use of the intra-arterial infusion of activated Vα24 NKT cells and the submucosal injection of α-GalCer-pulsed APC induced significant anti-tumor immunity and had measurable beneficial clinical effects in the management of advanced head and neck squamous cell carcinoma [44].

Our recent work has demonstrated that α-GalCer-activated NKT cells enhance malaria-specific T cell responses and, more importantly, protective anti-malarial immunity elicited by various immunogens, including a recombinant adenovirus expressing a malarial antigen [49]. The adjuvant effects of α-GalCer in this model require CD1d molecules, Vα14 NKT cells, and IFN-γ (Fig. 1). However, two recent studies have shown that α-GalCer or its analogues can display their adjuvant effects on T cell responses elicited by a soluble protein, possibly through CD40–CD40L interaction, as well as by cytokines such as type I interferon and IFN-γ [40], [50]. It was also shown that these interactions may ultimately lead to the full maturation of dendritic cells, and are therefore responsible for mediating the adjuvant activity of glycolipids [40], [41], [50], [51]. More recently, a number of studies have reported on the adjuvant effect displayed by α-GalCer and α-C-GalCer on vaccines against viruses and cancers in a mouse model [52], [53], [54], [55], [56]. Interestingly, a very recent study has shown that non-glycosidic analogues of α-GalCer could activate iNKT cells resulting in dendritic cell maturation and the priming of antigen-specific T and B cells, thereby acting as a vaccine adjuvant [57].

In an attempt to search for a more potent glycolipid, we synthesized a compound library of 25 glycolipids analogous to α-GalCer and evaluated their biological activity. The evaluation process included testing the ability of each glycolipid to stimulate human NKT cell lines and secrete cytokines including IFN-γ, as well as their ability to stimulate autologous DCs and secrete cytokines including IL-12. After selecting 5 glycolipids which displayed strong biological activity in in vitro assays, we carefully assessed the binding affinity of selected glycolipids to the human CD1d molecule, as well as to the invariant TCR (invTCR) of human NKT cells in vitro. The binding affinity of selected compounds to invTCR has been shown to have a strong correlation with its biological activity. Thus, after evaluating the biological activity of these glycolipids, we identified 7DW8-5 as a lead compound for further adjuvant testing [58].

After selecting 7DW8-5 as a lead compound, we compared its adjuvant effect with that of α-GalCer against various HIV vaccine platforms, namely Ad5–p24 and DNA–p24. For this purpose, different doses of each compound were co-administered intramuscularly with a sub-optimal dose of either Ad5–p24 or DNA–p24. Two weeks after a single immunizing dose of Ad5–p24 co-administered with each glycolipid, 7DW8-5 was shown to enhance the most the level of p24-specific CD8+, as well as CD4+ T cell responses that secrete IFN-γ, as determined by ELISpot assay [58]. Similarly, when mice were primed with a DNA–p24 vaccine plus either 7DW8-5 or α-GalCer, and then boosted with the DNA–p24 vaccine alone, 7DW8-5 displayed a stronger adjuvant effect than α-GalCer, eliciting significantly higher p24-specific CD8+ T cell and humoral responses. Next, in order to test whether 7DW8-5 displays its adjuvant effect on “protective” immunity induced by vaccines, we used Ad5 expressing a malarial antigen, Ad5–PyCS, as a vaccine platform. Two weeks after vaccinating mice with Ad5–PyCS together with or without a glycolipid, we challenged the vaccinated mice with live rodent malaria parasites. Then, we determined the amounts of parasite loads in the liver of infected mice. Non-immunized mice were used as a control. Through this challenge experiment, we confirmed that 7DW8-5 displays a more potent adjuvant effect than α-GalCer, leading to a significantly higher degree of “protection” against malaria in mice [58]. Finally, to ensure that the adjuvant effect of 7DW8-5 was mediated by CD1d molecules, we immunized mice lacking CD1d, as well as wild-type mice, with Ad5–p24 co-administered with 7DW8-5, and measured the level of p24-specific T cell responses. Not surprisingly, the adjuvant effect of 7DW8-5 was totally abolished in mice lacking CD1d molecules, confirming the CD1d mechanism-based adjuvant effect of 7DW8-5. These studies have led us to select 7DW8-5 as the lead compound for further clinical development [58].

Section snippets

NMRC-M3V-Ad-PfCA candidate malaria vaccine

AdPfCA is a non-replicating adenovirus serotype 5 based malaria vaccine candidate developed by the US Military Malaria Vaccine Program (USMMVP), GenVec, Inc. and USAID, and expresses two Plasmodium falciparum antigens–circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) [59], [60]. The CSP is expressed by pre-erythrocytic stages, whereas AMA1 antigen is expressed by both pre-erythrocytic and erythrocytic stages [61], [62], of the Plasmodium parasite life cycle. Thus, a combined

Manufacturing

Unlike laboratory reagents for use in small animal studies, all biologic substances must be manufactured under Good Manufacturing Practice (GMP) conditions (Code of Federal Regulations Title 21, Part 211, April 2009) before use in humans. This requires that compound synthesis be reproducible and scalable at large quantities. One of the practical advantages of glycolipids is their straightforward synthesis pathway from chemical compounds that are readily available at relatively low cost. In

Clinical testing of the safety and immunogenicity of 7DW8-5 as a vaccine adjuvant

Although the ability of glycolipids to act as vaccine adjuvants has been well established in the animal model, neither α-GalCer nor any compound derivatives have been tested in combination with vaccines in clinical trials. In a Phase 1 clinical trial conducted by the USMMVP, one dose of the unadjuvanted vaccine elicited strong cellular immunogenicity as measured by ex vivo IFN-γ ELISpot assay [60]. In a follow-on clinical trial, in which NMRC-M3V-Ad-PfCA was given as a boost sixteen weeks after

Conclusions

While glycolipid compounds have been studied as a potential cancer treatment in a number of trials to date, the use of glycolipid compounds as vaccine adjuvants has thus far been limited to research in small animals, with promising results to date. Clinical development of a novel glycolipid compound, 7DW8-5, is underway to determine whether it can provide an adjuvant effect on an adenoviral-based malaria vaccine in healthy human volunteers. Should this concept translate to humans, glycolipids

Acknowledgments

The authors wish to thank Captain Thomas Richie, Noelle Patterson and Dr. Martha Sedegah, US Military Malaria Vaccine Program at the United States Naval Medical Research Center/Walter Reed Army Institute of Research, Dr. David Ho, the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery, and the Malaria Vaccine Initiative.

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