A Randomized, Blinded, Placebo-Controlled Phase 1 Study to Evaluate the Safety and Immunogenicity of GLA in Healthy Volunteers

Vaccines against infectious diseases have been instrumental to the improvement of human health and remain a pillar of modern public health strategies. Yet serious life threatening infections including HIV, malaria and tuberculosis remain a global problem and pandemic diseases like influenza continue to threaten human life. As well, vaccines are now being pursued in the areas of cancer prevention and treatment. A fundamental barrier that has prevented effective vaccines for many diseases is that conventional protein based vaccines do not elicit the critical requirement of T-cell mediated immunity. One approach to generating an improved T-cell response to a vaccine has been to identify new protein target candidates. This approach has not resulted in significant advances in vaccine development. We are interested in a novel approach that combines specific immune stimulants, or adjuvants, with a vaccine target to optimize and enhance the desired T-cell immune response. The development and study of novel adjuvants like GLA will allow us to further our investigation into dendritic cell targeted vaccines leading to improved vaccines for many diverse diseases.

Toll-Like Receptor (TLR) Background The TLR's are type 1 transmembrane receptors that share a leucine-rich repeat domain (LRR) in the extracellular loop and a Toll/IL-1 receptor (TIR) homology domain in the intracellular tail. The importance of TLRs to host defense and immunity was first appreciated in Drosophila that became susceptible to fungal infections following genetic deletion of the toll genes. Mammalian homologs were identified soon after using a mouse strain that was well known to be highly susceptible to Gram-negative infections and were hyporesponsive to lipopolysaccharide (LPS). These mice were found to have a mutational change on the TLR4 gene, rendering it non-functional and this discovery solidified the existence and importance of the Toll proteins in mammals. Mice with genetic deletions of TLR4 demonstrated the importance of TLR to bacterial infections and provided clear evidence that TLR4 was specific for Gram negative infections. Currently 10 functional TLRs have been discovered in humans and extensive research has identified a number of pathogen-derived agonists for specific TLRs. It is generally accepted that the TLR's function by recognizing conserved structures of an organism or pathogen associated molecular patterns (PAMPs). Clearly these innate receptors are critical to surviving a microbial insult as they provide a first line of defense that is not dependent on generating a specific T-cell and B-cell response, a process that can take weeks. Despite their clear role in innate immunity, evidence is accumulating that TLR stimulation has potent influence on the development of T and B cell mediated immunity through the activation and maturation of dendritic cells (DC). This knowledge has led to the development of TLR-based vaccine adjuvants that activate and mature DC.

Monocyte and Dendritic Cell Overview Dendritic cells (DC) are part of the innate and adaptive immune systems and reside at host-microbial interfaces including the surfaces of the gut, lung and within the skin. These cells constantly survey their environment in search of microbial pathogens through the expression of several families of pattern recognition receptors (PRR) including the Toll-like receptors (TLR). Immature DC are particularly efficient in antigen uptake and processing, while activated DC mature and become potent antigen presenting cells to T-lymphocytes. Importantly, immature DC have the potential to induce immune tolerance by deleting antigen specific T cells, resulting in no immune response. Activated DC however direct the immune response through the release of particular cytokines. Different initial stimuli will influence DC to drive CD4+ T-cells to differentiate along very divergent functional pathways including Th1, Th2, Th17 and Treg. Therefore, DC are critical to appropriate innate host defense as well as to orchestrate an adaptive response. Given their pivotal role in host defense, DC are relatively rare cells that, at steady state, develop independently from other blood cells. Enhancing and directing the function and quantity of DC will provide benefit to both host defenses as well as to vaccine science. The Steinman lab has pioneered a novel method of vaccination by targeting the antigen of interest specifically to the DC population. DC-targeted vaccines have shown promise in animal models and a DC-targeted HIV vaccine has recently been administered to humans for the first time.

Monocytes are a more abundant cell type that account for approximately 10% of blood leukocytes in humans and 4% in mice. Monocytes are peripheral blood effector cells that participate in host defense and are efficient scavenging cells. They serve as precursors to tissue macrophages. In humans, 3 subsets of monocytes have been proposed based on cell surface expression of CD14 and CD16, while 2 subsets exist in mice and are marked by CD115 and Ly6C. The role of these subsets during innate immunity requires further investigation. Evidence suggests that monocytes can be induced to differentiate into DC, however definitive in vivo evidence of this was previously lacking. Recent work in the Steinman lab has shown that in vivo authentic DC can be rapidly differentiated and mobilized from the larger monocyte pool in times of acute inflammation and infection. This rapid re-deployment could be induced by either gram-negative bacteria or LPS and was entirely dependent on TLR4. Similarly, human monocytes can de induced to differentiate into DC, however the understanding of this process is limited to in vitro cell culture with large gaps in our understanding of in vivo human monocyte derived-DC. Preliminary data suggests that GLA administration in mice, like LPS, can mobilize the monocyte pool into becoming Mo-DC. It is unknown if human monocytes in vivo rapidly expand the DC population following administration of TLR4 adjuvant GLA. Understanding the mechanisms of this re-deployment will further our understanding of how to rapidly expand and harness the utility of DC using adjuvants. Ultimately, we anticipate that adjuvant-specific DC responses will improve T-cell immunity and enhance vaccine effectiveness.

Rationale for Investigating an Adjuvant in Isolation The standard adjuvant trial consists of the adjuvant under investigation combined with a known licensed vaccine but not in isolation. However, as previously described, we are hopeful that GLA may become an important adjuvant of our DC-targeted HIV vaccine currently in development. The DC represents the most potent antigen-presenting cell and specifically targeting an antigen of interest to this cell generates improved immune responses. However there are important considerations when targeting antigen to DC. Immature unstimulated DC that encounter an antigen have the ability to delete antigen specific T cells thereby causing tolerance and a DC-targeted antigen vaccine alone may cause immune tolerance. In contrast DC that have matured are able to induce potent effector T cell responses to the targeted antigen. DC can be activated by TLR stimulation and therefore combining TLR stimulated DC maturation with a DC-targeted antigen would result in optimal T cell mediated immunity. In theory the DC-targeted vaccine could never be given without a DC maturing adjuvant and the specific effects and actions of GLA would not be known unless it was studied first in isolation. To plan an eventual DC-targeted HIV vaccine trial with an adjuvant, it will also be critical to know the temporal immune effects of isolated GLA.