The Effects of GLA on Human Volunteers
Condition: Healthy Volunteers
Study type: Interventional
Phase: Phase 1

Summary: 
      The advent of vaccines contributed to major improvements in human morbidity and mortality due
      to infectious diseases such as polio, small pox, measles and diphtheria. However infectious
      diseases like HIV, malaria and tuberculosis continue to be major causes of death worldwide
      and conventional vaccine strategies have not been successful. The fundamental problem is that
      current protein based vaccines do not elicit the necessary T-cell immunity. Experimentally,
      adjuvants can be given in conjunction with a vaccine to activate and mature the dendritic
      cell (DC), which can then direct an immune response to enhance T-cell immunity. One family of
      potential adjuvants functions through the activation of Toll-like receptors (TLR) on the DC.
      Major gaps exist in our understanding of adjuvant effects in humans. We hypothesize that a
      synthetic adjuvant directed to activate TLR4 (GLA) will safely stimulate the innate immune
      system when administered subcutaneously (SC) or intramuscularly (IM). Importantly, in
      contrast to other adjuvant trials in which adjuvant is combined with an antigen or vaccine,
      GLA will be tested in isolation. This is because we anticipate the future administration of
      GLA with our dendritic cell targeted HIV vaccine. A DC-targeted vaccine cannot be given
      without an immune stimulating adjuvant due to potential risk of inducing immune tolerance.
      Therefore, in order to understand the specific contributions of GLA versus the DC-targeted
      vaccine, we need to understand the GLA effects in isolation. The safety and tolerability of 2
      different formulations of GLA (GLA-SE vs. GLA-AF) administered by 3 different routes (SC, ID,
      IM) will be the major focus of this trial. The second focus will be characterizing the innate
      immune response by assessing systemic cytokine and chemokine levels and determining global
      gene regulation following GLA stimulation. The third focus will be on the cellular effects of
      GLA, specifically on blood monocytes and dendritic cells. Monocytes may represent a large
      pool of inducible potent DC (monocyte-derived DC), however these cells have not been well
      characterized in humans. We will investigate the effects of GLA stimulation on the peripheral
      blood monocyte subsets that might give rise to monocyte-derived DC.
    

Detailed description: 
      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.

      GLA Background Information GLA is a new completely synthetic lipid A (active component of
      natural LPS) molecule that combines 6 acyl chains with a single phosphorylation site. The
      advantage of this LPS-like molecule is that it is not purified from a bacterial source
      thereby allowing a homogenous solution only containing molecules with 6 acyl chains. This is
      important because 6 acyl chains results in maximal TLR4 activation while lipid A molecules
      with 5 or 7 acyl chains are 100 times less active and 4 acyl chains is immuno-inhibitory. MPL
      is an existing TLR4 vaccine adjuvant that is derived from salmonella LPS and represents a
      heterogeneous mixture of 3, 4, 5 and 6 acylated molecules. MPL induces effective humoral
      antibody response during vaccination, but it does not generate CD4 T-cell immunity, making it
      an undesirable adjuvant for many protein-based vaccines. In animal models GLA added to the
      Fluzone vaccine improves humoral and cellular immunity to influenza. GLA has been tested in
      humans for safety in combination with an influenza vaccine however its individual effects
      have not been studied. Particularly the isolated effects on cytokine, chemokine and gene
      regulation in humans is not known and monocytes subsets were not previously investigated.

      The optimal formulation and route of administration are not known; therefore a major
      component of this investigation will be to determine a safe and tolerable formulation and
      route. GLA has been administered to humans in combination with the Fluzone vaccine ranging in
      doses from 0.5μg to 5μg intramuscularly; however it has not been tested in isolation, and
      only the oil-in-water stable emulsion (GLA-SE) was used. Fluzone is a reactogenic vaccine and
      dose-limiting toxicity did occur in 3 of 4 patients who received Fluzone + high dose GLA-SE
      (5μg), but a good safety profile was observed between 0.5-2.5μg. The stable emulsion (SE)
      contains squalene and was used at 2% (vol:vol). We will continue to use this percentage in
      our GLA-SE formulation. A second formulation of GLA has subsequently been developed to avoid
      the need for an emulsion; GLA-AF is aqueous and should theoretically have a reduced
      side-effect profile and less reactogenicity than GLA-SE. We plan to compare the two
      formulations of GLA in isolation; GLA-SE (stable emulsion) and GLA-AF (aqueous formulation).
      GLA-AF has never been administered to humans and therefore this version represents a
      first-in-human trial. For safety reasons GLA-AF injections must be separated by a day and
      since it will not be possible to know who will receive GLA-AF, we will inject the first nine
      subjects in each cohort one day apart. Due to the complexity of the number of groups in this
      study, we will enroll and complete each route sequentially as a cohort. The subcutaneous
      cohort will be the first group screened, enrolled, randomized and injected, followed by
      intramuscular.

      Summary This study will be a phase-1, randomized, placebo-controlled, double-blinded clinical
      trial to compare GLA formulation and administration route in humans. Safety and tolerability
      of the different formulations (GLA-SE vs. GLA-AF) and routes (SC, IM, ID) will be the primary
      focus. Our second focus will be to detail the global immune response be measuring systemic
      cytokines, chemokines and global gene regulation. The third focus will be to investigate the
      effects of GLA on the peripheral blood immune cells including monocytes and dendritic cells.
      This study will add first-in-human data on GLA-AF and detail the isolated effects of GLA on
      the innate immune system. The results of this trial will lay the foundation for a true
      adjuvant trial using GLA + antigen and ultimately to use GLA to adjuvant our DC-targeted
      vaccine in humans.
    

Intervention name(s): GLA-AF; GLA-SE; Squalene
Intervention type(s): Drug; Drug; Other
Intervention description(s): GLA-AF contains GLA, a new synthetic lipid A molecule that combines 6 acyl chains with a single phosphorylation site. GLA-AF contains GLA in an aqueous solution. One 2 mcg injection will be given per patient in the upper arm, each randomized to either subcutaneous or intramuscular routes.; GLA-SE contains GLA, a new synthetic lipid A molecule that combines 6 acyl chains with a single phosphorylation site. GLA-SE contains GLA in a squalene oil emulsion. One 2 mcg injection will be given per patient in the upper arm, each randomized to either subcutaneous or intramuscular routes.; Squalene is a natural organic compound obtained from shark liver oil. In this study, it is used to solubilize GLA in the GLA-SE formulation. Patients randomized to receive the squalene will be given one 2mcg injection of the squalene oil in the upper arm, each patient randomized further to either subcutaneous or intramuscular routes.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Basic Science