The Effects of GLA on Human Volunteers
The Effects of GLA on Human Volunteers
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.
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.
Inclusion Criteria:

 1. Healthy adult males and females, as assessed by a medical history, physical exam, and
 laboratory tests

 2. Age of at least 18 years of age on the day of screening and no greater than 60 years
 at time of administration

 3. Willing to comply with the requirements of the protocol and available for follow-up
 for the planned duration of the study (screening plus 4 weeks)

 4. Willing to undergo HIV testing and counseling and receive HIV test results

 5. If a female of child bearing potential, must be willing to use two effective methods
 of contraception (combined oral contraceptive pill; injectable contraceptive;
 diaphragm; Intra Uterine Device (IUD); condoms; anatomical sterility in self or
 partner) throughout until 6 weeks after study drug administration. If a sexually
 active male, must be willing to use two effective methods of contraception (such as
 condoms, anatomical sterility) from screening until 6 weeks after study drug
 administration (same as above) and will be advised not to get his partner(s) pregnant
 during this time.

 Exclusion Criteria:

 1. Confirmed HIV-1 or HIV-2 infection

 2. Any clinically significant abnormality on medical history or physical examination
 including history of immunodeficiency or autoimmune disease

 3. Any use of systemic corticosteroids immunosuppressive anticancer medications

 4. Any clinically significant acute or chronic medical condition requiring care of a
 physician (e.g., diabetes, coronary artery disease, rheumatologic illness, malignancy,
 substance abuse) that in the opinion of the investigator would preclude participation

 5. Any laboratory value outside of reference range other than CRP, with the exception of
 any non-clinically significant Grade I elevations of liver function tests (AST, ALT,
 direct/total bilirubin), electrolytes (Na, K, Cl, CO2), CBC, urinalysis as determined
 by the Principal Investigator or his designee.

 6. Within the 12 months prior to enrollment, the subject self reports excessive daily
 alcohol use, frequent binge drinking or chronic marijuana abuse (defined as greater
 than 2 times a week) or any other use of illicit drugs

 7. Positive hepatitis B surface antigen, positive hepatitis C antibodies, or active
 syphilis infection based on clinical evaluation;

 8. If female, pregnant, planning a pregnancy during the trial period, or lactating

 9. Receipt of a live attenuated vaccine within 30 days or other vaccine within 14 days
 prior to study drug

 10. Participation in another clinical study of an investigational product currently or
 within past 12 weeks, or expected participation during this study

 11. In the opinion of the investigator, unlikely to comply with protocol due to medical,
 social or psychiatric reasons

 12. Allergy to eggs

 13. A glomerular filtration rate that is less than 60mL/min/1.73 m2 as calculated by study
 team based on laboratory creatinine values.