The Therapeutic Vaccine Potential of MIS416 Adjuvant

Abstract

There is accelerating interest in the use of nonspecific immunostimulants or adjuvants as a means of enhancing or inducing nonspecific immunity. A further requirement for the development of successful therapeutic vaccines is the upregulation or induction of intrinsic innate immune defenses that have the capacity to destroy the infectious agent or tumor cell directly, or to synergize with existing cytotoxic or immunologic treatment regimens. This article discusses the potential of MIS416 adjuvant, a novel vaccine adjuvant and immunogen co-delivery system, to fulfill this requirement.

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There is a great need for the development of therapeutic vaccines for patients with cancer or acquired chronic infections, such as HIV, hepatitis, tuberculosis, and malaria. Therapeutic vaccines aim to enhance the inherent capability of the patient's immune system to fight the disease, leading to a reduction in severity or complete elimination of the disease. To date, developments have fallen short of expectations. This is caused, in part, by the failure of chronically infected or immunosuppressed patients to respond adequately to the vaccine adjuvant component, which itself may not have the appropriate breadth of immunostimulatory activity.

A requirement underpinning the successful development of prophylactic and therapeutic vaccines lies in the ability to target adjuvant or antigen complexes to dendritic cells with the simultaneous induction of a coordinated and targeted inflammatory response in the secondary lymphoid tissues. In addition to providing adequate immunostimulation to overcome host factors that may limit the success of therapeutic vaccines, we believe a further requirement for the development of successful therapeutic vaccines is the upregulation or induction of intrinsic innate immune defenses that have the capacity to destroy the infectious agent or tumor cell directly, or to synergize with existing cytotoxic or immunologic treatment regimens.

MIS416 adjuvant, a novel vaccine adjuvant and immunogen co-delivery system designed by Virionyx Corporation, is well positioned to fulfill this requirement, acting to induce anti-infective mechanisms such as the production of anti-viral IFN-α, and activation of oxidative microbiocidal pathways, in addition to the activation of broad spectrum anti-tumor responses. MIS416 adjuvant-mediated co-stimulation of dendritic and accessory cell immune crosstalk, concomitant with MIS416-enhanced anti-infective or anticancer cytotoxic activity is likely to facilitate uptake and presentation of autologous viral, bacterial, or tumor antigens, enhancing the generation of vaccine induced immunity. The additional activation of broad spectrum host immune defenses by MIS416 vaccine adjuvant therefore presents an exciting strategy for developing a broad range of therapeutic vaccines.

Immunoadjuvant Platform

MIS416 comprises multiple immunostimulatory TLR9 and NOD-2 ligands that are cross linked in a manner that also permits the covalent attachment of a broad range of immunogens (Figure 1). MIS416 vaccine candidates incorporating a broad range of immunogens have been shown to act in a well-defined manner and have demonstrated significant potency for induction of protective, long-lived humoral immunity, and cellular adaptive immunity with no adverse events noted, even following multiple immunizations.

Figure 1. Graphical representation of MIS416. MIS416 is a semi-synthetic, nontoxic, nonimmunogenic, cage structured microparticle (2 microns) composed of muramyl dipeptide repeats covalently attached to a poly amino acid backbone principally composed of lysine and glycine amino acid repeats. N-acetyl glucosamine is incorporated as an oxidizable carbohydrate for use in linking amino containing ligands. Bacterial DNA is incorporated through amino linkages covalently attached to the poly amino backbone. Microparticle components are isolated from the cell wall of Propionibacterium acnes and ligands or immunogens are produced synthetically with recombinant technology or isolated from natural sources to produce the desired vaccine formulation or activate the desired immune pathway. Insertion and covalent linkage of ligands or immunogens is achieved using bifunctional cross linkers, which link through primary and secondary amino groups present in the microparticle or through carbohydrate moieties following oxidation. (IMAGE CREDIT: RUSSELL KIGHTLEY MEDIA)

Activating Host Antimicrobial Defense Mechanisms

The release of inflammatory mediators by first-line immune cells is followed by cell activation. These mediators act, in part, through autocrine and paracrine signaling loops to enhance innate immune activities, such as phagocytosis and intracellular killing. A key example of this is the ability of MIS416 to induce reactive oxygen species (ROS) and nitrogen monoxide (NO) production by monocytes and granulocytes (Figure 2).

Figure 2. MIS416 is a potent activator of reactive oxygen species and nitric monoxide in granulocytes and monocytes. (A) Human blood was cultured with 10, 5, or 1 µg/mL MIS416 for three hours at 37°C. Cells were loaded with 1 µM of cell permeant fluorescent ROS indicator 3´-(p-aminophenyl) fluorescein (APF) for the last 30 minutes. Reactions were quenched by the addition of excess ice-cold 1.5 mM EDTA and cells were labelled with anti-CD14-PerCP for identification of monocytes and PI viability dye (1 µg/mL). Cells were analyzed by flow cytometry of gated viable monocytes and granulocytes and the percent of cells expressing upregulated APF fluorescence was determined. (B) Whole diluted blood was incubated for two hours with 10, 5, or 1 µg/mL MIS416. 5 µM nitric oxide (NO) cell permeant indicator dye (4-amino-5-methylamino-2, 7-difluorofluorescein diacetate; DAF-FM) was added and cells incubated for a further 30 minutes to allow for dye loading. Cells were then washed and re-incubated for 20 minutes at 37°C to allow for de-esterification fo the dye, then samples were analyzed by flow cytometry. The percent of cells showing DAF-FM positive fluorescence are indicated.

The functional potential of MIS416 microbiocidal activity has been demonstrated in a mouse therapeutic vaccine model studying the ability of MIS416 and Titermax adjuvant to induce adaptive immunity against drug resistant Mycobacterium tuberculosis (MTb) and reduce the infectious bioburden (Figure 3). Immunization of mice three times over a period of 14 days led to a significant reduction of infectious MTb in the MIS416-treated group, which was greater than that observed in the Titermax treated group.

Figure 3. MIS416 enhances the immunogenicity of ESAT-6 and clearance of drug-resistant M. tuberculosis in mice. C57Bl/6 mice were intravenously infected with drug-resistant strain of MTb at 105 CFU per mouse and infection allowed to progress for 14 days. Mice were then treated with 25 µg per mouse of recombinant MTb ESAT-6 protein in either Titermax adjuvant, MIS416 adjuvant, or no adjuvant, given on days 14, 18, and 21 (3 doses per mouse). Mice were sacrificed on day 28 and lung CFU counts performed by plating organ homogenates on 7H11 agar. 3-mice per time point were analyzed (* denotes significance by t test).

Activating Host Antiviral Defenses

Several innate immune responses are considered to be able to contribute to the control of viral infections. These effector mechanisms are multifaceted and include direct antiviral activities and immunomodulatory effects on infected host immune cells that contribute to the elimination of these cells. Direct antiviral activity may comprise soluble factors, such as CD8 antiviral factor (CAF) and IFN-α, which have the capacity to directly affect viral transcription. Although a wide range of other innate cytokines can mediate biologic functions regulating aspects of antiviral immunity, high levels of IFN-α/β appear to be dominant in the context of viral infections and act to regulate other innate responses. The clinical use of IFN-α in treating a variety of viral disorders such as chronic hepatitis B and C, and a broad range of human cancers, lies in its ability to induce a dominant array of antiviral genes that drive pleiotropic host defense pathways that prevent viral replication. Virionyx's MIS416 has been designed to induce both high levels of IFN-α and other key pro-inflammatory cytokines that are clinically relevant to the induction of broad-spectrum innate antiviral immunity. Plasamocytoid dendritic cells demonstrate potent induction of IFN-α following MIS416 uptake (Figure 4). Preclinical studies demonstrate MIS416 is able to inhibit mortality and ameliorate morbidity in a mouse influenza A model when administered following viral exposure (Figure 5).

Figure 4. MIS416 induces pDC IFN-α production. Human pDCs were purified from PBMCs using magnetic bead selection of BDCA-2+ cells to high purity and viability. Sorted pDC were cultured at 2.6 x 105 cells/mL in the presence of assay positive control (TLR9 ligand (CpG A ODN 2216; 1 µm) or a dose-response of MIS416 for 24 hours. Supernatants were assayed for IFN-α content using flow cytometry cytokine bead array methodology.

Activating Broad-Spectrum, Tumor-Killing Mechanisms

Therapeutic vaccines administered to cancer patients are designed to treat cancer by stimulating the immune system to recognize and attack human cancer cells without harming normal cells, and to overcome the immune system's tolerance to cancer antigens.

Figure 5. MIS416 therapy suppresses weight loss and mortality after infection with type A influenza. (A) C57BL/6 mice (n = 10 per group) were infected with 0.3 LD-50 influenza A strain A/PR/8/34 (H1N1). The following day, animals received vehicle control (saline), 50 µg MIS416, or 250 µg of MIS416 intravenously. Weights were monitored daily. Where indicated (*), MIS416 therapy significantly reduced weight loss. (B) Mice were infected as described above. Animals that lost more than 30% of their initial weight were considered moribund and euthanized. Data are pooled from two independent experiments (n = 15 total mice per group). The reduced morbidity of animals treated with MIS416 was statistically significant (p = 0.035 by Log rank test).

Figure 6. Enhancement of purified human NK spontaneous killing activity following stimulation with MIS416. Human CD56+ cells were purified from whole blood to 99% and cultured for 40 hours with no stimulus, known NK activating agents IL-2 (500 U/mL) and IL-12 (50 ng/mL) or with MIS416 (40, 20, 10, and 5 µg/mL). Stimulated NK cells were subsequently tested for cytotoxicity against fluorescently labelled NK sensitive K562 tumor targets at effector:target ratios of 5:1, 2:1, and 1:1. Tumor cell killing was determined after four hours by flow cytometric analysis of viability dye uptake (propidium iodide) of gated, fluorescent K562 targets.

Tumor-specific killing that results in the induction of apoptosis leads to clearance of these cells through the phagocytic pathway, resulting in uptake and cross-presentation of apoptotic tumor cell antigens by dendritic cells, which is required for the development of cytotoxic CD8+ antitumor responses. The induction of antitumor activity by immunostimulatory compounds is a clinically relevant therapeutic approach to treating neoplastic disease, as is demonstrated by the usefulness of BCG in preventing recurrence of bladder cancer that has been surgically removed at early stages of cancer development. Although natural killer (NK) cells play a central role in tumor cell surveillance and destruction, NKTs and monocytes or macrophages represent additional nonredundant arms of innate antitumor responses. Together these cell subsets are known to kill tumour targets by several mechanisms including, but not limited to, granule, FAS L and tumor necrosis factor-alpha (TNFα)-mediated pathways. MIS416 activates both NK cell granule-mediated cytotoxicity (Figure 6) and the secretion of cytotoxic soluble factors such as FAS L (Figure 7). When MIS416-stimulated pooled NK/NKT cells are tested against a range of tumors that have known differential sensitivity to these mechanisms, reliable killing is detected (Figure 8).

Figure 7. Induction of FAS L secretion by MIS416 stimulated NK/NKT cells. Human NK and NKT cells were purified from whole blood to 99% and cultured for 24 hours with no stimulus, known NK/NKT activating agents IL-2 (500 U/mL) and IL-12 (50 ng/mL), or with MIS416 (20, 10, 5, and 1 µg/mL). Cell-free supernatants were assayed for FAS L content using flow cytometric bead array technology.

Figure 8. MIS416 activates PBMC NK granule and FAS L cytotoxicity. MIS416-activated PBMC were cultured with MIS416 at 20 and 5 µg/ml. Known NK cell-activating agents, IL-2 (500 U/ml), IL-12 (50 ng/mL), and TLR3 ligand, poly I:C (50 µg/ml) served as assay positive controls. Following a 46-hour culture, activated PBMCs were then washed into fresh medium and tested for cytotoxicity against fluorescently labelled tumor target cells at an effector:target ratio of 100:1. Tumor cell killing was determined after four hours by flow cytometric determination of viability dye uptake live/dead discrimination of gated, fluorescent tumor targets. It can be seen that MIS416 microparticle enhances human PBMC spontaneous killing activity against NK sensitive K562 (erythroleukemia) and DU-145 (prostate), as well as FAS L sensitive Daudi (Burkitt lymphoma) and T47D (breast) tumor cell targets.

Summary

The molecular and cellular basis for adjuvancy and immune-boosting therapeutics is overlapping. There is accelerating interest in the use of nonspecific immunostimulants or adjuvants as a means of enhancing or inducing nonspecific immunity, which is a key to successful therapeutic vaccine development. The rationale behind this approach exploits several aspects associated with the innate immune system such as its rapid activation, the diverse activated array of broad-spectrum innate defenses are less likely to suffer the development of resistance, and the coherent activation of innate immune responses promotes normal activation of adaptive immune responses, which prevents or limits recurrent or progressive infections. The potential of the innate immune system as a therapeutic target has been demonstrated in several infectious and cancer models using TLR based and nonTLR-based approaches of selectively boosting the innate antiviral and antibacterial immune responses¹ . As such, incorporation of this activity into vaccine designs is highly desirable from a therapeutic vaccine standpoint, and MIS416 is well placed to fulfill this role.

Gill A. Webster, PhD, is chief scientific officer and Frank B. Gelder, PhD, is founding scientist at Virionyx Corporation Ltd., Auckland, New Zealand, +64 (9) 6362500, g.webster@virionyx.com

REFERENCES

1. Brown KL, Cosseau C, Gardy JL, Hancock REW. Complexities of Targeting Innate Immunity to Treat Infection. Trends in Immun. 2007;28:6.