Christopher B. Fox

Key roles of adjuvants in modern vaccines

Vaccines containing novel adjuvant formulations are increasingly reaching advanced development and licensing stages, providing new tools to fill previously unmet clinical needs. However, many adjuvants fail during product development owing to factors such as manufacturability, stability, lack of effectiveness, unacceptable levels of tolerability or safety concerns. This Review outlines the potential benefits of adjuvants in current and future vaccines and describes the importance of formulation and mechanisms of action of adjuvants. Moreover, we emphasize safety considerations and other crucial aspects in the clinical development of effective adjuvants that will help facilitate effective next-generation vaccines against devastating infectious diseases.

Use of defined TLR ligands as adjuvants within human vaccines

Summary:  Our improved understanding of how innate immune responses can be initiated and how they can shape adaptive B‐ and T‐cell responses is having a significant impact on vaccine development by directing the development of defined adjuvants. Experience with first generation vaccines, as well as rapid advances in developing defined vaccines containing Toll‐like receptor ligands (TLRLs), indicate that an expanded number of safe and effective vaccines containing such molecules will be available in the future. In this review, we outline current knowledge regarding TLRs, detailing the different cell types that express TLRs, the various signaling pathways TLRs utilize, and the currently known TLRLs. We then discuss the current status of TLRLs within vaccine development programs, including the importance of appropriate formulation, and how recent developments can be used to better define the mechanisms of action of vaccines. Finally, we introduce the possibility of using TLRLs, either in combination or with non‐TLRLs, to synergistically potentiate vaccine‐induced responses to provide not only prophylactic, but therapeutic protection against infectious diseases and cancer.

Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant

Innate immune responses to vaccine adjuvants based on lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, are driven by Toll-like receptor (TLR) 4 and adaptor proteins including MyD88 and TRIF, leading to the production of inflammatory cytokines, type I interferons, and chemokines. We report here on the characterization of a synthetic hexaacylated lipid A derivative, denoted as glucopyranosyl lipid adjuvant (GLA). We assessed the effects of GLA on murine and human dendritic cells (DC) by combining microarray, mRNA and protein multiplex assays and flow cytometry analyses. We demonstrate that GLA has multifunctional immunomodulatory activity similar to naturally-derived monophosphory lipid A (MPL) on murine DC, including the production of inflammatory cytokines, chemokines, DC maturation and antigen-presenting functions. In contrast, hexaacylated GLA was overall more potent on a molar basis than heterogeneous MPL when tested on human DC and peripheral blood mononuclear cells (PBMC). When administered in vivo, GLA enhanced the immunogenicity of co-administered recombinant antigens, producing strong cell-mediated immunity and a qualitative TH1 response. We conclude that the GLA adjuvant stimulates and directs innate and adaptive immune responses by inducing DC maturation and the concomitant release of pro-inflammatory cytokines and chemokines associated with immune cell trafficking, activities which have important implications for the development of future vaccine adjuvants.

Squalene Emulsions for Parenteral Vaccine and Drug Delivery

Squalene is a linear triterpene that is extensively utilized as a principal component of parenteral emulsions for drug and vaccine delivery. In this review, the chemical structure and sources of squalene are presented. Moreover, the physicochemical and biological properties of squalene-containing emulsions are evaluated in the context of parenteral formulations. Historical and current parenteral emulsion products containing squalene or squalane are discussed. The safety of squalene-based products is also addressed. Finally, analytical techniques for characterization of squalene emulsions are examined.

Physicochemical characterization and biological activity of synthetic TLR4 agonist formulations

Immunostimulatory molecules such as monophosphoryl lipid A (MPL), a Toll-like receptor 4 (TLR4) agonist, can be formulated to enhance vaccine adjuvant effects and to promote a Th1-type immune response. This study compares the in vitro and in vivo potency of aqueous and emulsion formulations containing a synthetic MPL analogue. In addition, formulation structure and association of the synthetic TLR-4 agonist and antigen with the formulation are characterized using dynamic light scattering, zeta potential measurement, HPLC, and SDS-PAGE. The biological and biophysical effects of formulating the agonist with different oil and surfactant components from animal, plant, and synthetic sources are examined. These findings have important implications for the formulation of TLR4 agonists as well as the influence of formulation component substitution on adjuvant activity. The results indicate that (1) the agonist is associated with the oil droplets in emulsion formulations, (2) the emulsion formulations containing synthetic TLR4 agonist induce higher IgG2a/IgG1 antibody ratios than aqueous formulations or an emulsion formulation without the agonist, and (3) appropriate plant-derived components can be substituted for animal-derived components in oil-in-water emulsions without loss of biological activity.

New generation adjuvants – From empiricism to rational design

Adjuvants are an essential component of modern vaccine development. Despite many decades of development, only a few types of adjuvants are currently included in vaccines approved for human use. In order to better understand the reasons that development of some adjuvants succeeded while many others failed, we discuss some of the common attributes of successful first generation adjuvants. Next, we evaluate current trends in the development of second generation adjuvants, including the potential advantages of rationally designed synthetic immune potentiators appropriately formulated. Finally, we discuss desirable attributes of next generation adjuvants. Throughout, we emphasize that the importance of formulation and analytical characterization in all aspects of vaccine adjuvant development is often underappreciated. We highlight the formulation factors that must be evaluated in order to optimize interactions between vaccine antigens, immune potentiators, and particulate formulations, and the resulting effects on safety, biological activity, manufacturability, and stability.

An update on safety and immunogenicity of vaccines containing emulsion-based adjuvants.

With the exception of alum, emulsion-based vaccine adjuvants have been administered to far more people than any other adjuvant, especially since the 2009 H1N1 influenza pandemic. The number of clinical safety and immunogenicity evaluations of vaccines containing emulsion adjuvants has correspondingly mushroomed. In this review, the authors introduce emulsion adjuvant composition and history before detailing the most recent findings from clinical and postmarketing data regarding the effects of emulsion adjuvants on vaccine immunogenicity and safety, with emphasis on the most widely distributed emulsion adjuvants, MF59® and AS03. The authors also present a summary of other emulsion adjuvants in clinical development and indicate promising avenues for future emulsion-based adjuvant development. Overall, emulsion adjuvants have demonstrated potent adjuvant activity across a number of disease indications along with acceptable safety profiles.

Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens.

The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.

Monitoring the effects of component structure and source on formulation stability and adjuvant activity of oil-in-water emulsions

Oil-in-water emulsions have shown promise as safe and effective adjuvant formulations for vaccines. In particular, formulations consisting of metabolizable oils such as shark-derived squalene and detergents such as egg phosphatidylcholine have been used to produce stable vaccine emulsion formulations. However, there is an emphasis in pharmaceutical regulatory bodies on using synthetic or plant-derived components from sustainable sources instead of animal-derived components. This study compares the physicochemical properties and biological efficacy of emulsions consisting of oil and detergent components from animal, plant, and synthetic sources. In particular, effects of component structure and source on emulsion stability and biological activity are examined. It is shown that oil-in-water emulsions using animal-derived components can be substituted with synthetic or plant-derived materials while still exhibiting satisfactory physicochemical and biological properties.

Adjuvant formulation structure and composition are critical for the development of an effective vaccine against tuberculosis

One third of the world is infected with Mycobacterium tuberculosis (Mtb) with eight million new cases of active tuberculosis (TB) each year. Development of a new vaccine to augment or replace the only approved TB vaccine, BCG, is needed to control this disease. Mtb infection is primarily controlled by TH1 cells through the production of IFN-γ and TNF which activate infected macrophages to kill the bacterium. Here we examine an array of adjuvant formulations containing the TLR4 agonist GLA to identify candidate adjuvants to pair with ID93, a lead TB vaccine antigen, to elicit protective TH1 responses. We evaluate a variety of adjuvant formulations including alum, liposomes, and oil-in-water emulsions to determine how changes in formulation composition alter adjuvant activity. We find that alum and an aqueous nanosuspension of GLA synergize to enhance generation of ID93-specific TH1 responses, whereas neither on their own are effective adjuvants for generation of ID93-specific TH1 responses. For GLA containing oil-in-water emulsions, the selection of the oil component is critical for adjuvant activity, whereas a variety of lipid components may be used in liposomal formulations of GLA. The composition of the liposome formulation of ID93/GLA does alter the magnitude of the TH1 response. These results demonstrate that there are multiple solutions for an effective formulation of a novel TB vaccine candidate that enhances both TH1 generation and protective efficacy.