Ryan M. Kramer

Working together: interactions between vaccine antigens and adjuvants

The development of vaccines containing adjuvants has the potential to enhance antibody and cellular immune responses, broaden protective immunity against heterogeneous pathogen strains, enable antigen dose sparing, and facilitate efficacy in immunocompromised populations. Nevertheless, the structural interplay between antigen and adjuvant components is often not taken into account in the published literature. Interactions between antigen and adjuvant formulations should be well characterized to enable optimum vaccine stability and efficacy. This review focuses on the importance of characterizing antigen–adjuvant interactions by summarizing findings involving widely used adjuvant formulation platforms, such as aluminum salts, emulsions, lipid vesicles, and polymer-based particles. Emphasis is placed on the physicochemical basis of antigen–adjuvant associations and the appropriate analytical tools for their characterization, as well as discussing the effects of these interactions on vaccine potency.

Elimination of the cold-chain dependence of a nanoemulsion adjuvanted vaccine against tuberculosis by lyophilization

Next-generation rationally-designed vaccine adjuvants represent a significant breakthrough to enable development of vaccines against challenging diseases including tuberculosis, HIV, and malaria. New vaccine candidates often require maintenance of a cold-chain process to ensure long-term stability and separate vials to enable bedside mixing of antigen and adjuvant. This presents a significant financial and technological barrier to worldwide implementation of such vaccines. Herein we describe the development and characterization of a tuberculosis vaccine comprised of both antigen and adjuvant components that are stable in a single vial at sustained elevated temperatures. Further this vaccine retains the ability to elicit both antibody and TH1 responses against the vaccine antigen and protect against experimental challenge with Mycobacterium tuberculosis. These results represent a significant breakthrough in the development of vaccine candidates that can be implemented throughout the world without being hampered by the necessity of a continuous cold chain or separate adjuvant and antigen vials.

Modulating Potency: Physicochemical Characteristics are a Determining Factor of TLR4‐Agonist Nanosuspension Activity

Activity of adjuvanted vaccines is difficult to predict in vitro and in vivo. The wide compositional and conformational range of formulated adjuvants, from aluminum salts to oil-in-water emulsions, makes comparisons between physicochemical and immunological properties difficult. Even within a formulated adjuvant class, excipient selection and concentration can alter potency and physicochemical properties of the mixture. Complete characterization of physicochemical properties of adjuvanted vaccine formulations and relationship to biological response is necessary to move beyond a guess-and-check paradigm toward directed development. Here we present a careful physicochemical characterization of a two-component nanosuspension containing synthetic TLR-4 agonist glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at various molar ratios. Physicochemical properties were compared with potency, as measured by stimulation of cytokine production in human whole blood. We found a surprising, nonlinear relationship between physicochemical properties and GLA-DPPC ratios that corresponded well with changes in biological activity. We discuss these data in light of the current understanding of TLR4 activation and the conformation-potency relationship in development of adjuvanted vaccines.

Particle Sizing of Nanoparticle Adjuvant Formulations by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA)

Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are two orthogonal and complementary methods of measuring size of particles in a sample. These technologies use the theory of Brownian motion by analyzing the random changes of light intensity scattered by particles in solution. Both techniques can be used to characterize particle size distribution of proteins and formulations in the nanometer to low micron range.Each method has benefits over the other. DLS is a quick and simple measurement that is ideal for monodisperse particles and can also analyze a distribution of particles over a wide range of sizes. NTA provides a size distribution that is less susceptible to the influence of a few large particles, and has the added benefit of being able to measure particle concentration. Here we describe methods for measuring the particle size and concentration of an oil-in-water nanoemulsion.

Quantitative Measurement of Toll-like Receptor 4 Agonists Adsorbed to Alhydrogel® by Fourier Transform Infrared-Attenuated Total Reflectance Spectroscopy

Aluminum salts have a long history as safe and effective vaccine adjuvants. In addition, aluminum salts have high adsorptive capacities for vaccine antigens and adjuvant molecules, for example, Toll-like receptor 4 (TLR4) agonists. However, the physicochemical properties of aluminum salts make direct quantitation of adsorbed molecules challenging. Typical methods for quantifying adsorbed molecules require advanced instrumentation, extreme sample processing, often destroy the sample, or rely on an indirect measurement. A simple, direct, and quantitative method for analysis of adsorbed adjuvant molecules is needed. This report presents a method utilizing Fourier transform infrared spectroscopy with a ZnSe-attenuated total reflectance attachment to directly measure low levels (<30 μg/mL) of TLR4 agonists adsorbed on aluminum salts with minimal sample preparation.

Effective Combination Adjuvants Engage Both TLR and Inflammasome Pathways To Promote Potent Adaptive Immune Responses

The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain–containing adapter inducing IFN-β are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1β are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC−/− animals. Importantly, the early proliferation of Ag-specific CD4+ T cells was completely ablated after immunization in ASC−/− animals. Moreover, numbers of Ag-specific CD4+ T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC−/−, NLRP3−/−, and IL-1R−/− mice compared with wild-type mice and were completely ablated in TLR4−/− animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4+ T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.

Staining and Transfer Techniques for SDS-PAGE Gels to Minimize Oil-in-Water Emulsion Adjuvant Interference

Adjuvants in modern vaccines boost and shape immune responses and allow for antigen dose-sparing. Analysis of protein antigens in the presence of adjuvants can prove challenging, especially if the adjuvant interferes with visualization of the protein band on an SDS-PAGE gel. In this chapter, a variety of different techniques are presented to mitigate the interference of a nanoemulsion adjuvant, GLA-SE, with different recombinant proteins of varying molecular weight by addressing sample preparation and staining methods.

Lyophilization of Adjuvanted Vaccines: Methods for Formulation of a Thermostable Freeze-Dried Product.

Lyophilization of vaccines is advantageous for the distribution and storage of thermally labile products, particularly in regions where cold chain management is difficult. To date, current lyophilized vaccines do not contain an adjuvant. Instead, adjuvanted vaccines may be presented as a two vial system, that require bedside-mixing prior to immunization. Here we present an example of a lyophilization cycle that we have used to successfully freeze-dry an adjuvanted protein formulation in a single vial.

Lyophilization of an Adjuvanted Mycobacterium tuberculosis Vaccine in a Single-Chamber Pharmaceutical Cartridge

Although substantial effort has been made in the development of next-generation recombinant vaccine systems, maintenance of a cold chain is still typically required and remains a critical challenge in effective vaccine distribution. The ability to engineer alternative containment systems that improve distribution and administration represents potentially significant enhancements to vaccination strategies. In this work, we evaluate the ability to successfully lyophilize a previously demonstrated thermostable tuberculosis vaccine formulation (ID93 + GLA-SE) in a cartridge format compared to a traditional vial container format. Due to differences in the shape of the container formats, a novel apparatus was developed to facilitate lyophilization in a cartridge. Following lyophilization, the lyophilizate was assessed visually, by determining residual moisture content, and by collecting melting profiles. Reconstituted formulations were assayed for particle size, protein presence, and GLA content. Based on assessment of the lyophilizate, the multicomponent vaccine was successfully lyophilized in both formats. Also, the physicochemical properties of the major components in the formulation, including antigen and adjuvant, were retained after lyophilization in either format. Ultimately, this study demonstrates that complex formulations can be lyophilized in alternative container formats to the standard pharmaceutical glass vial, potentially helping to increase the distribution of vaccines.

Development of a thermostable nanoemulsion adjuvanted vaccine against tuberculosis using a design-of-experiments approach

Background Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). Materials and methods In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. Results We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. Conclusion These efforts will aid in the development of a platform formulation for use with other similar vaccines.