We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.
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.
BackgroundAdjuvants 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 methodsIn 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.ResultsWe 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.ConclusionThese efforts will aid in the development of a platform formulation for use with other similar vaccines.
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.
Effective in vitro evaluation of vaccine adjuvants would allow higher throughput screening compared to in vivo studies. However, vaccine adjuvants comprise a wide range of structures and formulations ranging from soluble TLR agonists to complex lipid-based formulations. The effects of formulation parameters on in vitro bioactivity assays and the correlations with in vivo adjuvant activity is not well understood. In the present work, we employ the Limulus amebocyte lysate assay and a human macrophage cellular cytokine production assay to demonstrate the differences in in vitro bioactivity of four distinct formulations of the synthetic TLR4 agonist GLA: an aqueous nanosuspension (GLA-AF), an oil-in-water emulsion (GLA-SE), a liposome (GLA-LS), and an alum-adsorbed formulation (GLA-Alum). Furthermore, we demonstrate the importance of the localization of GLA on in vitro potency. By comparing to previous published reports on the in vivo bioactivity of these GLA-containing formulations, we conclude that the most potent activators of the in vitro systems may not be the most potent in vivo adjuvant formulations. Furthermore, we discuss the formulation considerations which should be taken into account when interpreting data from in vitro adjuvant activity assays.
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