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.

Antimicrobial susceptibility testing to evaluate minimum inhibitory concentration values of clinically relevant antibiotics.

Antimicrobial susceptibility testing is used to determine the minimum inhibitory concentration (MIC), the standard measurement of antibiotic activity. Here, we present a protocol for evaluating MIC values of clinically relevant antibiotics against bacterial isolates cultured in standard bacteriologic medium and in mammalian cell culture medium. We describe steps for pathogen identification, culturing bacteria, preparing MIC plates, MIC assay incubation, and determining MIC. This protocol can potentially optimize the use of existing antibiotics while enhancing efforts to discover new ones. For complete details on the use and execution of this protocol, please refer to Heithoff et al.1.

Re-evaluation of FDA-approved antibiotics with increased diagnostic accuracy for assessment of antimicrobial resistance.

Accurate assessment of antibiotic susceptibility is critical for treatment of antimicrobial resistant (AMR) infections. Here, we examine whether antimicrobial susceptibility testing in media more physiologically representative of in vivo conditions improves prediction of clinical outcome relative to standard bacteriologic medium. This analysis reveals that ∼15% of minimum inhibitory concentration (MIC) values obtained in physiologic media predicted a change in susceptibility that crossed a clinical breakpoint used to categorize patient isolates as susceptible or resistant. The activities of antibiotics having discrepant results in different media were evaluated in murine sepsis models. Testing in cell culture medium improves the accuracy by which MIC assays predict in vivo efficacy. This analysis identifies several antibiotics for treatment of AMR infections that standard testing failed to identify and those that are ineffective despite indicated use by standard testing. Methods with increased diagnostic accuracy mitigate the AMR crisis via utilizing existing agents and optimizing drug discovery.

A broad-spectrum synthetic antibiotic that does not evoke bacterial resistance.

BACKGROUND: Antimicrobial resistance (AMR) poses a critical threat to public health and disproportionately affects the health and well-being of persons in low-income and middle-income countries. Our aim was to identify synthetic antimicrobials termed conjugated oligoelectrolytes (COEs) that effectively treated AMR infections and whose structures could be readily modified to address current and anticipated patient needs. METHODS: Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, and each variant was evaluated for broad-spectrum antibacterial activity and for in vitro cytotoxicity in cultured mammalian cells. Antibiotic efficacy was analyzed in murine models of sepsis; in vivo toxicity was evaluated via a blinded study of mouse clinical signs as an outcome of drug treatment. FINDINGS: We identified a compound, COE2-2hexyl, that displayed broad-spectrum antibacterial activity. This compound cured mice infected with clinical bacterial isolates derived from patients with refractory bacteremia and did not evoke bacterial resistance. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to negate bacterial cell viability and the evolution of drug-resistance. Disruption of these bacterial properties may occur through alteration of critical protein-protein or protein-lipid membrane interfaces-a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis. INTERPRETATION: The ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. These COE features enable the construction of a spectrum of compounds with the potential for development as a new versatile therapy for an imminent global health crisis. FUNDING: U.S. Army Research Office, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.

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.

Adjuvanted pandemic influenza vaccine: variation of emulsion components affects stability, antigen structure, and vaccine efficacy.

BACKGROUND: Adjuvant formulations are critical components of modern vaccines based on recombinant proteins, which are often poorly immunogenic without additional immune stimulants. Oil-in-water emulsions comprise an advanced class of vaccine adjuvants that are components of approved seasonal and pandemic influenza vaccines. However, few reports have been published that systematically evaluate the in vitro stability and in vivo adjuvant effects of different emulsion components. OBJECTIVES: To evaluate distinct classes of surfactants, oils, and excipients, for their effects on emulsion particle size stability, antigen structural interactions, and in vivo activity when formulated with a recombinant H5N1 antigen. METHODS: Emulsions were manufactured by high pressure homogenization and characterized alone or in the presence of vaccine antigen by dynamic light scattering, zeta potential, viscosity, pH, hemolytic activity, electron microscopy, fluorescence spectroscopy, and SDS-PAGE. In vivo vaccine activity in the murine model was characterized by measuring antibody titers, antibody-secreting plasma cells, hemagglutination inhibition titers, and cytokine production. RESULTS: We demonstrate that surfactant class and presence of additional excipients are not critical for biological activity, whereas oil structure is crucial. Moreover, we report that simplified two-component emulsions appear more stable by particle size than more complex formulations.Finally, differences in antigen structural interactions with the various emulsions do not appear to correlate with in vivo activity. CONCLUSIONS: Oil-in-water emulsions can significantly enhance antibody and cellular immune responses to a pandemic influenza antigen. The dramatic differences in adjuvant activity between squalene-based emulsion and medium chain triglyceride-based emulsion are due principally to the biological activity of the oil composition rather than physical interactions of the antigen with the emulsion.