Control of Heterologous Simian Immunodeficiency Virus SIVsmE660 Infection by DNA and Protein Coimmunization Regimens Combined with Different Toll-Like-Receptor-4-Based Adjuvants in Macaques.

We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against simian immunodeficiency virus (SIV) infection. To further optimize the DNA-protein coimmunization regimen, we tested a SIVmac251-based vaccine formulated with either of two Toll-like receptor 4 (TLR4) ligand-based liposomal adjuvant formulations (TLR4 plus TLR7 [TLR4+7] or TLR4 plus QS21 [TLR4+QS21]) in macaques. Although both vaccines induced humoral responses of similar magnitudes, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5α-resistant (TRIM-5α R) allele. Vaccinees were preferentially infected by an SIVsmE660 transmitted/founder virus carrying neutralization-resistant A/K mutations at residues 45 and 47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, the delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. The presence of mucosal V1V2 IgG binding antibodies correlated with a significantly decreased risk of virus acquisition in both TRIM-5α R and TRIM-5α-moderate/sensitive (TRIM-5α M/S) animals, although this vaccine effect was more prominent in animals with the TRIM-5α R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T cell responses correlated with viremia control. In conclusion, the combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to a lower risk of infection by heterologous SIV challenge.IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven to be an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccinations applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for a reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition, and genetic makeup (TRIM-5α) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.

A nanoliposome delivery system to synergistically trigger TLR4 AND TLR7.

BACKGROUND: Recent reports that TLR4 and TLR7 ligands can synergistically trigger Th1 biased immune responses suggest that an adjuvant that contains both ligands would be an excellent candidate for co-administration with vaccine antigens for which heavily Th1 biased responses are desired. Ligands of each of these TLRs generally have disparate biochemical properties, however, and straightforward co-formulation may represent an obstacle. RESULTS: We show here that the TLR7 ligand, imiquimod, and the TLR4 ligand, GLA, synergistically trigger responses in human whole blood. We combined these ligands in an anionic liposomal formulation where the TLR7 ligand is in the interior of the liposome and the TLR4 ligand intercalates into the lipid bilayer. The new liposomal formulations are stable for at least a year and have an attractive average particle size of around 140 nm allowing sterile filtration. The synergistic adjuvant biases away from Th2 responses, as seen by significantly reduced IL-5 and enhanced interferon gamma production upon antigen-specific stimulation of cells from immunized mice, than any of the liposomal formulations with only one TLR agonist. Qualitative alterations in antibody responses in mice demonstrate that the adjuvant enhances Th1 adaptive immune responses above any adjuvant containing only a single TLR ligand as well. CONCLUSION: We now have a manufacturable, synergistic TLR4/TLR7 adjuvant that is made with excipients and agonists that are pharmaceutically acceptable and will have a straightforward path into human clinical trials.

Optimizing manufacturing and composition of a TLR4 nanosuspension: physicochemical stability and vaccine adjuvant activity.

BACKGROUND: Nanosuspensions are an important class of delivery system for vaccine adjuvants and drugs. Previously, we developed a nanosuspension consisting of the synthetic TLR4 ligand glucopyranosyl lipid adjuvant (GLA) and dipalmitoyl phosphatidylcholine (DPPC). This nanosuspension is a clinical vaccine adjuvant known as GLA-AF. We examined the effects of DPPC supplier, buffer composition, and manufacturing process on GLA-AF physicochemical and biological activity characteristics. RESULTS: DPPC from different suppliers had minimal influence on physicochemical and biological effects. In general, buffered compositions resulted in less particle size stability compared to unbuffered GLA-AF. Microfluidization resulted in rapid particle size reduction after only a few passes, and 20,000 or 30,000 psi processing pressures were more effective at reducing particle size and recovering the active component than 10,000 psi. Sonicated and microfluidized batches maintained good particle size and chemical stability over 6 months, without significantly altering in vitro or in vivo bioactivity of GLA-AF when combined with a recombinant malaria vaccine antigen. CONCLUSIONS: Microfluidization, compared to water bath sonication, may be an effective manufacturing process to improve the scalability and reproducibility of GLA-AF as it advances further in the clinical development pathway. Various sources of DPPC are suitable to manufacture GLA-AF, but buffered compositions of GLA-AF do not appear to offer stability advantages over the unbuffered composition.

Semi-synthetic terpenoids with differential adjuvant properties as sustainable replacements for shark squalene in vaccine emulsions.

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.

Screening of Oligomeric (Meth)acrylate Vaccine Adjuvants Synthesized via Catalytic Chain Transfer Polymerization.

This report details the first systematic screening of free-radical-produced methacrylate oligomer reaction mixtures as alternative vaccine adjuvant components to replace the current benchmark compound squalene, which is unsustainably sourced from shark livers. Homo-/co-oligomer mixtures of methyl, butyl, lauryl, and stearyl methacrylate were successfully synthesized using catalytic chain transfer control, where the use of microwave heating was shown to promote propagation over chain transfer. Controlling the mixture material properties allowed the correct viscosity to be achieved, enabling the mixtures to be effectively used in vaccine formulations. Emulsions of selected oligomers stimulated comparable cytokine levels to squalene emulsion when incubated with human whole blood and elicited an antigen-specific cellular immune response when administered with an inactivated influenza vaccine, indicating the potential utility of the compounds as vaccine adjuvant components. Furthermore, the oligomers' molecular sizes were demonstrated to be large enough to enable greater emulsion stability than squalene, especially at high temperatures, but are predicted to be small enough to allow for rapid clearance from the body.

Intranasal delivery of a synthetic Entamoeba histolytica vaccine containing adjuvant (LecA + GLA-3 M-052 liposomes): In vitro characterization.

Amebiasis, a disease caused by the parasite Entamoeba histolytica, is estimated to cause millions of infections and at least 55,000 deaths globally each year. With no vaccine currently available, there is an urgent need for an accessible means of stimulating protective mucosal immunity. The objective of this study was to characterize the nasal spray of a novel amebiasis vaccine candidate from a syringe-based liquid atomization device, the Teleflex MAD Nasal™, in both adult and infant nasal airways. Human ergonomic testing was completed to determine realistic actuation parameters. Spray pattern, plume geometry, and droplet size distribution were measured to evaluate reproducibility of free plume characteristics. The Alberta Idealized Nasal Inlet (AINI) and three realistic infant nasal airways were used to determine the in vitro deposition profile in adult and infant airways, respectively. Collectively, in vitro results demonstrated the feasibility of delivering the vaccine candidate to target sites within the nasal airways. Penetration through the nasal airways that could lead to deposition in the lungs was below the limit of quantification for both adult and infant geometries, indicating a low likelihood of adverse events due to lung exposure. These results support continued investigation of intranasal delivery of the synthetic Entamoeba histolytica vaccine.

Effects of emulsifier concentration, composition, and order of addition in squalene-phosphatidylcholine oil-in-water emulsions.

Development and characterization of stable and biocompatible oil-in-water emulsions is important for improved drug and vaccine delivery. In this work, two-component emulsions consisting of squalene and phosphatidylcholine have been developed. The reproducibility of the manufacturing process is established and production efficiency is improved by altering the order of component addition. The effects of emulsifier concentration and composition on emulsion stability and biocompatibility are assessed through dynamic light scattering, zeta potential measurement, viscosity, and hemolytic activity. High concentrations of egg phosphatidylcholine emulsifier decreased initial particle size and increased initial size polydispersity. However, high emulsifier concentrations also appeared to decrease long-term emulsion stability as well as absolute zeta potential values. Substitution of naturally derived egg phosphatidylcholine with synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) produced an emulsion with similar physicochemical properties and stability.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

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.

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.

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.

Charged aerosol detection to characterize components of dispersed-phase formulations.

Colloidal formulations based on biocompatible phospholipids, emulsifiers, and oils are employed in a wide range of applications including medicine, food, and cosmetics. However, characterization of these dispersed-phase components may be difficult to analyze by traditional HPLC with UV, visible, or fluorescence detection modalities due to lack of chromophores or fluorophores. Charged aerosol detection (CAD) is increasingly used for analysis of dispersed-phase components due to its broad applicability and high sensitivity for non-chromophore containing components found in many colloidal systems, such as lipid-based molecules. In this review, we summarize the recent applications of CAD reported in the literature as well as our own laboratory for the analysis of widely used components of dispersed-phase systems. In particular, we discuss the advantages and disadvantages of CAD compared to other HPLC detection methods, as well as the various sample preparation methods suitable for colloidal formulations prior to HPLC-CAD analysis.

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.