ISCOM-Matrices Nanoformulation Using the Raw Aqueous Extract of Quillaja lancifolia (Q. brasiliensis).

Quillaja saponins have an intrinsic capacity to interact with membrane lipids that self-assembles in nanoparticles (immunostimulating complexes or ISCOM-matrices) with outstanding immunoadjuvant activity and low toxicity profile. However, the expensive and laborious purification processes applied to purify Quillaja saponins used to assemble ISCOM-matrices show an important drawback in the large-scale use of this vaccine adjuvant. Thus, in this study, we describe a protocol to appropriately formulate ISCOM-matrices using the raw aqueous extract (AE) of Quillaja lancifolia leaves. In the presence of lipids, AE was able to self-assemble in nanostructures that resembles immunostimulating complexes (ISCOM). These negatively charged nanoparticles of approximately 40 nm were characterized by transmission electron microscopy and dynamic light scattering. In addition, well-known saponins with remarkable immunoadjuvant activity, as QS-21, were detected into nanoparticles. Thus, the easier, robust, cheaper, and environmentally friendly method developed here may be an alternative to the classical methods for ISCOM-matrices production that use high-purified saponins.

Biocompatible Lipid Polymer Cationic Nanoparticles for Antigen Presentation.

Biocompatible lipid polymer nanoparticles (NPs) previously used as antimicrobial agents are explored here as immuno-adjuvants. Poly (methyl methacrylate) (PMMA)/dioctadecyldimethylammonium bromide (DODAB)/poly (diallyldimethylammonium chloride) (PDDA) nanoparticles (NPs) were prepared by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB and PDDA, with azobisisobutyronitrile (AIBN) as the initiator. NPs characterization after dialysis by dynamic light-scattering yielded 225 ± 2 nm hydrodynamic diameter (Dz), 73 ± 1 mV zeta-potential (ζ), and 0.10 ± 0.01 polydispersity (P). Ovalbumin (OVA) adsorption reduced ζ to 45 ± 2 mV. Balb/c mice immunized with NPs/OVA produced enhanced OVA-specific IgG1 and IgG2a, exhibited moderate delayed type hypersensitivity reaction, and enhanced cytokines production (IL-4, IL-10, IL-2, IFN-γ) by cultured spleen cells. There was no cytotoxicity against cultured macrophages and fibroblasts. Advantages of the PMMA/DODAB/PDDA NPs were high biocompatibility, zeta-potential, colloidal stability, and antigen adsorption. Both humoral and cellular antigen-specific immune responses were obtained.

Stability Studies of the Vaccine Adjuvant U-Omp19.

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens. To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates. This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

Biocompatible lipid polymer cationic nanoparticles for antigen presentation

Biocompatible lipid polymer nanoparticles (NPs) previously used as antimicrobial agents are explored here as immuno-adjuvants. Poly (methyl methacrylate) (PMMA)/dioctadecyldimethylammonium bromide (DODAB)/poly (diallyldimethylammonium chloride) (PDDA) nanoparticles (NPs) were prepared by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB and PDDA, with azobisisobutyronitrile (AIBN) as the initiator. NPs characterization after dialysis by dynamic light-scattering yielded 225 ± 2 nm hydrodynamic diameter (Dz), 73 ± 1 mV zeta-potential (ζ), and 0.10 ± 0.01 polydispersity (P). Ovalbumin (OVA) adsorption reduced ζ to 45 ± 2 mV. Balb/c mice immunized with NPs/OVA produced enhanced OVA-specific IgG1 and IgG2a, exhibited moderate delayed type hypersensitivity reaction, and enhanced cytokines production (IL-4, IL-10, IL-2, IFN-γ) by cultured spleen cells. There was no cytotoxicity against cultured macrophages and fibroblasts. Advantages of the PMMA/DODAB/PDDA NPs were high biocompatibility, zeta-potential, colloidal stability, and antigen adsorption. Both humoral and cellular antigen-specific immune responses were obtained.

In vitro Notch-mediated adjuvant immunogenic potency is induced by combining QS-21 and MPL in a co-culture model of PBMC and HUVEC cells.

Few vaccine adjuvants have been approved for human use although several are currently being studied in preclinical and clinical trial. MPL is a toll-like receptor agonist able to trigger a high and persistent antibody response via-TLR-4 while QS-21 activates the NLRP3 inflammasome. Data suggest that there is a cross-talk between Notch and TLR signaling pathways modulating the polarization of the immune response in a MyD88-dependent manner. However, the role of Notch on the mechanism action of immunogenic adjuvants has not been addressed yet. This study aims to evaluate the in vitro toxicity and inflammatory response triggered by MPL and QS-21 using an in vitro human cell co-culture model and to determine whether NFκB or Notch signaling pathways are involved in their mechanism of immunotoxicity. In order to do this, we evaluated the effect of QS- 21/MPL alone or in combination using a co-culture of PBMC and HUVEC using cytotoxicity, surface expression of ECAMs, cell adhesion and cytokine release, NF-κB activation and NOTCH1 expression as observation endpoints. We found that both MPL and QS-21 were cytotoxic at concentrations over 5 µg/mL. Both adjuvants were able to trigger the expression of ECAMs and induce firm adhesion of PBMC to the endothelium. QS-21 and MPL combination demonstrated a synergistic effect on cellular recruitment and cytokine release generating a switch from Th2 to Th1 cytokine profile. Both MPL and QS-21 by themselves were able to generate significant NF-κB activation. However, this effect was not observed when both adjuvants were combined. On the contrary, the adjuvants alone and combined induced an overexpression of NOTCH-1. This is an important finding, as it provides new evidence that these adjuvants could modulate reactogenicity of vaccines through Notch signaling.

A liquid chromatography coupled to quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS) method for identification analysis of saponins from Quillaja saponaria bark extracts in foot-and-mouth disease vaccines: Development, validation and applicability.

Saponins from Quillaja saponaria have been commonly used as immunomodulatory adjuvants in foot-and-mouth disease vaccines (FMDVs). However, due to the lack of consensus over the possible exacerbation of local inflammatory responses in cattle and its economic impacts, their use has been discouraged by Brazilian authorities. A qualitative method intended to determine the presence of saponins from Q. saponaria bark extracts in FMDVs was developed and validated. Instrumental analysis was performed using an liquid chromatography (LC) coupled to a quadrupole-time-of-flight-mass spectrometry (TOF-MS) system. The method was validated according to the International Conference on Harmonization Harmonized Tripartite Guideline Q2 (R1) and Brazilian Ministry of Agriculture, Livestock and Food Supply Analytical Quality Assurance Guidelines. Validation parameters were determined and considered suitable to the established criteria. The validated method has been applied in routine analysis in the National Agricultural Laboratory at Rio Grande do Sul (LANAGRO-RS). All results obtained were in agreement with the vaccine's composition described by the manufacturer. The method is easy and adequate for analysis in routine laboratories. To the best of the authors' knowledge, this is the first report of a method which intends to investigate the presence of saponins from Q. saponaria bark extracts in veterinary vaccines.

Plant Phenolics and Lectins as Vaccine Adjuvants.

BACKGROUND: The immune system is responsible for providing protection to the body against foreign substances. The immune system divides into two types of immune responses to study its mechanisms of protection: 1) Innate and 2) Adaptive. The innate immune response represents the first protective barrier of the organism that also works as a regulator of the adaptive immune response, if evaded the mechanisms of the innate immune response by the foreign substance the adaptive immune response takes action with the consequent antigen neutralization or elimination. The adaptive immune response objective is developing a specific humoral response that consists in the production of soluble proteins known as antibodies capable of specifically recognizing the foreign agent; such protective mechanism is induced artificially through an immunization or vaccination. Unfortunately, the immunogenicity of the antigens is an intrinsic characteristic of the same antigen dependent on several factors. CONCLUSION: Vaccine adjuvants are chemical substances of very varied structure that seek to improve the immunogenicity of antigens. The main four types of adjuvants under investigation are the following: 1) Oil emulsions with an antigen in solution, 2) Pattern recognition receptors activating molecules, 3) Inflammatory stimulatory molecules or activators of the inflammasome complex, and 4) Cytokines. However, this paper addresses the biological plausibility of two phytochemical compounds as vaccine adjuvants: 5) Lectins, and 6) Plant phenolics whose characteristics, mechanisms of action and disadvantages are addressed. Finally, the immunological usefulness of these molecules is discussed through immunological data to estimate effects of plant phenolics and lectins as vaccine adjuvants, and current studies that have implanted these molecules as vaccine adjuvants, demonstrating the results of this immunization.

Evaluation of different adjuvants formulations for bluetongue vaccine

This study investigated the adjuvant potential of W/O/W multiple emulsions and microemulsions, comparing them with traditional aluminum hydroxide and oil-in-water emulsion adjuvants against bluetongue vaccine (BTV). Local inflammatory reactions were assessed in rabbits by measuring the temperature of the animals and the skin thickness at the site of application. Antibodies titers were determined by serum-neutralization test. Histological analyses of lesions at the site of adjuvants application were done. Results showed that multiple emulsion and microemulsion maintained their stability even in the presence of complex components and presented adequate characteristics for subcutaneous administration. They were able to induce immune response against BTV, but it was smaller than the traditional adjuvants. Despite microemulsion adjuvant showed lower antibodies titre, it was easier to prepare more stable at 4°C and it was the only one that did not induce any local reaction.

Archaeosomes display immunoadjuvant potential for a vaccine against Chagas disease.

Archaeosomes (ARC), vesicles made from lipids extracted from Archaea, display strong adjuvant properties. In this study, we evaluated the ability of the highly stable ARC formulated from total polar lipids of a new Halorubrum tebenquichense strain found in Argentinean Patagonia, to act as adjuvant for soluble parasite antigens in developing prophylactic vaccine against the intracellular protozoan T. cruzi, the etiologic agent of Chagas disease. We demonstrated for the first time that C3H/HeN mice subcutaneously immunized with trypanosomal antigens entrapped in these ARC (ARC-TcAg) rapidly developed higher levels of circulating T. cruzi antibodies than those measured in the sera from animals receiving the antigen alone. Enhanced humoral responses elicited by ARC-TcAg presented a dominant IgG2a antibody isotype, usually associated with Th1-type immunity and resistance against T. cruzi. More importantly, ARC-TcAg-vaccinated mice displayed reduced parasitemia during early infection and were protected against an otherwise lethal challenge with the virulent Tulahuén strain of the parasite. Our findings suggest that, as an adjuvant, H. tebenquichense-derived ARC may hold great potential to develop a safe and helpful vaccine against this relevant human pathogen.