Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation.

BACKGROUND: The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21. RESULTS: The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined. CONCLUSIONS: The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.

Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.

Multifunctional PLGA-based nanoparticles encapsulating simultaneously hydrophilic antigen and hydrophobic immunomodulator for mucosal immunization.

We describe here the development of nanoparticles made from poly(lactic-co-glycolic acid) (PLGA) able to deliver an encapsulated antigen with a Toll-Like Receptor-7 (TLR-7) agonist as immunostimulatory signal and coated with a muco-adhesive chitosan-derivate layer. The potential to stimulate an immune response of these vaccine formulations in the absence or presence of the TLR-7 agonist at the systemic and mucosal level were evaluated in mice following subcutaneous or nasal administrations. Intranasally immunized mice developed a high systemic immune response equivalent to mice injected subcutaneously. However, mucosal immune responses were only induced at local and distal sites in mucosally immunized animals. The adjuvant effect of imiquimod on the polarization of the immune response was only detected at local sites, which tends to increase safety of this vaccine delivery system.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NP>chitosan DNA NP=DNA unloaded chitosan NP>control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release.

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Toll-like receptor-7 agonist decoration enhances the adjuvanticity of chitosan-DNA nanoparticles.

In order to provide an adjuvant-equipped carrier system for plasmid deoxyribonucleic acid (pDNA) vaccines, we grafted for the first time a Toll-like receptor (TLR)-7 agonistic moiety [9-benzyl-8-hydroxyadenine (HA)] through a poly(ethylene glycol) (PEG) spacer onto a water-soluble chitosan derivative [final copolymer: 6-0-carboxymethyl-N,N,N-trimethylchitosan (CTC)-graft-PEG-HA (CTCPHA)]. Successful grafting was confirmed by spectroscopic (H NMR, mass, ultraviolet-visible, and Fourier transform infrared spectroscopy) and chromatographic (size-exclusion chromatography-multi-angle laser light scattering) methods. In this article, TLR-7 agonist-decorated CTCPHA nanoparticles (NPs) were formulated by complex coacervation with pDNA expressing the green fluorescence protein. Resulting NPs had a size of around 200 nm with a positive surface charge and high DNA encapsulation efficiency. In contrast to the use of DNA alone, NP protected DNA against enzymatic degradation and enabled transfection of alveolar A549 cells. Interestingly, TLR-7 agonist decoration increased significantly the interleukin-8-related immune stimulatory capacity of polymeric chitosan and chitosan-based NP in human THP-1 macrophages when compared with controls. In summary, we demonstrate here the proof-of-principle that covalent TLR-7 agonist functionalization of chitosan-DNA NPs enhances the carrier's adjuvanticity, representing a valuable concept for future polymer-based DNA vaccination.

Stimulation of human macrophages (THP-1) using Toll-like receptor-2 (TLR-2) agonist decorated nanocarriers.

The purpose of this study was to prepare and characterize nanocarrier systems, which allow the application of pDNA vaccines and adjuvants to mucosal vaccination. Chitosan from a vegetal source (Agaricus bisporus) and of GMP quality was used to synthesize the derivative 6-O-carboxymethyl-N,N,N-trimethylchitosan (CM-TMC). Toll-like receptor-2 (TLR-2) agonist, Pam(3)Cys, was synthesized and coupled to CM-TMC through a polyethylene glycol (PEG) spacer. Successively, Pam(3)Cys decorated nanocarriers were prepared by complexation with plasmid DNA (pDNA) expressing green fluorescence protein (GFP), and characterized with respect to their physicochemical properties and protection of the included plasmid against DNase I enzymatic degradation. In vitro studies using phorbol 12-myristyl 13-acetate (PMA) stimulated macrophage-like THP-1 (mTHP-1) cells were focused on cytotoxicity of both polymers and particles, and their potential to stimulate IL-8 release via the TLR-2 pathway. Our results showed that the TLR-2 functionalized pDNA nanocarriers have the ability to complex and to protect pDNA against enzymatic degradation. pDNA nanocarriers were of around 400 nm in size, and displayed a positive zeta potential of 27.9 +/- 1.6 mV. Chitosan, CM-TMC, and Pam(3)Cys-functionalized CM-TMC polymers displayed cytotoxicity on mTHP1 cells in a concentration-dependent manner, which decreased by 50-fold on complexation with pDNA. In addition, decorated pDNA nanocarriers induced IL-8 secretion by mTHP-1 macrophages, which was increased by 10-fold as compared to nondecorated carriers.