Jina Kazzaz

Cationic microparticles are an effective delivery system for immune stimulatory cpG DNA.

It has been demonstrated that bacterial DNA, but not vertebrate DNA, has direct immunostimulatory effects on immune cells in vitro and in vivo (1,2). This is a consequence of the activation of pattern recognition receptors of the innate immune system, which distinguish prokaryotic DNA from vertebrate DNA and respond accordingly to a perceived bacterial infection (2). The immunostimulatory effect of bacterial DNA is mainly due to the presence of unmethylated CpG dinucleotides, which are under-represented in mammalian DNA and are mostly methylated (3). However, the mechanism of cellular uptake and activation for CpG DNA remains to be defined. Nevertheless, it has been reported that CpG are taken up by non-specific endocytosis and that endosomal maturation is necessary for the activation of stress kinase pathways (4). In addition, it has also been reported recently that CpG binds to Toll-like receptor 9, although the localization of the receptor remains to be established (5). Because exposure to CpG brings about conversion of immature DC’s to mature APC’s (6) and many other immune activation events, CpG oligonucleotides represent a promising new class of vaccine adjuvants. In vivo, phosphorothioate oligonucleotides containing CpG have been shown to be potent adjuvants for the induction of Th1 responses, mainly through stimulating TNFa, IL-1, IL-6, and IL-12, and through the expression of co-stimulatory molecules (2,6–8). In addition, it has been reported that the adjuvant effect of CpG can be further enhanced by covalent conjugation to protein antigens (9). In addition to their potential use as adjuvants in a broad range of vaccines, CpG also have significant potential to modulate existing immune responses, which may be useful for the treatment of allergic diseases (10). In the current studies, we sought to improve the potency of CpG as a vaccine adjuvant by using a delivery system to promote the uptake and delivery of CpG into APC’s. CpG was adsorbed onto the surface of cationic poly lactide-coglycolide microparticles (PLG/CpG), which have previously been shown to be effective for enhancing immune responses to adsorbed plasmid DNA (11). In addition, recent studies have begun to define the mechanism of enhancement and have confirmed that cationic microparticles enhance the delivery of adsorbed plasmid into DC’s (12). In the current studies we evaluated the potential of PLG/CpG to induce enhanced antibody and cytotoxic T lymphocyte (CTL) responses to p55 gag and gp120 env from HIV-1 following intramuscular immunization in mice.

Novel anionic microparticles are a potent adjuvant for the induction of cytotoxic T lymphocytes against recombinant p55 gag from HIV-1

Microparticles with entrapped antigens have recently been shown to possess significant potential as vaccine delivery systems and adjuvants. However, the potential of microparticles as adjuvants has been seriously limited by the common problem of degradation and denaturation of antigens following encapsulation and release. To overcome these problems, we have developed a novel way to use microparticles as adjuvants, by the adsorption of proteins onto their surface. Anionic microparticles were prepared through the inclusion of an anionic detergent, sodium dodecyl sulphate (SDS), in the microparticle preparation process. The anionic microparticles were capable of the efficient and reproducible adsorption of recombinant p55 gag protein from HIV-1. Microparticles with adsorbed p55 were capable of inducing potent cytotoxic T lymphocyte responses in mice following intramuscular immunization. In addition, the microparticles also exhibited a potent adjuvant effect for antibody induction against p55.

A cationic sub-micron emulsion (MF59/DOTAP) is an effective delivery system for DNA vaccines

A novel cationic emulsion was developed to adsorb plasmid DNA and improve intracellular delivery of plasmid DNA upon immunization. The emulsion developed, had a highly uniform particle and charge distribution. Based on observations with cationic microparticles, the cationic emulsion was evaluated in vivo in mice and rabbits with a model HIV-1 pCMVp55 gag DNA. In both these species, the cationic emulsion engendered higher antibody responses than those obtained with naked DNA. The cationic emulsion also maintained the cellular responses seen with naked DNA at the same doses.

The Preparation, Characterization, and Evaluation of Cationic Microparticles for DNA Vaccine Delivery

The use of DNA vaccines has become well established (1), and intramuscular injection has resulted in the induction of potent immune responses, including antibody and cytotoxic T lymphocytes (CTL) (1,2). However, although immune responses have been induced in primates, including humans, high doses of DNA on multiple occasions have been required (3–6). Therefore, several approaches are currently under evaluation to improve the potency of DNA vaccines, including vector modification to enhance antigen expression (7), physical delivery methods (8,9), and the use of vaccine adjuvants (10). As an alternative, we have developed cationic microparticles with adsorbed plasmids as a delivery system for DNA vaccines (11), using the biodegradable and biocompatible polymer, polylactide-co-glycolide (PLG) (12). PLG has previously been used for a wide range of biomedical purposes, including the preparation of drug delivery systems (13). Although PLG microparticles (14–20) have previously been described as a delivery system for DNA vaccines, these formulations had some inherent disadvantages over our proposed concept. The entrappment of DNA within the microparticles (14–16), results in significant degradation during encapsulation and release (16,18,20). An additional problem with DNA microencapsulation is that following administration, the DNA is released very slowly, limiting the amount of DNA available to transfect target cells and induce immune responses. In the current paper, we describe in detail the preparation and characterization of microparticles with adsorbed DNA, and show in vivo data to support this approach for the development of improved DNA vaccines. We show that the load of DNA on microparticles can be varied, and that more than one plasmid can be delivered simultaneously. In addition, we demonstrate the requirement for the preparation of cationic PLG microparticles with adsorbed DNA, to achieve potent immune responses. MATERIALS AND METHODS

A comparison of biodegradable microparticles and MF59 as systemic adjuvants for recombinant gD from HSV-2

A recombinant form of glycoprotein D from herpes simplex virus type-2 (gD2) was encapsulated into polylactide-co-glycolide (PLG) microparticles using a previously established solvent evaporation technique. The mean size of the microparticles was about 1 micron and high encapsulation efficiency of the antigen was achieved (70-80%). The microparticles were administered intramuscularly to Balb/C mice and the immune responses were compared with those obtained with the oil in water adjuvant MF59. The serum IgG response to gD2 induced by the microparticles was comparable with that induced by MF59. The serum neutralization titres were also comparable for microparticles and the emulsion. However, the microparticles induced a higher IgG2a isotype response and a more potent serum IFN-gamma response than MF59, suggesting a more Th1 type of response. The MF59 induced higher levels of serum IL-4 and IL-5 cytokines, suggesting a more Th2 type of response.

Charged polylactide co-glycolide microparticles as antigen delivery systems.

Polymeric microparticles with encapsulated antigens have become well-established in the last decade as potent antigen delivery systems and adjuvants, with experience being reported from many groups. However, the authors have recently shown that an alternative approach involving charged polylactide co-glycolide (PLG) microparticles with surface adsorbed antigen(s) can also be used to deliver antigen into antigen-presenting cell populations. The authors have described the preparation of cationic and anionic PLG microparticles that have been used to adsorb a variety of agents, to include plasmid DNA, recombinant proteins and adjuvant active oligonucleotides. These novel PLG microparticles were prepared using a w/o/w solvent evaporation process in the presence of the anionic surfactants, such as dioctyl sodium sulfosuccinate, or cationic surfactants, such as hexadecyl trimethyl ammonium bromide. Antigen binding to the charged PLG microparticles was influenced by both electrostatic interaction and other mechanisms, including hydrophobic interactions. Adsorption of antigens to microparticles resulted in the induction of significantly enhanced immune responses in comparison with alternative approaches. The surface adsorbed microparticle formulation offers an alternative way of delivering antigens as a vaccine formulation.

The effect of CTAB concentration in cationic PLG microparticles on DNA adsorption and in vivo performance.

AbstractPurpose. Cationic PLG microparticles with adsorbed DNA have previously been shown to efficiently target antigen presenting cells in vivo for generating higher immune responses in comparison to naked DNA. In this study we tried to establish the role of surfactant (CTAB) concentration on the physical behavior of these formulations.nMethods. Cationic PLG microparticle formulations with adsorbed DNA were prepared using a solvent evaporation technique. Formulations with varying CTAB concentrations and a fixed DNA load were prepared. The loading efficiency and 24 h DNA release was evaluated for each formulation. Select formulations were tested in vivo.nResults. Higher CTAB concentration correlated with higher DNA binding efficiency on the microparticles and lower in vitro release rates. Surprisingly though, the in vivo performance of formulations with varying CTAB concentration was comparable to one another.nConclusions. Cationic PLG microparticles with adsorbed DNA, as described here, offer a robust way of enhancing in vivo responses to plasmid DNA.

Adsorption of a Novel Recombinant Glycoprotein from HIV (Env gp120dV2 SF162) to Anionic PLG Microparticles Retains the Structural Integrity of the Protein, Whereas Encapsulation in PLG Microparticles Does Not

No HeadingPurpose.To evaluate the delivery of a novel HIV-1 antigen (gp120dV2 SF162) by surface adsorption or encapsulation within polylactide-co-glycolide microparticles and to compare both the formulations for their ability to preserve functional activity as measured by binding to soluble CD4.Methods.Poly(lactide-co-glycolide) microparticles were synthesized by a water-in-oil-in-water (w/o/w) emulsification method in the presence of the anionic surfactant dioctylsulfosuccinate (DSS) or polyvinyl alcohol. The HIV envelope glyocoprotein was adsorbed and encapsulated in the PLG particles. Binding efficiency and burst release measured to determine adsorption characteristics. The ability to bind CD4 was assayed to measure the functional integrity of gp120dV2 following different formulation processes.Results.Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as hydrophobic attraction and structural accommodation of the polymer and biomolecule. The functional activity as measured by the ability of gp120dV2 to bind CD4 was maintained by adsorption onto anionic microparticles but drastically reduced by encapsulation.Conclusions.The antigen on the adsorbed PLG formulation maintained its binding ability to soluble CD4 in comparison to encapsulation, demonstrating the feasibility of using these novel anionic microparticles as a potential vaccine delivery system.

Synergistic adjuvant activity of immunostimulatory DNA and oil/water emulsions for immunization with HIV p55 gag antigen

A synthetic oligonucleotide containing a previously identified adjuvant active CpG DNA sequence was evaluated for its ability to augment antibody and CTL responses to p55 gag from HIV-1 in mice. Surprisingly, the CpG oligonucleotide, although, it had previously been described as the most potent adjuvant sequence in mice for the particulate HbsAg, was ineffective when used in a simple combination with urea-solubilized p55 antigen. However, a potent adjuvant effect was observed with the CpG sequence when it was formulated with emulsions. Enhancement of antibody titer by CpG emulsion formulations was observed with urea-solubilized p55 antigen, however, significantly higher titers were obtained with p55 bound to polylactide-co-glycolide microparticles. In both cases IgG2a was enhanced in the presence of CpG. It appears likely that presentation of CpG with emulsions and particulate antigens enhances their delivery into antigen presenting cells (APC) and results in more effective presentation of antigen and adjuvant. To support this hypothesis, preliminary in vitro studies were undertaken to show upregulation of CD86 on mouse bone marrow-derived dendritic cells (BMDC) in vitro, following incubation with CpG formulations.

Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1.

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone + p24.