Gary Ott

Distribution of DNA Vaccines Determines Their Immunogenicity After Intramuscular Injection in Mice

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-μl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 μl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

MF59. Design and evaluation of a safe and potent adjuvant for human vaccines.

MF59 is a safe, practical, and potent adjuvant for use with human vaccines. The formulation is easily manufactured, may be sterilized by filtration, and is both compatible and efficacious with all antigens tested to date. MF59 has been shown to be a potent stimulator of cellular and humoral responses to subunit antigens in both animal models and clinical studies. Toxicology studies in animal models and Phase I-III studies in humans have demonstrated the safety of MF59 with HSV, HIV, and influenza vaccines.

Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells.

Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescent-labeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of the fluorescence was concentrated in endosomal compartments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was subsequently processed and presented, resulting in stimulation of an H-2kd-restricted, gag-specific T cell hybridoma. Activation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1–20 μg DNA (10–2000 μg PLG) and was sustained up to 5 days after transfection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells.

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.

Immunization with the adjuvant MF59 induces macrophage trafficking and apoptosis

The mechanisms associated with the immunostimulatory activity of vaccine adjuvants are still poorly understood. We have undertaken a study to determine whether antigen‐presenting cell trafficking is modified by administration of the submicron emulsion adjuvant MF59. We investigated the fate of inflammatory macrophages after intramuscular injection of the antigen herpes simplex virus gD2 with fluorescence‐labeled MF59. A homogenous population of macrophages infiltrated the muscle, internalized adjuvant and expressed markers characteristic of mature macrophages over a 48‐h period. Macrophage influx to the injection site was reduced by 70% in mice deficient for the chemokine receptor 2 (CCR2). Two distinct cell populations were shown to contain fluorescence‐labeled MF59 in the draining lymph node at 48 h post injection. The first population had a round morphology, exhibited bright fluorescence, was located in the subcapsular sinus, and was apoptotic. The second population had a dendritic morphology, was weakly fluorescent, and was located in the T cell area where adjuvant‐containing apoptotic bodies identified by TUNEL labeling were present. We propose that lymph node‐resident dendritic cells can acquire antigen and MF59 after intramuscular immunization by uptake of the apoptotic macrophages.

Distribution of adjuvant MF59 and antigen gD2 after intramuscular injection in mice

MF59, which is an adjuvant approved for human use, typically elicits higher antibody titers than alum when used in combination with a variety of recombinant and natural subunit antigens. The mechanisms responsible for the adjuvant action of MF59 are not fully understood. In particular, little is known about the in vivo distribution of MF59 and of antigen after intramuscular (i.m.) injection. The goal of the present study was to determine the distribution of MF59 injected with soluble antigen gD2 from type 2 herpes simplex virus (HSV) and to compare the distribution of gD2 injected with or without MF59. At 4 h, 36% of the injected dose of labeled MF59 was in the quadriceps muscle and about 50% was in the inguinal fat surrounding the muscle. Half of the initial amount of labeled MF59 in muscle was detected 42 h after injection. The amount of labeled MF59 in the draining lymph nodes was maximal 2 d after injection, which represented 0.1-0.3% of the injected dose. At 4 h, 12% of the injected dose of labeled gD2 was found in the muscle. The presence of MF59 did not significantly modify the distribution of gD2. The results indicate that MF59 and gD2 distribute and are cleared independently after i.m. injection. Importantly, MF59 is unlikely to have a repository effect, whereby it slowly releases the antigen.

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.