Derek T. O’Hagan

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 novel bioadhesive intranasal delivery system for inactivated influenza vaccines

The aim of the current studies was to evaluate a bioadhesive delivery system for intranasal administration of a flu vaccine, in combination with a mucosal adjuvant (LTK63). A commercially available influenza vaccine, containing hemagglutinin (HA) from influenza/A Johannesberg H1N1 1996, and LTK63 or LTR72 adjuvants, which are genetically detoxified derivatives of heat labile enterotoxin from Escherichia coli, were administered IN in a bioadhesive delivery system, which comprised esterified hyaluronic acid (HYAFF) microspheres, to mice, rabbits and micro-pigs at days 0 and 28. For comparison, additional groups of animals were immunized intranasally with the HA vaccine alone, with soluble HA+LTK63, or IM with HA. In all three species, the groups of animals receiving IN immunization with the bioadhesive microsphere formulations, including LT mutants, showed significantly enhanced serum IgG responses (P<0.05) and higher hemagglutination inhibition (HI) titers in comparison to the other groups. In addition, the bioadhesive formulation also showed a significantly enhanced nasal wash IgA response (P<0.05). Most encouragingly, in pigs, the bioadhesive microsphere vaccine delivery system induced serum immune responses following IN immunization, which were significantly more potent than those induced by traditional IM immunization at the same vaccine dose (P<0.05).

Microparticles for intranasal immunization.

Of the several routes available for mucosal immunization, the nasal route is particularly attractive because of ease of administration and the induction of potent immune responses, particularly in the respiratory and genitourinary tracts. However, adjuvants and delivery systems are required to enhance immune responses following nasal immunization. This review focuses on the use of microparticles as adjuvants and delivery systems for protein and DNA vaccines for nasal immunization. In particular we discuss our own work on poly(lactide co-glycolide) (PLG) microparticles with entrapped protein or adsorbed DNA as a vaccine delivery system. The possible mechanisms involved in the enhancement of immune responses through the use of DNA adsorbed onto PLG microparticles are also discussed.

Characterization of Hepatitis C Virus Core-Specific Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8+ CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in ∼1-μm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4+ and CD8+ T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30–0.71 and 0.32–2.21% of the circulating CD8+ and CD4+ T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.

Mucosal immunization with HIV-1 gag DNA on cationic microparticles prolongs gene expression and enhances local and systemic immunity.

There is an urgent need to develop vaccines against transmission of HIV through the vaginal and rectal mucosa. In the present study we tested the ability of DNA encoding HIV-1 gag adsorbed onto the surface of cationic polylactide co-glycolide microparticles (PLG-DNA) to induce local and systemic gag-specific immunity following mucosal delivery. We found gag-specific cell- and antibody-mediated responses in local as well as systemic lymphoid tissues following intranasal (IN) immunizations with PLG-DNA but not with naked DNA. IN immunizations with PLG-DNA, but not naked DNA, induced prolonged expression of gag protein in local and systemic lymphoid tissues. These data have important implications for DNA vaccine development.

Human Immunodeficiency Virus Type 1 Gag–Specific Vaginal Immunity and Protection after Local Immunizations with Sindbis Virus–Based Replicon Particles

The majority of human immunodeficiency virus (HIV) infections occur through vaginal and rectal transmission. In seeking a safe, nonreplicating gene-delivery vector that can induce mucosal and systemic immune responses and protection, Sindbis virus-based replicon particles expressing HIV-1 Gag (SIN-Gag) were developed. In mice, after nasal or intramuscular immunization with SIN-Gag and vaginal challenge with vaccinia virus (VV) expressing HIV-1 Gag (VV-Gag), CD8(+) T cell-mediated responses were detected locally, in the vaginal mucosa and in the draining iliac lymph nodes (ILNs), and systemically, in the spleen. However, the mice were not protected against VV-Gag replication in the ovaries. In contrast, after vaginal or rectal immunization with SIN-Gag and vaginal challenge with VV-Gag, despite lower local CD8(+) T cell-mediated responses in the vaginal mucosa and ILNs, the mice were protected against VV-Gag replication in the ovaries. Therefore, local immunization with SIN-Gag induced both local mucosal cell-mediated responses and protection.

Controlled release of recombinant insulin-like growth factor from a novel formulation of polylactide-co-glycolide microparticles

The purpose of the current study was to develop a controlled-release delivery system for recombinant insulin-like growth factor (rhIGF-I). Polylactide-co-glycolide (PLG) microparticles with entrapped rhIGF-I were prepared by a novel emulsion based solvent evaporation process. Microparticles with two loading levels of rhIGF-I were prepared (4 and 20% w/w). The integrity of released rhIGF-I was characterized by RP-HPLC, SDS-PAGE and a bioactivity assay. In vitro and in vivo release profiles of rhIGF-I from these microparticles were also evaluated. Reproducible batches of microparticles with 4% and 20% w/w loading of rhIGF-I were prepared, with excellent encapsulation efficiency (81 and 85% of total protein respectively entrapped). The protein retained integrity after the microencapsulation process as evaluated by RP-HPLC, SDS-PAGE and bioactivity assay. The in vitro profiles exhibited a significant burst release of rhIGF-I (20-30%), followed by controlled release of protein for up to 28 days. A similar level of burst release was observed in vivo, followed by controlled release of protein for 14-18 days. In addition, there was a surprisingly close correlation between in vitro and in vivo release rates. PLG microparticles with entrapped rhIGF-I are a promising delivery system which may allow rhIGF-I to be used for a broad range of therapeutic indications.

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.

Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice

A carcinoembryonic antigen (CEA)-based DNA vaccine, adsorbed onto cationic microparticles of poly(DL-lactide-co-glycolide) (PLG) induced tumor-protective immunity against a lethal challenge of MC38-CEA colon carcinoma cells in CEA-transgenic mice that was more potent than that of the corresponding naked DNA vaccine. Boosting with a plasmid encoding murine GM-CSF increased the vaccines efficacy leading to a complete rejection of tumor cells in 50% of mice. This effect was due to activation of MHC class I-restricted CD8(+) T cells coupled with an increased secretion of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-2. Also, specific activation of dendritic cells was indicated by a two-three-fold upregulation of their costimulatory CD80 and MHC class II molecules. This approach may be a promising new strategy for the rational design of cancer vaccines for future clinical applications.

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.