Elsevier

Journal of Controlled Release

Volume 110, Issue 3, 21 February 2006, Pages 566-573
Journal of Controlled Release

Encapsulation of the immune potentiators MPL and RC529 in PLG microparticles enhances their potency

https://doi.org/10.1016/j.jconrel.2005.10.010Get rights and content

Abstract

Purpose

Monophosphoryl lipid A (MPL) and the synthetic LPS mimetic RC529, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles, were evaluated as immune potentiators in the presence of either HIV-1 gp120 protein or antigen from Neisseria meningitidis serotype B (Men B). The immunogenicity of these formulations was evaluated in mice and compared to CpG containing oligonucleotide. This work was done as part of an ongoing effort to enhance the potency of vaccine candidates against HIV and Men B.

Methods

Microparticles were made by a solvent evaporation method. Blank microparticles as well as microparticles with encapsulated MPL or RC529 were made using the PLG polymer RG503 and the ionic surfactant Dioctylsulfosuccinate by the water-in-oil-in-water emulsion technique. Antigens from HIV-1 and Men B were adsorbed on the surface of these anionic microparticles and the final formulations characterized for protein loading, release, and integrity. The formulations were then tested in mice for their ability to elicit antibodies and bactericidal activity in comparison with CpG containing oligonucleotide.

Results

We have found that adding soluble immune potentiators to Men B antigen formulated on PLG microparticles significantly enhanced the immune response to a level comparable to that obtained using CpG. In a separate study, we found that encapsulating MPL or RC529 in PLG microparticles further enhanced the response in comparison to soluble CpG, which is our control group. Similarly, adding soluble immune potentiators to gp120 antigen formulated on PLG microparticles resulted in a significant enhancement of the immune response. Moreover, delivering MPL or RC529 encapsulated in PLG microparticles with gp120 adsorbed on PLG microparticles, resulted in even further enhancement of serum titers over those obtained with soluble immune potentiators. These titers were comparable to or greater than those obtained with soluble CpG, the control group. This effect was observed for both antigens regardless of whether or not the immune potentiator and the antigen were used with the same or with separate particles. In conclusion, the advantages of encapsulating MPL and RC529 lie not only in the enhanced immune response they elicit, but also in the convenience of handling these relatively insoluble compounds, and flexibility in vaccine design. The fact that MPL and RC529 are readily soluble in methylene chloride used for the manufacturing of PLG microparticles makes it easy to avoid solubility issues. Moreover, formulating antigen and immune potentiator with the same particle offers an attractive approach to vaccine delivery.

Introduction

The majority of vaccines currently under investigation represent highly purified subunit components of pathogens. Unfortunately, they lack most of the features of the original pathogens such as the ability to replicate and produce high levels of antigens and immunostimulatory components. Therefore, they are usually poorly immunogenic and need adjuvants and delivery systems to improve immunogenicity.

Aluminum-based mineral salts (generally called Alum) have been successfully used as adjuvants in licensed vaccines for many years [1], [2]. Alum typically induces a Th2 immune response. Although it has been shown to be safe and effective in traditional vaccines where eliciting antibody response is necessary, it is a weak adjuvant for protein subunits. Moreover, it fails to induce the Th1 responses associated with the induction of gamma interferon and cytotoxic T lymphocytes (CTL) required to clear the body of intracellular viral infections.

In the recent past, we have focused on an alternative delivery system for vaccines consisting of microparticles prepared from the biodegradable, biocompatible polyesters, poly(lactide-co-glycolide) (PLG). Microparticles represent an attractive approach to vaccine delivery since PLG has been used in humans for many years as resorbable suture material and as controlled-release drug delivery systems [3], [4], [5]. It has also been shown that microparticles (∼ 1 μm) are taken up efficiently by antigen-presenting cells (APC) in vitro [6], and in vivo [7] and are able to induce cytotoxic T lymphocyte (CTL) responses in rodents [8], [9]. We recently developed novel charged PLG microparticles prepared with an anionic surfactant as the particle stabilizer, capable of efficient adsorption of antigen onto their surface [10], [11]. This approach allows the preservation of antigen integrity and results in the presentation of multiple copies of the antigen to the APC, which is similar to the surface of a pathogen. In previous studies, we have reported that immunization with a recombinant antigen from HIV adsorbed to PLG microparticles induces potent antibody and CTL responses in mice [10] and in non-human primates [11]. We have also shown microparticles to be a potent vaccine delivery system for antigen from Neisseria meningitides serotype B (Men B) [12].

It is well known that molecules such as monophosphoryl lipid A (MPL), lipopolysaccharide (LPS), and bacterial DNA (CpG containing optimized oligo sequences) represent pathogen-associated molecular patterns (PAMPs). This allows the pathogen recognition receptors (PRR) present on innate immune cells to recognize them and become activated [13], [14], [15]. PAMPs and related compounds can be called immune potentiators, allowing a clear distinction between them and particulate adjuvants such as emulsions, liposomes, virus-like particles and microparticles.

The role of a ‘delivery’ system is to enhance the amount of antigen reaching the cells responsible for immune response induction. Immune potentiators activate the cells through interaction with their receptors. They can be formulated into delivery systems to focus their effects onto the APC, to maximize their potency, and to minimize their effects on immune cells. Optimal new generation vaccines are likely to comprise recombinant antigens used with immune potentiators and delivery systems for both antigens and adjuvants.

The lipopolysaccharide (LPS) component of Gram-negative bacteria has been shown to act as a potent immune potentiator [16], [17], [18], [19], [20]; however, the profound toxicity and pyrogenicity of LPS prevents its use in humans [21]. Alternatively, a chemically modified LPS derived from Salmonella minnesota R595, called monophosphoryl lipid A (MPL), exhibits potent adjuvant activity with essentially no toxicity [22]. MPL has been shown to be an effective immune potentiator for the induction of both humoral and cell-mediated immunity in which MPL can induce both Th1- and Th2-type immune responses in the systemic and mucosal compartments of the immune system [23], [24], [25]. It was also shown to be potent when formulated within PLG microparticles [26]. MPL has been approved in Europe for a hepatitis B vaccine and is currently being evaluated in phase III clinical trials with HSV2 [27]. In addition, MPL is in advanced clinical trials with malaria [28] and human papillomavirus (HPV) [29].

RC529, a synthetic form of MPL, is an equally potent immune potentiator, recently approved in Argentina for use in a vaccine with recombinant hepatitis B antigen [30]. The use of a synthetic compound is quite attractive due to the general tendency to avoid natural compounds. It can also be prepared with high purity and is cost-effective.

Oligonucleotides with specific sequence surrounding CpG, appear to be very effective for the induction of potent Th1 responses. In a previous study, we reported that the potency of antigens adsorbed onto PLG microparticles was improved by co-administration with an oligonucleotide-based immune potentiator, CpG DNA [31].

In this paper, we describe the delivery of antigens and immune potentiators using a particulate delivery system. MPL and RC529 were each encapsulated into anionic PLG microparticles. Antigens from either Neisseria meningitides B [32] or from human HIV-1 were adsorbed on the surface of either blank microparticles or microparticles with entrapped immune potentiator. These formulations were evaluated for immunogenicity in mice, and the responses induced were compared to those obtained with CpG oligonucleotide.

Section snippets

Materials

RG503, poly(d,l-lactide-co-glycolide) 50 : 50 co-polymer composition (intrinsic viscosity 0.4 from manufacturer's specifications) was obtained from Boehringer Ingelheim. Dioctylsulfosuccinate (DSS) was from Sigma Chemical (St. Louis, MO). Escherichia coli (E. coli)-derived recombinant meningococcal B protein (Men B) was obtained from Chiron Vaccines, Siena (IRIS, Chiron S.r.l., Via Fiorentina 1, 53100 Siena, Italy) and was isolated and purified as described previously [32] (protein GNA #2132).

Physical characterization of microparticle formulations

PLG/DSS microparticles were prepared with a mean size of 1 μm (size distribution of 0.5–1.9 μm expressed in vol.%) and had a zeta potential of − 55 mV. Following adsorption of Men B protein to the microparticles, the zeta potential was − 17 mV, and following adsorption of gp120 protein, the zeta potential was − 25 mV, while the size of adsorbed particles did not change. Following lyophilization and re-constitution, particle size distribution was 3–5 μm.

PLG microparticles made with

Conclusion

The most important aspect of these studies is the potent immunogenic effect achieved by formulating MPL and RC529 in PLG microparticles. The superior responses obtained over soluble formulated immune potentiators have great benefits. Moreover, the fact that formulating immune potentiator and antigen using the same or separate particles results in the same level of enhanced response allows great flexibility in vaccine design. A single-unit vaccine with antigen and immune potentiator on the same

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