Development of synthetic biodegradable microparticulate vaccines: a roller coaster story

Single-dose vaccines

The introduction of injectable, biodegradable microspheres made of poly(lactide-co-glycolic acid) (PLGA) in the mid-1980s for cancer therapy was a major pharmatechnological milestone [1]. PLGA microspheres were most attractive to enhance the bioavailability of drugs that have to be administered primarily by the parenteral route, such as therapeutic peptides and proteins. The enhanced bioavailability was mostly due to the prolongation of drug release over several weeks or even months and, at the same time, the protection of the encapsulated drug from degradation and metabolism. In these early days of parenteral drug delivery systems, the PLGA types available for controlled drug release from PLGA microspheres mostly produced a pulsatile release of the encapsulated peptides and proteins. This was considered to be a disadvantage for drug therapy. By contrast, the prospect of prolonged pulsatile release of peptides and proteins elicited the excitement of some vaccinologists. They wondered whether a properly timed pulsatile antigen release might mimic immunologically the conventional booster doses in childhood vaccination and whether this would allow the replacement of the conventional prime–boost regime with a single injection. Indeed, the necessity of multiple well-timed vaccinations during the first 2 years of life represents a major hurdle for vaccination programs in developing countries. For illustration, seven to ten doctor visits are required during the first 2 years of life in order to receive the recommended 16 vaccine injections against ten childhood infectious diseases (hepatitis B, diphtheria, tetanus, pertussis, Haemophilus influenzae type b, polio, measles, mumps, rubella and varicella). Therefore, it should not be a surprise that the WHO was very interested in the idea of a so-called single-dose vaccine. In 1988, the WHO started the Children Vaccine Initiative to support several research groups for developing and testing PLGA microspheres for a single-dose tetanus vaccine [2,3]. Tetanus toxoid was selected as the antigen owing to its relative stability, good immunogenicity and the urgent need for a better vaccine against neonatal tetanus [4]. The efficiency of the particulate tetanus formulations in eliciting protective antibodies was tested in mice, rats, guinea-pigs and even in nonhuman primates [5]. The preclinical data were promising and advocated clinical development. By the turn of the century, academic research groups, a vaccine control laboratory, a vaccine producer, a contract research organization and the United Nations Children’s Fund (UNICEF) began the planning and preparative work for a Phase I clinical trial with tetanus toxoid-containing microspheres [6]. However, due to several unfavorable circumstances, further developments were stopped.

There were several scientific, technological and economic reasons for the termination of the single-dose tetanus vaccine program. Both the upscaling of the production technology and its transfer to clean-room good manufacturing practice (GMP) facilities for aseptic processing were very complex and costly enterprises under the given circumstances. The candidate vaccines were prepared by space-demanding spray drying or by reactor-based coacervation or solvent-evaporation technologies, which were all difficult to accommodate in a conventional pilot GMP laboratory. For instance, the removal of organic solvents during the microparticle preparation process, alone, required special infrastructural adjustments of the clean room, which constituted serious financial hurdles. It became obvious that a single-dose tetanus vaccine would be ten- to 100-times more expensive than the available conventional vaccine, which only costs 10 US cents per dose. More generally, little if any profit was expected in producing and marketing childhood vaccines against diseases for which vaccines were already available. Being more an enforced act of solidarity than a calculated prospect for profit, the development and market introduction of vaccines that are primarily beneficial for developing countries faces important economic hurdles.

Past developments of PLGA microparticle-based vaccines included numerous different antigens. Although most of the formulations elicited a good immune response in small animals, strategies to further strengthen the immunogenicity included the use of supplementary adjuvants. Initially, for the single-dose tetanus vaccines, as well as for the single-dose multivalent vaccines that, besides tetanus toxoid, also contained diphtheria, pertussis and Haemophilus influenzae type b antigens [7–9], salts of aluminum (alum) were the preferred adjuvants [6]. For coformulating PLGA microspheres and alum, the antigen-containing microparticles were suspended in aqueous alum gel and then lyophilized, which enabled an improved reconstitution in water prior to use. However, since one of the ancillary objectives for the development of microparticle vaccines was to avoid the nonbiodegradable aluminum salts, their inclusion in polymer-based single-dose vaccines was not well endorsed. Hence, what initially started with a perception of revolution and great hopes for improving the health situation in developing countries ended in promising prototype products, which were unfortunately not validated in a human clinical trial. Subsequently, the WHO and UNICEF set other priorities for improving worldwide vaccination coverage.

New applications & hopes: cytotoxic T-cell vaccines & DNA vaccines

In the past 10 years, the focus of interest of polymeric microparticle-based vaccines has shifted from antibody induction with protein antigens toward novel, more challenging applications. In particular, microparticles have been studied extensively for their capacity to induce cytotoxic T-cell responses against a number of peptide antigens. Original studies were conducted to induce cellular immunity, including cytotoxic T-cell responses, against malaria, HIV and other infectious agents in various rodents and nonrodent animals [10–14]. Interestingly, the responses were, in part, protective against the disease or infection in question. However, the immune responses were generally more modest than those induced by formulations with novel and stronger adjuvants, such as the immunostimulatory sequences of CG-rich oligodeoxyribonucleotides and other Toll-like receptor ligands [15] or with nonreplicating viral vectors [16].

When plasmid DNA was first shown to be a potent means for inducing immune responses [17], this was welcomed as a promising vaccination tool, especially against not yet preventable diseases, such as malaria, HIV/AIDS, tuberculosis and pandemic influenza. A major strength of DNA-based vaccines is that they are capable of directly stimulating the CD8 T-cell-mediated response, which is important for mounting an effective counterattack against infection or tumor growth. However, it soon became clear that the strong immunity obtained in small rodents was difficult to achieve in larger animals, mainly because DNA degraded readily in vivo prior to its uptake in, or transfection of, antigen-presenting cells or other somatic cells, which could mediate immune responses through cross-presentation. Consequently, extremely high doses of naked DNA were required in larger animals and humans, or uptake and transfection efficiency had to be improved. The latter was originally obtained by the condensation or aggregation of DNA with polycations or by preparation of DNA-containing micro- or nanoparticles [13,18,19]. This improved the uptake and immunogenicity of DNA vaccines to some extent. Most of these studies made use of PLGA copolymers [20]. However, since PLGA polymers undergo bulk hydrolysis, resulting in a dramatic drop in pH that can significantly degrade the embedded DNA, novel materials were also introduced to overcome this problem [21]. Despite original optimism and continuous improvement of the immunogenicity of DNA vaccines in preclinical models, DNA vaccines have not generally fulfilled expectations in terms of potency in humans. Therefore, current developments rather exploit engineered virus and bacterial particles for the delivery of DNA vaccines [22]. However, polymeric microparticles have, so far, failed technology transition, clinical evaluation and commercial realization, as seen previously for single-dose childhood vaccines.

Allergy vaccines

IgE-mediated rhinoconjunctivitis and asthma are allergic diseases, with a prevalence of approximately 20% in the industrialized world. Although symptomatic treatments are available, the only treatment with a long-lasting effect for allergic patients is allergen-specific immunotherapy or desensitization, during which gradually increasing doses of the allergen are injected subcutaneously over the course of several years (Table 1). Hence, a significant disadvantage of immunotherapy is the high cost and the total of 30–80 injections required [23]. Therefore, a simplified immunotherapy with a reduced number of injections would be highly advantageous since it would improve patient compliance and provide socioeconomic benefits. Here, biodegradable materials, such as PLGA, represent a potential therapeutic alternative [24–26]. While particulate childhood vaccines failed mainly owing to the already available alternatives and particulate cytotoxic T-cell vaccines might fail due to low efficacy compared with other technologies under development, it is our opinion that biodegradable microparticle technology may have a realistic chance of success for allergy vaccines. Protein-containing microparticles are mostly quite immunogenic. This has been demonstrated for several protein antigens (compare this with childhood vaccines). Furthermore, PLGA microparticles can, to some extent, also induce cell-mediated immune responses. This is beneficial in allergen-specific immunotherapy, which partly aims to shift the nature of the allergen-induced immune response from a pathological Th2 response toward a therapeutic Th1 response. One major concern with regards to the encapsulation of proteins in polymeric microparticles has been the potential denaturing effects of the materials and processes involved on conformation-sensitive therapeutic proteins [27]. The problem of protein or peptide destabilization could be solved, to a large extent and case by case, by using stabilizing additives. Nonetheless, some disruption of the molecule may not be as critical for the activity of allergens as it may be for the activity of other proteins. In fact, many of the new approaches in allergy treatment use modified allergens with low IgE-binding capacity, such as recombinant proteins [28,29] or chemically cross-linked allergens [30]. Similarly, heat-denatured allergens have been shown to lose IgE-binding capacity and, at the same time, gain capacity in eliciting therapeutic antibody and cell-mediated immune responses [31].

Furthermore, the poor IgE-binding capacity is important with respect to the induction of local and systemic allergic adverse effects of allergen-specific immunotherapy. If the allergen cross-links IgE on the surface of mast cells resident in the skin, this may induce allergic adverse effects (wheal-and-flare reaction). If the same takes place on basophil cells in the blood, the patient is at risk of a systemic anaphylactic reaction. By contrast, if the allergen is encapsulated inside a polymeric particle, a much lower risk can be expected. First, the allergen may drain with the particle to a draining lymph node, thereby avoiding encountering mast cells or basophils. Second, if the allergen is released from the particles, a much lower concentration of allergen will be freely available compared with that produced by conventional immunotherapy with allergen bound to aluminum salts.

In conclusion, we think that polymeric microparticles may have potential applications in allergen-specific immunotherapy owing to the poor efficiency of the existing vaccines, their slow release and their safety properties, as well as the fact that protein stability is of minor importance (Table 1). Allergy is a disease associated with industrialized regions with superior purchasing power. Moreover, the frequency of the disease (one in four individuals is allergic) and the potentially low developmental costs (both allergens and additives are available and already well established) should also make allergy vaccines interesting for investors and industry, and the financial return of a marketed product is expected to be high.

Table 1. Features of conventional subcutaneous allergen-specific immunotherapy and specific immunotherapy based on particulate and slow-release vaccine delivery systems.

Feature	Conventional subcutaneous SIT	Microparticle-based SIT
Antibody response	Good	Good
T-cell response	Poor	Intermediate but improvable by the use of immune-stimulating additives
Safety	Freely accessible allergen may cause local allergic reaction due to cross binding to IgE on skin mast cells	Less local allergic reactions due to the encapsulated nature of the allergen
Number of doctor visits and injections	30–80	UnknownApproximately 6–10 injections may be anticipated
SIT duration	3–5 years	Unknown
Allergen stability	Stable	UnknownPotential instability may even be beneficial as the unfolded protein may lose IgE-binding capacity

SIT: Specific immunotherapy.

Financial disclosure

The authors have no relevant financial interests related to this manuscript, including employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.