Identification of adjuvants that enhance the therapeutic antibody response to host IgE

Abstract

In the development of a novel vaccine against atopic allergies, we have screened for adjuvants that enhance the therapeutic antibody response against self immunoglobulin E (IgE). The response against self IgE is induced by administration of a vaccine antigen, which contains both self and non-self IgE regions, together with an adjuvant. We evaluated five commonly used adjuvants; Freund’s, aluminium hydroxide, ISCOMs, Montanide ISA 51 and Montanide ISA 720, and found that the mineral oil-based adjuvants; Montanide ISA 51 and Freund’s induced at least 5–10-fold higher anti-self IgE titers than any of the other candidates. However, with one exception, Alum, the immune responses against the carrier, i.e. the non-self regions, were similar for all adjuvants, indicating that the ability to induce responses against self and non-self antigens differ among adjuvants. The responses against non-self IgE were more than 50-fold higher than antibody responses against self IgE in both the Freund’s and Montanide 51-administered animals, indicating that the response against self molecules is markedly inhibited by tolerance-inducing mechanisms. Co-administration of Montanide ISA 51 with immuno-stimulatory substances from bacteria; muramyldipeptide (MDP), monophosphoryl-lipid A (MPL) or a formyl-methionine-containing tripeptide (fMLP), did not elevate the anti-self IgE response. Hence, adjuvants based on pure mineral oil without additional immuno-stimulatory substances appear to be the best adjuvant candidates in therapeutic vaccines aimed at regulating the in vivo levels of self-proteins.

Introduction

Ever since Jenner’s first attempt in 1798, vaccination has been the preferred and also the most successful method of controlling most viral and many bacterial diseases. Successful vaccination programs have resulted in a tight control or even in the eradication of many of the major human infectious diseases. This has encouraged researchers in other fields to develop vaccines against other clinical conditions.

During the past few decades several diseases caused by malfunctions of our own immune system, such as the allergic and autoimmune diseases, have become some of the major challenges of modern day medicine. Allergic diseases have increased to epidemic proportions in the past 20–30 years and estimates suggest that 20–30% of the total population is affected. The dominant form is atopic allergies, or IgE-mediated allergies, which include hay fever, fur allergies, dust mite allergies, insect venom allergies, extrinsic asthma and many types of food allergies. One interesting question is whether vaccines can also be developed against this type of diseases. One form of vaccination, so called hyposensitization therapy or immunotherapy has been used to treat allergies since the beginning of the 20th century [1], [2]. This is an allergen dependent treatment strategy, which involves the use of allergen extracts to treat patients by injection. However, hyposensitiztion therapy has been questioned due to the often low efficacy and occasionally severe side effects, and this strategy is also limited by the fact that different extracts have to be used for each individual form of allergy. New strategies to treat allergies are therefore presently being evaluated. Vaccines aimed at modulating the levels of various self-antigens is an interesting area and IgE and IL-5 are two promising target molecules.

New and powerful molecular tools facilitate the production of recombinant proteins to be used as vaccine antigens. However, most of these highly purified antigens lack some of the characteristics of classical vaccine antigens, e.g. heat-killed or attenuated viruses and bacteria. The recombinant antigens are usually less potent in inducing strong immune responses and they therefore need to be used together with strong immuno-potentiating agents, so called adjuvants. In an attempt to develop a vaccine against atopic allergies by targeting IgE, the central molecule in this type of disease, we have encountered some of these challenges [3], [4], [5], [6]. In addition, the induction of immune responses against self-proteins poses additional challenges not normally seen with entirely foreign antigens, like bacterial or viral proteins. Due to tolerance induction self-proteins are usually much less immunogenic. In order to address these questions we have screened a panel of different adjuvants for their potency in inducing an antibody response against self IgE. We have also compared the response against self IgE to the response with the foreign carrier molecule to obtain an estimate of the difference in antigenicity between self and non-self epitopes.

Agents that enhance the immune response to an antigen beyond the level achieved by the antigen alone are referred to as immunological adjuvants, reviewed in [7]. Freund’s complete and incomplete adjuvants (FCA and FIA, respectively) are the most widely used adjuvants for studies in laboratory animals. FCA is a water-in-mineral oil emulsion containing heat-killed Mycobacterium tuberculosis while FIA contains only mineral oil and the emulsifier, a lipophilic surfactant named mannide monooleate. Freund’s adjuvant, which was used as a standard in this analysis is known to be a very potent adjuvant. However, due to the low purity of the oil and the emulsifier, FCA is considered too reactogenic for use in humans [8]. Aluminum compounds are the only commonly used adjuvants for clinical use. However, for most antigens, aluminum hydroxide is a relatively poor adjuvant (reviewed in [9]).

Due to the need for novel safe and potent adjuvants for human and veterinarian use many additional adjuvants are under active evaluation [8], [10], [11]. Highly purified mineral oils are one interesting set of candidates. Several companies are producing various combinations of well-defined mineral oils and emulsifiers [8]. In addition, combinations of mineral and biodegradable oils, primarily squalene or plant oils, are currently under evaluation. Several of these adjuvants are already in clinical trials [8]. One additional active area of adjuvant research is the use of immuno-stimulating complexes (ISCOMs) [12], especially the development of small well-defined particles made up of cholesterol and saponins from the bark of a tropical tree, Quillaja saponaria. ISCOMs are presently used in several veterinary applications, for example a commercial horse influenza vaccine [13]. A recent interesting development is the incorporation of specific affinity tags in the ISCOM, which allows more efficient binding of recombinant proteins to the ISCOM surface, e.g. incorporation of Cu²⁺ chelating agents on the ISCOM that interacts with poly-histidine tags on the protein [14].

Many bacterial components have been shown to have strong immuno-potentiaiting effects. However, most of the crude extracts used to date are also rather toxic. To solve these problems, considerable effort has been devoted to isolating and purifying the specific components, and to identify regions within these bacterial components, that exert the immuno-stimulatory effect. Several such compounds have been identified and these are now available for a more detailed analysis of their potency as adjuvants. One such compound is muramyldipeptide (MDP) a component of the cell wall of bacteria [15]. It has been shown not only to enhance the potency of weak antigens, but also to increase resistance to bacterial, fungal and viral infections. A second highly interesting molecule is lipid A, a structural component of the lipopolysaccharide (LPS) located in the outermost of the double lipid bilayers that constitute the cell membrane of Gram-negative bacteria. It has been shown that the toxic and adjuvant properties of LPS is due to the lipid A portion. By the removal of a phosphate group from a sugar moiety, Ribi et al. [16] managed to decrease the toxicity 100- to 1000-fold. However, the immune stimulatory properties of the molecule were maintained in the molecule. Baldridge and Crane [17] reduced the toxicity further by the removal of an ester-linked fatty acid group. A third class of molecules of interest is formyl-methionin-containing tripeptides (fMLP), a biproduct of bacterial protein synthesis that has also been shown to have immuno-stimulatory effects probably due mainly to its chemotactic activity on several immune cells.

As a further step in the development of novel strategies to treat atopic allergies in man, we present here an analysis of the capacity of these immuno-stimulatory substances to induce both an anti-self IgE and an anti-non-self IgE response in an animal model, the Wistar rat.