Generation of monoclonal antibodies against covalently linked cell wall proteins of Candida albicans

Abstract

Fungal infections have been known for centuries, but their clinical relevance has not been recognized until the last century. The latter half of the twentieth century has seen enormous advancement in the field of medicine and surgery. This has led to the emergence of various forms of organ transplantation and cancer chemotherapy as essential medical treatment, automatically involving immunosupression as part of the procedure. Although many health problems have been adequately addressed in this manner, the ceaseless development in modern medicine has led to the creation of “at risk individuals”, who are extremely susceptible to infections. The increasing number of immunodeficient individuals due to rapid increase in the incidence of AIDS has resulted in an epidemic of diseases caused by opportunistic fungal pathogens. Fungal infections are classified broadly into four groups: invasive infections, lifethreatening infections (e.g. aspergillosis and candidiasis), mucosal infections, skin and allergic infections. The members of the genus Candida are known to be involved in all these four groups of diseases which are commonly known as candidiasis. The genus Candida belongs to the phylum Ascomycetes that has approximately 200 species. So far, 13 species are considered to be pathogenic, of which Candida albicans is the most pathogenic and the most frequently encountered fungus in clinical specimens (1, 2). The main reason for this relies on its common niche, as this fungus inhabits the human gastrointestinal and urogenital tract in a significant part of the population, where it behaves as a harmless commensal organism (2). However, upon alteration of the host defences, C. albicans disseminates within the human body gaining access to internal organs and causing severe infections (called candidiasis) and appears as an opportunistic pathogen. The ability of this fungus to change its morphology from a yeast-like (unicellular) to a filamentous (hyphal and pseudohyphae) form (a property called polymorphism) is regulated by environmental factors, such as the temperature, the pH or the availability of nutrients, and plays a major, albeit non-exclusive role in its ability to produce disease (3, 4, 5, 6). While the biology (life cycle, metabolism and morphogenesis) of all these Candida species greatly differs, they also share certain common features that enable a successful colonization of the human host and are able to counteract its defence mechanisms. Such features are frequently called virulence factors that include metabolic, structural and morphological features (7). Among others, it has been proposed that only those involved in the direct interaction with host cells should be considered as true virulence factors (8). Identification of virulence factors is an active area of research as they may provide the basis for the development of novel therapies to treat fungal infections (9, 10). While several virulence traits have been identified in many fungal species, the cell wall is still the most promising target in drug discovery for different reasons. First, it is unique to the fungus, and therefore, fulfils a basic requirement of selectivity for drug discovery. Second, it is an essential structure to the cell, whose inhibition leads to cell death (most frequently due to cell lysis). Third, and most importantly, it is the most external structure present in the fungal cell and therefore, mediates the interaction of the fungus with the mammalian host cells. As a consequence, it is involved in adhesion, colonization, signalling and immune recognition, and therefore plays a major role during infection (11). There are a number of antifungal agents available in the market to combat the fungal infections and most of them are having fungistatic effect barring amphotericin B which is a cidal drug. Moreover these antifungals are not free from side effects and the situation is further complicated due to emergence of resistance among the patients. Most commonly used antifungal agents like azoles, allylamines and morpholines inhibit the ergosterol biosynthetic pathway. Others, such as polyenes and echinocandins, impair membrane barrier function and glucan synthesis respectively (12). The incidences of C. albicans cells acquiring resistance to antifungal agents like azoles and its derivatives has increased considerably in recent years which have posed serious problems towards its successful chemotherapy, probably due to widespread use of azole drugs in cancer and AIDS patients suffering from secondary fungal infections (13, 14, 15, 16). Amphotericin B, a fungicidal drug in nature has also been described to pose the problem of resistance among some Candida species and various fungi including several Aspergillus species (15, 16). To overcome these problems, new antifungal agents targeting C. albicans and other opportunistic pathogens are urgently needed. Therefore, it is essential to dissect the infectious process of C. albicans to explore new target molecules and to search new effective, safe and broad spectrum antifungal strategies. Production of antibodies against fungal antigens/molecules is one of the significant outcomes of the host/fungal interaction, which are important contributors to host immunity during the fungal infections. Recently, a number of monoclonal antibodies have been developed for therapeutic use against fungal infections including candidiasis for effective and safe treatment. Two monoclonal antibody based therapeutics are under clinical evaluation for treatment of fungal infections. Antibodies offer protection against candidiasis by a variety of mechanisms like opsonisation-mediated phagocytosis, inhibition of germ tube formation, inhibition of attachment of pathogen with host tissue, and direct candidacidal activity (17, 18). The development of new technologies, such as hybridoma technology for generation of monoclonal antibodies, antibody engineering and proteomic techniques that facilitated the identification of target molecules present in complex proteome, have led to a renewed interest in the study of antibody responses against fungal infections. Now a days, monoclonal antibodies are frequently used in rapid identification of C. albicans and other related species, characterization of target molecules /epitopes in the process of cell wall assembly and in diagnosis of candidiasis by detection of circulating antigens in patients (19, 20). Cell wall is the outermost cellular structure, which determines the shape of fungal cell. Cell wall remodelling is required for the morphological conversion of C. albicans from the yeast to the filamentous form, which involves alteration in its composition and organization. It indicates plastic and dynamic nature of the cell wall, which changes constantly in response to environmental signals, and the different stages of the fungal cell cycle. Because of its privileged location within the cell, cell wall is also the initial point of contact between the cell and the environment and thus contributes to host fungus interaction. In addition given that mammalian cells lack a cell wall, this cellular compartment could be a promising molecular site to search for new specific antifungal drug targets. Further, the cell wall proteins play a key role in morphogenesis and pathogenesis (21, 22, 23). The cell wall of Candida albicans consists of an internal skeletal layer and an external protein envelop. This layer has a mosaic-like nature, containing approximately 20 different protein species covalently linked to the skeletal layer. Most of them are glycosylphosphatidylinositol (GPI) proteins. Covalently linked cell wall proteins vary widely in function and many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell-cell aggregation. An important role in the fitness and virulence of C. albicans is reserved for those cell wall proteins that are covalently linked to the skeletal cell wall polysaccharides. Covalently linked cell wall proteins play a crucial role in the capability of C. albicans to survive and grow in the host and to cope with the stress conditions associated with the host infections (24, 25). The main objective of the present work was to generate monoclonal antibodies against covalently linked proteins of C. albicans cell wall and to evaluate their therapeutic and/or diagnostic value for candidiasis. In this piece of work, isolation and characterization of covalently linked proteins from cell wall of both yeast and hyphae of C. albicans was done. The covalently linked cell wall proteins were characterized by peptide mass fingerprinting using MALDI-TOF-MS technique. As GPI cell wall proteins consist of largest fraction in covalently linked proteins, hyphal GPI cell wall proteins were used for generation and evaluation of monoclonal antibodies for their therapeutic and diagnostic potential in vitro and also against in vivo model of murine candidiasis. Paratope derived ppeptides were designed from the sequences obtained by reverse transcription and cDNA sequencing of hybridoma line showing the most protective response. Along with generation of monoclonal antibodies, amphotericin B resistant strain of C. albicans was developed and characterized on the basis of various virulence factors and at genetic level too. Monoclonal antibodies generated against covalently linked cell wall proteins were also evaluated for their efficacy on amphotericin B resistant strain.