Stefania Conti

Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria

Two recombinant strains of Streptococcus gordonii, secreting or displaying a microbicidal single-chain antibody (H6), and stably colonizing rat vagina, were used to treat an experimental vaginitis caused by Candida albicans. A post-challenge intravaginal delivery of the H6-secreting strain was as efficacious as fluconazole in rapidly abating the fungal burden. Three weeks after challenge, 75% and 37.5% of the rats treated with the H6-secreting or displaying bacteria, respectively, were cured of the infection, which persisted in 100% of the animals treated with a S. gordonii strain expressing an irrelevant single-chain antibody. Thus, a human commensal bacterium can be suitably engineered to locally release a therapeutic antibody fragment.

A Monoclonal Antibody Directed against a Candida albicans Cell Wall Mannoprotein Exerts Three Anti-C. albicans Activities

ABSTRACT Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.

Therapeutic activity of an engineered synthetic killer antiidiotypic antibody fragment against experimental mucosal and systemic candidiasis.

ABSTRACT Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exerted a strong candidacidal activity in vitro, with a 50% inhibitory concentration of 0.056 μM, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a β1-3 glucan molecule, but not by pustulan, a β1-6 glucan molecule. KP also competed with the binding of a KT-like monoclonal IdAb to germinating cells of the fungus. In a rat model of vaginal candidiasis, local, postchallenge administration of KP was efficacious in rapidly abating infections caused by fluconazole-susceptible or -resistant C. albicans strains. In systemic infection of BALB/c or SCID mice preinfected intravenously with a lethal fungal load, KP caused a highly significant prolongation of the median survival time, with >80% of the animals still surviving after >60 days, whereas >90% of control mice died within 3 to 5 days. KP is therefore the first engineered peptide derived from a recombinant IdAb retaining KT microbicidal activity, probably through the interaction with the β-glucan KT receptor on target microbial cells.

Interplay between Protective and Inhibitory Antibodies Dictates the Outcome of Experimentally Disseminated Candidiasis in Recipients of a Candida albicans Vaccine

ABSTRACT Mice immunized with heat-inactivated, whole yeast-form cells (Y cells) of Candida albicans developed intense, specific humoral and cell-mediated immune responses. However, they were modestly protected against a lethal challenge by the fungus, and their sera did not confer passive protection upon nonimmunized animals. Surprisingly, this immune serum conferred an elevated degree of passive protection to normal and SCID mice when preadsorbed on whole C. albicans cells. After adsorption, no antibodies specific to mannoprotein (MP)-rich extracts or secretions were detected by indirect enzyme-linked immunosorbent assay and no serum reaction with the fungal cell surface was seen in immunofluorescence assays. However, this serum had totally preserved the level of other antibodies, in particular those reacting with β-1,3 and β-1,6 glucan (GG). The hypothesis that anti-GG antibodies contributed to the passive protection was suggested by the following circumstantial evidence: (i) mice immunized with C. albicans cells treated with dithiothreitol and protease (YDP cells), which exposed GG on their surfaces and generated anti-GG but not anti-MP antibodies, were substantially protected against a lethal fungus challenge; (ii) the sera, and their immunoglobulin fractions, of mice immunized with YDP cells transferred protection to nonimmune animals; and (iii) this passive protection was substantially abolished by preadsorption on GG but not on intact cells. Overall, our findings demonstrate that some anti-Candida antibodies can block the protective potential of immune serum, a potential to which anti-GG antibodies appear to contribute. Our observations may also help explain why subjects with elevated anti-Candida antibody titers, inclusive of anti-MP and anti-GG antibodies, remain nonetheless susceptible to invasive candidiasis.

Antibodies, killer toxins and antifungal immunoprotection: a lesson from nature?

Abstract Cellular immunity if usually considered the principal defence mechanism against human opportunistic fungi. Recently, detection of protective antibodies has provoked a resurgence of interest in humoral immunity as critical to antifungal protection. In particular, the discovery of human natural fungicidal antibodies acting as internal images of yeast killer toxins has highlighted the importance of the idiotypic network and has suggested new immunotherapeutic approaches, against fungal pathogens.

Protection of Killer Antiidiotypic Antibodies against Early Invasive Aspergillosis in a Murine Model of Allogeneic T-Cell-Depleted Bone Marrow Transplantation

ABSTRACT Antiidiotypic monoclonal antibodies (MAbs) representing the internal image of a yeast killer toxin (KT) have therapeutic potential against several fungal infections. The efficacy of KT MAbs against Aspergillus fumigatus was investigated in a mouse model of T-cell-depleted allogeneic bone marrow transplantation (BMT) with invasive pulmonary aspergillosis. Mice were highly susceptible to infection at 3 days post-BMT, when profound neutropenia was observed both in the periphery and in the lungs. Treatment with KT MAbs protected the mice from infection, as judged by the long-term survival and decreased pathology associated with inhibition of fungal growth and hyphal development in the lungs. In vitro, similar to polymorphonuclear neutrophils, KT MAbs significantly inhibited the hyphal development and metabolic activity of germinated Aspergillus conidia. These results indicate that mimicking the action of neutrophils could be a strategy through which KT MAbs exert therapeutic efficacy in A. fumigatus infections.

Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities.

Background Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. Methodology/Principal Findings CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. Conclusions/Significance The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Idiotypic vaccination: immunoprotection mediated by anti-idiotypic antibodies with antibiotic activity.

Anti‐Id antibodies were raised in mice against a monoclonal antibody (MoAb KT4) that neutralized the in vitro activity of a Pichia anomala yeast killer toxin. Monoclonal antibody was administered to BALB/C syngeneic mice with different schedules of immunization before intravenous challenge with increasing amounts of yeast killer toxin‐sensitive Candida alhicans cells. The course of candidosis was studied in comparison with mice non‐immunized and immunized with an isotypc‐matched unrelated MoAb subdivided into control groups. Protection was reflected by statistically significant increases in survival rate of mice immunized with MoAb KT4 which showed variable serum levels of yeast killer toxin‐like anti‐Id antibodies. MoAb KT4 affinity chromatography purified mouse anti‐Id antibodies were capable of killing in vitro the yeast ceils of the Candida albicans strain used for the experimental infection.

Mycobactericidal Activity of Human Natural, Monoclonal, and Recombinant Yeast Killer Toxin-like Antibodies

Human natural (KTAb), murine monoclonal (KTMAb), and single-chain recombinant (KTScFv) candidacidal antibodies representing the internal image of a killer toxin from the yeast Pichia anomala (KT), characterized by a wide spectrum of antibiotic activity, exerted a lethal effect against a KT-sensitive multidrug-resistant isolate of Mycobacterium tuberculosis. KTMAb and KTScFv were produced by the hybridoma and DNA technologies, respectively, from the spleen lymphocytes of animals immunized with the idiotype of a KT-neutralizing MAb (MAb KT4), while KTAb were purified against MAb KT4 from the vaginal fluid of women infected with Candida albicans cells bearing an idiotype-like KT cell wall receptor. Mycobactericidal activity was related to the binding of KTAb, KTMAb, and KTScFv to the cell surface of KT-sensitive bacterial cells and was prevented by specific absorption of KT-like antibodies onto MAb KT4. These data identify a novel potentially useful immunotherapeutic approach to tuberculosis.

From yeast killer toxins to antibiobodies and beyond.

Antibiobodies are paradigmatic of yeast killer toxin (KT)-like antibodies (KAbs) mimicking the antimicrobial activity of KTs in the frame of the yeast killer phenomenon. Polyclonal, monoclonal and recombinant anti-idiotypic antibiobodies (anti-idiotypic KAbs), internal images of a wide-spectrum KT produced by the yeast Pichia anomala (PaKT), have been produced by immunization with the idiotype of a PaKT-neutralizing monoclonal antibody. Anti-idiotypic KAbs showed microbicidal activity against eukaryotic and prokaryotic pathogenic agents through the interaction with specific KT receptors (KTRs), putatively constituted by beta-glucans. Natural KAbs have been found in animals and humans experimentally or naturally infected by KTR-bearing microorganisms. Recombinant KAb-derived synthetic killer peptides showed further antiviral and immunomodulatory activities. The perspectives of KAbs and killer peptides as potential sources of novel therapeutic agents, and of KTRs and idiotypes as vaccines against infectious diseases are discussed.