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 microM, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a beta1-3 glucan molecule, but not by pustulan, a beta1-6 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 beta-glucan KT receptor on target microbial cells.

FIG. 1.

The ScFv H6 gene nucleotide and translated amino acid sequences. P6 amino acids are shown in italics. For other details, see the text.

FIG. 2.

Clearance of vaginal candidiasis in rats intravaginally administered KP, SP, or fluconazole or untreated. All rats (five per group) were challenged intravaginally with 10⁷ C. albicans cells (strain SA-40) in 0.1 ml of physiological saline on day 0 and then sampled for initial intravaginal CFU. The therapeutics (KP, SP, and fluconazole; 50 μg each) were administered 1, 24, and 48 h after the challenge. Starting on day 1, there was always a statistically significant difference (P < 0.05) in the vaginal CFU counts between untreated or SP-treated and KP- or fluconazole-treated rats. No statistically significant difference was at any time point found between SP-treated and untreated animals (except at the last day of measurement), and no statistically significant difference was similarly found at any time point between KP- and fluconazole-treated rats. Statistical significance was assessed by using Student's t test (two-tailed).

FIG. 3.

BALB/c (A) or SCID (B) mice (eight per group) were infected intravenously on day 0 with 10⁷ C. albicans cells (strain SA-40; predetermined to correspond to five LD₅₀'s) and then intraperitoneally injected with 50 μg of KP, SP, or fluconazole in saline at 1, 24, and 48 h after the infectious challenge. Untreated mice received saline only. Mortality was evaluated for 60 days, and dead mice were subjected to necropsy to assess the presence of the fungus in one target organ (kidney). The differences in the median survival time (in days) or in the ratio of dead versus total animals on day 60 were assessed by using the Mann-Whitney U test or by using Fisher's exact test, respectively. For both endpoint parameters, there was a highly significant (P < 0.01) statistical difference between untreated (or SP-treated) and KP-treated (or fluconazole-treated) mice. No statistical difference was found between KP- and fluconazole-treated SCID mice or between untreated and SP-treated mice.