Preclinical assessment of the efficacy of mycograb, a human recombinant antibody against fungal HSP90.

Mycograb (NeuTec Pharma plc) is a human genetically recombinant antibody against fungal heat shock protein 90 (HSP90). Antibody to HSP90 is closely associated with recovery in patients with invasive candidiasis who are receiving amphotericin B (AMB). Using in vitro assays developed for efficacy assessment of chemotherapeutic antifungal drugs, Mycograb showed activity against a wide range of yeast species (MICs against Candida albicans [fluconazole [FLC]-sensitive and FLC-resistant strains], Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, 128 to 256 microg/ml). Mycograb (4 or 8 microg/ml) showed synergy with AMB, the fractional inhibitory index being 0.09 to 0.31. Synergy was not evident with FLC, except for FLC-sensitive C. albicans. Murine kinetics showed that Mycograb at 2 mg/kg produced a maximum concentration of drug in serum of 4.7 microg/ml, a half-life at alpha phase of 3.75 min, a half-life at beta phase of 2.34 h, and an area under the concentration-time curve from 0 to t h of 155 microg. min/ml. Mycograb (2 mg/kg) alone produced significant improvement in murine candidiasis caused by each species: (i). a reduction (Scheffe's test, P < 0.05) in the mean organ colony count for the FLC-resistant strain of C. albicans (kidney, liver, and spleen), C. krusei (liver and spleen), C. glabrata (liver and spleen), C. tropicalis (kidney), and C. parapsilosis (kidney, liver, and spleen) and (ii). a statistically significant increase in the number of negative biopsy specimens (Fisher's exact test, P < 0.05) for C. glabrata (kidney), C. tropicalis (liver and spleen), and C. parapsilosis (liver). AMB (0.6 mg/kg) alone cleared the C. tropicalis infection but failed to clear infections caused by C. albicans, C. krusei, C. glabrata, or C. parapsilosis. Synergy with AMB, defined as an increase (Fisher's exact test, P < 0.05) in the number of negative biopsy specimens compared with those obtained using AMB alone, occurred with the FLC-resistant strain of C. albicans (kidney), C. krusei (spleen), C. glabrata (spleen), and C. parapsilosis (liver and spleen). Only by combining Mycograb with AMB was complete resolution of infection achieved for C. albicans, C. krusei, and C. glabrata.

Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy.

The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain. Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P<0.05). Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ. The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis. A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B. subtilis. Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands. Direct amino acid sequencing identified this as a possible ABC transporter. Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site cross-reacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively. This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen. Epitope mapping of this antigen with seropositive patients' sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI). The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library. An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection. This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections.

The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex.

Biosynthesis of the Escherichia coli K5 polysaccharide requires the KfiA, KfiB, KfiC and KfiD proteins. The subsequent transport of the polysaccharide onto the cell surface requires the KpsC, KpsD, KpsE, KpsM, KpsS and KpsT proteins, which are conserved between different group II capsular polysaccharides. The KfiA and KfiC, together with the KpsC, KpsS and KpsT proteins, were purified and polyclonal antisera to each protein generated. These antisera, together with one previously generated (by others) against the purified KfiD protein, were used in Western blot analysis to locate the corresponding proteins within the cell. Analysis of membrane fractions revealed that KfiA (involved in initiation of polysaccharide synthesis), KfiC (K5 glycosyl transferase) and the KfiD protein (UDP-glucose dehydrogenase) were associated with the inner membrane. The KpsC, KpsS, and KpsT proteins involved in polysaccharide transport were associated with the inner membrane and this membrane association occurred in the absence of any other capsule-related proteins. The effect of mutations in individual kps genes on the localization of each protein was determined. Mutations in the kpsC, kpsM, kpsS and kpsT genes resulted in a loss of membrane targeting for KfiA and KfiC, suggesting some form of hetero-oligomeric membrane-bound biosynthetic complex. Osmotic shock caused the release of KfiA, KfiC, KpsC and KpsS from the inner membrane into the periplasm, suggesting that the polysaccharide biosynthetic complex may be associated with sites of adhesion between the inner and outer membrane.