Human recombinant antibody to HSP90: a natural partner in combination therapy.

Recent years have seen the development of the concept of combination therapy for treating severe fungal sepsis. The advantages of this approach are a potential improvement in patient survival and a reduction in the chance of resistance developing to each of the single agents. The disadvantage is that combining drugs may increase the chance of toxicity. Mycograb is a genetically recombinant antibody against fungal heat shock protein 90 (hsp90) which is poised to become the mainstay of combination therapy. This paper presents data on how hsp90 is important to fungi and what role it might play in human disease with possible interactions with interleukin 6 and nitric oxide. There is discussion of preclinical data demonstrating synergy in vitro between Mycograb and amphotericin B and caspofungin. The progress of Mycograb through a Phase II pharmacokinetic study when used in escalating doses with a liposomal amphotericin B preparation has also been reviewed. The concepts behind a Phase II pivotal study, where Mycograb or a placebo was given in combination with a liposomal amphotericin B drug for five days for the treatment of disseminated candidiasis are discussed.

Differential growth of epidemic methicillin-resistant Staphylococcus aureus in vancomycin.

This study examines the ability of isolates representing the 17 epidemic methicillin-resistant strains of Staphylococcus aureus to grow in increasing levels of vancomycin. Only EMRSAs 1, 2, 8, 11, 12 and 15 showed any growth and were designated EMRSAs 1A, 2A, 8A, 11A, 12A and 15A. On population analysis, these strains all produced clones that grew on 32 microg/mL vancomycin, while EMRSA 12A and 15A grew at 128 microg/mL. This was associated with increased resistance to lysostaphin and teicoplanin, a loss of phage sensitivity and an increase in cell wall diameter. Typing by pulsed-field gel electrophoresis following SmaI digestion showed no change in EMRSA 8A and 15A, while the other EMRSAs all lost or gained at least one band. EMRSAs 1A, 2A and 15A became more resistant to methicillin, and EMRSAs 8A, 11A and 12A became less resistant to methicillin. These results suggest that more than one mechanism is responsible for this phenomenon.

Immunoblot analysis: a new method for fingerprinting hospital pathogens.

Immunoblotting has recently become popular as a way of fingerprinting those hospital pathogens where other more conventional typing systems are deficient. Extracts of microorganisms are prepared by chemical or enzymic means, run on a standard SDS-PAGE gel and transferred onto nitrocellulose membrane. They are then probed either by a hyperimmune antiserum raised in a rabbit or by serum from a patient who has been previously infected by the organism. The pattern of antigenic bands which stain forms the basis of the method. This article discusses the limitations of the system, makes recommendations for further systems and outlines a typical fingerprinting protocol.

Immunoblot fingerprinting Aspergillus fumigatus.

A new technique for typing Aspergillus fumigatus is presented. This is based on immunoblot fingerprinting each isolate against a rabbit hyperimmune antiserum raised against A. fumigatus NCTC 2109. All isolates were typable and reproducibility for the 16 antigenic bands which formed the basis of the system was excellent. Discrimination was better than silver staining and revealed 11 types among the 21 isolates from eight patients with an aspergilloma. Each aspergilloma could be due to either a single or multiple types.

Restriction endonuclease analysis of Aspergillus fumigatus DNA.

AIMS: To develop a genome based DNA fingerprinting system for Aspergillus fumigatus mould. METHODS: DNA was extracted from 21 isolates obtained from eight patients with an aspergilloma. This was with a freeze-dried mycelial extract fragmented in liquid nitrogen. DNA was subsequently purified by phenol-chloroform extraction followed by ultracentrifugation on a caesium chloride gradient. The DNA was restricted by EcoRI and Xba I. RESULTS: All isolates were identical when cut by EcoRI; Xba I delineated six DNA types. CONCLUSIONS: DNA fingerprinting can be used to type isolates of A fumigatus. Strains from within an aspergilloma which were morphologically distinct could either have the identical DNA fingerprint or produce a unique type.

A polymerase chain reaction enzyme immunoassay for diagnosing infection caused by Aspergillus fumigatus.

AIM: To develop a polymerase chain reaction enzyme immunoassay (PCR-EIA) to measure levels of circulating aspergillus DNA in invasive aspergillosis caused by Aspergillus fumigatus. METHODS: The PCR reaction was based on primers from the 18s rRNA gene. Binding of the product to a streptavidin coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR and this was the tag to which antibody was bound in the subsequent EIA. RESULTS: The optical density (OD) endpoint was < 0.1 in 10 sera from neutropenic patients with no evidence of invasive aspergillosis, and in 10 sera from nonneutropenic patients with bacterial pneumonia (group 1). The OD from five of 12 patients with allergic bronchopulmonary aspergillosis (ABPA) (group 2), three with an aspergilloma (group 3), and five with possible invasive aspergillosis (group 4) was > or = 0.1. In 63 sera from 33 cases of proven invasive aspergillosis (group 5) an OD > or = 0.1 was achieved in 48 sera from 30 patients. The maximum OD was 0.510. The level fell in survivors and gradually rose in fatal cases. CONCLUSIONS: This assay validated the concept of diagnosing invasive aspergillosis by measuring levels of circulating fungal DNA in serum.

Immunoblot analysis of serological response to Pseudomonas aeruginosa septicaemia in man.

The occurrence of an outbreak of Pseudomonas aeruginosa septicaemias on an oncology ward permitted an analysis of antibody responses in patients who were all orally exposed to the same source of infection. Seven patients became septicaemic. Serial serum samples were immunoblotted against the homologous strain. Responses were compared with those of 16 other patients with septicaemias caused by other strains and 10 healthy controls. All 18 survivors produced increasing IgG or IgA antibody, or both, against a 35,000 dalton band, whereas these antibodies were usually absent or fell in titre in the five fatal cases. These antibodies were also lacking in sera taken just before a patient became septicaemic. This band had the electrophoretic characteristics of the outer membrane porin protein F.

Role of immunoblotting in the diagnosis of culture negative and enterococcal endocarditis.

Serum samples from patients with endocarditis and septicaemia due to Enterococcus faecalis, Enterococcus faecium, Streptococcus bovis, and Streptococcus sanguis were immunoblotted against antigenic extracts from all four species. In E faecalis endocarditis there was a strong IgM response to E faecalis antigenic bands of 112, 88-90, and 45-47 Kd and a strong IgG response to 88-90 and 45-47 Kd bands. In E faecium endocarditis there was a pronounced IgG response to an E faecium band of 82-90 Kd. For S bovis endocarditis, there was a strong IgG response to several components of S bovis including bands of 66, 58, 52 and 4 Kd. For S sanguis, there was a strong IgG response to bands of 80-82, 76, 60 and 45 Kd. These patterns of antibody production were absent in patients with uncomplicated septicaemia and in controls. The delineation of these patterns enabled confirmation of the final diagnosis in seven patients initially suspected of having culture negative endocarditis.

Use of immunoblotting to identify antigenic differences between the yeast and mycelial phases of Candida albicans.

Western blotting was applied to the analysis of Candida albicans in the yeast and mycelial phases in an attempt to recognise mycelial specific antigens which might be of serodiagnostic value. The antisera were prepared in rabbits by immunising them with pressates of C albicans type A NCTC 3153 in the yeast phase or the mycelial phase. These were blotted against C albicans in the yeast and mycelial phases and the yeast phase of C parapsilosis, C krusei, C tropicalis, and Torulopsis glabrata. Cross reactivity was greatest against C parapsilosis. One yeast specific mannoprotein was identified with a molecular weight of 49 000. No mycelial specific antigens could be identified.

The 14th C. L. Oakley Lecture. Candida albicans HSP 90: link between protective and auto immunity.

Heat shock proteins (HSP) are thought to play a role in the aetiology of autoimmune diseases, but are also common targets for the immune response to many infections. Patients recovering from systemic candidosis produce antibodies to Candida albicans HSP 90, both to species-specific epitopes and, more commonly, to epitopes shared with human HSP 90. One such autoreactive antibody was protective in a mouse model of systemic candidosis.

Application of the polymerase chain reaction to the diagnosis of candidosis by amplification of an HSP 90 gene fragment.

A 317-base pair (bp) fragment of the Candida albicans heat shock protein 90 (HSP 90) gene was amplified by the polymerase chain reaction (PCR) for detection of C. albicans DNA in clinical specimens. One hundred specimens were examined including swabs (39), urines (36), peritoneal fluid (9), pus (8) and blood or serum (8): 23% gave positive results with routine culture, 31% with extended broth culture and 37% with PCR. The amplified product was identified by hybridisation with a radiolabelled internal probe and their restriction enzyme digest patterns (SspI, HaeIII, EcoRI, RsaI and XhoI), which could be predicted from the known sequence of HSP 90. C. albicans DNA gave the characteristic 317-bp band and specifically hybridised with restriction enzyme-digested candidal DNA. DNA from other sources intermittently gave multiple faint bands especially in the presence of high concentrations of DNA, but these could be readily distinguished. The method was sensitive to 50 pg of DNA (5 pg with radiolabelled probing) and 100 cfu of C. albicans.

A comparison of immunoblot and DNA restriction patterns in characterising methicillin-resistant isolates of Staphylococcus aureus.

The ability of EcoR1 restriction enzyme fragmentation patterns and of immunoblotting to differentiate methicillin-resistant isolates of Staphylococcus aureus were compared. All isolates examined were typable by both methods and the reproducibility of each was excellent. Immunoblotting differentiated eight types and DNA restriction patterns four. The former technique was of value in characterising methicillin-resistant isolates of S. aureus and controlling an outbreak due to them.

Epitope mapping Candida albicans proteinase (SAP 2).

The continuous epitopes of Candida albicans proteinase SAP 2 were derived by epitope mapping with sera from patients with oral candidiasis (n = 3), necropsy-proven disseminated candidiasis (n = 5), paired sera from patients who had recovered from blood culture-proven disseminated candidiasis (n = 3) and infection due to Candida parapsilosis (n = 2) and Candida tropicalis (n = 2). In C. albicans infection, IgM identified epitopes in amino acid positions 57-61 (QAVPV), 146-151 (SQGTLY) and 346-351 (PYDKCQ) and IgG at position 386-390 (VKYTS). For C. tropicalis IgM and IgG were positive for the same epitopes whilst IgG also detected epitopes at 78-83 (SNNQKL) and 159-164 (GVSIKN). For C. parapsilosis, IgM was positive for SNNQKL and IgG detected no epitopes. Reactivity of two of the epitopes as peptides KTSKRQAVPVTL and SLAQVKYTSASSI was confirmed in an indirect ELISA. At a cut-off optical density of 0.4, IgM against either peptide was associated with survival but present in only about half of the sera (n = 60) from patients who recovered from disseminated candidiasis whilst IgG levels were disappointing. Human recombinant antibodies from a patient who had recovered from disseminated candidiasis against either of these peptides had no activity in a lethal mouse model of candidal infection.

Epitope mapping human heat shock protein 90 with sera from infected patients.

Epitope mapping with sera from a range of infected patients showed that antibodies are commonly produced which cross-react with a number of epitopes on human heat shock protein 90 (HSP 90). Such autoreactive antibodies were particularly frequent in patients suffering from systemic candidiasis (9 patients), invasive aspergillosis (6 patients), ABPA (2 patients), a patient with aspergilloma and one with malaria. The patient with malaria recognized similar epitopes to those with invasive aspergillosis and candidiasis including the highly conserved epitope LKVIRKVIRK and an epitope NNLGTI which was otherwise only recognised by patients with candidiasis. Crossreacting antibodies to relatively few epitopes occurred in patients with Enterococcus faecalis and Corynebacterium jeikeium endocarditis. This was contrasted with the results from 6 patients with systemic lupus erythematosus (SLE) who were positive on immunoblot against fungal HSP 90. These did not react with the above epitopes but reacted with other areas within human HSP 90.

Defining potential targets for immunotherapy in Burkholderia cepacia infection.

Burkholderia cepacia has become a serious source of infection in patients with cystic fibrosis. Antibiotic therapy is difficult as the bacteria are intrinsically resistant to most antibiotics. The present study compared the antibody response by immunoblot of 50 negative control sera, 22 patients with cystic fibrosis and no evidence of B. cepacia, 9 clinically well patients with cystic fibrosis colonised by B. cepacia and 5 patients with cystic fibrosis and deteriorating or fatal B. cepacia infection. Nineteen antigenic bands varying in apparent molecular weights from 19 to 170 kDa were identified. Two bands at 19 and 21 kDa were only present when the organism was grown in an iron-deficient medium. The band at 30 kDa was identified as a porin and the possession of IgG antibody carried a statistically significant (P = 0.00003) better prognosis. This antigen was thus a potential target for immunotherapy.

Hospital outbreaks with yeasts.

Five previous outbreaks of disseminated candidosis due to Candida albicans are reviewed and a new outbreak on a neonatal unit in Belfast presented. This involved four disseminated cases. The control and definition of outbreaks by morpho-, immunoblot- and DNA-typing is discussed. An outbreak of Torulopsis glabrata infection involving 23 patients is described. This was defined by DNA fingerprinting with the enzyme Xba. There were five deaths attributable either completely or in part to the yeast infection.

A comparison of the DNA fragment patterns of the mouse-virulent challenge strains and clinical isolates of Bordetella pertussis.

The DNA fragment patterns of two mouse-virulent strains of Bordetella pertussis, commonly used in animal protection tests, were compared to those of clinical isolates. Strain W.18-323 and six variants were examined together with strain 353/Z and two variants. Chromosomal DNA was digested with the rare-cutting enzyme Xba I in order to produce relatively large fragments resolved by pulsed-field gel electrophoresis. The resulting DNA fragment patterns of strain 353/Z and those of its variants were indistinguishable from those of the commonest DNA type among clinical isolates. In contrast, strain W.18-323 and its variants showed a distinct genetic type, different from clinical isolates of B. pertussis, B. parapertussis or B. bronchiseptica. This finding, together with some phenotypic anomalies of this strain, raises doubts as to the suitability of W.18-323 as a challenge strain for assessing the efficacy of vaccines in the mouse model.

Transient abrogation of immunosuppression in a patient with chronic mucocutaneous candidiasis following vaccination with Candida albicans.

A patient with long-standing chronic mucocutaneous candidiasis had reversed T helper/suppressor (TH/TS) cell ratios, hypergammaglobulinaemia E and serum inhibitors of lymphocyte transformation to mitogens and candida antigens. Following vaccination with whole heat-killed yeasts of Candida albicans, temporary clinical improvement coincided with the return of the TH/TS cell ratio to normal, reduction in concentration of IgE and reduced serum inhibitory activity to Concanavalin A and candida antigens. These changes were not permanent and 6 months after vaccination all indices had reverted to their pretreatment values. The production of antibody to a 47 kDa antigen of C. albicans has been shown to coincide with recovery from systemic disease. High concentrations of this antibody were demonstrated initially in the patient's serum and were unaffected by vaccination. If the 47 kDa antibody is protective, the results of this study suggest that separate immune responses may protect against mucocutaneous and systemic candidiasis and that defective immune responses against mucocutaneous disease need not imply lack of protection against systemic spread.

Analysis of the antibody response developing in an infant with Candida albicans meningitis.

A case of meningitis due to Candida albicans in a neonate is described. This is the first report of an immunoblot analysis of both serial samples of serum and cerebrospinal fluid in relation to C. albicans antigens. The western blot technique demonstrated that the mother had IgM antibody against five candidal antigens, including one of molecular weight 47 kDaltons, but only a small amount of IgG antibody against two antigens of molecular weight 104 kDaltons and 60 kDaltons respectively. The baby produced IgM antibody and then IgG antibody against an antigen of 47 kDaltons molecular weight, and this was associated with recovery.

Analysis of antibody response to Cryptococcus neoformans in five patients with AIDS and cryptococcosis by immunoblotting.

OBJECTIVES: The serological response of five patients with AIDS and cryptococcosis to non capsular antigens from Cryptococcus neoformans var. neoformans has been investigated. METHODS: Pressates of different isolates of C. neoformans were used as antigenic preparation for immunoblotting of patient samples. RESULTS: Multiple sera and cerebrospinal fluids sequentially collected from five AIDS patients with cryptococcosis showed a wide heterogeneity in antibody response with bands at 48. 43, 38 and 26 kD being present in all clinical samples of all five patients. The variation in banding patterns of the sequential samples from three patients was correlated with a decrease of the antigen titre and with an amelioration of the cryptococcal infection. CONCLUSIONS: We identified antibodies to four immunodominant non-capsular antigens, which might represent major target molecules of the humoral response of patients with cryptococcosis.