Isolation of Human Fabs With Unique Binding Specificities to Candida albicans Serotypes A and B Mannan, Cloned From A Combinatorial Phage Display Library Joshua C. Steichen Department of Microbiology, California State University, Long Beach, Long Beach, 90840

OpeningOpening  Introduction including background information on C. albicans and the previous experiments involving this pathogenic yeast.  An discussion of our experiment including the materials and methods used.  An overview of combinatorial phage display library construction and use.  The results and observations.  Discussion of conclusions and possible future experiments.

Introduction The yeast, Candida albicans in an opportunistic pathogen that causes disseminated candidiasis, a disease which often results in the death of immuno-compromised patients. Conventional use of antifungal agents have had minimal effects against candidiasis. Therefore, alternative methods of treatment are being researched. Previous studies have shown that anti-C.albicans antibodies can be used to pass passive immunity in mice. Active immune responses in mice have also been induced by vaccination with mannan, a highly studied cell surface constituent of C. albicans, which has strong antigenic properties. It is hoped that future research will lead to the development of a treatment method by which passive or active immunization can be achieved in humans.

Experiment The goal of this project was to purify mannan, and to isolate human anti-mannan Fabs from a combinatorial phage display library. C. albicans, strains 36801C (serotype A) and A9 (serotype B) were grown up in YBP broth. Mannan was extracted and purified from each of the cell types by removing the cell lipids with acetone and then extracting the water soluble cell wall components by dialysis. Mannan was precipitated with the addition of Fehling solution. C. albicans, strains 36801C (serotype A) and A9 (serotype B) were grown up in YBP broth. Mannan was extracted and purified from each of the cell types by removing the cell lipids with acetone and then extracting the water soluble cell wall components by dialysis. Mannan was precipitated with the addition of Fehling solution. Cell prep. and mannan purification

Experiment Phage display library construction Human lymphocytes from bone marrow were used to generate a cDNA library. The library was screened with primers designed to amplify sequences of light and heavy chains that are commonly utilized in immunoglobulin producing B cells. The purified sequences were then ligated into a pComb 3H phagemid vector. The light chains were digested with SacI and XbaI, the heavy chains were digested with XhoI and SpeI, and the vector was cut with all four restriction enzymes. The inserted sequences were ligated downstream of lac Z promoter and the heavy chain sequence was ligated to phage DNA, gene III. Gene III encodes for a phage coat protein. E. coli transformed by the phagemid and phage particles are produced with the help a helper phage VCSM13. The helper phage contains the proteins necessary for phage assembly. Human lymphocytes from bone marrow were used to generate a cDNA library. The library was screened with primers designed to amplify sequences of light and heavy chains that are commonly utilized in immunoglobulin producing B cells. The purified sequences were then ligated into a pComb 3H phagemid vector. The light chains were digested with SacI and XbaI, the heavy chains were digested with XhoI and SpeI, and the vector was cut with all four restriction enzymes. The inserted sequences were ligated downstream of lac Z promoter and the heavy chain sequence was ligated to phage DNA, gene III. Gene III encodes for a phage coat protein. E. coli transformed by the phagemid and phage particles are produced with the help a helper phage VCSM13. The helper phage contains the proteins necessary for phage assembly.

Cloning human genes for antimannan Fab fragments from a combinatorial phage display library Library Human lyphocytes from bone marrow RNA  cDNA, PCR cloning V L -C L V H -C H stop Phagemid pComb 3H lac Z promoter amp R Light ChainHeavy Chaingene III Sac IXba IXho ISpe I Transform E. coli & make phage particles with Helper phage

Experiment Enrichment of anti-C.albicans Fab displaying phages by panning Phage clones retrieved from E. coli cells were incubated in wells arranged in columns containing either C. albicans serotype A, serotype B, or with no cells at all. The cells were washed, centrifuged and resuspended several times & were finally resuspended in trypsin/TBS solution to elute the phages. The phages eluted from each column were allowed to infect E. coli for amplification. The phages produced by each bacterial colony were retrieved and incubated with the C. albicans strains, and the cells were washed and phages were extracted just as before. This enrichment cycle was preformed for 4 rounds. Phage clones retrieved from E. coli cells were incubated in wells arranged in columns containing either C. albicans serotype A, serotype B, or with no cells at all. The cells were washed, centrifuged and resuspended several times & were finally resuspended in trypsin/TBS solution to elute the phages. The phages eluted from each column were allowed to infect E. coli for amplification. The phages produced by each bacterial colony were retrieved and incubated with the C. albicans strains, and the cells were washed and phages were extracted just as before. This enrichment cycle was preformed for 4 rounds.

Detection of anti-mannan Fab displaying phages by ELISA Experiment Enzyme linked immunosorbent assay (ELISA) allowed us to detect different anti-mannan Fabs. The wells of the ELISA plate were coated with mannan serotype A, mannan serotype B, with cell wall fraction of S. cerevisiae, or with nothing at all. 50 l aliquots of each of the purified phage clones was added to each of the four types of wells and incubated for 2 37  C. The plate was washed and incubated with anti-M13 conjugated antibody. O-phenylenediamine was added to obtain a colored product, and absorbance was measured at 492nm. -Absorbencies obtained for the negative control wells were subtracted from the test wells to give final corrected O.D. readings. Enzyme linked immunosorbent assay (ELISA) allowed us to detect different anti-mannan Fabs. The wells of the ELISA plate were coated with mannan serotype A, mannan serotype B, with cell wall fraction of S. cerevisiae, or with nothing at all. 50 l aliquots of each of the purified phage clones was added to each of the four types of wells and incubated for 2 37  C. The plate was washed and incubated with anti-M13 conjugated antibody. O-phenylenediamine was added to obtain a colored product, and absorbance was measured at 492nm. -Absorbencies obtained for the negative control wells were subtracted from the test wells to give final corrected O.D. readings.

Cloning human genes for antimannan Fab fragments from a combinatorial phage display library Screening A phage displaying a Fab ELISA plate wells coated with mannan serotype A, serotype B, or cell wall fraction from S. cerevisiae (control); incubated with Fab displaying phages mannan Amannan Bcontrol The plate is then incubated with a second, enzyme conjugated, anti-M13 Ab

Experiment Fab production Anti-mannan Fab displaying phages identified by ELISA were amplified in E. coli and the phagemids of each clone were recovered by using a Mini Plasmid Kit (Qiagen, Chatsworth, CA, U.S.A.) The phagemids were cut using Nhe1 and Spe1, resulting in the excision of gene III from each phagemid. The vector was religated and E. coli was transformed by electroporation. The cells were incubated in liquid culture containing ampicillin for 37  C. Fab production is increased by the addition of Isopropyl  - D - thiogalactoside (IPTG), which increased the activity of the lac Z promoter.

Cloning human genes for antimannan Fab fragments from a combinatorial phage display library Phagemid DNA is extracted stop lac Z promoter amp R Light ChainHeavy Chaingene III Sac IXba IXho ISpe INhe I E. coli is transformed Fab Produced stop lac Z promoter amp R Light ChainHeavy Chain Sac IXba IXho ISpe INhe I & Gene III is removed using restriction enzymes

Results Isolation and enrichment of anti-C. albicans Fab displaying phages The panning process was affective in in isolating anti-C.albicans Fab displaying phages from the library, & an approximate 12 fold increase of anti-C.albicans phages was observed after 4 rounds of enrichment. The reported yields, as shown in the graph, were corrected for background phage binding. The panning process was affective in in isolating anti-C.albicans Fab displaying phages from the library, & an approximate 12 fold increase of anti-C.albicans phages was observed after 4 rounds of enrichment. The reported yields, as shown in the graph, were corrected for background phage binding.

Results Detection of unique anti-mannan Fab displaying phage clones by ELISA ELISA was used to identify Fabs with different binding specificities to mannan. Each graph shows the binding characteristics of a particular Fab displaying phage clone (reported in corrected O.D.). Differences in each clone’s affinity to the different serotypes of mannan allowed us to identify unique anti-mannan Fabs. Three anti-mannan Fabs were detected by this procedure.

Discussion/Summary Panning of the library over intact C. albicans cells produced reasonable enrichment yields of human Fabs with high affinities for C. albicans antigens. While this procedure does not elude to the antigen specificity of the Fabs, these enriched anti-C.albicans Fabs may be used in future studies involving treatment of disseminated candidiasis. ELISA was successfully used to detect differences in each Fabs affinity to the different mannan serotypes, resulting in the identification and production of three unique human anti-mannan Fabs. Future studies may involve the full characterization of these Fabs, and because the fragments that we cloned were Fabs, as opposed to scFv’s, we may easily produce whole antibody molecules.

Discussion/Summary An antigen binding fragments with high binding affinities to both mannan serotypes would probably be the most promising when considering development of a treatment involving passive immunity because they would, theoretically, be useful in fighting disseminated candidiasis caused by either Candida strain. Finally, the mannan that purified in this project can be further characterized in future studies. As we learn more about the different epitopes of this antigen we come closer to understanding how it may be used in human vaccination/active immunization.

Acknowledgements Dr. Mason Zhang Department of Microbiology, CSULB Dr. Andrew Mason Department of Biology, CSULB Dr. Jim Archie Department of Biology, CSULB The Howard Hughes Medical Institute Dr. Mason Zhang Department of Microbiology, CSULB Dr. Andrew Mason Department of Biology, CSULB Dr. Jim Archie Department of Biology, CSULB The Howard Hughes Medical Institute

References Burton, D. R Antibody Libraries, In G.J. Stanton, C.F. Barbas III, D.R. Burton, J.K. Scott, Phage Display: A Lab Manual. Cold Spring Harbor Laboratory Press, New York. Haidaris, C. G., J. Malone, L. A. Sherrill, J. M. Bliss, A. A. Gaspari, R. A. Insel, M. A. Sullivan Recombinant human antibody single chain variable fragments with Candida albicans surface antigens. J. Immunol.. Methods 257: Han, Y., J. E. Cutler Antibody Response That Protects against Disseminated Candidiasis. Infec. Immun. 63: Han Y., M. H. Riesselman, J. E. Cutler Protection against Candidiasis by an Immunoglobin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody. Infec. Immun. 68: Han Y., T. Kanbe, R. Cherniak, J. E. Cutler Biochemical Characterization of Candida albicans Epitopes That Can Elicit Protective and Nonprotective Antibodies. Infec. Immun. 65: Jacquinot P. M., Y. Plancke, B. Sendid, G. Strecker, D. Poulain Nature of Candida albicans-derived Carbohydrate Antigen Recognized by a Monoclonal Anitbody in Patient Sere and Distribution Over Candida Species. FEMS Microbiol Lett. 169: Jarvis, W. R Epidemiology of nosocomial fungal infections, with emphasis on Candida species. Clin Infect. Dis. 20: Komshian, S. V., A. K. Uwaydah, J. D. Sobel, L. R. Crane Fungemia caused by Candida species and Torulopsis grabrata in hospitalized patient: frequency, characteristics, and evaluation of factors influencing outcome. Rev. Infect. Dis. 11: Rader, C., P. Steinberger, C. F. BarbasIII Selection Form Antibody Libraries, In G.J. Stanton, C.F. Barbas III, D.R. Burton, J.K. Scott, Phage Display: A Lab Manual. Cold Spring Harbor Laboratory Press, New York.. Webster, R Filamentous Phage Biology, In G.J. Stanton, C.F. Barbas III, D.R. Burton, J.K. Scott, Phage Display: A Lab Manual. Cold Spring Harbor Laboratory Press, New York.