Patent ID: 12247067

EXAMPLE 1—GENERATION OF FULLY HUMAN ANTI-CANDIDAMABS BY SINGLE B CELL CLONING

The generation of recombinant mAbs through direct amplification of VH and VL genes from single B cells produces fully human, affinity matured mAbs with the native antibody heavy and light chain pairing intact (14). We employed this technology to generate human recombinant anti-CandidamAbs to a definedC. albicansantigen—the morphogenesis-regulated protein 1 (Hyr1) protein expressed only in the hyphal cell wall (40), and toC. albicanswhole cell wall preparations. Hyr1 protein was selected based on its role in proposed role in resisting phagocyte killing and pre-clinical data demonstrating that a recombinant N-terminal fragment of Hyr1 confers protection in a murine model of disseminated candidiasis (23, 29, 41). Furthermore, because Hyr1 is expressed solely onC. albicanshyphal cells so mAbs generated against this protein would serve asC. albicans-specific markers. In addition we usedC. albicanswhole cell wall extracts as a target to screen against allows for the isolation of mAbs that bind to an array of different antigens, anticipating that some of the resulting mAbs would be pan fungal and therefore possess a broad spectrum of therapeutic activity and pan-Candidadiagnostic specificity.

To enhance the likelihood of isolatingCandida-related antibodies, the class switched memory (CSM) B cells used in this study were isolated from the blood of individuals who had recovered from a superficialCandidainfection within a year of sampling. Donors were selected from a panel of volunteers and the levels of target-specific circulating IgG in the donor plasma was assessed via ELISA. In this screen, donor 85 demonstrated the greatest IgG activity againstC. albicanswhole cell and donor 23 had the highest IgG titre against Hyr1 (FIG.2A). These donors were selected to provide the source of B cells to use for the generation ofCandida-specific recombinant antibodies. After the isolation of CSM B cells from a donor, approximately 80000-150000 cells were plated out at 5 cells/well and activated with a cocktail of cytokines and supplements to promote secretion of IgG into the supernatant. A high throughput screening platform was then employed to facilitate the detection of IgG in the B cell supernatant against target antigens by ELISA. Positive ELISA hits enabled identification of wells containing B cells secreting antigen-specific IgG into the supernatant. Typically, approximately 0.05% wells/screen were positive (OD>4×background). Non-specific hits were identified and eliminated by performing an ELISA screen against two unrelated proteins—human serum albumin (HSA) and human embryonic kidney nuclear antigen (HEK NA). CSM B cells from wells that were positive for the antigen screen and negative for the unrelated protein screen were then lysed and used as the source for VH, Vκ-Cκ and Vλ-Cλ gene amplification via RT-PCR and nested PCR (FIGS.2B, C). VH, Vκ-Cκ and Vλ-Cλ genes were sub cloned into the pTT5 mammalian expression vector and the sequences analysed (data not shown). Corresponding heavy and light chains originating from the same hit well were co-transfected into Expi293F cells for small scale whole IgG1 expression. From these co-transfections, recombinant mAbs that demonstrated binding to the original target were selected for large scale recombinant expression. These were then purified via affinity-based FPLC using a protein A resin and quality control checked via analytical mass spectrometry, SDS-PAGE gel analysis and analytical SEC (FIGS.2D-G).

In total, 18 purified recombinant IgG1 mAbs were generated using the single B cell technology described above. Five of these mAbs bound to purified Hyr1 protein and 13 bound toC. albicanswhole cells (Table S3).

EXAMPLE 2—PURIFIED RECOMBINANT ANTI-CANDIDAMABS EXHIBIT SPECIFIC TARGET BINDING

Purified anti-Hyr1 mAbs were primarily assessed for functionality through binding to the purified recombinant N-terminus of Hyr1 protein via ELISA. Four of the five mAbs demonstrated strong binding to the purified antigen with EC50values of 104 ng/ml, 76.5 ng/ml, 49.6 ng/ml and 53.3 ng/ml for AB120, AB121, AB122 and AB123 (FIG.3A) respectively. AB124 bound to Hyr1 with a lower affinity with an EC50value of 1050 ng/ml. To examine the specificity of these mAbs for the target protein, all five were tested against the unrelated antigens HSA and HEK nuclear antigen as negative controls and demonstrated no binding (FIG.13).

The purified recombinant anti-whole cell mAbs were originally screened and isolated againstC. albicansovernight culture. As such, the initial QC of these mAbs was to assess their binding toC. albicanswhole cells via ELISA. The majority of purified anti-whole cell mAbs boundC. albicansyeast cells with high affinity with EC50values ranging from 2.8 to 31.1 ng/ml (FIGS.3B, C). AB134 and AB135, which have similar amino acid sequences, both demonstrated slightly lower affinity for the target with EC50values of 1060 and 224 ng/ml respectively (FIG.3C).

Purified anti-whole cell mAbs exhibited a variety of affinities when binding toC. albicanscells where both yeast and hyphal morphologies were present (FIGS.3D, E). The majority bound these cells with high affinity with EC50values ranging between 3 and 50 ng/ml. As observed withC. albicansyeast cell binding, AB134 and AB135 demonstrated slightly lower affinities with EC50values of 684 and 69.4 ng/ml. EC50values were used here as a simple comparison to demonstrate the variability in anti-whole cell mAbs binding toC. albicanscell surface antigens. Therefore this methodology generated a panel of mAbs which bound to a variety of specific cell targets. Specificity of the anti-whole cell mAbs for a targetC. albicansantigen was assessed through binding to the two unrelated antigens HSA and HEK NA. All mAbs demonstrated no binding to these antigens confirming their specificity for the fungal cells (FIG.14).

EXAMPLE 3—PURIFIED RECOMBINANT ANTI-CANDIDAMABS SHOW DISTINCT BINDING PATTERNS TOC. ALBICANSAND OTHER FUNGAL SPECIES

The recombinant anti-Hyr1 mAbs generated by single B cell technology were initially isolated by screening against N-terminus of Hyr1 protein and, following purification, demonstrated binding to this recombinant antigen (above). We then visualized binding of these mAbs to Hyr1 protein expressed on theC. albicanscell surface by immunofluorescent staining using a fluorescently labelled secondary anti-human IgG mAb for detection. It was observed that the anti-Hyr1 mAbs bound to the predicted cellular location on the hyphae, and not the WTC. albicansyeast cells grown in different culture conditions (FIG.4A). We verified that the anti-Hyr1 mAbs did not bind to hyphae of a Δhyr1 null mutant (FIG.4B) and that binding was restored in aC. albicansstrain containing a single reintegrated copy of the deleted HYR1 gene (FIG.4C).

Next we visualised binding to WTC. albicansfor the anti-whole cell mAbs via indirect immunofluorescent staining. The anti-whole cell mAbs demonstrated a range of binding profiles to WTC. albicans(FIG.5). mAbs AB118, AB119, AB129, AB130, AB133, AB134, AB135, AB139, AB140 bound strongly to bothC. albicansyeast and hyphae (FIG.5A). AB132 bound to both yeast and hyphae but exhibited stronger binding to hyphae (FIG.5B). AB126 and AB131 appeared to be hypha-specific (FIG.5C) and AB127 stained the mother yeast cell and the tip of the growing hyphae (FIG.5D). Therefore the panel of antibodies apparently detected both morphology specific and morphology-independent epitopes.

C. albicanscells were enzymatically treated with proteinase K, endoglycosidase H (endo-H) and zymolyase 20T and assessed for mAb binding. Proteinase K treatment reduced AB120 (anti-Hyr1) but not anti-whole cell mAbs binding toC. albicansconfirming that anti-Hyr1 antibody recognised a protein epitope (FIG.15a). Following zymolyase 20T and endo-H treatments, binding of other anti-whole cell mAbs decreased suggesting that the cognate epitopes might be β-glucan or N-mannan respectively (FIG.15b, c). Some anti-whole cell mAbs demonstrated increased fluorescence after enzymatic treatment suggesting that their epitopes might be located deeper in the cell wall.

Commensurate with theC. albicans-specific nature of HYR1, anti-Hyr1 mAbs only bound toC. albicansand not to a range of otherCandidaspecies (FIG.6a). In contrast, a range of binding patterns were observed for the binding of anti-whole cell mAbs to other pathogenic fungal species. The majority of mAbs bound strongly to the closely related speciesC. dubliniensis, C. tropicalis, C. parapsilosisandC. lusitaniae. There was little binding of mAbs to the more distantly relatedC. glabrataandC. krusei. Only the homologous AB131 and AB132 antibodies demonstrated some weak binding toC. krusei(FIG.6b).

To assess for pan-fungal binding activity, all the anti-whole cell mAbs were tested againstA. fumigatus. C. neoformans, C. gattii, P. carinii, M. circinelloidesandM. dermatisbut no binding was observed (FIG.6b). Therefore the anti-Hyr1 mAbs areC. albicans-specific and the anti-whole cell mAbs demonstrate a variety of binding patterns to WTC. albicansand other pathogenicCandidaspecies, indicating that they target a range of different antigens and the expression levels of these antigens varies from species to species.

In conclusion, all purified recombinant mAbs generated by this single B cell technology bound specifically to their target antigens with high affinity. As expected, the anti-whole cell mAbs demonstrated distinct binding patterns to WTC. albicansand other pathogenic fungi, indicating that they target a range of different antigens and the expression levels of these antigens varies from species to species.

EXAMPLE 4—PURIFIED RECOMBINANT ANTI-CANDIDAMABS OPSONISEC. ALBICANSFOR PHAGOCYTOSIS BY MACROPHAGES

Phagocytic cells of the innate immune system are the first line of defence against fungal pathogens. Antibody binding enhances phagocytic clearance of pathogens. We utilised a live cell phagocytosis assay to examine whether the anti-CandidamAbs generated in this study opsonizedC. albicansfor phagocytosis by J774.1 macrophages and human monocyte-derived macrophages. The macrophages were challenged with live,C. albicansCAI4-CIp10 which had been pre-incubated with an anti-CandidamAb, an isotype control mAb or saline for 1 h. Live cell video microscopy using our standard phagocytosis assay (42, 43) was employed to determine the degree of opsonisation. No significant difference was observed between the saline control and anti-CandidamAb groups in terms of the overall number ofC. albicanscells taken up during the 3 h by macrophages. However, there was a difference in the time by which the majority of uptake events had occurred (FIG.7A).C. albicanscells that had been pre-incubated with either AB118, AB119 or AB140 (anti-whole cell mAbs) were taken more rapidly compared to the saline control-treated fungal cells, the IgG1 control pre-incubated fungal cells or AB120 pre-incubated fungal cells. The percentage of uptake events occurring by 20 min was 21±10, 54±9, 22±5 and 68±2, 44.3±0.6 and 7±2 (mean±SD) for saline control, AB118, AB120, AB140, AB119 and isotype control respectively (FIG.7A). A majority ofC. albicanscells pre-incubated with AB118, AB119 or AB140 were taken up as yeast cells and the majority of cells taken up by the saline control group, AB120 group and isotype control group, were hyphal cells (FIG.7B).

EXAMPLE 5—MACROPHAGES RAPIDLY ENGULF MAB-BOUNDC. ALBICANSCELLS THROUGH FCγR BINDING

Next we used live cell video microscopy and image analysis to examine whether there was any difference in the rate of engulfment betweenC. albicanscells pre-incubated with saline compared toC. albicanscells pre-incubated with selected anti-CandidamAbs. As shown previously we defined the rate of engulfment as the time taken from establishment of cell-cell contact to the time at which aC. albicanscell had been completely engulfed by a macrophage as indicated by its loss of FITC green fluorescence (42, 43) (FIGS.8A-C). WhenC. albicansyeast cells were pre-incubated with AB120 (anti-Hyr1 mAb) there was no difference in the rate of engulfment from the saline control or IgG1 control mAb however, in the presence of either AB118, AB119 or AB140 (anti-whole cell mAbs), fungal cells were engulfed at a significantly faster rate compared to the saline control and IgG1 control mAb, (FIG.8D). The hypha-specific mAb AB120 stimulated faster macrophage engulfment ofC. albicanshyphal cells by macrophages—taking an average of 5.8±0.3 min to engulf opsonised hyphae compared to 8.8±0.8 min for the control (FIG.8E).

Similar observations were obtained using human monocyte-derived macrophages (FIG.16).

Blocking FcγRs on the surface of the macrophage decreased the rate of engulfment of AB140-boundC. albicanscompared to that of the saline control (FIG.9) indicating that the increased rate of engulfment of mAb-boundCandidacells is, at least in part, due to uptake through the FcγRs.

EXAMPLE 6—MACROPHAGES MIGRATE FURTHER, FASTER AND MORE DIRECT TOWARDS ANTI-CANDIDA MAB BOUND C. ALBICANSCELLS

We showed that antibody-boundC. albicanscells were cleared earlier by macrophages than control cells. To determine the effect of antibody binding on uptake dynamics, we used imaging analysis to digitise the migration of macrophages until their first uptake event, measuring the distance travelled, directionality and velocity of the macrophage towards control or antibody-bound fungal cells. Macrophages travelled further and at a greater velocity towardsC. albicansyeast cells that had been pre-incubated with a whole-cell mAb (AB140) compared to control fungal cells or those pre-incubated with IgG1 control mAb (FIG.10A,B). Furthermore we observed that macrophages moved in a more directional manner towards antibody-boundC. albicanscells compared to control cells or those pre-incubated with IgG1 control mAb (FIGS.10C, D and E).

EXAMPLE 7—ANTI-WHOLE CELL MAB REDUCES FUNGAL BURDEN IN A MODEL OF DISSEMINATED CANDIDIASIS

To determine whether the anti-CandidamAbs possessed therapeutic potential in vivo, their action was assessed in a murine model of systemic candidiasis (44).C. albicansSC5314 yeast cells were pre-incubated for 1 h with either saline, an IgG1 isotype control mAb, AB119 (anti-whole cell) or AB120 (anti-Hyr1) before iv injection into the mouse lateral tail vein. Disease progression was monitored by weight change and kidney fungal burdens at day 3 which together generated an overall outcome score for disease progression (44). When SC5314 was pre-incubated with AB120 there was no decrease in fungal burden compared to the saline control or the IgG1 control mAb (FIG.11A). However, when AB119 was pre-incubated with SC5314, there was a significant decrease in kidney fungal burden compared to the saline control (FIG.11A, p<0.01). This was also considerably less than the kidney fungal burden for the IgG1 isotype control. By weight change there was no significant difference in disease outcome score between AB120 and the saline control and isotype control (FIG.11B). However, mice that had been injected with SC5314 pre-incubated with AB119 had a significantly lower disease outcome score than both the saline control group (p<0.01) and the isotype control group (p<0.05) indicating that when AB119 is present, the mice are able to clear infection more quickly and disease progression is limited (FIG.11B). Therefore exposure to antibody improved the survival of mice in a systemic disease model.

EXAMPLE 8—DISCUSSION OF EXAMPLES 1-7

Monoclonal antibodies (mAbs) have the potential to be used in multiple fungal therapy and disease management situations. Here we describe and use for the first time a novel technology facilitating the isolation of fully human anti-CandidamAbs against whole cells and a specific cellular target. These mAbs were derived directly from single B cells from donors with a history of mucosalCandidainfection and demonstrated distinct binding profiles toC. albicansand other pathogenic fungi, as well as the ability to opsonise fungal cells and to enhance phagocytosis and show partial protection in a murine model of disseminated candidiasis.

mAbs-based agents have been identified as an alternative strategy to complement the medical gaps associated with current antifungal treatments and diagnostics (13, 45, 46). In this study we generated 18 fully human recombinant anti-CandidamAbs through the direct amplification of mRNA isolated from VH and VL antibody genes produced naturally in vivo in response to aCandidainfection. By employing this method, the purified, affinity matured recombinant mAbs generated were less likely to be immunogenic, had importantly retained their native antibody heavy and light chain pairings, and therefore are more likely to be of therapeutic benefit (35). IgG1 was selected as the antibody scaffold because this isotype makes up the majority of mAbs in the clinic and so is the best characterised in terms of drug development (47, 48). Thirteen of the mAbs generated bound toC. albicanswhole cell and 5 bound to recombinant purified Hyr1 protein—a protein which is considered to be important forC. albicansresistance to phagocytosis and is currently in development as an experimental vaccine (29, 41) demonstrating that this novel technology can be utilised for screening against a wide range of specific antigens.

An antibody that recognises an antigen expressed across different fungal species could be highly beneficial as a pan-fungal therapeutic. At the same time, one of the major contributors to poor prognosis in the clinic is the lack of accurate and timely diagnostics with a knock on delay in appropriate treatment (6, 7, 49). In this case, it would be more beneficial to have a species-specific antibody which recognises an antigen only expressed on one species. As such, we assessed binding of our panel of mAbs to a number of emerging and resistant pathogenic fungi. We observed that anti-Hyr1 mAbs bound solely toC. albicanshyphae, correlating with findings that have reported that Hyr1 is only expressed onC. albicanshyphal cells (29, 40, 50). The binding pattern of anti-whole cell mAbs was more varied with the majority of mAbs binding strongly to the species that are closely related toC. albicanssuch as the emerging pathogensC. tropicalisandC. parapsilosis(51). As expected, little or no binding was observed to the more evolutionarily distinct speciesC. glabrataandC. krusei. Altogether this demonstrates that the novel technology employed here can be utilised to generate species-specific as well as pan fungal mAbs, which has great implications in terms of anti-fungal drug discovery and diagnostics. Furthermore, these mAbs could be utilised to isolate and identify protective antigens for development as fungal vaccines.

One of the many ways mAbs exert their protective effects is through opsonizing cells for phagocytosis (15). We have shown previously that by employing live cell imaging we can breakdown this process down into its component parts, thus allowing us to do a more in-depth analysis on the effect of mAbs on the individual stages of phagocytosis (42, 43). Here we observed that when yeast and hyphal cells were coated with an anti-whole cell mAb or a hyphal cell was coated with an anti-Hyr1 mAb, cells were engulfed at a significantly faster rate compared to unopsonized cells, and this was through engagement of the FcγR. Furthermore, macrophages migrated further, faster and in a more direct manner towards opsonizedC. albicanscells and this contributed to earlier clearance of fungal cells.

A number of invasive infections occur in the immunocompetent patient population as a consequence of severe trauma, and in these situations opsonizing mAbs could be a viable treatment option. The majority of antibody therapeutics in the clinic are hIgG1 so this isotype has been routinely tested pre-clinically in murine models of disease (47). Furthermore, the literature shows that hIgG1 binds to all activating mFcγRs with a similar profile to the most potent IgG isotype in mice, mIgG2a, validating the use of mouse models to assess Fc-mediated effects of hIgG1 mAbs (47). As such, we utilised an established three-day murine model of disseminated candidiasis (44, 52) to assess the efficacy of anti-CandidamAbs in vivo and observed a significant decrease in kidney fungal burden and overall disease outcome score whenC. albicanswas pre-incubated with an anti-whole cell mAb.

We have generated fully human antibodies from single B-cells to create reagents that have high specificity for targets with utility in the antifungal diagnostic and therapeutic markets. The antibodies are of high affinity and are and can be synthesised in milligram quantities under defined conditions for heterologous protein expression.

The relative by which these antibodies can be produced means that they could be used singly or in multiplex formats to create novel polyvalent diagnostic tests, as vaccine Candidates or as therapeutic delivery systems to target toxic molecules to specific microbial or cellular targets.

EXAMPLE 9—CIE ANALYSIS

FIG.17shows the results of counterimmunoelectrophoresis (CIE) analysis. This shows selected mAbs were able to detectC. albicansantigens in a format commonly used for the diagnosis of patients with aCandidainfection.

EXAMPLE 10—TEM ANALYSIS

FIG.18shows transmitting electron microscopy (TEM) images illustrating the binding of a select panel (one mAb from each CDR3 amino acid sequence cluster) of the anti-whole cell mAbs toC. albicansyeast and hyphal cell walls via immunogold labelling. The images show that the mAbs are very specific to the cell wall and that there are a variety of binding targets, for example AB126, AB127 and AB131 appear mainly to bind to hypha, whereas AB118C101 S, AB119, AB140 and AB135 appear to bind to more abundantly expressed targets in both yeast and hyphal cells.

General Methods

CandidaStrains and Growth Conditions

C. albicansserotype A strain CAI4+CIp10 (NGY152) was used as a control and its parent strain CAI4, used to construct the Δhyr1 null mutantC. albicansstrain (40) and the hyr1 re-integrant strain (unpublished). The clinical isolatesC. albicansSC5314,C. glabrataSC571182B,C. tropicalisAM2005/0546,C. parapsilosisATCC22019,C. lusitaniaeSC5211362H,C. kruseiSC571987M,C. dubliniensisCD36 are shown in Table S1. All strains were obtained from glycerol stocks stored at −80° C. and plated onto YPD plates (2% (w/v) mycological peptone (Oxoid, Cambridge, UK), 1% (w/v) yeast extract (Oxoid), 2% (w/v) glucose (Fisher Scientific, Leicestershire, UK) and 2% (w/v) technical agar (Oxoid)).Candidastrains tested were routinely grown in YPD (see above without the technical agar) except in the in vivo experiments where strains were grown in NGY medium (0.1% (w/v) Neopeptone (BD Biosciences), 0.4% (w/v) glucose (Fisher Scientific), 0.1% (w/v) yeast extract (Oxoid).Aspergillus fumigatusclinical isolate V05-27 was cultured on Potato Dextrose Agar slants for seven days before the spores were harvested by gentle shaking with sterile 0.1% Tween 20 in PBS. Harvested spores were purified, counted and re-suspended at a concentration of 1×108spores/ml. Swollen spores were generated by incubation in RPMI media for 4 h at 37° C.

Malassezia dermatisCBS9169 was cultured on Modified Dixon agar (3.6% (w/v) Malt extract (Oxoid), 1% (w/v) Bacto peptone (BD Biosciences), 2% (w/v) Bile salts (Oxoid), 1% (w/v) Tween40 (Sigma), 0.2% (w/v) Glycerol (Acros Organics), 0.2% (w/v) Oleic acid (Fisher Scientific), 1.5% technical Agar (Oxoid)) supplemented with chloramphenicol (0.05% (w/v) Sigma) and cycloheximide (0.05% (w/v) Sigma)). Overnight culture ofM. dermatiswas grown in Modified Dixon Medium.Mucor circinelloidesCBS277.49 was grown on Potato Dextrose Agar for 7 days before spores were harvested in PBS and filtered through 40 μm Nylon Cell Strainer (BD Biosciences).Cryptococcus neoformansKN99α andCryptococcus gattiiR265 were grown in YPD overnight, washed in PBS and 1×107cells were added to 6 ml RPMI+10% FCS in 6 well-plates. Plates were incubated at 37° C.+5% CO2for 5 days to induce capsule formation. Harvested cells were washed in PBS. Rat lung tissue isolates ofPneumocystis cariniiM167-6 were washed in PBS and immunostained.

Generation of Recombinant Hyr1 N-Protein

The recombinant N-terminus of the Hyr1 protein (amino acids 63 to 350—Table S2) incorporating an N-terminal 6×His tag was expressed in HEK293F cells and purified by nickel-based affinity chromatography using a nickel NTA superflow column (QIAGEN, USA). Fractions containing the recombinant N-terminus of the Hyr1 protein were pooled and further purified via Analytical Superdex 200 gel filtration chromatography (GE Healthcare, USA) in PBS. QC of the recombinant protein via SDS-PAGE gel analysis, analytical size exclusion chromatography (SEC) and Western blot (using an anti-His antibody for detection) confirmed a protein of 32 kDa (data not shown).

Identification of Human Anti-Hyr1 and Anti-Whole Cell mAbs from Donor B Cells PBMC Isolation

In brief, peripheral venous blood from donors who had recovered from aCandidainfection within the last year was collected in EDTA-coated vacutainers tubes and pooled. PBMCs and plasma were separated from the whole blood suspension via density gradient separation using Accuspin System-Histopaque-1077 kits (Sigma-Aldrich) according to manufacturer's instructions. Following separation, the plasma layer was aspirated and stored at 4° C. for later analysis of antibody titre and the PBMC layer was aspirated and washed in PBS and centrifugation at 250×g for 10 min three times before final resuspension at a concentration of 1×107cells/mi in R10 media (RPMI 1640 (Gibco, Life Technologies), 10% FCS, 1 mM sodium pyruvate (Sigma), 10 mM HEPES (Gibco, Life Technologies), 4 mM L-glutamine (Sigma), 1× penicillin/streptomycin (Sigma)) containing additional 10% FCS and 10% DMSO. PBMCs were split into 1 ml aliquots and stored in liquid nitrogen until they were required.

Purification of Donor Plasma

IgG was purified from donor plasma using VivaPure MaxiPrepG Spin columns (Sartorius Stedman) according to manufacturer's instructions. In brief, plasma sample was applied to the spin column to facilitate IgG binding. The column was washed twice in PBS and then bound IgG was eluted in an amine buffer, pH 2.5 and neutralized with 1 M Tris buffer, pH8. Eluted IgG concentration was measured by absorbance at 280 nm using a NanoVue Plus Spectrophotometer (GE Healthcare).

Circulating IgG Enzyme-Linked Immunosorbent Assay (ELISA) to Identify Donors with B Cells to Take Forward

To identify the donor to use for subsequent class switched memory (CSM) B cell isolation and activation, ELISAs were carried out against the target antigens using IgG purified from donor plasma. NUNC maxisorp 384-well plates (Sigma) were coated withC. albicansovernight culture (whole cell) or 1 μg/ml purified, recombinant N-terminus hyr1 protein antigen in 1×PBS and incubated at 4° C. overnight. The next day, wells were washed three times with wash buffer (1×PBS+0.05% Tween) using a Zoom Microplate Washer (Titertek). Wells were then blocked with block buffer (1×PBS+0.05% Tween+0.5% BSA) for 1 h at room temperature with gentle shaking to inhibit non-specific binding. After three washes (as above), titrated purified IgG or IVIG in block buffer was added in duplicate, and the plates were incubated for 2 h at room temperature with gentle shaking. Wells were washed with wash buffer as above before addition of goat anti-human IgG, HRP conjugated (ThermoScientific) secondary antibody at 1:5000 dilution in blocking buffer. Plates were incubated for 45 min at room temperature with gentle shaking. To develop the ELISA, wells were washed three times with wash buffer (as above) before the addition of TMB (Thermo Scientific). Plates were incubated at room temperature for 5 min to allow the blue colour to develop and the reaction was quenched by the addition of 0.18 M sulphuric acid. The plates were then read at an OD of 450 nm on an Envision plate reader (PerkinElmer). Labstats software in Microsoft Excel was used to generate concentration-response curves for EC50determination and donor selection for subsequent CSM B cell isolation and activation.

Isolation of Class Switched Memory B Cells

The PBMCs from donors who displayed a strong IgG response to the antigen of interest in the screening ELISA were taken forward for CSM B cell isolation and activation. The process of generating recombinant mAbs from a single donor's B cells to one particular antigen, beginning with the isolation of CSM B cells all the way through to expression and purification of recombinant mAbs, was termed an ‘Activation’. For each Activation, 5×107PBMCs were removed from the liquid nitrogen store and thawed by adding pre-warmed R10 media drop wise to the cells. The diluted cell suspension was then transferred into a fresh polypropylene tube containing pre-warmed R10, resulting in a final cell dilution of approximately 1:10. Benzonase nuclease HC, purity>99% (Novagen) was added at a 1:10000 dilution (to ensure any lysed cells and their components didn't interfere with the live cells), and the cells were centrifuged at 300×g for 10 min at room temperature and the supernatant removed. PBMCs were then washed again in R10 before final resuspension in 1 ml R10 for PBMC cell number and viability determination.

Isolation of class switched memory B cells from PBMCs was carried out by magnetic bead separation using a Switched Memory B cell isolation kit with Pre-Separation Filters and LS columns (MACS Miltenyi Biotec) according to manufacturer's instructions. In brief, counted PBMCs were incubated with a cocktail of biotin-conjugated antibodies against CD2, CD14, CD16, CD36, CD43, CD235a (glycophorin A), IgM and IgD. Cells were then washed and incubated with anti-biotin microbeads. Following another wash step, the suspension was passed through a Pre-Separation Filter (to remove cell aggregates) before applying it to an LS column where the magnetically labelled cells were retained in the column and the unlabelled CSM B cells passed through and could be collected in the flow-through for determination of cell number and viability.

Activation of CSM B Cells

To activate CSM B cells and promote antibody secretion into the supernatant, a mixture of cytokines, mAb, TLR agonist and a supplement were added to the R10 media (see above) to make complete R10 media. CSM B cells were resuspended in complete R10 media at 56 cells/ml and then plated out at 90 μl/well (5 cells/well) in ThermoFisher Matrix 384 well plates using a Biomek FX (Beckman Coulter). Cells were incubated at 37° C. 5% CO2for seven days. On day 7, 30 μl/well of supernatant was removed and replaced with 30 μl fresh complete R10. On day 13, all the supernatant was harvested from all plates and screened against the antigen of interest via ELISA. B cell activation and culturing was monitored by measuring IgG1 concentrations in B cell supernatants at day 7 and day 13.

B Cell Supernatant Screen Against Target Antigens Via ELISA

For B cell supernatant screening against target antigens, NUNC maxisorp 384-well plates (Sigma) were coated withC. albicansovernight culture (whole cell) or 1 μg/ml purified, recombinant N-terminus hyr1 protein antigen in 1×PBS and incubated at 4° C. overnight. Wells were washed three times with wash buffer using a Zoom Microplate Washer (Titertek) as above before incubation with blocking buffer for 1 h at room temperature with gentle shaking. After another three washes (as above), B cell supernatant was added and the plates incubated for 2 h at room temperature with gentle shaking. Wells were washed with wash buffer as above before addition of goat anti-human IgG, HRP conjugated (ThermoScientific) secondary antibody at 1:5000 dilution in blocking buffer and incubation for 45 min at room temperature with gentle shaking. ELISAs were developed and plates read at an OD of 450 nm on an Envision plate reader (PerkinElmer).

Positive hits were defined as wells with an OD450reading >4×background. B cells in ‘positive hit’ wells were resuspended in lysis buffer (ml DEPC-treated H2O (Life Technologies), 10 μl 1 M Tris pH 8, 25 μl RNAsin Plus RNAse Inhibitor (Promega)) and stored at −80° C.

Generation of Recombinant Anti-Hyr1 and Anti-Whole Cell IgG1 mAbs: Amplification of VH, Vκ-Cκ and Vλ-Cλ Genes—cDNA Synthesis and PCR

A schematic of the cloning protocol is shown inFIG.12. Primers used for the RT-PCR reaction were based on those used by Smith et. al., (36). To ensure all possible VH germline families were captured during the amplification, four forward primers specific to the leader sequences encompassing the different human VH germline families (VH1-7) were used in combination with two reverse primers; both placed in the human CgCH1 region. For the RT-PCR of human Vκ-Cκ genes, three forward primers specific to the leader sequences for the different human Vκ germline families (Vκ1-4) were used with a reverse primer specific to the human kappa constant region (Cκ) and two further reverse primers which were specific to the C- and N-terminal ends of the 3′ untranslated region (UTR). To capture the repertoire of human Vλ genes, 7 forward primers capturing the leader sequences for the different human Vλ germline families (Vλ1-8) were used in a mixture with two reverse primers which were complementary to the C- and N-terminal ends of the 3′ UTR and another reverse primer specific to the human lambda constant region (Cλ).

Prior to cDNA synthesis, B cell lysates were thawed and diluted 1:5, 1:15 and 1:25 in nuclease-free H2O (Life Technologies) before addition of oligodT20(50 μM) (Invitrogen, Life Technologies) and incubation at 70° C. for 5 min. Reverse transcription and the first PCR reaction (RT-PCR) were done sequentially using the QIAGEN OneStep RT-PCR kit according to manufacturer's instructions. For this step and the subsequent nested PCR step, amplification of the variable domain of human Ig heavy chain genes (VH), the variable and constant domains of human Ig kappa light chain genes (Vκ-Cκ) and the variable and constant domains of human Ig lambda light chain genes (Vλ-Cλ), were done in separate reactions. In brief, a reaction mixture was prepared containing QIAGEN OneStep RT-PCR Buffer 5x, dNTPs (10 mM), gene-specific forward and reverse primer mixes (10 μM), QIAGEN OneStep RT-PCR Enzyme Mix and nuclease-free H2O. Reaction mixture was then added to wells of a 96-well PCR plate before addition of neat or diluted (1:5, 1:15, 1:25) B cell lysate as the template, resulting in a final reaction volume of 50 μl/well. The following cycling conditions were used for the RT-PCR reaction; 50° C. for 30 min, 95° C. for 15 min then 35-40 cycles of (94° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min) with a final extension at 72° C. for 10 min.

Amplification of VH, Vκ-Cκ and Vλ-Cλ Genes—Nested PCR Reaction

Nested PCR reactions were carried out using the PCR products from the RT-PCR reaction as the template, nested gene-specific primers based on Smith et al. (36) and Platinum PCR SuperMix High-Fidelity (Invitrogen, Life Technologies). A total of 27 forward primers specific for the VH framework 1 (FW1) sequence were used together with two reverse primers specific for the framework 4 (FW4) region of the VH gene. For nested PCR of the Vκ-Cκ gene, a mixture of 18 forward primers specific for human Vκ FW1 sequence were used with a reverse primer specific to the human kappa constant region 3′ end. For amplification of the Vλ-Cλ gene, a mixture of 31 forward primers specific for human Vλ FW1 sequences were used together with a reverse primer that was placed at the 3′ end of the human lambda constant region. The primers used to generate the PCR fragments in these nested PCR reactions contained 15 bp extensions which were complementary to the target downstream pTT5 expression vector. Reaction mixtures containing Platinum PCR SuperMix High Fidelity, gene-specific forward primer mix (10 μM) and gene specific reverse primer mix (10 μM) was added to wells in a 96-well PCR plate before addition of cDNA template. Amplification of VH genes, Vκ-Cκ genes and Vλ-Cλ genes, were done in separate reactions. After the nested PCR reaction, samples were analysed via agarose gel electrophoresis and positive hits identified and taken forward for downstream InFusion cloning with pTT5 mammalian expression vector.

pTT5 Mammalian Expression Vector Preparation

The pTT5 mammalian expression used for mAb expression (licensed from the National Research Council of Canada (NRCC)) (53). The pTT5 vector plasmid contained an IgG1 heavy chain gene in the multiple cloning site so digestion to generate the heavy chain (HC) backbone for downstream sub cloning of VH was done by double digestion using FastDigest Restriction enzymes (Thermo Scientific) with BssHII before the leader sequence of the VH region and SalI restriction after the FW4 of the VH domain. This yielded the heavy chain constant region in the vector backbone. For double digestion of the vector to generate the light chain (LC) backbone, the whole IgG1 heavy chain gene was with BssHII and BamHI astDigest Restriction enzymes (Thermo Scientific) to generate the vector ready for insertion of either κ-Cκ or Vλ-Cλ. Digestion reactions to generate HC and LC backbones were carried out separately. Following confirmation of digestion, samples were run on a 1% agarose gel and bands were excised from the gel and purified using the QIAquick Gel Extraction kit (QIAGEN). DNA was quantified on a NanoVue Plus Spectrophotometer (GE Healthcare). To prevent vector self-ligation, the 3′- and 5′-termini of the linearized plasmids were dephosphorylated using FastAP Thermosensitive Alkaline phosphatase (Thermo Scientific). Reaction mixtures were cleaned up using the MinElute Reaction Cleanup Kit (QIAGEN) and then run on a 1% agarose gel. Bands corresponding to dephosphorylated HC and LC backbones were excised from the gel and purified using the QIAQuick Gel Extraction kit (QIAGEN) as above. Dephosphorylated linearized vector DNA was quantified on a NanoVue Plus spectrophotometer (GE Healthcare).

In-Fusion Cloning

The In-Fusion HD Cloning Kit (Clontech, USA) was used to clone the IgG VH, Vκ-Cκ and Vλ-Cλ genes into a pTT5 mammalian expression vector. To avoid the need for nested PCR product purification before cloning, cloning enhancer (Clontech, USA) was added to each nested PCR product in a 96-well PCR plate and incubated at 37° C. for 15 min, then 80° C. for 15 min. The cloning enhancer-treated PCR product was then added to the In-Fusion Enzyme Premix and linearized vector DNA (˜5-10 ng). Reactions were made up to 10 μl with nuclease-free H2O and incubated for 15 min at 50° C. Samples were then either stored at −20° C. or placed on ice before transformation of Stellar Competent cells (Clontech). For transformation, 2 μl of each In-Fusion reaction mixture was added to cells in a 96-well plate format, and left on ice for 30 min before heat shock at 42° C. for 40 sec and then returning to ice for 2 min. Cells were then recovered in SOC medium (Clontech, USA) with gentle shaking at 37° C. for 45-60 min before plating out onto LB agar plates (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, 1.5% (w/v) agar) containing 100 μg/ml ampicillin. Plates were incubated at 37° C. overnight and single colonies picked the next day.

Plasmid DNA Generation for Transfection

Following transformation, 8-16 single colonies per initial hit well for VH, Vκ and Vλ were picked and used to inoculate 2×TY media containing 100 μg/ml ampicillin in a Greiner deep well, 96-well plate (Sigma). VH, Vκ and Vλ plates were set up separately with the same plate layout to facilitate visual screening. Cells were grown at 37° C., 200 rpm overnight, and glycerol stocks were made the following day and stored at −80° C. To ensure accurate tracking of DNA sequences for downstream sequencing and transfections, each well inoculated by a single colony was given a unique ID based on the colony's original hit well and its position in the deep well 96 well plate following transformations. To obtain plasmid DNA for gene sequencing and small scale mammalian transfections, DNA minipreps from the overnight cultures were carried out in a 96-well plate format using the EPmotion (Eppendorf), according to manufacturer's instructions. DNA not taken for gene sequencing was stored at −20° C. until required for small scale transfections. Sequence data was analysed for CDR diversity and comparisons to germline sequences and used to identify clones to take forward for small scale transfection.

Small Scale Expression of Recombinant mAbs

Following VH, Vκ and Vλ gene sequencing, a file was generated containing all possible VH and Vκ/Vλ combinations resulting from the original hit wells from the primary ELISA screen. Automated mixing of the native heavy and light chain DNA pairing combinations (1.5 μg of HC plasmid DNA and 1.5 μg of LC plasmid DNA) into a new 96-well plate was facilitated through a HAMILTON MICROLAB® Starline liquid handling platform (Life Science robotics, Hamilton Robotics). Subsequent mixed DNA was used for small scale transient transfection of 3 ml of suspension cultured Expi293F cells (Life Technologies, USA) at a density of 2.5×106cells/ml in 24-well tissue culture plates using the Expifectamine 293 Transfection kit (Life Technologies, USA) in accordance with manufacturer's instructions. Expi293F cells were maintained in pre-warmed (37° C.) sterile Expi293 expression media (Invitrogen) without antibiotics at 37° C., 7% CO2, 120 rpm shaking. Supernatants were harvested on day 6 and recombinant mAb expression was quantified using anti-human IgG Fc sensors on an Octet QKe(ForteBio, CA, USA) for identification of mAbs to upscale.

Large Scale Expression, Purification and QC of Recombinant mAbs

For downstream large scale mammalian transfections, where a greater amount of DNA was required, DNA was prepared using a QIAGEN Plasmid Maxi Kit (QIAGEN, USA) according to manufacturer's instructions with typical yields of 1.5 μg/μl.

For large scale mAb expression, 100 μg of total DNA (50 μg of HC plasmid DNA and 50 μg LC plasmid DNA) was used to transiently transfect 100 ml of suspension cultured Expi293F cells (Life Technologies, USA) at a density of 2.5×106cells/ml using the Expifectamine 293 Transfection Kit (Life Technologies, USA) in accordance with the manufacturer's instructions. Supernatants were harvested on day 6 and recombinant mAb expression was quantified as above using an Octet QKe(ForteBio). Recombinant mAbs were purified via affinity based Fast Protein Liquid Chromatography using HiTrap Protein A HP columns on an ÄKTA (GE Healthcare) and eluted in 20 mM citric acid, 150 nM NaCl (pH2.5) before neutralisation with 1 M Tris buffer (pH8). Purified mAbs were dialysed in PBS overnight and IgG concentration was quantified on a NanoVue Spectrophotometer (GE Healthcare).

All purified recombinant mAbs were quality control checked via SDS-PAGE gel analysis using 4-12% Bis-Tris SDS-PAGE gels under reducing and non-reducing conditions to confirm mass, analytical size exclusion chromatography (SEC) to check for protein aggregation/degradation and analytical mass spectrometry to confirm the amino acid sequence identity of each mAb. Purified recombinant mAbs were also tested for functionality by binding to target antigen/whole cell via ELISA.

ELISA with Purified Recombinant mAbs

For confirmation of binding to target as purified recombinant mAbs an ELISA was carried out using the protocol for B cell supernatant screen. The only change was that titrated purified recombinant mAb was added in place of B cell supernatant.

Immunofluorescence Imaging of Anti-Hyr1 and Anti-Whole Cell mAbs Binding to Fungal Cells

Indirect immunofluorescence was performed using purified recombinant mAbs. A singleCandidacolony was used to inoculate 10 ml YPD medium and incubated at 30° C., 200 rpm overnight. Overnight cultures were diluted 1:1333 in milliQ water and then added to a poly-L-lysine coated glass slide (Thermo Scientific, Menzel-Gläser) and incubated for 30 min at room temperature to allow for adherence of yeast cells to the slide. To induce filamentation, cells were incubated in pre-warmed RPMI+10% FCS at 37° C. for 90 min-2 h (this step was omitted for staining of yeast cells), after which they were washed in Dulbecco's Phosphate Buffered Saline (DPBS) and fixed with 4% paraformaldehyde. Cells were washed again and blocked with 1.5% normal goat serum (Life Technologies) before staining with an anti-CandidamAb at 1-10 μg/ml for 1 h at room temperature. After three PBS washes, cells were stained with Alexa Fluor® 488 goat anti-human IgG antibody (Life Technologies) at a 1:400 dilution and incubated at room temperature for 1 h in the dark. For additional staining of fungal cell wall chitin, Calcofluor White (CFW) was added at 25 μg/ml and cells were incubated for 10 min at room temperature in the dark and washed with DPBS. Slides were left to air dry before adding one drop of Vectashield mounting medium (Vector Labs) and applying a 20 mm×20 mm coverslip to the slide. Cells were imaged in 3D on an UltraViEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK).

Preparation of Human Monocyte-Derived Macrophages

Human monocyte-derived macrophages were isolated from the blood of healthy volunteers. In brief, the PBMC layer was isolated as described above and was then washed and re suspended in DMEM medium (Lonza, Slough, UK) supplemented with 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM L-glutamine (Invitrogen, Paisley, UK). Serum was separated from blood using standard methods and heat-inactivated at 56° C. for 20 min before use. Monocytes were isolated from PBMCs via positive selection using CD14 microbeads (MACS, Miltenyi Biotec) according to manufacturer's instructions. PBMCs were incubated with MicroBeads conjugated to monoclonal anti-human CD14 antibodies. Cells were then washed and run through an LS column in a magnetic field causing the CD14+cells to be retained in the column and the unlabelled cells to run through. The CD14+cells were then eluted and resuspended in supplemented DMEM containing 10% donor-specific serum, for determination of cell count and viability. Monocytes were then plated out at a density of 1.2×105cells/well in an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated at 37°, 5% CO2for 7 days. Cells were used in imaging experiments on day 7. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with pre-warmed supplemented CO2-independent media (Gibco, Invitrogen, Paisley, UK) containing 1 μM LysoTracker Red DND-99 (Invitrogen, Paisley, UK). LysoTracker Red is a fluorescent dye that stains acidic compartments in live cells, enabling tracking of these cells during phagocytosis and phagolysosome maturation.

Preparation of J774.1 Mouse Macrophage Cell Line

J774.1 macrophages (ECACC, HPA, Salisbury, UK) were maintained in tissue culture flasks in DMEM medium (Lonza, Slough, UK) supplemented with 10% (v/v) FCS (Biosera, Ringmer, UK), 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM L-glutamine (Invitrogen, Paisley, UK) and incubated at 37° C., 5% CO2. For phagocytosis assays, macrophages were seeded in 300 μl supplemented DMEM at a density of 1×105cells/well in an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated overnight at 37° C., 5% CO2. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with 300 μl pre-warmed supplemented CO2-independent media (Gibco, Invitrogen, Paisley, UK) containing 1 μM LysoTracker Red DND-99 (Invitrogen, Paisley, UK).

Preparation of Fluorescein Isothiocyanate (FITC)-StainedC. albicans

C. albicanscolonies were grown in YPD medium and incubated at 30° C., 200 rpm overnight. LiveC. albicanscells were stained for 10 min at room temperature in the dark with 1 mg/ml FITC (Sigma, Dorset, UK) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) (BDH Chemicals, VWR International, Leicestershire, UK). Following the 10 min incubation, in phagocytosis assays usingC. albicansFITC-labelled yeast, the cells were washed three times in 1×PBS to remove any residual FITC and finally re-suspended in 1×PBS or 1×PBS containing purified anti-CandidamAb at 1-50 μg/ml. For assays where pre-germinatedC. albicanswas to be added to immune cells, cells were washed and re-suspended in supplemented CO2-independent media with or without anti-CandidamAb at 1-50 μg/ml and incubated at 37° C. with gentle shaking for 45 min.

Live Cell Video Microscopy Phagocytosis Assays

Phagocytosis assays were performed using our standard protocol with modifications (42, 43, 54). Following pre-incubation with/without anti-CandidamAb, live FITC-stained wild typeC. albicans(CAI4-CIp10) yeast or hyphal cells were added to LysoTracker Red DND-99-stained J774.1 murine macrophages or human monocyte-derived macrophages in an 8-well glass based imaging dish (Ibidi) at a multiplicity of infection (MOI) of 3. Video microscopy was performed using an UltraVIEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK) in a 37° C. chamber and images were captured at 1 min intervals over a 3 h period. At least three independent experiments were performed for each antibody and at least 2 videos were analysed from each experiment using Volocity 6.3 imaging analysis software (Improvision, PerkinElmer, Coventry, UK). Twenty five macrophages were selected at random from each experiment and analysed individually at 1 min intervals over a 3 h period. Measurements taken included:C. albicansuptake—defined as the number ofC. albicanscells taken up by an individual phagocyte over the 3 h period;C. albicansrate of engulfment—defined as the time point at which cell-cell contact was established until the time point at whichC. albicanswas fully engulfed (a fungal cell was considered to have been fully ingested when its FITC-fluorescent signal was lost, indicating that the fungal cell was now inside the phagocyte and not merely bound to the phagocyte cell surface) and finally Volocity 6.3 imaging analysis software was used to measure the distance travelled, directionality and velocity of macrophages at 1 min intervals during the first hour of the assay which provided a detailed overview of macrophage migration towardsC. albicanscells.

Mean values and standard deviations were calculated. One- or two-way ANOVA followed by Bonferroni multiple comparison tests or unpaired, two-tailed t tests were used to determine statistical significance.

Systemic Candidiasis Infection Model

A well-established three-day model of disseminated candidiasis was employed to assess the efficacy of anti-CandidamAbs in vivo (44, 52). On day 0, ˜3.2×105C. albicansSC5314 yeast cells were pre-incubated at RI with 7.5 mg/kg purified recombinant anti-CandidamAb for 60 min to allow binding of the antibody to theCandidacell surface before administration intravenously via the lateral tail vein. Assessment of disease progression was carried out by observation and weighing on successive days from day 0 up to and including day 3, at which point the animals were culled and the kidneys harvested for analysis of fungal burden. Fungal burdens were quantitated by homogenising the organ, and plating out serial dilutions on Sabouraud dextrose agar plates (1% mycological peptone (w/v), 4% glucose (w/v), 2% agar (w/v)) before incubation at 35° C. overnight. Colonies were counted the next day and fungal burden expressed as log CFU per gram of infected organ. An overall disease outcome score devised from the combination of 3-day weight loss and kidney burden data was also generated to assess disease progression.

Enzymatic Modification ofCandida albicansCell Wall

For proteinase K treatment, single colonies ofCandidawere inoculated into 10 ml YPD medium and incubated at 30° C., 200 rpm overnight. Cultures were diluted in milliQ water and then adhered on poly-L-lysine coated glass slides. To induce filamentation, cells were incubated in pre-warmed RPMI+10% FCS at 37° C. for 90 min-2 h. Slides were washed with DPBS and cells were treated with 50 μg/ml proteinase K at 37° C. for 1 h. For Endo-H and zymolyase 20T treatments,C. albicansovernight yeast cells were washed and resuspended in DPBS. Filamentous cells were induced as above. Cells were washed in DPBS and resuspended in Glycobuffer and Endoglycosidase H (10 U/μl; NEB) or Buffer S and Zymolyase 20T (50 U/g wet cells; MPBIO) at 37° C. for 2 h. Cells were then washed in DPBS and fixed with 4% paraformaldehyde, washed and blocked with 1.5% normal goat serum (Life Technologies) before staining with an anti-CandidamAb at 1 μg/ml for 1 h at room temperature. After 3 washes with DPBS, cells were stained with Alexa Fluor® 488 goat anti-human IgG antibody (Life Technologies) at a 1:400 dilution and incubated at room temperature for 1 h prior to imaging in 3D on an UltraVIEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK).

Preparation of Human Monocyte-Derived Macrophages

Human macrophages were derived from monocytes isolated from the blood of healthy volunteers. PBMCs were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Slough, UK) supplemented with 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM L-glutamine (Invitrogen, Paisley, UK). Serum isolated from blood was heat inactivated for 20 min at 56° C. PBMCs were seeded at 6×105in 300 μl/well supplemented DMEM medium containing 10% autologous human serum, onto an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated at 37° C. with 5% CO2for 1 h 45 min to facilitate monocyte adherence to the glass surface. Floating lymphocytes in the supernatant were aspirated and the same volume of fresh pre-warmed supplemented DMEM containing 10% autologous human serum added to the well. Cells were incubated at 37° C., 5% CO2for 7 days with media changed on days 3 and 6. Cells were used in imaging experiments on day 7. Supplemented DMEM was replaced with pre-warmed supplemented CO2-independent media containing 1 μM LysoTracker Red DND-99 (Invitrogen) immediately prior to phagocytosis experiments.

Counterimmunoelectrophoresis

Agar gels were prepared (Veronal buffer+0.5% (w/v) purified agar+0.5% (w/v) LSA agarose+0.05% (w/v) sodium azide, pH 8.2) and wells were cut out using a cutter. Into one column of wells, 10 μl of neat anti-CandidamAb was added. The same volume of antigen (crudeC. albicansyeast or hyphal preparation (following glass bead disruption of cells and 1 min centrifugation at 13000 rpm to generate disrupted cell wall/glass bead slurry and cell supernatant antigenic preparations)) was added to the second column of wells and gels were placed into an electrophoresis tank containing veronal buffer. Gels were oriented so that the antibody wells were lined up alongside the anode and the antigen wells alongside the cathode due to antibody migration towards the cathode via electroendosmosis and antigen migration towards the anode due to lower isoelectric points than the buffer pH. The gels were run at 100V for 90 min before removal and immersion in saline-trisodium citrate overnight. The following day the gels were rinsed with water and covered with moistened filter paper and left to dry in an oven for 2 h. Once dried, the filter paper was moistened and removed and the gels put back into the oven for a further 15 min to dry completely. Gels were then immersed in Buffalo black solution (0.05% (v/v) Buffalo black, 50% (v/v) distilled water, 40% (v/v) methylated spirit, 10% (v/v) acetic acid) for 10 min before destaining in destaining solution (45% (v/v) industrial methylated spirits, 10% (v/v) acetic acid, 45% (v/v) distilled water) for 10 min. Gels were then dried and examined for the formation of precipitin lines. The results are shown inFIG.17.

High-Pressure Freezing (HPF) of Samples for Immunogold Labelling ofC. albicansCells with Anti-CandidamAbs for Transmission Electron Microscopy (TEM).

C. albicansyeast and hyphal cell samples were prepared by high-pressure freezing using an EMPACT2 high-pressure freezer and rapid transport system (Leica Microsystems Ltd., Milton Keynes, United Kingdom). Using a Leica EMAFS2, cells were freeze-substituted in substitution reagent (1% (w/v) OsO4 in acetone) before embedding in Spurr resin and polymerizing at 60° C. for 48 h. A Diatome diamond knife on a Leica UC6 ultramicrotome was used to cut ultrathin sections which were then mounted onto nickel grids. Sections on nickel grids were blocked in blocking buffer (PBS+1% (w/v) BSA and 0.5% (v/v) Tween20) for 20 min before incubation in incubation buffer (PBS+0.1% (w/v) BSA) for 5 min×3. Sections were then incubated with anti-CandidamAb (5 μg/ml) for 90 min before incubation in incubation buffer for 5 min a total of 6 times. mAb binding was detected by incubation with Protein A gold 10 nm conjugate (Aurion) (diluted 1:40 in incubation buffer) for 60 min before another six 5 min washes in incubation buffer followed by three 5 min washes in PBS and three 5 min washes in water. Sections were then stained with uranyl acetate for 1 min before three 2 min washes in water and then left to dry. TEM images were taken using a JEM-1400 Plus using an AMT UltraVUE camera. The results are shown inFIG.18.

TABLE S1Clinical isolates and strainsStrain nameGenotypeReferenceCA14 + Clp10ura3Δ::λimm434/ura3Δ::λimm434Brand et al. 2004(NGY152)RPS1/rps1::URA3hyr1Δhyr1Δ::hisG/hyr1Δ:Bailey et al. 1996hisG-URA-3-hisGhyr1Δ + HYR1hyr1::hisG/hyr1::hisG/BelmonteRPS1/rps1::HYR1(unpublished)tup1Δtup1Δ::hisG/tup1Δ::Fonzi & Irwin 1993hisG-URA3-hisGC. albicansClinical isolateGillum et al. 1984SC5314C. glabrataClinical isolateOdds et al. 2007SCS71182BC. tropicalisClinical isolateClinical isolate fromAM2005/0546AberdeenC. lusitaniaeClinical isolateOdds et al. 2007SCS211362HC. kruseiClinical isolateOdds et al. 2007SCS71987MC. parapsilosisClinical isolateRudek 1978ATCC22019C. dubliniensisClinical isolateMoran et al. 1998CD36A. fumigatusClinical isolateNetea et al. 2003V05-27C. aurisClinical isolateSatoh et al. 2009CBS 109131C. haemuloniiClinical isolateKhan et al. 2007CBS 51491C. neoformansH99 mating type αNielsen et al. 2003KN99αC. gattiiR265Clinical isolateFyfe et al. 2002P. cariniiIsolated from rat lung tissue—M167-6M. dermatisCBSSugita et al. 2002CBS 9169M. circinelloidesCBSLi et al. 2011CBS 277.49

TABLE S2Recombinant Hyr1 protein amino acid sequence.The leader sequence is underlined and the 6xHis tag is in italics,and is followed by the linker ‘G’. Hyr1 proteinamino acids 63-350 make up the remainder of the sequence.Recombinant proteinAmino acid sequenceantigen name(amino acids 63-350)SEQ ID NO:Recombinant Hyr1 N-METDTLLLWVLLLWVPGSTGGSGHHHHHHG1terminus fragmentEVEKGASLFIKSDNGPVLALNVALSTLVRPVINNGVISLNSKSSTSFSNFDIGGSSFTNNGEIYLASSGLVKSTAYLYAREWTNNGLIVAYQNQKAAGNIAFGTAYQTITNNGQICLRHQDFVPATKIKGTGCVTADEDTWIKLGNTILSVEPTHNFYLKDSKSSLIVHAVSSNQTFTVHGFGNGNKLGLTLPLTGNRDHFRFEYYPDTGILQLRAAALPQYFKIGKGYDSKLFRIVNSRGLKNAVTYDGPVPNNEIPAVCLIPCTNGPSAPESESDLNTPTTSSIGT

TABLE S3Purified recombinant human IgG1 mAbsgenerated using the single B cell technology.AntibodyYield (mg)TargetAB-12012Hyr1 proteinAB-12128.5Hyr1 proteinAB-12267.9Hyr1 proteinAB-12367.3Hyr1 proteinAB-12438.9Hyr1 proteinAB-1187.5C. albicans‘whole cell’AB-11913.5C. albicans‘whole cell’AB-12660.9C. albicans‘whole cell’AB-12724.5C. albicans‘whole cell’AB-1292.3C. albicans‘whole cell’AB-1301.1C. albicans‘whole cell’AB-13124.1C. albicans‘whole cell’AB-1329.3C. albicans‘whole cell’AB-13319C. albicans‘whole cell’AB-1347.7C. albicans‘whole cell’AB-13516.5C. albicans‘whole cell’AB-13912.2C. albicans‘whole cell’AB-14019.5C. albicans‘whole cell’

TABLE VHSEQABVHIDnameVH FW1CDR1VH FW2VH CDR2VH FW3VH CDR3VH FW4NO:06-VH3QVTLKESGGGLVQPGRTYWVRQDPGRLDEVGRLTRFTISRDNAKNILYLQMNDLSGSADYWGQGTLV2AB-GSLRLSCVASGFTFWMHKGLVWVSSYADSVNGSLRAEDTGVYYCARTVSS11906-VH3EVQLVESGGGLVQPGSNYWVRQVPGRINEDGSVTRFTISRDNAKNTLYLQMDLCGERDDWGQGTLV3AB-GSLRLSCSASQFILWVHEGLVWVSSYADSVKGNSLRVDDTAVYYCVRSVSS11806-VH1EVQLVQSGGGLVQPGTSYWVRQAPGVITGNVGTSRFTISRDNSKKTVSLQMTRYDFSSGYYWGQGTLV4AB-GSLGLSCAASGFIFAMTKGLEWVSYYADSVKGNSLRAEDTAIYYCVKFDDSVSS12006-VH3EVQLVESGGILVQPGSDYWVRQAPGNIKQDGSEKRVTISRDNAQNSVFLQMDGYTFGPATTWGRGTLV5AB-GSLRLSCAASGFTFWMNKGLEWVAYYVDSLRGHSLSVEDTAVYYCARELDHSVSS12106-VH3EVQLVQSGGGLAQPGDDFWVRQPPGGLTINNGGSIRFTISRDNAKNSLFLQMGLSGGTMAPFWGQGTMV6AB-RSLRLSCAASGFGFAMHKGLEWVSDYAGSVRGNSLRAEDTALYYCAKDISVSS12206-VH3EVQLLESGGGVVQPGSNYWVRQAPGVVWFDGSYRFTISRDNSKSTLYLQMPIMTSAFDIWGPGTMV7AB-RSLRLSCAASGFTFGMHKGLEWVAKYYTDSVKGNSLRAEDTAVYYCVSSVSS12306-VH3EVQLVESGGGVVQPGSNYWVRQAPGVVWLDGSYRFTISRDNSKSTLYLQMPIMTSAFDIWGPGTMV8AB-RSLRLSCAASGFTFGMHKGLEWVAKYYTGSVKGNSLRAEDTAAYYCVSTVSS12406-VH3EVQLVESGGGLAQPGAGNWVRQAPGAIGGSDDRTRFTISRDKSKNTLSLQMDIWRWAFDYWGQGTLV9AB-GSLRLSCEASGFHLAMAKGLEWVADYADSVKGNSLRVEDTAVYYCAKSVSS12606-VH3EVQLVESGGGLVNPGSNYWVRQAPGSISRSGDYIYRSTISRDNAKNSLFLQMDWGRLGYCSSWGQGTRV10AB-GSLRLSCAASGFTFAMNKGLEWVSYADSLKGNSLRAEDSAVYYCARNNCPDAFDVSVSS12706-VH3QVQLVESGGGLVQPGSNYWVRQVPGRINEDGSVTRFTISRDNAKNTLYLQMDLCGERDDWGQGTLV11AB-GSLRLSCSASQFILWVHEGLVWVSSYADSVKGNSLRVDDTAVYYCVRTVSS12906-VH3QLQLQESGGGLVQPGSNYWVRQVPGRINEDGSVTRFTISRDNAKNTLYLQMDLCWERDDWGQGTLV12AB-3GSLRLSCSASQFILWVHEGLVWVSSYADSVKGNSLRVDDTAVYYCVRSVSS13006-VH3QVQLVQSGGGVVQPGKISIWVRQAPGAMSYDGFSKRLTISRDSSTNTLYLEMNEAYTSGRAGCWGQGVLV13AB-GSLRLSCAASPFTFLHKGLEWVSYYADSVKGSLRFEDTALYFCARFNPSVSS13106-VH3QVLKESGGGVVQPGGETSIWVRQAPGAMSYDGFSKRLTISRDSSTNTLYLEMNEAYTSGRAGCWGQGVLV14AB-SLRLSCAASPFTFLHKGLEWVSYYADSVKGSLRFEDTALYFCARFDPSVSS13206-VH3EVQLVESGGGLVQPGNTYWVRQAPGRINEDGTTISRFTISRDNAENTLYLQMDFTGPFDSWGQGTLV15AB-GSLRVSCAASGFTLWMHKGLVWVSYADSVRGHSLRAEDTGVYYCARSVSS13306-VH3QLQLQESGGGLVQPGSSHWVRQAPGSISISGGDTFRFTIFRDNSKNTVYLQMETSPNDYWGQGTLV16AB-GSLRLSCVVSGFTFAMSKGLEWVSYADSVRGNSLRAEDTAVYYCATSVSS13406-VH3EVQLVETGGGLVQPGSSHWVRQAPGSISISGGDTFRFTIFRDNSKNTVYLQMETSPNDYWGQGTLV17AB-GSLRLSCVVSGFTFAMSKGLEWVSYADSVRGNSLRAEDTAVYYCATTVSS13506-VH3EVQLVESGGGLVQPGNTYWVRQAPGRINEDGTTISRFTISRDNAENTLYLQMDFTGPFDSWGQGTLV18AB-GSLRVSCAASGFTLWMHKGLVWVSYADSVRGHSLRAEDTGVYYCARSVSS13906-VH3EVQLVESGGGLVQPGNTYWVRQAPGRINEDGTTISRFTISRDNAENTLYLQMDFTGPFDSWGQGTLV19AB-GSLRVSCAASGFTLWMHKGLVWVSYADSVRGHSLRAEDTGVYYCARSVSS140

TABLE VLSEQABVLIDnameVL FW1VL CDR1VL FW2CDR2VL FW3VL CDR3VL FW4NO:06-AB-VK2DVVLTQSPLFLPVTRSSQSLLHSWYLQKPGQSSVFNGVPDRFSGSGSGTDFTLMQALEPPYTFGQGTKLE20119PGEPASISCRGHTSLHPHLLIYRASKISRVEAEDVGVYYCIK06-AB-VK2DIVMTQSPLSLPVTRSSQSLLHRWYLQKPGQSLGSNGVPDRFSGSGSGTDFTLMQGLQTPYFGQGTKLE21118PGEAASISCNGKTFFAPQILIYRASKISRVEAEDVGIYYCTIK06-AB-VK1DIVMTQSPSSVSASRASQGISRWWYQQKPGEAAASSGVPSRFSGSGSGTDFTLQQANSFPITFGQGTRL22120VGDKVTITCLAPELLIYLQSTISSLQPEDFATYYCQIK06-AB-VL3QLVLTQPPSVSVSPSGDELRNKYWYQQKSGQSQDNNGIPERFSGSQSGDTATLTQAWVSQTLFGGGTKLT23121GQTASITCTSPVLVIYRPSISGTQAVDEADYYCVVL06-AB-VL3QAGLTQPPSVSVAGGNNIGSKHWYQQKPGQADDSDGVPERFSGSNSGNTATLQVWDRSSDFGGGTRLT24122PGQTATIPCVHPVAVVYRPSTISSVEAGDEADYYCHFWLVL06-AB-VL2QLVLTQPPSASGSTGTSSDVGGWYQHHPGKAEVSQGVPDRFSGSKSGNTASLSSYAGSVVLFGGGTKLT25123PGQSVTISCSNFVSPKLMIYRPSTVSGLQADDEADYYCVL06-AB-VL2QLVLTQPPSASGSTGTSSDVGGWYQHHPGKAEVSQGVPDRFSGSKSGNTASLSSYAGSVVLFGGGTKLT26124PGQSVTISCSNFVSPKLMIYRPSTVSGLQADDEADYYCVL06-AB-VK3DIVMTQSPATLSLSWASQYINTYWYQHKPGQADASKGIPARFSGSGSGTDFTLTQQGSNWPLFGQGTRL27126PGERATLSCVNPRLLIYRATISSLEPEDFAVYYCTEIK06-AB-VK1EIVMTQSPSFVSASRASQDISNWWYQQKPGKAASSNGVPSRFSGSGSGTDFALQQENSFPYFGQGTKLE28127VGDRVTITCLVPKLLIYLQSTIISLQPEDFATYYCTIK06-AB-VK2VIWMTQSPLSLPVTRSSQSLLHRWYLQKPGQSLGSNGVPDRFSGSGSGTDFTLMQGLQTPYFGQGTKLE29129PGEAASISCNGRTFFAPQILIYRAFKISRVEAEDVGIYYCTIK06-AB-VK2VIWMTQSPLSLPVTRSSQSLLHRWYLQKPGQSLGSNGVPDRFSGSGSGTDFTLMQGLQTPYFGQGTKLE30130PGEAASISCNGRTFFAPQILIYRAFKISRVEAEDVGIYYCTIK06-AB-VK1DIVMTQTPSTQSASRASQSISIWLWYQQKPGKADASTGVPSRFSGSGSGTEFTLQRYNDYPPFGPGTKVE31131VGDRVTITCAPKLLIHLESTISSLQPDDSATYYCTIK06-AB-VK1EIVMTQSPSTQSASRASQSISIWLWYQQKPGKADASTGVPSRFSGSGSGTEFTLQRYNDYPPFGPGTKVE32132VGDRVTITCAPKLLIHLESTISSLQPDDSATYYCTIK06-AB-VL1QSVLTQPPSVSGTSGSNSNAGWYQQVPGTAKNNQGVPDRFSGSKSGTSASLIVWDGSLSGFGTGTKVT33133PGQRVTISCRDYVSPKLLIYRPSAISGLRSEDDGDYYCYVVL06-AB-VL7SYELTQPSSLTVSPGLSSGAVTSWFQQKPGQADTSRWTPARFSGSLLGGKAALLLACNGACVFGGGTKLT34134GGTVTLTCGHYPYPKTLIFKHSTLSGAQPEDDADYYCVL06-AB-VL7SYELTQPSSLTVSPGLSSGAVTSWFQQKPGQADTSRWTPARFSGSLLGGKAALLLACNGACVFGGGTKLT35135GGTVTLTCGHYPYPKTLIFKHSTLSGAQPEDDADYYCVL06-AB-VL1QSVLTQPPSVSGTSGSNSNVGWYQQVPGTAKNNRGVPDRFSGSKSGTSASLIVWDGSLSGFGTGTKVT36139PGQRVTISCRDYVSPKLLIYRPSAISGLRSEDDGDYYCYVVL06-AB-VL1QLVLTQPPSVSGTSGSNSNVGWYQQVPGTAKNNQGVPDRFSGSKSGTSASLIVWDGSLSGFGTGTKVT37140PGQRVTISCRDYVSPKLLIYRPSAISGLRSEDDGDYYCYVVL
Antibody Sequences and Seq ID No.s

TABLE AAntibody AB11906-AB-SEQ119SequenceID NO:VH FW1QVTLKESGGGLVQPGGSLRLSCVASGFTF38VH CDR1RTYVVMH39VH FW2WVRQDPGKGLVWVS40VH CDR2RLDEVGRLTSYADSVNG41VH FW3RFTISRDNAKNILYLQMNSLRAEDTGVYYCAR42VH CDR3DLSGSADY43VH FW4WGQGTLVTVSS44VL FW1DVVLTQSPLFLPVTPGEPASISC45VL CDR1RSSQSLLHSRGHTSLH46VL FW2WYLQKPGQSPHLLIY47VL CDR2SVFNRAS48VL FW3GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC49VL CDR3MQALEPPYT50VL FW4FGQGTKLEIK51

TABLE BAntibody AB11806-AB-SEQ118SequenceID NO:VH FW1EVQLVESGGGLVQPGGSLRLSCSASQFIL52VH CDR1SNYWVH53VH FW2WVRQVPGEGLVWVS54VH CDR2RINEDGSVTSYADSVKG55VH FW3RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR56VH CDR3DLCGERDD57VH FW4WGQGTLVSVSS58VL FW1DIVMTQSPLSLPVTPGEAASISC59VL CDR1RSSQSLLHRNGKTFFA60VL FW2WYLQKPGQSPQILIY61VL CDR2LGSNRAS62VL FW3GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC63VL CDR3MQGLQTPYT64VL FW4FGQGTKLEIK65

TABLE CAntibody AB12006-AB-SEQ120SequenceID NO:VH FW1EVQLVQSGGGLVQPGGSLGLSCAASGFIF66VH CDR1TSYAMT67VH FW2WVRQAPGKGLEWVS68VH CDR2VITGNVGTSYYADSVKG69VH FW3RFTISRDNSKKTVSLQMNSLRAEDTAIYYCVK70VH CDR3TRYDFSSGYYFDD71VH FW4WGQGTLVSVSS72VL FW1DIVMTQSPSSVSASVGDKVTITC73VL CDR1RASQGISRWLA74VL FW2WYQQKPGEAPELLIY75VL CDR2AASSLQS76VL FW3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC77VL CDR3QQANSFPIT78VL FW4FGQGTRLQIK79

TABLE DAntibody AB12106-AB-SEQ121SequenceID NO:VH FW1EVQLVESGGTLVQPGGSLRLSCAASGFTF80VH CDR1SDYWMN81VH FW2WVRQAPGKGLEWVA82VH CDR2NIKQDGSEKYYVDSLRG83VH FW3RVTISRDNAQNSVFLQMHSLSVEDTAVYYCAR84VH CDR3DGYTFGPATTELDH85VH FW4WGRGTLVSVSS86VL FW1QLVLTQPPSVSVSPGQTASITC87VL CDR1SGDELRNKYTS88VL FW2WYQQKSGQSPVLVIY89VL CDR2QDNNRPS90VL FW3GIPERFSGSQSGDTATLTISGTQAVDEADYYC91VL CDR3QAWVSQTLV92VL FW4FGGGTKLTVL93

TABLE EAntibody AB12206-AB-SEQ122SequenceID NO:VH FW1EVQLVQSGGGLAQPGRSLRLSCAASGFGF94VH CDR1DDFAMH95VH FW2WVRQPPGKGLEWVS96VH CDR2GLTWNGGSIDYAGSVRG97VH FW3RFTISRDNAKNSLFLQMNSLRAEDTALYYCAK98VH CDR3GLSGGTMAPFDI99VH FW4WGQGTMVSVSS100VL FW1QAGLTQPPSVSVAPGQTATIPC101VL CDR1GGNNIGSKHVH102VL FW2WYQQKPGQAPVAVVY103VL CDR2DDSDRPS104VL FW3GVPERFSGSNSGNTATLTISSVEAGDEADYYC105VL CDR3QVWDRSSDHFWL106VL FW4FGGGTRLTVL107

TABLE FAntibody AB12306-AB-SEQ ID123SequenceNO:VH FW1EVQLLESGGGVVQPGRS108LRLSCAASGFTFVH CDR1SNYGMH109VH FW2WVRQAPGKGLEWVA110VH CDR2VVWFDGSYKYYTDSVKG111VH FW3RFTISRDNSKSTLYLQM112NSLRAEDTAVYYCVSVH CDR3PIMTSAFDI113VH FW4WGPGTMVSVSS114VL FW1QLVLTQPPSASGSPGQS115VTISCVL CDR1TGTSSDVGGSNFVS116VL FW2WYQHHPGKAPKLMIY117VL CDR2EVSQRPS118VL FW3GVPDRFSGSKSGNTASL119TVSGLQADDEADYYCVL CDR3SSYAGSVVL120VL FW4FGGGTKLTVL121

TABLE GAntibody AB12406-AB-SEQ ID124SequenceNO:VH FW1EVQLVESGGGVVQPGRS122LRLSCAASGFTFVH CDR1SNYGMH123VH FW2WVRQAPGKGLEWVA124VH CDR2VVWLDGSYKYYTGSVKG125VH FW3RFTISRDNSKSTLYLQM126NSLRAEDTAAYYCVSVH CDR3PIMTSAFDI127VH FW4WGPGTMVTVSS128VL FW1QLVLTQPPSASGSPGQS129VTISCVL CDR1TGTSSDVGGSNFVS130VL FW2WYQHHPGKAPKLMIY131VL CDR2EVSQRPS132VL FW3GVPDRFSGSKSGNTASL133TVSGLQADDEADYYCVL CDR3SSYAGSVVL134VL FW4FGGGTKLTVL135

TABLE HAntibody AB12606-AB-SEQ ID126SequenceNO:VH FW1EVQLVESGGGLAQPGGS136LRLSCEASGFHLVH CDR1AGNAMA137VH FW2WVRQAPGKGLEWVA138VH CDR2AIGGSDDRTDYADSVKG139VH FW3RFTISRDKSKNTLSLQM140NSLRVEDTAVYYCAKVH CDR3DIWRWAFDY141VH FW4WGQGTLVSVSS142VL FW1DIVMTQSPATLSLSPGE143RATLSCVL CDR1WASQYINTYVN144VL FW2WYQHKPGQAPRLLIY145VL CDR2DASKRAT146VL FW3GIPARFSGSGSGTDFTL147TISSLEPEDFAVYYCVL CDR3QQGSNWPLT148VL FW4FGQGTRLEIK149

TABLE IAntibody AB12706-AB-SEQ ID127SequenceNO:VH FW1EVQLVESGGGLVNPGGS150LRLSCAASGFTFVH CDR1SNYAMN151VH FW2WVRQAPGKGLEWVS152VH CDR2SISRSGDYIYYADSLKG153VH FW3RSTISRDNAKNSLFLQM154NSLRAEDSAVYYCARVH CDR3DWGRLGYCSSNNCPDAF155DVVH FW4WGQGTRVSVSS156VL FW1EIVMTQSPSFVSASVGD157RVTITCVL CDR1RASQDISNWLV158VL FW2WYQQKPGKAPKLLIY159VL CDR2ASSNLQS160VL FW3GVPSRFSGSGSGTDFAL161TIISLQPEDFATYYCVL CDR3QQENSFPYT162VL FW4FGQGTKLEIK163

TABLE JAntibody AB12906-AB-SEQ ID129SequenceNO:VH FW1QVQLVESGGGLVQPGGS164LRLSCSASQFILVH CDR1SNYWVH165VH FW2WVRQVPGEGLVWVS166VH CDR2RINEDGSVTSYADSVKG167VH FW3RFTISRDNAKNTLYLQM168NSLRVDDTAVYYCVRVH CDR3DLCGERDD169VH FW4WGQGTLVTVSS170VL FW1VIWMTQSPLSLPVTPGE171AASISCVL CDR1RSSQSLLHRNGRTFFA172VL FW2WYLQKPGQSPQILIY173VL CDR2LGSNRAF174VL FW3GVPDRFSGSGSGTDFTL175KISRVEAEDVGIYYCVL CDR3MQGLQTPYT176VL FW4FGQGTKLEIK177

TABLE KAntibody AB13006-AB-SEQ ID130SequenceNO:VH FW1QLQLQESGGGLVQPGGS178LRLSCSASQFILVH CDR1SNYWVH179VH FW2WVRQVPGEGLVWVS180VH CDR2RINEDGSVTSYADSVKG181VH FW3RFTISRDNAKNTLYLQM182NSLRVDDTAVYYCVRVH CDR3DLCWERDD183VH FW4WGQGTLVSVSS184VL FW1VIWMTQSPLSLPVTPGE185AASISCVL CDR1RSSQSLLHRNGRTFFA186VL FW2WYLQKPGQSPQILIY187VL CDR2LGSNRAF188VL FW3GVPDRFSGSGSGTDFTL189KISRVEAEDVGIYYCVL CDR3MQGLQTPYT190VL FW4FGQGTKLEIK191

TABLE LAntibody AB13106-AB-SEQ ID131SequenceNO:VH FW1QVQLVQSGGGVVQPGGS192LRLSCAASPFTFVH CDR1KTSILH193VH FW2WVRQAPGKGLEWVS194VH CDR2AMSYDGFSKYYADSVKG195VH FW3RLTISRDSSTNTLYLEM196NSLRFEDTALYFCARVH CDR3EAYTSGRAGCFNP197VH FW4WGQGVLVSVSS198VL FW1DIVMTQTPSTQSASVGD199RVTITCVL CDR1RASQSISIWLA200VL FW2WYQQKPGKAPKLLIH201VL CDR2DASTLES202VL FW3GVPSRFSGSGSGTEFTL203TISSLQPDDSATYYCVL CDR3QRYNDYPPT204VL FW4FGPGTKVEIK205

TABLE MAntibody AB13206-AB-SEQ ID132SequenceNO:VH FW1QVLKESGGGVVQPGGSL206RLSCAASPFTFVH CDR1ETSILH207VH FW2WVRQAPGKGLEWVS208VH CDR2AMSYDGFSKYYADSVKG209VH FW3RLTISRDSSTNTLYLEM210NSLRFEDTALYFCARVH CDR3EAYTSGRAGCFDP211VH FW4WGQGVLVSVSS212VL FW1EIVMTQSPSTQSASVGD213RVTITCVL CDR1RASQSISIWLA214VL FW2WYQQKPGKAPKLLIH215VL CDR2DASTLES216VL FW3GVPSRFSGSGSGTEFTL217TISSLQPDDSATYYCVL CDR3QRYNDYPPT218VL FW4FGPGTKVEIK219

TABLE NAntibody AB13306-AB-SEQ ID133SequenceNO:VH FW1EVQLVESGGGLVQPGGS220LRVSCAASGFTLVH CDR1NTYWMH221VH FW2WVRQAPGKGLVWVS222VH CDR2RINEDGTTISYADSVRG223VH FW3RFTISRDNAENTLYLQM224HSLRAEDTGVYYCARVH CDR3DFTGPFDS225VH FW4WGQGTLVSVSS226VL FW1QSVLTQPPSVSGTPGQR227VTISCVL CDR1SGSNSNAGRDYVS228VL FW2WYQQVPGTAPKLLIY229VL CDR2KNNQRPS230VL FW3GVPDRFSGSKSGTSASL231AISGLRSEDDGDYYCVL CDR3IVWDGSLSGYV232VL FW4FGTGTKVTVL233

TABLE OAntibody AB13406-AB-SEQ ID134SequenceNO:VH FW1QLQLQESGGGLVQPGGS234LRLSCVVSGFTFVH CDR1SSHAMS235VH FW2WVRQAPGKGLEWVS236VH CDR2SISISGGDTFYADSVRG237VH FW3RFTIFRDNSKNTVYLQM238NSLRAEDTAVYYCATVH CDR3ETSPNDY239VH FW4WGQGTLVSVSS240VL FW1SYELTQPSSLTVSPGGT241VTLTCVL CDR1GLSSGAVTSGHYPY242VL FW2WFQQKPGQAPKTLIF243VL CDR2DTSRKHS244VL FW3WTPARFSGSLLGGKAAL245TLSGAQPEDDADYYCVL CDR3LLACNGACV246VL FW4FGGGTKLTVL247

TABLE PAntibody AB13506-AB-SEQ ID135SequenceNO:VH FW1EVQLVETGGGLVQPGGS248LRLSCVVSGFTFVH CDR1SSHAMS249VH FW2WVRQAPGKGLEWVS250VH CDR2SISISGGDTFYADSVRG251VH FW3RFTIFRDNSKNTVYLQM252NSLRAEDTAVYYCATVH CDR3ETSPNDY253VH FW4WGQGTLVTVSS254VL FW1SYELTQPSSLTVSPGGT255VTLTCVL CDR1GLSSGAVTSGHYPY256VL FW2WFQQKPGQAPKTLIF257VL CDR2DTSRKHS258VL FW3WTPARFSGSLLGGKAAL259TLSGAQPEDDADYYCVL CDR3LLACNGACV260VL FW4FGGGTKLTVL261

TABLE QAntibody AB13906-AB-SEQ ID139SequenceNO:VH FW1EVQLVESGGGLVQPGGS262LRVSCAASGFTLVH CDR1NTYWMH263VH FW2WVRQAPGKGLVWVS264VH CDR2RINEDGTTISYADSVRG265VH FW3RFTISRDNAENTLYLQM266HSLRAEDTGVYYCARVH CDR3DFTGPFDS267VH FW4WGQGTLVSVSS268VL FW1QSVLTQPPSVSGTPGQR269VTISCVL CDR1SGSNSNVGRDYVS270VL FW2WYQQVPGTAPKLLIY271VL CDR2KNNRRPS272VL FW3GVPDRFSGSKSGTSASL273AISGLRSEDDGDYYCVL CDR3IVWDGSLSGYV274VL FW4FGTGTKVTVL275

TABLE RAntibody AB14006-AB-SEQ ID140SequenceNO:VH FW1EVQLVESGGGLVQPGGS276LRVSCAASGFTLVH CDR1NTYWMH277VH FW2WVRQAPGKGLVWVS278VH CDR2RINEDGTTISYADSVRG279VH FW3RFTISRDNAENTLYLQM280HSLRAEDTGVYYCARVH CDR3DFTGPFDS281VH FW4WGQGTLVSVSS282VL FW1QLVLTQPPSVSGTPGQR283VTISCVL CDR1SGSNSNVGRDYVS284VL FW2WYQQVPGTAPKLLIY285VL CDR2KNNQRPS286VL FW3GVPDRFSGSKSGTSASL287AISGLRSEDDGDYYCVL CDR3IVWDGSLSGYV288VL FW4FGTGTKVTVL289

TABLE VH-CDR3-mod(VariantSEQofLight orIDSEQ IDHeavy CDR3NO:NO:)06-AB-118.HeavyC101ADLAGERDD2905706-AB-118.HeavyC101SDLSGERDD2915706-AB-127.HeavyWYDWGRLGYWSSNNY292155PDAFDV06-AB-127.HeavyAADWGRLGYASSNNA293155PDAFDV06-AB-131.HeavyWEAYTSGRAGWFNP29419706-AB-131.HeavyAEAYTSGRAGAFNP29519706-AB-132.HeavyWEAYTSGRAGWFDP29621106-AB-132.HeavyAEAYTSGRAGAFDP29721106-AB-129.HeavyWDLWGERDD29816906-AB-129.HeavyADLAGERDD299169

TABLE VL-CDR3-mod06-AB-134.LightYWLLAYNGAWV30024606-AB-134.LightAALLAANGAAV30124606-AB-135.LightYWLLAYNGAWV30226006-AB-135.LightAALLAANGAAV303260

REFERENCES

1. Brown G D. Innate antifungal immunity: The key role of phagocytes.Annu. Rev. Immuol.29, 1-21 (2011).2. Lockhart S R. Current epidemiology ofCandidainfection.Clin. Microbiol. News.36, 131-136 (2014).3. Kim J, Sudbery P.Candida albicans, a major human fungal pathogen.J. Microbiol.49, 171-177 (2011).4. Gow N A R, Van De Veerdonk F L, Brown A J P, Netea M G.Candida albicansmorphogenesis and host defence: Discriminating invasion from colonization.Nat. Rev. Microbiol.10, 112-122 (2012).5. Ellepola A N B, Morrison C J. Laboratory diagnosis of invasive candidiasis.J. Microbiol.43, 65-84 (2005).6. Ostrosky-Zeichner L. Invasive mycoses: Diagnostic challenges.Am. J. Med.125, S14-24 (2012).7. Perfect J R. Fungal diagnosis: How do we do it and can we do better?Curr. Med. Res. Opin.29, 3-11 (2013).8. Pfaller M A. Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment.Am. J. Med.125(1 SUPPL.), S3-S13 (2012).9. Rader C. Chemically programmed antibodies.Trends. Biotechnol.32, 186-97 (2014).10. Yoon S, Kim Y-, Shim H, Chung J. Current perspectives on therapeutic antibodies.Biotechnol. Bioprocess Eng.15, 709-15 (2010).11. Carter P J. Introduction to current and future protein therapeutics: a protein engineering perspective.Exp. Cell Res.317, 1261-1269 (2011).12. Berry J D, Gaudet R G. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology.New Biotech.28, 489-501 (2011).13. Saylor C, Dadachova E, Casadevall A. Monoclonal antibody-based therapies for microbial diseases.Vaccine.27, G38-46 (2009).14. Wilson P C, Andrews S F. Tools to therapeutically harness the human antibody response.Nat. Rev. Immunol.12, 709-19 (2012).15. Casadevall A, Pirofski L A. Immunoglobulins in defense, pathogenesis, and therapy of fungal diseases.Cell Host Microbe.11, 447-56 (2012).16. Dromer F, Salamero J, Contrepois A, Carbon C, Yeni P. Production, characterization, and antibody specificity of a mouse monoclonal antibody reactive withcryptococcus neoformanscapsular polysaccharide.Infect. Immun.55, 742-748 (1987).17. Gigliotti F, Hughes W T. Passive immunoprophylaxis with specific monoclonal antibody confers partial protection againstPneumocystis cariniipneumonitis in animal models.J. Clin. Invest.81, 1666-1668 (1988).18. Brena S, Cabezas-Olcoz J, Moragues M D, Fernández De Larrinoa I, Dominguez A, Quindós G, et al. Fungicidal monoclonal antibody C7 interferes with iron acquisition inCandida albicans. Antimicrob. Agents Chemother.55, 3156-3163 (2011).19. Moragues M D, Omaetxebarria M J, Elguezabal N, Sevilla M J, Conti S, Polonelli L, et al. A monoclonal antibody directed against aCandida albicanscell wall mannoprotein exerts three anti-C. albicansactivities.Infect. Immun.71, 5273-5279 (2003).20. Torosantucci A, Bromuro C, Chiani P, De Bernardis F, Berti F, Galli C, et al. A novel glyco-conjugate vaccine against fungal pathogens.J. Exp. Med.202, 597-606 (2005).21. Torosantucci A, Chiani P, Bromuro C, De Bernardis F, Palma A S, Liu Y, et al. Protection by anti-ß-glucan antibodies is associated with restricted ß-1,3 glucan binding specificity and inhibition of fungal growth and adherence.PLoS ONE.4, e5392 (2009).22. Xin H, Cutler J E. Vaccine and monoclonal antibody that enhance mouse resistance to candidiasis.Clin. Vaccine Immunol.18, 1656-1667 (2011).23. Cassone A. VulvovaginalCandida albicansinfections: Pathogenesis, immunity and vaccine prospects.BJOG. doi:10.1111/1471-0528.12994. (2014).24. Cassone A. Development of vaccines forCandida albicans: fighting a skilled transformer.Nat. Rev. Microbiol.11, 884-91 (2013).25. Bromuro C, Romano M, Chiani P, Berti F, Tontini M, Proietti D, et al. Beta-glucan-CRM197 conjugates as Candidates antifungal vaccines.Vaccine.28, 2615-23 (2010).26. Schmidt C S, White C J, Ibrahim A S, Filler S G, Fu Y, Yeaman M R, et al. NDV-3, a recombinant alum-adjuvanted vaccine forCandidaandStaphylococcus aureus, is safe and immunogenic in healthy adults.Vaccine.30, 7594-7600 (2012).27. Han Y, Morrison R P, Cutler J E. A vaccine and monoclonal antibodies that enhance mouse resistance toCandida albicansvaginal infection.Infect. Immun.66, 5771-5776 (1998).28. Ibrahim A S, Luo G, Gebremariam T, Lee H, Schmidt C S, Hennessey J P, et al. NDV-3 protects mice from vulvovaginal candidiasis through T- and B-cell immune response.Vaccine.31, 5549-56 (2013).29. Luo G, Ibrahim A S, French S W, Edwards Jr. J E, Fu Y. Active and passive immunization with rHyr1p-N protects mice against hematogenously disseminated candidiasis.PLoS ONE.6, e25909. (2011).30. Thornton C, Johnson G, Agrawal S. Detection of invasive pulmonary aspergillosis in haematological malignancy patients by using lateral-flow technology.J. Vis. Exp.61, pii: 3721. doi: 10.3791/3721 (2012).31. Thornton C R. Development of an immunochromatographic lateral-flow device for rapid serodiagnosis of invasive aspergillosis.Clin. Vaccine Immunol.15, 1095-1105 (2008).32. Martinez-Jiménez M C, Muñoz P, Guinea J, Valerio M, Alonso R, Escribano P, et al. Potential role ofCandida albicansgerm tube antibody in the diagnosis of deep-seated candidemia.Med. Mycol.52, 270-275 (2014).33. Jarvis J N, Percival A, Bauman S, Pelfrey J, Meintjes G, Williams G N, et al. Evaluation of a novel point-of-care cryptococcal antigen test on serum, plasma, and urine from patients with HIV-associated cryptococcal meningitis.Clin. Infect. Dis.53, 1019-1023 (2011).34. Carter P J. Potent antibody therapeutics by design.Nat. Rev. Immunol.6, 343-357 (2006).35. Tiller T. Single B cell antibody technologies.N. Biotechnol.28, 453-457 (2011).36. Smith K, Garman L, Wrammert J, Zheng N Y, Capra J D, Ahmed R, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.Nat. Protoc.4, 372-384 (2009).37. Liao H X, Levesque M C, Nagel A, Dixon A, Zhang R, Walter E, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.J. Virol. Methods.158, 171-179 (2009).38. Huang J, Doria-Rose N A, Longo N S, Laub L, Lin C-, Turk E, et al. Isolation of human monoclonal antibodies from peripheral blood B cells.Nat. Protoc.8, 1907-1915 (2013).39. Jiang X, Suzuki H, Hanai Y, Wada F, Hitomi K, Yamane T, et al. A novel strategy for generation of monoclonal antibodies from single B cells using R T-PCR technique and in vitro expression.Biotechnol. Prog.22, 979-88 (2006).40. Bailey D A, Feldmann P J F, Bovey M, Gow N A R, Brown A J P. TheCandida albicansHYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins.J. Bacteriol.178, 5353-5360 (1996).41. Luo G, Ibrahim A S, Spellberg B,NobileC J, Mitchell A P, Fu Y.Candida albicansHyr1p confers resistance to neutrophil killing and is a potential vaccine target.J. Infect. Dis.201, 1718-1728 (2010).42. Lewis L E, Bain J M, Lowes C, Gillespie C, Rudkin F M, Gow N A R, et al. Stage specific assessment ofCandida albicansphagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants.PLoS Pathog.8, e1002578 (2012).43. Rudkin F M, Bain J M, Walls C, Lewis L E, Gow N A R, Erwig L P. Altered dynamics ofCandida albicansphagocytosis by macrophages and PMNs when both phagocyte subsets are present.mBio.4, e00810-13. doi: 10.1128/mBio.00810-13 (2013).44. MacCallum D M, Coste A, Ischer F, Jacobsen M D, Odds F C, Sanglard D. Genetic dissection of azole resistance mechanisms inCandida albicansand their validation in a mouse model of disseminated infection.Antimicrob. Agents Chemother.54, 1476-1483 (2010).45. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future.Br. J. Pharmacol.157, 220-233 (2009).46. Beyda N D, Regen S, Lewis R E, Garey K W. Immunomodulatory agents as adjunctive therapy for the treatment of resistantCandidaspecies.Curr. Fungal Infect. Rep.7, 119-125 (2013).47. Overdijk M B, Verploegen S, Buijsse A O, Vink T, Leusen J H W, Bleeker W K, et al. Crosstalk between human IgG isotypes and murine effector cells.J. Immunol.189, 3430-3438 (2012).48. Reichert J M. Marketed therapeutic antibodies compendium.mAbs.4, 413-415 (2012).49. Brown G D, Denning D W, Gow N A R, Levitz S M, Netea M G, White T C. Hidden killers: human fungal infections.Sci. Transl. Med.4, 165rv13 (2012).50. d'Enfert C, Goyard S, Rodriguez-Arnaveilhe S, Frangeul L, Jones L, Tekaia F, et al.CandidaDB: A genome database forCandida albicanspathogenomics.Nucleic Acids Res.33(DATABASE ISS.), D353-357 (2005).51. Yapar N. Epidemiology and risk factors for invasive candidiasis.Ther. Clin. Risk Manag.10, 95-105 (2014).52. MacCallum D M. Hosting infection: experimental models to assayCandidavirulence.Int. J. Microbiol.2012, 363764 (2012).53. Durocher Y, Perret S, Kamen A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.Nucleic Acids Res.30, e9 (2002).54. McKenzie C G J, Koser U, Lewis L E, Bain J M, Mora-Montes H M, Barker R N, et al. Contribution ofCandida albicanscell wall components to recognition by and escape from murine macrophages.Infect. Immun.78, 1650-1658 (2010).

ADDITIONAL REFERENCES FOR TABLE S1

Brand A, MacCallum D M, Brown A J, Gow N A, Odds F C. Ectopic expression of URA3 can influence the virulence phenotypes and proteome ofCandida albicansbut can be overcome by targeted reintegration of URA3 at the RPS10 locus.Eukaryot Cell3: 900-909. doi: 10.1128/ec.3.4.900-909.2004 (2004).Bailey D A, Feldmann P J F, Bovey M, Gow N A R, Brown A J P. TheCandida albicansHYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins.J. Bacteriol.178, 5353-5360 (1996).Fonzi, W. A., Irwin M. Y. Isogenic strain construction and gene mapping inCandida albicans. Genetics134, 717-728 (1993).Gillum A M, Tsay E Y, Kirsch D R. Isolation of theCandida albicansgene for orotidine-5′-phosphate decarboxylase by complementation ofS. cerevisiaeura3 andE. colipyrF mutations.Mol Gen Genet.198:179-8 (1984).Odds F C, Hanson M F, Davidson A D, Jacobsen M D, Wright P, Whyte J A, Gow N A, Jones B L. One year prospective survey ofCandidabloodstream infections in Scotland.J Med Microbiol.56: 1066-1075 (2007).Rudek W. Esterase activity inCandidaspecies.J. Clin. Microbiol.8: 756-759 (1978). Moran G P, Sanglard D, Donnelly S M, Shanley D B, Sullivan D J, Coleman D C. Identification and expression of multidrug transporters responsible for fluconazole resistance inCandida dubliniensis. Antimicrob. Agents Chemother.42:1819-1830 (1998).Moran, G. P., Sanglard D., Donnelly S. M., Shanley D. B., Sullivan D. J., Coleman D. C. Identification and expression of multidrug transporters responsible for fluconazole resistance inCandida dubliniensis. Antimicrob. Agents Chemother.42, 1819-1830 (1998).Netea, M. G. et al.Aspergillus fumigatusevades immune recognition during germination through loss of toll-like receptor-4-mediated signal transduction.J. Infect. Dis.188, 320-326 (2003).Satoh, K., Makimura K., Hasumi Y., Nishiyama Y., Uchida K., Yamaguchi H.Candida aurissp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital.Microbiol. Immunol.53, 41-44 (2009).Khan, Z. U. et al. Outbreak of fungemia among neonates caused byCandida haemuloniiresistant to amphotericin B, itraconazole, and fluconazole.J. Clin. Microbiol.45, 2025-2027 (2007).Nielsen, K., Cox G. M., Wang P., Toffaletti D. L., Perfect J. R., Heitman J. Sexual cycle ofCryptococcus neoformansvar.grubiiand Virulence of congenic a and a isolates.Infect. Immun.71, 4831-4841 (2003).Fyfe, M., W. Black and M. Romney. Unprecedented outbreak ofCryptococcus neoformansvar.gattiiinfections in British Columbia, Canada. Abstracts of the 5th International Conference onCryptococcusand Cryptococcosis (2002).Sugita, T. et al. New yeast species,Malassezia dermatis, isolated from patients with atopic dermatitis.J. Clin. Microbiol.40, 1363-1367 (2002).Li, C. H. et al. Sporangiospore size dimorphism is linked to virulence ofMucor circinelloides. PLoS Pathogens7, e1002086 (2011).