ANTIBODY MOLECULES AND USES THEREOF

Abstract

This invention relates to recombinant human antibody molecules for use in a method of treatment of Acinetobacter infection. The antibodies bind Acinetobacter antigens, for example from Acinetobacter spp. Human antibody encoding genes targeting clinically relevant Candida epitopes have been isolated from single B cells from carefully selected donors and screened with specified types of protein or cell wall extract. The panel of purified, fully human recombinant IgG1 mAbs generated displayed a diverse range of specific binding profiles and demonstrated efficacy in a disease model. The fully human mAbs and derivatives thereof have utility in the generation of diagnostics, therapeutics and vaccines.

Claims

1. An isolated recombinant human anti-Candida antibody molecule derived from single B cells for use in a method of treatment of an Acinetobacter bacterial infection, wherein the antibody molecule comprises a VH domain comprising a HCDR3 having the amino acid sequence of SEQ ID NO: 6x or the sequence of SEQ ID NO: 6x with 1, 2, or 3 amino acid substitutions, deletions or insertions, wherein ‘x’ is one letter from F, E, D and C, and said sequence is as shown in Table ‘x’ herein.

2. An antibody molecule for use according to claim 1 wherein the antibody molecule comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 4x or the sequence of SEQ ID NO: 4x with 1, 2, or 3 amino acid substitutions, deletions or insertions.

3. An antibody molecule for use according to claim 1 or claim 2 wherein the antibody molecule comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 2x or the sequence of SEQ ID NO: 2x with 1, 2 or 3 amino acid substitutions, deletions or insertions.

4. An antibody molecule for use according to any one of claims 1-3 wherein the antibody molecule comprises a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 2x, 4x and 6x respectively.

5. An antibody molecule for use according to any one of claims 1-4 wherein the antibody molecule comprises a VH domain comprising one or more or all of a FW1, a FW2, a FW3 and a FW4 having the sequences of SEQ ID NOs lx, 3x, 5x and 7x respectively.

6. An antibody molecule for use according to any one of claims 1-5 wherein the antibody molecule comprises a VH domain having an amino acid sequence at least about 80% identical to SEQ ID NO: 15x and\or having the amino acid sequence of SEQ ID NO: 15x and\or the sequence of SEQ ID NO: 15x with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or insertions in SEQ ID NO: 15x.

7. An antibody molecule for use according to any one of claims 1-6 wherein the antibody molecule comprises a VL domain comprising LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 9x, 11x and 13x respectively, or the sequences of SEQ ID NOs 9x, 11x and 13x respectively with, independently, 1, 2 or 3 or more amino acid substitutions, deletions or insertions.

8. An antibody molecule for use according to claim 7 wherein the antibody molecule comprises a VL domain comprising LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 9x, 11x and 13x respectively.

9. An antibody molecule for use according to any one of claims 1-8 wherein the antibody molecule comprises a VL domain comprising one or more or all of a FW1, a FW2, a FW3 and a FW4 having the sequences of SEQ ID NOs 8x, 10x, 12x and 14x respectively.

10. An antibody molecule for use according to any one of claims 1-9 wherein the antibody molecule comprises a VL domain having an amino acid sequence at least about 80% identical to SEQ ID NO: 16x and\or having the sequence of SEQ ID NO: 16x and\or the sequence of SEQ ID NO: 16x with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 16x.

11. An antibody molecule for use according to any one of claims 1-10 wherein the antibody molecule comprises a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 2x, 4x, and 6x, respectively, and a VL domain comprising a LCDR1, a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 9x, 11x and 13x, respectively.

12. An antibody molecule for use according to any one of claims 1-11 wherein the antibody molecule comprises VH and VL domains having the amino acid sequences of SEQ ID NO: 15x and SEQ ID NO: 16x respectively.

13. An antibody molecule for use according to any one of claims 1-12, wherein the bacterial infection is an A. baumannii infection.

14. An antibody molecule for use according to any one of claims 1-12, wherein the bacterial infection is an A. Baumannii infection, an A. pittii infection, an A. nosocomialis infection, an A. calcoaceticus infection, an A. seifertii infection or an A. dijkshoorniae infection.

15. An antibody molecule for use according to anyone of claims 1-13, wherein the antibody molecule binds A. baumannii with an EC.sub.50 value of (a) 1 to 1500 ng/mL; or (b) less than 20 ng/mL.

16. An antibody molecule for use according to any one of claims 1-15 wherein the antibody molecule is a whole antibody or a scAb.

17. An antibody molecule for use according to any one of claims 1-16 wherein the antibody molecule comprises a payload which is cytotoxic.

18. A pharmaceutical composition for use in a method of treatment of an Acinetobacter bacterial infection, the composition comprising an antibody molecule as defined in any one of claims 1-17 and a pharmaceutically acceptable excipient.

19. A composition of matter for use in in a method of treatment of an Acinetobacter bacterial infection, the composition comprising (1) a pharmaceutical composition as defined in claims 18 and (2) a further antibacterial agent.

20. A method of identifying or labelling an Acinetobacter cell, the method comprising contacting the cell with an antibody molecule as defined in any one of claims 1-16.

21. A method of opsonising, or increasing the rate of opsonisation of, an Acinetobacter cell, the method comprising contacting or pre-incubating the cell with an antibody molecule as defined in any one of claims 1-16.

22. A method of increasing the rate of engulfment of an Acinetobacter cell, the method comprising contacting the cell with an antibody molecule as defined in any one of claims 1-16.

23. A method of treatment of an Acinetobacter bacterial infection, comprising administering an antibody molecule as defined in any one of claims 1-17, or a composition as defined in claim 18 or claim 19, to an individual in need thereof.

24. The method of claim 23, wherein the Acinetobacter bacterial infection is an A. baumannii bacterial infection.

25. The method of claim 23, wherein the bacterial infection is an A. Baumannii infection, an A. pittii infection, an A. nosocomialis infection, an A. calcoaceticus infection, an A. seifertii infection or an A. dijkshoomiae infection.

26. The method of claims 23-25, wherein the treatment comprises administering a second antibacterial agent, wherein the second antibacterial agent is optionally: (a) a cephalosporin; (b) a combination beta-lactam/beta-lactamase inhibitor (optionally sulbactam); (c) a carbapenem (optionally meropenem, doripenem, or imipenem); (d) a polymyxin (optionally colistin or polymixin B); (e) tigecycline; or (f) minocycline.

27. Use of an antibody molecule as defined in any one of claims 1-17 in the manufacture of a medicament for use in treating or preventing an Acinetobacter infection.

28. An antibody molecule for use according to any one of claims 1-17, a composition for use according to any one of claim 18 or claim 19, a method according to any one of claims 23-26, or a use according to claim 27, wherein the Acinetobacter bacterial infection is in an immunosuppressed individual.

29. A method for detecting the presence or absence of a bacterium which is Acinetobacter spp, the method comprising (i) contacting a sample suspected of containing the bacterium with an antibody molecule as defined in any one of claims 1-16, and (ii) determining whether the antibody molecule binds to the sample, wherein binding of the antibody molecule to the sample indicates the presence of the bacterium.

30. A method for diagnosing a bacterial infection in an individual which is caused by Acinetobacter spp, the method comprising (i) contacting a biological sample obtained from the individual with an antibody molecule as defined in any one of claims 1-16, and (ii) determining whether the antibody molecule binds to the biological sample, wherein binding of the antibody molecule to the biological sample indicates the presence of a bacterial infection.

31. A linear flow device (LFD) for detecting an analyte which is a bacterial pathogen in a sample fluid, wherein said LFD comprises: (i) a housing, and (ii) at least one flow path leading from a sample well to a viewing window, wherein said flow path comprises one or more carriers along which the sample fluid is capable of flowing by capillary action, the or each carrier comprising an analyte-detecting means; wherein the presence of analyte produces a line in the viewing window which indicates an analyte concentration, wherein the bacterial pathogen is Acinetobacter spp. and the at least one analyte-detecting means is an antibody molecule as defined in any one of claims 1-16.

32. A device as claimed in claim 31 which further comprises a control zone capable of indicating the assay has been successfully run.

33. A device as claimed in claim 31 or claim 32 having a plurality of analyte-detecting means capable of distinguishing between multiple bacterial pathogens, wherein one of the analyte-detecting means is an antibody molecule as defined in any one of claims 1-16.

34. A device as claimed in claim 33 wherein the multiple bacterial pathogens comprise A. baumannii, plus one or more or all of Pseudomonas aeruginosa, Escherichia coli and Serratia marcescens.

35. A device as claimed in claim 33 wherein the multiple bacterial pathogens comprise A. pittii, A. nosocomialis, A. calcoaceticus, A. seifertii or A. dijkshoomiae, plus one or more or all of Pseudomonas aeruginosa, Escherichia coli and Serratia marcescens.

36. The device of any one of claims 31-35, for use in a method of any one of claims 23-26.

37. Use of an antibody molecule as defined in any one of claims 1-16 for the prevention of biofilm formation between A. baumannii and C. albicans.

Description

FIGURES

[0207] FIG. 1—Workflow for the generation of human monoclonal antibodies from single B cells. Class-switched memory B cells were isolated from individuals and microcultured in activating media to promote IgG secretion for screening against target antigens. VH and VL genes from B cells positive for the target were amplified and cloned into a mammalian expression vector for expression and purification via fast protein liquid chromatography. Following QC, recombinant mAbs were assessed for functional activity in vitro and in vivo. Adapted from Huang et al. 2013 (38).

[0208] FIG. 2—Representative images from the process employed to generate fully human anti-Candida mAbs. FIG. 2A shows the ELISA screening of purified donor circulating IgG against the antigen C. albicans ‘whole cell’ yeast and target antigen hyphae, and purified Hyr1 protein, to select the donors to take forward for B cell isolation. FIG. 2B and 2C are representative agarose gel images following RT-PCR and nested PCR of VH and Vk-Ck genes respectively. FIGS. 2D and 2E are analytical mass spectrometry and analytical SEC traces of one of the purified recombinant IgG1 mAbs. Further quality control was carried out by SDS-PAGE gel analysis under non-reducing and reducing conditions as shown in FIGS. 2F and 2G.

[0209] FIG. 3—Concentration response curves showing anti-Candida mAbs binding to target antigens. FIG. 3 shows purified anti-Hyr1 mAbs binding to purified recombinant Hyr1 protein in a concentration-dependent manner via ELISA. Values represent mean±SEM (n=2-4).

[0210] FIG. 4. Anti-Candida Hyr1 mAbs bind selectively to the gram negative bacteria, Acinetobacter baumannii. The anti-Hyr1 mAbs, AB120 (A), AB121 (B), AB122 (C), AB123 (D) and anti-Candida control AB as a negative control (E) were screened for their binding to whole cells of the gram negative bacteria: Acinetobacter baumannii, Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa via ELISA. Values represent mean±SEM (n=4).

[0211] FIG. 5. Indirect immunofluorescence and TEM images of anti-CaHyr1 mAbs binding to A. baumannii cells. (A) Immunofluorescent images of anti-Hyr1 mAbs and negative control against A. baumannii using an alexa-488 conjugated secondary goat anti-human IgG antibody. (B) TEM images showing the ultrastructural binding of anti-Hyr1 mAbs to the cell surface of A. baumannii cells. A colloidal gold (10 nm) secondary goat anti-human IgG antibody was used to detect anti-Hyr1 mAb binding. Scale bars represent 10 μm on immunofluorescent images and 100 nm on TEM images.

[0212] FIG. 6. Treatment with anti-Hyr1 mAbs promote phagocytosis by macrophages and protects against A. baumannii infection. (A) Percentage of uptake events that occurred within the first 30 min of the assay following pre-incubation of A. baumannii with saline, an IgG control antibody or an anti-Hyr1 mAb. *p<0.005 compared to saline and control AB. (B) Images taken from live cell microscopy capturing images of phagocytosis of A. baumannii pre-incubated with 50 μg/ml control mAb or 50 μg/ml anti-Hyr1 mAb (AB123) at 30 min and 2 h.

[0213] FIG. 7. Treatment with anti-Hyr1 mAbs protects against A. baumannii infection. Galleria mellonella were infected with 1×10.sup.6 cells of A. baumannii following pre-incubation with (A) saline or 50 μg/ml anti-Hyr1 mAbs (B) AB120, (C) AB121, (D) AB122, (E) AB123, and their survival monitored every 12 h. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, n =30.

[0214] FIG. 8—Indirect immunofluorescence of AB120 binding to Hyr1 protein expressed on C. albicans hyphal cells. Indirect immunofluorescence with anti-Hyr1 mAb AB120 against WT CAl4-Clp10 (A), Hyr1 null mutant (B) and a Hyr1 re-integrant strain (C). A fluorescently conjugated secondary goat anti-human IgG antibody was used to detect anti-Hyr1 mAb binding. Scale bars represent 15 μm.

[0215] FIG. 9—Schematic of VH, Vκ-Cκ and Vλ-Cλ cloning into pTT5 expression vector. B cells positive for antigen binding in the initial ELISA screen were lysed. mRNA in B cell lysate was used as a template for VH, Vκ-Cκ and Vλ-Cλ gene amplification via RT-PCR. RT-PCR was carried out using forward primers specific to human V domain leader sequences and reverse primers specific for human IgCH1, Cκ or Cλ regions or light chain UTR. To increase the specificity of gene amplification, nested PCR was carried out using RT-PCR products as the template. Forward primers specific for human VH FW1 sequences and reverse primers specific for human VH FW4 sequences were used to amplify VH genes. To capture Vκ-Cκ and Vλ-Cλ genes, forward primers specific to human Vκ and human Vλ FW1 sequences were used in combination with reverse primers specific to the 3′ end of the human Cκ or human Cλ regions. Primers used in nested PCR reactions contained 15 bp extensions which were complementary to the pTT5 expression vector to facilitate downstream Infusion cloning. Amplification of VH, Vκ-Cκ and Vλ-Cλ genes were done in separate reactions. RT-PCR—reverse transcriptase polymerase chain reaction; UTR untranslated region; L—leader sequence; V.sub.H—heavy chain variable domain; Vκ—kappa chain variable domain; Vλ—lambda chain variable domain; C.sub.H—heavy chain constant domain; Cκ—kappa chain constant domain; Cλ—lambda chain constant domain.

[0216] FIG. 10—Concentration response curves of purified anti-Hyr1 mAbs screened for binding to unrelated proteins. (a, b) Purified anti-Hyr1 mAbs screened against HSA and HEK NA respectively via ELISA. Values represent mean (n=2-4).

[0217] FIG. 11—Indirect immunofluorescence of mAbs binding to WT CAl4-Clp10 before and after enzymatic modification of the cell wall. Proteinase K treatment was used to reduce protein residues. Decrease in indirect immunofluorescence after enzymatic treatments suggested the nature of the mAb epitopes. Scale bars represent 4 μm.

[0218] FIG. 12. Treatment with anti-Hyr1 mAb prevents biofilm formation between C. albicans and A. baumannii. C. albicans was germinated in 10% serum for 2 h to form hyphae and then treated with either (A) 50 μg/ml of IgG1 control mAb or (B) 50 μg/ml of AB123 for 1 h, followed by co-incubation with A. baumannii for 3 h. Scale bars are 5 μm.

[0219] FIG. 13. Anti-Candida Hyr1 mAbs bind selectively to clinical isolates of Acinetobacter baumannii from different anatomical locations. The anti-Hyr1 mAb, AB123 and a control mAb were screened for their binding to whole cells of A. baumannii isolated from (A) blood, (B) respiratory samples, (C) tissues samples, (D) urine and (E) wound samples.

EXAMPLES

Example 1

Generation of Fully Human Anti-Candida mAbs by Single B Cell Cloning

[0220] 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-Candida mAbs to a defined C. albicans antigen—the morphogenesis-regulated protein 1 (Hyr1) protein expressed only in the hyphal cell wall (40).

[0221] To enhance the likelihood of isolating Candida-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 superficial Candida infection 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 23 had the highest IgG titre against Hyr1 (FIG. 2A). This donor was selected to provide the source of B cells to use for the generation of Candida-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).

[0222] The IgG1 mAbs generated using the single B cell technology described above bound to purified Hyr1 protein (Table S3).

Comparative Example 2

Purified Recombinant Anti-Candida mAbs Exhibit Specific Target Binding

[0223] Purified anti-Hyr1 mAbs were primarily assessed for functionality through binding to the purified recombinant N-terminus of Hyr1 protein via ELISA. Four mAbs demonstrated strong binding to the purified antigen with EC.sub.50 values of 104 ng/ml, 76.5 ng/ml, 49.6 ng/ml and 53.3 ng/ml for AB120, AB121, AB122 and AB123 (FIG. 3) respectively. To examine the specificity of these mAbs for the target protein, all four were tested against the unrelated antigens HSA and HEK nuclear antigen as negative controls and demonstrated no binding (FIG. 10).

Comparative Example 3

Purified Recombinant Anti-Candida mAbs Show Distinct Binding Patterns to C. albicans and Other Fungal Species

[0224] 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 the C. albicans cell 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 WT C. albicans yeast cells grown in different culture conditions (FIG. 8A). We verified that the anti-Hyr1 mAbs did not bind to hyphae of a Δhyr1 null mutant (FIG. 8B) and that binding was restored in a C. albicans strain containing a single reintegrated copy of the deleted HYR1 gene (FIG. 8C).

[0225] C. albicans cells 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) (FIG. 11).

[0226] Commensurate with the C. albicans-specific nature of HYR1, anti-Hyr1 mAbs only bound to C. albicans and not to a range of other Candida species (results not shown).

[0227] In conclusion, all purified recombinant mAbs generated by this single B cell technology bound specifically to their target antigens with high affinity.

Example 4

Anti-Candida Hyr1 mAbs Bind Selectively to the Gram Negative Bacteria, Acinetobacter baumannii

[0228] Purified anti-Hyr1 mAbs were assessed for binding to a panel of Gram negative bacteria via ELISA. All four mAbs demonstrated strong binding to A. baumannii whole cells with EC.sub.50 values of 6.4 ng/ml, 5.8 ng/ml, 4.3 ng/ml and 11.7 ng/ml for AB120, AB121, AB122 and AB123 respectively (FIG. 4 A-D). This binding was specific to A. baumannii and no cross reactivity to other Gram negative bacteria tested (E. coli, S. marcescens, and P. aueruginosa) was observed (FIG. 4 A-D). The IgG1 negative control mAb did not bind to any of the Gram negative bacteria tested (FIG. 4E).

Example 5

Indirect Immunofluorescence and TEM Images of Anti-CaHyr1 mAbs Binding to A. baumannii Cells

[0229] Binding of the four mAbs AB120-AB123 to A. baumannii was visualised by immunofluorescent staining using a fluorescently labelled alexa-488 conjugated secondary goat anti-human IgG antibody for detection. It was observed that all four anti-Hyr1 mAbs demonstrated binding to a target that was abundantly expressed on the A. baumannii cell surface (FIG. 5A). Transmission electron microscopy (TEM) revealed the ultrastructural binding of AB120-AB123 to the A. baumannii cell surface (FIG. 5B).

Example 6

Treatment with Anti-Hyr1 mAbs Promote Phagocytosis by Macrophages and Protects Against A. baumannii Infection

[0230] Phagocytic cells of the innate immune system are the first line of defence against microbial pathogens. Antibody binding enhances phagocytic clearance of pathogens. A live cell phagocytosis assay was utilised to examine whether the anti-Hyr1 mAbs generated in this study opsonized A. baumannii for phagocytosis by J774.1 macrophages. A. baumannii cells that had been pre-incubated with either AB120, AB121, AB122 or AB123 were taken up significantly more rapidly than cells which had been pre-incubated with saline or IgG1 control mAb. This was demonstrated through analysing the percentage of uptake events that had occurred within the first 30 min of the assay (FIG. 6A). Snapshots taken from live cell imaging compare the phagocytosis of A. baumannii cells by macrophages at 30 min and 2 h when bacterial cells had been pre-incubated with either 50 μg/ml control mAb or 50 μg/ml anti-Hyr1 mAb AB123 (FIG. 6B).

Example 7

Treatment with Anti-Hyr1 mAbs Protects Against A. baumannii Infection

[0231] To determine the therapeutic potential of the anti-Hyr1 mAbs for treating A. baumannii infection, their activity was assessed in a Galleria mellonella model of systemic infection (FIG. 7). Galleria mellonella were infected with 1×10.sup.6 cells of A. baumannii following pre-incubation with saline (A) or 50 μg/ml IgG1 control mAb or anti-Hyr1 mAb AB120 (B), AB121 (C), AB122 (D), or AB123 (E), and their survival monitored every 12 h. Galleria mellonella in the saline and IgG1 control groups did not survive more than 24 hours whereas anti-Hyr1 mAb-treated groups survived significantly longer (p<0.005, p<0.05, p<0.01 and p<0.0001 for AB120, AB121, AB122 and AB123 vs IgG1 control, respectively). Galleria mellonella incubated with AB123 displayed the greatest survival, with around 60% surviving for up to 48 hours (p<0.0001 vs IgG1 control).

Example 8

Prevention of Biofilms

[0232] FIG. 12 demonstrates that treatment with mAb AB123 prevents biofilm formation between C. albicans and A. baumannii.

Example 9

Binding to Diverse Isolates

[0233] FIG. 13 is an expanded ELISA screen demonstrating mAb AB123 binding to a panel of diverse A. baumannii isolates isolated from different body sites.

[0234] General Methods

[0235] Candida and Gram Negative Bacteria Strains and Growth Conditions

[0236] C. albicans serotype A strain CA14+Clp10 (NGY152) was used as a control and its parent strain CA14, used to construct the Δhyr1 null mutant C. albicans strain (40) and the hyr1 re-integrant strain (unpublished). The clinical isolates C. albicans SC5314, C. glabrata SC571182B, C. tropicalis AM2005/0546, C. parapsilosis ATCC22019, C. lusitaniae SC5211362H, C. krusei SC571987M, C. dubliniensis CD36 are shown in Table 51. 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)). Candida strains tested were routinely grown in YPD (see above without the technical agar). Acinetobacter baumannii strain ATCC 19606 is shown in Table 51. It was obtained from glycerol stocks stored at −80° C. and plated onto Mueller-Hinton agar and routinely grown in Mueller-Hinton broth. Isolates of Pseudomonas aeruginosa ATCC27853, Serratia marcescensDb10 and Escherichia coli AM2002/0068 (Table 51) were all obtained from −80° C. glycerol stocks and maintained on LB agar (1% (w/v) tryptone (Oxoid), 1% (w/v) NaCl (Fisher), 0.5% (w/v) yeast extract and 2% (w/v) technical agar) and routinely grown in LB broth at 37° C.

[0237] Generation of Recombinant Hyr1 N-Protein

[0238] The recombinant N-terminus of the Hyr1 protein (amino acids 63 to 350—Table S2) incorporating an N-terminal 6×xHis 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).

[0239] Identification of Human Anti-Hyr1 mAbs from Donor B Cells PBMC Isolation

[0240] In brief, peripheral venous blood from donors who had recovered from a Candida infection 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 1×10.sup.7 cells/ml 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.

[0241] Purification of Donor Plasma

[0242] 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, pH 8. Eluted IgG concentration was measured by absorbance at 280 nm using a NanoVue Plus Spectrophotometer (GE Healthcare).

[0243] Circulating IgG Enzyme-linked Immunosorbent Assay (ELISA) to Identify Donors with B Cells to Take Forward

[0244] To identify the donor to use for subsequent class switched memory (CSM) B cell isolation and activation, ELISAs were carried out against the target antigen using IgG purified from donor plasma. NUNC maxisorp 384-well plates (Sigma) were coated with 1 μg/ml purified, recombinant N-terminus hyrl 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.

[0245] 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 EC.sub.50 determination and donor selection for subsequent CSM B cell isolation and activation.

[0246] Isolation of Class Switched Memory B Cells

[0247] 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, 5×10.sup.7 PBMCs 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.

[0248] 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.

[0249] Activation of CSM B Cells

[0250] 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% CO.sub.2 for 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.

[0251] B Cell Supernatant Screen Against Target Antigens Via ELISA

[0252] For B cell supernatant screening against target antigens, NUNC maxisorp 384-well plates (Sigma) were coated with 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 450nm on an Envision plate reader (PerkinElmer). Positive hits were defined as wells with an OD.sub.450 reading>4×background. B cells in ‘positive hit’ wells were resuspended in lysis buffer (ml DEPC-treated H.sub.2O (Life Technologies), 10 μl 1 M Tris pH 8, 25 μl RNAsin Plus RNAse Inhibitor (Promega)) and stored at −80° C.

[0253] Generation of Recombinant Anti-Hyr1 mAbs: Amplification of VH, Vκ-Cκ and Vλ-Cλ Genes—cDNA Synthesis and PCR

[0254] A schematic of the cloning protocol is shown in FIG. 9. 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 (VK1-4) were used with a reverse primer specific to the human kappa constant region (CK) 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 (VA1-8) 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 (CA).

[0255] Prior to cDNA synthesis, B cell lysates were thawed and diluted 1:5, 1:15 and 1:25 in nuclease-free H.sub.2O (Life Technologies) before addition of oligodT.sub.20 (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 (Vθ-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 5×, dNTPs (10 mM), gene-specific forward and reverse primer mixes (10 μM), QIAGEN OneStep RT-PCR Enzyme Mix and nuclease-free H.sub.2O. 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.

[0256] Amplification of VH, Vκ-Cκ and Vλ-Cλ Genes—Nested PCR Reaction

[0257] 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.

[0258] pTT5 Mammalian Expression Vector Preparation

[0259] The pTT5mammalian 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 BssHll before the leader sequence of the VH region and Sall 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 BssHll and BamHl astDigest Restriction enzymes (Thermo Scientific) to generate the vector ready for insertion of either K-Cκ or VA-CA. 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 QlAquick 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).

[0260] In-Fusion Cloning

[0261] 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 H.sub.2O 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.

[0262] Plasmid DNA Generation for Transfection

[0263] Following transformation, 8-16 single colonies per initial hit well for VH, Vκ and Vλ were picked and used to inoculate 2xTY 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.

[0264] Small Scale Expression of Recombinant mAbs

[0265] 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 2.5×10.sup.6 cells/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% CO.sub.2, 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.

[0266] Large Scale Expression, Purification and QC of Recombinant mAbs

[0267] 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 2.5×10.sup.6 cells/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 AKTA (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.

[0268] ELISA with Purified Recombinant mAbs

[0269] 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. For assessment of mAb binding to A. baumannii, A. baumannii overnight culture was coated onto plates in place of 1 μg/ml purified, recombinant N-terminus hyrl protein antigen.

[0270] Immunofluorescence Imaging of Anti-Hyr1 mAbs Binding to Fungal Cells

[0271] Indirect immunofluorescence was performed using purified recombinant mAbs. For A. baumannii staining, a single A. baumannii colony was used to inoculate 10 ml Mueller-Hinton medium and incubated at 37° C., 200 rpm overnight. For imaging of Candida cells, a single Candida colony was used to inoculate 10 ml YPD medium and incubated at 30° C., 200 rpm overnight. Overnight Candida cultures were diluted 1:1333 in milliQ water and then added to a poly-L-lysine coated glass slide (Thermo Scientific, Menzel-Glaser) 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). A. baumannii and Candida cells were then washed in Dulbecco's Phosphate Buffered Saline (DPBS) and fixed with 4% paraformaldehyde for 30 min. After fixing, cells were washed again and blocked with 1.5% normal goat serum (Life Technologies) before staining with an anti-Hyr1 mAb at 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 in Candida cells, 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 with Candida cells were left to air dry, A. baumannii cells were washed three time with DPBS and 5 μl of cell suspension added to a poly-L-lysine coated glass slide. One drop of Vectashield mounting medium (Vector Labs) was added to the slides and a 20 m×20 mm coverslip applied. Cells were imaged in 3D on an UltraVIEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK).

[0272] High-Pressure Freezing (HPF) of Samples for Immunogold Labelling of A. baumannii Cells with anti-Hyr1 mAbs for Transmission Electron Microscopy (TEM)

[0273] A. baumannii 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). The frozen samples were then fixed in an automatic temperature controlled Leica AFS freeze substitution system in dried acetone containing 2% (w/v) OsO.sub.4, 1% (w/v) uranyl acetate, 1% (v/v) methanol and 5% (v/v) water in acetone at −90° C. for 48 h (Walther & Ziegler, 2002). Samples were then warmed to −30° C. and processed in a Lynx tissue processor with 1:2 acetone: resin and embedding in TAAB812 (TAAB Laboratories, Aldermaston, UK) epoxy resin. One hundred nm sections were cut with a Leica ultracut E and placed on nickel grids. Sections 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-Candida mAb (5 μg/ml) for 90 min before incubation in incubation buffer for 5 min, for a total of 6 times. mAb binding was detected by incubation with Protein A conjugated to 10 nm gold (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 left to dry. TEM images were taken using a JEM-1400 Plus using an AMT UltraVUE camera.

[0274] 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% CO.sub.2. For phagocytosis assays, macrophages were seeded in 300 μl supplemented DMEM at a density of 2×10.sup.5 cells/well in an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated overnight at 37° C., 5% CO.sub.2. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with 300 μl pre-warmed supplemented CO.sub.2-independent media (Gibco, Invitrogen, Paisley, UK) containing 1 μM LysoTracker Red DND-99 (Invitrogen, Paisley, UK).

[0275] Preparation of Fluorescein Isothiocyanate (FITC)-Stained A. baumannii

[0276] A. baumannii colonies were grown in Mueller-Hinton medium and incubated at 370° C., 200 rpm overnight. Live A. baumannii cells 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 using A. baumannii FITC-labelled, the cells were washed three times in 1 x PBS to remove any residual FITC and finally re-suspended in 1×PBS or 1×PBS containing purified anti-Candida mAb at 1-50 μg/ml.

[0277] Live Cell Video Microscopy Phagocytosis Assays

[0278] Phagocytosis assays were performed using our standard protocol with modifications (42, 43, 54). Following pre-incubation with/without anti-Hyr1 mAb, live FITC-stained wild type A. baumannii 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 10. 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 4 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 4 h period. Measurements taken included: A. baumannii uptake—defined as the number of A. baumannii cells taken up by an individual phagocyte over the 4 h period.

[0279] Mean values and standard deviations were calculated. One- or two-way ANOVλ followed by Bonferroni multiple comparison tests or unpaired, two-tailed t tests were used to determine statistical significance.

[0280] Enzymatic Modification of Candida albicans Cell Wall

[0281] For proteinase K treatment, single colonies of Candida were 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/mI proteinase K at 37° C. for 1 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-Hyr1 mAb at 1 μg/mI 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).

[0282] Galleria mellonella Infection with A. baumannii and Treatment with Anti-Hyr1 mAbs

[0283] Sixth instar larvae of Galleria mellonella (Livefoodsbypost, Isle of Wight, England) were separated into 10 healthy larvae per treatment group. Overnight cultures of A. baumannii were diluted 1 in 100 in either PBS or PBS with 10 μg/ml of anti-Hyr1 mAb for 1 h at room temperature. Bacterial cells were then standardised to an OD.sub.600 of 0.5, which corresponded to 10.sup.6 bacterial cells/ml. Larvae were inoculated with 10 μl of each culture through the last left pro-leg and incubated at 37° C. Survival of larvae was recorded every 12 h.

[0284] Biofilm Assay

[0285] Biofilms were formed by diluting an overnight culture of C. albicans yeast cells in pre-warmed RPMI-1640 +10% FCS to a concentration of 1×10.sup.6 cells/ml. The C. albicans culture was added to a poly-L-lysine coated glass slide (Thermo Scientific, Menzel-Glaser) and incubated for 2 h at 37° C. to induce hyphal formation. After incubation cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS) and treated with either 50 μg/ml of control IgG1 mAb or 50 μg/ml of AB123 for 1 h at RT. Cells were then washed three times with DPBS. Overnight cultures of A. baumannii were washed three times with DPBS and diluted to 1×10.sup.5 cells/ml in pre-warmed RPMI-1640 +10% FCS and added to the C. albicans hyphae. Cells were then co-incubated at 37° C. for 3 h. Cells were then washed three times with DPBS and fixed with 4% paraformaldehyde and imaged on n UltraVIEW VoX spinning disk confocal microscope (Nikon, Surrey, UK).

TABLE-US-00002 TABLE S1 Clinical isolates and strains Strain name Genotype Reference ATCC 19606 Clinical isolate ATCC stock hyr1Δ hyr1Δ::hisG/ Bailey et al. 1996 hyr1Δ:hisG-URA-3-hisG hyr1Δ + HYR1 hyr1::hisG/ Belmonte hyr1::hisG/RPS1/ (unpublished) rps1::HYR1 C. albicans Clinical isolate Gillum et al. 1984 SC5314 C. glabrata Clinical isolate Odds et al. 2007 SCS71182B C. tropicalis Clinical isolate Clinical isolate from AM2005/0546 Aberdeen C. lusitaniae Clinical isolate Odds et al. 2007 SCS211362H C. krusei Clinical isolate Odds et al. 2007 SCS71987M C. parapsilosis Clinical isolate Rudek 1978 ATCC22019 C. dubliniensis Clinical isolate Moran et al. 1998 CD36 P. aeruginosa Clinical isolate ATCC stock ATCC27853 E. coli Clinical isolate Clinical isolate from AM2002/0068 Aberdeen S. marcescens Clinical isolate Flyg et al. 1980 Db10

TABLE-US-00003 TABLE S2 Recombinant 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 protein amino acids 63-350 make up the remainder of the sequence. Recombinant protein Amino acid sequence antigen name (amino acids 63-350) Recombinant Hyr1 METDTLLLWVLLLWVPGSTGGSGHHHHHHG N-terminus EVEKGASLFIKSDNGPVLALNVALSTLVRP fragment VINNGVISLNSKSSTSFSNFDIGGSSFTNN GEIYLASSGLVKSTAYLYAREWTNNGLIVA YQNQKAAGNIAFGTAYQTITNNGQICLRHQ DFVPATKIKGTGCVTADEDTWIKLGNTILS VEPTHNFYLKDSKSSLIVHAVSSNQTFTVH GFGNGNKLGLTLPLTGNRDHFRFEYYPDTG ILQLRAAALPQYFKIGKGYDSKLFRIVNSR GLKNAVTYDGPVPNNEIPAVCLIPCTNGPS APESESDLNTPTTSSIGT

TABLE-US-00004 TABLE S3 Purified recombinant human IgG1 mAbs generated using the single B cell technology. Antibody Yield (mg) Target AB-120 12 Hyr1 protein AB-121 28.5 Hyr1 protein AB-122 67.9 Hyr1 protein AB-123 67.3 Hyr1 protein

TABLE-US-00005 TABLE VH SEQ AB VH ID name VH FW1 CDR1 VH FW2 VH CDR2 VH FW3 VH CDR3 VH FW4 NO 06-AB- VH1 EVQLVQSGGGLVQPG TSYA WVRQAPGK VITGNVGTSY RFTISRDNSKKTVSLQMNS TRYDFSSGYY WGQGTLVS 15C 120 GSLGLSCAASGFIF MT GLEWVS YADSVKG LRAEDTAIYYCVK FDD VSS 06-AB- VH3 EVQLVESGGTLVQPG SDY WVRQAPGK NIKQDGSEKY RVTISRDNAQNSVFLQMHS DGYTFGPATT WGRGTLVS 15D 121 GSLRLSCAASGFTF WMN GLEWVA YVDSLRG LSVEDTAVYYCAR ELDH VSS 06-AB- VH3 EVQLVQSGGGLAQPG DDFA WVRQPPGK GLTWNGGSID RFTISRDNAKNSLFLQMNS GLSGGTMAPF WGQGTMVS 15E 122 RSLRLSCAASGFGF MH GLEWVS YAGSVRG LRAEDTALYYCAK DI VSS 06-AB- VH3 EVQLLESGGGVVQPG SNYG WVRQAPGK VVWFDGSYK RFTISRDNSKSTLYLQMNS PIMTSAFDI WGPGTMVS 15F 123 RSLRLSCAASGFTF MH GLEWVA YYTDSVKG LRAEDTAVYYCVS VSS

TABLE-US-00006 TABLE VL SEQ AB VL ID name VL FW1 VL CDR1 VL FW2 CDR2 VL FW3 VL CDR3 VL FW4 NO 06-AB- VK1 DIVMTQSPSSVS RASQGIS WYQQKPGE AASSL GVPSRFSGSGSGTDFTLTISS QQANSFPIT FGQGTRLQI 16C 120 ASVGDKVTITC RWLA APELLIY QS LQPEDFATYYC K 06-AB- VL3 QLVLTQPPSVSV SGDELRN WYQQKSGQ QDNN GIPERFSGSQSGDTATLTISG QAWVSQTLV FGGGTKLTV 16D 121 SPGQTASITC KYTS SPVLVIY RPS TQAVDEADYYC L 06-AB- VL3 QAGLTQPPSVSV GGNNIGS WYQQKPGQ DDSD GVPERFSGSNSGNTATLTISS QVWDRSSDH FGGGTRLTV 16E 122 APGQTATIPC KHVH APVAVVY RPS VEAGDEADYYC FWL L 06-AB- VL2 QLVLTQPPSASG TGTSSDV WYQHHPGK EVSQR GVPDRFSGSKSGNTASLTVS SSYAGSVVL FGGGTKLTV 16F 123 SPGQSVTISC GGSNFVS APKLMIY PS GLQADDEADYYC L

[0286] Antibody Sequences and Seq ID No.s

TABLE-US-00007 TABLE C Antibody AB120 Seq. 06-AB-120 Sequence ID No. VH FW1 EVQLVQSGGGLVQPGGSLGLSCAASGFIF 1C VH CDR1 TSYAMT 2C VH FW2 WVRQAPGKGLEWVS 3C VH CDR2 VITGNVGTSYYADSVKG 4C VH FW3 RFTISRDNSKKTVSLQMNSLRAEDTAIYYCVK 5C VH CDR3 TRYDFSSGYYFDD 6C VH FW4 WGQGTLVSVSS 7C VL FW1 DIVMTQSPSSVSASVGDKVTITC 8C VL CDR1 RASQGISRWLA 9C VL FW2 WYQQKPGEAPELLIY 10C  VL CDR2 AASSLQS 11C  VL FW3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 12C  VL CDR3 QQANSFPIT 13C  VL FW4 FGQGTRLQIK 14C 

TABLE-US-00008 Table D Antibody AB121 Seq. 06-AB-121 Sequence ID No. VH FW1 EVQLVESGGTLVQPGGSLRLSCAASGFTF 1D VH CDR1 SDYWMN 2D VH FW2 WVRQAPGKGLEWVA 3D VH CDR2 NIKQDGSEKYYVDSLRG 4D VH FW3 RVTISRDNAQNSVFLQMESLSVEDTAVYYCAR 5D VH CDR3 DGYTFGPATTELDH 6D VH FW4 WGRGTLVSVSS 7D VL FW1 QLVLTQPPSVSVSPGQTASITC 8D VL CDR1 SGDELRNKYTS 9D VL FW2 WYQQKSGQSPVLVIY 10D  VL CDR2 QDNNRPS 11D  VL FW3 GIPERFSGSQSGDTATLTISGTQAVDEADYYC 12D  VL CDR3 QAWVSQTLV 13D  VL FW4 FGGGTKLTVL 14D 

TABLE-US-00009 TABLE E Antibody AB122 Seq. 06-AB-122 Sequence ID No. VH FW1 EVQLVQSGGGLAQPGRSLRLSCAASGFGF 1E VH CDR1 DDFAMH 2E VH FW2 WVRQPPGKGLEWVS 3E VH CDR2 GLTWNGGSIDYAGSVRG 4E VH FW3 RFTISRDNAKNSLFLQMNSLRAEDTALYYCAK 5E VH CDR3 GLSGGTMAPFDI 6E VH FW4 WGQGTMVSVSS 7E VL FW1 QAGLTQPPSVSVAPGQTATIPC 8E VL CDR1 GGNNIGSKHVH 9E VL FW2 WYQQKPGQAPVAVVY 10E  VL CDR2 DDSDRPS 11E  VL FW3 GVPERFSGSNSGNTATLTISSVEAGDEADYYC 12E  VL CDR3 QVWDRSSDHFWL 13E  VL FW4 FGGGTRLTVL 14E 

TABLE-US-00010 TABLE F Antibody AB123 Seq. 06-AB-123 Sequence ID No. VH FW1 EVQLLESGGGVVQPGRSLRLSCAASGFTF 1F VH CDR1 SNYGMH 2F VH FW2 WVRQAPGKGLEWVA 3F VH CDR2 VVWFDGSYKYYTDSVKG 4F VH FW3 RFTISRDNSKSTLYLQMNSLRAEDTAVYYCVS 5F VH CDR3 PIMTSAFDI 6F VH FW4 WGPGTMVSVSS 7F VL FW1 QLVLTQPPSASGSPGQSVTISC 8F VL CDR1 TGTSSDVGGSNFVS 9F VL FW2 WYQHHPGKAPKLMIY 10F  VL CDR2 EVSQRPS 11F VL FW3 GVPDRFSGSKSGNTASLTVSGLQADDEADYYC 12F VL CDR3 SSYAGSVVL 13F VL FW4 FGGGTKLTVL 14F

REFERENCES

[0287] 9. Rader C. Chemically programmed antibodies. Trends. Biotechnol. 32, 186-97 (2014).

[0288] 10. Yoon S, Kim Y-, Shim H, Chung J. Current perspectives on therapeutic antibodies. Biotechnol. Bioprocess Eng. 15, 709-15 (2010).

[0289] 11. Carter P J. Introduction to current and future protein therapeutics: a protein engineering perspective. Exp. Cell Res. 317, 1261-1269 (2011).

[0290] 12. Berry J D, Gaudet RG. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology. New Biotech. 28, 489-501 (2011).

[0291] 13. Saylor C, Dadachova E, Casadevall A. Monoclonal antibody-based therapies for microbial diseases. Vaccine. 27, G38-46 (2009).

[0292] 14. Wilson P C, Andrews S F. Tools to therapeutically harness the human antibody response. Nat. Rev. Immunol. 12, 709-19 (2012).

[0293] 34. Carter P J. Potent antibody therapeutics by design. Nat. Rev. Immunol. 6, 343-357 (2006).

[0294] 35. Tiller T. Single B cell antibody technologies. N. Biotechnol. 28, 453-457 (2011).

[0295] 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).

[0296] 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).

[0297] 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).

[0298] 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 RT-PCR technique and in vitro expression. Biotechnol. Prog. 22, 979-88 (2006).

[0299] 40. Bailey D A, Feldmann P J F, Bovey M, Gow N A R, Brown A J P. The Candida albicans HYR 1 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).

[0300] 42. Lewis L E, Bain J M, Lowes C, Gillespie C, Rudkin F M, Gow N A R, et al. Stage specific assessment of Candida albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants. PLoS Pathog. 8, e1002578 (2012).

[0301] 43. Rudkin F M, Bain J M, Walls C, Lewis L E, Gow N A R, Erwig L P. Altered dynamics of Candida albicans phagocytosis by macrophages and PMNs when both phagocyte subsets are present. mBio. 4, e00810-13. doi: 10.1128/mBio.00810-13 (2013).

[0302] 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).

[0303] 54. McKenzie C G J, Koser U, Lewis L E, Bain J M, Mora-Montes H M, Barker R N, et al. Contribution of Candida albicans cell wall components to recognition by and escape from murine macrophages. Infect. Immun. 78, 1650-1658 (2010). Additional references for Table 51:

[0304] 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 of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot Cell 3: 900-909. doi: 10.1128/ec.3.4.900-909.2004 (2004).

[0305] Bailey D A, Feldmann P J F, Bovey M, Gow N A R, Brown A J P. The Candida albicans HYR1 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).

[0306] Gillum A M, Tsay E Y, Kirsch D R. Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet. 198:179-8 (1984).

[0307] 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 of Candida bloodstream infections in Scotland. J Med Microbiol. 56: 1066-1075 (2007).

[0308] Rudek W. Esterase activity in Candida species. J. Clin. Microbiol. 8: 756-759 (1978).

[0309] 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 in Candida dubliniensis. Antimicrob. Agents Chemother. 42, 1819-1830 (1998).

[0310] Flyg, C. et al. Insect Pathogenic Properties of Serratia marcescens: Phage-resistant Mutants with a Decreased Resistance to Cecropia Immunity and a Decreased Virulence to Drosophila. J Gen. Microb. 120(1): 173-181 (1980)