Monoclonal IgM Antibodies Targeting Candida albicans Hyr1 Provide Cross-Kingdom Protection Against Gram-Negative Bacteria

Abstract

Recent years have seen an unprecedented rise in the incidence of multidrug-resistant (MDR) Gram-negative bacteria (GNBs) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent, and treat infections by GNBs are urgently needed. Previously, we have reported that the Candida albicans hypha-regulated protein Hyr1 shares striking three-dimensional structural homology with cell surface proteins of Acinetobacter baumannii. Moreover, active vaccination with rHyr1p-N or passive immunization with anti-Hyr1p polyclonal antibody protects mice from Acinetobacter infection. In the present study, we use molecular modeling to guide design of monoclonal antibodies (mAbs) generated against Hyr1p and show them to bind to priority surface antigens of Acinetobacter and Klebsiella pneumoniae. The anti-Hyr1 mAbs block damage to primary endothelial cells induced by the bacteria and protect mice from lethal pulmonary infections mediated by A. baumannii or K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAbs as a cross-kingdom immunotherapeutic strategy against MDR GNBs.

Keywords: Acinetobacter baumannii; Candida Hyr1; Klebsiella pneumoniae; cross-kingdom immunotherapy; molecular modeling; monoclonal antibodies; passive vaccine.

Figure 1

Localization of Hyr1 peptide #5 and putative cross-reactive epitopes in modeled Klebsiella pneumoniae target sequences. (A) Sequence alignments between Hyr1 peptide #5 and putative cross-reactive motifs within K. pneumoniae FhaB sequence; identical residues are boxed. (B) van der Waals space-filling models illustrating conservation of amino acid physicochemistry in the highly homologous motifs; coloration is a modified RasMol schema (Gly, Ala–cream; Asn, Gln–khaki; Thr–orange; Val–green; Asp–red). (C) Comparative models of Candida albicans Hyr1 and K. pneumoniae FhaB showing homologous cross-reactive domains in red van der Waals space-filling spheres within the homologous domains of the two proteins. (D) Superimposition overlay of the homologous regions of Hyr1 and FhaB showing strong 3-D homology in antiparallel β-sheet facets and overall structures in which the conserved target motifs exist. Peptide #5 is shown in red as van der Waals space-filling spheres in the comparative Hyr1 and FhaB models. (E) Individual models for four targeted cross-reactive antigens of K. pneumoniae (OmpA, TonB, Fmp, and ExbD) are shown with domains homologous to Hyr1 peptide #5 in red. Sequence alignments between Hyr1 peptide #5 and cross-reactive target motifs with identical residues are boxed; also shown are domains showing identical and/or physicochemically conserved residues as space-filling spheres; coloration is a modified RasMol schema (Gly, Ala–cream; Asn, Gln–khaki; Thr, Ser–orange; Val, Ile, Leu, Met, Cys–green; Trp, Tyr, Phe–olive green; Asp, Glu–red; Arg, Lys–blue; His–sky blue; Pro–chartreuse).

Figure 2

Binding of monoclonal antibodies (mAbs) targeting Hyr1 peptide #5 to Gram-negative bacteria (GNBs). MAb clones (and isotype-matched control) IgM were evaluated for binding to Acinetobacter baumannii (HUMC-1), Klebsiella pneumoniae-QR (KP-QR), and K. pneumoniae-RM (KP-RM) at a concentration of 100 μg/ml. The extent of binding was quantified by flow cytometry after staining the bound antibodies with Alexa 488-conjugated secondary antibody. Data were represented as mean fluorescence intensity of the Ab-bound bacteria (A). The degree of binding was also visualized by a shift in the peaks in the anti-Hyr1 IgM binding conditions vs. the control antibodies (B).

Figure 3

Monoclonal antibody (mAb) clone H3 targeting Hyr1 peptide #5 binds Gram-negative bacteria (GNBs) in a dose-dependent manner. MAb clone H3 (and IgM isotype control) were evaluated for binding to HUMC-1, KP-QR, and KP-RM at concentrations ranging from 30 to 0.1 μg/ml. The extent of binding was quantified by flow cytometry after staining the antibodies with Alexa 488-conjugated secondary antibody. Data were represented in a scatter plot highlighting the percentage of bacteria that were bound by the Abs.

Figure 4

Monoclonal antibodies (mAbs) targeting Hyr1 peptide #5 prevents HUMC-1-, KP-QR-, and KP-RM-induced A549 lung alveolar epithelial cells and primary human umbilical vein endothelial cells (HUVECs) damage. HUMC-1-, KP-QR-, and KPC-RM-induced A549 cell injury in the presence of 15 or 30 μg/ml of an isotype-matched IgM, mAb H3, or mAb H4 (A). Damage to HUVECs by HUMC-1 and KP-QR (in the presence of 15 μg/ml of H3 and H4), also KPC-RM (30 μg/ml of the mAbs) (B). Cell damage was determined by ⁵¹Cr-release assay after 48 and 24 h for HUMC-1 and KP, respectively. Percentage damage was normalized to IgM isotype-matched control after subtracting spontaneous cell damage. *P < 0.001 vs. control IgM. N = 12 per group from three independent experiments.

Figure 5

Monoclonal antibody (mAb) clones H3 and H4, targeting Hyr1 peptide #5, protect mice from HUMC-1- or KP-QR- induced pneumonia. Immunosuppressed CD-1 male mice (n = 10/group from two experiments) were infected with HUMC-1 via inhalation [average 5 × 10¹⁰ colony-forming units (CFU)] (A). Immunocompetent mice (n = 10/group from two experiments) were infected intratracheally with KP-QR (average 3.4 × 10⁷ CFU) (B). Intraperitoneal (i.p.) treatment with mAb H3, H4, or isotype-matched control IgM started 24 h and repeated at 96 h post infection (30 μg/mouse each dose). *P < 0.05, P = 0.06, and **P < 0.001 vs. control IgM by log-rank test. For CFU measurement, H3 and control IgM were administered 6 h and 3 days post infection, and lungs harvested from mice at Day +4 (for HUMC-1) (C) and at Day +2 (for KP-QR) post infection (D).