Glycan microarray analysis of Candida-related antibodies in human and mice sera guides biomarker discovery and vaccine development

Abstract

Invasive, high-mortality yeast infections caused by pathogenic Candida species are the most common fungal nosocomial bloodstream infections. The World Health Organization (WHO) has called for improved prevention and diagnostic strategies for fungal pathogens. Here, we demonstrate that Candida-related antibodies can be detected using glycan microarrays containing synthetic glycans to guide the discovery of oligosaccharide epitopes for diagnostics and vaccine design. Pure, synthetic mannans and β-glucans, resembling carbohydrates found on the surface of Candida spp., were employed to screen and compare sera from infected humans and mice with noninfected individuals. IgM antibodies directed toward β-glucans were observed shortly after infection, and after a longer time of infection, IgM and IgG antibodies that preferentially recognize mannans. Phosphodiester-linked mannosides and the β-(1,2)-mannose monomer help to distinguish different Candida spp. The tetrasaccharide antigen β-(1,2)Man-α-(1,2)Man-α-(1,2)Man-α-(1,2)Man, and the pentasaccharide antigens α-(1,2)Man-α-(1,3)Man-α-(1,2)Man-α-(1,2)Man-α-(1,2)Man and β-(1,3)Glc-β-(1,3)Glc-β-(1,3)Glc-[β-(1,6)Glc]-β-(1,3)Glc were identified as potential epitopes for detection, e.g., through monoclonal antibody lateral flow tests, and the development of glycoconjugate or monoclonal antibody vaccines against Candida.

Keywords: Candida; antibody; glycan; microarray.

Fig. 1.

Architecture of the Candida cell wall. Adapted and modified from (14, 88).

Fig. 2.

Synthetic glycans in glycan microarray analysis. Schematic representation of the synthetic glycans (A), glycan microarray printing pattern (B) and exemplary binding pattern of human serum to immobilized synthetic glycans (C).

Fig. 3.

Mean fluorescence intensity of IgG antibody binding to synthetic glycans in humans. Mean fluorescence intensity of IgG antibodies in sera derived from humans with invasive candidiasis, binding to synthetic mannans (A) or β-glucans (B). Mean fluorescence intensity of IgG antibodies in sera derived from humans with different Candida spp. infections, binding to synthetic mannans (C) or β-glucans (D). A serum dilution of 1:100 was used. Values represent mean. Differences were tested for significance to noninfected controls (A–D) using multiple Mann–Whitney test with ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig. 4.

Mean fluorescence intensity of IgG antibody binding to synthetic glycans in humans monitored over time. Mean fluorescence intensity of IgG antibodies in sera derived from humans before (baseline) or at an early (1 d), medium (5 to 8 d) or late (12 to 16 d) timepoint after positive blood culture with invasive Candida spp., binding to synthetic mannans (A) or β-glucans (B) or glycan M8 of individual patients infected with Candida dubliniensis (red), Candida albicans (orange), Candida glabrata (green), or Candida parapsilosis (blue) (C). Two patients with invasive C. glabrata infection were not screened after the first positive blood culture, and three were not screened before the first positive blood culture. A serum dilution of 1:100 was used. Values represent mean (A and B) with SEM (C). Differences were tested for significance to baseline using multiple Mann–Whitney test with ***P < 0.001, **P < 0.01 and *P < 0.05 (A and B) or to the first timepoint of sampling of the individual patients using Welch’s t test with ***P < 0.001, **P < 0.01 and *P < 0.05 (C).

Fig. 5.

Mean fluorescence intensity of IgM and IgG antibody binding to synthetic glycans in mouse models of invasive candidiasis. Mean fluorescence intensity of IgM antibodies (A) and IgG antibodies (B) binding to synthetic β-glucans after three days or seven days of infection with live C. albicans (CWZ 10061110), and IgM antibodies (C) and IgG antibodies (D) binding after three days or seven days of infection with live C. auris (CWZ 10051896) (belonging to clade I). Mean fluorescence intensity of IgM antibodies binding to synthetic mannans after one month or two months of inoculation with killed C. auris NCPF13001#16 (clade 1) (E) or C. auris VPCI479/P/13 (clade 1) (F). A serum dilution of 1:100 was used. Values represent mean. Differences were tested for significance to three days of infection (A–D) or preinoculation (E and F) using multiple Mann–Whitney test with ***P < 0.001, **P < 0.01 and *P < 0.05.