Bifunctional chemokine-nanobody fusion protein enhances neutrophil recruitment to impede Acanthamoeba immune evasion.

BACKGROUND: Acanthamoeba keratitis (AK) is a severe infectious disease that causes serious visual impairment and low quality of life. This study aims to investigate the immune landscape in AK, with the goal of improving treatment outcomes through immunotherapy. METHODS: We conducted single-cell transcriptome sequencing on corneal tissues from nine patients (3 AK patients, 3 patients with fungal keratitis and 3 patients with bacterial keratitis). Bioinformatic analysis calculated the cell subsets and their proportions within different infectious keratitis. CellChat analysis elucidated the differential expression of chemokines in keratitis. After that, screening amebic nano-antibodies, synthesizing antibody-chemokine fusion proteins, and validated their affinity and chemotactic abilities in vitro and in vivo. And assessing of the therapeutic efficacy of antibody-chemokine fusion proteins. FINDINGS: The UMAP plot demonstrated the 13 major cell clusters in infectious keratitis. Compared with non-AK group, the neutrophil proportion of AK group is markedly reduced. Cell communication indicated a diminished CXCL pathway in AK. Acanthamoeba-specific antibodies were obtained by screening a natural antibody library derived from alpacas. The amoeba-specific antibodies were conjugated with the CXCL1 chemokine, and this fusion protein exhibited robust binding affinity to Acanthamoeba and chemotactic capacity both in vitro and in vivo. Furthermore, in vivo animal investigations indicated that the fusion protein presented excellent therapeutic effect and could effectively eliminate the Acanthamoeba burden. INTERPRETATION: This study revealed an immune evasion mechanism employed by Acanthamoeba and offered a therapeutic approach. It presents promising potential for enhancing the treatment of infectious diseases by targeting and overcoming challenges posed by immune evasion. FUNDING: This work was funded by National Natural Science Foundation of China (grant number 82171017 and 82471041) and the Beijing Municipal Public Welfare Development and Reform Pilot Project for Medical Research Institutes (PWD&RPP-MRI, JYY2023-6).