Abstract
Invasive aspergillosis (IA) is a life-threatening, acute and rapidly progressing infectious disease. It is frequently caused by Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. The morbidity of IA worldwide continues to increase due to the expanding number of patients being treated with immune suppressive regimens. Because of the lack of sensitivity and specificity of current IA diagnostic methods, and toxicity and various adverse effects caused by current anti-Aspergillus drugs, it is important to attempt novel avenues to diagnose and treat IA in a more accurate and timely manner. A monoclonal antibody (mAb) is produced by a single B-lymphocyte clone with highly specific binding to the matching antigen. Therefore, mAb-based approaches are being developed and regarded as a new alternative in the specific diagnosis and accurate management of IA. As such, the overall aim of this thesis is to explore the diagnostic and therapeutic value of two novel mAbs (1D2 and 4E4) against hyphal antigens produced by A. fumigatus in IA.
The immunoreactivity and specificity of 1D2 and 4E4 was tested by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF). In addition, the characteristics of antigens recognised by these two antibodies were analysed by ELISA and western blot techniques. Clinical human samples were also tested in this study. On top of the in vitro experiments, an IA mouse model was established to investigate the in vivo value of 1D2 and 4E4 mAb-based methods in IA diagnosis.
In addition, the in vitro anti-Aspergillus activities of mAb 1D2 was detected by treating the Aspergillus conidia or hyphae with antibody at different growth stages. Moreover, the in vitro anti-fungal activity of 1D2 mAb against A. fumigatus in combination with antifungal drugs was performed by checkerboard assay. Finally, the in vivo prophylactic effect of mAb 1D2 was explored by injecting the antibody into IA mice prior to Aspergillus inoculation.
These two antibodies sensitively identified the antigens on the cell wall of A. fumigatus and A. flavus. The antigens identified by 1D2 and 4E4 were shown to be glycoproteins with a molecular weight between 30 kDa and 100 kDa.
A final IA mouse model was induced by a single inoculation of Aspergillus with 2×105 conidia in the neutropenic mice. The mAb-based approaches could detect circulating antigens as early as 18 hours in mice infected with A. fumigatus and 48 hours after inoculation with A. flavus. Furthermore, both antibodies could also identify hyphae or swollen conidia in liver, kidney and spleen from IA mice. Additionally, IF staining with 1D2 and 4E4 were both sensitive for A. fumigatus hyphae in human tissues.
In addition, mAb 1D2 had promising in vitro growth suppression activity against the important invasive Aspergillus species that cause IA in humans. It inhibited the attachment of Aspergillus on a composite surface and fibronectin and has an inhibitory effect on Aspergillus conidia swelling and germination, predominantly in the first two hours of incubation. Furthermore, 1D2 mAb was able to damage the hyphae of A. fumigatus, A. flavus, A. niger and A. terreus.
Finally, we demonstrated that 1D2 had an additive activity in vitro against A. fumigatus in combination with voriconazole, posaconazole, amphotericin B and caspofungin. In addition, there was no significant cross-reactivity between mAb 1D2 and those tested antifungals and antibiotics.
Importantly, 1D2 inhibited the Aspergillus growth in mice challenged with A. fumigatus. The administration of 1D2 before A. fumigatus inoculation lessened mouse clinical infection signs. In addition, 1D2 mitigated mouse liver injury and reduce the fungal burden in various tissues, thus impeding the inflammation reaction in IA mice.
In conclusion, in this thesis, we report two novel monoclonal antibodies 1D2 and 4E4 against cell wall glycoproteins of A. fumigatus and A. flavus. These two antibodies can be used to detect the antigens in blood and Aspergillus pathogens in tissues from IA mice in the early stage. In addition, both antibodies detect A. fumigatus in stored tissues from patients with invasive fungal infections. In vitro studies demonstrated 1D2 had promising activity against A. fumigatus, A. flavus, A. niger and A. terreus, as well as the additive activity of 1D2 against A. fumigatus in combination with voriconazole, posaconazole, amphotericin B and caspofungin. There is no significant cross-reactivity between mAb 1D2 and those tested antifungals and antibiotics. Finally, in vivo studies showed prophylactic administration of 1D2 reduced injury of IA in a mouse model using A. fumigatus. In summary, this study highlights that these novel monoclonal antibodies, 1D2 and 4E4, may serve as new alternatives for IA diagnosis and treatment.