Production and characterization of anti-Cryptococcus neoformans monoclonal Antibodies using the multiple tolerization subtractive immunization protocol (MTSI) and epitope masking

• Grant number: 24/23484-8
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: June 01, 2025
• End date: February 28, 2030
• Field of knowledge: Health Sciences - Pharmacy
• Principal Investigator: Andrei Moroz
• Grantee: Rafaela Cristine dos Santos
• Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Associated research grant: 23/03240-4 - Study of the therapeutic potential of peptides generated by Galleria mellonella using the epitope masking technique, AP.R

Abstract

Cryptococcosis is a serious fungal infection caused by the pathogens Cryptococcus neoformans and Cryptococcus gattii, mainly affecting immunocompromised individuals. Infection occurs by inhalation of yeast spores present in contaminated organic materials (C. neoformans) or tree bark (C. gattii). The main symptoms include cryptococcal meningitis, a condition with a high mortality rate. Cryptococcus sp. has several serotypes and molecular types, with variations in ecology and response to treatment. Advances in genomics, proteomics and metabolomics have facilitated the identification of biomarkers and the development of diagnostic and therapeutic strategies. The immune system faces challenges in defending against cryptococcosis due to the galactoxylomannan (GalXM), glucuroxylomannan (GXM) capsule and other virulence factors associated with it, such as melanin, helping the fungus to evade the immune response. Techniques such as subtractive immunization, developed to overcome the difficulty of isolating antibodies against poorly immunogenic antigens, and epitope masking, which seeks to direct the immune response to less dominant epitopes, are innovative techniques to improve antibody specificity. This study seeks to develop specific monoclonal antibodies against Cryptococcus neoformans antigens, using the Multiple Tolerization Subtractive Immunization (MSTI) protocol, through the use of capsular wild-type strains (H99) and acapsular mutant strains (Cap67), aiming to obtain at least two unique clones. The objective is to contribute to the advancement of clinical research, since there are few monoclonal antibodies available, targeting components other than GXM, generating impacts on diagnosis and public health. In addition, the two best candidates will be tested for their possible therapeutic action, at in vitro analyses, verifying the action of the antibodies in immune tests and in tests to study different virulence factors of the fungus. The best ones will be humanized in the laboratory led by Prof. Dr. Cory Brooks (University of the State of California - Fresno).