Publicação
New generation antigen delivery system for Candida albicans recombinant proteins as a vaccination strategy against systemic fungal infections
| Resumo: | Over the past five decades, a rise in numbers of immunocompromised individuals has led to a significant increase in invasive fungal infections, including those caused by Candida albicans. This has created a pressing need for innovative approaches to combat these fungal pathogens. While several immunotherapeutic strategies and vaccines are in development, none have reached clinical use yet. Previous work emphasised the potential of liposomal formulations containing C. albicans cell wall surface proteins (ADS1) as a vaccine candidate, highlighting chitinase 3 (Cht3) as a promising antigen. A monovalent formulation incorporating DODAB:MO liposomes with purified, recombinant, P. pastoris-produced, Cht3 was prepared. Cht3 displayed stability and activity across a wide pH range (pH 3-9) and at elevated temperatures (up to 50-60 ºC), making it suitable for liposomal inclusion. Cht3 hydrolysed chitin and chito-oligomers into smaller chito-oligomers, with chitobiose being the principal end-product but with chitotriose and the monomer N-acetylglucosamide being also produced. Cht3 liposomes (DMC) showed enhanced antigen delivery and cellular internalisation compared to ADS1, eliciting a Th1/Th17 immune response and generating substantial levels of opsonising antigen-specific IgG and IgG1 antibodies. A divalent formulation (DMCS) including Cht3 liposomes and a new antigen, the C. albicans protease Sap2, was also prepared. DMCS maintained liposome characteristics and induced cytokine production similar to DMC but surprisingly provided greater protection against systemic infection, likely due to a stronger Th1/Th2 immune response, with a higher IFN-γ/IL-4 ratio, trained immunity, and higher antigen-specific IgG response. The role of Cht3 in C. albicans virulence was explored using knockout mutants, revealing a decrease in virulence with CHT3 deletion. In vitro studies showed no significant differences in physiological characteristics, but the release of small chito oligomers by Cht3 with potential immunomodulatory effects could play a role in virulence. The results obtained in this thesis signify a significant advancement in the field by introducing novel specific antigen formulations and confirming DODAB:MO as an effective delivery system and adjuvant. DMCS emerges as a promising candidate, demonstrating enhanced protection and immune response. Additionally, it highlights chitinase activity as a virulence factor in C. albicans infections and the complex interaction between chitinases and chito-oligomers. Further studies will delve into these interactions and their implications for fungal infection therapies. |
|---|---|
| Autores principais: | Barbosa, Augusto Alexandre Costa |
| Assunto: | Candida albicans Vaccine Chitinase 3 Liposomes Virulence Vacina Quitinase 3 Lipossomas Virulência |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Over the past five decades, a rise in numbers of immunocompromised individuals has led to a significant increase in invasive fungal infections, including those caused by Candida albicans. This has created a pressing need for innovative approaches to combat these fungal pathogens. While several immunotherapeutic strategies and vaccines are in development, none have reached clinical use yet. Previous work emphasised the potential of liposomal formulations containing C. albicans cell wall surface proteins (ADS1) as a vaccine candidate, highlighting chitinase 3 (Cht3) as a promising antigen. A monovalent formulation incorporating DODAB:MO liposomes with purified, recombinant, P. pastoris-produced, Cht3 was prepared. Cht3 displayed stability and activity across a wide pH range (pH 3-9) and at elevated temperatures (up to 50-60 ºC), making it suitable for liposomal inclusion. Cht3 hydrolysed chitin and chito-oligomers into smaller chito-oligomers, with chitobiose being the principal end-product but with chitotriose and the monomer N-acetylglucosamide being also produced. Cht3 liposomes (DMC) showed enhanced antigen delivery and cellular internalisation compared to ADS1, eliciting a Th1/Th17 immune response and generating substantial levels of opsonising antigen-specific IgG and IgG1 antibodies. A divalent formulation (DMCS) including Cht3 liposomes and a new antigen, the C. albicans protease Sap2, was also prepared. DMCS maintained liposome characteristics and induced cytokine production similar to DMC but surprisingly provided greater protection against systemic infection, likely due to a stronger Th1/Th2 immune response, with a higher IFN-γ/IL-4 ratio, trained immunity, and higher antigen-specific IgG response. The role of Cht3 in C. albicans virulence was explored using knockout mutants, revealing a decrease in virulence with CHT3 deletion. In vitro studies showed no significant differences in physiological characteristics, but the release of small chito oligomers by Cht3 with potential immunomodulatory effects could play a role in virulence. The results obtained in this thesis signify a significant advancement in the field by introducing novel specific antigen formulations and confirming DODAB:MO as an effective delivery system and adjuvant. DMCS emerges as a promising candidate, demonstrating enhanced protection and immune response. Additionally, it highlights chitinase activity as a virulence factor in C. albicans infections and the complex interaction between chitinases and chito-oligomers. Further studies will delve into these interactions and their implications for fungal infection therapies. |
|---|