Immunomodulation via carbohydrate ligands in a vaccine strategy helps in the control of Cryptococcus gattii infection

Author(s)

Patrícia Kellen Martins Oliveira Brito

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-12-08

Examining board members

Thiago Aparecido da Silva; José Carlos Farias Alves Filho; Paulo Sergio Rodrigues Coelho; David Nascimento Silva Teixeira

• Advisor: Thiago Aparecido da Silva

Abstract

Fungal infections affect millions of lives each year and, in this case, cryptococcosis has an important relevance in reaching immunocompromised and healthy individuals, representing one of the main causes of morbidity and mortality worldwide among HIV+ individuals. The low efficacy and side effects associated with antifungal agents have highlighted the importance of developing new immunotherapeutic approaches for the treatment of Cryptococcus gattii infection. In the studies that constitute the present thesis, an immunization strategy against C. gattii infection was developed and, for this, selective agonists of the Dectin-1 receptor or TLR2/CD14 agonist were used, which were as an adjuvant in the vaccine protocol. Initially, BALB/c mice received the administration of adjuvant ArtinM previously the immunization protocol, and this protocol did not significantly alter the cytokine levels and concentration of CD3+ and CD11b+ cells in the lung tissue of the mice treated with ArtinM, compared to with the immunized group. This immunization protocol associated with the adjuvant ArtinM did not contribute to the reduction of pulmonary fungal burden and the survival curve of mice infected with C. gattii. This led to a change in the immunization protocol that modified to a high concentration of inactivated C. gattii yeast, with longer intervals between immunizations. Then, C57BL/6 mice that received the adjuvant ArtinM, prior to immunization, showed elevated levels of IL-10 and an increased relative expression of IL-23 in the lungs, and a reduction in the burden of C. gattii in the lung tissue was detected. In parallel, the above-cited immunization protocol was evaluated in the administration of dectin-1 agonists as adjuvants. For this, BALB/c or C57BL/6 mice received curdlan or peptide β-glucans (BGP), prior to immunization with C. gattii, and 14 days after the last immunization they were infected with C. gattii. After 14 days of infection, the animals were euthanized for evaluation. The adjuvant curdlan restored the levels of TNF-α that were reduced in the group of animals submitted only to the immunization protocol with C. gattii. The mean area and relative frequency of C. gattii titan cells in the lungs of BALB/c mice treated with curdlan were reduced. However, the pulmonary fungal burden was not reduced and the inflammatory infiltrate in the parenchyma of BALB/c mice was not reduced. On the other hand, the adjuvant curdlan induced high levels of IFN-γ and IL-10 and decreased the burden of C. gattii in the lungs of C57BL/6 mice, which was not replicated in mice treated with β-glucan peptides. Finally, the evaluation of AMJ2-C11, a alveolar macrophages cell line from C57BL/6 mice, which demonstrated a low expression of the dectin-1, were used in the incubation with curdlan that resulted in the polarization of AMJ2-C11 macrophages to the M1 profile, increased expression of TLR2, and increased phagocytic activity on C. gattii. However, curdlan did not induce greater fungicidal activity of AMJ2-C11 on C. gattii. The use of Syk inhibitors and tyrosine kinases of the Src family demonstrated the specificity of the action of curdlan in AMJ2-C11 cells via the dectin-1 receptor. Thus, the adjuvant ArtinM and the adjuvant curdlan favored the control of C. gattii infection, especially for the model of infection with C. gattii in C57BL/6 mice. The present study will have important implications for the development of new immunotherapeutic approaches to treat C. gattii infection. (AU)

• FAPESP's process: 18/19949-4 - Immunotherapy against experimental Cryptococcosis: a vaccine strategy associated with immunostimulators that recognize carbohydrate
• Grantee: Patrícia Kellen Martins Oliveira Brito
• Support Opportunities: Scholarships in Brazil - Doctorate