The study of the polyamine transporter, PotD, and it hybrids as vaccine antigens against Streptococcus pneumoniae.

Author(s)

Thiago Rojas Converso

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2017-02-10

Examining board members

Luciana Cezar de Cerqueira Leite; Luiza Guilherme Guglielmi; Gabriel Padilla Maldonado; Marilis do Valle Marques; Rita de Cássia Ruiz

• Advisor: Luciana Cezar de Cerqueira Leite

Abstract

Polyamine Transporter D (PotD) is an important antigen for Streptococcus pneumoniae virulence in vivo, protecting immunized mice against systemic infection and reducing the bacterial load in the nasopharynx of immunized animals. However, in order to extend vaccine coverage, the combination of PotD with other antigens of the bacterium is required. The present study aimed at expanding the investigation of the immune response generated against PotD alone or fused with two other pneumococcal proteins: the Pneumolysin derivative, PdT and Pneumococcal Surface Protein A (PspA). Therefore, the potD, pdt and pspA genes were cloned and expressed, either alone or in fusion, generating the hybrid proteins rPotD-PdT and rPspA-PotD. The recombinant proteins and hybrids were used for subcutaneous immunization of BALB/c mice, generating high levels of antibodies. Sera from immunized animals were able to recognize and bind onto the surface of different pneumococcal strains, and to enhance phagocytosis of the bacterium in vitro. In all tests, the hybrids were more effective than the isolated proteins. The cellular immune response was characterized by the production of INF-γ, IL-2 and IL-17 by splenocytes and increased production of NO by peritoneal cells of the immunized animals. Despite promising results in vitro, rPotD-PdT protein was not able to induce protection in any of the tested challenge models. In contrast, rPspA-PotD fusion was able to protect mice against sepsis with two virulent isolates of pneumococcus and led to reduction in bacterial loads in the nasopharynx of challenged animals. Finally, we demonstrate that the addition of exogenous polyamines, spermidine, and putrescine, in the pneumococcal culture interfered with biofilm formation in vitro. Considering the important role of biofilm formation for successful colonization, this result suggests a possible mechanism of action of PotD during colonization by pneumococcus. Taken together, the results suggest that the use of the hybrid rPspA-PotD comprises a promising vaccine strategy, able to protect against colonization and pneumococcal sepsis, through the production of opsonizing antibodies and activation of protective cytokines, such as IL-17. (AU)

• FAPESP's process: 12/04286-3 - The study of polyamines transporter, PotD, as a vaccine antigen against Streptococcus pneumoniae
• Grantee: Thiago Rojas Converso
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)