Extracellular vesicles secreted by Talaromyces marneffei enriched with a mannoprotein constitute an effective vaccine

Abstract

Talaromyces marneffei (T. marneffei), formerly known as Penicillium marneffei, is the causative agent of talaromycosis (penicilliosis) which presents with respiratory, skin or systemic infections. T. marneffei is a thermally dimorphic fungus and is endemic mainly in Southeast Asia. It is regarded as one of the world’s top ten most feared fungi. So far there are only limited antifungals and no vaccine available for the intervention of talaromycosis. Fungal extracellular vesicles (EVs) contain multiple components including proteins, nucleic acids, polysaccharides and lipids, and were shown to modulate the host immunity and were used as potential vaccine candidates for preventing fungal diseases. This led to the hypothesis that T. marneffei could release EVs which are immunoregulatory and are capable of inducing protective immunity against T. marneffei infection.

In the study, EVs from T. marneffei yeast were isolated, purified and characterized by SEM, TEM and nanoparticle tracking analysis. Label-free quantitative proteomics work suggested that T. marneffei EVs contain multiple proteins with the majority not containing a signal peptide (95%). Potential T. marneffei EV positive and negative protein markers were defined. Membrane-associated proteins and antigenic proteins, especially a conserved mannoprotein, Mplp6, were enriched in EVs compared to the yeast cell proteins. T. marneffei EVs were found to be immunoreactive with sera from talaromycosis patients. These EVs also enhanced macrophage phagocytotic and fungicidal activities by inducing the expression of pro-inflammatory cytokines.

T. marneffei EVs were non-cytotoxic and conferred full immune protection in a mice vaccination model against T. marneffei infection by inducing specific serum IgG and T cell responses. IL-4 CD4+ T cells (Th2) and IFN-γ CD8+ T cells (Tc1), but not IFN-γ CD4+ T cells (Th1) and IL-17 CD4+ T cells (Th17), were significantly increased in EV immunized mice after challenging with T. marneffei when compared to the PBS control. Furthermore, Mplp6 was demonstrated EV membrane-associated with multiple molecules per EV and immunostimulatory. The protective immunity of EVs against T. marneffei infection was greatly reduced when mice were immunized with Mplp6-depleted EVs. While no protective immunity was observed when EV-immunized mice were challenged with heterogeneous T. marneffei strains, immunization with a mixture of T. marneffei EVs and recombinant Mplp6 protein (rtMplp6) provided partial protection with a delay of abnormal symptoms and prolonged mice survival, suggesting potential for vaccination against different T. marneffei strains.

This study characterized T. marneffei EVs multidimensionally and for the first time investigated their potency for being used as a vaccine against talaromycosis in an animal model, which encourages further studies on the use of EVs as vaccine candidates in fungal infections. As most talaromycosis patients are either HIV positive or immunosuppressed, further studies are required to assess whether T. marneffei EVs or their combination with rtMplp6 can protect mice with CD4+ T cell deficiency.