Vaccination mediates protection against Penicillium marneffei in BALB/c mice

Abstract

P. marneffei is the unique thermal dimorphic fungus among Penicillium spp. which grows as mycelium at 25 ºC and as yeast at 37 ºC. Intravascular injection of lethal dose of P. marneffei spores might lead to systemic infections in BALB/c mice, and the hepatic tissue seemed to suffer from the most severe infection with significant high CFU value than other organs. In this study, we aimed at generating a candidate vaccine for P. marneffei using two independent approaches, recombinant Mp1p protein and live-attenuated strain vaccination. Antigenic mannoprotein Mp1p of P. marneffei strain PM1 was cloned and expressed in E. coli and Pichia pastoris, and these two types of recombinant Mp1p was used to perform vaccination study. Both subcutaneous administration of recombinant Mp1p with Freund’s Adjuvant and intraperitoneal injection of Mp1p with Sigma Adjuvant System elicited efficacious resistance against lethal dose challenge of PM1 in BALB/c mice. Four different strains PM1, PM18, PM25 and PM36 were chosen to detect the protective breadth of strain coverage of recombinant Mp1p and results showed that recombinant Mp1p only generated protection against strain PM1 but failed to resist against the infections of other P. marneffei isolates. Fungal burden and histopathological examination elucidated that recombinant Mp1p with SAS had efficacy in suppression and clearance of hepatic and splenic PM1 yeast cells, but failed to control the dissemination of PM25 which caused invasive hepatic and splenic infection and malignant inflammatory response. Thus, we hypothesized that recombinant Mp1p elicited a narrow breadth of strain coverage due to the high degree of polymorphism of MP1 and its homologues. Depletion of virulence gene MP1 from strain PM1 yielded one live-attenuated P. marneffei strain BC122. BC122 vaccinated mice were able to resist infections due to different P. marneffei isolates, including strain PM1, PM18, PM25 and PM36. Thus, it displayed wide strain coverage. Fungal burden analysis elucidated that both PM1 and PM25 yeast cells in mice spleens and livers presented a trend of clearance. H&E and GMS staining of vaccinated mice hepatic tissues founded that both PM1 and PM25 yeast cells were fast sequestered in granulomas and killed progressively, and many empty granulomas were seen since days 6 post-infection. Intracellular staining showed that the frequency of IFN-γ producing CD4+ T cells in hepatic tissue was significant higher than the peripheral blood, which might elucidate the influx of functional hepatic Th1 cells. The absolute number of hepatic Th1 cells in immunized mice were also significant greater than that of PBS control group. In addition, CD4 deficient mice seemed to be vulnerable even to the virulence attenuated strain BC122. Furthermore, the percentage of hepatic Th17 cells rocketed at days 2 post-infection then fast decreased to bottom out which was not consistent with the tendency of fungal clearance. Thus, we concluded that vaccine BC122 had elicited efficacious protection through hepatic Th1 cell immunity in BALB/c mice model. This preliminary study will help us to characterize the mechanisms of exclusive immune response against P. marneffei and provide informative reference for preventive or therapeutic strategies against penicilliosis.