Paracoccidioidomycosis (PCM) is a granulomatous fungal disease caused by the dimorphic fungal species of Paracoccidioides, which mainly affects the lungs. Modern strategies for the treatment and/or prevention of PCM are based on a Th1-type immune response, which is important for controlling the disease. One of the most studied candidates for a vaccine is the P10 peptide, derived from the 43 kDa glycoprotein of Paracoccidioides brasiliensis. In order to improve its immune modulatory effect, the P10 peptide was associated with a chitosan-conjugated nanoparticle. The nanoparticles presented 220 nm medium size, poly dispersion index (PDI) below 0.5, zeta potential of +20 mV and encapsulation efficiency around 90%. The nanoparticles’ non-toxicity was verified by hemolytic test and cell viability using murine macrophages. The nanoparticles were stable and presented physicochemical characteristics desirable for biological applications, reducing the fungal load and the usual standard concentration of the peptide from 4 to 20 times.

The Cryptococcus neoformans capsular extracellular polysaccharide (EPS) plays an essential role in virulence and host immunomodulation. In this study, we analyzed the virulence of four serotype A C. neoformans strains with different polysaccharide structures: two multi-motif (H99 and KN99α) and two single-motif (Mu-1 and ATCC-24064 (24064)). Mice were infected by the intranasal (IN) or intravenous (IV) route. Survival, fungal load (colony-forming unit), cell lung population, cytokines, and histology were analyzed. No differences in cell size or capsule size were found between the four strains. However, polysaccharide sizes were highly variable, and no significant differences were observed for all other virulence factors. Nitrite production in cell culture dendritic cells was reduced for the H99 or KN99α strains of EPS. The reactive oxygen species in cell culture M1 macrophages and dendritic cells were increased for the H99 or KN99α strain’s EPS. Cytokines differed from the IN and IV infections. Lung histology showed increased inflammatory cell presence after infection with Mu-1 or 24064 strains, with no cryptococcal cells observed in the lung parenchyma or alveoli, whereas the opposite pattern was seen after challenges with H99 or KN99α. There were differences in lung neutrophil infiltration between the analyzed strains, and a higher presence of activated macrophages and CD4+ lymphocytes for the Mu-1 or 24064 strains. Polysaccharides from single and multi-motif strains elicited different effects on macrophage-like cells in vitro . These results suggest that variation in polysaccharide composition and structure can translate into differences in virulence and pathogenicity. IMPORTANCE: Cryptococcosis is a systemic fungal infection that causes approximately 1 million cases globally, leading to approximately 625,000 annual deaths. Two species are responsible for the majority of cases, Cryptococcus neoformans and Cryptococcus gattii . C. neoformans usually causes disease in immunosuppressed hosts, whereas C. gattii can infect and cause disease in immunocompetent hosts. The capsule of Cryptococcus spp. is one of its major virulence factors, due to its immunomodulation abilities. The capsule is composed of three major different molecules: glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins. GXM is composed of six different structures called motifs. The exact mechanism and structures associated with its immunomodulation are still not well elucidated. Here, we looked at different immune responses based on the capsule composition. Our results strongly suggest that the capsule motif composition can affect the virulence, pathogenicity, and immunomodulatory capabilities of the Cryptococcus capsule.

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated by CD4⁺ Th₁ lymphocytes that was associated with the increased production of IFN-γ in mice challenged with a virulent isolate of Paracoccidoides brasiliensis. Cationic liposomes formulated with dioctadecyldimethylammonium and trehalose dibehenate (DDA/TDB, termed also CAF01–cationic adjuvant formulation) have been developed for safe administration in humans and CAF01 liposomes are utilized as an adjuvant for modulating a robust Th₁/Th₁₇ cellular response. We evaluated the efficacy of the adsorption of peptide P10 to CAF01 cationic liposomes and used the generated liposomes to vaccinate C57Bl/6 mice infected with P. brasiliensis. Our results showed that P10 was efficiently adsorbed onto CAF01 liposomes. The vaccination of infected mice with cationic liposomes formulated with DDA/TDB 250/50 µg/mL and 20 µg of P10 induced an effective cellular immune response with increased levels of Th₁₇ cytokines, which correlated with significant decreases in the fungal burdens in lungs and protective granulomatous tissue responses. Hence, cationic liposomes of DDA/TDB 250/50 µg/mL with 20 µg of P10 are a promising therapeutic for safely and effectively improving the treatment of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a fungal infection caused by the thermodimorphic Paracoccidioides sp. PCM mainly affects the lungs, but, if it is not contained by the immune response, the disease can spread systemically. An immune response derived predominantly from Th1 and Th17 T cell subsets facilitates the elimination of Paracoccidioides cells. In the present work, we evaluated the biodistribution of a prototype vaccine based on the immunodominant and protective P. brasiliensis P10 peptide within chitosan nanoparticles in BALB/c mice infected with P. brasiliensis strain 18 (Pb18). The generated fluorescent (FITC or Cy5.5) or non-fluorescent chitosan nanoparticles ranged in diameter from 230 to 350 nm, and both displayed a Z potential of +20 mV. Most chitosan nanoparticles were found in the upper airway, with smaller amounts localized in the trachea and lungs. The nanoparticles complexed or associated with the P10 peptide were able to reduce the fungal load, and the use of the chitosan nanoparticles reduced the necessary number of doses to achieve fungal reduction. Both vaccines were able to induce a Th1 and Th17 immune response. These data demonstrates that the chitosan P10 nanoparticles are an excellent candidate vaccine for the treatment of PCM.

Cryptococcus neoformans is an important fungal pathogen that causes cryptococcosis, a life-threatening disease predominantly affecting immunocompromised individuals. Current estimates suggest that cryptococcosis is responsible for over 150,000 deaths annually, highlighting the need for the development of effective preventive measures. Early research utilizing polysaccharide filtrates (CneF) laid the groundwork for polysaccharide-containing vaccines, demonstrating partial protection in murine infection models. Further vaccine constructs made with purified extracellular polysaccharides (EPS) conjugated to protein carriers elicited strong antibody responses and enhanced survival. Recently, more attention was given to the purified capsule components glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal), with studies indicating that GXM conjugates can elicit protective effects. The development of semisynthetic polysaccharide-based vaccines offers a new approach to addressing lot-to-lot variability, enhancing consistency. In conclusion, strategies that advance from early crude formulations to more sophisticated conjugate and synthetic vaccines hold great promise for developing an effective vaccine to prevent and treat cryptococcal diseases.