Immunoproteomic analysis of Cryptococcus neoformans antigens reactive with sera from patients with cryptococcosis in Colombia

Introduction. Cryptococcus neoformans (Cn) is the main etiological agent of cryptococcosis, a life-threatening systemic mycosis and one of the leading secondary infections among HIV-positive patients. In Colombia, cryptococcosis has an annual incidence of 2.3 cases per million inhabitants, affecting predominantly patients with AIDS, and with a mortality of up to 47.5% of the cases. Despite the benefits of available antifungal drugs, their use is limited by the emergence of drug-resistant fungal strains and several side-effects resulting from long term medication. It is also known that while a T-helper (Th)1 response elicits protective immunity during cryptococcosis, a Th2 response assists fungal growth leading to peripheral dissemination. Therefore, adjunctive immunotherapy, addressed to skew a detrimental response towards a protective response, together with antifungal treatment, is a promising option for the future. Materials and methods. Serum samples from Cn-cryptococcosis patients (n=113) and control patients with other mycoses (n=3) from Colombia were included in this study. Using ELISA, total levels of Th2-dependent immunoglobulin (Ig)E and IgG4 and levels of Cn-specific IgG were determined in sera. Protein extracts from C. neoformans H99 were separated by 1D-SDS-PAGE and transferred to a nitrocellulose membrane by Western blot. Membranes were subsequently incubated with sera to detect antigens reactive with IgG antibodies. Results. Increased total IgE levels were detectable in cryptococcosis patients (up to 43.875 µg/ml) with HIV+ patients showing a significant higher total IgE production compared with HIV- patients (p<0.05). Conversely, total IgG4 levels did not differ among patients with different HIV status (p>0.05), and neither IgG4 nor IgE levels differed between male and female patients (p>0.05). Cn-specific IgG titers were higher in HIV- patients (p<0.01) compared to HIV+ patients, but did not differ with the patients’ gender (p>0.05). Preliminary immunoblot analysis of two sera from HIV+ patients revealed several immunoreactive cryptococcal antigens, including 130, 75, 40, 35 and 18 kDa proteins, which were determined to be IgG-immunoreactive. Conclusions. Compared to healthy people, who have estimated IgE levels <0.72 µg/ml, patients with cryptococcal meningitis presented with markedly elevated IgE production. Specific cryptococcal antigens that were recognized by reactivity with IgG were for the first time detected using human serum samples. To identify infection-specific cryptococcal antigens that are involved in the pathogenesis of C. neoformans, further analyses including healthy Colombian control patients and HIV+ positive patients, not infected with C. neoformans or any other fungal agent, need to be done. Additional mass spectrometry analyses are also required to identify the immunoreactive cryptococcal proteins and their possible roles. The identification of proteins that are major targets of the humoral response of patients with cryptococcosis greatly extends the analysis of sera from mice with pulmonary cryptococcal infection done earlier by our group, which may contribute to vaccine development or adjunctive immunotherapy against C. neoformans infection.