An immunoinformatics and extensive molecular dynamics study to develop a polyvalent multi-epitope vaccine against cryptococcosis

Abstract

Cryptococcosis is a lethal mycosis instigated by the pathogenic species Cryptococcus neoformans and Cryptococcus gattii, primarily affects the lungs, manifesting as pneumonia, and the brain, where it presents as meningitis. Mortality rate could reach 100% if infections remain untreated in cryptococcal meningitis. Treatment options for cryptococcosis are limited and and there are no licensed vaccines clinically available to treat or prevent cryptococcosis. Our study utilizes an integrated bioinformatics approaches to develop a polyvalent multiepitope subunit vaccine focusing on the key virulent proteins Heat shock transcription factor and Chaperone DnaK of both C. neoformans and C. gatti. Then in silico analysis was done to predict highly antigenic epitopes by assessing antigenicity, transmembrane topology screening, allergenecity, toxicity, and molecular docking approaches. Following this analysis, we designed two vaccine constructs integrating a compatible adjuvant and suitable linkers. These constructs exhibited notable characteristics including high antigenicity, non-toxicity, solubility, stability, and compatibility with Toll-like receptors (TLRs). The interaction between both vaccine constructs and TLR2, TLR3, and TLR9 was assessed through molecular docking analysis. Molecular dynamics simulations and MM-PBSA calculations suggest the substantial stabilizing property and binding affinity of Vaccine Construct V1 against TLR9. Both the vaccines revealed to have a higher number of interchain hydrogen bond with TLR9. These findings serve as a crucial stepping stone towards a comprehensive solution for combating cryptococcus infections induced by both C. neoformans and C. gattii. Further validation through in vivo studies is crucial to confirm the effectiveness and potential of the vaccine to curb the spread of cryptococcosis. Subsequent validation through in vivo studies is paramount to confirm the effectiveness and potential of the vaccine in reducing the spread of cryptococcosis.

Fig 1. Schematic (A) and Constructive (B) representation of vaccine construct 1 and vaccine construct 2.

Fig 2. The best docked complex with their respective docking energy.

(A) V1-TLR9 complex (B) V2-TLR9 complex (C) V1-TLR3 complex (D) V2-TLR3 complex.

Fig 3. The analysis of RMSD, RMSF, and Rg.

A) RMSD in TLR3 and TLR9 (left side panel) and RMSD in vaccine constructs V1 and V2 (right side panel), B) RMSF in side chain atoms of TLR3 (left side panel), TLR9 (right side panel), C) RMSF in side chain atoms of vaccine construct V1 and vaccine construct V2, D) Radius of gyration in TLR3 and TLR9 (left side panel) and RMSD in vaccine constructs V1 and V2 (right side panel).

Fig 4. The analysis of solvent accessible surface area and interaction energies.

A) Total solvent aceesible surface area. Interaction energies between B) TLR3 and vaccine construct V1, C) TLR3 and vaccine construct V2, D) TLR9 and vaccine construct V1, and E) TLR9 and vaccine construct V2.

Fig 5. Hydrogen bond analysis between TLR3 and vaccine construct V1.

The top panel plot the number of hydrogen bonds against simulation time. In other panels, the surface view of respective TLR (blue surface) and vaccine construct (pink surface). In the ribbon representations, the blue ribbons represent TLRs, while the orange ribbons represent vaccine constructs.

Fig 6. Hydrogen bond analysis between TLR3 and vaccine construct V2.

(Refer to color schemes in Fig 4).

Fig 7. Hydrogen bond analysis between TLR9 and vaccine construct V1.

(Refer to color schemes in Fig 4).

Fig 8. Hydrogen bond analysis between TLR9 and vaccine construct V2.

(Refer to color schemes in Fig 4).

Fig 9. Gibb’s free energy landscape.

A) TLR3-vaccine construct V1 complex, B) TLR3-vaccine construct V2 complex, C) TLR9-vaccine construct V1 complex, and D) TLR9-vaccine construct V2 complex.

Fig 10. DCC analysis for A) TLR3-vaccine construct V1 complex, B) TLR3-vaccine construct V2 complex, C) TLR9-vaccine construct V1 complex, and D) TLR9-vaccine construct V2 complex.