Proteome-based Computational Identification of Cathepsin B as a TLR1/2-binding Virulence Factor in Naegleria fowleri and Design of a Novel Multi-epitope Vaccine Candidate.

Primary Amoebic Meningoencephalitis (PAM), also known as Naegleriasis, has historically been rare but is re-emerging as a major public health concern. This acute, often fatal infection is caused by a free-living freshwater amoebic protozoan Naegleria fowleri, which invades the host central nervous system (CNS), inducing severe neuroinflammation. The immunopathogenesis of PAM remains poorly understood. Herein, the entire N. fowleri proteome was computationally analyzed, revealing Cathepsin B as a putative virulence factor with a potential to form a stable complex with human TLR1/2. This finding was further supported by molecular dynamic simulation analyses of the complex. Interestingly, Cathepsin B has been reported to mediate amoebic invasion of the host nasal mucosa, which reinforces our prediction. Based on these findings, a novel vaccine construct was designed using immunoreactive T and B cell epitopes from Cathepsin B and was coupled with different TLR adjuvants. Computational analyses demonstrated the strong binding affinity of TLR4 adjuvant-coupled multi-epitope vaccine with TLR4, forming the most stable interactions. Theoretical immune simulation studies revealed that this vaccine elicits robust IgM and IgG responses upon triple administration, maintains B cell memory and triggers the expression of IFN- γ. Our data thus provide an insight on the pathogenesis of PAM and offer a promising lead toward the development of an effective vaccine to combat the disease, which currently lacks reliable and effective treatments.