# A novel mRNA-based multiepitope vaccine candidate against Cryptosporidium hominis and Cryptosporidium parvum employing reverse-vaccinology and immunoinformatics approaches

## TL;DR

This paper presents a new mRNA vaccine design for Cryptosporidium parasites using computational methods, aiming to combat a disease with no approved vaccine.

## Contribution

The novel contribution is an in-silico multiepitope mRNA vaccine candidate targeting both C. hominis and C. parvum using reverse vaccinology and immunoinformatics.

## Key findings

- The vaccine showed structural stability and strong receptor-binding affinity in simulations.
- Immune simulations indicated robust immune response amplification upon repeated exposure.
- Codon optimization for E. coli expression suggested high potential for vaccine expression.

## Abstract

The parasite Cryptosporidium spp. causes cryptosporidiosis, a diarrheal disease in humans and animals. This study describes the development of mRNA vaccine targeting antigens from C. hominis and C. parvum, important gut pathogens. This vaccine was designed with reverse vaccinology and immunoinformatics as no FDA-approved vaccine exists for cryptosporidiosis.

Initially, a thorough literature review was conducted to identify five pathogenic proteins (aminopeptidase, heat shock protein, P23, serine protease, and sporozoite glycoproteins) associated with these two parasites. Next, a multiple sequence alignment was conducted, and the conserved sequences were used to design a novel multiepitope mRNA vaccine against these two parasites, combining the best CD8+, CD4+, and continuous B-cell epitopes. Additionally, structural prediction, docking, dynamics, and immune simulation, as well as cloning, were conducted.

The vaccine demonstrated standard biophysical properties, indicating that the protein is soluble and stable. Both two-dimensional (substantial alpha helix, beta sheet, and coil structures) and three-dimensional structures (Ramachandran score of 83.1% and a Z score of −7.39) of the vaccine were standard. The docking energy for TLR-2 (−1151.9) and TLR-4 (−1028.3) exhibited significant interactions. Furthermore, MM-GBSA and dynamics simulation both verified their stability, compactness, and flexibility. Next, codon optimization for Escherichia coli expression yielded promising results, with the vaccine demonstrating substantial expression, as evidenced by a GC content of 46.97% and a CAI of 0.988. Afterwards, immune simulation demonstrated robust immune response amplification upon repeated exposures. In addition, the vaccine exhibited stability in its mRNA structures.

This study developed an in-silico multiepitope novel mRNA vaccine candidate for C. hominis and C. parvum with excellent structural stability, antigenicity, receptor-binding affinity, and expected immune responses. These findings offer a novel approach due to numerous species target but with significant drawbacks like no validation beyond simulation, uncertainty of long-term immunity, protein quality, stability and safety, requiring experimental validation.