Evaluation of genetic diversity

In order to develop a broad-spectrum T. cruzi vaccine, the prioritized proteins were scrutinized for their genetic diversity among fully annotated proteomes of 12 T. cruzi strains and different species (Supplementary Table 10). Protein sequences from these strains which are positive for that particular protein were downloaded from NCBI RefSeq (Pruitt et al., 2007) and aligned to predict conserved regions using CLC Main Work (Workbench et al., 2010). Evolutionary distances (p-distances) among variant sites were also calculated for prioritized proteins using Mega 6.0 (Tamura et al., 2013). The predicted epitopes were also checked for their sequence divergence among different strains and species of Trypanosoma. Each predicted epitope was further checked for antigenicity using VaxiJen (threshold value = 0.4) (Nas et al., 2015). For epitope mapping, we first reverse translated all the epitope sequences and evaluated for homology using BLASTn with the genome of different strains and species of T. cruzi. Epitope conservancy for all 24 predicted epitopes were also evaluated using the IEDB conservancy analysis tool (Bui et al., 2007).

Supplementary Table 10: Different strains and species of T. cruzi

• References:

• Pruitt KD, Tatusova T, Maglott DR. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2007 Jan;35(Database issue):D61-5. doi: 10.1093/nar/gkl842. Epub 2006 Nov 27. PMID: 17130148; PMCID: PMC1716718.

• Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013 Dec;30(12):2725-9. doi: 10.1093/molbev/mst197. Epub 2013 Oct 16. PMID: 24132122; PMCID: PMC3840312.

• Bui HH, Sidney J, Li W, Fusseder N, Sette A. Development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines. BMC Bioinformatics. 2007 Sep 26;8:361. doi: 10.1186/1471-2105-8-361. PMID: 17897458; PMCID: PMC2233646.

• Naz, A., Awan, F. M., Obaid, A., Muhammad, S. A., Paracha, R. Z., Ahmad, J., & Ali, A. (2015). Identification of putative vaccine candidates against Helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach. Infection, Genetics and Evolution, 32, 280-291.