Phage display identification of vaccine candidates for schistosomiasis mansoni based on self-healing of rhesus monkeys (Macaca mulatta)

Individuals of the genus Schistosoma are hemoparasites that cause schistosomiasis, a worldwide distributed disease that affects more than one hundred million people. In Brazil, the etiologic agent of schistosomiasis is Schistosoma mansoni. Since the publication of the species transcriptome in 2003, there has been little success in testing vaccine candidates, pointing to the need of new methods to recognize new candidate targets. One approach is to use rhesus monkeys as a study model, which is able to heal spontaneously from Schistosoma infection. This work involves the development of an essential bioinformatics pipeline to explore the data obtained with a new phage display technology with synthetic oligonucleotides, capable of screening the antibodies produced by infected and self-cured rhesus monkeys; we used the rhesus monkey as an animal model for the first time in conjunction with this new technology. The approximately 120,000 synthetic oligonucleotides used to build the phage display library represent, with equal abundance, the coding sequences that cover all 58-amino-acid fragments across all parasite proteins that have a known sequence, allowing for unbiased screening of all antibodies of rhesus monkeys that mediate self healing. For the phage display assay, large-scale sequencing of the phages captured by serum samples from each of 12 infected monkeys, collected at times 0 and 8 weeks after infection, was used. The bioinformatics pipeline developed here identified that 98.3% of the oligonucleotide sequences in the library were detected at least once among the 63 sequenced samples. In this pipeline, we use the zero-inflated generalized Poisson distribution and the Negative binomial distribution as two statistical approaches to calculate the significance of enrichment of the peptides captured by the serum of each monkey at each time point. 421 peptides were identified as significantly enriched by both statistical methods, and from this result 61 peptides were enriched in at least four serum samples from week 8 after infection, with 9 peptides belonging to 8 protein coding genes (annotated as Smps). The remaining 52 peptides represent different alternative splicing isoforms of 2 microexon genes (annotated as micro-exon genes, MEGs). Among Smp proteins, only one protein is described as intracellular, while the others are described as expressed at the host-parasite interface. The antibodies developed by monkeys against these proteins may be involved in the parasite\'s death process. With the application of the enrichment analysis pipeline to the sequencing data of phages captured by monkeys\' serum collected between weeks 10 and 62 after infection, which are still being processed, we expect to continue identifying the same peptides already recognized in week 8, in addition to identifying new antigenic peptides from the parasite, which can be tested in future studies for the development of a vaccine against schistosomiasis. (AU)