African trypanosomiasis is a vector-borne neglected tropical disease caused by parasites of the Trypanosoma genus that are cyclically transmitted through the bite of an infected tsetse fly. Two forms of the disease are endemic to sub-Saharan Africa: Human African Trypanosomiasis, also known as sleeping sickness, and Animal African Trypanosomiasis, commonly known as nagana (Aksoy et al., 2014). Nagana, caused by Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense, is considered to be the main disease that limits the trade of livestock in sub-Saharan Africa and kills approximately 3 million cattle annually, with an estimated loss of US

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPI-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate’s skin.