Brine Shrimp Lethality Test and Characterization of Snail Soluble Proteins of Biomphalaria pfeifferi as a Candidate for Vaccine Development against Schistosoma Mansoni

Abstract

Schistosomiasis infects about two hundred million people around the world. Currently, treatment for Schistosoma mansoni infection is by use of Praziquantel™. Challenges associated with Praziquantel™ use include drug resistance, rapid re-infection and high cost. A longer lasting solution would be a vaccine to enhance the use of drugs. Unfortunately, no human vaccine for schistosomiasis is available. Snails that are the intermediate host of S. mansoni have been discovered to have common proteins with the schistosome worms. A research conducted in mouse model showed that two candidate vaccines derived from Biomphalaria pfeifferi, RT (soluble proteins from the rest of snail tissue) and DG (soluble proteins from the digestive gland), were protective against S. mansoni based on increased immune responses, worm reduction and reduced pathology. Both of them met the World Health Organization criteria of over 40% protection. It is important to ensure that substances being used as vaccines are both efficacious and non-toxic and therefore are safe. There are a number of common product safety tests and the simplest being brine shrimp lethality test. This work tested for in vivo Brine Shrimp Lethality Test (BSLT) of DG and RT. For BSLT, DG and RT were processed and their concentration determined by microtitre technique. Concentrations for both DG and RT were 1.4 mg/ml. Evaluation of the cytotoxicity of DG and RT was done in in terms of Lethality concentration (LCD₅₀) using10μg/ml, 100μg/ml and 1000μg/ml concentrations of the proteins. Ten Brine Shrimps larvae (nauplii) were placed in duplicate tubes of each concentration. After 24 hours the surviving Brine Shrimps larvae were counted and LCD₅₀ was determined by Finney computer program at 95% confidence interval. The LCD₅₀ for DG was 3988.73 µg/ml and RT was 4158.06 µg/ml. The average percentage mortality in all the three different concentrations for the both soluble proteins was less than 50%. DG and RT had a LCD₅₀ of over 1000µg/ml. The toxicity results for the two soluble proteins at the three different concentrations shows that both proteins are non-toxic and are therefore safe vaccines. After a product is found safe and efficacious, it is essential to determine its structure. This study also characterized DG and RT using Gas chromatography-mass spectrometry (GC-MS) and ultraviolet (UV) spectroscopy. Chemical identification and characterization of DG and RT using the GC-MS spectrum established the presence of different chemical compounds with varied retention times. The results from the GC-MS were confirmed by UV spectrum results which confirmed the presence of proteins.

Keywords: Schistosomiasis, Toxicity, vaccine, characterization