The Human Immunodeficiency Virus (HIV) epidemic still poses a problem with approximately 2 million new infections reported worldwide in 2014. New strategies are required to alleviate this burden. Our laboratory has previously shown that crude saliva and purified mucins from cervical plug mucin, saliva and breast milk inhibit HIV-1 infection in vitro. This project investigates purified mucins sourced from pig and horse mucus, as an alternative and abundant source of material for anti-HIV-1 research. Pig gastric and cervico-vaginal mucus was collected and stirred overnight in 6M guanidine hydrochloride with 10mM Na₂HPO4, 10mM EDTA, 1mM PMSF and 5mM NEM. Gastric and cervicovaginal mucus was purified by density gradient ultracentrifugations in CsCl at 105 000g for 48 hours, twice, and mucin rich fractions were separated by size exclusion column chromatography. Mucin-rich materials eluting in the void volume (V₀) were reduced with 10mM dithithreitol (DTT) or subjected to proteolysis with trypsin. Pig saliva was collected in 0.2M NaCl:0.02% sodium azide and horse saliva (due to its viscous nature) was collected and stirred overnight in 6M guanidine hydrochloride with 10mM Na₂HPO4, 10mM EDTA, 1mM PMSF and 5mM NEM. Pig and horse saliva samples underwent size exclusion column chromatography, where the V₀ fractions of both were purified with one density gradient ultracentrifugation and then dialysed and freeze dried, after which aliquots were treated with either DTT or trypsin. At every stage of purification, lyophilized aliquots of all mucin sources were tested on a luciferase based replication defective HIV neutralization assay on a CD4 expressing HeLa cell line. Luciferase expression quantified as relative light units by a luminometer was used to calculate percentage neutralization. Log dose response curves were constructed to extrapolate the half maximal inhibitory concentrations (IC₅₀) on GraphPad Prism. Samples were tested on an MTT cell toxicity assay. Pig gastric and cervicovaginal mucins were added to a simulated vaginal fluid to make gels (at a concentration of 30mg of mucin per ml of buffer). These gels were tested on the neutralization, MTT assays and the pig gastric mucin gel then underwent particle tracking and nanoparticle diffusion assays at varying pH. Pig gastric and cervicovaginal mucin showed good inhibition and low toxicity, with pig gastric mucin V₀ having the best IC₅₀ (1.668μg/ml). Pig and horse saliva showed inhibition but low cell viability. Pig gastric and cervicovaginal mucin gels exhibited good IC₅₀'s but pig gastric mucin had the best neutralization and lowest toxicity (PGM in Gel Solution 4 IC₅₀: 20.23μg/ml). HIV particle tracking and nanoparticle diffusion assays showed that the pig gastric mucin gel inhibited HIV-1 at low pH and existed as a soft gel. This project shows the efficacy of pig gastric mucin to possibly being a component of an anti-HIV-1 vaginal microbicide.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/26949 |
| Date | January 2017 |
| Creators | Pillay, Santhoshan Thiagaraj |
| Contributors | Mall, Anwarul Haq Suleman |
| Publisher | University of Cape Town, Faculty of Health Sciences, Department of Surgery |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, Doctoral, PhD |
| Format | application/pdf |
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