| Summary: | A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine
JOHANNESBURG, 2015 === Directing antibodies against CD4, the primary receptor for HIV entry on its host T-cells, has been explored as an alternative way of inhibiting viral infection. Here, a panel of 40 CD4 targeting monoclonal antibodies (MAbs) were generated from mice immunized with bacterially expressed recombinant 2-domain CD4 (2dCD4). Firstly, the first two individual domains of CD4, termed D1 and D2, were generated. Whilst the mutant D1 generated was based on a previous work, the expression and characterisation of individual wild-type domain D2 has not yet been reported in detail. D1 and D2 cassettes were cloned and expression in E.coli was optimised. After the establishment of an optimised purification protocol, the purified protein were assessed by means of basic biophysical analyses (analytical SDS-PAGE, CD Spectroscopy), which allowed us to gain insights into the structural and functional integrity of the purified products. Recombinant D1 and D2 were then used to define the domain specificities of the MAbs by ELISA. We also demonstrated the ability of the MAbs to inhibit CD4-gp120 binding in vitro by ELISA, and inhibit viral infection by a pseudovirion inhibition assay. These preliminary results reinforce the potential of CD4-directed MAbs as candidate anti-HIV agents, and provide a solid platform for further pre-clinical development of this class of antiviral compounds.
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