A thesis submitted to Faculty of Health Sciences,
University of the Witwatersrand,
in fulfillment of the requirements for the degree of
Doctor of Philosophy
Johannesburg, March 2014 / The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine targeting the human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the surface of the virion, thereby preventing cell entry. Previous work in our laboratory focused on the generation of a 2dCD4S60C molecule (a variant of the CD4 primary Env receptor) with higher affinity for HIV-1 Env through targeted disulphide exchange. This study reports on the design and construction of an HIV-1 subtype C founder virus consensus Env immunogen derived from newly transmitted/founder virus sequences, and the ability of the purified recombinant Env proteins (2dCD4S60C-liganded and unliganded) to induce a broadly neutralizing antibody response in small animals. A total of 1894 founder sequences from 80 HIV-1 subtype C infected patients were available and downloaded from the databases. A consensus sequence was generated for each of the patients, and this alignment was subsequently used to generate a founder virus consensus env sequence. The env sequence was used to create codon-optimized constructs encoding monomeric (gp120FVCm), dimeric (gp120FVCGCN4d) and trimeric (gp140FVCGCN4t(+) and gp140FVCGCN4t(-) founder virus conformations cloned into the pcDNA3.1(-) mammalian expression vector. All four Env constructs were successfully expressed in HEK293T mammalian cell culture. The 2dCD4S60C was expressed in E. coli BL21 (DE3) and purified by nickel affinity chromatography. Large scale expression and purification of the gp120, gp120GCN4 and gp140GCN4 +/- in the unliganded or 2dCD4S60C liganded state were purified by lectin affinity chromatography, followed by conformation and complex purification using size exclusion chromatography. Immunogens/immune complexes were evaluated by ELISA, SDS-PAGE, native PAGE and surface plasmon resonance, and confirmed they were functional and conformationally intact. Immunogenicity of each conformation alone or complexed to 2dCD4S60C was evaluated in rabbits. Breadth and potency of the rabbit sera was tested against 12 pseudoviruses (Tiers 1-3), derived from HIV-1 subtype B and C Env, using the PhenoSense Neutralizing antibody assay (Monogram Bioscience, Inc.). Minimal neutralizing breadth was obtained from animals immunized exclusively with Env conformations. However, animals that received the Env/2dCD4S60C complex showed extensive neutralizing capacity against all 12 viruses tested, including the tier 2 and 3 virus strains. End-point ELISA titer results revealed that the rabbits that were immunized with Env/2dCD4S60C produced both Env and 2dCD4S60C specific titers, but those directed towards 2dCD4 were on average 10x lower than the 2dCD4S60C control group. This implies a proportion of the NAb activity is directed towards conserved epitopes exposed on
the Env/2dCD4S60C immunogens. Overall, these results show that the use of founder Env/2dCD4S60C complexes as vaccine immunogens dramatically improves the antibody neutralization breadth and magnitude as compared to founder Env or 2dCD4S60C alone. This level of broad neutralization has not been previously reported in the literature, and these results provide encouraging data to inform us of the best envelope vaccine immunogen to include in a preventative vaccine.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/17462 |
| Date | 21 April 2015 |
| Creators | Killick, Mark Andrew |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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