Sequential adeno-associated virus deliverd HIV-1 subtype C ENV glycoproteins as novel vaccine immunogens

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dc.contributor.authorMoodie, Melanie
dc.date.accessioned2019-05-17T07:49:49Z
dc.date.available2019-05-17T07:49:49Z
dc.date.issued2018
dc.descriptionA Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg In fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, October 2018en_ZA
dc.description.abstractAn efficacious HIV-1 vaccine that can elicit broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) is likely to provide protection from infection. To date, no vaccine immunogen has elicited bNAbs against HIV-1. Recent evidence from infected individuals suggests that bNAbs develop over time in response to constant antigenic challenge provided by an evolving Env that drives the somatic hypermutation of these antibodies and is necessary for their development. Env sequences required to engage the unmutated germline Bcells and drive the somatic hypermutation required to develop a bNAb response are available. Recombinant adeno-associated viral (rAAV) vectors present an attractive novel strategy for in vivo delivery and long-lived expression of sequential HIV-1 Env immunogens. Thus, this study aimed to provide proof-of-concept that rAAV can be used for the delivery and expression of sequentially-derived HIV-1 Env sequences in mammalian cells. Five sequential Env sequences (from a total of 292), over an infection period of 78 weeks, replicating in vivo viral evolution from patient CH505 who was infected with HIV-1 subtype C, were selected, codon optimized, and cloned into pcDNA3.3 for mammalian expression of Env. Recombinant Env glycoproteins were transiently expressed in HEK293 T cells, purified, and biochemically characterized by SDS-PAGE, Western Blotting and ELISA. The matched env sequences were also sub-cloned into the rAAV-Luc vector, allowing for the production of rAAV following a polyethylenimine (PEI)-based triple transfection protocol of HEK293 T cells. rAAVs were purified via polyethylene glycol (PEG) precipitation, followed by iodixanol gradient centrifugation, and quantified via droplet-digital PCR (ddPCR). These rAAVs were used to transduce HEK293 T cells and characterize the expression of Env in vitro by ELISA. Consensus Env sequences were generated from all time points, and following bioinformatics analyses, sequences from weeks 4, 20, 30, 53, and 78 post infection were selected. All pcDNA3.3-CH505 Env constructs were successfully expressed and purified, apart from the week 4 Env. Binding of the recombinant Envs to a panel of bNAbs, as determined by ELISA, confirmed they were functional and conformationally intact. Interestingly, differences in antibody binding were noted between weeks 20 – 78, highlighting potential changes in epitope exposure. All 5 envs were successfully sub-cloned into the pAAV backbone vector with intact ITRs, as confirmed by restriction enzyme digest analysis. The env transgenes were then packaged into serotype 2 rAAV vectors, and rAAVs were successfully produced and purified, with an average yield of 1,4 x 1011 genome copies/ml, as quantified by digital droplet PCR. Transduction assays and characterization revealed that they were functional and produced recombinant Env glycoproteins, with the exception of week 4, with optimal expression between 6-8 days posttransduction. Overall, these results provide the first evidence that HIV-1 Env can be successfully cloned into rAAV, and the associated recombinant proteins can be expressed in HEK293 T cells. Moreover, we provide reagents for future prime-boost immunizations using a panel of sequential Env expressing AAVs with matched recombinant proteins for testing in small animal models.en_ZA
dc.description.librarianMT 2019en_ZA
dc.identifier.urihttps://hdl.handle.net/10539/27024
dc.language.isoenen_ZA
dc.subjectAdeno-Associated Virus
dc.subject.meshHIV-1
dc.subject.meshAntibodies, Neutralizing
dc.titleSequential adeno-associated virus deliverd HIV-1 subtype C ENV glycoproteins as novel vaccine immunogensen_ZA
dc.typeThesisen_ZA
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