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This dissertation, Exploring the Human Genomic DNA Antibody Repertoire for Development of an Effective HIV-1 Vaccine by Yu, Zhang, 張宇, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: HIV-1 broadly neutralizing antibodies (bnAbs) with great potency and breadth have been identified from approximately 10 to 30% HIV-1 infected individuals with protection against viral infections in vitro and in vivo. However, no vaccine candidates have induced antibodies comparable to known HIV-1 bnAbs in neutralization so far. Besides the envelope plasticity, glycan shield and other tricky viral strategies, lack of detectable binding of bnAbs' predicted germline antibodies to HIV-1 envelope proteins (Env)could account for the failure. After endeavoring to obtain engineered Env proteins capable of triggering the initiation of B cell receptor (BCR) maturation of some known germline bnAbs, it has not been successful to mature them in vitro. It is unknown whether these putative germline antibodies or some unknown germline antibodies could mature to bnAbs. It is also unknown the frequency of these rearranged germline bnAbs in human antibody gene repertoire and the mechanisms of antibody somatic maturation. This study focuses on the exploration of genomic DNA antibody repertoires to help answer these questions, which may ultimately helpHIV-1 vaccine development. The unusual characteristics of the known HIV-1 bnAbs such as extremely high somatic mutation level, long CDR3s and polyreactivity which may halt their maturation process, the preference in lineage usageof currently known bnAbs, as well as the prevalent events of clonal deletion, anergy, and other negative regulations in immune system led us hypothesize that genomic DNA antibody repertoire, before and impervious to any transcriptional or translational regulation, may be more suitable than cDNA antibody repertoire for studying the development of bnAbs. The genomic DNA antibody repertoire may be more diverse than the cDNA antibody repertoire in antibody gene lineage utilization and may have more antibodies or germline antibodies with potential to mature to bnAbs. To investigate human genomic DNA antibody repertoire, large genomic DNA and cDNA antibody libraries from both healthy and HIV-1 infected individuals'peripheral blood mononuclear cells were constructed. 454 deep sequencing result showed that genomic DNA antibody libraries were more diverse than cDNA libraries in lineage usage, yet with extremely low frequencies of the exactly recombined germline genes of bnAbs which may be one of the reasons for the failure in eliciting bnAbs. However, there were relatively higher frequencies of antibodies using other germline gene recombinations with the potential to develop to bnAbs. These findings were further confirmed by the result from panning genomic DNA antibody libraries. A panel of germline and germline-like antibodies, that use diverse V(D)J recombinations have good binding affinity for various HIV-1 envelope proteins were isolated from the genomic DNA antibody libraries but not the corresponding cDNA libraries. They showed improved binding affinity and obtained neutralization ability following the very early steps of in vitro maturation, indicating their potential development to broadly neutralizing antibodies. These results indicate that exploring the genomic DNA antibody repertoire may facilitate the isolation of germline antibodies with potential to mature to bnAbs and the understanding of the mechanisms for the maturation of bnAbs in vivo, which may eventually help HIV-1 vaccine development.

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