More than 30 years has passed since the discovery of Human Immunodeficiency Virus (HIV) yet it remains one of the most important current threats to global public health. HIV is a T-lymphotrophic retrovirus that is the causative agent of Acquired Immune Deficiency Syndrome, and despite decades of research, there remains no cure. Vaccines are most effective when they are able to induce broadly neutralizing antibodies at concentrations capable of blocking viral infection. Notwithstanding all of the effort, a successful vaccine that is capable of inducing complete protection from the immune system has yet to be found. In this thesis, the first chapter provides a history of the discovery of HIV, the origins of the virus, description of the HIV genome, focusing primarily on the envelope glycoprotein, a trimeric spike on the surface of the HIV virion necessary for viral fusion and the sole epitope for broadly neutralizing antibodies. Lastly, the first chapter reviews an overview of the antiviral immune response specifically the role of humoral immune branch and broadly neutralizing antibodies, as well as their limitations in protection against HIV. Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. In addition to structural features of HIV\u2019s envelope spike that facilitate antibody evasion, we proposed that the low-density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The work described in subsequent chapters attempts to overcome HIV\u2019s evasion strategy of low spike density through the design of novel antibody architectures.
\r\n\t\r\nWe postulated that anti-HIV-1 spike antibodies primarily bind monovalently because HIV\u2019s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike trimer structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and neutralization potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a \u201cmolecular ruler\u201d to measure intra-epitope distances on virion-bound spikes and to construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude of increased potency (>100-fold average increases across a virus panel) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection. These results shed light on dynamic spike conformations and are relevant to therapeutic interventions.
\r\n", "doi": "10.7907/Z9QC01FR", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8504", "collection": "thesis", "collection_id": "8504", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072014-140700155", "type": "thesis", "title": "Biophysics of V(D)J Recombination and Genome Packaging: In Singulo Studies on RAG, HMGB1, and TFAM", "author": [ { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-Geoffrey-A" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.\r\n", "doi": "10.7907/Z9W9573H", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" } ]