Microbiology & Immunology - Theses
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ItemThe antibodies involved in the antibody dependent cellular cytotoxicity immune response in HIV( 2012)Background: Antibodies that mediate killing of Human Immunodeficiency Virus (HIV) infected cells by NK cells could be a very useful component of a successful HIV vaccine strategy. Antibody dependent cellular cytotoxicity (ADCC) targets HIV-infected cells by utilising the Fc region of specific antibodies bound to NK cells. However, the role of ADCC responses in preventing HIV-1 remains controversial. Previous studies of ADCC activity have been hampered by difficulties identifying and mapping these responses. An intracellular cytokine staining (ICS) technique developed in our laboratory allows us to detect and map HIV-specific ADCC via detecting activation of NK-cells by ADCC on small volumes of blood. There is an imperative to study the antibodies involved in inducing a HIV-specific ADCC response and use this knowledge to develop effective HIV vaccines. Methods: ADCC responses to overlapping HIV-1 consensus peptide pools were analysed using an ICS assay measuring NK cell activation in 83 anti-retroviral therapy (ART)-naïve HIV-infected subjects followed prospectively for 3 years. We mapped 32 responses to individual consensus HIV subtype B 15mer peptides within the Pol protein. The ADCC-assay was also used to analyse autologous virus-derived peptide epitope responses and compared to consensus derived peptides sequence, across a titration of peptide concentrations, to study escape from ADCC immune response. Isolation of the antibodies responsible for Env and Vpu-specific ADCC responses were purified using affinity chromatography and assessed for their viral inhibition activity, purity and neutralisation activity. We also evaluated bulk antibody production methods using EBV transformation of B cells, Mass Spectrometry of the purified ADCC antibodies and the use of fluorochrome bound peptides to identify and select the ADCC-specific B cells. Results: From the 83 subjects 32 Pol-specific responses were identified. Of these 12 have been mapped to regions of Pol and 2 subjects were identified with ADCC-specific responses to 2 different highly conserved Pol epitopes. Fifty-four subjects recognised Env peptides. From 11 mapped Env responses studied, 6 showed a loss of recognition of autologous virus-derived peptides. This suggested a potential pressure ADCC responses apply on HIV. Purification of Env and Vpu-specific ADCC antibodies proved that ADCC inducing antibodies inhibited viral replication in the Antibody-Dependent Cellular Viral Inhibition (ADCVI) assay and they did not involve neutralising activity. Experiments to determine the most optimal ADCC-specific B cell isolation technique indicated that the use of dual fluorochrome labelled peptides offered the most promising results. Conclusions: Targeting ADCC to more conserved proteins, such as Pol and Vpu proteins may be a more effective ADCC-based vaccine approach. Escape from Env-specific ADCC may, unless broad or directed to rare conserved regions within Env, limit the utility of Env-specific ADCC in controlling or preventing HIV. Development of methods to isolate and generate large amounts of ADCC-specific antibodies will assist in defining the utility of HIV-specific ADCC responses and their role in limiting S/HIV infection. The most effective ADCC epitopes can then be engineered into novel HIV vaccines.