Investigating the role of HIV broadly neutralizing antibodies on co-receptor expansion.

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HIV-1 Envelope (Env) is the sole target for neutralizing antibodies and therefore the primary candidate for vaccine development. HIV-1 rapidly mutates in response to the host immune system leading to a high-diversity in Env sequence which represents a major challenge for vaccine development. However, 10-30% of HIV-1 infected patients after 2-3 years of infection will develop broadly neutralizing antibodies (bnAbs) that are capable of neutralizing a wide number of HIV-1 strains offering some promise that an HIV-1 vaccine with broad protection might be achieved. Isolation of bnAbs from chronically infected patients has allowed identification and characterisation of the neutralization epitopes on HIV-1 Env. This knowledge has been applied to rationale design of HIV-1 vaccines. More recently, there has been an increasing interest in using HIV-1 bnAbs as an alternative treatment of HIV-1 infected patients and therefore a better understanding of the selective pressures afforded by bnAbs on Env is needed. HIV-1 requires interaction with the CD4 receptor and either co-receptor CCR5 (R5) or CXCR4 (R4). Most transmitted viruses are R5-tropic but over the course of infection X4 viruses can arise which coincide with a decline in CD4+ T cell levels and progression to AIDS. The driving force of this change is not well understood but mutations in the V3 loop are thought to facilitate co-receptor expansion. My lab has previously identified an HIV-1 infected donor (donor SUT) that developed an bnAb response targeted at the N332/V3 epitope and where the circulating viruses have undergone a co-receptor expansion such that at later timepoints R5 and X4 viruses are co-circulating. Analysis of Env sequences reveals that mutations in the V3 loop that are predicted to be important for co-receptor specificity are overlapping with the epitope of the bnAb response. Therefore, this donor offers a unique opportunity to study the co-evolution of HIV-1 bnAbs and viral escape in the context of co-receptor expansion. Aim of the investigation (up to 150 words) We hypothesis that the host adaptive immune response acts as a selective pressure driving HIV-1 co-receptor switching via mutation to V1/V3 regions in the SUT donor. Using clinical samples from HIV-1 infected donors this project aims are: 1: To determine coreceptor usage in relation to V1/V3 regions in HIV-1 Envs isolated from SUT donor. 2: To isolation of mAbs from SUT donor across multiple stages of infection. 3: To characterise isolated mAbs, including HIV-1 neutralisation of R5 and X4 viruses and epitope binding. 4: To investigate the selective pressure of SUT mAbs on co-receptor expansion. Proposed plan of work (up to 1000 words) To address the project aims we will conduct the following experiments: 1: To determine coreceptor usage in relation to V1/V3 regions in HIV-1 Envs isolated from SUT donor. We will identify Env mutations that facilitate co-receptor expansion in the SUT donor. This will involve site-directed mutagenesis of SUT Envs and measurement of cell entry in R5 or X4 expressing cells. To gain further in depth knowledge on the pathways leading to co-receptor expansion, we will also conduct next generation sequencing on the V1-V3 loops of Env at time points prior to co-receptor expansion. 2: To isolation of mAbs from SUT donor across multiple stages of infection. We will use antigen-specific B cell sorting to isolate Env specific monoclonal antibodies from different stages of infection. These techniques have been extensively used in my lab. We will analyze gene usage, somatic hypermutation and CDRH3/L3 lengths. Antigen baits will include gp120 proteins from autologous viruses and heterologous viruses to allow a full assessment of the antibody repertoire in the SUT donor. 3: To characterise isolated mAbs, including HIV-1 neutralisation of R5 and X4 viruses and epitope binding.