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

HIV-1包膜(Env)是中和抗体的唯一靶点，因此是疫苗开发的主要候选者。HIV-1在宿主免疫系统的作用下发生快速变异，导致env序列高度多样性，这对疫苗开发构成了重大挑战。然而，10-30%的HIV-1感染者在感染2-3年后会产生广谱中和抗体(BNAbs)，这些抗体能够中和大量的HIV-1毒株，这为实现具有广泛保护作用的HIV-1疫苗提供了一些希望。从慢性感染患者中分离出bNAbs，可以鉴定和鉴定HIV-1env的中和表位。这一知识已被应用于HIV-1疫苗的基本设计。最近，人们对使用HIV-1bNAbs作为HIV-1感染者的替代治疗越来越感兴趣，因此需要更好地了解bNAbs对环境的选择压力。HIV-1需要与CD4受体以及共同受体CCR5(R5)或CXCR4(R4)相互作用。大多数传播的病毒是R5嗜性病毒，但在感染过程中可能会出现X4病毒，这与CD4+T细胞水平下降和发展为艾滋病不谋而合。这一变化的驱动力尚不清楚，但V3环中的突变被认为促进了辅助受体的扩张。我的实验室之前已经确认了一名HIV-1感染捐赠者(捐赠者SUT)，他产生了针对N332/V3表位的bNab反应，并且循环中的病毒经历了共同受体的扩张，从而在稍后的时间点R5和X4病毒共同循环。对env序列的分析表明，V3环中被预测为对协同受体特异性重要的突变与bNab反应的表位重叠。因此，这位捐赠者提供了一个独特的机会来研究HIV-1bNAbs的共同进化和在共同受体扩大的背景下的病毒逃逸。研究目的(150字)我们假设宿主获得性免疫反应作为一种选择性压力，通过突变将HIV-1辅受体切换到SUT供体的V1/V3区。利用HIV-1感染供者的临床样本，本项目的目的是：1：确定从SUT供者分离的HIV-1 env中与V1/V3区域相关的辅助受体的使用。2.从不同感染阶段的SUT供者中分离单抗。3：鉴定分离的单抗，包括HIV-1对R5和X4病毒的中和作用和表位结合。4.研究SUT单抗对辅受体扩增的选择性压力。拟议的工作计划(最多1000字)为解决该项目的目标，我们将进行以下实验：1：确定从SUT供体分离的HIV-1环境中与V1/V3区域相关的辅助受体使用情况。我们将确定在SUT供体中促进共同受体扩展的Env突变。这将涉及SUT环境的定点突变和R5或X4表达细胞中细胞进入的测量。为了进一步深入了解导致辅助受体扩展的途径，我们还将在辅助受体扩展之前的时间点对Env的V1-V3环进行下一代测序。2.从不同感染阶段的SUT供者中分离单抗。我们将使用抗原特异性B细胞分选法从感染的不同阶段分离出Env特异性单抗。这些技术在我的实验室中得到了广泛的应用。我们将分析基因使用、体细胞超突变和CDRH3/L3长度。抗原诱饵将包括来自自体病毒和异源病毒的gp120蛋白，以允许对SUT供体的抗体谱系进行全面评估。3：鉴定分离的单抗，包括HIV-1对R5和X4病毒的中和作用和表位结合。

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.