CHIP-Seq analysis of CREB1 binding after ALVAC vaccination in Human PBMC

The RV144 HIV vaccine trial remains the only study to demonstrate significant protection from future HIV-1 acquisition. One of the key components of the RV144 vaccine was the use of the canarypox vector ALVAC as the priming component. Since AIDSVAX, the booster component, alone failed to provide protection we hypothesized that the ALVAC prime contributed significantly to the generation of protection. To test this, we designed a NHP immunogenicity trial to mechanistically link ALVAC vaccination with the magnitude of V1V2 titers, the most significant immune correlate of reduced HIV-1 acquisition in RV144. Our objective was to use a systems biology approach to identify the transcription factors, target genes and immune pathways which were being induced by ALVAC vaccination and associated with higher V1V2 titers. We identified the transcription factor CREB1 and its target genes as rapidly induced by ALVAC in multiple immune subsets and that CREB1 drives the expression and activation of a network of other TFs which are critical for modulating immune responses. Pathways induced by this ALVAC-CREB1 axis include lymphocyte/leukocyte migration, lymphocyte differentiation, antigen processing and presentation, T cell co-stimulation and cytokine signaling. Overall design: PBMCs were infected in vitro with an MOI 10:1 of ALVAC-HIV, with media cultured cells as a control. ALVAC-infected PBMCs were compared with media condition at 24 hours after CREB1 chromatin immunoprecipitation.

RV144 HIV疫苗试验是目前唯一被证实可显著降低HIV-1新发感染风险的临床研究。RV144疫苗的关键组分之一为金丝雀痘病毒载体ALVAC作为初免组分。鉴于加强组分AIDSVAX单独使用时无法提供保护，我们推测ALVAC初免在保护效力的产生中发挥了关键作用。为验证这一假说，我们设计了一项非人灵长类动物（Non-human Primate, NHP）免疫原性试验，以从机制层面关联ALVAC疫苗接种与V1V2抗体滴度水平——该指标是RV144试验中与HIV-1感染风险降低关联最显著的免疫相关性标志物。本研究的目标是通过系统生物学方法，识别ALVAC疫苗接种所诱导、且与更高V1V2滴度相关的转录因子、靶基因及免疫通路。我们发现转录因子CREB1及其靶基因可在多种免疫细胞亚群中被ALVAC快速诱导，且CREB1可驱动一系列其他关键转录因子的表达与激活，而这些转录因子对免疫应答的调控至关重要。该ALVAC-CREB1信号轴所诱导的通路包括淋巴细胞/白细胞迁移、淋巴细胞分化、抗原加工与呈递、T细胞共刺激以及细胞因子信号传导。整体实验设计：以感染复数（Multiplicity of Infection, MOI）10:1的ALVAC-HIV体外感染外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs），以仅添加培养基的细胞作为空白对照。在感染后24小时进行CREB1染色质免疫沉淀实验，随后对比ALVAC感染组与培养基对照组的相关样本特征。