Innate immune activity correlates with suppression of HIV replication and CD4+ T cell-associated HIV-1 DNA decline during treatment with pegylated IFN-α2a in chronic ART-suppressed HIV infection. Innate immune activity correlates with suppression of HIV replication and CD4+ T cell-associated HIV-1 DNA decline during treatment with pegylated IFN-α2a in chronic ART-suppressed HIV infection

In study NCT00594880, we successfully administered weekly doses of pegylated interferon-α-2a (Peg-IFN-α2a) with antiretroviral therapy (ART) for 5 weeks, before continued Peg-IFN-α2a monotherapy to 12 weeks (primary endpoint) in chronic HIV-infected subjects. Subjects maintaining HIV viral load <400 copies/ml by primary endpoint were defined as responders. We now describe innate immune correlates and transcriptional profiles associated with viral control and decrease in integrated HIV DNA after Peg-IFN-α2a immunotherapy. Peripheral blood samples were obtained prior to Peg-IFN-α2a administration (ART), after 5 weeks of ART+Peg-IFN-α2a dual treatment, and after 12 weeks of Peg-IFN-α2a monotherapy. Cell subset modulation, natural killer cell (NK) function and signaling, as well as inflammatory mediators and gene expression were assessed. Results were analyzed using R.2.5.1 or MATLAB 7.10.0. Five weeks of ART+Peg-IFN-α2a preserved the frequency of immune subsets and NK cytotoxicity, while increased the levels of inflammatory mediators, and decreased cell-responsiveness to in vitro IFN-α re-stimulation. Gene expression analysis showed that induction of host restriction factors after ART+Peg-IFN-α2a was not solely predictive of a virologic response, and revealed a 108 gene signature that identified subjects who did not modulate genes after ART+Peg-IFN-α2a. A 29 gene signature along with higher NK cell activation and IFN-γ-induced protein 10 (IP-10) levels on ART, as well as higher in vitro responses to IFN-γ-induced NK cytotoxicity, and decrease in the frequency of NK bearing inhibitory receptors [i.e. killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail 1 (KIR2DL1) or KIR2DL2/DL3] and C-C chemokine receptor type 7+ (CCR7) myeloid dendritic cells after ART+Peg-IFN-α2a distinguished responders from non-responders. Reductions in integrated HIV DNA after immunotherapy were associated with expression patterns of genes that are associated with activated cell-mediated response and NK cytotoxicity. In summary, innate activation and NK cell cytotoxicity are identified as correlates of HIV control and reduction after Peg-IFN-α2a immunotherapy in ART-suppressed subjects. Overall design: Expression from cryo-preserved PBMC for patients on pegylated interferon-α-2a (Peg-IFN-α2a) with antiretroviral therapy (ART) treatment Please note that [1] the visit 3, 5, and 8 represents; Visit 3: prior to Peg-IFN-α2a administration (ART) Visit 5: after 5 weeks of ART+Peg-IFN-α2a dual treatment Visit 8: after 12 weeks of Peg-IFN-α2a monotherapy [2] the 'characteristics: response' values represent; RC: response based on gene expression to 5 weeks of ART+Peg-IFN-α2a treatment, controlled plasma viral load RN: response based on gene expression to 5 weeks of ART+Peg-IFN-α2a treatment, do not control plasma viral load NN: no response based on gene expression to 5 weeks of ART+Peg-IFN-α2a treatment, do not control plasma viral load

在临床试验NCT00594880中，我们对慢性HIV感染者给予每周一次的聚乙二醇化干扰素α-2a（pegylated interferon-α-2a, Peg-IFN-α2a）联合抗反转录病毒治疗（antiretroviral therapy, ART），持续5周，随后继续给予Peg-IFN-α2a单药治疗至12周（主要终点）。将主要终点时HIV病毒载量<400拷贝/mL的受试者定义为应答者。本研究旨在描述与Peg-IFN-α2a免疫治疗后病毒控制及整合型HIV DNA（integrated HIV DNA）降低相关的先天免疫相关性指标及转录组特征。

分别于Peg-IFN-α2a给药前（仅ART治疗阶段）、ART联合Peg-IFN-α2a双治疗5周后、以及Peg-IFN-α2a单药治疗12周后采集外周血样本。本研究评估了细胞亚群调控、自然杀伤细胞（natural killer cell, NK）功能与信号通路、炎症介质及基因表达情况，研究结果采用R 2.5.1及MATLAB 7.10.0进行分析。

分析结果显示：5周的ART联合Peg-IFN-α2a治疗可维持免疫细胞亚群比例及NK细胞杀伤活性，同时升高炎症介质水平，并降低细胞对体外干扰素α再刺激的应答能力。基因表达分析表明，ART联合Peg-IFN-α2a治疗后宿主限制因子（host restriction factors）的诱导并不能单独预测病毒学应答，且鉴定出一组包含108个基因的特征标签（gene signature），可区分出经ART联合Peg-IFN-α2a治疗后未发生基因表达调控的受试者。此外，一组包含29个基因的特征标签，结合ART阶段较高的NK细胞活化水平及干扰素γ诱导蛋白10（IFN-γ-induced protein 10, IP-10）水平、体外对干扰素γ诱导的NK细胞杀伤活性的更强应答，以及ART联合Peg-IFN-α2a治疗后携带抑制性受体[即杀伤细胞免疫球蛋白样受体2DL1（killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail 1, KIR2DL1）或KIR2DL2/DL3]的NK细胞比例降低、CC趋化因子受体7阳性（C-C chemokine receptor type 7+, CCR7+）髓系树突状细胞比例降低，可有效区分应答者与非应答者。免疫治疗后整合型HIV DNA的降低与活化细胞介导的免疫应答及NK细胞杀伤活性相关基因的表达模式显著相关。综上，在接受ART抑制治疗的受试者中，先天免疫活化及NK细胞杀伤活性可作为Peg-IFN-α2a免疫治疗后HIV病毒控制与载量降低的相关性指标。

总体实验设计：本研究对接受聚乙二醇化干扰素α-2a（Peg-IFN-α2a）联合抗反转录病毒治疗（ART）的患者的冻存外周血单个核细胞（cryo-preserved peripheral blood mononuclear cell, PBMC）进行基因表达分析。

请注意：
[1] 访视3、5及8分别代表：
访视3：Peg-IFN-α2a给药前（仅ART治疗阶段）
访视5：ART联合Peg-IFN-α2a双治疗5周后
访视8：Peg-IFN-α2a单药治疗12周后
[2] “特征：应答”的取值含义如下：
RC：经5周ART联合Peg-IFN-α2a治疗后基于基因表达判定为应答，且血浆病毒载量得到控制
RN：经5周ART联合Peg-IFN-α2a治疗后基于基因表达判定为应答，但血浆病毒载量未得到控制
NN：经5周ART联合Peg-IFN-α2a治疗后基于基因表达判定为无应答，且血浆病毒载量未得到控制