Ahmed Tina, Borthwick Nicola J, Gilmour Jill, Hayes Peter, Dorrell Lucy, Hanke Tomáš
The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.
Human Immunology Laboratory, International AIDS Vaccine Initiative, London SW10 9NH, United Kingdom; Faculty of Medicine, Imperial College, London SW7 2AZ, United Kingdom.
Vaccine. 2016 Feb 24;34(9):1215-24. doi: 10.1016/j.vaccine.2015.12.021. Epub 2016 Jan 16.
The specificity of CD8(+) T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8(+) effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4(+) cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication.
CD8(+) T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates.
Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells.
We formally demonstrated that the vaccine-elicited inhibitory human CD8(+) T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells.
These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of conserved immunogens.
CD8(+) T细胞的特异性对于早期控制初始/传播及再激活的HIV-1至关重要。为应对HIV-1的变异性和逃逸，我们设计了由主要Gag和Pol蛋白的14个高度保守区域组装而成的疫苗免疫原HIVconsv。在HIV-CORE 002试验中给HIV-1阴性人类志愿者接种时，HIVconsv疫苗引发了CD8(+)效应T细胞，这些细胞可抑制多达8种HIV-1分离株在自体CD4(+)细胞中的复制。这种抑制作用与针对Gag和Pol肽库产生的干扰素-γ相关，但缺乏抑制特异性的直接证据。在此，我们旨在通过识别这些效应细胞抑制HIV-1复制的表位来进行明确。
从23名疫苗接种者中选出3名对HIV-1抑制作用最广泛的个体，其CD8(+) T细胞通过Gag或Pol肽再刺激在培养中进行扩增，并使用HIV-1 B亚型和A亚型分离株在病毒抑制试验（VIA）中进行测试。
首先使用来自Gag或Pol保守区域的肽库扩增冷冻的外周血单核细胞（PBMC），并在VIA中对HIV-1感染细胞进行测试，或通过单个肽测试其效应功能。然后通过在HIV-1感染细胞上测试单个肽扩增的效应细胞来确认负责抑制HIV-1复制的单肽特异性。
我们正式证明了疫苗引发的抑制性人类CD8(+) T细胞识别Pol和Gag蛋白的保守表位。我们确定了7个最小表位，其中3个是新的，可能是天然亚显性的。效应细胞具有多效性，可产生多种细胞因子和趋化因子，并杀伤肽脉冲靶细胞。
这些结果表明，除了广泛使用的Gag外，在T细胞疫苗设计中还可使用Pol的功能保守区域。产生这些效应细胞的志愿者比例及其在循环PBMC中的频率是不同的问题，如果需要，可通过更有效的载体和保守免疫原给药方案来解决。
