Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.
Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.
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工程化良好表达的V2免疫聚焦HIV-1包膜糖蛋白膜三聚体用于异源初免-加强疫苗方案
DOI：
10.1371/journal.ppat.1009807
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发表时间：
2021-10
期刊：
影响因子：
6.7
通讯作者：
Binley JM
中科院分区：
文献类型：
--
作者：
Crooks ET;Almanza F;D'Addabbo A;Duggan E;Zhang J;Wagh K;Mou H;Allen JD;Thomas A;Osawa K;Korber BT;Tsybovsky Y;Cale E;Nolan J;Crispin M;Verkoczy LK;Binley JM
HIV-1 疫苗免疫聚焦策略可能能够诱导广泛反应的中和抗体 (NAb)。在这里，我们设计了一组多样化的、膜驻留的天然 HIV-1 三聚体，易受两个广泛靶标的影响——V2 顶端和融合肽 (FP)。选择标准包括 i) 高表达和 ii) 感染功能，以便可以在假病毒 (PV) 测定中分析三聚体中和敏感性。最初，我们通过截短 gp41 并引入 gp120-gp41 SOS 二硫键以防止 gp120 脱落来增强 17 个候选三聚体的表达。进行“修复”是为了填充聚糖孔并消除其他菌株特异性畸变。当我们的标准测定不足时，新的中和测定允许 PV 感染。带有暴露的 V3 环的三聚体（非 NAb 的靶标）被丢弃。为了尝试增加 V2 敏感性，我们去除了冲突聚糖并修改了 C 链。值得注意的是，D167N 突变在一些情况下提高了 V2 敏感性。 JR-FL 三聚体的糖肽分析显示序列子几乎完全占据，并且填充 N197 聚糖孔的耐受性良好。相比之下，序列子优化和在其他位置插入/去除聚糖经常对整个 gp120 外部结构域和 gp41 的聚糖成熟和序列子占据产生全局“涟漪”效应。 V2 MAb CH01 选择性地与 V1 环基部附近具有小高甘露糖聚糖的三聚体结合，从而避免冲突。研究发现，敲入一种罕见的 N49 聚糖会扰乱 gp41 聚糖，从而增加 FP NAb 的敏感性，有时甚至会改善表达。最后，VLP 的生物物理分析表明，i) 约 25% 的颗粒带有 Env 尖峰，ii) 自发颗粒出芽很高，并且在 Gag 转染后仅增加 4 倍，iii) Env+ 颗粒表达约 30-40 个尖峰。总而言之，我们鉴定了 7 种不同的三聚体，它们对两个靶标具有一系列敏感性，以便能够严格测试免疫聚焦疫苗概念。尽管经过近 40 年的创新，诱导抗体阻止 HIV 感染的疫苗仍然难以捉摸。挑战包括艾滋病毒表面刺突无与伦比的序列多样性及其限制抗体进入的致密糖衣。越来越多来自艾滋病毒感染者的单克隆抗体提供了疫苗蓝图，但由于其不寻常的特征，很难通过疫苗接种来诱导。然而，两个目标，一个位于病毒尖峰顶端，另一个位于尖峰侧面，对不寻常抗体的“需求”更加宽容。在这里，我们在这两个位点制作了一组易受攻击的艾滋病病毒尖峰，用作疫苗，试图将抗体集中在这些目标上。我们对这些刺突的选择标准是：i) 当在颗粒上表达时，它们具有传染性，使我们能够使用由相同三聚体制成的颗粒来评估免疫原和疫苗血清，ii) 细胞很容易产生足以用于疫苗使用的数量的刺突。最终，我们选择了 7 个三聚体，这将使我们能够探索使我们更接近 HIV 疫苗的概念。
HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts. Despite almost 40 years of innovation, a vaccine to induce antibodies that block HIV infection remains elusive. Challenges include the unparalleled sequence diversity of HIV’s surface spikes and its dense sugar coat that limits antibody access. A growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints, but have been difficult to induce by vaccination, due to their unusual features. However, two targets, one at the viral spike apex and another at the side of the spikes are more forgiving in their ’demands’ for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites to be used as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that when expressed on particles, they are infectious, allowing us to evaluate immunogens and vaccine sera using particles made with the same trimers, ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.
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影响因子：
7
作者：
Andrabi R;Bhiman JN;Burton DR
通讯作者：
Burton DR
影响因子：
64.5
作者：
Briney, Bryan;Sok, Devin;Jardine, Joseph G.;Kulp, Daniel W.;Skog, Patrick;Menis, Sergey;Jacak, Ronald;Kalyuzhniy, Oleksandr;de Val, Natalia;Sesterhenn, Fabian;Le, Khoa M.;Ramos, Alejandra;Jones, Meaghan;Saye-Francisco, Karen L.;Blane, Tanya R.;Spencer, Skye;Georgeson, Erik;Hu, Xiaozhen;Ozorowski, Gabriel;Adachi, Yumiko;Kubitz, Michael;Sarkar, Anita;Wilson, Ian A.;Ward, Andrew B.;Nemazee, David;Burton, Dennis R.;Schief, William R.
通讯作者：
Schief, William R.
影响因子：
16.6
作者：
Cao L;Pauthner M;Andrabi R;Rantalainen K;Berndsen Z;Diedrich JK;Menis S;Sok D;Bastidas R;Park SR;Delahunty CM;He L;Guenaga J;Wyatt RT;Schief WR;Ward AB;Yates JR 3rd;Burton DR;Paulson JC
通讯作者：
Paulson JC
影响因子：
5.4
作者：
Chuang, Gwo-Yu;Lal, Yen-Ting;Kwong, Peter D.
通讯作者：
Kwong, Peter D.
影响因子：
5.4
作者：
Binley, JM;Cayanan, CS;Burton, DR
通讯作者：
Burton, DR