Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA.
J Virol. 2023 Mar 30;97(3):e0185722. doi: 10.1128/jvi.01857-22. Epub 2023 Feb 23.
Human immunodeficiency virus (HIV-1) entry into cells involves triggering of the viral envelope glycoprotein (Env) trimer ([gp120/gp41]) by the primary receptor, CD4, and coreceptors, CCR5 or CXCR4. The pretriggered (State-1) conformation of the mature (cleaved) Env is targeted by broadly neutralizing antibodies (bNAbs), which are inefficiently elicited compared with poorly neutralizing antibodies (pNAbs). Here, we characterize variants of the moderately triggerable HIV-1 Env on virions produced by an infectious molecular proviral clone; such virions contain more cleaved Env than pseudotyped viruses. We identified three types of cleaved wild-type AD8 Env trimers on virions: (i) State-1-like trimers preferentially recognized by bNAbs and exhibiting strong subunit association; (ii) trimers recognized by pNAbs directed against the gp120 coreceptor-binding region and exhibiting weak, detergent-sensitive subunit association; and (iii) a minor gp41-only population. The first Env population was enriched and the other Env populations reduced by introducing State-1-stabilizing changes in the AD8 Env or by treatment of the virions with crosslinker or the State-1-preferring entry inhibitor, BMS-806. These stabilized AD8 Envs were also more resistant to gp120 shedding induced by a CD4-mimetic compound or by incubation on ice. Conversely, a State-1-destabilized, CD4-independent AD8 Env variant exhibited weaker bNAb recognition and stronger pNAb recognition. Similar relationships between Env triggerability and antigenicity/shedding propensity on virions were observed for other HIV-1 strains. State-1 Envs on virions can be significantly enriched by minimizing the adventitious incorporation of uncleaved Env; stabilizing the pretriggered conformation by Env modification, crosslinking or BMS-806 treatment; strengthening Env subunit interactions; and using CD4-negative producer cells. Efforts to develop an effective HIV-1 vaccine have been frustrated by the inability to elicit broad neutralizing antibodies that recognize multiple virus strains. Such antibodies can bind a particular shape of the HIV-1 envelope glycoprotein trimer, as it exists on a viral membrane but before engaging receptors on the host cell. Here, we establish simple yet powerful assays to characterize the envelope glycoproteins in a natural context on virus particles. We find that, depending on the HIV-1 strain, some envelope glycoproteins change shape and fall apart, creating decoys that can potentially divert the host immune response. We identify requirements to keep the relevant envelope glycoprotein target for broad neutralizing antibodies intact on virus-like particles. These studies suggest strategies that should facilitate efforts to produce and use virus-like particles as vaccine immunogens.
人类免疫缺陷病毒 (HIV-1) 进入细胞涉及到主要受体 CD4 和辅助受体 CCR5 或 CXCR4 触发病毒包膜糖蛋白 (Env) 三聚体 ([gp120/gp41])。成熟 (切割) 的 Env 的预触发 (State-1) 构象被广泛中和抗体 (bNAb) 靶向，与低效中和抗体 (pNAb) 相比，bNAb 的诱导效率较低。在这里，我们对来自感染性分子前病毒克隆产生的病毒粒子上的中度可触发 HIV-1 Env 变体进行了表征；这样的病毒粒子比假型病毒含有更多切割的 Env。我们在病毒粒子上鉴定了三种类型的切割野生型 AD8 Env 三聚体：(i) 优先被 bNAb 识别且表现出强烈的亚基结合的 State-1 样三聚体；(ii) 被针对 gp120 辅助受体结合区的 pNAb 识别且表现出弱、去污剂敏感的亚基结合的三聚体；和 (iii) 少数 gp41 仅存在的群体。通过在 AD8 Env 中引入稳定 State-1 的改变或通过用交联剂或 State-1 偏好的进入抑制剂 BMS-806 处理病毒粒子，第一类 Env 群体被富集，而其他 Env 群体减少。这些稳定的 AD8 Envs 也更能抵抗由 CD4 模拟物诱导的 gp120 脱落或在冰上孵育。相反，State-1 失稳、不依赖 CD4 的 AD8 Env 变体表现出较弱的 bNAb 识别和较强的 pNAb 识别。对于其他 HIV-1 株，在病毒粒子上观察到 Env 触发能力与抗原性/脱落倾向之间存在类似的关系。通过最小化未切割 Env 的偶然掺入，可以显著富集 State-1 Env；通过 Env 修饰、交联或 BMS-806 处理稳定预触发构象；增强 Env 亚基相互作用；并使用 CD4 阴性的生产细胞。开发有效的 HIV-1 疫苗的努力受到无法诱导识别多种病毒株的广泛中和抗体的挫败。这些抗体可以结合 HIV-1 包膜糖蛋白三聚体的特定形状，因为它存在于病毒膜上，但在与宿主细胞上的受体结合之前。在这里，我们建立了简单但强大的测定法来在天然环境中对病毒粒子上的包膜糖蛋白进行特征描述。我们发现，根据 HIV-1 株的不同，一些包膜糖蛋白会改变形状并解体，形成可能会转移宿主免疫反应的诱饵。我们确定了保持广谱中和抗体在病毒样颗粒上针对相关包膜糖蛋白靶标的要求。这些研究提出了一些策略，应该有助于生产和使用病毒样颗粒作为疫苗免疫原。
