Knock-in mice expressing germline-reverted broadly neutralizing HIV antibodies

这是一个更新R01应用程序，用于开发敲入小鼠模型和超突变人类B细胞系 携带针对HIV Env的生殖系回复的广泛中和抗体（gl-bNAbs），目的是加速 HIV疫苗研究。开发艾滋病毒疫苗是一个主要的全球卫生目标，因为没有有效的疫苗 仍然存在针对HIV Env糖蛋白的bnAb可能可以控制感染，但通过疫苗接种引发bnAb是不可能的。 具有挑战性，因为Env遗传变异性和对保守Env表位的应答是 弱. bnAb在一些患者中自然产生，但通常只有在感染多年后，当它们提供 没有真实的保护。然而，许多单独的bnAb可以在动物模型中提供被动保护， 这表明这些抗体的诱导可能是保护性的，特别是如果针对几种保守的 网站可以同时发布。为了开始研究通过疫苗接种诱导bnAb的最佳方法，我们 先前已经产生了B细胞系和携带针对VRC01，PGT 121， b12、4E10和其他特异性。gl-bnAb小鼠模型对于抗原设计和疫苗接种是有价值的 因为它们携带B细胞，在适当刺激后有可能成为bnAb， 体细胞突变将V（D）J基因置于生理性IG基因座中允许敲入的B细胞 进行正常的H链类别转换和V区超突变。此外，基因敲入小鼠允许 在一个方便的，相对低成本的小鼠模型中进行疫苗接种研究。我们可以从这些老鼠身上接种B细胞 在生理上低的数量，否则野生型小鼠，并评估他们的反应，在面对竞争 来自非bnAb克隆。在这些细胞系和小鼠中，我们一直在进行研究，以评估是否 估算的gl-bnAb正常发育并评估各种候选疫苗和免疫 战略布局我们已经测试了所谓的生殖细胞靶向免疫原，这些免疫原被设计成更好地结合gl-bnAb 比来自WT Env.在本提案中，我们关注疫苗设计的两个方面： 使用经验证而非推断的gl-bnAb前体，并评估选择性的容易程度 使用体细胞突变的人gl-bnAb细胞系和gl-bnAb小鼠的有益突变。我们 假设通过采用这些方法，我们将鉴定最有效成熟优选gl-bnAb 成为bnAb和优选的免疫原或选择途径，其应最有效地促进bnAb 在灵长类动物中测试时的产量。由于前体对抗原亲和力差， 通过体细胞突变使亲和力成熟的途径是对所有免疫应答的限制， 这里应该适用于各种疫苗靶点。

This is a renewal R01 application to exploit knock-in mouse models and hypermutating human B cell lines carrying germline-reverted broadly neutralizing antibodies (gl-bNAbs) against HIV Env with the goal to speed HIV vaccine research. Developing an HIV vaccine is a major global health objective as no effective vaccine yet exists. bnAbs to HIV Env glycoprotein can likely control infection but eliciting bnAbs by vaccination is challenging because of Env genetic variability and the fact that responses to conserved Env epitopes are weak. bnAbs are made naturally in some patients, but usually only after years of infection, when they provide no real protection. However, many individual bnAbs can provide passive protection in animal models, suggesting that elicitation of these antibodies could be protective, particularly if bnAbs to several conserved sites can be raised simultaneously. To begin to investigate the best ways to elicit bnAbs by vaccination, we have previously generated B cell lines and knock-in mice carrying inferred gl-bnAbs for VRC01, PGT121, b12, 4E10 and other specificities. gl-bnAb mouse models are valuable for antigen design and vaccination studies because they carry B cells with the potential to become bnAbs after appropriate stimulation and somatic mutation. Placement of the V(D)J genes into the physiological Ig loci allows the knock-in B cells to undergo normal H-chain class switching and V-region hypermutation. Moreover, knock-in mice allow vaccination studies in a convenient, relatively low-cost mouse model. One can seed B cells from these mice at physiologically low numbers in otherwise WT mice and assess their responses in the face of competition from non bnAb clones. In these cell lines and mice, we have been carrying out studies to evaluate whether the imputed gl-bnAbs develop normally and to assess various vaccine candidates and immunization strategies. We have tested so-called germline-targeting immunogens engineered to bind better to gl-bnAbs than immunogens derived from WT Env. In the present proposal we focus on two aspects of vaccine design: the use of validated, rather than inferred, gl-bnAb precursors and an assessment of the ease of selectability of beneficial mutations using somatically-mutating human gl-bnAb cell lines and gl-bnAb mice. We hypothesize that by taking these approaches we will identify preferred gl-bnAbs that most effectively mature to become bnAbs and preferred immunogens or selection paths that should most effectively promote bnAb production when tested in primates. Because the problem of poor precursor affinity to antigen and uncertain paths to affinity maturation by somatic mutation are limitations to all immune responses, knowledge obtained here should be applicable to a variety of vaccine targets.