Rational design of a safe recombinant Candid#1 vaccine

阿雷纳病毒在啮齿动物种群中是地方性的，可以传播给人类导致严重的生命- 危险的出血热。美国相关机构(国土安全部、国土安全部和国防部)和世界卫生组织承认这些 病毒被列为对公众健康和国家安全构成严重威胁的高度优先的病原体。现场直播- Junín病毒1号减毒株(JUNV)目前在阿根廷用于预防阿根廷人 出血热，但这种病毒具有明显的易感性，即衰减仅依赖于单一的 JUNV的GP2融合亚基中第427(F427I)位的苯丙氨酸到异亮氨酸的取代 包膜糖蛋白(GPC)。事实上，在细胞培养中和在细胞培养中，很容易发生衰减位置的逆转。 实验动物。目前的建议旨在利用我们对GPC结构和 设计稳定保持衰减而不损害的重组坦诚1号(Rcan)病毒的功能 保护功效。我们发现GP2中减弱的F427I突变之间存在上位性相互作用 和GPC稳定信号肽(SSP)亚单位第33位(K33S)的赖氨酸到丝氨酸突变，该突变 提供了一种进化障碍，防止回复到病原体F427。初步研究表明，K33S Rcan确实在豚鼠身上减弱，并能够诱导对致命性攻击的保护性免疫 和JUNV一起。我们进一步假设，K33S rcan疫苗的安全性可以通过以下方式额外增强 整合了具有良好特性和遗传稳定性的GPC缺失。通过将rcan病毒的特征描述为 为了体现这些策略，我们的目标是提高第二代rcan疫苗的安全性。为了实现这一目标， 我们将追求以下具体目标：目标1.确定衰减程度和遗传稳定性 K33S rcan在小鼠体内的表达。我们将利用成熟的小鼠模型来确定 Rcan变异体，并确认减弱的F427I突变的遗传稳定性。目标2.评估平衡 K33S rcan对致死性豚鼠模型的减毒作用、免疫原性和保护性免疫的研究 JUNV感染。豚鼠是评估坦率第一衰减的黄金标准模型 保护功效。我们将对我们的试验结果进行扩展，以优化衰减和 保护功效。我们将评估病毒中和抗体的生产，这是公认的病毒中和抗体的替代品 保护，以及疫苗诱导病毒特异性CD8+T细胞的能力。目标3.设计和表征 Rcan变异体携带多余的和遗传稳定的突变，促进衰减。我们有 确定了GPC中支持rcan传染性的两个缺失。我们将描述带有这些的rcan变体的特征 删除以整合其他衰减层。总而言之，我们的努力将建立一个最佳的 在第二代rcan疫苗中的衰减、遗传稳定性和有效性之间的平衡，并阐明 衰减的分子基础和保护的免疫学相关性。这些策略也可能是 适用于开发急需的拉萨病毒减毒活疫苗。

Arenaviruses are endemic in rodent populations and can be transmitted to humans to cause severe life- threatening hemorrhagic fevers. Relevant US agencies (DHHS, DHS and DoD) and the WHO recognize these viruses as high priority pathogens that pose a serious threat to public health and national security. The live- attenuated Candid#1 strain of Junín virus (JUNV) is currently used in Argentina to protect against Argentine hemorrhagic fever, but this virus carries the distinct liability that attenuation is solely dependent on a single phenylalanine-to-isoleucine substitution at position 427 (F427I) in the GP2 fusion subunit of the JUNV envelope glycoprotein (GPC). Indeed, reversion at the attenuating position occurs readily in cell culture and in laboratory animals. The current proposal seeks to capitalize on our understanding of GPC structure and function to design recombinant Candid#1 (rCan) viruses that stably maintain attenuation without compromising protective efficacy. We have discovered an epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 (K33S) in the stable signal peptide (SSP) subunit of GPC that provides an evolutionary barrier against reversion to the pathognomonic F427. Pilot studies indicate that K33S rCan is indeed attenuated in guinea pigs and capable of eliciting protective immunity against lethal challenge with JUNV. We hypothesize further that safety in a K33S rCan vaccine can be additionally enhanced by incorporating well-characterized and genetically stable GPC deletions. By characterizing rCan viruses that embody these strategies, we aim to enhance safety in a second-generation rCan vaccine. Towards this goal, we will pursue the following specific aims: Aim 1. Determine the degree of attenuation and genetic stability of K33S rCan in mice. We will utilize well-established mouse models to determine the degree of attenuation in rCan variants and confirm the genetic stability of the attenuating F427I mutation. Aim 2. Assess the balance of attenuation, immunogenicity and protective immunity of K33S rCan in the guinea pig model of lethal JUNV infection. Guinea pigs serve as the gold-standard model for assessing Candid#1 attenuation and protective efficacy. We will expand upon our pilot findings to optimize the balance between attenuation and protective efficacy. We will evaluate production of virus-neutralizing antibodies, an accepted surrogate of protection, and the ability of the vaccine to elicit virus-specific CD8+ T cells. Aim 3. Design and characterize rCan variants bearing redundant and genetically stable mutations that promote attenuation. We have identified two deletions in GPC that support rCan infectivity. We will characterize rCan variants bearing these deletions to integrate additional layers of attenuation. Taken together, our efforts will establish an optimal balance of attenuation, genetic stability and efficacy in a second-generation rCan vaccine, and elucidate the molecular basis for attenuation and the immunologic correlates of protection. These strategies may also be applicable towards the development of an urgently needed live-attenuated Lassa virus vaccine.