Rational design of a safe recombinant Candid#1 vaccine

沙粒病毒在啮齿动物种群中流行，并可传播给人类，导致严重的生命- 威胁出血性发热美国相关机构（卫生和公众服务部、国土安全部和国防部）和世卫组织认识到， 病毒作为高度优先的病原体，对公共卫生和国家安全构成严重威胁。活的- Junín病毒的减毒的RID#1株（JUNV）目前在阿根廷用于预防阿根廷 出血热，但这种病毒携带明显的责任，即减毒是完全依赖于一个单一的 在JUNV的GP 2融合亚基中的位置427处的苯丙氨酸至异亮氨酸取代（F427 I） 包膜糖蛋白（GPC）。事实上，在细胞培养物中和在细胞培养物中容易发生在减毒位置的逆转。 实验室动物目前的建议旨在利用我们对GPC结构的理解， 功能，以设计稳定维持减毒而不损害 保护功效我们已经发现GP 2中的减弱F427 I突变之间存在上位相互作用， 和GPC的稳定信号肽（SSP）亚基中第33位的赖氨酸-丝氨酸突变（K33 S）， 提供了一个进化障碍，以防止回复到特异性F427。初步研究表明，K33 S rCan在豚鼠中确实是减毒的，并且能够引起针对致死性攻击的保护性免疫 关于JUNV我们进一步假设，K33 S rCan疫苗的安全性可以通过以下方式进一步增强： 掺入了充分表征的和遗传稳定的GPC缺失。通过表征rCan病毒， 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， 我们会致力达致以下目标：目标1。确定衰减程度和遗传稳定性 K33 S rCan在小鼠中的表达。我们将利用完善的小鼠模型来确定在 rCan变体，并确认减毒F427 I突变的遗传稳定性。目标二。评估平衡 K33 S rCan在致死性豚鼠模型中的减毒、免疫原性和保护性免疫 JUNV感染。豚鼠作为评估PD#1衰减的金标准模型， 保护功效我们将扩大我们的试点研究结果，以优化衰减和 保护功效我们将评估病毒中和抗体的生产， 保护，以及疫苗引发病毒特异性CD 8 + T细胞的能力。目标3：设计和表征 携带促进减毒的冗余和遗传稳定突变的rCan变体。我们有 在GPC中发现了两个支持rCan感染性的缺失。我们将表征带有这些的rCan变体 删除以整合额外的衰减层。综合考虑，我们的努力将建立一个最佳的 第二代rCan疫苗中减毒、遗传稳定性和效力的平衡，并阐明 减毒的分子基础和保护的免疫学相关性。这些战略也可能是 适用于开发迫切需要的减毒拉沙病毒活疫苗。

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.