A Novel Multi-Epitope-Based Universal Vaccine Against Multiple Coronavirus Variants of Concern

摘要 在过去的两年里，人类一直面临着由新的冠状病毒2号引起的新冠肺炎大流行 (SARS-CoV-2)感染。重大缺口：SARS-CoV-2基因组经常发生突变和缺失 (主要是在Spike蛋白中)导致更具传播性和致病性的“关注变种”(VOCs) 这可以逃脱第一代新冠肺炎疫苗赋予的免疫。因为大多数突变和 产生20种已知VOCs的缺失集中在Spike蛋白上，目前存在这样的风险 基于Spike蛋白的新冠肺炎亚单位疫苗尽管诱导产生VOC，但仍无法预防未来的VOC 针对原始病毒株的强病毒特异性中和抗体。在80个突变/缺失中 在Omicron变体中，仅Spike蛋白序列就集中了32个突变/缺失。这 强调现有疫苗的两大局限性：第二代通用疫苗的必要性 冠状病毒疫苗：(1)除高度可变的刺突蛋白以外的靶抗原(AGS)；以及(2) 整合来自Spike和Non-Spike Ags的B细胞和T细胞表位，这些表位在所有20种VOC中都高度保守 这将引发强烈的体液免疫和细胞免疫反应。我们的长期目标是开发一种 有效的第二代通用冠状病毒疫苗可阻止/减少SARS-CoV-2感染和由 多个VOC。初步结果：我们：(1)确定了高免疫原性的人类B和T细胞靶表位 从SARS-CoV-2全基因组；(2)从SARS-CoV-2全基因组中鉴定人类T细胞表位 针对无症状新冠肺炎患者的“保护性”免疫系统选择性靶向的基因组； 和(3)利用验证后的mRNA制得第一个原型多表位通用冠状病毒候选疫苗 传递系统平台，以及(4)创造了新颖的人性化易感HLA-DR/HLA-A*0201/hACE2三元组 转基因小鼠模型，用于检测另外7种多表位通用冠状病毒候选疫苗 跨越整个冠状病毒基因组的不同高度保守的人类B和T细胞表位。我们假设 我们的7种通用候选疫苗中的一种或多种将保护“人性化”小鼠免受感染和疾病。 由鼻腔接种SARS-CoV-2a、b、g、d和Omicron VOCs引起。我们的具体目标是：目标 1：设计和构建另外7个基于mRNA的通用候选疫苗，这些候选疫苗将高度整合 从20个VOC中选择保守的B和T细胞表位。目的2：确定其安全性、免疫原性和 7种多表位通用冠状病毒疫苗对SARS-2a、b、g、d或Omicron VOCs的保护作用 在“人源化”的人类白细胞抗原-DR/人类白细胞抗原A*0201/hACE2小鼠模型中经鼻腔给药。耐用性 保护作用及其与封闭/中和抗体和冠状病毒特异性抗体的数量和功能的相关性 驻留在肺和脑中的CD4+和CD8+TRM细胞将被确定。如果成功，领头羊环球 预防5种VOCs中的大多数的CoV疫苗可能会迅速进入FDA的第一阶段临床试验

SUMMARY Over the last 2 years humanity has been confronting COVID-19 pandemic caused by the new Corona Virus 2 (SARS-CoV-2) infection. Major gaps: Mutations and deletions often occur in the genome of SARS-CoV-2 (predominantly in the Spike protein) resulting in more transmissible and pathogenic “variants of concern” (VOCs) that can escape immunity conferred by first generation COVID-19 vaccines. Because most mutations and deletions that produced the 20 known VOCs are concentrated on the Spike protein, there is a risk that current COVID-19 sub-unit vaccines based on the Spike protein will fail to protect against future VOCs despite inducing strong virus-specific neutralizing antibodies against the original virus strain. Among the 80 mutations/deletions present in OMICRON variant, 32 mutations/deletions are concentrated in the sequence Spike protein alone. This emphasizes two major limitations of currently available vaccines: The need for second-generation universal coronavirus vaccines that (1) target antigens (Ags) other than the highly variable Spike protein; and (2) incorporate both B- and T-cell epitopes from Spike and non-Spike Ags that are highly conserved in all 20 VOCs and that will induce strong humoral and cell-mediated immune responses. Our long-term goal is to develop a potent second generation universal CoV vaccine to stop/reduce SARS-CoV-2 infections and disease caused by multiple VOCs. Preliminary Results: We: (1) Identified highly immunogenic human B and T cell target epitopes from the whole SARS-CoV-2 genome; (2) Characterized human T cell epitopes from the whole SARS-CoV-2 genome that are selectively targeted by the “protective” immune system from asymptomatic COVID-19 patients; and (3) Produced a first prototype multi-epitope universal CoV vaccine candidate using the validated mRNA delivery system platform, and (4) Created novel “humanized” susceptible HLA-DR/HLA-A*0201/hACE2 triple transgenic mouse model with which to test 7 additional multi-epitope universal CoV vaccine candidates that bear different highly conserved human B and T cell epitopes spanning the entire CoV genome. We hypothesize that one or more of our 7 universal vaccine candidates will protect “humanized” mice from infection and disease caused by intranasal inoculation with SARS-CoV-2 a, b, g, d and Omicron VOCs. Our Specific Aims are: Aim 1: To design and construct 7 additional mRNA-based universal vaccine candidates that will incorporate highly conserved B and T cell epitopes selected from 20 VOCs. Aim 2: To determine the safety, immunogenicity, and protective efficacy against SARS-CoV-2 a, b, g, d or Omicron VOCs of 7 multi-epitope universal CoV vaccine candidates delivered intranasally in the “humanized” HLA-DR/HLA-A*0201/hACE2 mouse model. The durability of protection and its correlation with blocking/neutralizing antibodies and the number and function of CoV-specific CD4+ and CD8+ TRM cells that reside in the lungs and brains will be determined. If successful, the lead universal CoV vaccine that protects against most of the 5 VOCs could proceed quickly into an FDA Phase 1 clinical trial