Countermeasures against COVID-19

针对 COVID-19 的对策

• 批准号: 10692240
• 负责人: Andrea Marzi
• 金额: $ 4.65万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692240
• 关键词: 2019-nCoV; ACE2; Acute; Animal Model; Animals; Antibodies; Antibody Response; Antibody-Dependent Enhancement; Antigens; Biological Assay; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; COVID-19; COVID-19 detection; COVID-19 pandemic; COVID-19 patient; COVID-19 pneumonia; COVID-19 vaccine; Cell Culture Techniques; Cells; Clinical; Complement; Complement 1q; Containment; Control Groups; Data; Data Set; Development; Disease; Dose; Ebola virus; Ebola virus envelope glycoprotein; Enzyme-Linked Immunosorbent Assay; Euthanasia; Exhibits; Fc Receptor; Gene Expression Profile; Gene Expression Profiling; Genes; Growth; Hamsters; Histologic; Human; Immune; Immune response; Immunobiology; Immunoglobulin G; Immunology; Indiana; Infection; Inflammatory; Interstitial Pneumonia; Intramuscular; Japan; Kinetics; Knowledge; Laboratories; Lung; Macaca mulatta; Mediating; Mesocricetus auratus; Microbiology; Modeling; Molecular Virology; Mutation; Oral; Pathogenesis; Pathway interactions; Patients; Performance; Plasma; Principal Component Analysis; Proteins; Pulmonary Pathology; Research; Risk; Route; SARS-CoV-2 B.1.1.7; SARS-CoV-2 B.1.351; SARS-CoV-2 B.1.617.2; SARS-CoV-2 antigen; SARS-CoV-2 infection; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Sampling; Serum; Severities; South Africa; Structure of parenchyma of lung; Swab; Symptoms; T cell response; Testing; Therapeutic; Time; Tropism; United Kingdom; Universities; Vaccinated; Vaccination; Vaccines; Variant; Vesicular stomatitis Indiana virus; Viral Load result; Virus; Virus Diseases; Virus Shedding; base; chemokine; cohort; cytokine; design; differential expression; efficacy study; frontier; immunogenicity; immunological status; immunopathology; immunoregulation; innovation; lung injury; lung lesion; neutralizing antibody; novel coronavirus; pandemic disease; pathogen; pathogenic virus; protective allele; protective efficacy; receptor binding; response; sample collection; severe COVID-19; transcriptome sequencing; transcriptomics; vaccination outcome; vaccine development; vaccine efficacy; vaccine immunogenicity; variants of concern; viral genomics

Summary 1. Intranasal single-dose vaccination with VSV-based COVID-19 vaccines protects hamsters from disease within 10 days We designed, cloned, and recovered two replication-competent VSV-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the EBOV GP (VSV-SARS2-EBOV). EBOV GP was included to enable ACE2-independent vaccine replication in the host and to possibly expand target cell tropism. Growth kinetics in cell culture confirmed superior replication of the VSV-SARS2-EBOV vaccine in VeroE6 cells. Using the well-established Syrian golden hamster model of COVID-19, we compared vaccine immunogenicity after a single intranasal (IN) or intramuscular (IM) dose of 105 PFU. IN vaccination resulted in an early CD8+ T cell response in the lungs and a greater total antigen-specific IgG response by ELISA compared to IM-vaccinated and control hamsters. In addition, the neutralizing response was also increased after IN vaccination compared to IM. In contrast, IM-vaccinated hamsters developed an early CD4+ T cell response. For the protective efficacy study, we vaccinated hamsters IN or IM 28 days prior to challenge with four SARS-CoV-2 VOC: the ancestral, alpha, beta, and delta variants. IN vaccination with either the VSV-SARS2 or VSV-SARS2-EBOV resulted in the highest protective efficacy, as demonstrated by decreased virus shedding and lung viral load in vaccinated hamsters. Histopathologic analysis of the lungs revealed the least amount of lung damage in the IN-vaccinated animals regardless of the challenge virus. In addition, IN-vaccination resulted in protection within 10 days and the animals did not show any clinical or significant histopathologic signs of COVID-19 pneumonia when challenged with the ancestral SARS-CoV-2 strain or the alpha or beta variants. These data demonstrate the ability of VSV-based vaccines to not only protect from disease caused by SARS-CoV-2 VOC, but also to reduce viral shedding (ODonnell et al., Frontiers in Immunology 2021; ODonnell et al., Vaccines 2022). 2. VSV-SARS2-EBOV protected NHPs within 10 days when administered IM but not IN This project investigated the protective efficacy of a single dose of the VSV-SARS2-EBOV vaccine in rhesus macaques based on the superior performance of this vaccine after IM administration -the approved route for VSV-EBOV vaccination in humans - in hamsters as demonstrated above. We vaccinated NHPs with a single dose of 107 PFU of the vaccine by the IM or IN route, while NHPs vaccinated IN or IM with 107 PFU VSV-EBOV served as controls. Challenge with SARS-CoV-2 WA1 by multiple routes as described previously occurred 10 days later. At 7 days after challenge, all NHPs were euthanized for sample collection. NHPs IM-vaccinated with VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, IN vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to controls. The IN-vaccinated NHPs exhibited lung lesions and presented with more lung infiltrates compared to the IM and control groups at the time of euthanasia. Histological examination of the lung tissue revealed immunopathology that was most significant in IN-vaccinated animals. Importantly, IM-vaccinated animals did not develop signs of interstitial pneumonia, nor could we detect SARS-CoV-2 antigen in the lungs. Indeed, at the time of euthanasia, lung lesions were apparent in animals from the IN and control groups, but not in the IM group (Furuyama et al., mBio 2022). 3. SARS-CoV-2 VOC infection induces similar disease but distinct humoral responses and transcriptomic changes in Syrian golden hamsters There was a critical need to understand the impact of the emerging SARS-CoV-2 VOC on host response and disease dynamics to facilitate the development of vaccines and therapeutics. Syrian golden hamsters are the leading small animal model recapitulating key aspects of severe COVID-19. In this project, we demonstrated that IN inoculation of 105 TCID50 of SARS-CoV-2 into hamsters with the ancestral virus (nCoV-WA1-2020) or VOC first identified in the United Kingdom (alpha, B.1.1.7) and South Africa (beta, B.1.351) led to similar gross and histopathologic pulmonary lesions 4 days after challenge. Although differences in viral genomic copy numbers were noted in the lungs and oral swabs of infected hamsters, infectious titers in the lungs were comparable. At 14 days after challenge, lung pathology was mostly resolved for all challenged hamsters in this non-lethal model. While there was no difference in the S-specific IgG titers at either 14 or 28 days after challenge among the groups, IgG specific to the S receptor-binding domain (RBD) was significantly increased in hamsters infected with B.1.351 compared to B.1.1.7. Antibody neutralization capacities varied, dependent on the original challenge virus and cross-variant protective capacity. Bulk RNA sequencing of lung samples collected 4 days after challenge was performed. Principal component analysis revealed distinct separation between uninfected and infected hamsters, with the B.1.1.7 infection resulting in the most distinct transcriptional profile and the largest number of differentially expressed genes (DEGs)(n=1,277) while infection with B.1.351 resulted in the smallest number of DEGs (n=395). Transcriptional profiling indicated significant induction of antiviral pathways in response to all three challenges with a more robust inflammatory signature in response to B.1.351 infection. Furthermore, no additional mutations in the spike protein were detected at peak disease. These observations document differences in acute early responses or alterations in immune modulation by VOC potentially impacting the efficacy of existing vaccines and therapeutics. 4. Analysis of human COVID-19 serum samples reveals In order to expand our understanding of human cytokine responses during COVID-19, we analyzed human serum samples collected over time from patients from Indiana. SRAS-CoV-2 infection results in a variety of clinical symptoms ranging from no or mild to severe disease. Currently, there are multiple postulated mechanisms that may push a moderate to severe disease into a critical state. Human serum contains abundant evidence of the immune status following infection. Cytokines, chemokines, and antibodies can be assayed to determine the extent to which a patient responded to a pathogen. We examined serum and plasma from a cohort of patients infected with SARS-CoV-2 early in the pandemic and compared them to negative-control sera. Cytokine and chemokine concentrations varied depending on the severity of infection, and antibody responses were significantly increased in severe cases compared to mild to moderate infections. Neutralization data revealed that patients with high titers against an early 2020 isolate had detectable but limited neutralizing antibodies against the emerging SARS-CoV-2 Alpha, Beta and Delta variants. This study highlights the potential of re-infection for recovered COVID-19 patients (Griffin et al., Sci Rep 2021). We also supported a study by Prof. Ayato Takada (Hokkaido University, Japan) exploring the potential risks of antibody-dependent enhancement (ADE) in COVID-19. Clinical importance of ADE is unclear since the proposed ADE mechanism mostly depends on the Fc receptor (FcR) expressed exclusively on immune cells which are not primary targets of SARS-CoV-2. We show that SARS-CoV-2 utilizes at least two distinct ADE mechanisms, FcR-mediated and FcR-independent complement component C1q-mediated pathways. We found that both FcR- and C1q-mediated ADE activities were detectable in the sera of acute and convalescent COVID-19 patients at a high rate (Okuya et al., Microbiology Spectrum 2022).

概括 1. 鼻内单剂量接种基于 VSV 的 COVID-19 疫苗可保护仓鼠在 10 天内免受疾病侵害 我们设计、克隆和回收了两种可复制的基于 VSV 的疫苗，它们单独编码 SARS-CoV-2 刺突蛋白 (VSV-SARS2) 或与 EBOV GP 联合编码 (VSV-SARS2-EBOV)。包含 EBOV GP 是为了能够在宿主中实现不依赖于 ACE2 的疫苗复制，并可能扩大靶细胞向性。细胞培养物的生长动力学证实 VSV-SARS2-EBOV 疫苗在 VeroE6 细胞中具有优异的复制能力。使用完善的 COVID-19 叙利亚金仓鼠模型，我们比较了单次鼻内 (IN) 或肌内 (IM) 剂量 105 PFU 后的疫苗免疫原性。与 IM 疫苗接种的仓鼠和对照仓鼠相比，IN 疫苗接种导致肺部出现早期 CD8+ T 细胞反应，并且通过 ELISA 检测显示更高的总抗原特异性 IgG 反应。此外，与 IM 相比，IN 疫苗接种后中和反应也有所增加。相比之下，IM 疫苗接种的仓鼠出现了早期 CD4+ T 细胞反应。 为了进行保护功效研究，我们在攻击前 28 天对仓鼠进行了 IN 或 IM 疫苗接种，使用了四种 SARS-CoV-2 VOC：祖先、α、β 和 δ 变体。接种 VSV-SARS2 或 VSV-SARS2-EBOV 的 IN 疫苗接种可产生最高的保护功效，接种疫苗的仓鼠的病毒脱落和肺部病毒载量减少就证明了这一点。肺部组织病理学分析显示，无论攻击病毒如何，接种 IN 疫苗的动物的肺部损伤最少。此外，IN 疫苗接种可在 10 天内产生保护，并且当受到祖先 SARS-CoV-2 毒株或 α 或 β 变体的攻击时，动物没有表现出任何 COVID-19 肺炎的临床或显着组织病理学迹象。这些数据表明，基于 VSV 的疫苗不仅能够预防 SARS-CoV-2 VOC 引起的疾病，还能减少病毒脱落（ODonnell 等人，Frontiers in Immunology 2021；ODonnell 等人，Vaccines 2022） 。 2. VSV-SARS2-EBOV 在肌内注射但不能注射时在 10 天内保护 NHP 该项目研究了单剂 VSV-SARS2-EBOV 疫苗对恒河猴的保护功效，基于该疫苗在仓鼠肌内注射（经批准的人类 VSV-EBOV 疫苗接种途径）后的优异性能，如上所述。我们通过 IM 或 IN 途径对 NHP 进行单剂 107 PFU 疫苗接种，而用 107 PFU VSV-EBOV 进行 IN 或 IM 疫苗接种的 NHP 作为对照。 10 天后，如前所述，通过多种途径进行 SARS-CoV-2 WA1 攻击。攻击后 7 天，对所有 NHP 实施安乐死以收集样本。肌肉注射 VSV-SARS2-EBOV 疫苗的 NHP 在 10 天内受到保护，并且没有表现出 COVID-19 肺炎的迹象。相比之下，与对照组相比，IN 疫苗接种导致免疫原性有限，并增强了 COVID-19 肺炎的发生。在安乐死时，与 IM 组和对照组相比，IN 疫苗接种的 NHP 表现出肺部病变，并出现更多的肺部浸润。肺组织的组织学检查揭示了免疫病理学，这在接种 IN 疫苗的动物中最为显着。重要的是，IM 疫苗接种的动物没有出现间质性肺炎的症状，我们也无法在肺部检测到 SARS-CoV-2 抗原。事实上，在安乐死时，IN 组和对照组的动物肺部病变很明显，但 IM 组的动物则不然 (Furuyama et al., mBio 2022)。 3. SARS-CoV-2 VOC感染在叙利亚金仓鼠中诱发类似的疾病，但有不同的体液反应和转录组变化 迫切需要了解新出现的 SARS-CoV-2 VOC 对宿主反应和疾病动态的影响，以促进疫苗和治疗方法的开发。叙利亚金仓鼠是概括严重 COVID-19 关键方面的主要小动物模型。在该项目中，我们证明了将 105 TCID50 的 SARS-CoV-2 接种到仓鼠中，其中携带祖先病毒 (nCoV-WA1-2020) 或首次在英国 (alpha, B.1.1.7) 和南方发现的 VOC Africa（β，B.1.351）在攻击后 4 天导致类似的肉眼和组织病理学肺部病变。尽管在受感染仓鼠的肺部和口腔拭子中发现病毒基因组拷贝数存在差异，但肺部的感染滴度相当。攻击后 14 天，在该非致死模型中，所有受到攻击的仓鼠的肺部病理学大部分得到解决。虽然攻击后 14 或 28 天各组之间的 S 特异性 IgG 滴度没有差异，但与 B.1.351 感染的仓鼠相比，感染 B.1.351 的仓鼠中 S 受体结合域 (RBD) 特异性的 IgG 显着增加。 1.1.7。抗体中和能力各不相同，取决​​于原始攻击病毒和跨变体保护能力。对攻击后 4 天收集的肺样本进行批量 RNA 测序。主成分分析显示未感染和感染的仓鼠之间存在明显的区别，B.1.1.7 感染导致最独特的转录谱和最大数量的差异表达基因 (DEG) (n=1,277)，而 B.1.351 感染则导致在最少数量的 DEG 中（n = 395）。转录分析表明，针对所有三种挑战，显着诱导了抗病毒途径，并针对 B.1.351 感染产生了更强烈的炎症特征。此外，在疾病高峰期没有检测到刺突蛋白的额外突变。这些观察结果记录了 VOC 引起的急性早期反应或免疫调节变化的差异，可能影响现有疫苗和治疗方法的功效。 4. 对人类 COVID-19 血清样本的分析表明 为了扩大我们对 COVID-19 期间人类细胞因子反应的了解，我们分析了一段时间内从印第安纳州患者收集的人类血清样本。 SRAS-CoV-2 感染会导致各种临床症状，从无疾病或轻度疾病到严重疾病。目前，有多种假设机制可能会将中度至重度疾病推向危急状态。人血清含有丰富的感染后免疫状态的证据。可以通过分析细胞因子、趋化因子和抗体来确定患者对病原体的反应程度。我们检查了大流行早期感染 SARS-CoV-2 的一组患者的血清和血浆，并将其与阴性对照血清进行了比较。细胞因子和趋化因子浓度根据感染的严重程度而变化，与轻度至中度感染相比，重度病例的抗体反应显着增加。中和数据显示，2020 年初分离株具有高滴度的患者体内可检测到但有限的针对新出现的 SARS-CoV-2 Alpha、Beta 和 Delta 变种的中和抗体。这项研究强调了康复的 COVID-19 患者再次感染的可能性（Griffin 等人，Sci Rep 2021）。 我们还支持 Ayato Takada 教授（日本北海道大学）的一项研究，探讨了抗体依赖性增强 (ADE) 在 COVID-19 中的潜在风险。 ADE 的临床重要性尚不清楚，因为拟议的 ADE 机制主要取决于仅在免疫细胞上表达的 Fc 受体 (FcR)，而免疫细胞不是 SARS-CoV-2 的主要目标。我们发现 SARS-CoV-2 利用至少两种不同的 ADE 机制，即 FcR 介导的途径和 FcR 独立的补体成分 C1q 介导的途径。我们发现，在急性和恢复期 COVID-19 患者的血清中，FcR 和 C1q 介导的 ADE 活性可高检测到（Okuya 等人，Microbiology Spectrum 2022）。