Preclinical Studies of Vaccines for Pandemic Influenza

大流行性流感疫苗的临床前研究

• 批准号: 7592290
• 负责人: Kanta Subbarao
• 金额: $ 254.96万
• 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/7592290
• 关键词: Adult; American; Animal Model; Antibodies; Asia; Attenuated; Attenuated Live Virus Vaccine; Avian Influenza; Avian Influenza A Virus; B-Lymphocytes; Base Sequence; Biological Assay; Birds; Canada; Cell Line; Cessation of life; Chickens; Clinical; Clinical Trials; Collaborations; Cooperative Research and Development Agreement; Data; Deposition; Development; Disease; Disease Outbreaks; Domestic Fowls; Dose; Epidemic; Equilibrium; Evaluation; Event; Exhibits; Ferrets; Formalin; Gene Proteins; Genes; Genetic; Genetic Techniques; Geographic Locations; Goals; H7N7; Health; Hemagglutinin; Human; Human Herpesvirus 4; Immune Sera; Immune response; In Vitro; Inbred BALB C Mice; Indonesia; Infection; Inflammation; Inflammatory Response; Influenza; Influenza A Virus, H5N1 Subtype; Influenza A virus; Intranasal Administration; Kinetics; Licensing; Life; Location; Lower respiratory tract structure; Lung; Membrane Glycoproteins; Memory B-Lymphocyte; Modeling; Mus; Mutation; Nature; Needles; Netherlands; Neuraminidase; Pathogenesis; Pathogenicity; Patients; Pattern; Phase I Clinical Trials; Phenotype; Phylogenetic Analysis; Plasmids; Population; Prevention; Prophylactic treatment; Public Health; Range; Readiness; Research Personnel; Safety; Scientist; Seeds; Serum; Simian B disease; Specific qualifier value; Statistically Significant; Switzerland; Temperature; Testing; Therapeutic; Toxic effect; Treatment Efficacy; Ursidae Family; Vaccines; Vietnam; Virulence; Virus; Week; aquatic bird; attenuation; base; concept; cross reactivity; human monoclonal antibodies; immunogenic; immunogenicity; in vivo; influenza virus vaccine; influenzavirus; mouse model; neutralizing antibody; pandemic disease; pandemic influenza; peripheral blood; positional cloning; pre-clinical; preclinical study; programs; prophylactic; response; vaccine development; vaccine evaluation

LID scientists are collaborating with scientists from Medimmune, Inc., under a CRADA to generate candidate vaccines against H5N1 viruses that have caused human infections in Asia in 1997, 2003 and 2004. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian H5N1 influenza virus and six internal gene segments from the AA ca virus. H5N1 ca vaccine candidates are attenuated in ferrets, chickens and mice. The vaccines are immunogenic and efficacious in mice and ferrets. The toxicity of the vaccine candidates at doses comparable to seasonal LAIV and at doses up to 100-fold higher was characterized in the ferret model. The results demonstrate that H5N1 ca vaccines are restricted in replication in the lower respiratory tract of ferrets, even when administered at high doses. However, intranasal administration of 0.5 mL can result in deposition of H5N1 vaccine in the ferret lung, where it induces a pulmonary inflammatory response in the absence of significant local replication of the vaccine virus. Based on promising preclinical data in mice and ferrets, clinical lots of the 2004 and 2003 H5N1 ca vaccines were generated and Phase I clinical trials of the safety and immunogenicity of the vaccines for healthy adults were undertaken under an IND. Avian influenza A H7 subtype viruses also pose a significant threat to human health because of their ability to transmit directly from domestic poultry to humans and to cause disease and sometimes death. Although it is important to develop vaccines against viruses of this subtype, very limited information is available on the immune response and pathogenesis of H7 viruses in animal models such as mice and ferrets. A group of 10 H7 viruses were selected for possible vaccine development based on their phylogenetic relationships and geographical location. The virulence of the 10 viruses for mice was determined, and the immunogenicity of the viruses in mice and ferrets were evaluated to study the extent of antigenic relatedness and the level of cross-reactivity of the antibodies that were induced by infection. Most of the viruses showed similar patterns of cross-reactivity with mice and ferret antisera. The Eurasian viruses elicited broadly cross-reactive antibodies that neutralized viruses from both Eurasian and North American lineages but the converse was not true. A subset of the viruses was also evaluated for their ability to replicate and cause disease in BALB/c mice following intranasal administration. H7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication indicating that the mouse model can be used for preclinical evaluation of vaccines against H7 subtype viruses. We have selected A/Netherlands/219/03 (H7N7) and A/chicken/BC/CN-7/04 (H7N3) viruses for vaccine development based on phylogenetic data, the induction of broadly cross-neutralizing antibodies in mouse and ferret antisera, and the ability to replicate in mice. We applied plasmid-based reverse genetics techniques to generate an H7N3 ca vaccine virus that derived its HA and NA genes from the low pathogenicity influenza A/chicken/BC/CN-6/04 (H7N3) virus that was the precursor of the highly pathogenic influenza A A/chicken/BC/CN-7/04 (H7N3) virus that caused the outbreak of disease in poultry and 2 human infections in Canada in 2004. The candidate H7N3 ca vaccine virus possessed the ts and att phenotypes specified by the internal protein genes of the AA ca virus, and the vaccine virus was immunogenic in mice. Four weeks after receiving a single dose of vaccine, mice developed neutralizing antibodies in the serum and they were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wild-type highly pathogenic H7 viruses isolated from diverse locations. Four weeks after receiving two doses of the vaccine, mice and ferrets were fully protected from pulmonary replication of the highly pathogenic homologous wild-type H7N3 virus. Based on promising preclinical data in mice and ferrets, a clinical lot of the 2004 H7N3 ca vaccine was generated and an IND was submitted. In addition to the development and evaluation of vaccines against avian influenza viruses, we have investigated the prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. In collaboration with investigators from Vietnam and Switzerland, we generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. Memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses were immortalised using Epstein Barr virus (EBV) and supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vivo, the mAbs conferred protection from lethality in mice challenged with A/Viet Nam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of 3 mAbs provided robust protection from lethality at least up to 72 h postinfection with A/Viet Nam/1203/04 (H5N1) and were also therapeutically active in vivo against the A/Indonesia/5/2005 (H5N1), a virus that belongs to a different genetic clade. These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.

LID 科学家正在与 Medimmune, Inc. 的科学家合作，在 CRADA 下生产针对 H5N1 病毒的候选疫苗，这些病毒曾于 1997 年、2003 年和 2004 年在亚洲引起人类感染。这些疫苗是使用基于质粒的反向遗传学生产的，每种疫苗都含有血凝素和来自禽类 H5N1 流感病毒的神经氨酸酶基因以及来自 AA ca 病毒的六个内部基因片段。 H5N1 ca 候选疫苗在雪貂、鸡和小鼠体内的效果较弱。该疫苗对小鼠和雪貂具有免疫原性且有效。在雪貂模型中表征了候选疫苗在剂量与季节性 LAIV 相当以及高出 100 倍的剂量下的毒性。结果表明，即使以高剂量施用，H5N1 ca 疫苗在雪貂下呼吸道中的复制也受到限制。 然而，鼻内施用 0.5 mL 可能会导致 H5N1 疫苗沉积在雪貂肺部，在疫苗病毒没有显着局部复制的情况下，它会诱导肺部炎症反应。 基于小鼠和雪貂的有希望的临床前数据，产生了 2004 年和 2003 年 H5N1 ca 疫苗的临床批次，并根据 IND 开展了疫苗对健康成人的安全性和免疫原性的 I 期临床试验。 甲型H7亚型禽流感病毒也对人类健康构成重大威胁，因为它们能够直接从家禽传播给人类并导致疾病，有时甚至导致死亡。尽管开发针对该亚型病毒的疫苗很重要，但有关小鼠和雪貂等动物模型中 H7 病毒的免疫反应和发病机制的信息非常有限。根据系统发育关系和地理位置，选择了一组 10 种 H7 病毒用于可能的疫苗开发。测定了10种病毒对小鼠的毒力，并评估了病毒在小鼠和雪貂体内的免疫原性，以研究抗原相关性的程度以及感染诱导的抗体的交叉反应水平。大多数病毒与小鼠和雪貂抗血清表现出相似的交叉反应模式。欧亚病毒引发了广泛的交叉反应抗体，中和了欧亚和北美谱系的病毒，但反之则不然。还评估了一部分病毒在鼻内给药后在 BALB/c 小鼠中复制和引起疾病的能力。 H7亚型病毒能够在不适应的情况下感染小鼠，并表现出不同水平的致死率和复制动力学，表明小鼠模型可用于针对H7亚型病毒的疫苗的临床前评估。我们根据系统发育数据、在小鼠和雪貂抗血清中诱导广泛交叉中和抗体，选择了 A/Netherlands/219/03 (H7N7) 和 A/chicken/BC/CN-7/04 (H7N3) 病毒用于疫苗开发，以及在小鼠中复制的能力。 我们应用基于质粒的反向遗传学技术来生成 H7N3 ca 疫苗病毒，该病毒的 HA 和 NA 基因源自低致病性甲型流感/鸡/BC/CN-6/04 (H7N3) 病毒，该病毒是高致病性流感的前身。甲型流感 A/chicken/BC/CN-7/04 (H7N3) 病毒，2004 年在加拿大引起家禽疾病爆发和 2 例人类感染。候选 H7N3 ca疫苗病毒具有AA ca病毒内部蛋白基因指定的ts和att表型，并且该疫苗病毒在小鼠中具有免疫原性。接受单剂量疫苗 4 周后，小鼠在血清中产生了中和抗体，并且在受到从不同地点分离的同源和抗原不同的异源野生型高致病性 H7 病毒攻击后，它们得到了充分的保护，免于死亡。接受两剂疫苗 4 周后，小鼠和雪貂完全免受高致病性同源野生型 H7N3 病毒肺部复制的影响。基于小鼠和雪貂的有前景的临床前数据，2004 H7N3 ca 疫苗的临床批次已生成并提交了 IND。 除了针对禽流感病毒的疫苗的开发和评估外，我们还研究了针对H5N1流感的人单克隆抗体的预防和治疗效果。我们与越南和瑞士的研究人员合作，制备了中和性抗 H5N1 人类单克隆抗体 (mAb)，并在小鼠感染模型中测试了其预防和治疗的功效。使用 Epstein Barr 病毒 (EBV) 使从 HPAI H5N1 病毒感染中恢复的越南成年人的记忆 B 细胞永生化，并在病毒中和测定中筛选 B 细胞系的上清液。克隆分泌中和抗体的 B 细胞系并纯化 mAb。通过中和试验在体外测试了这些抗体对不同H5N1毒株的交叉反应性，并在小鼠体内测试了它们的体内预防和治疗功效。在体内，单克隆抗体以剂量依赖性方式为经 A/Viet Nam/1203/04 (H5N1) 攻击的小鼠提供免于致死的保护。单克隆抗体预防可以显着降低肺部病毒滴度，减少肺部相关炎症，并限制病毒的肺外传播。治疗剂量的 3 种 mAb 在感染 A/Viet Nam/1203/04 (H5N1) 后至少 72 小时内提供了强大的致死保护，并且在体内对 A/Indonesia/5/2005 (H5N1) 也具有治疗活性。属于不同遗传分支的病毒。这些研究提供了概念证明，即具有中和活性的全人单克隆抗体可以从恢复期患者的外周血中快速产生，并且这些单克隆抗体可有效预防和治疗小鼠模型中的 H5N1 感染。一组具有中和性、交叉反应性的单克隆抗体可能有助于预防或辅助治疗人类 H5N1 流感病例。