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 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来自AA CA病毒的段。 H5N1 Ca疫苗候选物在雪貂，鸡和小鼠中减弱。疫苗在小鼠和雪貂中具有免疫原性和有效性。在雪貂模型中，疫苗候选剂量的毒性剂量可与季节性LAIV相当，剂量高达100倍。结果表明，即使以高剂量给药，H5N1 CA疫苗在下呼吸道的复制中也受到限制。 然而，鼻内给药0.5 mL可能导致雪貂肺中H5N1疫苗的沉积，在没有明显的局部复制疫苗病毒的情况下，它会诱导肺部炎症反应。 基于小鼠和雪貂的有前途的临床前数据，生成了2004年和2003年H5N1 Ca疫苗的临床批次，并根据IND进行了健康疫苗的安全性和免疫原性的I期临床试验。 禽流感A H7亚型病毒也对人类健康构成了重大威胁，因为它们能够直接从家居家禽传播到人类，并导致疾病，有时甚至导致死亡。尽管针对该亚型的病毒开发疫苗很重要，但在动物模型（例如小鼠和雪貂）中，有关H7病毒的免疫反应和发病机理的信息非常有限。根据其系统发育关系和地理位置选择了10种H7病毒以进行疫苗开发。确定了10种小鼠病毒的毒力，并评估了小鼠和雪貂病毒的免疫原性，以研究抗原相关性的程度以及通过感染引起的抗体的交叉反应水平。大多数病毒显示出与小鼠和雪貂抗血清的交叉反应模式相似的模式。欧亚病毒引起了广泛的交叉反应性抗体，这些抗体从欧亚和北美谱系中中和病毒，但相反并非如此。还评估了病毒的一部分，其在鼻内给药后在BALB/C小鼠中复制和引起疾病的能力。 H7亚型病毒能够不适应而感染小鼠，并表现出不同水平的复制性和动力学，表明小鼠模型可用于对H7亚型病毒的临床前评估疫苗。我们已经根据系统发育数据选择了A/荷兰/219/03（H7N7）和A/Chicken/BC/CN-7/04（H7N3）病毒来开发疫苗的开发，从而诱导小鼠和垂坠抗的小鼠和小鼠的能力。 我们应用了基于质粒的反向遗传学技术来生成H7N3 CA疫苗病毒，该病毒从低致病性流感A/Chicken/BC/CN-6/04（H7N3）病毒中得出其HA和NA基因，该病毒是高度致病的A a/Chicke a/Chicke a a/bc/cn-cn-n3的先前（H7N3）病毒。 2004年，加拿大的家禽和2种人类感染。候选H7N3 CA疫苗病毒具有由AA CA病毒的内部蛋白质基因指定的TS和ATT表型，而疫苗病毒在小鼠中是免疫原性的。在接受单剂​​量的疫苗接种后四个星期后，小鼠在血清中产生了中和抗体，并在同源性和抗原上不同的异源异源野生型高度致病的H7病毒中完全保护了致死性，并从不同位置分离出来。接受两剂疫苗后四周，将小鼠和雪貂完全保护不受高度致病的同源性野生型H7N3病毒的肺复制。基于小鼠和雪貂的有希望的临床前数据，生成了2004 H7N3 CA疫苗的临床批次，并提交了IND。 除了针对禽流感病毒的疫苗开发和评估外，我们还研究了针对H5N1流感的人类单克隆抗体的预防和治疗功效。与越南和瑞士的研究人员合作，我们在鼠类感染模型中产生了中和中和抗H5N1人类单克隆抗体（MAB），并测试了其预防和治疗的功效。使用爱泼斯坦Barr病毒（EBV）使从HPAI H5N1病毒感染中恢复过的越南成年人的记忆B细胞被永生，并在病毒中和测定中筛选了B细胞系的上清液。将分泌中和抗体的B细胞系克隆并纯化mAb。这些抗体对不同菌株H5N1的交叉反应性通过中和测定在体外测试，并在小鼠中测试了它们在体内的预防性和治疗功效。在体内，mAb以剂量依赖性方式赋予了A/越南/1203/04（H5N1）的小鼠的杀伤力。 MAB预防可提供肺部病毒滴度的统计学显着降低，减少肺部相关炎症，并受到病毒外肺外传播的限制。治疗剂量的3 MAB可提供强大的保护性，可通过A/越南/1203/04（H5N1）感染后至少72小时（至少在72小时）进行致死性，并且在体内也具有治疗性活性，对A//5/2005（H5N1），一种属于不同遗传外包的病毒。这些研究提供了概念证明，表明具有中和活性的完全人物受体可以由康复患者的周围血液迅速产生，并且这些mAB在小鼠模型中有效预防和治疗H5N1感染。一个中和，交叉反应性mAB的面板可能有助于预防或辅助治疗人类的H5N1流感病例。