Paramyxoviruses as Vaccine Vectors Against Highly Pathogenic Viruses

副粘病毒作为高致病性病毒的疫苗载体

• 批准号: 7964502
• 负责人: PETER LEON COLLINS
• 金额: $ 142.51万
• 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/7964502
• 关键词: Adult; Aerosols; Affect; Animals; Antibodies; Antibody Formation; Antigens; Attenuated; Avian Influenza A Virus; Avulavirus; Biological Assay; Birds; Cause of Death; Cavia; Cells; Cercopithecus pygerythrus; Chickens; Childhood; Clinical; Control Animal; Coronavirus; DNA; Development; Disease; Disease Outbreaks; Distal; Dose; Ebola virus; Ebola virus envelope glycoprotein; Engineering; Enzyme-Linked Immunosorbent Assay; Evaluation; Experimental Animal Model; Face; Gene Expression; Genes; Genetic; Genetic Recombination; Genetic Variation; Genome; Glycoproteins; Goals; Hemagglutinin; Human; Human Virus; Human Volunteers; Immune; Immune response; Immunity; Immunization; Immunoglobulin A; Immunoglobulin G; In Vitro; Incidence; Infection; Infectious Agent; Influenza A Virus, H5N1 Subtype; Intranasal Administration; Irrigation; Laboratories; Life; Lung; Macaca mulatta; Measles virus; Membrane Proteins; Methods; Modeling; Natural History; Nebulizer; Neuraminidase; Newcastle disease virus; Nose; Para-Influenza Virus Type 3; Paramyxovirus; Pathogenicity; Property; Proteins; RNA; RNA Viruses; Recombinants; Respiratory System; Respiratory tract structure; Reverse Transcriptase Polymerase Chain Reaction; Route; Safety; Sequence Analysis; Serotyping; Serum; Severe Acute Respiratory Syndrome; Site; Surface Antigens; Swab; Syndrome; System; Testing; Time; Tissue Harvesting; Tissues; Vaccines; Vertebral column; Viral; Viral Hemorrhagic Fevers; Viral Vector; Virulence; Virus; attenuation; base; clinically relevant; design; egg; experience; immunogenic; immunogenicity; improved; neutralizing antibody; nonhuman primate; pathogen; positional cloning; protective efficacy; prototype; recombinant virus; respiratory; response; vaccine development; vaccine safety; vector; vector vaccine; virus genetics

This project involves evaluating common human and animal paramyxoviruses as potential human vaccine vectors against highly pathogenic viruses. We previously evaluated human parainfluenza virus type 3 (HPIV3) as a vector to express the spike glycoprotein of Severe Acute Respiratory Syndrome Coronavirus (SARS). A single dose of the HPIV3-S construct administered by the combined intranasal (IN) and intratracheal (IT) routes was immunogenic and protective against SARS challenge in African green monkeys (AGM). We also previously evaluated HPIV3 as a vector to express the single glycoprotein GP of Ebola virus (EBOV). This construct was highly immunogenic and completely protective in guinea pigs against an adapted strain of EBOV. A single IN/IT inoculation of rhesus monkeys was moderately immunogenic against EBOV and protected 88% of the animals against severe hemorrhagic fever and death caused by EBOV challenge. Two doses were highly immunogenic and all of the animals were free of disease signs and detectable EBOV challenge virus. Since HPIV3 is a common human pathogen and essentially all adults have a history of natural infection with HPIV3, it was important to determine whether previous infection with HPIV3 would restrict the replication and immunogenicity of the HPIV3 vector. In guinea pigs that were infected with HPIV3 and challenged 40 days later with HPIV3/EboGP, replication of the vector could not be detected, indicating a high level of restriction. Surprisingly, however, the immune response to EBOV GP was almost equivalent to that achieved in control animals that had not been previously infected with HPIV3. Next, rhesus monkeys were infected twice with HPIV3 and, 11 months following the second infection, were immunized with two doses of HPIV3/EboGP given 4 weeks apart. ELISA assay of EBOV-specific serum IgG and IgA showed that the level of EBOV-specific serum antibodies following the first dose was reduced 10-15 fold compared to the response in control animals that were HPIV3-nave. However, the serum antibody responses following the second dose were indistinguishable in HPIV3-immune versus HPIV3-naive animals. Thus, an HPIV3-based vector was substantially immunogenic even in the face of strong pre-existing immunity to the vector. Next, we deleted the F and HN genes from HPIV3 and replaced them with EBOV GP to create a virus, HPIV3/delF-HN/EboGP, in which GP would be the sole viral transmembrane surface protein. This virus was attenuated in vitro but eventually reached titers comparable to those of HPIV3. Following IN infection of guinea pigs, this virus was highly attenuated and completely restricted to the respiratory tract but nonetheless was highly immunogenic. A single IN dose provided complete protection of guinea pigs against an otherwise lethal challenge of guinea pig-adapted EBOV. Lacking the HPIV3 neutralization antigens, HPIV3/delF-HN/EboGP was insensitive to neutralization by HPIV3-specific antibodies in vitro. In addition, there was no significant difference in its immunogenicity in guinea pigs that were HPIV3-naive versus HPIV3-immune. Thus, HPIV3/delF-HN/EboGP provides an alternative to HPIV3/EboGP that is very highly attenuated, is insensitive to HPIV3-neutralizing antibodies, and nonetheless is nearly as immunogenic. We also are investigating the use of the avian Newcastle disease virus (NDV) as a human vaccine vector. NDV is antigenically distinct from common human pathogens and thus should not be affected by pre-existing immunity. In addition, there is anecdotal evidence that NDV is highly restricted in humans and does not cause significant disease. We confirmed that NDV is very highly attenuated following IN/IT inoculation of rhesus monkeys and AGM. We found that both low-virulence (lentogenic) and intermediate-virulence (mesogenic) strains replicated to similar low titers in non-human primates, suggesting that either backbone should be suitable for human vaccine purposes. Despite the high level of attenuation, which would be predictive of a high level of vaccine safety, expressed foreign proteins were moderately-to-highly immunogenic. For example, AGM that were immunized IN and IT with two doses of NDV expressing the SARS S protein developed a high titer of SARS-neutralizing serum antibodies and were strongly protected against challenge with a high dose of SARS. Another NDV was engineered to express the hemagglutinin HA glycoprotein of highly pathogenic avian H5N1 influenza virus (HPAIV) (NDV-HA). The NDV-HA virus was highly attenuated in AGM as well as in eggs and chickens. In AGM, two doses of NDV-HA induced a substantial titer of HPAIV-neutralizing serum antibodies; in addition, a substantial respiratory mucosal IgA response was induced following one and two doses, which would be particularly important in controlling a respiratory pathogen. We established a challenge model using AGM and showed that two doses of NDV-HA conferred essentially complete protection against challenge with a high dose (7.2 log10 PFU) of HPAIV. The high level of restriction of HPAIV challenge virus was established by assay of nasal swabs and tracheal lavages for virus by infectious virus assay and RT-PCR, by direct assay for infectious virus in harvested tissue, by immunohistochemical analysis of harvested tissue, and by profiling challenge-induced pulmonary host gene expression. Replacement of the polybasic cleavage site of the HA insert with the monobasic site from a low pathogenicity strain, an expedient designed to preclude any possibility of introduction of the polybasic site into circulating viruses by genetic exchange, resulted in improved immunogenicity and protective efficacy in this small study. In addition, immunization with NDV expressing the other major HPAIV surface antigen, the neuraminidase (NA) protein, also was highly immunogenic and protective. This was somewhat surprising, since the NA protein had not been considered to be a potent neutralization or protective antigen. These results showed that the modified HA gene and the NA gene are the genes of choice for inclusion in a vectored vaccine for human use. IN administration would be feasible in humans, but IT administration would not. We evaluated IN administration of the NDV construct expressing the SARS S protein and found it was not very immunogenic or protective, presumably because of insufficient vector replication in the nasal passages. Whether or not this will be predictive of replicative capability and immunogenicity in humans is unclear and can only be determined by administration to human volunteers. In the meantime, we explored an additional method of administration, namely by aerosol generated using a nebulizer. This method has been successfully and safely used in large-scale immunization against measles virus. This method of administration proved to be immunogenic and highly protective in AGM, providing a clinically relevant alternative to IN administration. In summary, NDV has considerable potential for further development as a highly attenuated vector for human vaccine use. NDV represents serotype 1 of the avian paramyxoviruses (APMV). There are 8 other serotypes, namely APMV2-9. We have initiated antigenic and sequence analysis of these as a prelude to their evaluation for attenuation and safety in non-human primates as potential vectors. Complete sequences have been determined for representatives of APMV2, 3, 4, 7, 8, and 9. In some cases, complete sequences are available for more than one strain of a serotype, namely APMV2 (2 strains), APMV3 (2 strains), and APMV8 (2 strains). Sequences already were available for two strains of APMV6: we have analyzed 2 more in addition. Also, sequencing of APMV5 is almost complete. The purpose in analyzing multiple strains is to investigate genetic diversity suggested by observed phenotypic and/or genetic diversity

该项目涉及评估常见的人类和动物副粘病毒作为针对高致病性病毒的潜在人类疫苗载体。我们之前评估了人类副流感病毒 3 型 (HPIV3) 作为表达严重急性呼吸系统综合症冠状病毒 (SARS) 刺突糖蛋白的载体。通过鼻内 (IN) 和气管内 (IT) 联合途径施用的单剂量 HPIV3-S 构建体在非洲绿猴 (AGM) 中具有免疫原性和针对 SARS 攻击的保护作用。我们之前还评估了 HPIV3 作为表达埃博拉病毒（EBOV）单糖蛋白 GP 的载体。该构建体具有高度免疫原性，并且对豚鼠具有完全保护作用，可抵抗埃博拉病毒适应株。对恒河猴进行单次 IN/IT 接种对 EBOV 具有中等免疫原性，并保护 88% 的动物免受 EBOV 攻击引起的严重出血热和死亡。两种剂量均具有高度免疫原性，所有动物均未出现疾病症状，也未检测到埃博拉病毒攻击病毒。 由于HPIV3是一种常见的人类病原体，并且基本上所有成年人都有HPIV3的自然感染史，因此确定先前的HPIV3感染是否会限制HPIV3载体的复制和免疫原性非常重要。在感染 HPIV3 并在 40 天后用 HPIV3/EboGP 攻击的豚鼠中，无法检测到载体的复制，表明高度限制。然而，令人惊讶的是，对 EBOV GP 的免疫反应几乎与先前未感染 HPIV3 的对照动物所达到的免疫反应相同。接下来，恒河猴感染 HPIV3 两次，并在第二次感染后 11 个月，间隔 4 周注射两剂 HPIV3/EboGP 进行免疫。 EBOV 特异性血清 IgG 和 IgA 的 ELISA 测定表明，与未接种 HPIV3 的对照动物相比，首次给药后 EBOV 特异性血清抗体水平降低了 10-15 倍。然而，在 HPIV3 免疫动物和未接触过 HPIV3 的动物中，第二次给药后的血清抗体反应没有区别。因此，基于HPIV3的载体即使面对对该载体的强预先存在的免疫也具有显着的免疫原性。 接下来，我们删除了 HPIV3 中的 F 和 HN 基因，并用 EBOV GP 替换它们，创建了病毒 HPIV3/delF-HN/EboGP，其中 GP 是唯一的病毒跨膜表面蛋白。该病毒在体外被减毒，但最终达到与 HPIV3 相当的滴度。豚鼠感染 IN 后，该病毒高度减毒并完全局限于呼吸道，但仍然具有高度免疫原性。单剂量注射可以完全保护豚鼠免受豚鼠适应的埃博拉病毒的致命攻击。由于缺乏 HPIV3 中和抗原，HPIV3/delF-HN/EboGP 在体外对 HPIV3 特异性抗体的中和不敏感。此外，其在未接触 HPIV3 的豚鼠与未接种 HPIV3 的豚鼠中的免疫原性没有显着差异。因此，HPIV3/delF-HN/EboGP 提供了 HPIV3/EboGP 的替代方案，其减毒程度非常高，对 HPIV3 中和抗体不敏感，但几乎具有同样的免疫原性。 我们还正在研究使用禽新城疫病毒（NDV）作为人类疫苗载体。新城疫病毒在抗原上与常见的人类病原体不同，因此不应受到预先存在的免疫力的影响。此外，有轶事证据表明，新城疫病毒在人类中受到高度限制，不会引起重大疾病。我们证实，在对恒河猴进行 IN/IT 接种和 AGM 后，新城疫病毒得到了高度减毒。我们发现低毒力（弱毒力）和中毒力（中等毒力）毒株在非人类灵长类动物中复制到类似的低滴度，这表明任一主链都应适合人类疫苗目的。尽管高水平的减毒预示着高水平的疫苗安全性，但表达的外源蛋白具有中度至高度的免疫原性。例如，用两剂表达 SARS S 蛋白的 NDV 进行 IN 和 IT 免疫的 AGM 产生了高效价的 SARS 中和血清抗体，并受到强有力的保护，免受高剂量 SARS 的攻击。另一种NDV被设计用于表达高致病性禽H5N1流感病毒（HPAIV）的血凝素HA糖蛋白（NDV-HA）。 NDV-HA 病毒在 AGM 以及鸡蛋和鸡中高度减毒。在 AGM 中，两剂 NDV-HA 诱导产生大量 HPAIV 中和血清抗体；此外，一剂和两剂后可诱导显着的呼吸道粘膜 IgA 反应，这对于控制呼吸道病原体尤为重要。我们使用 AGM 建立了攻击模型，结果表明，两剂 NDV-HA 基本上能提供完全的保护，以抵抗高剂量 (7.2 log10 PFU) HPAIV 的攻击。通过传染性病毒测定和 RT-PCR 对鼻拭子和气管灌洗液中的病毒进行测定，通过直接测定收获的组织中的传染性病毒，通过对收获的组织进行免疫组织化学分析，并通过分析，建立了对 HPAIV 攻击病毒的高水平限制。攻击诱导的肺宿主基因表达。用来自低致病性菌株的一元位点替换HA插入物的多碱基切割位点，这是一种权宜之计，旨在排除通过基因交换将多碱基位点引入循环病毒的任何可能性，从而提高了这种小病毒的免疫原性和保护功效。学习。此外，用表达另一种主要 HPAIV 表面抗原、神经氨酸酶 (NA) 蛋白的 NDV 进行免疫也具有高度免疫原性和保护性。这有点令人惊讶，因为 NA 蛋白不被认为是有效的中和或保护性抗原。这些结果表明，修饰的HA基因和NA基因是包含在人用载体疫苗中的选择基因。 IN 管理对于人类来说是可行的，但 IT 管理则不然。我们评估了表达 SARS S 蛋白的 NDV 构建体的 IN 给药，发现它的免疫原性或保护性不太好，可能是因为鼻道中的载体复制不足。这是否可以预测人类的复制能力和免疫原性尚不清楚，只能通过对人类志愿者施用来确定。与此同时，我们探索了另一种给药方法，即使用雾化器产生的气雾剂。该方法已成功、安全地应用于麻疹病毒的大规模免疫接种。这种给药方法被证明在 AGM 中具有免疫原性和高度保护性，为 IN 给药提供了临床相关的替代方案。 总之，NDV 作为人类疫苗用途的高度减毒载体，具有进一步开发的巨大潜力。 NDV 代表禽副粘病毒 (APMV) 血清型 1。还有其他8种血清型，即APMV2-9。我们已经开始对它们进行抗原和序列分析，作为评估它们作为潜在载体在非人类灵长类动物中的减毒和安全性的前奏。已确定 APMV2、3、4、7、8 和 9 代表的完整序列。在某些情况下，完整序列可用于一种以上血清型毒株，即 APMV2（2 个毒株）、APMV3（2 个毒株）和 APMV8（2 株）。两种 APMV6 毒株的序列已经可用：我们还分析了另外 2 种。此外，APMV5 的测序也已基本完成。分析多个菌株的目的是研究观察到的表型和/或遗传多样性所暗示的遗传多样性