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帕托病毒作为针对高致病性病毒的潜在人疫苗向量。我们先前评估了3型人类副氟氟扎病毒（HPIV3）作为表达严重急性呼吸综合症冠状病毒（SARS）的尖峰糖蛋白的载体。由鼻内（IN）和气管内（IT）途径组合给予的单剂量HPIV3-S构建体具有免疫原性和保护性，可抵抗非洲绿猴（AGM）中的SARS挑战。我们先前还评估了HPIV3作为载体，以表达埃博拉病毒（EBOV）的单糖蛋白GP。该构建体具有高度免疫原性，并且在豚鼠中完全具有保护性，以针对适应的EBOV菌株。对恒河猴的单一接种/IT接种对EBOV的免疫原性是中等免疫原性的，并保护了88％的动物免受严重的出血热和EBOV挑战引起的死亡。两种剂量具有高度免疫原性，所有动物都没有疾病体征和可检测的EBOV挑战病毒。 由于HPIV3是一种常见的人类病原体，本质上所有成年人都有HPIV3自然感染的史，因此确定先前对HPIV3的先前感染是否限制HPIV3载体的复制和免疫原性很重要。在感染HPIV3并在40天后用HPIV3/ebogp挑战的豚鼠中，无法检测到载体的复制，表明很高的限制。然而，令人惊讶的是，对EBOV GP的免疫反应几乎等同于先前未感染HPIV3的对照动物中的免疫反应。接下来，用HPIV3感染了两次恒河猴，第二次感染后11个月，用两剂HPIV3/EBOGP进行免疫，相隔4周。 EBOV特异性血清IgG和IgA的ELISA分析表明，与HPIV3-Nave的对照动物的反应相比，第一次剂量后的EBOV特异性血清抗体水平降低了10-15倍。然而，第二剂量后的血清抗体反应在HPIV3-免疫与HPIV3的动物中是无法区分的。因此，即使面对强烈的对载体的免疫力，基于HPIV3的载体也具有基本的免疫原性。 接下来，我们从HPIV3中删除了F和HN基因，并用EBOV GP代替它们，以创建病毒HPIV3/DELF-HN/EBOGP，其中GP将是唯一的病毒跨膜表面蛋白。该病毒在体外减弱，但最终达到了与HPIV3相当的滴度。在感染豚鼠后，该病毒被高度减弱，完全局限于呼吸道，但仍具有高度免疫原性。单一的剂量为豚鼠提供了完全保护，以防止豚鼠适应的EBOV挑战。缺乏HPIV3中和抗原，HPIV3/DELF-HN/EBOGP在体外对HPIV3特异性抗体中和不敏感。此外，在HPIV3-Neive与HPIV3-免疫的豚鼠中，其免疫原性没有显着差异。因此，HPIV3/DELF-HN/EBOGP提供了HPIV3/eBOGP的替代方法，该替代品非常衰减，对HPIV3中和抗体不敏感，尽管如此，几乎是免疫原性的。 我们还正在研究使用鸟类纽卡斯尔病毒（NDV）作为人类疫苗载体的使用。 NDV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，还有轶事证据表明，NDV在人类中受到高度限制，不会引起重大疾病。我们证实，在恒河猴和股东大会接种/接种时，NDV非常严重。我们发现，在非人类灵长类动物的类似低滴度中，低率（遗传源）和中等率（中源）菌株均表明，这两种骨架都应适合人类疫苗的目的。尽管衰减水平很高，这将预测高水平的疫苗安全性，但表达的外蛋白具有适中的免疫原性。例如，已免疫的AGM，并用两种表达SARS蛋白的NDV剂量产生了高滴度的SARS中和血清抗体，并受到高剂量的SARS受到强烈保护。另一项NDV的设计是表达高度致病性鸟类H5N1流感病毒（HPAIV）（NDV-HA）的血凝素HA糖蛋白。 NDV-HA病毒在AGM以及鸡蛋和鸡中受到高度减弱。在AGM中，两剂NDV-HA引起了HPAIV中和血清抗体的大量滴度。此外，一剂和两剂诱导了大量的呼吸粘膜IgA反应，这对于控制呼吸道病原体尤为重要。我们使用AGM建立了一个挑战模型，并表明两剂NDV-HA通过HPAIV的高剂量（7.2 log10 pfu）实质上完全保护了挑战。通过传染性病毒测定和RT-PCR对HPAIV挑战病毒的高度限制是通过对收获组织中感染性病毒的直接测定，通过对收获组织中的感染性病毒的直接测定来确定病毒，并通过对收获组织的免疫组织化学分析以及通过培训挑战挑战诱导的肺部宿主的宿主表达进行了传染性病毒。从低致病性菌株中，用单摩托部位代替了HA插入物的多性裂解位点，这是一种方便的，旨在通过遗传交换将多重性位点引入循环病毒的任何可能性，从而提高了这项小型研究的免疫原性和保护功效。另外，用NDV表达其他主要HPAIV表面抗原，神经氨酸酶（Na）蛋白的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蛋白没有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基因是纳入人类使用的矢量疫苗中的首选基因。 在管理中，在人类中是可行的，但IT管理是不可行的。我们评估了表达SARS蛋白的NDV构建体的给药，发现它不是很免疫原性或保护性，这可能是由于鼻腔中的载体复制不足。这是否可以预测人类复制能力和免疫原性尚不清楚，只能通过给人类志愿者来确定。同时，我们探索了一种额外的给药方法，即使用雾化器生成的气溶胶。该方法已成功，安全地用于针对麻疹病毒的大规模免疫。事实证明，这种给药方法在AGM中具有免疫原性和高度保护性，提供了一种临床相关的替代方案。 总而言之，作为人类疫苗的高度减弱，NDV具有进一步发展的潜力。 NDV代表禽paramyxoviruses（APMV）的血清型1。还有其他8种血清型，即APMV2-9。我们已经开始对它们的抗原和序列分析，以此作为其评估非人类灵长类动物作为潜在载体的衰减和安全性的序言。已经确定了APMV2、3、4、7、8和9的代表的完整序列。在某些情况下，可用于多种血清型的完整序列，即APMV2（2菌株），APMV3（2个菌株）（2个菌株）和APMV8（2株）。序列已适用于两种APMV6菌株：我们还分析了2个。同样，APMV5的测序几乎是完整的。分析多种菌株的目的是研究观察到的表型和/或遗传多样性提出的遗传多样性