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: 9566628
• 负责人: PETER LEON COLLINS
• 金额: $ 231.32万
• 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/9566628
• 关键词: Adult; Aerosols; Alphavirus; Animals; Antibodies; Antibody titer measurement; Antigens; Apoptosis; Attenuated; Avian Influenza A Virus; Avulavirus; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Candidate Disease Gene; Cattle; Cavia; Cell Culture Techniques; Cells; Cercopithecus pygerythrus; Child; Childhood; Chimera organism; Clinical; Clinical Research; Clinical Trials; Codon Nucleotides; Comparative Study; Coronaviridae; Coronavirus; Coronavirus spike protein; Cytoplasmic Tail; Development; Devices; Dose; Ebola virus; Ebola virus envelope glycoprotein; Effectiveness; Engineering; Evaluation; Family member; Gene Order; Generations; Genes; Genetic Transcription; Genome; Genomics; Glycoproteins; Helper-Inducer T-Lymphocyte; Human; Immune response; Immunity; Immunize; Immunoglobulin A; Immunoglobulin G; Impairment; Infant; Infection; Interferons; Intranasal Administration; Laboratories; Length; Lung; Macaca; Macaca fascicularis; Macaca mulatta; Measles virus; Mediating; Medical; Membrane Glycoproteins; Membrane Proteins; Messenger RNA; Modification; Mucosal Immunity; Mutation; Newcastle disease virus; Nucleotides; Open Reading Frames; Para-Influenza Virus Type 1; Para-Influenza Virus Type 3; Paramyxoviridae; Paramyxovirus; Pathogenicity; Phase I Clinical Trials; Plaque Assay; Polymerase; Population; Primates; Proteins; Quantitative Reverse Transcriptase PCR; RNA; Reporting; Respiratory System; Respiratory tract structure; Rodent; Route; Safety; Scientist; Series; Serotyping; Serum; Severe Acute Respiratory Syndrome; Signal Transduction; Staining method; Stains; Structure of respiratory epithelium; Surface Antigens; System; TRIP10 gene; Testing; Texas; Time; Tropism; United States National Institutes of Health; Universities; Upper respiratory tract; Vaccination; Vaccines; Vertebral column; Viral; Viral Vaccines; Virion; Virus; Virus Replication; Virus Shedding; Work; attenuation; base; cohort; conjunctiva; design; expression vector; immunogenic; immunogenicity; in vivo; neutralizing antibody; nonhuman primate; open label; parainfluenza virus; particle; pathogen; programs; research clinical testing; respiratory; response; reverse genetics; safety study; seropositive; tissue tropism; vaccine candidate; vaccine delivery; vaccine development; vaccine trial; vector; vector vaccine; vector-based vaccine; volunteer

We are developing human parainfluenza viruses (HPIVs) as vaccine vectors for human use against highly pathogenic emerging viruses, with a present focus on HPIV serotypes 1 and 3. This strategy takes advantage of the natural respiratory tract tropism of HPIVs to provide respiratory administration and stimulate strong systemic immune responses as well as local mucosal immunity that is important for restricting pathogens that infect and are spread via the respiratory tract and conjunctiva. HPIV3 vector: We previously constructed a 1st generation construct called HPIV3/EboGP, in which the complete HPIV3 genome was modified by the addition of the EBOV GP gene in the third gene position, between the HPIV3 P and M genes. EBOV GP is the sole EBOV virion surface protein and is the sole EBOV neutralization antigen and major protective antigen. The EBOV GP gene was engineered to have the appropriate HPIV3 transcription signals for it to be expressed as a separate mRNA by the HPIV3 polymerase. HPIV3/EboGP was substantially immunogenic and protective when given to non-human primates by combined intranasal (IN) and intratracheal (IT) administration, even in animals previously infected with HPIV3. However, immunogenicity depended on IT delivery of vaccine: IN delivery alone was insufficient. This suggested that vector expression beyond the upper respiratory tract was necessary for immunogenicity. IT delivery in humans would not be feasible, but might be substituted by aerosol delivery. The aerosol route has been used by others to immunize humans in large vaccine trials, such as with the measles virus vaccine, and suitable delivery devices exist. We explored delivery of the HPIV3/EboGP construct by the aerosol route in rhesus macaques. The aerosol route was generally more immunogenic and protective than the combined IN/IT route. This included generally higher serum and mucosal EBOV-specific IgG, IgA, and neutralizing antibody titers, as well as EBOV-specific cellular responses in the lungs, including polyfunctional CD8+ T cells and CD4+ T helper cells that were predominately Th1. In addition, the HPIV3/EboGP vaccine produced more robust cell-mediated and humoral immune responses than an alphavirus vaccine delivered parenterally in parallel. One aerosol dose of HPIV3/EboGP conferred 100% protection to macaques against EBOV challenge We performed (with JHU clinical collaborators) an open label phase 1 clinical trial to determine the safety, tolerability, and immunogenicity of HPIV3/EboGP delivered IN in healthy adults (NCT025645750), which was intended to be a safety study prior to evaluating aerosol delivery. Ten subjects received 2 doses (4- to 8-week interval) of 6.0 log10 PFU of vaccine. The first dose was moderately infectious (7/10 subjects shed virus detected by qRT-PCR, mean peak titer 3.8 log10 genomic equivalents/ml, mean duration of shedding 7.9 days). Little shedding was detected after the 2nd dose. A second cohort (n=20) received 1 of 2 planned doses of 7.0 log10 PFU of vaccine. Shedding was similar but of shorter duration (mean of 3.7 days). The vaccine was well tolerated, with the exception that asymptomatic ALT elevations were noted in 5 volunteers (3 mild, 2 moderate) in cohort 2 after vaccination and associated with shedding. All resolved by day 28. The study was halted due to these elevations of ALTs, but their significance is unclear. Because of this, this vaccine will not be administered further at this time. Induction of serum antibodies was poor (mucosal antibodies not yet analyzed), but this was expected since, as noted above, we had previously observed that administration by the IN route alone was poorly immunogenic in rhesus monkeys. We also previously developed a 2nd generation version of HPIV3/EboGP in which the HPIV3 F and HN genes were deleted, leaving EBOV GP as the sole viral surface glycoprotein. A large comparative study in cynomolgus monkeys by our collaborator Alexander Bukreyev at the University of Texas Medical Branch, Galveston, (who made the construct while a Staff Scientist in this laboratory) showed that this 2nd generation version is even more protective than the 1st generation even though it is very highly restricted for replication (much more than the 1st generation construct). We have manufactured clinical trial material of this construct for evaluation in adult volunteers. HPIV1 vector: We previously developed a series of attenuating mutations for human parainfluenza virus type 1 (HPIV1) as part of our program to develop live-attenuated intranasal pediatric HPIV1 vaccines. One of these mutations was a 6-nucleotide deletion in the P/C ORF (C170) that impaired the ability of HPIV1 to inhibit host interferon and apoptosis responses. We used HPIV1 bearing this attenuating mutation as a vector to express a codon-optimized version of the EBOV GP gene, which was inserted either at HPIV1 gene position 1, preceding the N gene (pre-N), or at HPIV1 gene position 2, between the N and P genes (N-P). In addition, EBOV GP was expressed either as the full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were substituted with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and increase immunogenicity. The four resulting constructs grew to high titers in cell culture and efficiently and stably expressed EBOV GP. When administered to African green monkeys by the combined intranasal (IN) and intratracheal (IT) routes, the HPIV1 constructs were attenuated, replicating at low titers over several days. They were substantially more attenuated than the HPIV3/EboGP construct (see HPIV3 vector, above) administered in parallel, which did not contain specific attenuating mutations. The presence of the EBOV GP gene in the HPIV1 backbone increased its attenuation in addition to the C170 mutation. A single dose of the various HPIV1 constructs was poorly immunogenic. Two doses of the HPIV1 candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P candidates, and the TMCT modification did not increase immunogenicity. Even though the HPIV1 constructs were much more attenuated than the HPIV3/EboGP construct included as a positive control, the titers of EBOV-neutralizing serum antibodies achieved following the second dose of the pre-N HPIV1 constructs were similar to that of HPIV3/EboGP. Unmodified EBOV GP was packaged in substantial quantities into the HPIV1 particle. Replacement of the EBOV GP TMCT with that of the HPIV1 vector F protein did not increase packaging. Evaluation of the stability of expression of the EBOV GP protein following in vivo replication by a double-staining immuno-plaque assay showed that expression of the unmodified form of GP was relatively stable, while that of the TMCT forms was substantially less stable. In conclusion, we identified an attenuated and immunogenic HPIV1-based vaccine candidate expressing EBOV GP from the pre-N position. Because of its substantial level of attenuation, this construct is expected to be well-tolerated in humans and is available for clinical evaluation.

我们正在开发人类副磷素病毒（HPIVS）作为对人类使用的疫苗向量，以对抗高度致病的新兴病毒，目前侧重于HPIV血清型1和3。该策略利用了自然呼吸道的热情，利用了HPIV的自然呼吸道对呼吸道的影响，并刺激了强大的系统性免疫和局部质量，并具有重要的系统性免疫效果，并激发了局部质量的质量，并激发了ISASS的影响力，并激发了ISASS的影响。通过呼吸道和结膜。 HPIV3向量： 我们先前构建了一种称为HPIV3/eBOGP的第一代构建体，其中通过在HPIV3 P和M基因之间在第三个基因位置中添加EBOV GP基因来改变完整的HPIV3基因组。 EBOV GP是唯一的EBOV病毒表面蛋白，是唯一的EBOV中和抗原和主要保护性抗原。 EBOV GP基因经过设计，具有适当的HPIV3转录信号，以通过HPIV3聚合酶表示为单独的mRNA。当通过鼻内（IN）和气管内（IT）给药给予非人类灵长类动物时，HPIV3/EBOGP也具有基本的免疫原性和保护性，即使在先前感染了HPIV3的动物中也是如此。但是，免疫原性取决于IT疫苗的输送：仅输送不足。这表明超出上呼吸道的载体表达对于免疫原性是必需的。人类中的IT分娩是不可行的，但可能被气溶胶递送代替。 其他人已经使用了气溶胶途径在大型疫苗试验中免疫人类，例如与麻疹病毒疫苗，并存在合适的递送装置。我们探索了恒河猕猴中的气溶胶路线的HPIV3/eBOGP结构。与/IT路线中的组合相比，气溶胶途径通常更具免疫原性和保护性。这通常包括较高的血清和粘膜EBOV特异性IgG，IgA和中和抗体滴度，以及肺中EBOV特异性的细胞反应，包括多功能的CD8+ T细胞和主要Th1的CD4+ T辅助细胞。此外，与平行于寄生的α病毒疫苗相比，HPIV3/EBOGP疫苗产生的细胞介导的和体液免疫反应更强。 HPIV3/EBOGP的一种气溶胶剂量授予猕猴针对EBOV挑战的100％保护 我们（与JHU临床合作者一起）进行了开放标签1期临床试验，以确定健康成年人提供的HPIV3/eBOGP的安全性，耐受性和免疫原性（NCT025645750），该研究旨在在评估气溶胶交付之前是一项安全性研究。十名受试者接受了2剂（4至8周间隔）为6.0 log10 pfu的疫苗。第一个剂量是中等感染的（7/10受试者脱离了QRT-PCR检测到的病毒，平均峰值滴度3.8 Log10基因组等效物/mL，平均脱落的持续时间为7.9天）。第二剂剂量后检测到很少的脱落。第二个队列（n = 20）接受了2种计划的7.0 log10 pfu疫苗的1个。脱落相似，但持续时间较短（平均为3.7天）。疫苗的耐受性良好，除了在疫苗接种后在队列2中发现5名志愿者（3个温和，2个中度）的无症状ALT升高，并且与脱落有关。所有这些都在第28天解决了。由于这些Alts的这些升高，该研究已停止，但其意义尚不清楚。因此，目前将不进一步施用该疫苗。血清抗体的诱导很差（粘膜抗体尚未分析），但是这是预期的，如上所述，我们先前已经观察到，仅在恒河猴中，仅通过途径给药是免疫原性的。 我们先前还开发了第二代HPIV3/EBOGP，其中删除了HPIV3 F和HN基因，使Ebov GP成为唯一的病毒表面糖蛋白。我们的合作者亚历山大·布克里耶夫（Alexander Bukreyev）在加尔维斯顿（Galveston）的德克萨斯大学医学分公司（他在该实验室中的一名员工科学家制作了构造时）在Cynomolgus Monkeys进行了一项大型比较研究表明，该第二代版本比第一代比第一代人更具保护性，即使对重复的重复性更大（比第一代组成更大）。我们已经制造了这种结构的临床试验材料，以评估成人志愿者。 HPIV1向量： 我们以前曾开发了一系列针对人类副菌病毒1型病毒（HPIV1）的衰减突变，作为我们开发实时肌内鼻内小儿HPIV1疫苗的计划的一部分。这些突变之一是P/C ORF（C170）中的6-核苷酸缺失，它损害了HPIV1抑制宿主干扰素和凋亡反应的能力。我们使用携带这种衰减突变的HPIV1作为矢量来表达EBOV GP基因的密码子优化版本，该版本是在N基因（pre-n）之前或在N基因位置2，N和P基因之间的HPIV1基因位置1插入的。此外，将EBOV GP表示为全长蛋白，或者是工程嵌合形式，在该形式中，其跨膜和细胞质尾巴（TMCT）结构域被HPIV1 F蛋白取代，以增强载体包装到载体中并增加免疫原性。 所得的四个构建体在细胞培养中增长到高滴度，并有效，稳定地表达了EBOV GP。当通过鼻内（IN）和气管内（IT）路线组合向非洲绿色猴子施用时，HPIV1构建体被减弱，在几天内以低滴度复制。与HPIV3/eBOGP构建体相比，它们的衰减要大得多（请参见上面的HPIV3载体）并行给药，该构建体不包含特定的衰减突变。除C170突变外，HPIV1主链中EBOV GP基因的存在增加了其衰减。单剂量的各种HPIV1构建体的免疫原性很差。与N-P候选物相比，从前N位置表达GP的两剂HPIV1候选者会引起更高的GP中和血清抗体滴度，并且TMCT修饰并没有增加免疫原性。即使HPIV1构建体的衰减比HPIV3/EBOGP构建体的衰减得多，但在第二次剂量的NHPIV1构建体后，获得了EBOV中和中和中和的血清抗体的滴度与HPIV3/eboGP的滴度相似。 将未修饰的EBOV GP大量包装到HPIV1粒子中。用HPIV1载体F蛋白的EBOV GP TMCT替换不会增加包装。通过双重染色的免疫斑测定在体内复制后对EBOV GP蛋白表达的稳定性的评估表明，未修饰的GP形式的表达相对稳定，而TMCT形式的表达却大大稳定。 总之，我们确定了一种从前N位置表达EBOV GP的衰减和免疫原性的基于HPIV1的疫苗。由于其大量的衰减水平，预计该结构将在人类中得到良好的耐受性，可用于临床评估。