Paramyxoviruses as Vaccine Vectors Against Highly Pathogenic Viruses

我们之前构建了第一代构建体HPIV3- ebovz GP，其中通过在HPIV3 P和M基因之间的第三个基因位置添加Ebov GP基因来修饰HPIV3 JS株的全基因组。根据以前的临床研究，JS毒株被认为是一种减毒的HPIV3病毒，尽管这种减毒的基础尚不清楚。Ebov GP是唯一的Ebov病毒粒子表面蛋白，是唯一的Ebov中和抗原，也是主要的保护性抗原。Ebov GP基因被设计成具有适当的HPIV3转录信号，以便通过HPIV3聚合酶作为单独的mRNA表达。通过鼻内（IN）和气管内（IT）联合给药给非人类灵长类动物时，HPIV3- ebovz GP具有显著的免疫原性和保护性，即使在先前感染过HPIV3的动物中也是如此。然而，免疫原性取决于疫苗的信息技术递送：单独递送免疫球蛋白是不够的。这表明，载体在上呼吸道以外的表达对于免疫原性是必要的。我们还探索了通过气溶胶途径在恒河猴中递送HPIV3-EbovZ GP结构。气雾剂途径的免疫原性和保护作用一般比IN/IT联合途径更强。这导致血清和粘膜ebov特异性IgG、IgA和中和抗体滴度普遍升高，以及肺中ebov特异性细胞反应，包括以Th1为主的多功能CD8+ T细胞和CD4+ T辅助细胞。此外，HPIV3-EbovZ GP疫苗诱导的细胞介导和体液免疫应答比平行注射的甲病毒疫苗更强。一剂气溶胶剂量的HPIV3-EbovZ GP对猕猴具有100%的保护作用，免受EBOV的攻击。

We previously constructed a first-generation construct called HPIV3-EbovZ GP, in which the complete genome of the JS strain of HPIV3 was modified by the addition of the Ebov GP gene in the third gene position, between the HPIV3 P and M genes. The JS strain is thought to be an attenuated HPIV3, based on previous clinical studies, although the basis of this attenuation is unknown. Ebov GP is the sole Ebov virion surface protein, the sole Ebov neutralization antigen, and the 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-EbovZ GP 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. We had also explored delivery of the HPIV3-EbovZ GP construct by the aerosol route in rhesus macaques. The aerosol route was generally more immunogenic and protective than the combined IN/IT route. This induced 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-EbovZ GP vaccine induced more robust cell-mediated and humoral immune responses than an alphavirus vaccine delivered parenterally in parallel. One aerosol dose of HPIV3-EbovZ GP conferred 100% protection to macaques against EBOV challenge. We performed (with clinical collaborators at the Johns Hopkins Bloomberg School of Public Health) an open label phase 1 clinical trial to determine the safety, tolerability, and immunogenicity of HPIV3-EbovZ GP delivered IN in healthy adults in an inpatient setting (NCT025645750), which was intended to be a safety study prior to evaluating aerosol delivery. Ten subjects received two 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 second dose. A second cohort (n=20) received one of two 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 to be 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 developed a second-generation version of this vector, called HPIV3/delHNF/EbovZ-GP, in which the HPIV3 F and HN genes were deleted, leaving Ebov GP as the sole viral surface glycoprotein. We have initiated a Phase 1 study to evaluate the safety, infectivity, and immunogenicity of this second-generation HPIV3/delHNF/EbovZ GP vaccine candidate when administered intranasally in healthy adults in an inpatient setting (NCT03462004). This vaccine candidate contains EbovZ GP as the sole envelope glycoprotein. Participants are being enrolled sequentially in two cohorts. Participants in Cohort 1 have been randomly assigned to receive two doses of either 6.0 log10 PFU/mL of HPIV3/delHNF/EbovZ-GP vaccine or placebo. The first dose was given on Day 0 and the second dose was given 35 days later. Vaccine replication was evaluated by nasal wash and RT-qPCR and infectivity assays, and serum antibody responses will be measured. As expected, at the 6.0 log10 PFU dose, the HPIV3/delHNF/EbovZ-GP vaccine was marginally infectious, and adverse events were generally mild to moderate. The study was deemed safe to proceed to the evaluation of the higher 7.0 log10 PFU dose. Participants in Cohort 2 will be randomly assigned to receive two doses of either 7.0 log10 PFU/mL of HPIV3/delHNF/EbovZ-GP vaccine or placebo on Days 0 and 28. We recently collaborated with Alexander Bukreyev and colleagues (UTMB Galveston, TX) to compare the qualitative and quantitative humoral parameters of different intranasal vector vaccines expressing EbovZ-GP in cynomolgus macaques. Of the five Ebov GPexpressing mucosal vaccines derived from human and avian paramyxoviral vectors (based on versions of HPIV1, HPIV3, or Newcastle disease virus), the HPIV3/delHNF/EbovZ GP vaccine conferred NHPs with the best protection, leaving them free of disease with near-sterilizing immunity against Ebov. Thus, the continued evaluation of the HPIV3/delHNF/EbovZ GP vaccine candidate in the Phase 1 study (described above) is warranted.