CAP: Trivalent Filovirus Vaccine for Pre- and Post-Exposure Vaccination

我们的主要疫苗平台是基于重组水疱性口炎病毒(RVSVs)，这是一种减毒活疫苗方法。多年来，我们已经产生了几个表达所有种类埃博拉病毒代表性分离株的糖蛋白(GP)的rVSVs：苏丹埃博拉病毒(Sebov)、扎伊尔埃博拉病毒(ZEBOV)、泰林埃博拉病毒(TFEBOV)、本迪布乔埃博拉病毒(Bebov)和雷斯顿埃博拉病毒(ReBOV)。此外，我们还构建了表达马尔堡病毒两个分离株：维多利亚湖马尔堡病毒分离株Musoke和安哥拉分离株的rVSVs。所有疫苗载体在细胞培养中都有广泛的特征，它们的保护效果已经在动物模型(啮齿动物、非人灵长类动物)中进行了评估，以抵御同源挑战。为了破译rVSV疫苗载体的保护机制，我们以rVSV/ZEBOVgp为模型。我们可以在非人类灵长类动物身上证明，针对外来免疫原的抗体在保护中发挥着关键作用。最近的类似工作也证实了抗体在rVSV疫苗载体介导的针对Marv的保护机制中的作用。总体而言，我们假设抗体(总抗体和中和抗体)在保护所有rVSV候选疫苗的机制中发挥关键作用。 为了应对最近在西非爆发的埃博拉疫情，rVSV/ZEBOVgp候选疫苗被快速追踪，并被证明对人类是安全的和具有免疫原性的。在几内亚、塞拉利昂和利比里亚启动了该候选疫苗的第三阶段临床试验。为了支持临床试验，我们已经证明，在西非使用的GMP生产的rVSV/ZEBOVgp疫苗批次可以抵御最近一种当地分离株的挑战，这一证据在试验开始时就缺失了。最近发表在《柳叶刀》上的几内亚人体试验的初步报告报告说，rVSV/ZEBOVgp疫苗在环状疫苗接种方法中取得了成功。这一非凡的结果得到了我们团队最近对非人类灵长类动物进行的另一项研究的支持，该研究探讨了保护所需的最短时间。当给予rVSV/ZEBOVgp至少一周时，我们可以表现出完全保护，而当接近于激发前三天给予时，我们可以表现出部分保护。总体而言，这是埃博拉对策制定过程中的一项里程碑式的成就。 不同的埃博拉和马尔堡病毒物种之间的交叉保护是一个重要的考虑因素，但由于相对较高的遗传变异性和普遍缺乏交叉保护抗体，特别是在属之间，而且在单一属内的物种之间，人们认为很难实现交叉保护。在解决这个问题的第一次尝试中，我们以前使用了三种混合疫苗载体(rVSV/SEBOVgp、rVSV/ZEBOVgp和rVSV/MARVgp)的单次注射方案，并展示了对三种同源病毒的攻击完全保护。我们还进行了另一项概念验证研究，在该研究中，我们评估了用单一疫苗载体(rVSV/ZEBOVgp或rVSV/CIEBOVgp)免疫后的交叉保护，并展示了对异源病毒(Bebov)攻击的部分交叉保护。这表明，基于单价rVSV的疫苗对新出现的丝状病毒物种可能是有用的；然而，跨物种的异源保护仍然具有挑战性，可能取决于通过加强免疫或通过包括多种免疫原来增强免疫反应。总体而言，我们可以得出结论，单价rVSV疫苗载体可以在基因上更接近的挑战病毒物种的情况下提供部分交叉保护。 如上所述，克服这一限制的一种方法是使用混合的单价rVSV疫苗载体，它提供了更广泛的保护，以抵御同源和部分保护，以抵御某些异源挑战。克服交叉保护局限性的另一种方法是使用多价rVSV疫苗载体。在啮齿动物模型的概念验证研究中，分别使用同时表达ZEBOVgp和ANDV糖蛋白或ZEBOVgp和H5血凝素的单个rVSV载体，证明了对ZEBOV和ANDV(ANDV)或ZEBOV和H5N1流感病毒的攻击具有保护作用。这一数据表明，使用双价rVSV载体是一种可行的接种多种病原体的方法。 基于上述结果，在过去的两个财政年度，我们成功地产生了额外的二价和三价rVSV载体，表达两到三种不同的丝状病毒GP，一个作为跨膜蛋白(取代VSV糖蛋白)，以及一个或两个作为可溶性糖蛋白，在载体复制过程中将被分泌。恢复这些重组疫苗病毒被证明是困难的，但最近取得了成功。这些载体的体外特性，包括病毒生长曲线和外源免疫原表达的验证已经完成。在埃博拉和马尔堡仓鼠模型中进行的疗效测试取得了令人振奋的结果；非人类灵长类动物的研究正在进行中。

Our main vaccine platform is based on recombinant vesicular stomatitis virus (rVSVs), a live-attenuate vaccine approach. Over the years we have generated several rVSVs expressing the glycoproteins (GP) of representative isolates of all species of Ebola virus: Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Tai forest ebolavirus (TFEBOV), Bundibugyo ebolavirus (BEBOV) and Reston ebolavirus (REBOV). Additionally, we generated rVSVs expressing the GPs of two isolates of Marburg virus: Lake Victoria marburgvirus isolate Musoke and Angola. All vaccine vectors have been extensively characterized in cell culture and their protective efficacy has been evaluated in animal models (rodents, nonhuman primates) against homologous challenges. In an effort to decipher the mechanism of protection of the rVSV vaccine vectors we used the rVSV/ZEBOVgp as a model. We could demonstrate in nonhuman primates that antibodies specific to the foreign immunogen play a critical role in protection. Recent similar work also confirmed a role of antibodies for the mechanism of protection mediated by the rVSV vaccine vector against MARV. Overall, we postulate that antibodies (total and neutralizing IgG) play a key role for the mechanism of protection for all rVSV-based vaccine candidates. In response to the recent Ebola outbreak in West Africa, the rVSV/ZEBOVgp vaccine candidate was fast-tracked and shown to be safe and immunogenic in humans. Phase III clinical trials with this vaccine candidate were initiated in Guinea, Sierra Leone and Liberia. To support the clinical trials we have shown that the GMP-produced rVSV/ZEBOVgp vaccine lot used in West Africa protects against challenge with a recent local isolate, a proof that had been missing at trial start. A recent preliminary report published in Lancet from the human trial in Guinea reports success of the rVSV/ZEBOVgp vaccine in a ring vaccination approach. This remarkable outcome is supported by another recent study of our group in nonhuman primates looking into the minimum time needed for protection. We could demonstrate complete protection when rVSV/ZEBOVgp was administered at least one week and partial protection when administered as close as three days prior to challenge. Overall, this is a milestone achievement in the development of Ebola countermeasures. Cross-protection among the different Ebola and Marburg virus species is an important consideration, but is thought to be difficult to achieve due to relatively high genetic variability and the general lack of cross-protective antibodies among genera in particular, but also among species within a single genus. In a first attempt to address this issue, we previously used a single-injection protocol with three blended vaccine vectors (rVSV/SEBOVgp, rVSV/ZEBOVgp and rVSV/MARVgp) and demonstrated complete protection against challenge with the three homologous virus species. We have also performed another proof-of-concept study, in which we evaluated cross-protection following immunization with a single vaccine vector (rVSV/ZEBOVgp or rVSV/CIEBOVgp) and demonstrated partial cross-protection against challenge with a heterologous virus species (BEBOV). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging filovirus species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens. Overall, we can conclude that single monovalent rVSV vaccine vectors can provide partial cross-protection in cases of challenge virus species that are genetically more closely related. As mentioned above, one approach to overcome this limitation is the use of blended monovalent rVSV vaccine vectors, which provide broader protection against homologous and partial protection against certain heterologous challenges. Another approach to overcome the limitations in cross-protection is the use of multivalent rVSV vaccine vectors. In a proof-of-concept study in rodent models protection against ZEBOV and Andes virus (ANDV) or ZEBOV and influenza virus (H5N1) challenge was demonstrated using a single rVSV vector expressing both the ZEBOVgp and the ANDV glycoprotein or ZEBOVgp and a H5 hemagglutinin, respectively. This data showed that the use of bivalent rVSV vectors are a feasible approach to vaccination against multiple pathogens. Based on the results described above, we have over the past two fiscal year successfully generated additional bivalent and trivalent rVSV vectors expressing two or three different filovirus GPs, one as a transmembrane protein (replacing the VSV glycoprotein) and one or two as soluble glycoproteins that will be secreted during vector replication. Recovery of these recombinant vaccine viruses turned out to be difficult but has recently been successful. In vitro characterization of these vectors, including viral growth curves and verification of foreign immunogen expression has been completed. Efficacy testing in the Ebola and Marburg hamster models has resulted in promising results; nonhuman primate studies are scheduled.