Laboratory And Pre-clinical Studies Of Parainfluenza Viruses

近年来，我们专注于使用基于PIV 3的载体表达RSV抗原，提供针对两种最重要的儿科病毒性呼吸道病原体的二价疫苗。该载体是我们以前开发的一种载体，称为rB/HPIV 3，它由牛PIV 3组成，其中F和HN基因已被HPIV 3的基因取代。这导致嵌合病毒由于BPIV 3骨架而在非人灵长类动物和人中减毒，并且其携带HPIV 3的中和和主要保护性F和HN抗原。空B/HPIV 3载体和表达未修饰的RSV F蛋白的B/HPIV 3之前均显示在婴儿和幼儿中耐受良好。因此，该矢量似乎在期望的衰减范围内。 在前几年继续进行的工作中，我们继续专注于表达RSV融合F糖蛋白，因为它通常被认为是最重要的RSV中和和保护性抗原。RSV F在RSV毒株中也比附着G蛋白高度保守，附着G蛋白是另一种中和抗原和第二重要的保护性抗原。我们继续评估了许多策略，以优化表达RSV F蛋白的rB/HPIV 3的免疫原性。其中一个修改是通过引入NIH疫苗研究中心和其他地方的同事报告的突变来增加F蛋白融合前构象的稳定性-这是诱导RSV中和抗体最有效的构象。最成功的突变涉及添加二硫键（称为DS突变）与两个空腔填充错义突变（称为Cav 1）的组合。另一种修饰是将RSV F工程化以有效包装在B/HPIV 3载体颗粒中。这是通过用BPIV 3 F的跨膜和胞质尾区（TMCT）结构域替换RSV F的跨膜和胞质尾区（TMCT）结构域完成的。这两种修饰DS-Cav 1和TMCT中的每一种导致血清RSV中和抗体的诱导显著增加，特别是在体外有效中和RSV而不添加补体的抗体，因此在中和中非常有效。这些主要候选药物的临床研究材料已经准备和表征，并将在2024年的1期研究中进行评估。在过去的一年中，已经编写了监管文件，目前正在编写临床研究方案。

In recent years, we have focused on using a PIV3-based vector to express RSV antigen, providing a bivalent vaccine against the two most important pediatric viral respiratory pathogens. The vector is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3. This results in a chimeric virus that is attenuated in non-human primates and humans due to the BPIV3 backbone, and which bears the neutralization and major protective F and HN antigens of HPIV3. Both the empty B/HPIV3 vector and B/HPIV3 expressing the unmodified RSV F protein were previously shown to be well-tolerated in infants and young children. Therefore, this vector appears to be in the desired range of attenuation. In work continuing from previous years, we continued to focus on expressing the RSV fusion F glycoprotein because it generally is considered to be the most important RSV neutralization and protective antigen. RSV F also is much more highly conserved among RSV strains than the attachment G protein, which is the other neutralization antigen and the second most important protective antigen. We continued to evaluate a number of strategies to optimize the immunogenicity of rB/HPIV3 expressing RSV F protein. One modification was to increase the stability of the pre-fusion conformation of the F protein - the conformation that is the most effective in inducing RSV-neutralizing antibodies - by introducing mutations that have been reported by colleagues in the NIH Vaccine Research Center and elsewhere. The most successful mutations involved addition of a disulfide bond (called the DS mutation) in combination with two cavity-filling missense mutations (called Cav1). The other modification was to engineer RSV F to be efficiently packaged in the B/HPIV3 vector particle. This was done by replacing the transmembrane and cytoplasmic tail (TMCT) domains of RSV F with those of BPIV3 F. Each of these two modifications, DS-Cav1 and TMCT, resulted in a substantial increase in the induction of serum RSV-neutralizing antibodies, and in particular antibodies that neutralized RSV efficiently in vitro without added complement and thus are highly effective in neutralization. Clinical study material of these lead candidates has been prepared and characterized and will be evaluated in a Phase 1 study in 2024. In the past year, regulatory documents have been prepared, and a clinical study protocol is currently in preparation.