Laboratory And Pre-clinical Studies Of Parainfluenza Viruses

近年来，我们致力于利用基于piv3的载体表达RSV抗原，提供针对两种最重要的儿科呼吸道病毒病原体的二价疫苗。该载体是我们以前开发的一种，称为rB/HPIV3，它由牛PIV3组成，其中F和HN基因已被HPIV3基因所取代。这就产生了一种嵌合病毒，这种病毒在非人灵长类动物和人类中由于BPIV3骨干而被减毒，并且具有HPIV3的中和和主要保护性F和HN抗原。空的B/HPIV3载体和表达未修饰的RSV F蛋白的B/HPIV3在婴儿和幼儿中均表现出良好的耐受性。因此，这个矢量似乎在期望的衰减范围内。

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is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3.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 2023.