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is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3.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. This involved increasing both the quantity and the quality of the expressed RSV F antigen. Evaluation of several different positions for the RSV F gene in the vector genome identified the second gene position as generally being optimal. Evaluation of several versions of codon-optimization of the RSV F ORF identified the most efficient one, provided by GenScript (GS). The RSV F protein was modified with two missense mutations (called HEK) to be identical to an early-passage isolate of this RSV strain, which reduced fusion and stabilized the trimer. Two additional modifications in particular substantially increased the immunogenicity of vector-expressed F protein: (i) 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). (ii) 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. In work continuing from previous years, we constructed and evaluated more than 35 versions of rB/HPIV3-RSV-F in pre-clinical studies. These constructs included a variety of missense mutations identified by workers in the field as well as with deletion of the F cleavage site, resulting in a single chain protein. These studies resulted in the identification of two lead versions. One is called rB/HPIV3-F2/HEK/GS-opt/DS-Cav1 and has the following characteristics: insertion of RSV F at the second gene position (F2), an early-passage amino acid sequence (HEK), GenScript optimization (GS-opt), and the DS-Cav1 pre-F stabilization. The second lead version, called rB/HPIV3-F2/HEK/GS-opt/DS-Cav1/B3TMCT, is identical except that it also contains the TMCT modification. These candidates presently are being manufactured into clinical trial material for pediatric clinical evaluation. We also used HPIV3 as a vector to express the RSV fusion protein. One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of a modified RSV F gene, which may occur with rB/HPIV3. This project built on scientific insights obtained with B/HPVI3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.