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双价疫苗。这个载体是我们之前开发的一个称为RB/HPIV3的载体，它由牛PIV3组成，其中F和HN基因已经被HPIV3取代。这导致了一种嵌合病毒，由于BPIV3主干，它在非人类灵长类动物和人类中减弱，并携带HPIV3的中和和主要保护性F和HN抗原。空的B/HPIV3载体和表达未经修饰的RSV F蛋白的B/HPIV3载体先前被证明在婴幼儿中都具有良好的耐受性。因此，该向量似乎在所需的衰减范围内。 在前几年的继续工作中，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蛋白保守得多，附着G蛋白是另一种中和抗原，也是第二重要的保护性抗原。我们继续评估一些策略来优化表达RSV F蛋白的Rb/HPIV3的免疫原性。这包括提高表达的RSV F抗原的数量和质量。对RSV F基因在载体基因组中的几个不同位置的评估表明，第二个基因位置通常是最佳的。对RSV F ORF密码子优化的几个版本的评估确定了最有效的密码子优化版本，由GenScrip(GS)提供。RSV F蛋白被两个错义突变(称为HEK)修饰为与该RSV毒株的早期传代分离株相同，这减少了融合并稳定了三聚体。另外两种修饰尤其显著地提高了载体表达的F蛋白的免疫原性：(I)一种修饰是通过引入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--这种构象是诱导RSV中和抗体最有效的构象。最成功的突变包括添加一个二硫键(称为DS突变)和两个充满空洞的错义突变(称为Cav1)。(Ii)另一种修饰是将RSV F有效地包装在B/HPIV3载体颗粒中。这是通过用BPIV3 F替换RSV的跨膜和细胞质尾部(TMCT)结构域来实现的。DS-Cav1和TMCT这两种修饰都能显著增加血清中和RSV抗体的诱导，特别是在体外无需添加补体就能有效中和RSV的抗体，从而具有很高的中和效率。 在延续前几年的工作中，我们在临床前研究中构建和评估了超过35个版本的RB/HPIV3-RSV-F。这些结构包括现场工作人员发现的各种错义突变以及F裂解位点的缺失，从而产生单链蛋白。这些研究的结果是确定了两个主要版本。一种命名为RB/HPIV3-F2/HEK/GS-opt/ds-Cav1，具有以下特征：在第二基因位置插入RSV F(F2)、早期传递氨基酸序列(HEK)、GenScrip优化(GS-opt)和DS-Cav1前F稳定。第二个铅版本称为RB/HPIV3-F2/HEK/GS-OPT/DS-CAV1/B3TMCT，除了它还包含TMCT修改外，它是相同的。这些候选药物目前正在被制成临床试验材料，用于儿科临床评估。 我们还利用HPIV3作为载体表达了RSV融合蛋白。一个优点是rHPIV3表达所有的HPIV3抗原，而Rb/HPIV3只表达两个。此外，使用rHPIV3作为载体应避免添加修改的RSV F基因后的过度衰减，这种情况可能发生在Rb/HPIV3中。该项目建立在通过B/HPVI3获得的科学见解的基础上。为了增强其免疫原性，对RSV F进行了以下两方面的修饰：(1)提高了预融合(Pre-F)构象的稳定性；(2)用HPIV3F(H3TMCT)的跨膜(TM)和胞浆尾部(CT)结构域取代了其跨膜区(TM)和细胞质尾区(CT)，以增加对载体病毒粒子的掺入。RSV F(+/-H3TMCT)在rHPIV3基因的第一位(F/pren)或第二位(F/N-P)表达。H3TMCT的修饰显著增加了RSV F在载体病毒粒子中的包装，在仓鼠中，除了F前稳定带来的增加外，还导致高质量血清中和RSV抗体的滴度显著增加。只有F-H3TMCT/pren在鼻甲中的复制通过RSV F插入而显著减弱。F-H3TMCT/PREN、F/N-P和F-H3TMCT/N-P可完全抵抗野生型呼吸道合胞病毒的攻击。F-H3TMCT/N-P是RSV F最稳定和最高表达的载体，为其进一步发展提供了动力。

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is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3.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