A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats

Abstract

Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants.However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (⌬SH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-⌬SH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log 10 total PFU and 2.7 log 10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. IMPORTANCERSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codondeoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. R espiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants (1). Globally, RSV causes an estimated 3.4 million (1) hospitalizations and 234,000 deaths per year in children under the age of 5 years (2). Almost all children have been infected with RSV by the age of 2 years, with clinical manifestations ranging from upper respiratory tract infections to pneumonia with respiratory failure. Despite the striking burden of RSV disease in children worldwide, no RSV-specific therapies or vaccines are commercially available. The development of a safe and effective RSV vaccine is therefore a public health priority.The initial attempt to develop an RSV vaccine by formalin inactivation (FI-RSV) not only failed to protect ...

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation

“…We then introduced K357 and Y371 into the F of a genetically divergent vaccine strain DB1, which expresses a consensus F gene of the antigenic subgroup B ‘Buenos Aires' (BAF) clade. We previously described the generation of DB1, which also contains codon-deoptimized non-structural protein genes and deleted SH gene, with a genotype RSV-A2-dNS1-dNS2-ΔSH-BAF9. DB1 expressed low levels of pre-F antigen and was thermally unstable; however, incorporation of the K357 and Y371 residues to generate DB1–357/371 enhanced MPE8 binding (Fig.…”

“…The coding region of codon-deoptimized G (dG) was synthesized by GenScript and cloned by restriction digestion and ligation into the A2-K-dNSh-ΔSH-line19F(I557V) BAC yielding A2-K-dNSh-ΔSH-dG-line19F(I557V) yielding the recovery BAC for OE4. The rescue of DB1 was previously described9. DB1–357/371 was generated through introduction of the line 19F residues K357 and Y371 into the DB1 coding sequence.…”

“…The G and F gene segments were cloned into the A2-K BAC by restriction digestion and ligation, and the reporter viruses were recovered by transfection into BSR-T7/5 cells, followed by propagation of stocks in HEp-2 cells as was previously described9.…”

“…However, development of pediatric RSV LAV strains with sufficient attenuation and immunogenicity has been difficult6. To address these dual challenges, newer RSV LAVs have incorporated genetic modifications rationally designed to retain or enhance immunogenicity compared with wild-type virus789 because natural infection may be suboptimally immunogenic for LAVs derived by classic attenuation methods.…”

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

“…With regard to live-attenuated RSV strains, a number of candidates are presently in clinical trials and represent several different mechanisms of attenuation, specifically, temperature-sensitive mutations, which in some cases have been stabilized against deattenuation, combined with deletion of the SH surface protein gene (8,36); deletion of the transcription/replication regulatory protein M2-2, which appears to be very promising (10); and deletion of the interferon antagonist NS2 gene in combination with deletion of a codon in the L gene (48). Other attenuated RSV strains in preclinical development represent strategies including codon deoptimization of the NS1 and NS2 ORFs (49,50), codon-pair deoptimization of various RSV ORFs (51), and the use of less fusogenic F protein (49). An advantage of an attenuated RSV strain is that it provides all or nearly all of the RSV antigens and thus should induce a more complete immune response than a vector expressing one or several RSV ORFs.…”