Enhancing Anti-G Antibody Induction by a Live Single-Cycle Prefusion F—Expressing RSV Vaccine Improves In Vitro and In Vivo Efficacy

Abstract

The human respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease, and a vaccine is not available. We previously reported a novel live vaccine expressing prefusion-stabilized fusion protein (preF) in place of the native F protein (RSV-preFΔCT). As preF is non-functional, RSV-preFΔCT was amplified in a production line expressing a functional substitute, and exhibited a single-cycle replication phenotype, which holds several unique potential advantages. RSV-preFΔCT prevented shedding and lung pathology after viral challenge in mice, but induced low levels of anti-attachment protein (G) antibodies (Abs). Given the significant contributions of anti-G Abs toward disease prevention, we generated modifications to RSV-preFΔCT in an effort to induce higher anti-G Ab levels. The Ab levels were monitored after the prime-boost vaccination of mice with modified vaccines. The most successful modification for enhancing induced anti-G Abs was seen with the placement of G in the first genome position. This vaccine also reduced the pathology after challenge with a high dose of wt RSV, and outperformed the sera from wt RSV-vaccinated mice in in vitro neutralization. Thus, raising the anti-G Ab levels induced by RSV-preFΔCT enhanced efficacy in vitro and in vivo, and constitutes an important next step in developing a live, single-cycle, efficacious vaccine for the human population.

Intranasal Vaccination with a Respiratory-Syncytial-Virus-Based Virus-like Particle Displaying the G Protein Conserved Region Induces Severe Weight Loss and Pathology upon Challenge with Wildtype Respiratory Syncytial Virus

“…Mice vaccinated with wt RSV showed significant protection relative to mock-vaccinated mice. This is in agreement with our previous work [ 32 , 82 ], in which we also noted that interstitial pneumonia and edema scores correlated well with overall vaccine effectiveness [ 32 ]. These two disease parameters, as well as neutrophils and mucus, had a score of 0 for wt RSV-vaccinated challenged mice ( Figure 5 C).…”

“…After primary Ab incubation, plates were processed and developed as described above for cell ELISA. Note that the G, preF, and postF antigens and ELISAs were validated in previous work [ 32 , 82 ]. The N antigen and anti-N Ab ELISA were validated in Figure S4 .…”

“…Lungs were fixed in 10% neutral-buffered formalin and then routinely processed through graded alcohols and xylene, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) (Sigma, St. Louis, MO, USA. Slides were scored for parameters assessed for RSV-induced pathology, scoring from 0 (no lesions) to 3 (marked lesions), attributed to perivascular cuffing (PVC; leukocytes surrounding blood vessels), peribronchiolar cuffing (space and fluid surrounding bronchioles), interstitial pneumonia (thickness of alveolar septa; leucocytes in the alveolar space), edema (flooding of lymph vessels surrounding blood vessels and airways), mucus, eosinophil influx, and neutrophil influx [ 32 , 82 ]. Lesion scoring was performed by board-certified (ACVP) veterinary pathologists blinded to study groups.…”