Nonglycosylated G-Protein Vaccine Protects against Homologous and Heterologous Respiratory Syncytial Virus (RSV) Challenge, while Glycosylated G Enhances RSV Lung Pathology and Cytokine Levels

Abstract

New efforts are under way to develop a vaccine against respiratory syncytial virus (RSV) that will provide protective immunity without the potential for vaccine-associated disease enhancement such as that observed in infants following vaccination with formalin-inactivated RSV vaccine. In addition to the F fusion protein, the G attachment surface protein is a target for neutralizing antibodies and thus represents an important vaccine candidate. However, glycosylated G protein expressed in mammalian cells has been shown to induce pulmonary eosinophilia upon RSV infection in a mouse model. In the current study, we evaluated in parallel the safety and protective efficacy of the RSV A2 recombinant unglycosylated G protein ectodomain (amino acids 67 to 298) expressed in Escherichia coli (REG) and those of glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model. Vaccination with REG generated neutralizing antibodies against RSV A2 in 7/11 BALB/c mice, while RMG did not elicit neutralizing antibodies. Total serum binding antibodies against the recombinant proteins (both REG and RMG) were measured by surface plasmon resonance (SPR) and were found to be >10-fold higher for REG-than for RMG-vaccinated animals. Reduction of lung viral loads to undetectable levels after homologous (RSV-A2) and heterologous (RSV-B1) viral challenge was observed in 7/8 animals vaccinated with REG but not in RMG-vaccinated animals. Furthermore, enhanced lung pathology and elevated Th2 cytokines/chemokines were observed exclusively in animals vaccinated with RMG (but not in those vaccinated with REG or phosphate-buffered saline [PBS]) after homologous or heterologous RSV challenge. This study suggests that bacterially produced unglycosylated G protein could be developed alone or as a component of a protective vaccine against RSV disease. IMPORTANCENew efforts are under way to develop vaccines against RSV that will provide protective immunity without the potential for disease enhancement. The G attachment protein represents an important candidate for inclusion in an effective RSV vaccine. In the current study, we evaluated the safety and protective efficacy of the RSV A2 recombinant unglycosylated G protein ectodomain produced in E. coli (REG) and those of glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model (strains A2 and B1). The unglycosylated G generated high protective immunity and no lung pathology, even in animals that lacked anti-RSV neutralizing antibodies prior to RSV challenge. Control of viral loads correlated with antibody binding to the G protein. In contrast, the glycosylated G protein provided poor protection and enhanced lung pathology after RSV challenge. Therefore, bacterially produced unglycosylated G protein holds promise as an economical approach to a protective vaccine against RSV.

Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes

“…Therefore, it is unlikely that RSV G assumes distinct conformations without additional external stabilizing interactions. One form of stabilization may come from the oligomerization state of RSV G. It was previously suggested that RSV G exists as a trimer or tetramer (57,58). The extensive glycosylation of RSV G in the mucin-like regions flanking the CCD may also restrict RSV G flexibility.…”

Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

“…Lung tissues were evaluated for perivascular eosinophil infiltration. Briefly, vessels with cellular infiltration (n ϭ 20/lung) were randomly selected by a masked pathologist, and the number of eosinophils was enumerated and averaged for a final score for each lung (52).…”

Protective antigenic sites in respiratory syncytial virus G attachment protein outside the central conserved and cysteine noose domains

“…As a comparator, we used a G-ectodomain protein (residues 67–298) of RSV-A2 containing the CCD motif produced using E . coli (REG 67–298), which was previously found to generate protective immunity in mice and cotton rats [ 16 , 17 ]. We evaluated the immunogenicity of a CCD-deleted G-ectodomain [REG ΔCCD; with residues 172–186 replaced by a (Gly 4 Ser) 2 linker], and two large G subdomains covering the N-terminus (REG 67–163) and C-terminus (REG 187–298) upstream and downstream of the CCD, respectively, that were identified as immunodominant in the epitope profiling of post-RSV primary infection human sera ( Fig 1 panels A-B).…”

“…Neutralizing antibody titers represent 50% plaque inhibition. (F) SPR analysis of post-third vaccination serum antibodies (diluted 10-fold) binding to fully glycosylated G ectodomain RMG from RSV-A2 [ 16 ].…”

“…Since in vitro PRNT assays may not detect all antibodies, we also evaluated the binding of immune sera (post-third vaccination) to fully glycosylated G protein from RSV-A2 strain produced in mammalian 293 cells (RMG-A2) [ 16 ]. All four recombinant proteins generated antibodies in rabbits that bound to fully glycosylated G protein ( Fig 1F ).…”