Durr, E. | Search bioRxiv

bioRxiv2026

Structure-based design of soluble prefusion-stabilized herpes simplex virus type 2 glycoprotein B antigens

Herpes simplex virus type 2 (HSV-2) causes genital herpes through latent infection and periodic reactivation. Although antivirals alleviate symptoms, no prophylactic or therapeutic vaccines have been licensed. Glycoprotein B (gB) is a class III fusion protein that mediates entry by irreversibly transitioning from a metastable prefusion conformation to a stable postfusion conformation. Leveraging prior structure-based designs for human cytomegalovirus (HCMV) gB, we engineered amino acid substitutions in HSV-2 gB to stabilize its prefusion conformation. Cryo-EM of the engineered construct revealed a prefusion conformation and non-native dimers of gB trimers. Introduction of N-linked glycosylation sites resulted in the gB-G3 variant, which exhibited improved expression and reduced dimerization. Cryo-EM of gB-G3 bound to neutralizing antibodies yielded a 2.8 [A] resolution structure of the stabilized prefusion conformation in a closed state, which differs from the open states observed in recently published HSV gB structures. Both prefusion and postfusion gB variants were evaluated for immunogenicity in mice, delivered either as a protein subunit or mRNA vaccine. Each gB conformation elicited robust humoral and cellular responses. However, prefusion stabilization of gB did not improve neutralizing antibody titers relative to the postfusion construct, consistent with prior observations for HCMV gB. Collectively, these findings reveal insights into prefusion gB conformational dynamics, provide stabilized reagents for studying gB-directed immune responses and inform HSV-2 vaccine design.

microbiology↗

bioRxiv2022

The Respiratory Syncytial Virus G Protein Enhances the Immune Responses to the RSV F Protein in an Enveloped Virus-like Particle Vaccine Candidate

Respiratory syncytial virus (RSV) is a serious human respiratory pathogen, but no RSV vaccine has been licensed. Many of the vaccine candidates are focused on the viral F protein. However, it is the G protein that binds the likely receptor, CX3CR1, in human alveolar lung cells raising the question of the importance of the G protein in vaccine candidates. Using virus-like particle (VLP) vaccine candidates, we have directly compared VLPs containing only the pre-fusion F protein, only the G protein, or both glycoproteins. We report that VLPs containing both glycoproteins bind to anti-F protein specific monoclonal antibodies differently than VLPs containing only the pre-fusion F protein. Using RSV naive cotton rats as an animal model, we have found that VLPs assembled only with the pre-F protein stimulated extremely weak neutralizing antibody (NAb) titers as did VLPs assembled with G protein. However, VLPs assembled with both glycoproteins stimulated quite robust neutralizing antibody titers, titers that were significantly higher than the combined titers induced by pre-F only or G only VLPs. VLPs assembled with both glycoproteins induced improved protection of the animals from RSV challenge compared to pre-F VLPs and induced significantly higher levels of antibodies specific for F protein antigenic sites 0, site III, and AM14 binding site compared with VLPs containing only the pre-F protein. These combined results indicate that assembly of pre-F protein with G protein in VLPs further stabilized the pre-fusion conformation or otherwise altered the conformation of the F protein increasing the induction of protective antibodies. ImportanceRSV causes significant disease in infants, young children, and the elderly. Thus, development of an effective vaccine for these populations is a priority. Most ongoing efforts in RSV vaccine development have focused on the viral fusion (F) protein, however, the importance of inclusion of G in vaccine candidates is unclear. Here, using VLPs assembled with only the F protein or only the G protein or both glycoproteins, we show that VLPs assembled with both glycoproteins are a far superior vaccine, in a cotton rat model, than VLPs containing only F protein or only G protein. The results show that the presence of G protein in the VLPs influences the conformation of the F protein and the immune responses to F protein resulting in significantly higher neutralizing antibody titers and better protection from RSV challenge. These results suggest that inclusion of G protein in a vaccine candidate may improve its effectiveness.

microbiology↗