A second-generation molecular clamp stabilised bivalent candidate vaccine for protection against diseases caused by respiratory syncytial virus and human metapneumovirus

Abstract

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two medically important causes of respiratory tract infections and diseases. After more than five decades of research and development, vaccines have recently been approved for the prevention of lower respiratory tract disease caused by RSV. However, vaccines for hMPV remain in early-stage development. Here we describe the design and characterisation as well as pre-clinical development of a bivalent vaccine, VXB-241, comprised of the recombinantly expressed viral fusion proteins from both RSV and hMPV, stabilised in their pre-fusion conformation by combining the use of two technologies, the second-generation molecular clamp (MC2S) and key pre-fusion stabilizing mutations. Each of the two antigens were produced at high yield in a mammalian expression system and purified by an affinity capture resin specific to MC2S. Each antigen was demonstrated to adopt the pre-fusion conformation, which was stable for at least twelve months in liquid formulation at 2-8°C. Head-to-head evaluation in mouse immunogenicity studies showed that the VXB-241 candidate vaccine induced a neutralising immune response that was superior or equivalent to the pre-fusion stabilised comparator antigens for either RSV or hMPV, including the RSVPreF3 antigen of the licensed RSV vaccine, Arexvy (GSK). The results presented here have supported progression of VXB-241 into a Phase 1 clinical trial which commenced enrolment in August 2024 (ClinicalTrials.gov ID NCT06556147).

Fig 1. In vitro characterisation of RSV preF and hMPV preF candidate vaccine antigens.

(A-F) SEC analysis of RSV and hMPV antigen panels using a Superdex 200 Increase 10/300 GL (Cytiva). The UV absorbance profiles in mAU are subdivided as ‘aggregate’ (red), ‘trimer’ (blue) and ‘monomer’ (green), with the percentage of total antigen calculated by area under curve (AUC). (G-H) mAb dissociation constant (K_D) for the RSV and hMPV antigens determined by ELISA and calculated by one site specific binding and plotted with SE. Analysis was performed using GraphPad Prism version 10.0.2 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

Fig 2. Research-scale candidate vaccine immunogenicity in BALB/c mice.

(A) Serum IgG titre to the RSV F antigens (blue), hMPV F antigens (purple), and the IgG titre specific to either the foldon or MC2S trimer stabilising domain (orange). Titres were determined by ELISA and expressed as EC₅₀ values. (B) Relative proportion of foldon/MC2S reactivity calculated as a percentage of the total antigen specific titre. (C/D) Serum virus neutralisation titre determined in a plaque reduction neutralisation test (PRNT₅₀) against RSV A2 and hMPV (CAN97-83). Geometric means with geometric SD are plotted. Mann Whitney Test was performed to assess the statistical significance of differences between VXB-213 and comparator DSCav1 and between VXB-221 and comparator DSCavEs2. The limit of detection (LoD) is defined as half of the initial dilution factor used in the titration and is indicated with a dotted line. Analysis was performed using GraphPad Prism version 10.0.2 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

Fig 3. Immunogenicity of 50 L scale antigen preparations in BALB/c mice.

(A) Serum IgG titre to VXB-213 (blue), VXB-221 (purple) or Nipah F containing either foldon or MC2S(orange). (B) The EC₅₀ values from the serum titrations are expressed as percentages of total IgG to show the proportion of total IgG directed towards the trimer stabilising domains. ANOVA Dunn’s multiple comparisons test with only significant values >0.05 shown (C) Serum virus neutralisation titre (PRNT₅₀) determined against RSV A2. Mann Whitney Test was performed to assess statistically significant differences between VXB-213 and comparator RSVpreF3 antigen from Arexvy (GSK) at both the 0.5 and 3 µg dose levels. (D) Serum virus neutralisation titre (PRNT₅₀) determined against hMPV (strain CAN97-83). ANOVA Dunn’s multiple comparisons test was performed to compare between four active groups and found to be non-significant. In A-D geometric means with geometric SD are plotted. The limit of detection (LoD) is defined as half of the initial serum dilution factor. Analysis was performed using GraphPad Prism version 10.0.2 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.