A Computationally Designed Prefusion Stabilized Human Metapneumovirus Fusion Protein Vaccine Antigen Elicited a Potent Neutralization Response

Figure 1. Expression and characterization of human metapneumovirus (hMPV) fusion protein (F) designs. (A) Expression levels of the 20, prefusion (pre-F) stabilized, candidate designs and the benchmark. Colors indicate the stabilization strategy used. Dashed line indicates expression level of the D185P benchmark. (B) Size-exclusion chromatography (SEC) traces of the pre-F benchmark and the three highest yielding designs (N = 3). (C) Thermograms comparing the melting temperatures (Tm) of the benchmark and the two designs with >90% trimer. Tm and colloidal stability were determined by nano differential scanning fluorimetry (N = 3).

Figure 2. Immunogenicity of pre-F constructs and benchmarks in mice. Groups of eight mice were intramuscularly immunized with 0.5 µg of the indicated hMPV F constructs adjuvanted with Alhydrogel and boosted with the same regimens 21 days post-prime. Blood was collected just before the first immunization (Day 0) and 2 weeks after the second immunization (Day 35). (A) hMPV pre-F binding antibody (Ab) titers determined by ELISA. (B) Neutralizing Ab titers determined by microneutralization against hMPV subtype A2 strain expressing GFP. Day 0 samples were run as group pools. Dashed lines represent lower limits of quantification. Error bars represent one geometric standard deviation from the geometric mean. * = p < 0.05. Brackets below the graphs indicate the subtypes of the base strains of the constructs.

Figure 3. Site specific antibody competition of murine sera. ELISA plates coated with N46V_T160F were incubated with Day 35 serum pools from the indicated groups (eight mice per group). Plates were washed and incubated with biotinylated monoclonal Abs (mAbs), then washed and developed with horseradish peroxidase conjugated anti-Biotin. The 50% inhibitory concentrations were reported as decreases in optical density relative to the control wells receiving only the biotinylated mAbs. The dashed line represents the lower limit of quantification.

Figure 4. Cross-neutralization assessment of N46V_T160F immunized mouse sera. NAb titers in Day 35 sera of the eight were mice determined against prototype hMPV strains from subtypes (A) A1 (NL/1/00), (B) A2 (NL/17/00), (C) B1 (NL/1/99), and (D) B2 (NL/1/94) via PRNT60 assays. Four replicates of an in-house generated adult human sera pool were tested in each assay as an intra- and inter-assay control and internal neutralization benchmark. Dashed lines represent lower limits of quantification. Error bars represent one geometric standard deviation from the geometric mean.

Figure 5. Evaluation of the immune response to the N46V_T160F pre-F construct and additional benchmarks in mice. Groups of eight mice were intramuscularly immunized with 0.5 µg of the indicated hMPV F constructs adjuvanted with Alhydrogel and boosted with the same regimens 21 days post-prime. Blood was collected one day prior to the first (Day −1) and 2 weeks after the second (Day 35) immunization. (A) hMPV pre-F binding Ab titers were determined by ELISA. (B) NAb titers were determined by microneutralization against hMPV subtype A2 strain expressing GFP. Day −1 samples were run as group pools. Dashed lines represent lower limits of quantification. Error bars represent one geometric standard deviation from the geometric mean. * = p < 0.05, *** = p < 0.001.

Figure 6. Stability of the pre-F constructs. (A) Binding of 338 (site II), San27-14 (site V), and San32-2 (site Ø) IgG to D185P and N46V_T160F stored for up to 6 months at 4 °C measured by bio-later interferometry normalized to 100% at Day 0 (N = 3). (B) Binding of San32-2 (site Ø), San27-14 (site V), and 338 (site II) to heat-treated N46V_T160F (blue) and control D185P (green). Samples were heated to the different temperatures shown on the X-axis and binding levels were normalized to initial binding levels of untreated samples.