A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates

Abstract

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ~one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use including in infants.

Fig. 1. DCFHP design and validation.

A DCFHP schematic showing the modifications made to convert S∆C-Fer into DCFHP in red. Receptor binding domain (RBD), N-terminal domain (NTD), S1/S2 cleavage site, S2’ cleavage site, fusion peptide (FP), heptad repeat 1 (HR1), as annotated. B SDS-PAGE gel showing purified DCFHP running as a monomer at the anticipated kDa molecular weight (ladder, shown left). The gel is representative of the numerous DCFHP samples run by SDS-PAGE. C UV (yellow) and light scattering (gray) traces determined from SEC-MALS shows a homogenous nanoparticle peak with approximate molecular weight (dashed line) of 3.4 MDa. D 3D reconstructed cryo-EM density maps of DCFHP, refined with octahedral symmetry. E Similar robust neutralization of Wuhan-1 SARS-CoV-2 pseudovirus with day 21 serum from mice immunized with either S∆C-Fer or DCHFP formulated with 500 µg alum and 20 µg CpG 1826, following a single immunization. Neutralization titers were assessed in a HeLa cell line expressing ACE2 and TMPRSS2. Data for 10 mice are presented as geometric mean titer and standard deviation. Assay limit of quantitation (LOQ) is shown as a dotted horizontal line.

Fig. 2. Formulated DCFHP-alum is thermostable up to 37 °C for 14 days.

Neutralization titers against Wuhan-1 SARS-CoV-2 pseudovirus for serum obtained from individual animals 42 days following immunization with DCFHP-alum (10 µg DCFHP with 150 µg alum) that had been stored at a range of temperatures (bottom) for either 7 days or 14 days, compared to freshly formulated DCFHP-alum (left, black circles). Assay limits of quantitation is shown as a dotted horizontal line. A single representative experiment of samples run in technical duplicate is shown. GMT and STD are shown.

Fig. 3. DCFHP-alum immunized NHPs elicit cross-reactive neutralizing responses.

A Immunization scheme for NHPs immunized in either group A or group B with a 50 µg dose of DCFHP formulated with 750 µg alum (SI Table 2). Arrows indicate days of immunization. B Pseudoviral neutralization (plotted as the log of the neutralizing titer (reciprocal serum dilution)) of Wuhan-1 SARS-CoV-2 from NHP serum obtained 21 days following initial immunization are similar between groups A and B. C Cross-reactive pseudoviral neutralization by serum from NHPs isolated 14 days post boost. Means and standard deviations for biological replicates are plotted and noted for each animal (n = number of replicate neutralization assays conducted for these samples on independent days). D As in panel (C) for group B. E Limited dilution, neutralization values for authentic SARS-CoV-2 VOCs for serum samples obtained 14 days post-boost. NHP identification provided correlate with SI Table 1. Assay limits of quantitation indicated by horizontal dotted line. n is defined as individual replicates of the experiment, GMT and STD are shown.

Fig. 4. DCFHP-alum immunized NHPs elicit long-lived immunity against both Wuhan-1 and BA.4/5 pseudoviruses.

A Serum neutralizing titers were monitored over 337 days against Wuhan-1 SARS-CoV-2 pseudovirus for animals in group A; days of prime and boost indicated with arrows. B As in panel (A) but against BA.4/5 pseudovirus. C, D As in panels (A) and (B) but with group B NHPs, respectively. Averages and standard deviations for replicate neutralization assays are shown; for panels A–D: n = 3, n = 4, n = 2, and n = 3, respectively (n = number of replicate neutralization assays conducted for these samples on independent days). NHP identification provided correlate with SI Table 1. Assay limits of quantitation indicated by horizontal dotted lines.

Fig. 5. Robust serum neutralizing anamnestic responses following a second booster of DCFHP-alum after ~one year in NHPs.

A Pseudovirus neutralization against Wuhan-1 (A), BA.4/5 (B), SARS-1 (C), or BQ.1 (D) by antisera from NHPs in group A following a boost at day 381. Days shown on x axis. E–H As in panels (A–D) but with NHPs in group B. NHP identification provided correlate with SI Table 1 (n = 2, n = number of replicate neutralization assays conducted for these samples on independent days). Assay limits of quantitation indicated by horizontal dotted lines. Significance was tested between days 395 and days 381 using a nonparametric, one-way ANOVA comparing pre- and post-boost at day 381. ns = P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. GMT (bars) and STD (for each animal) are shown. P = < 0.0001, <0.0001, 0.0013, <0.0001, 0.0006, 0.0043, 0.0298, 0.0232 for A–H, respectively.

Fig. 6. DCFHP-alum immunized NHPs shows a balanced distribution of Th1 and Th2 CD4 + T cell responses.

T cells from animals in group A, isolated on the day shown on the x-axis, were stimulated with a peptide pool derived from Wuhan-1 (A) or Omicron BA.1 (B) spike protein and Th1 (top) and Th2 (bottom) cytokines were measured using flow cytometry (SI Fig. 9). Percent of CD4 + T cells that express either Th1 (IL-2, IFNg, or TNFa) (yellows and reds) or Th2 (IL-4) (grays) cytokines following stimulation shows both are elicited following vaccination with DCFHP-alum. Arrow denotes day of boost. C, D As in panels (A) and (B) but with NHPs in group B. NHP identification provided correlate with SI Table 1. Percentages are shown as difference between treated samples and DMSO-treated control samples for a single experiment.