A SARS-CoV-2 Vaccine Designed for Manufacturability Results in Unexpected Potency and Non-Waning Humoral Response

Abstract

The rapid development of several highly efficacious SARS-CoV-2 vaccines was an unprecedented scientific achievement that saved millions of lives. However, now that SARS-CoV-2 is transitioning to the endemic stage, there exists an unmet need for new vaccines that provide durable immunity and protection against variants and can be more easily manufactured and distributed. Here, we describe a novel protein component vaccine candidate, MT-001, based on a fragment of the SARS-CoV-2 spike protein that encompasses the receptor binding domain (RBD). Mice and hamsters immunized with a prime-boost regimen of MT-001 demonstrated extremely high anti-spike IgG titers, and remarkably this humoral response did not appreciably wane for up to 12 months following vaccination. Further, virus neutralization titers, including titers against variants such as Delta and Omicron BA.1, remained high without the requirement for subsequent boosting. MT-001 was designed for manufacturability and ease of distribution, and we demonstrate that these attributes are not inconsistent with a highly immunogenic vaccine that confers durable and broad immunity to SARS-CoV-2 and its emerging variants. These properties suggest MT-001 could be a valuable new addition to the toolbox of SARS-CoV-2 vaccines and other interventions to prevent infection and curtail additional morbidity and mortality from the ongoing worldwide pandemic.

Keywords: COVID-19; SARS-CoV-2; durable immunity; emerging variants; protection; vaccine.

Figure 1

Detail of the SARS-CoV-2 spike protein in the region 300–600 and MT-001 construct design. (A) Structure of MT-001 construct derived from PDB IDs 7BYR and 7KNE. The RBD construct is color-coded by annotated blocks of amino acid sequence (“regions”, see panel C and [45]). Cysteines are shown as yellow balls. The cell surface target of the RBM, ACE2 (from 7KNE), is shown as a gray molecular surface (left). (B) The MT-001 construct (ribbon) shown in the context of the full-length spike trimer (space-filling model). (C) Schematic of the regions shown in (A). Top: Color-coded region key for the MT-001 construct in (A). NT: N-terminal region (residues 316–332, red); CD1: “core domain 1” region (333–436, magenta); RBM: receptor binding motif (437–508, green); CD2: “core domain 2” region (509–527, cyan); CTD1: “C-terminal domain 1” region (528–594, blue). The 538–590 disulfide bond that stabilizes CTD1 is indicated by a red arrow. Middle: Black bars—Sequence identity per residue between SARS-CoV-2 spike and representative members of the coronavirus superfamily, demonstrating highly conserved regions N- and C-terminal to the RBM (Table S1). Orange bars—Sites of and frequency of mutations in characterized SARS-CoV-2 variants [3]. Lower: Schematic showing the secondary structure and post-translational modifications in the region from residues 300–600 in the SARS-CoV-2 spike protein. Alpha helices are shown as blue cylinders, beta sheets as red arrows, and turns as orange loops. Disulfide bonds are denoted with purple bridges, and N-linked glycosylation sites are denoted with green circles. Bottom: Alignment of the MT-001 construct with the visualized region.

Figure 2

Dose-response and durability of anti-RBD serum IgG levels in mice vaccinated intramuscularly with Alhydrogel-adjuvanted MT-001. (A) Immunization and bleeding schedule. Prime and secondary immunizations of the animals were at weeks 0 and 3, respectively; bleeds were performed on week 5, week 29, and week 52 to provide sera for analyses. Primary and secondary immunizations were with the same amount of MT-001 antigen per animal—1 µg, 3 µg, or 15 µg—for each group of 10 mice. (B) Midpoint (EC₅₀) geometric mean anti-RBD IgG ELISA titers for each dosage group at each time point. GMTs are indicated numerically below each cluster of points.

Figure 3

Immunization of mice with 3 µg of MT-001 antigen in Alhydrogel with or without the TLR-9 agonist co-adjuvant, CpG ODN1826. (A) Schematic illustrating the MT-001 prime-boost regimen and bleeding schedule for 8–10-week-old female BALB/cJ mice (n = 10). (B) RBD-specific IgG binding titers were assessed in mice immunized with 3 μg MT-001 adjuvanted with 500 μg Alhydrogel only (−, open circles) or with 500 μg Alhydrogel plus 20 μg CpG ODN1826 (+, closed circles). Binding antibody responses are displayed at 5, 15, 29, and 47 weeks post-primary immunization. The balanced Th1/Th2 response resulting from the addition of CpG ODN1826 is evidenced by increased RBD-specific IgG1 (C) and IgG2 (D) antibody titers. This corresponded to an increased ratio of IgG2 to IgG1 antibody levels in CpG ODN1826-adjuvanted animals (E). Each circle (open or solid) represents the half-maximal titer for each serum sample averaged across at least three independent ELISAs. Horizontal bars indicate geometric mean titers per dose. p values reflect unpaired t tests between groups (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 4

SARS-CoV-2 challenge of hamsters vaccinated with MT-001 adjuvanted with Alhydrogel and CpG ODN1826. (A) MT-001 prime-boost regimen and SARS-CoV-2 challenge schedule for Syrian golden hamsters. (B) Midpoint hamster RBD-specific IgG ELISA GMTs. (C) Lung viral load in hamsters vaccinated with 10 µg of MT-001 co-adjuvanted with 500 µg Alhydrogel + 100 µg CpG ODN1826, or mock-vaccinated with adjuvants alone, and infected with 10⁵ PFU of SARS-CoV-2 six weeks after the primary immunization (top). Individual data points and mean +/− S.D for MT-001 with adjuvants (open circles; n = 8) or adjuvants alone (open squares; n = 6) is shown. (D) Viral RNA copy numbers in the lungs of hamsters vaccinated with MT-001 or adjuvants alone four days after intranasal infection with SARS-CoV-2. Data were analyzed by non-parametric Mann–Whitney test (** p < 0.01). (E). Representative plates from lung viral load assessment. Lung homogenates from adjuvant-only (control) or MT-001-immunized (MT-001) hamsters were prepared 4 days post-infection and used to infect Vero E6 cells. No plaques are observed with the lung homogenates from MT-001 immunized hamsters even at a 1:10 dilution, while plaques are visible from the lungs of control animals even at a 1:1,000,000 dilution.

Figure 5

SARS-CoV-2 variant neutralization. BALB/cJ mice (n = 10 per group) were immunized twice at a three-week interval with 3 µg MT-001 and 500 µg Alhydrogel, with or without 20 µg ODN1826 (CpG), as indicated. Mice were bled at 29 and 47 weeks post-primary immunization (see Figure 3A), and sera were assayed for antibody binding and neutralization. (A) Anti-RBD midpoint ELISA titers at 29 weeks post-primary immunization were determined using the Wu-1 RBD (WT) or the Delta variant RBD (Delta) as a target. (B) Mouse serum-virus neutralization titers at approximately six months (29 weeks) and eleven months (47 weeks) post-primary immunization with MT-001 + Alhydrogel without (−CpG) or with (+CpG) ODN1826 were determined using SARS-CoV-2 USA-WA1/2020 (US/WA-1) or SARS-CoV-2 isolate hCoV-19/USA/MD-HP20874/2021 (Omicron BA.1) as described in Methods. Geometric mean titers are as indicated. Asterisks indicate statistical significance as determined by a two-tailed Kruskal–Wallis test with subsequent Dunn’s multiple-comparisons test (* p < 0.05; ** p < 0.01).