Toward a pan-SARS-CoV-2 vaccine targeting conserved epitopes on spike and non-spike proteins for potent, broad and durable immune responses

Abstract

Background: The SARS-CoV-2 non-Spike (S) structural protein targets on nucleocapsid (N), membrane (M) and envelope (E), critical in the host cell interferon response and memory T-cell immunity, are grossly overlooked in COVID vaccine development. The current Spike-only vaccines bear an intrinsic shortfall for promotion of a fuller T cell immunity. Vaccines designed to target conserved epitopes could elicit strong cellular immune responses that would synergize with B cell responses and lead to long-term vaccine success. We pursue a universal (pan-SARS-CoV-2) vaccine against Delta, Omicrons and ever-emergent new mutants.

Methods and findings: We explored booster immunogenicity of UB-612, a multitope-vaccine that contains S1-RBD-sFc protein and sequence-conserved promiscuous Th and CTL epitope peptides on the Sarbecovirus N, M and S2 proteins. To a subpopulation (N = 1,478) of infection-free participants (aged 18-85 years) involved in a two-dose Phase-2 trial, a UB-612 booster (third dose) was administered 6-8 months after the second dose. The immunogenicity was evaluated at 14 days post-booster with overall safety monitored until the end of study. The booster induced high viral-neutralizing antibodies against live Wuhan WT (VNT50, 1,711) and Delta (VNT50, 1,282); and against pseudovirus WT (pVNT50, 11,167) vs. Omicron BA.1/BA.2/BA.5 variants (pVNT50, 2,314/1,890/854), respectively. The lower primary neutralizing antibodies in the elderly were uplifted upon boosting to approximately the same high level in young adults. UB-612 also induced potent, durable Th1-oriented (IFN-γ+-) responses (peak/pre-boost/post-boost SFU/106 PBMCs, 374/261/444) along with robust presence of cytotoxic CD8+ T cells (peak/pre-boost/post-boost CD107a+-Granzyme B+, 3.6%/1.8%/1.8%). This UB-612 booster vaccination is safe and well tolerated without SAEs.

Conclusions: By targeting conserved epitopes on viral S2, M and N proteins, UB-612 could provide potent, broad and long-lasting B-cell and T-cell memory immunity and offers the potential as a universal vaccine to fend off Omicrons and new VoCs without resorting to Omicron-specific immunogens.

Trial registration: ClinicalTrials.gov ID: NCT04773067; ClinicalTrials.gov ID: NCT05293665; ClinicalTrials.gov ID: NCT05541861.

Fig 1. UB-612 induced T cell responses measured by ELISpot and ICS analyses.

T-cell responses to stimulation by epitope peptides (RBD + Th/CTL or Th/CTL alone) were analysed with PBMCs collected from 83 vaccinees from Immunogenicity group (n = 83) on Day 57 (28 days after 2^nd dose); and from 32 vaccinees from the Immunogenicity (n = 18) or Safety groups (n = 14) who joined the Phase-2 extension booster study to evaluate the T-cell responses in PBMCs on Days 197 to 242 (pre-boosting days) and Days 211 to 256 (14 days post-booster third dose). T-cell responses were measured by (A) ELISpot at 10-μg/mL per stimulator, in which the Spot-forming units (SFU) per 1×10⁶ PBMCs producing IFN-γ, IL-2 and IL-4 after stimulation with the RBD + Th/CTL peptide pool or the Th/CTL peptide pool are expressed. The PBMC samples stimulated with Th/CTL+RBD were also evaluated for T cell responses by (B) Intracellular Staining (ICS), by which the %CD4⁺ T cells producing IFN-γ, IL-2 and IL-4; and (C) %CD8⁺ T cells producing IFN-γ, IL-2 and CD107a⁺Granzyme B⁺ in response to the stimulation by RBD+Th/CTL peptide pool or the Th/CTL peptide pool are shown. (D) Summary of mean and 95% CI are presented for plots as shown in panels (A) to (C). Horizontal bars indicate mean with 95% CI.

Fig 2. Viral-neutralization effects of UB-612 booster vaccination against wild type, Delta, Omicron BA.1 and BA.2 variants.

Viral-neutralizing titers against SARS-CoV-2 wild-type, Delta, Omicron BA.1, and BA.2 variants were investigated during the infection pandemic that BA.1/BA.2 dominated. Serum samples from 41 participants (n = 27 for 18–65 years; n = 14 for 65–85 years) collected at 14 days post-booster were subjected to a live virus or pseudovirus-luciferase neutralization assay. (A) Live virus assay for Wuhan wild type WT vs. Delta for all ages and (B) live virus assay for WT vs. Delta for young adults and the elderly. (C) Pseudovirus assay for WT vs. BA.1 for all ages and (D) Pseudovirus assay WT vs. BA.1 for young adults and the elderly. (E) Pseudovirus assay for WT vs. BA.2 for all ages and (F) Pseudovirus assay WT vs. BA.2 for young adults and the elderly. The 50% viral-neutralizing antibody geometric mean titers (GMT, 95% CI) were measured, VNT₅₀ for live virus and pVNT₅₀. Statistical analysis was performed by the Student’s t-test (ns, p>0.05; ****, p<0.0001). No significant difference is notable between the two age groups in neutralization effect against WT, Delta, BA.1, and BA.2.

Fig 3. Comparative viral-neutralization effects against wild type strain, Omicron BA.1, BA.2, and BA.5 variants.

At the SARS-CoV-2 pandemic when the Omicron BA.5 variant dominates, viral-neutralizing titers against wild-type, Omicron BA.1, BA.2, and BA.5 variants were measured using pseudovirus assay for comparison. Serum samples from 12 participants (n = 7 for 18–65 years; n = 5 for 65–85 years) collected at 14 days post-booster were subjected to pseudovirus-luciferase neutralization assay. (A) Wuhan wild type WT, BA.1, BA.2, and BA.5 for study participants of all ages, (B) WT vs. BA.1 for young adults and the elderly, (C) WT vs. BA.2 for young adults and the elderly, and (D) WT vs. BA.5 for young adults and the elderly. The 50% viral-neutralizing antibody geometric mean titers (GMT, 95% CI) were measured, pVNT₅₀. Statistical analysis was performed by the Student’s t-test (ns, p>0.05; ****, p<0.0001). No significant difference is notable between the two age groups in neutralization effect against WT, BA.1, BA.2, and BA.5.

Fig 4. Functional correlations between ACE2:RBD_WT binding inhibition and viral-neutralization against Delta, Omicron BA.1, BA.2, and BA.5.

Of 871 participants enrolled in the Phase-2 primary 2-dose series and grouped for Immunogenicity investigation, serum samples from 87 participants who had received a booster 3^rd-dose of 100 μg UB-612 were collected at Day 1 (pre-dose 1), Day 57 (28 days post-dose 2), Day 220 (pre-booster between Days 197 to 242), Day 234 (14 days post-booster between Days 211 to 256). HCS from 20 SARS-CoV-2 infected individuals were also included for comparative testing by two functional assays: (A) viral-neutralizing titer (VNT₅₀) against live wild-type Wuhan strain (WT) by CPE method; and (B) the antibody concentration calibrated with an internal standard for ACE2:RBD_WT binding inhibition by ELISA. The correlations are explored between the two function assays, i.e., ACE2:RBD_WT binding inhibition ELISA and the viral-neutralizing titers against the live virus (VNT₅₀ for original wild-type and Delta strains) or the psuedovirus (pVNT₅₀ for Omicron BA.1 strain). The RBD_WT stands for the RBD binding protein domain bearing amino acid sequence of the original SARS-CoV-2 wild-type (WT) Wuhan strain. The correlations were explored for (C) ACE2:RBD_WT inhibition vs. anti-WT VNT₅₀, (D) ACE2:RBD_WT inhibition vs. anti-Delta VNT₅₀, (E) ACE2:RBD_WT inhibition vs. anti-Omicron BA.1 pVNT₅₀, (F) ACE2:RBD_WT inhibition vs. anti-Omicron BA.2 pVNT₅₀, and (G) ACE2:RBD_WT inhibition vs. anti-Omicron BA.5 pVNT₅₀. The correlation coefficients were evaluated by Spearman r with 95% CI. Statistical analysis was performed with the Student’s t-test (ns, p>0.05; ***, p<0.001; ****, p<0.0001). (H) Summary of geometric mean titer (GMT) and 95% CI are presented for plots as shown in panels (A) and (B).

Fig 5. Functional correlations between pseudovirus and live virus neutralizing titers, pVNT₅₀ and VNT₅₀, against Wild-type and Delta strains.

The correlations between two viral-neutralizing titer assays against pseudo-virus (pVNT₅₀) and live-virus (VNT₅₀) are explored. This case was only available from UB-612 vaccinees participated in the Phase-1 trial, whose serum samples were collected from primary series and booster vaccination and subjected to both neutralization assays against Wuhan wild-type virus (WT) and Delta strain. The correlation coefficients were evaluated by Spearman r with 95% CI.