A novel RBD-protein/peptide vaccine elicits broadly neutralizing antibodies and protects mice and macaques against SARS-CoV-2

Abstract

The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins (three from S2 subunit, one from membrane and one from nucleocapsid), and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague-Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster.

Keywords: SARS-CoV-2; neutralizing antibody; non-human primate; protection; subunit vaccine.

Figure 1.

Antibody responses in UB-612 immunized BALB/C mice. (A) Immunization and challenge schedule. (B) Temporal RBD-specific antibody titers measured by ELISA. Arrows indicate immunization timepoints. Statistical significance is indicated as * p < 0.05 between 3 and 9 µg groups; ** p < 0.01 between 9 and 30 µg groups; *** p < 0.005 between 3 and 30 µg groups. (C) RBD:hACE-2 binding inhibition titers at 2 weeks after the 2^nd immunization. Each bar represents GMT ± SD NAb titers with standard deviation. The red dash line indicates the assay cut-off. Statistical significance is indicated as * p < 0.05; ** p < 0.01 compared to Saline group. (D) NAb titers against SARS-CoV-2 WT virus at 2 weeks after the 2^nd immunization. Statistical significance is indicated as ****p < 0.0001 between each vaccine dose and Saline group.

Figure 2.

Protective immunity against SARS-CoV-2 challenge in AAV/hACE-transduced BALB/c mice. (A) Temporal body weight changes (%) to the baseline prior challenge. Each curve represents a group geometric mean with standard deviation. The statistical significance is indicated as * p < 0.05, each vaccine group compared to Saline group. (B) Lung viral RNA titers . (C) Lung virus TCID₅₀. (D) Lung pathology scores. Each bar represents GMT ± SD on Day 5 post-challenge in panels B, C and D. The red dash line indicates the assay cut-off. Statistical significance is indicated as * p < 0.05; ** p < 0.01 compared to Saline group.

Figure 3.

Antibody responses and protection in rhesus macaques. (A) Vaccine groups, dosing, and challenge schedule. (B) RBD-specific antibody responses at 2 or 6 weeks after the 2^nd immunization and 1 week after the 3^rd immunization. (C) CPE NAb titers against SARS-CoV-2 WT strain at 2 or 6 weeks after the 2^nd immunization and 1 week after the 3^rd immunization. (D) Lung viral RNA levels at 7 days post-challenge. Each bar represents GMT ± SD NAb titers or viral RNA loads. Statistical significance is indicated as * p < 0.05; ** p < 0.01 compared to Saline group.

Figure 4.

Serum NAb responses against SARS-CoV-2 VOCs and VOIs in UB-612 immunized rhesus macaques. (A) CPE NAb titers in group-pooled macaque sera against live viruses: D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.v2) in 30 and 100 μg dose groups at 1 week after the 3^rd immunization. The numbers on top of each bar represent the NAb titer in group-pooled sera. (B) NAb titers against SARS-CoV-2 pseudoviruses expressing S protein from original Wuhan strain (WT), 3 VOCs: Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1), and 2 VOIs: Epsilon (B.1.429) and Iota (B.1.526). Sera were collected at 2 or 6 weeks after the 2^nd immunization, and 1 week after the 3^rd immunization. Each bar represents GMT ± SD NAb titer, with GMT value above.

Figure 5.

Antibody responses in UB-612 immunized cynomolgus macaques. (A) UB-612 vaccine and saline immunization groups, dosing, and challenge schedule. (B) Temporal RBD-specific antibody responses. (C) Temporal NAb responses against SARS-CoV-2 WA strain. The arrows indicate the time points of immunization. Each curve represents GMT ± SD.

Figure 6.

NAb titers against SARS-CoV-2 WA stain, Delta (A) and Omicron (B) VOCs in cynomolgus macaque sera collected 3 weeks post the 2^nd immunization. A. CPE NAb titers; B. Microneutralization NAb titers. The bar represents group GMT ± SD, with GMT value above.

Figure 7.

SARS-CoV-2 S2/M/N peptide specific IFN-γ T-cell responses in cynomolgus macaque PBMCs collected at 3 weeks post the 2^nd UB-612 immunization. ELISpot (A) and intracellular cytokine staining of CD8 (B) and CD4 (C) T-cells. The IFN-γ T-cell ELISpots, CD8 and CD4 responses are presented as GMT ± SD. Statistical significance between groups is indicated, *p < 0.05; **p < 0.01.

Figure 8.

Protection against SARS-CoV-2 WA strain IT/IN challenge in cynomolgus macaques at 3 weeks post the 2^nd immunization. Animals were immunized with saline, 30 or 100 μg of UB-612 vaccine. The temporal viral sgmRNA levels detected in BAL (A) and nasal swabs (C) of individual animals. The solid curves represent individual macaques while the red dashed curves represent GMT. The peak sgmRNA levels detected in BAL (B) and nasal swab (D) samples of each group on Day 3 post-challenge. The peak viral loads are presented as GMT ± SD. (E) Examples of histopathology of lung samples collected on Day 8 post-challenge (magnitude x 40). (F) The lung pathology scores presented as GMT ± SD. Statistical significance is indicated in panels B, D and F for 30 or 100 μg UB-612 dose group compared to Saline group, *p < 0.05.