RBD-Protein/Peptide Vaccine UB-612 Elicits Mucosal and Fc-Mediated Antibody Responses against SARS-CoV-2 in Cynomolgus Macaques

Abstract

Antibodies provide critical protective immunity against COVID-19, and the Fc-mediated effector functions and mucosal antibodies also contribute to the protection. To expand the characterization of humoral immunity stimulated by subunit protein-peptide COVID-19 vaccine UB-612, preclinical studies in non-human primates were undertaken to investigate mucosal secretion and the effector functionality of vaccine-induced antibodies in antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) assays. In cynomolgus macaques, UB-612 induced potent serum-neutralizing, RBD-specific IgG binding, ACE2 binding-inhibition antibodies, and antibodies with Fc-mediated effector functions in ADMP and ADNKA assays. Additionally, immunized animals developed mucosal antibodies in bronchoalveolar lavage fluids (BAL). The level of mucosal or serum ADMP and ADNKA antibodies was found to be UB-612 dose-dependent. Our results highlight that the novel subunit UB-612 vaccine is a potent B-cell immunogen inducing polyfunctional antibody responses contributing to anti-viral immunity and vaccine efficacy.

Keywords: ADCP; ADMP; ADNKA; COVID-19; Fc-mediated effector function; RBD; SARS-CoV-2; antibody; non-human primates; subunit; vaccine.

Figure 1

Composition of subunit protein–peptide COVID-19 vaccine UB-612. Receptor-binding domain (RBD) of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc. Five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins: three from the S2 subunit, one from membrane, and one from nucleocapsid, and one universal Th peptide. Peptides are precipitated by charge interactions with CpG1 ODN to form a stable complex. The protein and peptides are adsorbed on aluminum phosphate adjuvant.

Figure 2

RBD-specific binding antibody responses in macaques three weeks after a 2-dose UB-612 immunization: RBD-binding IgG titers (A); Avidity of IgG binding to RBD, expressed as Avidity Index (AI% = OD mean value ratio between KSCN-treated and -non-treated sera) (B); and IgG isotypes (C). IgG titers and IgG isotypes data are expressed as the highest serum dilution with a reading of two standard deviations over the background cut-off). Presented are the individual value group means with standard deviations. Statistical significance is presented as **** p < 0.0001 to compare IgG1 with IgG2, IgG3, or IgG3.

Figure 3

Functional active anti-viral antibody responses three weeks after the 2-dose UB-612 vaccinations. (A) RBD:ACE2 binding-inhibition activities (serum dilution resulting in 50% binding inhibition). (B) Neutralizing antibody titers against SARS-CoV-2-WA (serum dilution resulting in 50% plaque reduction). Presented are the individual values and group means with standard deviations. Statistical significance between conditions determined by one-way ANOVA is presented as **** p < 0.0001.

Figure 4

Mucosal IgG (A) and IgA (B) titers (dilution resulting in EC50) in macaque bronchoalveolar lavage fluids (BAL) were collected three weeks after the 2-dose UB-612 vaccinations. Presented are the individual values and group mean with standard deviations. The IgA was measured only in n = 3 (saline) and n = 2 (UB-612) immunized NHPs.

Figure 5

Antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) in UB-612-immunized macaques. Sera were collected three weeks after the second immunization. (A) ADMP: change in the proportion (%) of GFP-positive monocytes due to ADMP activity in the presence of vaccine immune sera compared to the negative controls with no sera added. ADNKA (B–D) (B) Proportion (%) of CD107a-positive CD56 NK cells. (C) % IFN-γ-positive CD56 NK cells. (D) % TNF-α-positive CD56 NK cells. Presented are the individual values and group means with standard deviations. Statistical significance between conditions by using one-way ANOVA is presented as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.