Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

Keywords: Adjuvant effects; Cellular immunity; MERS-CoV neutralization; Subunit vaccine development.

Fig. 1

Characterization of recombinant trimeric MERS-CoV S1-Fd protein. (A) Schematic representation of the primary structure of the recombinant MERS-CoV S1 and S1-Fd proteins. S1 and S1-Fd proteins encode MERS-CoV spike protein subunit 1 (S1) from amino acid residues 1 to 747 with the V5 tag and Fd motif at the C-terminus, respectively, followed by His tag. SP: signal peptide, NTD: N-terminal domain, RBD: receptor binding domain, V5: V5 tag, His: hexahistidine tag, and Fd: T4 trimerization domain (foldon). (B) Analytical SEC-FPLC and SDS-PAGE. The affinity-purified S1-Fd protein was resolved by analytical SEC-FPLC (left; blue line) and its molecular weight was determined according to protein standards (Bio-Rad; red line). The protein expression profile and purity of the fractions 1 and 2 (elution volume of 10.5 mL and 11.0 mL, respectively) were confirmed on reducing SDS-PAGE stained with Coomassie blue (CB) (right). (C) Immunoblot analysis. S1-Fd and S1 proteins prior to (Input) and during cobalt affinity purification (Eluent and Beads) were subjected to SDS-PAGE and immunoblot analysis using anti-V5 antibody and MERS-CoV S1 antisera as indicated. Culture supernatant from non-infected Hi5 cells was used as a control. The molecular weight markers (kDa) are shown on the right. (D) EM micrograph of S1-Fd protein. The eluent fraction 2 of S1-Fd protein after SEC-FPLC was negatively stained with 2% uranyl acetate and examined by electron microscopy. Scale bars: 50 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Immunization of MERS-CoV S1-Fd protein with AddaVax adjuvant induces high levels of both nAbs and circulating S1-specific IgG and its isotypes. (A) Study design of mice immunization and sample collection. (B) End-point reciprocal dilution titers of MERS-CoV S1-specific antibodies. Total IgG, IgG1, and IgG2a antibodies in C57BL/6 mice (A1 to A4) immunized with S1-Fd as measured by ELISA were shown. Serum from unimmunized mouse was used as a control. (C, D) Neutralization activity. Sera collected from C57BL/6 mice (n = 4/group) immunized with S1 or S1-Fd (10 µg/mice) (panel C) and hDPP4-Tg mice (n = 8) immunized with S1-Fd (10 µg/mice) (panel D) were analyzed for neutralizing activity against live MERS-CoV in NT₅₀ assay. Symbols represent individual animals with S1 (blue closed circles) or S1-Fd (red closed squares) protein immunization. P-values were determined by Tukey's two-way ANOVA (in panel C) and unpaired Student’s t-tests (in panel D), respectively. *P < 0.05, **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

MERS-CoV S1-Fd protein with AddaVax adjuvant vaccination provides protection against MERS-CoV challenge in hDPP4-Tg mice. hDPP4-Tg mice were immunized with AddaVax (50 µL) mixed S1-Fd (10 µg/mice) or PBS-immunized (Control) followed by 100 LD₅₀ MERS-CoV challenge at 4 weeks after the last immunization. Survival rates (A) and body weight loss (B) were monitored for 13 days (n = 5/group). Symbols represent animals with (red squares) or without S1-Fd (grey triangles) vaccination. (C) Pulmonary viral loads were analyzed (n = 3/group) on day 3 post-infection using the TCID₅₀ assay. Error bars indicate mean ± SEM. P-values were determined by Mantel-Cox log-rank test (in panel A and B) and by unpaired Student’s t-test (in panel C). **P < 0.01. (D) Lung histopathology of immunized hDPP4-Tg mice at 3 days post-MERS-CoV challenge. (Upper panels) The lung histology from 1 of 3 mice immunized with MERS-CoV S1-Fd plus adjuvant AddaVax was examined 3 days after infection. Hematoxylin and eosin staining was performed. Representative images are shown. Mononuclear infiltration of lymphocytes and monocytes is present in the peribronchial, peribronchiolar, and perivascular areas, as well as a sparse amount of neutrophils (magnification, ×400). Arrows indicate the presence of eosinophils. (Lower panels) The lung histology of 1 of 3 mice inoculated with the chimpanzee adenovirus vector rAd5-S1/F/CD40L expressing MERS-CoV spike protein S1 and CD40L was examined 3 days after infection. Moderate peribronchial, peribronchiolar, and perivascular infiltration is present (magnification, ×400). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

MERS-CoV S1-Fd protein vaccination induces both MERS-CoV-specific CD4 and CD8 T cell responses. (A) Study design of mice immunization and sample collection. C57BL/6 mice (n = 5 for naïve group; n = 4 for each adjuvanted group) were immunized with S1-Fd (20 µg/mice) in 50 μL in the presence of adjuvant AddaVax (50 μL), QS-21 (50 μg), or AddaVax (50 μL) + QS-21 (50 μg). Sera and splenocytes from immunized mice were collected 4 weeks (i.e., week 12) and 17 weeks (i.e., week 25) after the last dose of immunization, respectively. (B) Serological assay of MERS-CoV S1-specific antibodies. Sera were collected at week 12 and the levels of IgG, IgG1 and IgG2a against the S1-Fd protein were analyzed. The data shown (mean ± SEM) represent absorbance at 450 nm for different reciprocal serum dilutions using ELISA. There was one mouse from the AddaVax and AddaVax + QS-21 groups shown undetectable IgG2a titers. Symbols represent immunization with different adjuvants: AddaVax (red open circles), QS-21 (blue open triangles), and AddaVax + QS-21 (orange open squares). P-values were determined by two-way ANOVA. *P < 0.05, **P < 0.01. (C–H) Detection of MERS-CoV-specific T cell responses. Splenocytes from immunized mice were harvested at week 25 and stimulated for 6 h in the presence of brefeldin A (5 μg/mL). The stimulations were as follows: (1) medium alone as the negative control group, (2) PMA (20 ng/mL) + ionomycin (1 μg/mL) as the positive control group that activate T cell responses, (3) ovalbumin-derived peptide OVA_257-264 (2 µM; red closed circles) as an irrelevant peptide control, (4) MERS-CoV spike reference CD8⁺ T cell epitopes S_395-402 (2 µM; blue closed triangles), (5) MERS-CoV spike reference CD8⁺ T cell epitopes S_434-441 (2 µM; orange closed squares), (6) S1-Fd protein (0.6 µM; green closed diamonds). Splenocytes harvested from naïve mice were used as controls. Stimulated cells were then subjected to intracellular cytokine staining. CD4⁺ and CD8⁺ T cells were identified as CD3⁺CD4⁺CD8⁻ and CD3⁺CD4⁻CD8⁺ cells, respectively. The percentages of TNFα-, IFNγ-, and TNFα/IFNγ double-producing CD8⁺ and CD4⁺ T cells among CD8⁺ (in panel C, D and E) and CD4⁺ T cells (in panel F, G and H), respectively, were shown. Each dot represents an individual data point. P-values were determined by Dunnett's two-way ANOVA. *P < 0.05, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)