The Potency of an Anti-MERS Coronavirus Subunit Vaccine Depends on a Unique Combinatorial Adjuvant Formulation

Abstract

Vaccination is one of the most successful strategies to prevent human infectious diseases. Combinatorial adjuvants have gained increasing interest as they can stimulate multiple immune pathways and enhance the vaccine efficacy of subunit vaccines. We investigated the adjuvanticity of Aluminum (alum) in combination with rASP-1, a protein adjuvant, using the Middle East respiratory syndrome coronavirus MERS-CoV receptor-binding-domain (RBD) vaccine antigen. A highly enhanced anti-MERS-CoV neutralizing antibody response was induced when mice were immunized with rASP-1 and the alum-adjuvanted RBD vaccine in two separate injection sites as compared to mice immunized with RBD + rASP-1 + alum formulated into a single inoculum. The antibodies produced also significantly inhibited the binding of RBD to its cell-associated receptor. Moreover, immunization with rASP-1 co-administered with the alum-adjuvanted RBD vaccine in separate sites resulted in an enhanced frequency of TfH and GC B cells within the draining lymph nodes, both of which were positively associated with the titers of the neutralizing antibody response related to anti-MERS-CoV protective immunity. Our findings not only indicate that this unique combinatorial adjuvanted RBD vaccine regimen improved the immunogenicity of RBD, but also point to the importance of utilizing combinatorial adjuvants for the induction of synergistic protective immune responses.

Keywords: MERS-CoV; T follicular helper cells; adjuvant combination; adjuvants; aluminum; functional antibody responses; germinal center B cells; rASP-1; receptor-binding domain; synergy.

Figure 1

Neutralizing antibody titers against the pseudotyped Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in sera of immunized mice: C57BL/6 mice were immunized i.m. with RBD with or without alum and rASP-1 alone or together using different combinations and/or formulations (Table 1 and X-axis legend). Sera samples were collected 7 days post-2nd immunization and analyzed for neutralization of the pseudotyped MERS-CoV. The data represents the mean and standard error (SEM) of NT₅₀ titers from at least two independent experiments with 3 to 5 mice per group. “+” indicates the presence and “−“ indicates the absence of the protein or adjuvants in the formulation. Statistics was performed using one-way ANOVA with Tukey’s multiple comparison. p < 0.001: ***, p < 0.0001: ****, ND: not detectable.

Figure 2

Inhibition of binding of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RBD-Fc to hDPP4 receptor by sera of immunized mice: C57BL/6 mice were immunized i.m. with MERS-RBD-Fd with or without alum and rASP-1 alone or together using different combinations and/or formulations (Table 1 and X-axis legend). Sera samples were collected on day 7 post-2nd immunization and assayed for inhibition of the binding of MERS-CoV RBD-Fc to Huh-7 cells expressing MERS-CoV receptor DPP4. The data represents the mean and standard error (SEM) of percentage inhibition of binding from at least two independent experiments with 3 to 5 mice per group. “+” indicates the presence and “−“ indicates the absence of the protein or adjuvants in the formulation. Statistics was performed using one-way ANOVA with Tukey’s multiple comparison. p < 0.05: *, p < 0.0001: ****. ND: not detectable.

Figure 3

Monocyte subsets in the lymph nodes (LNs) of immunized mice: C57BL/6 mice were immunized i.m. with MERS-RBD-Fd with or without alum and rASP-1 alone or together using different combinations and/or formulations (Table 1 and X-axis legend). The draining LNs were harvested 7 days post-2nd immunization and the number of cells per LN were analyzed. (A) Representative flow cytometry plot of CD40⁺ monocytes (upper panel) and CCR7 ⁺ monocytes (lower panel in each immunization group. (B) The number of CD40⁺ monocytes, and (C) CCR7⁺ monocytes per LN were analyzed. The experiment was done once, and the data presented is from left and right draining LNs of 5 mice per group: mean and standard error. “+” indicates the presence and “−“ indicates the absence of the protein or adjuvants in the formulation. Statistics was performed using one-way ANOVA with Tukey’s multiple comparison. p < 0.05: *, p < 0.001: ***.

Figure 4

Fold increase in the frequency of TfH cells in the lymph nodes (LNs) of immunized mice: C57BL/6 mice were immunized i.m. with MERS-RBD-Fd with or without alum and rASP-1 alone or together using different combinations and/or formulations (Table 1 and X-axis legend). The draining LNs were harvested on day 7 post-2nd immunization and the frequency of TfH cells per LN was analyzed. (A) Representative flow cytometry plot in each immunization group. (B) The fold increase in the frequency of TfH cells per LN in all the adjuvanted-vaccine groups was normalized against the RBD vaccine group (G2). (C) The fold increase in the frequencies of TfH cells per LN from groups G5 and G6 were correlated with the NT₅₀ titers determined in each serum sample of the corresponding mice. The experiment was repeated at least twice with 3 to 5 mice per group and the data are represented as mean and standard error of individual LN per mice. “+” indicates the presence and “−“ indicates the absence of the protein or adjuvants in the formulation. Statistics was performed using one-way ANOVA with Tukey’s multiple comparison. p < 0.05: *, p < 0.001: ***. Spearman correlation was performed to determine the association of TfH cells with neutralizing antibody titers.

Figure 5

Frequency of B cells and fold increase in the frequency of GC B cells in the lymph nodes (LNs) of immunize mice: C57BL/6 mice were immunized i.m. with MERS-RBD-Fd with or without alum and rASP-1 alone or together using different combinations and/or formulations (Table 1 and X-axis legend). The draining LNs were harvested on day 7 post-2nd immunization and the frequency of GC B cells per LN was analyzed. (A) Representative flow cytometry plot in each immunization group. (B) The frequency B cells per LN as well as (C) the fold-increase in the frequency of GC B cells per LN was analyzed. The frequency of GC B cells per LN in all the adjuvanted-vaccine groups was normalized against the RBD vaccine group (G2). (D) The fold increase in the frequencies of GC B cells per LN in groups G5 and G6 were correlated with the NT₅₀ titers determined in each serum sample of the corresponding mice. The experiment was repeated at least twice with 3 to 5 mice per group and the data are represented as mean and standard error. “+” indicates the presence and “−“ indicates the absence of the protein or adjuvants in the formulation. Statistics was performed using one-way ANOVA with Tukey’s multiple comparison. p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****. Spearman correlation was performed to determine the association of GC B cells with neutralizing antibody titers.