A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine

Abstract

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection as drift variants continue to emerge. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced T_H1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

Keywords: RIG-I agonist; SARS-CoV-2; cross-protection; intranasal vaccine; mucosal adjuvant; nanoemulsion (NE).

Figure 1

Acute phase cytokine response after immunization with NE and NE/IVT. Acute cytokines were assessed in the serum upon by measuring (A) IL-6, (B) TNF-α, (C) IL12p70, and (D) IFN-γ by multiplex immunoassay at 6 and 12 h post-IN immunization with 10 μg of RBD only, or with 20% NE, or 20% NE/0.5 μg IVT DI.

Figure 2

Serum S1-specific IgG titers induced by immunization with NE and NE/IVT and comparison of antibody avidity. Serum S1-specific IgG measured in mice immunized IN with 15 μg S1 alone, or with 20% NE/0.5 μg IVT DI. S1-specific IgG was measured 2 weeks after each immunization at (A) 2 wks post-initial immunization (prime), (B) 6 wks post-initial immunization (prime/boost), and (C) 10 wks post-initial immunization (prime/boost/boost). (*p < 0.05, **p < 0.01 by Mann-Whitney U test). (D) Antibody avidity for S1-specific IgG measured by NaSCN elution for a 1:1,250 dilution of serum for the 6 wk and 10 wk sera. The control group (ctrl) consisted of untreated mice shown as mean ± SEM (n=5/grp).

Figure 3

Serum S1-specific IgG subclass distributions induced by immunization. Levels of (A) IgG1, (B) IgG2b, and (C) IgG2c were measured in mice immunized IN with 15 μg S1 alone, or with 20% NE, or 20% NE/0.5 μg IVT DI after the last boost immunization, 10 wks post-initial immunization. (*p < 0.05, **p < 0.01 by Mann-Whitney U test).

Figure 4

Virus neutralization titers to homologous and heterologous SARS-CoV-2 following immunization. Neutralizing antibody titers in serum from mice receiving two (wk 6) or three (wk 10) immunizations were determined using microneutralization assays against the WT SARS-CoV-2 (2019-nCoV/USA-WA1/2020), pseudotyped lentivirus expressing the WT SARS-CoV-2 spike protein (Lenti-CoV2), and MA-SARS-CoV-2. Viral neutralization was plotted as percentage inhibition of viral infection in Vero E6 cells (for WT virus and MA-virus) relative to virus only (no serum) positive controls versus the inverse serum dilution. The titer at which 50% inhibition of infection was achieved (IC50) was determined for the (A, B) WT virus and the (D, E) MA virus. (C) The results were confirmed for the same week 10 serum samples using the Lenti-CoV2 pseudovirus expressing firefly luciferase with HEK-293T cells expressing hACE2. Microneutralization titers using the Lenti-CoV2 were determined by detecting viral infection by measuring luminescence (*p < 0.05, **p < 0.01 by Mann-Whitney U test). Pretreatment (pre) sera were obtained from the same set of mice before immunizations.

Figure 5

Protection offered by passive transfer of serum from vaccinated mice against heterologous challenge with MA-SARS-CoV-2. Naïve C57Bl/6 mice (n=3-4/group) each received 150 μL of pooled serum through the intraperitoneal route from donor mice given three IN immunizations of S1, NE/S1, or NE/IVT/S1. 2 h after serum transfer, mice were challenged IN with 10⁴ PFU of MA-SARS-CoV-2. (A) Body weight loss was measured over three days, and at 3 dpi (B) lung virus titers were determined in homogenate from one lobe of the isolated lungs by plaque assay (solid symbols). Passive transfer/challenge was repeated for the PBS control and S1 sera for verification, and virus titers were measured in whole lung in the replicated experiment (open square symbols). (*p < 0.05 by Mann-Whitney U test assessed for half lung data points).

Figure 6

Antigen recall response assessed in splenocytes from immunized animals. Splenocytes isolated from mice immunized IN with S1 alone, or with NE, or NE/IVT after the final boost immunization (10 weeks post-initial immunization) were stimulated ex vivo with 5 μg of recombinant S1 for 72 h, and levels of secreted cytokines (A) IFNγ, (B) IL2, (C) IP10, (D) TNFα, (E) IL6, (F) IL17A, (G) IL4, (H) IL5, (I) IL13 were measured in the supernatant relative to unstimulated cells by multiplex immunoassay. An unvaccinated control was included for comparison. (n = 5/grp; *p < 0.05, **p < 0.01 by Mann-Whitney U test).

Figure 7

Antigen recall response assessed in the draining lymph nodes (cervical LN). Lymphocytes isolated from the cLNs of mice immunized IN with S1 alone, or with NE, or NE/IVT after the final boost immunization (10 weeks post-initial immunization) were stimulated ex vivo with 5 μg of recombinant S1 for 72h, and levels of secreted cytokines (A) IFNγ, (B) IL2, (C) IP10, (D) TNFα, (E) IL6, (F) IL17A, (G) IL4, (H) IL5, (I) IL13 were measured in the supernatant relative to unstimulated cells by multiplex immunoassay. An unvaccinated control was included for comparison. (n = 5/grp; *p < 0.05, **p < 0.01 by Mann-Whitney U test).