Intranasal Delivery of Thermostable Subunit Vaccine for Cross-Reactive Mucosal and Systemic Antibody Responses Against SARS-CoV-2

Abstract

Despite the remarkable efficacy of currently approved COVID-19 vaccines, there are several opportunities for continued vaccine development against SARS-CoV-2 and future lethal respiratory viruses. In particular, restricted vaccine access and hesitancy have limited immunization rates. In addition, current vaccines are unable to prevent breakthrough infections, leading to prolonged virus circulation. To improve access, a subunit vaccine with enhanced thermostability was designed to eliminate the need for an ultra-cold chain. The exclusion of infectious and genetic materials from this vaccine may also help reduce vaccine hesitancy. In an effort to prevent breakthrough infections, intranasal immunization to induce mucosal immunity was explored. A prototype vaccine comprised of receptor-binding domain (RBD) polypeptides formulated with additional immunoadjuvants in a chitosan (CS) solution induced high levels of RBD-specific antibodies in laboratory mice after 1 or 2 immunizations. Antibody responses were durable with high titers persisting for at least five months following subcutaneous vaccination. Serum anti-RBD antibodies contained both IgG1 and IgG2a isotypes suggesting that the vaccine induced a mixed Th1/Th2 response. RBD vaccination without CS formulation resulted in minimal anti-RBD responses. The addition of CpG oligonucleotides to the CS plus RBD vaccine formulation increased antibody titers more effectively than interleukin-12 (IL-12). Importantly, generated antibodies were cross-reactive against RBD mutants associated with SARS-CoV-2 variants of concern, including alpha, beta and delta variants, and inhibited binding of RBD to its cognate receptor angiotensin converting enzyme 2 (ACE2). With respect to stability, vaccines did not lose activity when stored at either room temperature (21-22°C) or 4°C for at least one month. When delivered intranasally, vaccines induced RBD-specific mucosal IgA antibodies, which may protect against breakthrough infections in the upper respiratory tract. Altogether, data indicate that the designed vaccine platform is versatile, adaptable and capable of overcoming key constraints of current COVID-19 vaccines.

Keywords: COVID-19; CpG; Intranasal vaccination; Receptor binding domain (RBD); SARS-CoV-2; chitosan; mucosal immunity.

Figure 1

RBD-specific IgG antibody and isotype responses following s.c. vaccination. Mice (n=3 per group) were vaccinated once (A–C) or twice (D–F) with RBD alone, CS/RBD, IL-12/RBD or CS/IL-12/RBD. RBD-specific IgG (A, D), IgG1 (B, E), or IgG2a (C, F) levels in sera as a function of time after immunization were measured via ELISA. Vaccination schemas were created with BioRender.com.

Figure 2

Serum RBD-specific antibody titers and avidity. Mice (n=3 per group) were vaccinated s.c. with RBD alone, CS/RBD, IL-12/RBD or CS/IL-12/RBD. Serum anti-RBD IgG levels in serial dilutions were measured 21 days after one (A) or two (B) vaccinations. Anti-RBD avidity index (C) was calculated as the ratio of IgG binding to RBD in the presence vs. the absence of urea.

Figure 3

RBD-specific antibody and isotype responses comparing IL-12 and CpG as immunoadjuvants. Mice (n=3 per group) were vaccinated once (closed symbols) or twice (open symbols) with RBD alone, CS/RBD, CS/IL-12/RBD or CS/CpG/RBD. RBD-specific IgG (A) IgG1 (B) and IgG2a (C) levels in sera 21 days after the final vaccination were measured via ELISA. *p < 0.05 via one-way ANOVA with Tukey’s posttest, “ns” indicates “not significant”. The presence of neutralizing antibodies in sera of mice vaccinated twice were measured as the inhibition of RBD binding to ACE2 via a surrogate virus neutralization test (sVNT) (D). **p < 0.05 via two-way ANOVA with Tukey’s posttest.

Figure 4

Cross-reactivity and durability of antibody responses to diverse CS/CpG/RBD vaccinations. Mice (n=3 per group) were vaccinated s.c. twice with RBD alone, CS/RBD, CS/IL-12/RBD or CS/CpG/RBD and bled 21 days later. IgG specificity against RBD, RBD-N501Y, RBD-E484K and RBD-K417N was measured via ELISA (A). Mice (n=3 per group) were vaccinated s.c. twice with CS/CpG/RBD-N501Y, CS/CpG/RBD-E484K, CS/CpG/RBD-K417N or a CS/CpG formulation with RBD, RBD-N501Y, RBD-E484K and RBD-K417N combined (“combo”). IgG specificity against RBD, RBD-N501Y, RBD-E484K and RBD-K417N was measured via ELISA (B). Mice (n=3 per group) were vaccinated s.c. twice with CS/CpG/RBD-N501Y (C), CS/CpG/RBD-E484K (D), or CS/CpG/RBD-K417N (E). Durability of IgG responses were measured via ELISA plates coated with the vaccine antigen, i.e. RBD-N501Y (C), RBD-E484K (D) or RBD-K417N (E), using sera collected 2 weeks, 2 months and 5 months after booster vaccinations. *p < 0.05 via one-way ANOVA with Tukey’s posttest. **p < 0.05 via two-way ANOVA with Tukey’s posttest. ns, not significant.

Figure 5

Effect of vaccine storage on efficacy. Mice (n=3 per group) were vaccinated s.c. twice with CS/CpG/RBD prepared fresh, stored at 4°C for 1 month or stored at room temperature (21-22°C) and bled 21 days later. RBD- specific IgG levels in serially diluted sera were measured via ELISA.

Figure 6

Intranasal vaccination with CS/CpG/RBD results in mucosal and systemic immunity. Mice (n=4 per group) were vaccinated twice i.n with RBD alone, CS/RBD or CS/CpG/RBD. For comparison, an additional cohort (n=3) was vaccinated twice s.c. with CS/CpG/RBD. RBD-specific IgA responses in sera (A) and nasal rinses (B) 14 days after booster immunizations were detected via ELISA. Binding of serum IgG to RBD (C) or RBD-L452R-T478K (D) was measured via ELISA. Neutralizing antibody responses were determined by inhibition of RBD (E) or RBD-L452R-T478K (F) binding to ACE2 by diluted serum samples. *p < 0.05 via one-way ANOVA with Tukey’s posttest. ns, not significant.