Heterologous Systemic Prime-Intranasal Boosting Using a Spore SARS-CoV-2 Vaccine Confers Mucosal Immunity and Cross-Reactive Antibodies in Mice as well as Protection in Hamsters

Abstract

Background: Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are administered systemically and typically result in poor immunogenicity at the mucosa. As a result, vaccination is unable to reduce viral shedding and transmission, ultimately failing to prevent infection. One possible solution is that of boosting a systemic vaccine via the nasal route resulting in mucosal immunity. Here, we have evaluated the potential of bacterial spores as an intranasal boost. Method: Spores engineered to express SARS-CoV-2 antigens were administered as an intranasal boost following a prime with either recombinant Spike protein or the Oxford AZD1222 vaccine. Results: In mice, intranasal boosting following a prime of either Spike or vaccine produced antigen-specific sIgA at the mucosa together with the increased production of Th1 and Th2 cytokines. In a hamster model of infection, the clinical and virological outcomes resulting from a SARS-CoV-2 challenge were ameliorated. Wuhan-specific sIgA were shown to cross-react with Omicron antigens, suggesting that this strategy might offer protection against SARS-CoV-2 variants of concern. Conclusions: Despite being a genetically modified organism, the spore vaccine platform is attractive since it offers biological containment, the rapid and cost-efficient production of vaccines together with heat stability. As such, employed in a heterologous systemic prime-mucosal boost regimen, spore vaccines might have utility for current and future emerging diseases.

Keywords: COVID-19; SARS-CoV-2; nasal vaccine; prime boost.

Figure 1

Spore coat expression of SARS-CoV-2 proteins. Panel (A) PK120 (thyA::cotB-RBD^Wuh) and PK122 (thyA::cotC-HR1-HR2^Wuh) express the RBD and HR1-HR2 domains of Spike from the Wuhan-Hu-1 (2019-nCoV) variant when probed with anti-S (Wuhan-Hu-1) PAbs. Bands corresponding to CotB-RBD^Wuh (62 kDa) and CotC-HR1-HR2^Wuh (40 kDa) are shown. No bands were detectable in spores of the isogenic strain WT which expresses no heterologous antigens. Panel (B) PK230 (thyA::cotB-RBD^Omi) expresses the RBD domain of Omicron (B.1.1.529) fused to CotB (63 kDa) when probed with anti-S (Omicron) PAbs. No bands were detectable in spores of the strain WT.

Figure 2

Intranasal boosting of a subunit prime or AZD1222. Female BALB/C mice were primed (i.m.) with recombinant Spike (rS^Wuh) or AZD1222 (1.0 × 10⁸ IU) followed by two intranasal boosts with 1 × 10⁹ spores of either WT (naked spores, no antigen expression) or SporCoVax (1:1 mixture of CotB-RBD^Wuh) and CotC-HR1-HR2^Wuh spores) three and five weeks post prime. Panels show S^Wuh-specific responses determined by ELISA (OD_{450 nm}) 48 days post immunization. Panel (A,B) rS^Wuh-specific sIgA in longitudinal saliva samples, (C,D) rS^Wuh-specific IgA in the lungs, (E,F) rS^Wuh-specific IgG in serum. Mann–Whitney, ** p < 0.01.

Figure 3

Spore-induced mucosal Spike-specific sIgA cross-reacts with Wuhan and Omicron variants. Mice were primed (i.m.) with 1.0 × 10⁸ IU of AZD1222 followed by two intranasal boosts with spores (1 × 10⁹ CFU) of WT (naked) or SporCoVax (1:1 mixture of CotB-RBD^Wuh and CotC-HR1-HR2^Wuh spores) at three and five weeks post prime. rS^Omi-specific sIgA in the saliva 48 days post immunization is shown. Mann–Whitney, ** p < 0.01, *** p < 0.001.

Figure 4

Cytokine profiles. BALB/C mice (female, aged 8 weeks; n = 3/gp) were immunized with AZD1222 (i.m.; 1.0 × 10⁸ IU) followed by two intranasal boosts with spores (1 × 10⁹ CFU/dose) of WT or SporCoVax (a 1:1 mixture of CotB-RBD^Wuh and CotC-HR1-HR2^Wuh) three and five weeks post prime immunization. Control groups included untreated animals (naïve) and animals primed only (AZD1222). All mice were sacrificed 48 days post prime, and their spleens dissected and stimulated with 2.5 µg/mL rS^Wuh protein for 72 h, and levels of the cytokines IL-2 (panel (A)), TNF-α (panel (B)) and IL-5 (panel ((C)) determined by flow cytometry.

Figure 5

Protection in golden Syrian hamsters. Golden Syrian Hamsters were primed (i.m.) with recombinant spike (rS^Wuh) protein followed by two intranasal boosts with 2.5 × 10⁹ spores of SporCoVax (1:1 mixture of CotB-RBD^Wuh and CotC-HR1-HR2^Wuh spores) (Group 3). Negative, unvaccinated, (Group 2) and naive (Group 1) control groups received PBS in place of immunogens. 7-days after the final boost animals in Groups 2 and 3 were intranasally challenged with SARS-CoV-2 (D614G variant, 10⁴ TCID₅₀/animal). Weight loss post-challenge are shown (panel (A)). Animals were sequentially euthanized (2, 4, and 7 dpi), necropsies were performed, and viral load of SARS-CoV-2 were determined by qPCR in NT (panel (B0), OP swabs (panel (C)) and lungs (panel (D0). Mann-Whitney, * p < 0.05, ** p < 0.01.