An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection

Abstract

The development of more effective, accessible, and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector, and whole inactivated virus vaccines is essential to curtailing the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six-proline-stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to outer membrane vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells, and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum-neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination, hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs 7 days after challenge in OMV-mC-Spike-vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.

Keywords: COVID-19; Neisseria; intranasal; mucosal immunity; outer membrane vesicle; vaccine.

Figure 1

Schematic presentation of the spike molecule and OMV decoration with spike. (A) Schematic of 2019-nCoV S primary structure colored by domain. S, signal sequence; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1–HR2, heptad repeat 1 and 2; FC, disrupted S1/S2 furin cleavage site (R682G, R684S, R685S). Features that were added to the ectodomain (amino acids 1–1,208) expression construct are colored white gray gradient (amino acid 1,209–1,333). From light to dark gray: Foldon trimerization motif (F); HRV 3C site; 8xHis tag; Twin Strep tag; 3x GGGS repeat; mCramp (mC). Not shown: six stabilizing prolines in S2 at positions F817P, A892P, A899P, A942P, K986P, and V987P (27). The aspartic acid at position 614 in the original HexaPro (27) spike protein was replaced with a glycine (D614G). (B) Schematic overview of how antigens can associate with the OMVs. In the spike protein, an mCRAMP (antimicrobial peptide) motif was included which is depicted in light blue. This peptide associates spontaneously to the LPS of the OMV thereby attaching the spike protein (dark blue) to the OMV.

Figure 2

Mouse immunogenicity study. Balb/C mice were immunized intranasally or intramuscular on day 0 and day 21 with 15 µg OMV (control group) or 15 µg OMV combined with 15 µg Spike with the presence of mCRAMP or without mCRAMP (OMV+Spike). Sera were collected from all animals at day 35. (A) Experimental setup and timeline of the mouse immunogenicity experiment. (B) Total IgG antibody levels were measured in sera diluted at 1:50,000. (C) IgA levels were measured in nasal washes (1:1 dilution), lung (1:50 dilution), and serum (1:200 dilution). (D) Virus neutralization titers were determined in sera. Data are depicted as mean ± SD and are representative results of two independent experiments. Significance (E) is depicted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance of the difference was evaluated by a 2-way ANOVA test followed by the non-parametric Mann whitney test.

Figure 3

Hamster challenge study. Animals were immunized intranasally or intramuscularly on day 0 and day 21 with 15 µg OMV, 15 µg spike or 15 µg OMV, and 15 µg Spike combined with mCRAMP. In the control group, animals were immunized with 10 mM Tris-3% sucrose, which is the OMV buffer. Sera were collected from all hamsters at experimental days 0, 21, 42, 46, and 49. At day 42, all hamsters were challenged intranasally with 10^4.0 TCID50 SARS-CoV-2, strain BetaCoV/Munich/BavPat1/2020. At day 46, half of the animals per group (4 out of 8) were sacrificed and at day 49 the remaining 4 animals were sacrificed. (A) Experimental setup and timeline of the hamster challenge experiment. (B) Total IgG anti-spike antibody levels were measured in sera (dilution 1:4,000) from day 42 in an ELISA. (C) Virus neutralization was determined in sera from day 42. (D) When animals were sacrificed at day 46 (day 4 post challenge) and day 49 (day 7 post challenge), the percentage of the lung that presented lung lesions was quantified. The viral load was determined in throat swabs (E), lungs, and nasal turbinates (F). Data are depicted as mean ± SD. Significance (E) is depicted as *p < 0.05, **p < 0.01, ***p < 0.001. Statistical significance of the difference was evaluated by a 2-way ANOVA test followed by the non-parametric Mann whitney test.