Single-injection COVID-19 subunit vaccine elicits potent immune responses

Abstract

Current vaccination schedules, including COVID-19 vaccines, require multiple doses to be administered. Single injection vaccines eliciting equivalent immune response are highly desirable. Unfortunately because unconventional release kinetics are difficult to achieve it still remains a huge challenge. Herein a single-injection COVID-19 vaccine was designed using a highly programmable release system based on dynamic layer-by-layer (LBL) films. The antigen, S1 subunit of SARS-CoV-2 spike protein, was loaded in CaCO₃ microspheres, which were further coated with tannic acid (TA)/polyethylene glycol (PEG) LBL films. The single-injection vaccine was obtained by mixing the microspheres coated with different thickness of TA/PEG films. Because of the unique constant-rate erosion behavior of the TA/PEG coatings, this system allows for distinct multiple pulsatile release of antigen, closely mimicking the release profile of antigen in conventional multiple dose vaccines. Immunization with the single injection vaccine induces potent and persistent S1-specific humoral and cellular immune responses in mice. The sera from the vaccinated animal exhibit robust in vitro viral neutralization ability. More importantly, the immune response and viral inhibition induced by the single injection vaccine are as strong as that induced by the corresponding multiple dose vaccine, because they share the same antigen release profile. STATEMENT OF SIGNIFICANCE: Vaccines are the most powerful and cost-effective weapons against infectious diseases such as COVID-19. However, current vaccination schedules, including the COVID-19 vaccines, require multiple doses to be administered. Herein a single-injection COVID-19 vaccine is designed using a highly programmable release system. This vaccine releases antigens in a pulsatile manner, closely mimicking the release pattern of antigens in conventional multiple dose vaccines. As a result, one single injection of the new vaccine induces an immune response and viral inhibition similar to that induced by the corresponding multiple-dose vaccine approach.

Keywords: COVID-19 vaccines; Pulsatile release; Single-injection vaccines; Unconventional release kinetics.

Graphical abstract

Fig. 1

(A) Encapsulation of S1 subunit in CaCO₃ microspheres, followed by coating with TA/PEG films by LBL assembly. (B-D) SEM images of the empty CaCO₃ microspheres (B), BSA@CaCO₃ microspheres (C) and BSA@CaCO₃/(TA/PEG)₅ microspheres (D). (E, F) Size distribution of empty CaCO₃ (E) and the BSA@CaCO₃ (F) microspheres determined using dynamic light scattering (DLS). (G) Zeta potential of BSA@CaCO₃ microspheres, the same microspheres deposited sequentially with a PEI layer and a 5 bilayer TA/PEG film.

Fig. 2

(A) Release kinetics of TA from TA/PEG films-coated CaCO₃ microspheres plotted as cumulative release percentage. (B) Release kinetics of TA from TA/PEG films-coated CaCO₃ microspheres plotted as accumulative released amount of TA (denoted by accumulative adsorption increase at 221 nm). (C) Disintegration time of the TA/PEG films with different bilayer numbers.

Fig. 3

(A) Release kinetics of FITC-BSA from TA/PEG films-coated FITC-BSA@CaCO₃ microspheres. (B) 1st derivative of the release profiles. (C) Disintegration time and lag time as a function of bilayer number of the TA/PEG coatings, respectively. (D) Release profile of FITC-BSA from a mixture of FITC-BSA@CaCO₃/(TA/PEG)₅, FITC-BSA@CaCO₃/(TA/PEG)₁₀ and FITC-BSA@CaCO₃/(TA/PEG)₁₅. (E) 1st derivative of the release profile. (F) CD spectra of the pristine BSA and BSA released from BSA/CaCO₃/(TA/PEG)₁₅.

Fig. 4

Representative fluorescence images of C57BL/6 mice after receiving one administration of free Cy5-BSA (A), Cy5-BSA@CaCO₃ (B), Cy5-BSA@CaCO₃/(TA/PEG)₂₀ (C), Cy5-BSA@CaCO₃/(TA/PEG)₄₀ (D), and a mixture of Cy5-BSA@CaCO₃, Cy5-BSA@CaCO₃/(TA/PEG)₂₀ and Cy5-BSA@CaCO₃/(TA/PEG)₄₀ (E).

Fig. 5

Humoral immune response of the vaccines. (A) The schedule of vaccine immunization and blood collection. The single injection vaccine group receives one injection, while other groups receive three injection on Day 0, Day 14 and Day 28. (B, C) S1 subunit-specific IgM (B) and IgG (C) produced in sera of the immunized mice.

Fig. 6

Cellular immune response of the vaccines. (A) Proliferation index of splenocytes after 72 h re-stimulation with S1 subunit. (B-E) Cytokine (IFN-γ (B) and TNF-α (C), IL-4 (D), and IL-6 (E)), secretion by splenocytes after 24 h re-stimulation with S1 subunit. The cytokine concentrations in the supernatant were measured by ELISA. (F) Flow cytometry analysis of CD4⁺ and CD8⁺ T cells.

Fig. 7

(A, C) Schematic description of surrogate virus neutralization test (sVNT) (A) and pseudovirus virus neutralization test (pVNT) (C). (B, D) Inhibition of the sera from different groups measured by sVNT (B) or pVNT (D). The sera were 100-fold diluted.

Fig. 8

(A) Viability of BMDCs after 24 h coculture with CaCO₃/(TA/PEG)₄₀ microspheres. (B) Body weights of the immunized mice changing with time. (C) H&E stained tissues of major organs harvested from immunized mice 6 weeks post-vaccination. Scale bar: 100 µm.