Development and in vivo evaluation of a SARS-CoV-2 inactivated vaccine using high hydrostatic pressure

Abstract

Developing low-cost vaccine production strategies is crucial to achieving global health equity and mitigating the spread and impact of disease outbreaks. High hydrostatic pressure (HHP) technology is a widely used technology employed in the food industry for long-term preservation. This project aims at validating HHP as a cost-effective method for the production of highly immunogenic thermal stable whole-virus SARS-CoV-2 vaccines. Structural studies on HHP-inactivated viruses demonstrated pressure-dependent effects, with higher pressures (500-600 MPa) destabilizing viral morphology. Immunogenicity assessments, in animal models, revealed that 500 MPa treatment elicited the most robust humoral and cellular immune responses, outperforming heat inactivation. Additionally, HHP-inactivated viral preparation retained thermostability for 30 days at 4 °C, reducing cold-chain dependencies and enabling vaccine distribution also in low-resource settings. With its rapid, cost-effective, and scalable production process, HHP presents a transformative, equitable solution for global vaccine development, particularly for emerging pathogens.

Fig. 1. HHP treatment of viral isolates and its effects on viral structure and antigenicity.

a Pressure profile of high hydrostatic pressure (HHP) treatment applied over time. Each pressure was maintained for a fixed duration, followed by a rapid decompression phase to atmospheric pressure. After each pressurization cycle, the viral suspension subjected to the treatment was removed and replaced with a fresh suspension for the following cycle. The machine was maintained active throughout the entire series of treatments, continuously recording pressure, including during decompression phases when the pressure returned to ambient levels, resulting in the appearance of a sequential pressure profile in the figure, although each cycle was conducted separately. b Viral replication dynamics, measured as ΔCt values of Real-Time PCR, for HHP-treated and non-HHP treated B.1 and BQ.1.1 lineages over time. C. nsEM microscopy results for 400–600 MPa-treated SARS-CoV-2 isolates compared to the control (non-HHP-treated). d Results of western blot analyses conducted to test the ability of viral proteins to bind polyclonal and monoclonal antibodies after inactivation at high pressures.

Fig. 2. HHP-inactivated SARS-CoV-2 in vivo safety and immunogenicity testing and activation of specific humoral responses.

a Experimental timeline and protocol for mouse immunization, including priming (day 0), boosting (day 28), biological samples collection, and endpoint (day 53). b Immunization groups. c, e IgG titers measured via ELISA for B.1 (c) and BQ.1.1 (e) lineages. d, f Comparison of IgG titers at distinct time points for B.1 (d) and BQ.1.1 (f) lineages. Statistically significant differences are indicated: ***p < 0.001, **p < 0.05, ns = not significant.

Fig. 3. Induction of neutralizing antibody response in HHP-inactivated SARS-CoV-2 vaccinee.

a Western blot analysis of sera from mice immunized with B.1 and BQ.1.1 lineage viruses inactivated by 500 MPa, 600 MPa HHP, or heat. b–e Neutralizing antibody titers in immunized mice measured at different time points. Images B and D show longitudinal neutralization titers for B.1 (b) and BQ.1.1 (d) lineages. Images (c, d) show the comparison of neutralizing titers between immunization groups. Statistical significance: ***p < 0.001, **p < 0.05, ns not significant.

Fig. 4. Induction of T-cell response in HHP-inactivated SARS-CoV-2 vaccinee.

a, c IFN-γ ELISpot results for PBMCs from mice immunized with B.1 (a) and BQ.1.1 (c) lineage viruses inactivated by 500 MPa, 600 MPa HHP, or heat. b, d IFN-γ ELISpot results for splenocytes from mice immunized with B.1 (b) and BQ.1.1 (d) lineage viruses. Statistical significance: **p < 0.05, ns = not significant.

Fig. 5. Western blot analysis of the Spike protein signal from SARS-CoV-2 B.1 and BQ.1.1 variants inactivated at 500 MPa, stored at different temperatures (-80 °C, 4 °C, 25 °C).

Vaccine candidate aliquots stored under different conditions were analyzed over various time points (7, 14, and 30 days).