Since its first appearance in 2019, SARS-CoV-2 has spread rapidly worldwide and continues to circulate in many countries, causing symptoms and COVID-19 disease, despite an unprecedented, swift deployment of effective first-generation mRNA- and vector-based vaccines that target the viral Spike (S) protein. Since then, multiple virus variants have emerged, carrying escape mutations mainly in the S gene, which correlate with declining protection rates.
To combat new variants of the virus and induce a broad immune response to more viral proteins, promising recent vaccine approaches focus on attenuating the virus and favoring the intranasal route to induce stronger mucosal immunity. However, these recent approaches have not succeeded in inducing transmission-blocking immunity. Principal drawbacks of attenuation include residual infectivity or the risk of spontaneous reversion to virulence, i.e., causing the wild-type disease from which one would like to protect. This is particularly crucial for at-risk groups such as immunocompromised, transplanted, and elderly individuals or cancer patients.
To generate a safe and effective next-generation SARS-CoV-2 vaccine that induces a potent immune response, we developed the ‘single-cycle infection concept’. By deleting the essential structural Envelope (E) gene from the viral genome and combining it with a stable cellular trans-complementation system, as described for other coronaviruses, we produced an intact but propagation-defective vaccine virus to serve as a SARS-CoV-2 vaccine candidate.
E, the smallest essential structural viral gene involved in viral budding, was selected to render the vaccine virus single-cycle. With its small size, it is also well-suited for manipulation due to its minimal genetic burden. Additionally, the E gene encodes a low-abundance, membrane-embedded viroporin that is poorly immunogenic and contributes insignificantly to T-cell responses. For better traceability during initial research, the E gene was replaced with an eGFP reporter (ΔEG).
To simultaneously enhance immune functions, we deleted critical accessory genes: open reading frame 6 (ORF6) has been described to suppress the T cell response and eliminate the interferon (IFN) response in the infected cells. ORF8 has been shown to reduce the T-cell response in vivo. Moreover, ORF6 and ORF8 are non-structural cytoplasmic proteins that will not have a major impact on antibody responses to the virus. The elimination of ORF6 and ORF8 is thereby expected to increase the immunogenicity of single-cycle infection viruses (SCVs) beyond that of a natural SARS-CoV-2 infection while reinforcing the high level of safety.
This study investigates the properties of a single-cycle, triple-deletion vaccine virus (ΔEG68) and assesses the direct impact of eliminating ORF6 and ORF8 by comparing it to an “E-deleted only” virus (ΔEG). We show evidence of enhanced immune stimulation, the elicitation of full protection against challenge infection, and transmission-blocking immunity in the Syrian hamster model.