Development Strategies for Influenza Vaccines Utilizing Phage RNA Polymerase and Capping Enzyme NP868R

Abstract

Influenza remains a highly contagious respiratory disease with profound global health and economic implications. Although traditional vaccines, including inactivated influenza vaccines (IIVs), live attenuated influenza vaccines (LAIVs), and recombinant subunit influenza vaccines (RIVs), are widely available, their efficacy against emerging viral strains is often limited. This limitation underscores the urgent need for novel vaccine strategies. In this study, we explored both DNA and RNA vaccine platforms for influenza, utilizing phage RNA polymerase (RNAP) and the capping enzyme NP868R. For the influenza DNA vaccine strategy, we employed a phage RNAP-dependent positive feedback transcription system to achieve high-efficiency expression of the influenza hemagglutinin (HA) antigen. Utilizing the transcription mechanism dependent on phage RNAP polymerase, our DNA vaccine strategy confines antigen transcription and translation within the cytoplasm, thereby reducing the risk of genomic integration inherent to conventional DNA vaccines. In parallel, for the influenza RNA vaccine, we developed a replication-deficient vesicular stomatitis virus (rdVSV) expressing HA as a self-amplifying RNA vaccine. By replacing the traditional T7 vaccinia virus with T7 RNAP fused to a capping enzyme in the rdVSV rescue process, we achieved a high titer of 1.2 × 10⁷ PFU/mL in a single round of rescue. This modification not only shortened the time required for recombinant VSV (rdVSV) rescue but also mitigated the safety concerns associated with T7 vaccinia virus usage. Moreover, this innovation facilitates faster RNA vaccine production, reduces manufacturing costs, and relaxes environmental requirements for RNA vaccine production. In animal studies, BALB/c mice immunized with the DNA vaccine exhibited significantly enhanced HA protein expression and higher antibody titers when dendritic cells (DCs) were employed as delivery carriers. Similarly, RNA vaccine immunized mice exhibited robust humoral and cellular immune responses, marked by increased HA-specific IgG levels and elevated cytokine production. These findings highlight the potential of both platforms as versatile tools for rapidly responding to emerging pathogens and advancing vaccine design for infectious diseases and therapeutic applications. With further technological optimization and clinical validation, this strategy is expected to provide a promising new solution for influenza prevention and control.