To investigate the synergistic antitumor effect and immunological mechanisms of a recombinant oncolytic influenza virus carrying programmed cell death 1 (PD-1) antibody (OVFlu-αhPD1) combined with radiotherapy (RT) in hepatocellular carcinoma (HCC).

A recombinant oncolytic virus was successfully rescued in the early stage using reverse genetics(RG) technology, and designated OVFlu-αhPD1, which was comprehensively characterized via chicken red blood cell hemagglutination assay and detection of replication kinetics in human hepatocellular carcinoma cells Huh7 and murine hepatocellular carcinoma cells Hepa1-6. In vitro, CCK-8 assays were used to assess the effects of OVFlu-αhPD1 at different multiplicities of infection (MOI=0.1, 1, and 3), alone or combined with radiotherapy (RT; 8 Gy), on the viability of hepatoma cells (Huh7 and Hepa1-6) and normal hepatocytes (LX2 and AML12), and a colony formation assay was performed to evaluate the inhibitory effect of OVFlu-αhPD1 plus RT on Huh7 cell proliferation. For in vivo experiments, female C57 BL/6 mice (6 to 8 weeks old, weighing 17 to 19 g; n=45) were subjected to establish a subcutaneous tumor-bearing model of Hepa1-6 cells, and then the model mice were randomized into 5 groups(n=9): PBS, wild-type PR8, OVFlu-αhPD1, RT(24 Gy in 3 fractions), and OVFlu-αhPD1+RT to evaluate the antitumor efficacy of the combination therapy. Tumor volumes were measured every 2 days during treatment. After the final treatment, the spleens were havested for CD4⁺CD69⁺ and CD8⁺CD69⁺ T-cell frequencies by flow cytometry. Major organs (heart, liver, lung, kidneys, and brain) were harvested for HE staining to assess histopathological changes, and the activities of serum AST and ALT were measured to evaluate the safety of oncolytic virus therapy.

The recombinant oncolytic influenza virus OVFlu-αhPD1 showed a hemagglutination titer of 2⁷ to 2⁸, and replicated efficiently in Huh7 and Hepa1-6 cells, with viral titers reaching 7 to 8 lgTCID₅₀/mL at 72 h post-infection. CCK-8 assay demonstrated that OVFlu-αhPD1 inhibited HCC cell viability in a dose-dependent manner (MOI=3 significantly decreased viability in Huh7 and Hepa1-6 cells, P<0.01), and the inhibitory effect was further enhanced after combination with RT (P<0.01). Whereas no significant toxicity to normal hepatocytes was observed. Colony formation assay indicated that combined treatment significantly reduced colony numbers of Huh7 cells (P<0.01). In the mouse tumor-bearing model, the combination group exhibited significantly smaller tumor volumes than any monotherapy group (P<0.01), accompanied by significantly increased proportions of splenic CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells in spleen (P<0.01). HE staining revealed no evident pathological changes in major organs, and serum ALT and AST levels did not differ significantly among groups (P>0.05).

The combination of oncolytic influenza virus and radiotherapy significantly enhances the killing effect against HCC cells in vitro and markedly improves tumor suppression in HCC-bearing mice by activating antitumor immune responses, demonstrating a favorable synergistic antitumor effect and providing a theoretical basis for the clinical application of oncolytic virus-based combination therapy.

To construct a universal influenza mRNA vaccine and evaluate its immunogenicity.

The antigen sequence of hemagglutinin (HA), nucleoprotein (NP) and matrix protein 2 ectodomain (M2e) in influenza A/California/04/2009 was optimized. HA, NP and 3 tandem M2e (3M2e) were cloned into pcDNA3.1 vector, respectively. Then the mRNAs were synthesized by linearization, in vitro transcription, enzymatic capping and enzymatic tailing, and named as mRNA-HA, mRNA-NP and mRNA-3M2e, respectively. The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay. Comb-mRNA vaccine was prepared by enveloped mRNA-HA, mRNA-NP and mRNA-3M2e with lipid nanoparticles, respectively, and the particle size and potential were identified. Twenty-eight 6-week-old female BALB/c mice (18~22 g) were randomly divided into LNP group (n=14) and Comb-mRNA group (n=14). Hemagglutination inhibition (HI) method and microneutralization (MN) test were used to evaluate the serum antibody titer induced by Comb-mRNA vaccines. The mice were infected by 5LD₅₀ wild-type H1N1 influenza virus to evaluate the protective efficacy.

The mRNA-HA, mRNA-NP and mRNA-3M2e were successfully constructed, and the 3 mRNAs could be expressed in 293T cells. The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm, and the average potential was -8.23±1.3 mV. The geometric mean titer (GMT) of HI and MN in the Comb-mRNA group were 179.6 and 201.6, compared with the LNP group. The ratio of IFN-γ⁺CD4⁺/CD8⁺T cells was increased. The Comb-mRNA group could provide protection against 5LD₅₀ wild type influenza H1N1 virus after 2 weeks of booster immunization.

Comb-mRNA, an influenza vaccine candidate, can induce immune responses and protect mice from influenza virus challenge.