Influenza poses a significant global health threat, causing millions of severe infections and hundreds of thousands of deaths annually. Current control methods, including vaccines and neuraminidase inhibitors, have limitations. Vaccines' efficacy varies due to viral evolution, while neuraminidase inhibitors are most effective when administered early in the infection. This study proposes a novel therapeutic approach combining the antiviral activity of a broad-spectrum neuraminidase inhibitor with the immunologic power of a vaccine. The researchers hypothesized that conjugating zanamivir (a neuraminidase inhibitor effective against both influenza A and B viruses) with the highly immunogenic DNP hapten would create a molecule capable of both inhibiting viral replication and recruiting the immune system to eliminate infected cells. This dual mechanism of action was expected to enhance therapeutic efficacy, particularly in treating advanced infections, and overcome the limitations of existing treatments. The study aimed to synthesize this bifunctional conjugate, characterize its binding affinity and antiviral activity, and evaluate its therapeutic efficacy in a mouse model of lethal influenza infection. The success of this approach would provide a promising universal anti-influenza therapy.

The paper reviews the existing approaches to influenza treatment: vaccines and neuraminidase inhibitors. It highlights the limitations of vaccines due to the rapid antigenic drift of the influenza virus and the narrow therapeutic window for neuraminidase inhibitors, which are most effective when administered within 48 hours of symptom onset. The emergence of neuraminidase inhibitor-resistant viral strains further underscores the need for novel therapeutic strategies. The researchers point out that influenza virus-infected cells express increased viral proteins on their surfaces, creating a target for therapeutic intervention. While the use of DNP as a hapten has shown some evidence of inducing ligand-targeted cytotoxicity, it's noted that the data on its antigenic abilities requires further validation.

The researchers synthesized a zanamivir-DNP conjugate (zan-DNP) by linking zanamivir to DNP via a PEG linker. They characterized zan-DNP's binding affinity to neuraminidase from various influenza A and B virus strains using binding assays, including analysis of the dissociation constant (Kd). The antiviral activity of zan-DNP was evaluated by assessing its ability to suppress the cytopathic effect of influenza A and B viruses in MDCK cell cultures. The ability of zan-DNP to recruit anti-DNP antibodies to virus-infected cells was investigated using in vitro assays on MDCK cells and in vivo studies on mice infected with influenza A and B viruses. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays were conducted to determine the mechanisms by which zan-DNP mediates infected cell killing. In vivo therapeutic efficacy was assessed in mice challenged with lethal doses of influenza A and B viruses. Mice were treated with zan-DNP, zanamivir, or a control, and their body weight and survival were monitored. The timing of treatment administration was varied to assess the effectiveness of zan-DNP in treating advanced infections. To explore the possibility of parenteral administration, the researchers synthesized a zanamivir-99mTc conjugate for biodistribution studies and SPECT-CT imaging in mice. Finally, experiments were conducted to assess the role of exogenous anti-DNP antibodies in mediating the therapeutic effect of zan-DNP in mice lacking sufficient endogenous anti-DNP antibodies. Statistical analyses were performed using appropriate methods to compare the results of different treatment groups.

Zan-DNP retained high binding affinity for neuraminidase from various influenza A and B virus strains. It effectively inhibited neuraminidase activity and recruited anti-DNP antibodies to virus-infected cells in vitro. In vivo, zan-DNP treatment significantly improved survival and reduced weight loss in mice infected with lethal doses of influenza A and B viruses. This effect was observed even when treatment was delayed by up to 72 hours post-infection, unlike the limited effectiveness of zanamivir in advanced infection. The protective effect of zan-DNP was dependent on the presence of anti-DNP antibodies. Zan-DNP mediated both CDC and ADCC. Biodistribution studies using a zanamivir-99mTc conjugate demonstrated that intravenously administered zan-DNP accumulated specifically in the lungs of virus-infected mice. A single dose of zan-DNP was highly effective, showing 100% survival in lethally infected mice across various strains of influenza A and B viruses. Even in mice lacking sufficient endogenous anti-DNP antibodies, co-administration of exogenous anti-DNP antibodies restored the therapeutic efficacy of zan-DNP.

The study demonstrated that zan-DNP, a novel bifunctional conjugate, effectively combats influenza infection through a dual mechanism of action. By inhibiting neuraminidase activity and recruiting the immune system to eliminate both free virus and infected cells, zan-DNP exhibits superior therapeutic efficacy compared to zanamivir alone, especially in treating advanced infections. The study suggests that the dual mechanism enhances efficacy beyond what is observed with either strategy alone. The fact that a single dose of zan-DNP achieved complete recovery in mice with severe infections further highlights its potential as a universal anti-influenza therapy. The successful use of both intranasal and intraperitoneal administration provides flexibility in treatment options. The observed tissue-specific accumulation of zan-DNP in the lungs of infected mice, evidenced by SPECT-CT imaging, confirms targeted drug delivery. The results underscore the importance of considering the immune response in the development of anti-influenza therapeutics.

This study demonstrates the significant therapeutic potential of zan-DNP, a dual-mechanism anti-influenza agent. Its ability to effectively treat advanced infections, its broad-spectrum activity against both influenza A and B viruses, and its flexibility in administration routes make it a promising candidate for further development as a universal anti-influenza therapy. Future research could focus on optimizing the drug's pharmacokinetic and pharmacodynamic properties, exploring alternative targeting ligands and haptens, and conducting clinical trials to assess its safety and efficacy in humans.

The study was conducted in a mouse model, and the results may not fully translate to human clinical settings. The specific mechanism of action and the contribution of CDC and ADCC need further investigation. While the use of exogenous anti-DNP antibodies restored efficacy in mice lacking sufficient endogenous antibodies, the prevalence of sufficient endogenous anti-DNP antibodies in humans requires further research. The long-term effects and potential for drug resistance also warrant further investigation.