A universal dual mechanism immunotherapy for the treatment of influenza virus infections

The study addresses the need for more effective influenza therapeutics capable of treating severe and late-stage infections. Annual influenza causes substantial morbidity, mortality, and economic burden. Existing control strategies—vaccines, neuraminidase inhibitors, and other antivirals—have limitations: vaccine mismatch due to rapid viral evolution, reduced efficacy of neuraminidase inhibitors when treatment starts more than 48 hours after symptom onset, and emergence of drug-resistant variants. The authors hypothesize that combining a broad-spectrum neuraminidase inhibitor with an immunogenic hapten to decorate virus and infected cells could both inhibit viral release and recruit endogenous antibodies to mediate immune clearance, thereby achieving superior efficacy across influenza A and B strains, including in advanced infections.

The paper reviews: (1) Vaccine performance varies annually due to antigenic drift and strain prediction challenges, leading to breakthrough infections despite vaccination. (2) Approved neuraminidase inhibitors (zanamivir, oseltamivir, peramivir, laninamivir) block viral neuraminidase and reduce illness when given early but provide limited benefit if started beyond two days post-symptom onset; resistance has emerged at low levels. (3) Broadly acting antivirals and combination approaches are being explored; zanamivir binds neuraminidases across influenza A (groups 1 and 2) and B, offering a potential universal target. (4) Naturally occurring anti-DNP antibodies exist in humans and have been used in hapten-targeted immunotherapies in oncology and infectious disease models, suggesting a strategy to recruit immune effector mechanisms against pathogen-decorated targets.

Design and synthesis: Zanamivir was conjugated at the solvent-exposed C-7 hydroxyl via a PEG linker to dinitrophenyl (DNP) to create zan-DNP (therapeutic) and to rhodamine (zan-rhodamine) for binding assays. A 99mTc-chelated zanamivir conjugate (zan-99mTc) was synthesized for biodistribution and imaging. Binding and inhibition assays: Saturation binding assays were performed against neuraminidases from multiple influenza strains: A/Wisconsin/629-D0015/2009 (H1N1)pdm09, A/Netherlands/22/2003 (H3N2), B/Florida/4/2006 (Yamagata), and B/Brisbane/60/2008 (Victoria). Binding to neuraminidase expressed on virus-infected MDCK cells and on primary normal human bronchial epithelial (NHBE) cells grown at air–liquid interface (ALI) was measured. Antiviral potency was assessed by cytopathic effect (CPE) reduction in MDCK–influenza co-cultures to determine EC50 values versus H1N1 and H3N2. Antibody recruitment: In vitro, biotinylated anti-DNP antibodies with streptavidin-PE were used to quantify antibody binding to zan-DNP-decorated infected MDCK cells by confocal microscopy and flow cytometry as a function of zan-DNP concentration. In vivo, BALB/c mice infected with 100× MLD50 A/California/07/2009 (H1N1)pdm09 received zan-DNP and exogenous anti-DNP antibodies; lung cells were analyzed by flow cytometry for hemagglutinin positivity and anti-DNP opsonization. Effector function assays: HEK293 cells transduced to express influenza A N1 neuraminidase were used to assess complement-dependent cytotoxicity (CDC) by incubation with zan-DNP, anti-DNP antibodies, and complement. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using engineered Jurkat effector cells or human PBMCs in the presence of anti-DNP antibodies and varying zan-DNP concentrations. Mouse infection and therapy studies: BALB/c mice (often immunized with DNP-KLH to raise anti-DNP titers; non-immunized cohorts used with exogenous anti-DNP supplementation) were challenged intranasally with lethal influenza doses (typically 100× MLD50; some text mentions higher loads). Therapeutic regimens compared zan-DNP to zanamivir or DNP alone. Dosing included single intranasal (IN), intravenous (IV), or intraperitoneal (IP) administration at various time points (24, 48, 72, 96 hours post-infection). Outcomes: body weight monitoring, survival (euthanasia threshold 25% weight loss or moribund), and lung viral titers by RT-qPCR in select studies. Biodistribution/imaging: Virus-infected and control mice received IV zan-99mTc; organ radioactivity (%ID/g) was quantified and SPECT-CT imaging performed. Competition with excess zanamivir assessed target specificity. Statistics: GraphPad Prism used. Unpaired two-sided t-tests for group comparisons in antibody recruitment and lung titers; two-sided log-rank tests for survival analyses. Replicates typically n=3 for in vitro; mouse group sizes varied (commonly 5 per group; details in figure legends).

- Zan-DNP maintains high-affinity binding to influenza neuraminidase: - A/Wisconsin/629-D0015/2009 (H1N1)pdm09: Kd zan-DNP 0.8 nM (zanamivir 0.3 nM; zan-rhodamine 4.9 nM). - A/Netherlands/22/2003 (H3N2): Kd zan-DNP 1.1 nM (zanamivir 1.0 nM; zan-rhodamine 10.0 nM). - B/Florida/4/2006 (Yamagata): Kd zan-DNP 21.5 nM (zanamivir 2.4 nM; zan-rhodamine 25.9 nM). - B/Brisbane/60/2008 (Victoria): Kd zan-DNP 58.4 nM (zanamivir 7.2 nM; zan-rhodamine 43.2 nM). - Binding to neuraminidase on NHBE ALI cultures: apparent Kd ~1.4 nM (influenza A, N1). - Antiviral activity in CPE assays: zan-DNP reduced cytopathic effect with potencies similar to zanamivir: EC50 ~1.7 nM (H1N1) and ~7.6 nM (H3N2). - Antibody recruitment: - In vitro: zan-DNP mediated saturable recruitment of anti-DNP antibodies to infected MDCK cells; binding blocked by excess zanamivir. - In vivo: In H1N1-infected mice treated with zan-DNP plus anti-DNP antibodies, 24.4% of hemagglutinin-positive lung cells were opsonized versus 0.8–1.7% in controls (zanamivir or zan-DNP alone) and 0.4% without anti-DNP. - Effector functions: - CDC: >60% killing of neuraminidase-expressing HEK293 cells at 100 nM zan-DNP; killing required concurrent zan-DNP, anti-DNP antibodies, and complement; reduced or abrogated when any component was omitted or blocked. - ADCC: Potent ADCC observed with engineered Jurkat effectors or PBMCs when zan-DNP bridged anti-DNP antibodies to neuraminidase-expressing cells; bell-shaped dose–response consistent with bridging saturation at high concentrations. - Therapeutic efficacy in vivo: - Zan-DNP outperformed zanamivir in mice lethally infected with influenza A (H1N1). Standard zanamivir dosing (0.5 μmol/kg, IV, BID ×5 days) had minimal effect on weight loss and survival when started 24 h post-infection, whereas a single dose of zan-DNP led to full recovery in DNP-immunized mice. - Late treatment: A single zan-DNP dose remained effective when initiated 48–72 h post-infection (100% survival at 48 h; high survival at 72 h; reduced efficacy by 96 h), while zanamivir was ineffective even at 48 h. - Route flexibility: Parenteral administration was effective. Zan-99mTc localized specifically to infected lungs with highest %ID/g in lungs; uptake was blocked by 100× excess zanamivir and absent in uninfected mice, demonstrating target specificity. Apparent Kd of zan-99mTc binding to infected MDCK cells ~15.1 nM. - Single IP dose (1.5 or 4.5 μmol/kg) of zan-DNP at 24 h post-infection yielded 100% survival and weight recovery across multiple strains: A/PR/8/1934 (H1N1), X-31 (H3N2), A/California/07/2009 (H1N1)pdm09, and B/Florida/4/2006. Zanamivir did not confer similar survival. - In non-immunized mice with low endogenous anti-DNP, co-administration of exogenous anti-DNP antibodies (e.g., 10 mg/kg IV) with zan-DNP rescued lethally infected animals; controls lacking zan-DNP or antibodies did not survive. - Overall, zan-DNP’s dual mechanism—neuraminidase inhibition plus immune recruitment to kill free virus and infected cells—produced complete responses in severe, advanced murine influenza models across A and B lineages.

The findings validate a dual-mechanism therapeutic strategy that addresses key shortcomings of current influenza treatments. By targeting neuraminidase with zanamivir’s broad binding spectrum and simultaneously decorating virions and infected cells with DNP, zan-DNP not only inhibits viral egress but also recruits endogenous (or supplemented) anti-DNP antibodies to trigger CDC and ADCC. This combination explains the superior efficacy observed, including successful rescue when treatment is delayed up to 72 hours, a window where standard neuraminidase inhibitor monotherapy is typically ineffective. The biodistribution data and therapeutic outcomes via intranasal and parenteral routes highlight practical flexibility in clinical administration. The ability to eliminate both free virus and infected cells likely contributes to the rapid viral clearance and survival benefits. Conceptually, the platform could be generalized to other exposed viral targets (e.g., hemagglutinin, M2) and alternative haptens to optimize immune engagement. These results suggest a path toward a universal, strain-agnostic influenza therapy leveraging innate antibody repertoires, with potential to complement or surpass current standards in severe disease settings.

This work introduces zan-DNP, a zanamivir–dinitrophenyl conjugate that couples potent neuraminidase inhibition with immune recruitment to eradicate influenza infections. Zan-DNP retains nanomolar affinity for neuraminidases across influenza A and B, recruits anti-DNP antibodies to virions and infected cells, and activates CDC and ADCC. In murine models challenged with lethal viral doses, a single intranasal or parenteral dose produced complete survival, even when treatment was delayed, and outperformed zanamivir monotherapy. Radiolabeled analog studies confirmed specific accumulation in infected lungs. Co-administration of exogenous anti-DNP antibodies restored efficacy in non-immunized mice with low endogenous titers. These data support further development of zan-DNP as a universal anti-influenza therapy. Future research should evaluate safety and immunogenicity profiles, pharmacokinetics, optimal dosing and routes in larger animals and humans, explore alternative haptens and viral ligands, assess combination regimens with other antivirals, and investigate applicability to other respiratory pathogens using analogous hapten-targeting strategies.

- Translation to humans remains untested; efficacy and safety were demonstrated in mouse models only. - Therapeutic efficacy depends on sufficient anti-DNP antibody levels; some subjects may require vaccination or exogenous antibody supplementation, adding complexity. - Comprehensive toxicity, immunopathology, and off-target immune activation (e.g., complement-mediated damage) were not extensively characterized. - Some reported in vivo experiments had small group sizes, and authors note that several in vivo experiments were performed once; reproducibility across independent cohorts and laboratories is needed. - Binding affinity to influenza B neuraminidases was reduced relative to zanamivir (though still in low-to-moderate nanomolar range); clinical relevance of this difference remains to be established. - Potential for viral escape or alterations in neuraminidase antigenicity under selective pressure was not assessed. - Some methodological descriptions in the excerpt are partially incomplete/garbled, but core findings are supported by multiple assays.