A Molecularly Engineered Antiviral Banana Lectin Inhibits Fusion and is Efficacious Against Influenza Virus Infection in Vivo

There is a strong need for a new broad-spectrum antiinfluenza therapeutic, as vaccination and existing treatments are only moderately effective. We previously engineered a lectin, H84T banana lectin (H84T), to retain broad-spectrum activity against multiple influenza strains, including pandemic and avian, while largely eliminating the potentially harmful mitogenicity of the parent compound. The amino acid mutation at position 84 from histidine to threonine minimizes the mitogenicity of the wild-type lectin while maintaining antiinfluenza activity in vitro. We now report that in a lethal mouse model H84T is indeed nonmitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus–endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a broad-spectrum antiinfluenza agent

COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus - and R. arrhizus -induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections

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Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs’ intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.</div

mtROS induction stimulates antimicrobial responses.

(A) Representative fluorescence images primary tracheal epithelial cells harvested from mt-roGFP mice, pre-treated (or not) with TTFA and FCCP, then treated with PBS or ODN. Images shown as gradient of color intensity from the reduced (blue) form to the oxidized (green) form of roGFP. Scale bar, 50 μm. (B) Ratio of the fluorescence intensity of the oxidized:reduced roGFP from A, quantified at 488 nm and 405 nm, respectively. (C) Bacterial burden of HBEC3-KT cells treated with the indicated ligands with or without TTFA-FCCP treatment. (D) Survival of wild type mice challenged with P. aeruginosa one day after nebulized treatment with PBS or Pam2 and ODN with or without TTFA-FCCP (n = 15 mice/group). (E) Survival of Tlr9-/- mice challenged with P. aeruginosa one day after nebulized treatment with PBS or Pam2 and ODN with or without TTFA-FCCP (n = 15 mice/group). Bacterial burden of primary mouse tracheal epithelial cells (F), primary mouse alveolar cells (G), or primary human alveolar cells (H) after treatment with Pam2 and ODN. Bacterial burden of HBEC3-KT cells treated with Pam2 and erastin or ODN (I) or Pam2 and VBIT-4 or ODN (J). (K) Mouse survival of P. aeruginosa challenge given one day after nebulized treatment with the indicated agents (n = 15 mice/group). (L) Mouse lung bacterial burden immediately after P. aeruginosa challenge following treatment with the indicated agents (n = 4 mice/group). * p <0.02 vs PBS, † p< 0.05 vs ODN, ‡ p < 0.05 vs same ligand without TTFA-FCCP, ¶ P <0.0001 vs. PBS.</p

Uncut immunoblots for mitochondria mass analysis.

HBEC3-KT cells were exposed to ODN for 0, 50, or 100 min. Shown are the uncut immunoblots for the bands shown in Fig 2B. These include (A) SDHB, succinate dehydrogenase subunit B; (B) COX4, cytochrome c oxidase subunit IV; (C) ATP5A, ATP synthase alpha-subunit; (D) CS, citrate synthase; (E) VDAC1, voltage dependent anion channel 1; and (F) β-Actin, used as a loading control. (EPS)</p

Effect of TCA cycle metabolites on ODN-induced mtROS generation.

mtROS dose response to ODN in HBEC3-KT cells supplemented with the TCA metabolites or metabolite analogues (A) citrate, (B) pyruvate, (C) α-ketoglutarate, (D) dimethyl succinate, (E) dimethyl malonate, (F) dimethyl fumurate or (G) oxaloacetate. * p≤0.003 vs. 0 μM ODN treated with no metabolite pretreatment by one-way ANOVA using Holm-Sidak method. † p≤0.05 vs. same ODN dose with no metabolite pretreatment by one-way ANOVA using Holm-Sidak method. ‡ p (EPS)</p

VBIT-4 increases epithelial mtROS generation and Δ_Ψm.

MitoSOX fluorescence (A) and JC-1 ratio (B) of HBEC3-KT cells treated for 100 min with PBS, ODN or VDAC1 inhibitor VBIT-4. * p (EPS)</p

Correlation between ODN and VDAC1 pixel intensity by linear regression.

Pixel intensity values for FITC-labeled ODN and Alexa Fluor 555-labeled anti-VDAC1 antibody were plotted against each other and a simple linear regression model was fit to the data. Pearson’s correlation coefficients were calculated to determine whether VDAC1 pixel intensity tended to accumulate with ODN1 pixel intensity. FITC, Fluorescein isothiocyanate; VDAC1, voltage dependent anion channel 1. (EPS)</p