The Interferon-Lambda Family Celebrates 20 Years of Scientific Discovery.

It has now been 20 years since the original discovery of the interferon λ (IFN-λ) family (Kotenko et al., 2003; Sheppard et al., 2003) and 10 years since the subsequent discovery of IFN-λ4 (Prokunina-Olsson et al., 2013). The IFN-λ family (type III IFNs) includes 4 members: IFN-λ1, 2, 3, and 4, and all 4 of these proteins signal through the same heterodimeric receptor complex: IFN-λR1 plus IL-10R2. Throughout the past 20 years, much has been learned about the IFN-λ family and the important role of these cytokines in antiviral responses against viruses such as hepatitis C virus, influenza A virus, and SARS-CoV-2. This special issue of the Journal of Interferon & Cytokine Research (JICR) features a group of new reports that highlight recent developments regarding various aspects of IFN-λ-mediated responses. Many of these reports were first presented during the Interferon Lambda 2022 Satellite Meeting after the "Cytokines 2022" meeting in Hawaii. These articles underscore the fact that our understanding of the IFN-λ family continues to evolve and remains a critical subject area for additional future research.

Impact of Baloxavir Resistance-Associated Substitutions on Influenza Virus Growth and Drug Susceptibility.

Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. IMPORTANCE Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.

Characterization of Monoclonal Antibodies to Measure Cell Surface Protein Levels of Human Interferon-Lambda Receptor 1.

Type III interferons (IFN-lambdas, IFN-λs) are important antiviral cytokines that can also modulate immune responses by acting through a heterodimeric receptor composed of the specific and limited expressed IFN-λR1 chain and the ubiquitous IL-10R2 chain, which is shared with IL-10 family cytokines. Conflicting data have been reported regarding which cells express the IFN-λR1 subunit and directly respond to IFN-λs. This is, in part, owing to transcript levels of the IFN-λR1 gene, IFNLR1, not always correlating with cell surface protein levels. In this study, we tested a panel of novel monoclonal antibodies (mAbs) that specifically recognize human IFN-λR1. Initially, antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA), from which a subset of antibodies was selected for additional flow cytometry and neutralization assays. We further characterized two antibodies based on their strong ELISA binding activity (HLR1 and HLR14) and found only HLR14 could reliably detect cell surface IFN-λR1 protein on a variety of cell lines by flow cytometry. HLR14 could also detect IFN-λR1 protein on certain primary human blood cells, including plasmacytoid dendritic cells and B cells from peripheral blood. Availability of the HLR14 mAb will enable the quantification of IFN-λR1 protein levels on cells and better characterization of the cell specificity of the IFN-λ response.

Comparison of the Antiviral Activity of Remdesivir, Chloroquine, and Interferon-ß as Single or Dual Agents Against the Human Beta-Coronavirus OC43.

The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-ß, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-ß exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-ß plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-ß alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-ß plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.

Identification of a pharmacological approach to reduce ACE2 expression and development of an in vitro COVID-19 viral entry model.

Because of rapid emergence and circulation of the SARS-CoV-2 variants, especially Omicron which shows increased transmissibility and resistant to antibodies, there is an urgent need to develop novel therapeutic drugs to treat COVID-19. In this study we developed an in vitro cellular model to explore the regulation of ACE2 expression and its correlation with ACE2-mediated viral entry. We examined ACE2 expression in a variety of human cell lines, some of which are commonly used to study SARS-CoV-2. Using the developed model, we identified a number of inhibitors which reduced ACE2 protein expression. The greatest reduction of ACE2 expression was observed when CK869, an inhibitor of the actin-related protein 2/3 (ARP2/3) complex, was combined with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of sodium-hydrogen exchangers (NHEs), after treatment for 24 h. Using pseudotyped lentivirus expressing the SARS-CoV-2 full-length spike protein, we found that ACE2-dependent viral entry was inhibited in CK869 + EIPA-treated Calu-3 and MDA-MB-468 cells. This study provides an in vitro model that can be used for the screening of novel therapeutic candidates that may be warranted for further pre-clinical and clinical studies on COVID-19 countermeasures.

Generation and characterization of interferon-beta-resistant H1N1 influenza A virus.

Interferons (IFNs) mediate innate antiviral activity against many types of viruses, including influenza viruses. In light of their potential use as anti-influenza agents, we examined whether resistance to these host antiviral proteins can develop. We generated IFN-ß-resistant variants of the A/California/04/09 (H1N1) virus by serial passage in a human airway epithelial cell line, Calu-3, under IFN-ß selective pressure. The combination of specific mutations (i.e., L373I in PB1, K154E1, D222G1, I56V2, and V122I2 in HA, and M269I in NA) correlated with decreased ability of the virus to induce expression of IFN (IFNB1, IFNL1, and IFNL2/3) and IFN-stimulated genes (IFIT1, IFIT3, OAS1, IRF7, and MX1) by target respiratory epithelial cells. In addition, the IFN-induced mutations were associated with decreased HA binding affinity to α2,6 sialyl receptors, reduced NA enzyme catalytic activity, and decreased polymerase transcription activity. Our findings demonstrate that the mutations in the influenza HA, NA, and PB1 proteins induced by IFN-b selective pressure significantly increase viral ability to productively infect and replicate in host cells. Keywords: influenza A virus; interferon-ß; lung epithelial cells; interferon response.

Pleiotropic Effects of Influenza H1, H3, and B Baloxavir-Resistant Substitutions on Replication, Sensitivity to Baloxavir, and Interferon Expression.

Baloxavir is an anti-influenza endonuclease inhibitor that targets the polymerase acidic (PA) protein of influenza A and B viruses. Our knowledge regarding the pleiotropic effects of baloxavir resistance-associated substitutions is limited. We generated recombinant A/California/04/09 (H1N1)-, A/Hong Kong/218849/2006 (H3N2)-, and B/Victoria/504/2000-like viruses that contained PA substitutions identified in baloxavir clinical trials and surveillance that could potentially be associated with baloxavir resistance. We characterized their susceptibility to baloxavir, impact on polymerase activity, viral growth, and ability to induce interferon (IFN) and IFN-stimulated genes expression in vitro. Four PA substitutions, H1N1 I38L/T, E199D, and B G199R, significantly reduced the sensitivity of the recombinant viruses to baloxavir (14.1-fold). We confirmed our findings by using the luciferase-based ribonucleoprotein minigenome assay and by using virus yield reduction assay in Calu-3 and normal human bronchial epithelial (NHBE) cells. We observed that I38L and E199D resulted in decreased viral replication of the H1N1 wild-type virus (1.4-fold) but the H1N1 I38T and B G199R substitutions did not significantly alter replication capacity in Calu-3 cells. In addition, H1N1 variants with PA I38L/T and E199D induced significantly higher levels of IFNB1 gene expression compared to the wild-type virus (4.2-fold). In contrast, the B variant, G199R, triggered the lowest levels of IFN genes in Calu-3 cells (1.6-fold). Because baloxavir is a novel anti-influenza therapeutic agent, identifying and characterizing substitutions associated with reduced sensitivity to baloxavir, as well as the impact of these substitutions on viral fitness, is paramount to the strategic implementation of this novel countermeasure.

Effect of Fatty Acid Composition in Polysorbate 80 on the Stability of Therapeutic Protein Formulations.

PURPOSE: Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions. METHODS: Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity. RESULTS: The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions. CONCLUSIONS: These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

Adaptation of influenza B virus by serial passage in human airway epithelial cells.

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. To understand their adaptation capability, we examined the genetic changes that occurred following 15 serial passages of two influenza B viruses, B/Brisbane/60/2008 and B/Victoria/504/2000, in human epithelial cells. Thirteen distinct amino acid mutations were found in the PB1, PA, hemagglutinin (HA), neuraminidase (NA), and M proteins after serial passage in the human lung epithelial cell line, Calu-3, and normal human bronchial epithelial (NHBE) cells. These changes were associated with significantly decreased viral replication levels. Our results demonstrate that adaptation of influenza B viruses for growth in human airway epithelial cells is partially conferred by selection of HA1, NA, and polymerase mutations that regulate receptor specificity, functional compatibility with the HA protein, and polymerase activity, respectively.

Laninamivir-Interferon Lambda 1 Combination Treatment Promotes Resistance by Influenza A Virus More Rapidly than Laninamivir Alone.

Each year, 5% to 20% of the population of the United States becomes infected with influenza A virus. Combination therapy with two or more antiviral agents has been considered a potential treatment option for influenza virus infection. However, the clinical results derived from combination treatment with two or more antiviral drugs have been variable. We examined the effectiveness of cotreatment with two distinct classes of anti-influenza drugs, i.e., neuraminidase (NA) inhibitor, laninamivir, and interferon lambda 1 (IFN-λ1), against the emergence of drug-resistant virus variants in vitro We serially passaged pandemic A/California/04/09 [A(H1N1)pdm09] influenza virus in a human lung epithelial cell line (Calu-3) in the presence or absence of increasing concentrations of laninamivir or laninamivir plus IFN-λ1. Surprisingly, laninamivir used in combination with IFN-λ1 promoted the emergence of the E119G NA mutation five passages earlier than laninamivir alone (passage 2 versus passage 7, respectively). Acquisition of this mutation resulted in significantly reduced sensitivity to the NA inhibitors laninamivir (∼284-fold) and zanamivir (∼1,024-fold) and decreased NA enzyme catalytic activity (∼5-fold) compared to the parental virus. Moreover, the E119G NA mutation emerged together with concomitant hemagglutinin (HA) mutations (T197A and D222G), which were selected more rapidly by combination treatment with laninamivir plus IFN-λ1 (passages 2 and 3, respectively) than by laninamivir alone (passage 10). Our results show that treatment with laninamivir alone or in combination with IFN-λ1 can lead to the emergence of drug-resistant influenza virus variants. The addition of IFN-λ1 in combination with laninamivir may promote acquisition of drug resistance more rapidly than treatment with laninamivir alone.

The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

Influenza A virus hemagglutinin mutations associated with use of neuraminidase inhibitors correlate with decreased inhibition by anti-influenza antibodies.

BACKGROUND: Vaccination and the use of neuraminidase inhibitors (NAIs) are currently the front lines of defense against seasonal influenza. The activity of influenza vaccines and antivirals drugs such as the NAIs can be affected by mutations in the influenza hemagglutinin (HA) protein. Numerous HA substitutions have been identified in nonclinical NAI resistance-selection experiments as well as in clinical specimens from NAI treatment or surveillance studies. These mutations are listed in the prescribing information (package inserts) for FDA-approved NAIs, including oseltamivir, zanamivir, and peramivir. METHODS: NAI treatment-emergent H1 HA mutations were mapped onto the H1N1 HA1 trimeric crystal structure and most of them localized to the HA antigenic sites predicted to be important for anti-influenza immunity. Recombinant A/California/04/09 (H1N1)-like viruses carrying HA V152I, G155E, S162 N, S183P, and D222G mutations were generated. We then evaluated the impact of these mutations on the immune reactivity and replication potential of the recombinant viruses in a human respiratory epithelial cell line, Calu- 3. RESULTS: We found that the G155E and D222G mutations significantly increased viral titers ~ 13-fold compared to the wild-type virus. The hemagglutination and microneutralization activity of goat and ferret antisera, monoclonal antibodies, and human serum samples raised against pandemic A(H1N1)pdm09 viruses was ~ 100-fold lower against mutants carrying G155E or D222G compared to the wild-type virus. CONCLUSIONS: Although the mechanism by which HA mutations emerge during NAI treatment is uncertain, some NAI treatment-emergent HA mutations correlate with decreased immunity to influenza virus.

Effect of influenza H1N1 neuraminidase V116A and I117V mutations on NA activity and sensitivity to NA inhibitors.

Neuraminidase inhibitors (NAIs) play a key role in the management of influenza. Given the limited number of FDA-approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority. Two NA mutations, V116A and I117V, are found in ∼0.6% of human, avian, and swine N1 isolates. Using the A/California/04/09-like (CA/04, H1N1) background, we examined the impact of V116A and I117V NA mutations on NAI susceptibility, substrate specificity, and replicative capacity in normal human bronchial (NHBE) cells and a human respiratory epithelial cell line (Calu-3). We compared the impact of V116A and I117V on the functional properties of NA and compared these mutations with that of previously reported NAI-resistant mutations, E119A, H275Y, and N295S. All NA mutations were genetically stable. None of the viruses carrying NA mutations grew to significantly lower titers than CA/04 in Calu-3 cells. In contrast, V116A, I117V, E119A, and N295S substitutions resulted in significantly lower viral titers (1.2 logs) than the parental CA/04 virus in NHBE cells. V116A conferred reduced sensitivity to oseltamivir and zanamivir (13.7-fold). When MUNANA, 3'SL, and 6'SL substrates were applied, we observed that V116A reduced binding ability for all substrates (13.9-fold) and I117V led to the significantly decreased affinity for MUNANA and 6'SL (4.2-fold). Neither mutation altered the catalytic efficiency (kcat/KM) in catalyzing 3'SL, but the efficiency in catalyzing MUNANA and 6'SL was significantly decreased: only ∼34.7% compared to the wild-type NA. The efficiencies of NAs with E119A, H275Y, and N295S mutations to catalyze all substrates were ∼19.4% of the CA/04 NA. Our study demonstrates the direct effect of drug-resistant mutations located inside or adjacent to the NA active site on NA substrate specificity.

Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm.

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.

Meeting Overview: Interferon Lambda-Disease Impact and Therapeutic Potential.

A meeting entitled, "Interferon Lambda: Disease Impact and Translational Potential," was held on the campus of the National Institutes of Health in Bethesda, Maryland, on October 25-26, 2018. To our knowledge, this was the first meeting that focused exclusively on interferon lambda (IFN-λ). The meeting's purpose was to enhance interdisciplinary communication and promote new collaborations. The gathering brought together an international group of scientists from a wide range of disciplines. Sessions included: IFN-λ Biology, Therapy and Genetic Variation; IFN-λ and Hepatitis C Virus Infection; IFN-λ in Other Infections; and IFN-λ-Hepatic Fibrosis and Cancer. The next meeting on IFN-λ is planned for 2020.

A comparison of interferon gene expression induced by influenza A virus infection of human airway epithelial cells from two different donors.

Influenza is an acute respiratory disease that can cause local annual epidemics and worldwide pandemics of different morbidity and mortality. Our understanding of host factors that modulate the frequency and severity of influenza virus infections is less than complete. In this study, we examined the inter-individual variations in the innate immune responses to H1N1 and H3N2 influenza A viruses (IAV) using primary cultures of normal human bronchial epithelial (NHBE) cells derived from two different donors (D1 and D2). Although IAV replication kinetics were similar in cultures derived from these two donors, the levels of type III interferons (IFNs) were significantly higher in D1 cells compared to D2 cells (˜31-fold↑ in D1 cells versus D2 cells; P < 0.05). The levels of IFN-λ1 protein at individual time points as well as the total amounts of IFN-λ1 secreted over 72 h were also significantly higher in D1 than in D2 NHBE cells (0.7-7.7-fold↑, P < 0.05). The relative levels of IFN-stimulated gene (ISG) expression also differed significantly between D1 and D2 cells. Our data indicate that donor-specific differences can result in significant differences in IFN and ISG induction by human airway epithelium.