Influenza virus infection is responsible for hundreds of thousands of deaths annually. Current vaccination strategies and antiviral drugs provide limited protection; therefore, new strategies are needed. RNA interference is an effective means of suppressing virus replicationin vitro. Here we demonstrate that treatment with small interfering RNAs (siRNAs) specific for highly conserved regions of the nucleoprotein or acidic polymerase inhibits influenza A virus replicationin vivo. Delivery of these siRNAs significantly reduced lung virus titers in infected mice and protected animals from lethal challenge. This protection was specific and not mediated by an antiviral IFN response. Moreover, influenza-specific siRNA treatment was broadly effective and protected animals against lethal challenge with highly pathogenic avian influenza A viruses of the H5 and H7 subtypes. These results indicate that RNA interference is promising for control of influenza virus infection, as well as other viral infections.

Changes in influenza viruses require regular reformulation of strain-specific influenza vaccines. Vaccines based on conserved antigens provide broader protection. Influenza matrix protein 2 (M2) is highly conserved across influenza A subtypes. To evaluate its efficacy as a vaccine candidate, we vaccinated mice with M2 peptide of a widely shared consensus sequence. This vaccination induced antibodies that cross-reacted with divergent M2 peptide from an H5N1 subtype. A DNA vaccine expressing full-length consensus-sequence M2 (M2-DNA) induced M2-specific antibody responses and protected against challenge with lethal influenza. Mice primed with M2-DNA and then boosted with recombinant adenovirus expressing M2 (M2-Ad) had enhanced antibody responses that cross-reacted with human and avian M2 sequences and induced T-cell responses. This M2 prime-boost vaccination conferred broad protection against challenge with lethal influenza A, including an H5N1 strain. Vaccination with M2, with key sequences represented, may provide broad protection against influenza A.

Morbidity and mortality resulting from influenza-like disease are a threat especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics are urgently needed that are efficacious against major drivers of influenza-like disease including influenza viruses and respiratory syncytial virus (RSV). Using a dual-pathogen high throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N⁴-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses and IAVs of human, avian, and swine origin. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36-56%, sustained levels of the active 5′-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAV in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burden in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC a promising candidate for future clinical development as a treatment option for influenza-like diseases.

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG_35–55-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4⁺ Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4⁺ T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP_178–191. Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG_35–55, comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG_35–55-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.