Viral- and host-targeted interventions to improve influenza disease outcomes
Despite decades of work by scientists and public health officials, influenza viruses continue to cause widespread morbidity and mortality. Each year it is estimated that these pathogens infect up to 20% of the global population, leading to nearly a million deaths. In considering this, it is clear that additional research and intervention strategies are needed. When examining influenza virus infection and subsequent outcomes, it is easiest to look at the disease from two perspective; the virus and the host. The influenza virus, a sort of obligate intracellular parasite, must coopt a myriad of host cell proteins and processes in order to complete its lifecycle, and produce infectious progeny virions in hopes of spreading to and infecting new cells and hosts. At the same time, the infected host must both detect this viral invader, as well as mount an effective response, in the form of both innate and adaptive immune pathways, to try and limit and eventually clear the viral pathogen. In considering how important these two sides of the same coin were, we decided to take a two-pronged approach and focus our research efforts on both the virus and host in the hopes of improving influenza infection outcomes. Seasonal vaccination has been, and still remains, the best strategy for preventing influenza infection. Each year, a new formulation of the seasonal influenza vaccine is designed and manufactured with the hopes of inducing protective immunity, in the form of neutralizing antibodies, against the major circulating influenza strains. Seasonal vaccine efficacy (VE), however, remains low; averaging close to fifty percent over the past two decades. Recent work has demonstrated that this is in large part due to adaptive mutations that occur during growth of the virus in embryonated chicken eggs, the major manufacturing platform in which most vaccine doses are made, due to conformational differences between eggs and the human respiratory tract in the influenza host cell receptor, sialic acid. Many of these mutations, which often occur in the major antigenic protein hemagglutinin (HA), have been shown to drastically reduce the antigenic match to circulating strains, leading to vaccination with misrepresentative antigens. In order to combat this, we took advantage of influenza based reverse-genetic systems to design a vaccine platform resistant to these mutations. In order to combat this, we utilized reverse genetics to develop an influenza virus capable of expressing two hemagglutinins, termed a Dual-HA virus. These Dual-HA viruses encode a “helper HA” that is adapted to growth in embryonated chicken eggs, alongside the HA from a clinically relevant circulating strain. Together, we demonstrated that the Dual-HA design allows for the virus to successfully grow to high titers using the “helper HA” while simultaneously stabilizing the antigenicity of the clinically relevant HA for vaccination, theoretically preventing future adaptive mutations and enhancing seasonal vaccine efficacy. While seasonal vaccination is the best strategy for preventing influenza infections, effective therapeutics will always be needed to treat those who eventually contract the virus. To date, there have been several classes of effective antivirals that have been developed, FDA-approved and are currently used to treat infected patients. All classes of these drugs, unfortunately, elicit their effect by directly targeting influenza proteins and preventing their function. While effective in the short-term, this strategy of directly targeting viral proteins is especially risky when considering the rate at which these viruses mutate and their potential to develop resistance. Amantadines for example, the first-class of influenza antivirals developed, are no longer used in the clinic due to nearly 100% of influenza strains now possessing resistant mutations. In fact, influenza strains have been isolated possessing resistant mutations to every class of influenza antivirals we possess, highlighting the need for a more universal strategy that would be theoretically very difficult to for influenza viruses to adapt to. Once again we turned to the virus and used reverse genetics to target one of the most universally conserved processes of the influenza lifecycle, genomic segment packaging. In order to produce infectious progeny virions, influenza viruses must package their genomic segments into budding virions using conserved motifs in these segments termed packaging signals. Taking advantage of this, we engineered an influenza virus that packages and propagates two additional genomic segments missing key viral proteins termed “decoy segments”. We demonstrated that these “decoy segments” can be packaged by wild-type influenza viruses during coinfection, and that this is capable of not only interfering with replication and spread of a variety of viruses but is also capable of rescuing animals from a lethal infection when administered therapeutically. Given the near universal conservation of these packaging signals, this therapeutic strategy should be effective against a wide-range of influenza viruses, as well as be near impossible for viruses to develop resistant mutations to.Having addressed issues with both vaccination and therapeutic strategies, we next turned to looking at the host immune response during influenza infection. During the early period of infection, it is appreciated that innate immune signaling, in particular type I IFN signaling, is primarily responsible for response to and control of influenza virus infections. Interestingly, it is well appreciated that pregnant women are up to four times more likely to develop severe influenza infections. This finding become even more important when considering recent work has demonstrated that severe infections lead to systemic type I IFN signaling, and that this form of IFN signaling leads to fetal malformations and even fetal demise. Despite these findings, however, influenza infections have never been shown to significantly increase the risk of fetal demise or birth defects. Taken together, this suggests that some unidentified regulator of type I IFN signaling must be protecting the developing fetus from this harmful systemic IFN response during severe influenza infections. Using CRISPR/Cas9 screening techniques, and a type I IFN reporter system, we identified G-protein coupled estrogen receptor 1 (GPER1) as a candidate regulator. Our subsequent experiments demonstrated that blocking GPER1 function with a selective antagonist lead to prolonged IFN signaling, whereas activation with a selective agonist was capable of suppressing signaling. Furthermore, using a murine pregnancy model, we demonstrated that the loss of GPER1 function lead to drastic increase in fetal demise during influenza infection, and that GPER1 function was dispensable if maternal IFN signaling was blocked. This work suggests that GPER1 is a critical regulator of type I IFN signaling, and that modulating its function could be used to toggle innate immune responses to potentially improve infection outcomes. Taken together, these studies describe new technologies and findings that could be used to improve influenza infection outcomes. By focusing our efforts on both the virus and the host we have covered an array of areas relevant to human health including vaccination, antiviral therapeutics, and regulation of potentially dangerous host immune responses.
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